DIAGNOSTIC PROCEDURES AND REAGENTS Copyright, 1963 AMERICAN PUBLIC HEALTH ASSOCIATION, INC. Fourth Edition First Printing Library of Congress catalog card number: 63-14119 za S DIAGNOSTIC PROCEDURES AND REAGENTS TECHNICS FOR THE T.ABORATORY DIAGNOSIS AND CONTROL OF THE COMMUNICABLE DISEASES FOURTH EDITION Edited by Harris and Coleman This report was prepared by the Subcommittee on Diagnostic Procedures and Reagents. It has been reviewed by the Coordinating Committee on Laboratory Methods and recommended for publication. Publication has been authorized by the Committee on Evaluation and Standards of the American Public Health Association. Published by the American Public Health Association, Inc. 1790 Broadway, New York 19, N. Y. 1963 PUBLIC HEALTH H ea \ Hy Libre v FOREWORD TO FOURTH EDITION The Fourth Edition of Diagnostic Procedures and Reagents is being published under the time-honored title, although the content of the book calls for a more descriptive title, such as Laboratory Diag- nostic Procedures in Bacteriology, Mycology, Parasitology, and Im- munology. The primary purpose of the book remains the same as in previous editions—to serve as a practical guide to the bench workers in public health laboratories. ) The Subcommittee undertook to establish one selected method, representing the best, or one of the best methods, for each type of examination as determined by the consensus of individual experts in the various fields. Committees were invited to contribute and were charged with the task of reaching agreements and of writing the com- posite, detailed procedures. Several years of committee meetings, correspondence, preparation of manuscripts, and revisions have cul- minated in a book which does credit to the members of the author groups, who, resolving differences in the spirit of give and take, selected and described the recommended methods. Changes are occurring rapidly in the diagnostic laboratory field, with the inevitable result that newer knowledge unfolding between the dates of comple- tion of the various manuscripts and the publication of this edition will not appear. The problems confronting the Subcommittee and the author groups were considerable. The mechanics involved in the joint preparation of manuscripts by scientists often in widely separated localities can readily be appreciated. Each chapter was reviewed by two or more members of the Subcommittee. The Subcommittee acknowledges with grateful appreciation the work of those primarily responsible for the preparation of the volume for the printer, among them Edmund K. Kline, Dr.P.H., Col. Francis B. Elder, M.S.P.H., and Helen F. Hough. 785 J vi FOREWORD The Subcommittee hopes that the volume will justify the enormous amount of thought, effort, and time expended by the leaders in diag- nostic laboratory work who have contributed to its preparation without remuneration, If it raises the standards of performance in public health laboratories, it will be serving its purpose. SUBCOMMITTEE ON DIAGNOSTIC PROCEDURES AND REAGENTS Avert H. Harris, M.D., Chairman Marion B. CoLEMAN, Co-Chairman Howarp L. Bobiry, PH.D. Evan T. Bynog, Pu.D. GeorrFrey Epsarr, M.D. Rapa S. MuckeNFuss, M.D. CONTRIBUTORS AjeLro, Lisero, Pu.D. (24) Chief, Mycology & Parasitology Sec., Lab. Branch, DC, Atlanta 22, ALEXANDER, AARON D., Pu.D. 19) Chief, WHO/FAO Leptospirosis_ Refer- ence "Lab., Walter Reed Army Inst. of Med. Research, Washington, D. C., 20012 ALEXANDER, HarTIE, E., M.D. (14,15,16) Professor of Pediatrics, Columbia Univ. Coll. Dljyslcjans & Surgeons, New York AverBacH, Oscar, M.D. (9) Senior Medical Investigator, VA Hospital, East Orange, N. J AvusTRIAN, RoBeErT, M.D. Professor and Chairman, Dept. of Re- search Med., Univ. of Pennsylvania Sch. of Med., Philadelphia 6 BarrHOLOMEW, JAMES W., Pa.D. (1) Professor of Bacteriology, Univ. of Southern California, Los Angeles 7 Bauer, Henry, Pa.D. (12) Director, Div. of Med. Laboratories, Minnesota State Board of Health, Univ. of Minnesota Campus, Minneapolis 14 Beaver, Pauvr C., Pu.D. (26) . William Vincent Professor of Trop. Dis. & Hyg., Tulane Univ. Sch. of Med., New Orleans 12, La. “BENHAM, Ruopa W., Pu.D. (24) Formerly Assoc. Professor of Dermatology, Columbia Univ. Coll. Physicians & Surgeons, New York, . Brair, Eugene B. (May. MSC), Pu.D. (4) Asst. Chief, Dept. of Bacteriology, Walter Reed Army Inst. of Research, Washington 12 % Bars, Joun E., Pu.D. (6) Head, Dept. of Bacteriology, Hospital for Joint Diseases, New York 35, N. Y. BooiLy, Howarp L., Pu.D (7) Member, Subcommittee on Diagnostic Pro- cedures & Reagents, APHA; and Chief, Div. of Laboratories, Dept. of Pub. Health, Berkeley 4, Calif. Borman, Earre K. (12) Director, Lab. Div., Connecticut Dept. of Health, Hartford 1 Bors, I. H.,, M.D. (12) Director, State Hygienic Lab., State Univ. of Towa, Iowa City BozicevicH, JouN (26) Senior Investigator, Dept. of Immunol Microbiological Associates, Inc., Bethes a 14, Md. Brapororp, WirLriam L., M.D. (14,15) Professor, Dept. of Pediatrics, Univ. of Rochester Sch. of Med., Seong Memorial Hospital, Rochester, *BranHAM, SARA E., So PD. (16) Formerly with NIH, Bethesda, Md. State Brooke, M.M., Sc.D. (26) Chief, Lab. ’ Consultation & Development Sec., USPHS, CDC, Atlanta 22, Ga. Brown, HaroLp W., M.D. 29 . " Professor of Parasitology, Columbia Univ. Coll, Physicians & Surgeons, New York 2, N.Y. *BROWNE, ALcor S., Pu.D. (10) Former] Chief, Microbiology Lab. Cali- fornia Dent, of Pub. Health, Berkeley Burrows, WirLLiaMm, Pu.D. (10) Professor of Microbiology, Univ. of Chi- cago, Chicago, Ill Bynog, Evan T., Pu.D. (2,8) Member, Subcommittee on Diagnostic Pro- cedures & Reagents, APHA; and Director, Lab. of Hyg., Dept. of Nat’l Health & Welfare, Ottawa, Ont., Canada Cameron, T. W, M., M.D., Pu.D., Sc.D. (26) Macdonald Coll, Inst. of Parasitology, Ste Anne de Bellevue, Quebec, Que., Canada CARPENTER, CHARLES M., M.D., D.V.M,, Pu.D. 12 ’ ) Professor of Infectious Diseases, Sch. of Pub. Health Center for Health Sci., Univ. of California, Los Angeles CHrisTENSEN, C. W., Pu.D. (4) Director, Microbiology, Difco Laboratories, Detroit 1, Mich. 7CoLEMAN, MarION B. (10) Co-chairman, Subcommittee on Diagnostic Procedures & Reagents, APHA; formerly with the New York State Dept. "of Health at Albany Conant, Norman F., (24) James B. Duke 250 of Vierohiolozys and Chairman, Dept. of Microbiolos Duke Univ, Sch. of Med., Durham, N. C. Co~nNELL, RoBErT, D.V.M. (22-1) Director, Vet. Sci. Dotty Univ. of Sas- katchewan, Saskatoon, Sask., Canada Cravirz, Leo, Dr.P.H. (21) Microbiologist in Charge, Rodlisster Gen- eral Hospital, Rochester 8, N. Crecerivus, H. GiLsert, PH.D. Director of Laboratories, es State Dept. of Health, Phoenix 7 Crort, CuArLes C., Sc.D. (1 hief, Div. of ILaboratories, Dept. of Health, Columbus 10 CuMmMiINGs, MARTIN M., M.D. (9) Assoc. Director for Research Grants, NIH, Bethesda 14, Md. Dack, Gan. M., M.D., Pu.D. (11) Director, Food Research Inst.; and Pro- fessor, Dept. of Mistoblelogy, Univ. of Chicago, Chicago 37, Ill. iDamon, SAMUEL R., Pu.D. % 12) Formerly Director, Bureau o Laboratories, Indiana State Board of Health at In- dianapolis Ohio State Note A: Chapter or chapters of authorship will be indicated by the number(s) in parenthesis following name and postgraduate degrees. Note. B: Chapter 22 (Miscellaneous Infections) consists of 10 separate sections indicated by a roman numeral following the chapter number. * Deceased. T Retired. vii viii Dosyam Crauvpe E., M.B.,, Pu.D.,, D.P.H. 11 Professor and Head, Dept. of Bacteriology & Immunology, Univ. of British Columbia, Vancouver, B. C., Canada Drake, CuarLes H., Pu.D (22-VI) Professor of Bacteriology & Pub. Health, Washington State Univ., Pullman EpsaLr, GeorrFrey, M.D. Member, Subcommittee on Diagnostic Pro- cedures & Reagents, APHA; and Super- intendent, Inst. of Laboratories, Dept. of Pub. Health, Jamaica Plain 30, Mass. *EISENBERG, GEORGE M., Sc.D. (22-11) Formerly of the Div. of Bacteriology & Serology, Philadelphia General Hospital, Philadelphia, Pa. ELpErING, GRACE, Sc.D. (14,15) Assoc. Director, Div, of Laboratories for Western Michigan, Michigan State Dept. of Health, Grand Rapids Emmons, Custer W., Pa.D. (24) Chief, Med. = Mycology Sec., Bethesda 14, Md. *Evans, Ropert E.,, Pu.D. (2) Formerly with the Iowa State Dept. of Health, Iowa City tFALK, Mrs. CAroLYN R. (27,16) Formerly with the New York City Dept. of Health, New York, N. Y. FeLoMmaN, Harry A.,, M.D. (22-IX) Professor and Chairman, Dept. of Prev. Med., Upstate Medical Center, State Univ. of New York, Syracuse 10 FeLomanN, WirLiam F., D.V.M. (9) Chief, Lab. Research in Pulmonary Dis., VA, Washington 25, D. C. FercusoN, WiLLiam W., Pa.D. (10) Chief, Microbiology Sec., Div. of Labora- tories, Michigan State Dept. of Health, Lansing Fink, Freperick C., Pu.D. (27) Director, Hospital Lab. Advisor: os Pfizer Co., Inc., New NIH, Svce, ork 17, FinLaND, MaxweLL, M.D. (7) Professor of Medicine, Harvard Univ. Sch. of Med.; and Thorndike Memorial Lab., Boston City Hospital, Boston, Mass. Forster, GeorGE F., Pu.D. (2) hief, Bureau of Diagnostic Svces, Illinois State Dept. of Pub. Health, Chicago 12 Frank, Paur F. (5) Naval Med. Research Unit 4, Naval Training Center, Great Lakes, IIL TFROBISHER, MARTIN, JR., Sc.D. (8) Formerly hief, ~ Bacteriology Sec, Chi USPHS, CDC, Atlanta 22, Ga. GarsoN, WarriELD, M.D., M.P.H. (18) Chief, Career Do Div., Office of Personnel, USPHS, Washington 25, D. C. Goesgsous, WiLrLiam S., Jr., (Cor. USA) Walter Reed Army Inst. of Research, Walter Reed Army Med. Center, Wash- ington, D. C., 20012 GORESLINE, Harry E., Pu.D. (11) Deputy Scientific Director, Armed Forces od & Container Institute, Chicago 9, 11. GREENBERG, Louis, Pu.D (27) Chief, Biologics Control ILab., Lab. of Hygiene, Ottawa, Ont., Canada * Deceased. T Retired. CONTRIBUTORS GROVE-RASMUSSEN, Morten, M.D. (28) Director, Blood Bank & Transfusion Svce, Massachusetts General Hospital, Boston 14 tHaas, Vicror H., M.D. (25) Formerly Medical Director, Bethesda, Md. *Hacan, WiLLiam, D.Sc, D.V.M. (20,21) Formerly Director, Nat'l Animal Disease Lab., Agricultural Research Svce, USDA, Ames, Iowa FHaMiLTON, Joun H., M.D. (2) Formerly Director, State Lab. of Hyg., North Carolina State Board of Health, Raleigh Hanks, Joun H., Pu.D. (9) Bacteriologist, Leonard Wood Memorial; and Professor of Pathology, Johns Hop- kins Sch. of Hyg., Baltimore 5, Md. Harpy, Arserr V., M.D., Dr.P.H. (10) Asst. State Health Officer, Florida State Board of Health, Jacksonville THarris, Ap (18) Formerly Director, VDRL, USPHS, CDC, Atlanta 22, Ga. Harris, Avert H., M.D. Chairman, Subcommittee on Diagnostic Procedures Reagents, AP san Asst. Director, Div. of Laboratories & Research, New York State Dept. of Health, Albany 1 HausLer, WiLrLiam J., Jr., Pu.D. (2) Asst, Director, State Hyg. Lab.; and Asst. Professor of Hyg. Prev. Med., State Univ. of Iowa, Iowa City Hinman, E. HaroLp, M.D., Pu.D. (25) Professor and Head, Dept. of Prev. Med., Jetfusan Medical College, Philadelphia Pa. Hogesy, Grabys L., Pu.D. (27) Director, Special Research Lab., VA Hos- pital, East Orange, N. J. Horr, J. C, Pu.D. (22.V]) Research Microbiologist, USPHS Shellfish Sanitation Lab., Gig Harbor, Wash. Horringer, NLL F., Pu.D. (5 ssoc. Professor, Univ. o Sch. of Pub. Health, Berkeley Hoop, Marion, Pu.D., D.Sc. (22-111) ssoc. Clinical Professor, Dept. of Mi- crobiology and Dept. of Trop. Med. & Parasitol., Louisiana State Univ. Sch. of Med., New Orleans; Microbiologist, Dept. of . Pathology, Charity Hospital of Louisi- ana, New Orleans *HumpHREYS, F. A.,, D.V.Sc. (13) Formerly with the Dept. of Nat’l Health & Welfare, Kamloops, B. C., Canada tHUNTER, CHARLES A., Pu.D. (12) Formerly Director of Laboratories, Kansas State Board of Health, Topeka Jackson, GeorGe GEE, M.D. (6) Professor of Medicine, Univ. of Illinois, Chicago JeLLisoN, WirLLiam L., Pu.D. (22-VII) Parasitologist, Nat'l Inst. of Allergy & Infect. Dis., Rocky Mountain Laboratory, Hamilton, Mont. Herserr G., M.D., P=a.D. USPHS, California *JOHNSTONE, Formerly Professor of Parasitology, Univ. of California Sch. of Med., Berkeley KELLNER, AARON, M.D. (28) Director, Central Laboratories, The New York Hospital-Cornell Medical Center, New York 21, N. Y. CONTRIBUTORS TKENDRICK, PEARL L., Sc.D. (10,14,15) Resident Lecturer Emeritus, * Univ. of Michigan Sch. of Pub. Health, Ann Arbor Kent, Joun F., Pu.D. (18) Assoc. Research Scientist, Div. of Labora- tories & Research, New York State Dept. of Health, Albany 1 King, Miss Evizasere O. (22-IV Gen’l Bacteriology Unit, Bacteriology Sec., USPHS, CDC, Atlanta 22, Ga. iKring, Epmunp K., Dr.P.H. Indexer Diagnostic Procedures & Reagents (4th ed.); formerly Director, attaraugus County Laboratories, Olean, N. Y Knapp, WERNER, M.D. (13) Hygiene Institute, Tuebingen, Germany Kn~iguT, VERNON, M.D. (27) Clinical Director, Nat’l Inst. of Allergy & Infect. Dis., NIH, Bethesda 14, Md. Read L. Roranp (Cor., MSC), Pu.D. Med. Research & Nutrition Lab., Fitzsim- mons General Hospital, Denver 30, Colo. KurrNER, ANN G., M.D., Pu.D. (5) Professor of Pediatrics, Bellevue Hospital, ew York, N. LancerieLp, Resecca C., Pu.D. (5) Professor and Member, The Rockefeller Institute, New York 21, N. Y. Larson, CarL L., M.D., D.Sc. (13) Professor of Microbiology and Director, Stella Duncan Research Inst.,, Montana State Univ., Missoula Lawrence, C. A., Pu.D. (1) Director, Bureau of Laboratories, Los Angeles County Health Dept.; and Assoc. Clinical Professor of Infect. Dis., Univ. of California, Los Angeles Lepper, Mark H., M.D. (16) Professor of Preventive Medicine and Head, Dept. of Prev. Med., Univ. of Illinois Coll. of Med., Chicago 12 Levine, PaIiLip, M.D. (28) Director, Div. of Immunohematology, Ortho Research Foundation, Raritan, fLonG, Esmond R., M.D., Sc.D. (3) Formerly Director, Henry Phipps Institute, Philadelphia, Pa. MacCreapy, Roserr A., M.D. (1,8) Director, Div. of Diagnostic Laboratories, Massachusetts Dept. of Pub. Health, Jamaica Plain 02130 *MAcLENNAN, Joun D., M.D. (23) Formerly with the Dept. of Microbiology, Columbia Univ. Coll. Physicians & Surgeons, New York, N. Y. MacLeop, Corin M., M.D. (7) Professor of Medicine, New York Univ. Sch. of Med., New York 16, N. Y. MarTIN, DonaLp S., M.D., Dr.P.H. (24) Chief, Training Branch, USPHS, CDC, Atlanta 22, Ga. McCarty, MacLyn, M.D. (5) Professor and Member, and Physician-in- hief, The Rockefeller Institute, New York21, N. VY. McCrung, L. S.,, Pa.D. (23) Professor of Bacteriology and Chairman, Dept. of Bacteriology, Indiana Univ., Bloomington McGee, Roser T. (28) Product Service Manager, Ortho Pharma- ceutical Corp., Raritan, N. J * Deceased. 1 Retired. 1x *MEeDLAR, Epcar M., M.D. (9) Formerly with H. M. Biggs Memorial Hospital Laboratories, Ithaca, N. Y. Meyer, K. F., M.D., Pu.D., D.V.M. (13) Director Emeritus, The George Williams Hooper Foundation for Med. Research, Univ. of California, San Francisco Medi- cal Center, San Francisco 22, Calif. iMickLE, Frienp Leg, Sc.D. (2) Formerly Director, Bureau of Labora- tories, Connecticut State Dept. of Health, Hartford 1 Moun, James F., M.D. (28) Professor of Bacteriology & Immunology, State Univ. of New York Sch. of Med., Buffalo Moopby, Max D., Pa.D. (1) Chief, Staphylococcus & Streptococcus Unit, Bacteriology Sec., Lab. Branch, USPHS, CDC, Atlanta 22, Ga. Morse, ErskiNE V., D.V.M., Pu.D, (22-X) Professor of Microbiology and Dean of Veterinary Med., Purdue Univ. Sch. of Vet. Sci. & Med., Lafayette, Ind. Morton, Harry E., Sc.D. (17,22-V) : Professor of Bacteriology, Dept. of Mi- crobiology, Univ. of Pennsylvania Sch. of Med., Philadelphia 4 Moss, Taeas, M.D.,, D.M.S.,, D.T.M. & H. Professor of Prev. Med., New York Univ. 5 Med., NYU Medical Center, New ork 1 TMvuckeNFUSs, Rarer S., M.D. Member, Subcommittee on Diagnostic Pro- cedures & Reagents, APHA: formerl Technical Director, Naval Med. Researc Inst, Nat’l Naval Medical Center, Bethesda 14, Md. NeTer, Erwin, M.D. (10) Director of Bacteriology, Children’s Hos- ital; and Assoc. Professor of Bacteriology Pediatrics, State Univ. of New York Sch. of Med., Buffalo Niven, CHARLES F., Jr., Pu.D. (11) Scientific Director, American Meat Insti- tute Foundation, Chicago 37, IIL Packer, Henry, M.D., Dr.P.H. (17) Professor of Preventive Medicine, Dept. of Prev. Med., Univ. of Tennessee Coll. of Med., Memphis PerraN, EvizaBera I., Pa.D. (4) hief, Div. of Microbiology, Bureau of Laboratories, Maryland State Dept. of Health, Baltimore Pike, Rosert M., PH.D. (3) Professor of Microbiology, Univ. of Texas, Southwestern Med. Sch., Dallas PirTMAN, MARGARET, PH.D. (14,15) Chief, Lab. of Bacterial Products, Div. of Biologics Standards, NIH, Bethesda ’ PorriTzer, R., M.D. (13) Research Associate of Fordham Univ., Pn Library of Medicine, Bethesda 14, PorrTNoOY, Josepu, Pu.D. (18) Director of Immunological Research, Hynson, Westcott & Dunning, Inc., Balti- more 1, Md. Quan, S. F,, Pu.D. (13) Bacteriologist, San Francisco Field Sta- Hon USPHS, CDC, San Francisco 18, alif. RamMmeLkaMP, CuarcEs H.,, M.D. (5) Professor of Medicine, Western Reserve Univ., Cleveland, Ohio fRANDALL, Raymond (Cor., USA), D.V.M. (20,21) Assoc. Frofesar, Dept. of Med. Univ. of Maryland Sch. of Med., Baltimore 1 RanTz, LoweLL A., M.D. (5) Stanford Medical Center, Palo Alto, Calif. ReinuArD, Karr R., D.V.M., Pu.D. (19) Div. of Research Grants, Review Branch, NIH, Bethesda 14, RoBiNsON, LucIiLLE Ty (22- VIII) Research Microbiologist, TB Cooperative Shady Control Lab., VA Hospital, Atlanta a, RosenrieLp, R. E., M.D. (28) Attending Hematologist and Director, Blood Bank, The Mount Sinai Hospital, New York 29, N. Y. SANDERS, ArVEY (CoL., USA) (17) of the Chief, Research & Develop- ment Group, USA, Washington 25, D. C. ScueErAGO, Morris, D.V.M. (2) Professor and ead, Dept. of Micro- biology, Univ. of Kentucky, Lexington SeastoNng, C. V., M.D. (10) Professor and Chairman, Dept. of Med. Microbiology, Univ. of Wisconsin Med. Sch., Madison SuavcuNEssEy, Howarp J., Pu.D. (2) Chief, Div, of Laboratories, Illinois Dept. of Pub. Health; Professor and Head, Dept. of Pub. Health, Univ. of Illinois Coll. of Med., Chicago SuookHOFF, Howarp B., M.D. (26) Chief, Div. of Trop. 'Dis., City of New York Dept. of Health, Sai Health Center, New York 56, N. Y. Sige, M. MicuaeL, Pua.D. (3) Professor of Microbiology, Univ. of Miami Sch. of Med.; and Research Di- rector, Variety Child Research Founda- tion, Coral Gables, Fla. Srocum, GLENN G., Pr.D. (11) Director Div. of Microbiology, FDA, Dept. of Health, Ed., & Welfare, Wash: ington 25, D. C. Smita, C. Ricuarp, M.D. (9) Director of boratory, Barlow Sane torium; and Assoc. Professor of Pathog nly, ‘of Southern Calif. Sch. of Los Angeles Smit, Cartes E.,, M.D., D.P.H. (3,24) Dean, Univ. of California Sch. of Pub. Health, Berkeley Smita, Louis D. S., Pua.D. (23) Dept. of Botany & Bacteriology, Montana State Coll., Bozeman StaviTsky, AsraM B., V.M.D., Pu.D. (19) Professor of Microbiology, Western Re- serve Univ., Cleveland, Ohio STEENKEN, WiLLiaM, Jr., Sc.D. (9) Member, Trudeau Foundation, Saranac Lake, N. Y. Inc., * Deceased. 1 Retired. CONTRIBUTORS iStEIN, CLARENCE D., V.M.D. (20) Formed with the Agriculture Research Svce, USDA, Washington, D. C. Began Kurt, M.D. {2 8) Professor, Dept. of Pathology, Univ. of Illinois, Chicago SuLkiN, S. Epwarp, Pu.D. (3) Professor and Chairman, Dept. of Micro- biology, Univ, of Texas Southwestern Med. Sch., Dallas 35 1Sunkes, E. J., Dr.P.H. Director, Lab. Branch, con Dept. of Pub. Health, Atlanta 3 TuaL, E.,, V.M.D. (13) State Veterinary Medical Institute, Stock- holm, Sweden Urppyke, ELaiNe L., Sc.D. (5) Chief, Bacteriology Training Unit, Lab. Consultation & Development fo USPHS CDC, Atlanta 22, Ga. VERA, HarriETTE D., PH.D. (4) Director, Quality Control, B-D Labora- tories, Inc., Subsid. of Becton, Dickinson Co., Baltimore, Md. WALTER, Carr, M.D. (1) Surgeon, Peter Bent Brigham Hospital; and Clinical Professor of Surgery, Har- vard Univ. Sch. of Med., Boston, Ma Warp, MarTtHA K., Sc.D. (19) Chief, Bacteriology Div.,, USA Med. Unit, Fort Detrick, Frederick, Md. WepuM, ArNoLp G., M.D. (3) Safety Director, Safety Div.,, USA Med. Unit, Fort Detrick, Frederick, WEINSTEIN, Louts, M.D., Pu.D. (1,16) Professor of Medicine, Tufts Univ. Sch. of Med.; and Assoc. Physician-in-Chief and Chief, Infect. Dis. Svce, Pratt Clinic- Sw England Center Hospital, Boston, Mass. WibeLock, Danier, PH.D. (18) Deputy Director, Bureau of Laboratories, City of New York Dept, of Health, New York13,'N. Y. TWiLcox, Miss AiMEE (25) Formerly with NIH at Columbia, S. C. WiLson, ARMINE T., M.D. (5) Chief of Microbiology, Alfred I, duPont Inst., The Nemours Foundation, Wilming- ton 99, Del. Wisg, Ropert I, M.D., Pu.D. (6) Magee Professor of Med. and Head, Dept, of Med., Jefferson Medical Coll., Philadelphia, Pa. YAGER, RoserT H. (CoL., USA) (19) Director, Div. of Vet. Med., Walter Reed Army Inst. of Research, Walter Reed Army Medical Center, Washington, D. C., 20012 YAriNsky, ALLEN, PH.D. (26) Research Scientist, Parasitology Sec., Bureau of Laboratories, New York oy Dept. of Health, New York 32, N. Foreword Contributors Chapter 1 General Procedures . : % 3 3 2 Mailing, Receiving and Provesiing Specimens : 3 Laboratory Infections and Accidents . 4 Culture Media . 5 Streptococcus Infections 6 Staphylococcus Infections . 7 Pneumococcus Infections . 8 Diphtheria . gs 9 Tuberculosis and Leprosy : 10 Bacterial Infections of the Gastrointestinal Tract : 11 Bacterial Food Poisoning . 12 Brucellosis . 13 Pasteurella Ttortions 14 Whooping Cough 15 Hemophilus Infections . 16 Bacterial Meningitis . . 17 The Venereal Diseases, Trdtudire of Syphilis ‘ 18 Syphilis . 19 Leptospirosis 20 Anthrax : : 21 Glanders and Melioidosis ‘ 22 Miscellaneous Infections 23 Anaerobic Infections Fungus Infections Malaria . ea Helminthiasis and Intestinal Protozoiasis Antimicrobial Susceptibility Tests . Blood Grouping and Rh Typing . Index . xi CONTENTS Page Vv vii 71 89 105 187 207 222 231 261 287 319 337 357 398 414 426 462 502 544 578 592 599 662 699 739 777 821 834 869 Tr - a = , : . “0 ® a wh va weed dress vg 2 cn leoqunpe Fa u vs Ny 5 : . - | . ye: a . WI pan - a» - * 2 Jue a ow Eevee ) ) TL ve FU . . teeny ap . hg » % 3 wy So " a ) ¥ Tet L Bo, EL, BT, - a 3 a . 5 : cp 4h BT i # Hand “ . 3 &, i tip a 0 os a0 7% peut ® PGT od FEY : 3 “* He & % Tu Ee * - = ue Biv mg at vb GER i x SE, v5 a B75 v 2 “ . 3 oF sg vy 3 BE ew ¥ . B ke oe mart CL | EE iF @ CHAPTER 1 GENERAL PROCEDURES I. Suggestions to the Clinician and the Health Officer A. General Remarks i. B. Collection and Submission of Specimens TI. General Administration of the Laboratory A. Liaison with Clinicians and Health Officers B. Laboratory Standards and Methodology ITI. Selection and Use of Supplies and Equipment Microscopes Incubators Water Baths Refrigerators, Cold Rooms, Freezers Centrifuges Rotators and Shaking Machines Glassware and Plastics Other Equipment Water Stills and Demineralizers Glassware Washing and Handling Hot-Air Sterilizers and Sterilization Steam Sterilization Chemical, Filtration and Ultraviolet Sterilization IV. Hazards in the Laboratory A. Physical Hazards B. Hazards in the Use of Chemicals C. Treatment of Accidental Poisoning from Hazardous Chemicals V. Inoculation of Media and Fishing of Colonies Inoculation and Fishing of Culture Plates Inoculation of Culture Tubes Recognition of Blood Culture Contamination Incubation in Increased Carbon Dioxide Incubation under Anaerobic Conditions Biochemical Tests VI. Stains Blood Film Stains Acid-Fast Stains Gram Stain Loeffler’s Methylene Blue Stain Malachite Green Stain Wayson’s Stain G. Dienes Stain H. Macchiavello Stain VII. Test Procedures A. Hydrogen Sulfide Production “ B. ' Indole Production oe B afad . Hi C. Voges-Proskauer Test 5 y ZRER-EOERDOE> IHgOwe AmgOwe 2 GENERAL PROCEDURES Sorensen’s pH Buffers Dichromate Cleaning Fluid D. Methyl Red Test E. Reduction of Nitrates F. Catalase Test G. Coagulase Test H. Oxidase Test 1 KX. VIII. Laboratory Animals Care and Feeding Identification of Test Animals Injections of Guinea Pigs Injections of Mice Injections of Rabbits Post-Mortem Examinations lool alte IX. Fluorescent Antibody Technics in Diagnostic Bacteriology A. Fluorescent Microscopy and Photography B. Labeling Antibody Solutions with Fluorescent Dyes C. Use of Fluorescent Antibody Solutions X. Interpretation of Laboratory Results References I. SUGGESTIONS TO THE CLINICIAN AND THE HEALTH OFFICER A. General Remarks This mantal is written essentially for the diagnostic bacteriologists who operate our public health laboratories. We address these first remarks, however, to the clinicians and the health officers who use the laboratory services, because only when the laboratory is used with intelligence and understanding can its work yield the most rewarding information. Laboratories are expensive to operate. Reasonable re- straint is therefore necessary. Otherwise, the efforts that should be spent on important tests will be dissipated on quite needless tests. When a large outbreak of enteric disease occurs in a mental institution, for example, it is usually necessary to obtain stools from only a rep- resentative sampling of the ill patients to determine that Shigella sonnei, say, is involved. The other patients with the same symptoms do not require stool tests before treatment can be commenced. When symptoms subside, the required release specimens from these patients can still be obtained, yet the load on the laboratory during the crisis of outbreak will have been kept within reasonable limits. As a result, time will be gained for a more efficient investigation in the first place of the cause of the outbreak. GENERAL PROCEDURES 3 While in the presence of such an outbreak the examination of food handlers is certainly indicated, in the absence of an outbreak the routine examination of food handlers and other groups of individuals for carriers of pathogenic bacteria is contraindicated, so small is the yield in terms of the outlay involved. The immediate submission of throat cultures from an entire school because one or two cases of diphtheria have occurred in one of the classes is another example of wasteful use of the public health laboratory. Despite the great amount of work involved, no very useful knowledge is obtained, since the carrying of diphtheria organisms in the throat is often erratic and transient, with little predictable relation to the onset of clinical illness. Daily examination of the immediate contacts by the school physician or nurse, followed by submission of throat cultures from those who develop sore throat, temperature ele- vation, nasal discharge or other signs, is distinctly more rewarding. In such a scheme, the cultures are taken at a diagnostically ad- vantageous time, and useless effort is avoided. The public health laboratory should be employed judiciously—at times intensively—but always with the value of the tests weighed against the cost in human effort. When outbreaks or unusual situations arise, the laboratory director should be consulted. He can advise what specimens to send and their proper scheduling. The clinician and the health officer should appreciate, too, the necessary limitations of the laboratory with reference to the commonly performed diagnostic procedures. For instance, throat cultures sent to the public health laboratory are of diagnostic value chiefly in diphtheria and in streptococcal infections. When the laboratory work can be done at the hospital, tests for pneumonia and laryngitis incited by Hemophilus influenzae may be added. In pulmonary infections, also, if no sputum is being expectorated, the throat swab may be used to culture the small amounts of sputum that generally appear at the epiglottis after a cough. Under such circumstances, throat swabs may yield important information. However, the use of throat cultures to detect other predominating microorganisms is likely to be misleading, because the organism recovered may not be the cause of the patient’s symptoms, or even the one that predominates in the throat. Cultures should be taken before antibacterial treatment is given, because it is not unusual to fail to culture highly sensitive bacteria from specimens collected within 6 to 12 hr after beginning appro- priate antibacterial treatment, even though the bacteria may still be visible in direct films. If the culture is not taken before antibacterial treatment is begun, the laboratory should be informed of the anti- 4 GENERAL PROCEDURES bacterial drugs being used. Then, the laboratory can take specific inhibitory measures, such as p-aminobenzoic acid for sulfonamides, or penicillinase for penicillin, or even a simple dilution, which may make possible the recovery of bacteria that otherwise could not be cultured. From time to time unusual laboratory tests may be indicated. In such cases it is wise to obtain advice directly from the laboratory to verify the value of the procedure being considered and to learn the proper collection and shipment of specimen material. Otherwise, an unsatisfactory specimen may be submitted and much useless effort spent. In this connection public health laboratories distribute appro- priate pamphlets of explanation and instruction. B. Collection and Submission of Specimens It is highly important that the laboratory forms which accompany specimens be filled in carefully and completely. Additional pertinent information about the case is often of great help to the laboratory and should certainly be added whenever it might possibly be of value. Checking to be certain that the specimens have been properly collected and identified, with full information given, is the clear responsibility of the clinician or health officer who is submitting the specimens. In the collection of blood for films, counts, blood grouping and cross matching, cultures or serologic tests, the danger of transmitting homologous serum hepatitis is ever present. Indeed, this grave and quite unnecessary disease is widespread enough to demand the utmost care in the taking of blood specimens. It has been spread from patient to patient by the improper use of a single lancet sterilized between applications only by immersion in 70 per cent alcohol. Be sure that all lancets, needles and syringes are properly sterilized. Neither chemical disinfection nor less than prolonged boiling or autoclaving can assure destruction of the virus. For the obtaining of blood, use either the small separately wrapped sterile disposable lancets, syringes and nee- dles now available commercially; or individual lancets, needles and syringes, sterilized by autoclaving for 30 min at 121.5° C (250.7° F).2 From time to time difficulty is experienced in the performance of venipunctures for blood cultures or serology. Attention to the sugges- tions below will frequently obviate such difficulties: 1. For a small vein, particularly in a child, use a finer needle than usual—for example, a 25 gauge needle. 2. To make the arm veins prominent, “milk” the arm vigorously toward the tourniquet with the hand, meanwhile having the patient open and close his fist. 3. Re sterilizing or using a syringe, move the plunger back and forth within the barrel a few times, with the needle attached, in order to make perfectly sure that the lumen of the needle is patent. GENERAL PROCEDURES 5 4. In the collection of blood for cultures, observe the most rigid aseptic technic. Otherwise, contaminating microorganisms may spoil the culture. An effective technic is the one utilized by the American Red Cross Donor Service: Using forceps and sterile gauze, scrub the skin with 5 per cent coconut oil soap. Remove the lather at the end of the scrubbing with a sterile applicator saturated in 70 per cent alcohol. Then apply 2 per cent aqueous iodine with sterile applicator, allowing the iodine to remain on the skin until dry. Finally, remove the iodine with one or more sterile applicators saturated in 70 per cent alcohol. 5. For most effective results, draw blood for culture, when possible, just as the patient’s temperature begins to rise—during or just after a chill. Review ap- propriate chapters of this volume for further information on the blood cul- tures indicated for the various specific etiologic agents. Many blood specimens are received in the laboratory unfit for serologic examination because of hemolysis. This troublesome condi- tion can usually be avoided by careful adherence to the following rules: a. Use an absolutely dry syringe, since even a little moisture may cause hemolysis. Sterilization by autoclaving is best. Syringes sterilized by boiling are usually not perfectly dry when used. Boiled syringes are satisfactory, however, if rinsed with sterile physiological salt solution after removal from the sterilizer. b. Withdraw the blood under rigid aseptic precautions, since contamination may cause hemolysis. c. Empty the blood from the syringe into the test tube slowly, always first removing the needle. Roughly handled blood may hemolyze. Allow the blood to stand undisturbed at room temperature for at least an hour before refrigeration, to permit clot to form and retract. d. Send the specimen promptly, refrigerating it until mailing time. It is some- times best to use special delivery or a messenger, for the sooner the blood is received the less the danger of hemolysis. e. Mail bloods as early in the week as possible, since bloods received at the end of the week may hemolyze over the weekend. Avoid mailing bloods in very cold or very hot weather because extremes of temperature may cause hemolysis. Specific directions for the collection of specimens for the various diagnostic procedures will be found in the appropriate sections of this book. The clinician sheuld give careful directions to nurse and patient as indicated and should specifically check to see that the directions are followed. Once collected, the specimen should be transported or mailed to the laboratory as expeditiously as possible (see further discussion in Chapter 2, “Mailing, Receiving and Processing Specimens”). Il. GENERAL ADMINISTRATION OF THE LABORATORY A. Liaison with Clinicians and Health Officers A much closer acquaintance between clinicians and health officers than frequently exists is greatly to be desired. If the laboratory is to 6 GENERAL PROCEDURES render the most efficient service, its bacteriologists must learn from the clinician the problems of the field. The clinician, on the other hand, must learn from the bacteriologists more about the functions and interpretations of the various diagnostic laboratory tests. Informal consultations in the laboratory and over the telephone are therefore of great value. They should be encouraged in spite of their time- consuming nature. Of particular worth are scheduled conferences in which clinicians and health officers exchange their experiences and special knowledge with the information and special skills of the laboratory workers. The tests in the laboratory are done with greater interest and understanding when the laboratory worker knows about the case and understands why the test is required. The specimens themselves are selected and taken more intelligently and carefully when the physician understands clearly what the laboratory can do with them. B. Laboratory Standards and Methodology While variations are to be expected in the technics of different laboratory workers, reasonably close adherence to the procedures that are approved in a given laboratory is of paramount importance and must be accepted as a guiding principle by all workers. Research for improved methods and the trial of new methods described in the literature are definite responsibilities of the public health laboratory and are to be encouraged and fostered at every opportunity. Changes in the procedures, however, are to be made only with the approval of the responsible director or chief in charge and should be posted in writing. The unequivocal goal of every laboratory is to render as prompt and accurate service as humanly possible. In particular there should be a check of the work on every specimen, and a check for accuracy of every finished report. The integrity and discretion of every worker must be above reproach throughout the processing of the specimen; reports must be made only to the physician, the health officer or a designated representative. The laboratory and its workers should grow professionally—the laboratory by giving periodic seminars and in- service training, the workers by their attendance of professional meet- ings and refresher courses when these are available. Ill. SELECTION AND USE OF SUPPLIES AND EQUIPMENT The public health diagnostic laboratory will necessarily have a large amount of expensive equipment. Workers should be instructed in the reasonable care and maintenance of this equipment. There should be GENERAL PROCEDURES 7 one worker in every laboratory, preferably a competent mechanic, who will be responsible for the maintenance and periodic checking of all equipment. He will do well to maintain spare parts for some of the elements most commonly needing replacement. A. Microscopes®* The bacteriological laboratory requires a compound microscope with three objectives as a minimum. A binocular microscope is recom- mended in order to minimize eye fatigue. (See Fig 1.) Source: Bausch & Lomb Figure 1—The microscopic path of light. 8 GENERAL PROCEDURES The microscope consists of a stand; stage, substage condenser, mirror, body tubes, focusing adjustments, objective and ocular lenses. A mechanical stage is a useful accessory. The microscope must pro- vide magnification and resolution (ability of lens to distinguish fine detail in structure, or to image closely spaced objects so that they can be recognized as separate structures). It must also provide correction of spherical aberration (inability of lens to bring light rays from all points on a lens surface to a common focal point) and correction of chromatic aberration (inability of lens to focus light of different colors at a single point). Lenses—Objective lenses are supplied for general work as achromatic (free from chromatic aberration, hence converging light rays of different colors satisfactorily to a single focus). They are adequately corrected for all but the most critical work. Apochromatic objectives (most completely corrected) are used for advanced microscopy, and they approach perfect definition. The 10X, 16 mm objective is known as the low power. The 43X, 4 mm objective is the high, dry. The 97X, 1.8 mm objective is the oil-immersion, which is necessary for studying minute structures. The microscope may be ob- tained with coated optics for detailed work. The thin, tough coating used produces a sharper image by eliminating light scattering. Eyepiece lenses magnify and correct the image formed by the objective lens. Huygenian eyepieces are for achromatic objectives, and compensating eyepieces for apochromatic objectives. Wide-field eye- pieces extend the fields of vision and have high eyepoints, permitting wearers of spectacles to see the whole field. The usual eyepieces magnify 5 or 10 diameters. Final magnification is the product of objective and eyepiece magnification. For example, the combination of a 97X oil-immersion objective with a 10X eyepiece produces a magnification of 97 X 10, or 970 diameters. Condensing lenses concentrate light on the object at the proper angle. Modifications of standard microscopes may be obtained for special- ized examinations such as bacteria counts in milk or food (calibrated for standard counting factors) and photomicrography. Illumination—The basic requirements for a satisfactory illumi- nator are: 1. Condenser in light source 2. Iris diaphragm in light source 3. Means for focusing image of bulb filament on plane of microscope condenser (Koehler illumination) 4. Intensity control (transformer or neutral-density filters) 5. Provision for inserting filters into light path GENERAL PROCEDURES 9 The selection of a lamp meeting these requirements is most im- portant, for no microscope can be any better than its light source permits, The microscopist who wishes to operate efficiently should adhere to the following method of illumination: Use the plane surface of the mirror of a microscope equipped with condenser. Place the lamp 6 to 8 in. from the microscope mirror and, by focusing either the lamp condenser or the bulb base, focus the image of the bulb filament on the closed diaphragm of the microscope condenser (see by looking into substage mirror). Remove any ground glass from the lamp system when focusing. Maximum resolving power is available only when the back lens of the objective is evenly and completely filled with light. Remove eyepiece and look through the body tube to check for this condition. Only then can the full numerical aperture and resolving power of the objective be utilized for separation of finest details. It is often necessary to reduce light intensity to gain contrast, either by placing one or more neutral- density filters in the light path or by means of a transformer. When intensity controls are not available, the general practice is to reduce intensity by lowering the microscope condenser or by closing down the condenser iris diaphragm. Contrast is gained in this manner, but resolving power is lost in direct ratio to reduction of the numerical aperture. Adjust height of the microscope condenser for each slide by closing the lamp diaphragm and focusing the condenser to bring the image of the lamp diaphragm into focus with the specimen. Then open the lamp diaphragm just enough to clear the field. If maximum resolving power of the oil-immersion objective is desired, place im- mersion oil between the condenser and the slide, as well as between the slide and the objective. One eyepiece tube on the binocular body is focusable and should be adjusted to correct for minor differences in users’ eyes. Focus the microscope for the fixed eye tube, then adjust the other. Also, adjust the interpupillary distance on binocular microscopes for each individual. Care and precautions—Use improved immersion oil, such as Crown oil or Cargille’s. Cargille’s oil is available in two viscosities, the more viscous generally used for immersing the condenser. Keep the oil free from dust in a stoppered bottle. Carry microscopes with both hands and avoid jarring them. Keep microscopes covered when not in use. Keep microscopes clean. Do not blow breath on lenses to remove dust. Never wipe lenses until first removing dust with an air syringe or camel's hair brush, and then wipe only with lens paper or soft, clean, washed cloth. Optical glass is softer than window glass and is 10 GENERAL PROCEDURES easily scratched. Do not handle lenses. The inside lenses of objectives and eyepieces can be dusted with a brush or an air syringe, but objec- tives should not be taken apart. Leave such operations to the micro- scope repairman. The outside surfaces of the dry objectives, con- denser and oculars may be cleaned with distilled water. The outside surfaces of the oil-immersion objective and the top lens of the con- denser may be cleaned with xylene. Wipe dry immediately. Occa- sionally, clean slideways of the coarse adjustment and the mechanical stage with a cloth moistened in xylene. Remove any excess with a dry cloth and relubricate with vaseline. Dark-field microscopes—The dark-field microscope utilizes a special condenser which focuses light rays obliquely on the specimen, causing it to appear luminous. Clean, scratch-free slides for dark- field use should be of optimum thickness, as required by the condenser in use, and cover slips should be 0.13 to 0.17 mm in thickness. In all cases, manufacturer’s directions should be closely followed. In setting up a preparation, first focus the dark-field condenser by raising it until its surface is just below the level of the stage. At the proper level a small piece of paper can just be inserted between the condenser and a slide resting on the stage. Remove the paper and slide, and with 10X eyepieces, focus the 16 mm objective on the ring, if any, which is engraved on the surface of the condenser. Center this ring by means of the adjusting screws on the sides of the condenser. Place a drop of heavy immersion oil, free from bubbles, on the condenser. Then place the specimen slide on the stage, being sure to use a slide of proper thickness for the condenser. Place regular immersion oil on the top of the slide. Insert a funnel stop in the oil-immersion objective and focus on the specimen. Use the outer screws of the condenser to center the built-in lamp, if the instrument has one, or adjust the lamp diaphragm as necessary if you are using an outside lamp. All filters should, of course, first be removed from the outside lamp. The diaphragm should be adjusted to give as close to parallel rays of light as possible. This is done by imaging filaments on the wall about 15 ft away. The field should appear dark, and the object bright and clear. The following technic, with no funnel stop and using the high, dry objective, gives a larger field for examination and has been found quite satisfactory. Remove the filters from the microscope lamp. Lower the dark-field condenser and place a drop of oil in the center of its upper surface. Place the center of the film to be examined directly over the center of the oil and raise the substage condenser until the slide is almost lifted from the stage. There should be no air GENERAL PROCEDURES 11 bubbles. Next, gradually lower the high, dry objective until well- illuminated dancing particles are seen, then carefully adjust the light so that these particles are plainly visible. Particles may be seen when the objective is above or below this critical position, but they do not have characteristic motion or clear definition. With the microscope so focused, examine the slide, particularly those portions that show a predominance of the dark field and relatively few dancing particles, which are chiefly foreign matter. A 16 mm objective may be used with water rather than oil between the condenser and the slide, especially for observing agglutination of leptospirae. Stereoscopic microscopes—These microscopes are valuable in the study of colony characteristics, fishing of colonies, identification of parasites, microdissection, etc. They enable the observer to see clearly in three dimensions by using paired objectives. Stereoscopic microscopes .are available with single or multiple pairs of objectives. Recommended magnifications are 6.3X to 30X. Combinations are available for use with either transmitted or reflected light. Phase-contrast microscopes—Phase-contrast microscopes employ controlled illumination whereby slight changes in thickness and re- fractive index, which cause slight irregularities in wave fronts not visible in the bright-field microscope, are transformed into cor- responding variations of brightness so that the structure is rendered visible. Direct light and diffracted light are utilized. These microscopes enable the bacteriologist or parasitologist to examine unstained living material or structures which differ little in optical path (refractive index X thickness) from their surroundings by improving the contrast between the specimen and the mounting medium. Phase contrast may be advantageous for studying bacterial cultures, spores and flagellae, spirochetes, cell structures, protozoa, red blood cells, platelets, yeasts and molds. New applications may be found. To operate the phase microscope, rotate the nosepiece to place the proper objective in the optical path, rotate the turret to the correspond- ing annular stop, and center the condenser diaphragm image and phase- altering pattern by using the auxiliary telescope in place of the eye- piece. (Follow manufacturer’s detailed instructions.) Ultraviolet microscopes—The application of fluorescent antibody technics in the rapid identification of microorganisms has created a demand for ultraviolet illuminators. Almost any good monocular 12 GENERAL PROCEDURES microscope fitted with a cardioid type of dark-field condenser and with the proper filters will suffice. Special fluorite lenses are not re- quired if the optical system is free of inherent fluorescence. Illuminators may be purchased from either foreign or domestic suppliers. The light source emanates from a high-pressure mercury vapor lamp which delivers a high-intensity peak of energy in the near- ultraviolet spectrum, at a wave length of about 3800 A. The light is modified further by suitable ultraviolet pass filters. The carbon arc lamp formerly used creates maintenance difficulties which render it impractical. Equipment should be set up in a darkened room. (Caution: Ultraviolet rays are harmful to the naked eye and should therefore be passed through a filter opaque to the rays.) Fluorescence microscopy using fluorchromes has been applied to bacteriological and histological examinations such as the observation of microchemical processes in tissues. B. Incubators The need for adequate incubator space should be emphasized. Incubators equipped with thermal control units are necessary in most bacteriological operations to provide optimum constant tempera- tures for the growth of microorganisms. There are several types of incubators. Gravity-convection models provide for uniform temperatures inside the cabinets by natural cir- culation of air, whereas the mechanical-convection types circulate air by means of a blower or fan. Adequate insulation of these anhydric models by a material such as glass wool is important. In water-jacketed types, a water jacket surrounds the chamber on five sides and maintains a constant and uniform temperature by radiation. Most large laboratories have well-insulated walk-in incubator rooms with forced air circulation. Constant humidity near saturation is important to prevent drying of culture media, and where there is a flow of air, as in mechanical- convection types, it is often necessary to place a pan of water inside the chamber. Water dropping on a heating element maintains a con- dition of saturated humidity in wet-type incubators. Humidity in incubator rooms can be regulated by adding steam to provide moisture. The mechanism for steam injection may be controlled by a goldbeater’s skin regulator, which may be devised locally (see Fig 2). A membrane of this type elongates when moist and contracts when dry, operating an electric steam valve. Water-jacketed incubators have a relatively constant humidity because air flow is not necessary for heat distribution. GENERAL PROCEDURES 13 Steam Line M [o lrsokety Thermostat 10 Volt J Lie Carrer? Jor steam cut of Sepik erly “2.5% Nut 4 Stearn i ; Reduction Terminal fa | Clamp -S xX > N & RY § y~ 76 Incubator Micro Switch Condensate Funne/ § Drain Source: Wisconsin Laboratory of Hygiene Figure 2—Incubator humidifying equipment. The recommended standard incubator temperature for pathogenic microorganisms as well as for standard plate counts is 35° C (95° F).* This temperature has been generally accepted as being best for the cultivation of pathogenic microorganisms. A major reason for not recommending 37.5° C is the safety factor, for even a varia- tion of +2° C would result in a dangerously high temperature, and pathogens grow as well at 35° C as at 37.5° C. Equip all incubators with accurate thermometers and check oc- casionally against a Bureau of Standards thermometer, if available, or against several other thermometers, to eliminate those that are inaccurate. Check periodically to see that mercury columns are con- tinuous. In addition, maximum-minimum thermometers or recording thermometers are recommended for all incubators and are useful in determining variations, not otherwise detectable, which preferably should not exceed =£0.5° C in any part of the chamber, In order to detect areas where temperatures are above or below those recom- * Unless otherwise indicated, incubator temperature will be understood to be 35° C = 0.5° C throughout this publication. 14 GENERAL PROCEDURES mended, place at least one small thermometer on shelves where cul- tures are incubated. Immerse the bulb of the thermometer in liquid (glycerol, water or mineral oil) to provide temperature readings un- influenced by brief transient fluctuations of temperature on the maximum-minimum thermometer. Record daily readings. Uneven cabinet temperatures may be caused by improper venting, use of bottom heating elements only, or overloading. In general, incubators with inside dimensions smaller than 20 by 20 by 24 in. are not recommended. Stack petri plates with glass covers in the inverted position on shelves in piles not more than four deep, and separate one stack from another and from the walls to allow for air circulation, with piles placed directly above each other on successive shelves. Plates with porous tops or with metal tops and absorptive top liners need not be inverted. Room temperature variations or drafts contribute to uneven in- cubator temperatures. In cold climates do not place incubators against outside walls. Incubator chambers should be draft-free and dust-tight, have a steady flow of heat and rapid heat recovery. C. Water Baths Using a sensitive thermoregulator, a heating coil, a mechanical stirrer and an electronic relay, one may construct one’s own water bath in any suitable container. The bacteriological or serological laboratory usually provides itself with ready-made baths of stainless steel or copper, electrically heated and constructed to maintain a constant temperature with #0.5° C accuracy. The baths should be suitably insulated to provide this degree of temperature control, and the cause of any larger variation should be checked immediately. There is usually a temperature-adjustment knob which allows the bath thermostat to be set at any temperature between room tempera- ture (20° C to 22° C) and either 60° C or 100° C, depending on the maximum available. Intermittent-immersion heaters on water baths should respond quickly to the thermostatic control and reach the desired temperature within a short time. In areas where tap water is very hard, the water baths should be filled with distilled water in order to prevent scale. Even then, they will require occasional cleaning. The addition of 1 ml of 10 per cent benzalkonium chloride for each gallon of water will prevent growth of algae, bacteria, etc. GENERAL PROCEDURES 15 All water baths must be equipped with an accurate immersed ther- mometer. Covers for baths may be provided to prevent undue evaporation, but care must be exercised to prevent water of condensa- tion from dropping into tests. Place water baths in a draft-free area of the laboratory. D. Refrigerators, Cold Rooms, Freezers Every laboratory requires ample refrigeration facilities for the storage of reagents, media, stains, biologicals and other perishable items. Most of the larger laboratories provide walk-in refrigerator rooms with mechanical air circulation. Where these units are not required, the restaurant type of commercial box is more advantageous than the usual household-type refrigerator from the standpoint of increased space and variety of door arrangements. Glass doors enable more efficient location of items within the cabinet. Commercial boxes usually have mechanical air convection and yield uniform cabinet temperatures and high humidity, which prevents drying of media. Household refrigerators are efficient for small laboratories or for individual rooms and usually require less frequent servicing than com- mercial refrigerators. Household units are equipped with freezer space, which is not always available or necessary in the commercial types but is important in some laboratory operations. Set the temperature control at 6° C to 8° C. Keep preferably a maximum-minimum thermometer inside the cabinet for periodic checking. Individual freezer boxes are needed in many laboratories. These boxes may be the usual mechanical chest type or the upright variety and must maintain the necessary temperature for the work involved. The ordinary mechanical freezer holds temperatures of —10° C to —20° C, which are usually adequate for keeping sera and other materials frozen. For virus storage, special cabinets using stand- ardized refrigeration equipment can be purchased which will maintain a temperature of approximately —60° C, Below this temperature multistage or cascade equipment is required, which makes tempera- tures down to. —100° C available. Alternatively, dry carbon dioxide freezer chests may be used, for which dry ice must be supplied periodically. While the temperature of dry ice is —70° C to —75° C, there may be wide variations of temperature in the chest up to —45° C. Recommendations for the care of refrigerators and freezers are as follows: 16 GENERAL PROCEDURES a. Defrost as necessary for economy of operation. b. Clean and disinfect interiors periodically to combat psychrophilic bacteria, yeasts and molds. Solutions of formalin or a quaternary ammonium com- pound such as benzalkonium chloride may be used. c. Allow good circulation of air around the motor and compressor. Keep the condenser free of dust and lint. d. Locate units well away from heat pipes. E. Centrifuges® Theory of centrifugal force—Centrifugal force is the force which tends to impel an object, or parts of an object, outward from the center of rotation. Relative centrifugal force (RCFE) assumes a unit mass and uses the force of gravity as a base unit. It is a function of the speed of rotation and the radius from the center of rotation to the point at which the force is to be determined. The formula: RCF=KrN2 (expressed in gravities) K=0.00001118 (when r=centimeters) K=0.00002842 (when r=inches) r= rotating radius N = speed of rotation (rpm) Therefore, a description of centrifuging conditions should include, in addition to time of centrifuging, the radius as well as the speed of the centrifuge, or it should be given in terms of RCFE. Types of centrifugation—There are three ways of applying centrifugal force in laboratory centrifuges : horizontal swinging, angle and basket. 1. In horizontal centrifuging, a tube or bottle is held in a shield or cup which is so attached to a head that it is free to swing from a vertical position at rest to a horizontal position during rotation. Par- ticles travel perpendicularly to the axis of rotation through the full length of the liquid column. 2. In angle centrifuging, a tube or bottle is held at a fixed angle of 35° to 50° (manufacturer’s choice) in holes drilled in a conical head. Again particles travel perpendicularly to the axis of rotation but pass through only part of the liquid, striking the outer wall of the tube and sliding to the bottom. Both horizontal and angle centrifuging have their advantages and limitations. Windage or air friction is the main factor governing the speed of rotation, so that the streamlined angle heads reach higher speeds and consequently produce greater centrifugal forces. Hori- zontal heads are better for volumetric readings on graduated glass- ware, since the line of separation is always parallel to the axis of rota- tion. Liquid-from-liquid separations are also more satisfactory in GENERAL PROCEDURES 17 horizontal centrifugation; with angle heads gelatinous materials are apt to stick to the sides of the tube and prevent clean-cut separations. 3. Basket-type heads are mounted on the motor shaft surrounded by draining chambers which carry away the mother liquor. They are available for continuous extraction and clarification of bulk solids and liquids and are obtainable as perforated or solid baskets. Per- forated baskets are usually lined with a filter medium and are used for separating solids from liquids. Solid baskets are used for clarification to separate small amounts of a heavier solid or liquid from a liquid of lower specific gravity. Types of centrifuges—The average medical laboratory should have a versatile type of centrifuge, adaptable for all the methods of centrifugation noted here by changeable heads and accessories. This type of centrifuge is available in bench models with capacities up to 200 ml, and in various floor model sizes of much greater capacity and versatility. As new technics for routine work are developed, more specialized centrifuges are appearing in medical laboratories—for ex- ample, the small high-speed centrifuges for microchemical procedures. In the more specialized laboratories such as those for research and development, many variations of the standard centrifuge are used. Some of these special types may be listed : Heated Refrigerated Vapor-tight and explosion-proof Continuous-separation Very high-speed (60,000 rpm) Ultracentrifuge (with optical system) Manufacturers have also developed many special-purpose centri- fuge accessories such as sealed units that protect laboratory workers from dangerous aerosols. Safety (a) Protection from rotating parts: The high speeds and great forces generated by a laboratory centrifuge result in a tre- mendous store of kinetic energy. The laboratory supervisor must be certain that centrifuges are equipped with high impact strength guards to protect his personnel in the event of a release of this energy due to operator error or mechanical malfunction. He must recognize speed and overload limitations for particular accessories. b) Electrical safety: Underwriters’ Laboratories approval of a centrifuge and proper care and maintenance reduce the danger of electrical accidents. 18 GENERAL PROCEDURES Operation and precautions—The first step in operating a stand- ard centrifuge properly is to insure good accessory balance, an im- portant factor in prolonging the life of bearings and armatures. a. The following balancing technic renders the best possible weight distribution, providing as well maximum external support for the glassware : 1) Place opposite cups containing filled glassware on the two sides of a balance. 2) To the lighter centrifuge cup, add water around tube or bottle to the level of the liquid in the glassware but no closer to the top of the centrifuge cup than 74 in. 3) Add water to the other centrifuge cup until the two are in good balance. It is important not only to place equal weights on opposite sides, but also to have the same center of gravity. A thin-wall centrifuge bottle should therefore not be counterbalanced by one with heavy walls, even though the difference in weight is made up with water. A supporting liquid cushion surrounding the glass container will often offset internal pressure in large bottles and prevent breakage. When using multiple carriers it is not necessary to balance; how- ever, the same number of tubes should be placed in opposite carriers. Never use mercury for balancing purposes, as it may produce an amalgam with the metal of the accessories and weaken them. b. After carefully loading opposite positions of the head with equal loads, close and lock the cover. ¢. Turn the speed controller to the zero position ; turn the power on and advance it slowly to the desired setting. A rapid increase of power will send an excessive surge of current through the motor. Do not exceed the speed limits recommended by the manufacturer. d. Govern the speed of deceleration by the character of the pre- cipitate, If it is tightly packed, the brake can probably be used with- out disturbing the sediment. However, if the packing is light, as with a flocculent material, use the brush release to extend the deceleration time. Electric interval timers are available on some centrifuges and can be installed with any centrifuge. They are very useful devices, since they can be set to turn the centrifuge off automatically at the end of any prearranged period. Care and maintenance—Follow the manufacturer’s lubrication in- structions. If there is excessive sparking, clean the commutator with fine sandpaper and make sure brushes are in good contact. Change brushes when they become worn to about a third of their original length. Keep rubber mountings in good condition to insure smooth opera- tion. GENERAL PROCEDURES 19 Clean the centrifuge regularly, keeping the motor and frame free of excess oil and grease. Remove grit and broken glass from the guard bowl to prevent scouring of the head and shields. After breakage has occurred, be sure shields and cushions are free of glass. Glass em- bedded in cushions can cause additional glass breakage. F. Rotators and Shaking Machines Rotating machines are prescribed for serological technics and they may be used for a variety of other laboratory operations. The area of rotation on a horizontal plane and the speed are specified for each particular test, and these directions should be strictly followed. Auto- matic shut-off timing mechanisms are usually incorporated into the machines. It is advantageous to purchase machines for which both speed and time can be adjusted. Select machines for use with heavy- duty motors which are not markedly influenced by voltage fluctua- tions. Check the speed of rotation under load frequently each day. Slides as well as small flasks may be rotated, although it is often necessary to employ rubber or spring restrainers on the holding plat- form. Shaking machines such as Kahn-type shakers are also employed in serologic tests. These machines accommodate racks of tubes, and carriers for bottles or flasks may be attached for other procedures. Variable speed power units are advantageous for a variety of purposes. ; Heavy-duty shakers imparting varying motions suitable for con- tinuous duty are sometimes used for homogenization or dissolution of materials. Paint shakers have a particular application in homogeniz- ing specimens for culturing tubercle bacilli. An interval time switch may be installed to shut off any of the machines at a desired time. G. Glassware and Plastics The bacteriological or serological laboratory will require basic glassware items consisting of petri dishes, flasks, bottles, beakers, graduates, slides and test tubes in various sizes as considered standard for each laboratory. : A major consideration in the purchase of glassware is whether to obtain standard flint glass or one of the brands of borosilicate glass. The borosilicate products, which are preferred, have the advantage of possessing a lower coefficient of expansion, which permits heavy con- struction and reduces loss due to thermal shock, Borosilicate glass also has greater resistance to alkali, hot water and steam, resulting in a product which resists clouding and etching. Flint or soda-lime glass is 20 GENERAL PROCEDURES easily scratched and develops a milky appearance after prolonged washing in strong detergents or sterilization. Avoid rapid temperature changes in glass by heating and cooling slowly. Take care not to scratch or abrade surfaces. Discard tubes with nicked edges. Calibrated glassware products include pipettes, graduates and syringes and are used to measure volumes. Most items are calibrated at a temperature of 20° C and are usually marked as TC (to contain) or TD (to deliver), which indicates whether they contain or deliver the indicated volume. Pipettes are designated as: a. Volumetric or transfer, without subdivisions, designed to transfer accurately a single volumetric unit. b. Mohr or measuring, with subdivisions, not graduated to the tip, designed for slow delivery. ) c. Serological-bacteriological, which may or may not be graduated to the tip, designed for rapid delivery. No drainage period is allowed when emptying serological or measur- ing pipettes. These may vary from 0.5 per cent to 4.0 per cent in accuracy, depending on the volume. Some pipettes are intended to deliver the required volume only after the small drop remaining in the tip is blown out, and these have a narrow opaque or lined band at the mouth end. The accuracy limits on calibrated ware for quantita- tive chemical purposes (Class A ware) should not exceed those estab- lished by the National Bureau of Standards (Circular C434). Sero- logical-bacteriological pipettes may be obtained which have a con- stricted mouth end for containing a cotton protective plug. Various rubber bulb devices are available for attachment to pipettes to avoid mouth pipetting in microbiological work. (See Chapter 3 outline to locate discussion of pipetting hazards.) Polyethylene ware can be used to replace some glass containers such as beakers, bottles, cylinders, funnels and pipette jars. It has the ad- vantages of being unbreakable, light in weight, and resistant to chemi- cal action, but it cannot be used efficiently above 70° C. Such ware softens at 108° C to 111° C. It may be sterilized within limitations by means of a bland germicide or by ultraviolet light. Disposable petri plates are now on the market, and there will be other useful plastic products to come. H. Other Equipment Closures for bottles or tubes—Depending upon the use for which the bottle or tube is intended, the following closures are available, and judgment should be exercised in their choice: Cotton plugs Cork stoppers GENERAL PROCEDURES 21 Rubber or Neoprene stoppers Screw caps of metal or bakelite, with liners of paper, vinylite, teflon (preferred for general use), or rubber Aluminum, stainless steel or plastic caps Dissecting instruments (forceps, knife blades and handles, scissors )—Stainless steel products will usually prove of greater value than the nickel-plated varieties, which will rust in time, Instrument or needle sharpeners—Hand sharpening may be ac- complished by use of a Washita oil stone. Motor-driven sharpening and polishing wheels are of great value when there are a large number of needles to be processed. Thermoregulators—These instruments are supplied as integral parts of incubators, water baths and other heating or cooling equip- ment for use in controlling temperatures. The bimetallic type has contact points of tungsten or silver. These points may occasionally require cleaning with a smooth piece of writ- ing paper. Polishing with very fine emery paper will remove corro- sion, but care must be exercised to keep contact surfaces flat. Hydraulic regulators contain an expansive liquid which actuates a snap-action switch with silver contacts. These are said never to require attention, as they are completely sealed units. Other types have mercury-to-platinum contacts and do not require attention. J. Water Stills and Demineralizers Water stills—Distilled water is produced by the evaporation of water and convection of the chemically pure vapor to a chemically clean condenser where the condensate is collected. Two factors affect the quality of the condensate: the design of the still and the care and intelligence with which it is maintained and operated.® There are three types of still in use. The most popular arrange- ment is an evaporating pan heated by flame, electricity or a steam coil. Water is maintained over the heating elements by a constant-level device. A means may be provided to bleed off a portion of the boiling water which prevents concentration of impurities in the evaporator. The water vapor is usually conducted through a series of baffles, where entrained particles of water impinge and are removed from the current of steam. The clean steam strikes the surface of the water- cooled condenser tube, condenses, and is collected in a protected vessel. Condensers are usually lined with block tin or are fabricated of corrosion-resistant aluminum or stainless steel. Nonabsorbent glass is frequently used in laboratory condensers. 22 GENERAL PROCEDURES The distillate is best collected in nonabsorbent glass carboys. For biological purposes, only enough is made for immediate use unless it is sterilized and hermetically sealed to prevent contamination and the growth of pyrogen-producing bacteria. Maintenance of a still varies with the hardness and type of feed water used. Deposition of alkali earth salts in the evaporator and con- denser tubes occurs rapidly with some water. This may be in the form of a sludge or soft coating or as a hard scale that must be leached away periodically with dilute hydrochloric or nitric acid. It is often more economical to demineralize the water prior to distillation than to service the still frequently enough to assure reliable and efficient operation. Proper operation entails the maintenance of a water pressure at 4-8 1b per sq in. (psi) so that the rate of cooling is constant. Fluctuat- ing water pressure results in overheating and priming, with resultant chemical contamination of the condenser by droplets of raw water or foam. When the still is started, care must be taken to turn on the water before turning on the heat to prevent boiling the evaporator dry. The still is stopped by turning off the heat and permitting the evaporator to cool before turning off the water. The second type, the Selas still, utilizes steam generated in a high- pressure boiler. Oil and condensate are removed by passage through a separator, The clean steam is then filtered through a Selas ceramic polar filter before it is led to the condenser. A third type of still utilizes compression distillation. In single- effect evaporation, such as described, heat is applied to vaporize liquid. The vapors are condensed by transferring the latent heat to the cool- ing liquid in the condenser, where it is wasted. Latent heat of vaporization can be recycled continuously by mechanically com- pressing the vapor to supply the small increment of energy needed to increase vapor temperature and pressure to the point where the cooling water boils. The condenser thus becomes an evaporator and the latent energy is reused instead of being wasted. This technic of compression distillation is economical because once the cycle is begun, the latent heat is conserved. Demineralizers*—JIonizable alkali earth salts, present in all natural waters, can be removed by contact with appropriate ion exchange resins, Organic solutes (including pyrogens), crystalloids, and bac- teria remain in the water. The conductivity of the effluent is tested * Details are available from the Barnstead Still and Sterilizer Co., 2 Lanes- ville Terrace, Roslindale 31, Mass. GENERAL PROCEDURES 23 to indicate exhaustion of the resin. The resins are regenerated by flushing the cationic bed with dilute acid and vice versa. The resins in small columns may be dyed with an indicator to warn of exhaus- tion. Cartridges of fresh resin are often used in lieu of regeneration in such units. Resins are chosen to provide optimum service with local water conditions. Demineralization is an inexpensive way to produce water for applications where sterility and organic impurities are unimportant. The complexity of the cycle depends on the degree of purity re- quired for the water. Serial passage through cation and anion beds removes the anions (Ca, Na, K, Mg, Fe, Cu) with liberation of chloride from the cationic resin. This chloride is removed on the anion resin, along with any chloride and other cations (SO, HCO;, CO3) occurring in the water, liberating hydrogen. When water of greater resistivity is required, serial passage through two pairs of beds in tandem is arranged. Greatest purity is attained by passage through a bed of mixed anion and cation resins, the water contacting successive particles of resin with the result that the ionizable impurity content may be held to less than 1 ppm with a resistivity of 5 X 10-7 mhos. During regeneration the resins are separated by flowing water backward through the bed to float the lighter anionic resin to the top. The regenerating acid is drawn up through the layer of cationic resin, while the alkali filters down through the anionic bed. The spent regenerants are discharged from the interface between the resins. The resins are thoroughly remixed by blowing compressed air into the bed for a few seconds. Demineralizing equipment must be carefully fabricated to prevent corrosion of the columns, regenerant tanks, piping and valves. Rubber- lined steel, plastic-coated steel, plastics and the like are chosen. Successful design involves the proper layout of piping, valves, and flow meters to assure simple operation. Small units are usually manually operated. Larger units are either semiautomatic or com- pletely automatic. With all, monitoring of the effluent with a con- ductivity bridge is desirable. K. Glassware Washing and Handling” Before the final sterilization in preparation for diagnostic bac- teriological work, glassware should be scrupulously cleaned. Other- wise, the results of bacteriological and serological tests may not be completely reliable. The glassware to be prepared may be new glass- ware, or it may be used glassware requiring first the removal and decontamination of considerable specimen and medium material. 24 GENERAL PROCEDURES Cleansing of new glassware—The soaking of routine types of glassware used in microbiological procedures in a weak solution of hydrochloric acid for a few hours will usually suffice to remove alkali from the glass. A very effective cleaning fluid for new glassware, and occasionally for very dirty old glassware, is a sulfuric acid bichromate mixture. However, this compound is dangerously corro- sive; it should therefore be prepared and used only by skilled laboratory workers who are fully acquainted with the hazards in- volved. Wear rubber gloves, rubber aprons and goggles and avoid all contact with skin and clothing. Prepare the solution by dissolving 65 g of sodium bichromate by heating in 35 ml of water. Then allow to cool and very slowly, with stirring, add concentrated sulfuric acid to make 1 liter of solution. Always add the concentrated sul- furic acid to the water, never add the water to the acid; otherwise dangerous spattering may occur. Keep the mixture in a heavy re- ceptacle in a sink, preferably protecting it further by placing it inside an earthenware crock. With this added protection no serious damage will occur if the glass container cracks. First, wash thoroughly the glassware to be prepared, with de- tergents and hot water ; then rinse in hot water. Immerse in the clean- ing solution for one to several hours. Rinse thoroughly several times in warm tap water, followed by several rinses in distilled water. The glassware is now ready for sterilization as described elsewhere in this chapter. Cleansing of used glassware—Glassware holding potentially in- fectious material such as used culture media or old specimen material must first be decontaminated by autoclaving. When this procedure has been used on tubes containing residues of blood clots or serum from serological laboratories, the subsequent difficulties in washing are often compounded by the presence of coagulated proteins. Some laboratories prefer to wash these tubes without autoclaving, but in such cases the laboratory assistant should wear rubber gloves when emptying and rinsing the tubes to protect against accidental infection with hepatitis virus (see Chapter 3, “Laboratory Infections and Accidents”). The blood and serum tubes should first be emptied of clots and sera into a pan or bucket containing a germicide, which is subsequently autoclaved or emptied into a toilet bowl. In the washing process, the tubes must be subjected to steam or boiling water for 30 min in order to destroy any hepatitis virus. Today the modern public health laboratory should be equipped with a mechanical washing machine for glassware, preferably of an auto- GENERAL PROCEDURES 25 matic type. Directions of the manufacturer as to washing and rinsing should be scrupulously followed. Pretreatment by soaking in a special protein-digesting compound* for 24 hr will be required for glass- ware containing such media as Loeffler’s coagulated serum, coagulated egg media, etc. It may also be necessary to remove gross foreign matter manually before proceeding with machine washing or hand washing, using a good detergent followed by careful rinsing. A pipette washer which operates on the siphon principle should be available. Presoaking of pipettes in washing or protein-digesting compounds is often necessary. L. Hot-Air Sterilizers and Sterilization Dry heat is utilized for the sterilization of (1) articles which are corrosively attacked by steam; (2) anhydrous materials which are spoiled by moist heat; and (3) dehydrated substances which prevent the microbicidal action of moist heat. Cutting edges, surgical gut, ground glass surfaces and dry chemicals (such as powders), grease, oil or glycerol are examples. Dry heat has limitations which make its application quite difficult and time-consuming. When high tempera- tures are used to shorten the sterilizing period, fabric, rubber goods, plastics and chemicals are destroyed. Because heating is by conduction and radiation, penetration is slow as compared to rapid heating by convection of steam into packages. Furthermore, hot air must be circulated mechanically to prevent stratification and insure uniform temperature. The thermal death time for resistant dry spores is shown in Fig 3a. Even a small percentage of water decreases the exposure necessary to kill. Successful operation of a dry-heat sterilizer depends on the attainment of a lethal temperature throughout the load to permit timing exposure. One hour at 160° C or 4 hr at 121° C are common exposures for oil-free articles. The latter standard can be satisfied in the usual hospital dressing sterilizer by maintaining 15 psi steam pressure in the jacket to heat the wall of the chamber. The load can be left overnight to provide ample time for heating and lethal exposure. M. Steam Sterilization Saturated steam under pressure is the most frequently used micro- bicide. It is a physical entity with properties which can be measured conveniently. Its readily available store of latent energy is liberated * “Haemo-sol” (Meinecke and Co., Inc, New York) is a useful detergent. “BWC,” available from the Wyandotte Chemical Corp., Wyandotte, Mich., has been found effective, but care should be observed in its use because of its caustic nature. 26 GENERAL PROCEDURES 200 180 160 140 Temperature— Degrees Centigrade 12 0 40 80 120 160 200 240 Time in Minutes Figure 3a—Thermal death time of resistant dry spores in dry heat. simply by a change in state. This is of enormous advantage because it permits rapid heating. On contacting cold objects, saturated steam condenses, simultaneously heating the object and wetting it, providing both requisites for the thermal destruction of bacterial life—heat and moisture. Saturated steam penetrates readily for two reasons: First, the steam is less dense than air, which initially occupies sterilizers and packages. Mixture does not occur readily and the steam displaces the cold, heavy air from the interstices of packages and textiles by convection. Second, the abstraction of heat from steam causes a change in state with a simultaneous 99 per cent decrease in volume. The sudden collapse in volume results in the instantaneous development of local areas of negative pressure at the cold front, The negative pressure hastens penetration because more steam bearing its load of latent energy rushes in to overcome the low pressure, contacts cold surfaces and, in turn, condenses. Thus penetration by steam is a self- perpetuating process so long as steam can contact cold objects. Saturated steam destroys resistant dry spores upon relatively short exposure (Fig 3b). Several temperatures have come into common usage: Steam at 121° C, 15 psi, is widely used for sterilization of solutions, textiles and rubber goods. It is effective after 13 min exposure. Steam at 132° C, 27 psi, is used for emergency demands, 2 min sufficing for sterilization. GENERAL PROCEDURES 27 140 W0o— 0 20 36 40 50 6 70 Time in Minutes Figure 3b—Thermal death time of resistant spores in saturated steam. Saturated steam has several limitations which interfere with its microbicidal properties. When superheated, the steam is no more effective than dry air. Improper preparation of textiles and faulty operation of the sterilizers are common causes of superheating. The presence of air interferes with the temperature developed within a sterilizer. It also prevents any mechanical contact between bacteria and steam. A horizontal path must be provided for the escape of air by gravity. Oil and grease also prevent access of moist heat to bacteria and negate the effectiveness of steam. When properly used, a steam sterilizer is not destructive to the majority of instruments, solutions and materials, which, however, must be properly packaged in a porous wrapper, care being taken to assure loose arrangement of the contents to afford ready penetration. Packages must be arranged to provide a horizontal path for the escape of air and to permit prompt access by steam to all surfaces of the package. The control of sterilization in a properly loaded steam sterilizer is simple, requiring only the determination of the time when the temperature in the exhaust line at the bottom of the sterilizer registers the temperature characteristic of steam under the pressure being used. The exposure necessary for penetration of the largest package, plus a minimal thermal death time, are clocked from this 28 GENERAL PROCEDURES moment. Telltale indicators, recording thermometers, and periodic cultures may yield misleading information on a process which is dependent for its effectiveness on trained personnel using foolproof technics who can make accurate observations of time and temperature. At the completion of the sterilizing cycle, the steam pressure can be vented and the door of the sterilizer permitted to stand ajar while the load is dehydrated by the radiant energy supplied by the steam jacket. If exceptional dryness is wanted, a vacuum can be drawn to help dehydrate the contents. When solutions are sterilized, the steam supply to the sterilizer is turned off at the termination of the sterilizing cycle and the entire sterilizer is permitted to cool until the solutions reach a temperature below that of boiling water. Following this, the sterilizer can be opened without fear of ebullition of steam from superheated solutions. Steam sterilizers can be used for inspissation; that is, thickening or jelling and sterilization of heat-coagulable protein media. Success requires careful control of the rate of heating to prevent bubble formation due to the release of gas in the medium during heating. The temperature in such a sterilizer must be kept low because the heat- coagulable proteins are destroyed by excess exposure to heat. Heretofore this was accomplished by insulating the tubes in cloth or paper and retaining all the air in the sterilizer by closing a valve in the air and condensate discharge line. Once 15 psi was attained by the admission of steam, the air was slowly replaced with steam, main- taining the pressure in the autoclave constant, When the tempera- ture in the drain line reached 120° C, timing of the sterilizing period was begun. At the end of the standard 15 min period, all the ports of the autoclave were closed and the sterilizer was allowed to recool until atmospheric pressure was attained. Modern vapor-heat sterilizers provide slow uniform heating of culture tubes containing the coagulable protein media under cir- cumstances where the gas can be expelled slowly, without forming bubbles, and excessive heating of the media can be avoided. Screw-cap tubes or vials can be used for trapping air in the sealed tubes to assist in maintaining a smooth surface on top of the medium. N. Chemical, Filtration and Ultraviolet Sterilization®1© Germicides—The use of germicides for sterilization of articles that do not withstand heat is being abandoned because of their in- effectiveness against spores and the virus of homologous serum jaundice (see second paragraph under Section IB of this chapter, “Collection and Submission of Specimens”). GENERAL PROCEDURES 29 When the tubercle bacillus must be destroyed, any of the following tuberculocides may be used: 70 per cent ethyl alcohol; 70 per cent isopropyl alcohol ; substituted phenolic,* or an iodophor.} Gas sterilization—Ethylene oxide and formaldehyde are the chief gases used for sterilization. When applied as gas, the ethylene oxide is used in mixture with carbon dioxide to prevent explosive combus- tion. Because the carboxide gas does not penetrate readily, the sterilizing cycle includes the evacuation of air from the sterilizer and its load and its replacement by carboxide gas. The gas is not germicidal unless a relative humidity of 25 to 40 per cent can be maintained. This is accomplished by saturating loads of absorptive material, such as textile or paper, with steam. When performed properly, this step also raises the temperature of the ma- terials to the optimum for sterilization, namely 65° C. The usual cycle involves two exposures to a high degree of vacuum before flooding the sterilizing chamber with gas to provide a gaseous environment containing 400-600 mg ethylene oxide per liter. Steam is admitted to relieve the initial vacuum and carboxide introduced after the second vacuum cycle until a pressure of 27-30 psi is ob- tained. Exposure time under these conditions is 4 hr. At the end of the cycle the carboxide is vented, a vacuum is drawn, and the sterilizer is flooded with sterile air. Sterilization by formaldehyde gas—Formaldehyde gas is effec- tive when used in concentrations of 10 mg per liter in an environ- ment of 80 per cent relative humidity, at 26° C. The gas penetrates poorly, hence an initial 100 cu. mm vacuum must be drawn to rid the sterilizer of its load of air. Exposure for 30 min suffices to sterilize clean articles. A final vacuum is drawn to rid the sterilizer of irritat- ing fumes. Filtration—DBacteria-free filtrates can be obtained by passage through filters with a maximum pore size of 1.5u or less. The Seitz asbestos filter and the Berkefeld diatomaceous candle are both adsorptive. The Chamberland unglazed porcelain filter and the Corn- ing (UF) sintered glass filter are inert. The easiest filter to use is the the membrane of cellulose esters in a network of crossed, linked ele- ments forming pores approaching molecular dimension—for example, *(O-syl, 2 per cent, manufactured by Lehn & Fink Products Corp., 445 Park Ave., New York 22, can be used with soap or in routine cleansing. 7 Wescodyne 100 ppm iodine, 4 ml per liter, manufactured by the West Dis- infecting Div., West Chemical Products, 42-16 West St. Long Island City, N. Y,, is an efficient germicidal nonionizing detergent suitable for use with all water supplies. 30 GENERAL PROCEDURES the Millipore Filter,* available sterile and mounted in a plastic hous- ing suitable for filtration of laboratory quantities of fluid with the aid of vacuum. If the recovery of bacteria is sought, the membrane can be moistened with culture media and the colonies grown directly. Sterilization by ionizing radiation—Biologic materials, such as certain plastics, drugs and vaccines, and surgical gut, are advan- tageously sterilized by ionizing radiation. The source may be the Van de Graaff accelerator, cobalt 60, or an x-ray tube with a beryllium copper window. For sterilization, 3,000,000 to 4,000,000 roentgen equivalent physical (rep) are required. Ultraviolet radiation—The germicidal action of ultraviolet radia- tion (25374) is a surface effect only. The radiation is reflected from the surface of liquids, and organisms in shadows escape destruction. The intensity of the surface effect decreases as the square of the distance from the radiator, so that care must be taken to provide proper distribution for bactericidal action. The technic finds most use for the sterilization of droplet nuclei in clean air. Care must be exercised to protect the skin and eyes from excess exposure. IV. HAZARDS IN THE LABORATORY1-18 In a diagnostic laboratory there are always the dangers of labora- tory-acquired infections, physical hazards and hazards in the use of chemicals. Laboratory infections and their prevention are dis- cussed in Chapter 3. Physical and chemical hazards are discussed in the following. While there are no ready-made first-aid kits avail- able to handle all accidents, certainly a reasonably well-equipped first- aid box should be easily accessible to all laboratory workers. It should include Band-Aids, sterile gauze, adhesive tape and mild anti- septic solutions such as Zephiran 1:1000 or pHisoHex to clean out wounds or to irrigate an area exposed to highly irritating chemicals. The most important first-aid tool, however, is the knowledge and judgment of the individual who applies the first aid. The optimum number of laboratory workers should therefore attend local Red Cross first-aid refresher courses as available from time to time, A. Physical Hazards Physical hazards of the laboratory include the danger of electric shock and of fire. Such dangers are well known and in general are not peculiar to the laboratory, but a few cardinal principles should be emphasized. First, the danger of electric shock is greatly minimized * Manufactured by Millipore Filter Corp., type MH WHT GRD 037 MMDS. GENERAL PROCEDURES 31 when all machines are properly grounded; second, the danger is still further reduced if laboratory workers learn never to handle the electric machines or their switches and controls with wet hands. For the prevention of fire there should be strict rules against smoking around flammable materials, and persistent vigilance by every laboratory worker using a Bunsen burner or gas oven. Fire-resistant blankets should be located at strategic points to wrap at once about any victim whose clothes catch fire. There should be an adequate supply of properly placed fire extinguishers, which must be checked and weighed regularly at 6-month intervals. Fire drills, with emphasis on the turning off of gas burners and the closing of doors, should be held about twice a year to indoctrinate each worker with his proper duties in the event of fire. The fire department should be called at once for even small fires. Some of the worst fires have been devastat- ing largely because they were fought locally too long before the fire department was called. B. Hazards in the Use of Chemicals The laboratory worker is daily confronted with the potential hazards of toxic chemicals and poisons. Unlike the delayed response to accidental infections of microbial origin, most of the toxic reactions present their effects almost immediately after the accident has oc- curred. Such accidents can occur when a worker inhales toxic vapors, ingests chemicals by mouth in the act of pipetting, unexpectedly intro- duces the agent into the eye or spills it on the skin. In particular, the pipetting of chemicals must be done with the greatest caution. The laboratory technician, with his basic training in chemistry and re- lated sciences, should have at least fundamental information concern- ing the potential hazards that attend improper handling and disposal of poisonous, corrosive, flammable or explosive chemicals. Some general directions in the handling and storage of chemicals may be useful : 1. Store volatile, flammable and explosive materials in a metal cabinet or closed room. The storage place should be cool and well ventilated. 2. Keep volatile, flammable, explosive and corrosive materials in their original containers, in glass or in resistant plastic containers. 3. Use pumps in transferring large volumes of dangerous liquids. Transfer small volumes with a bulb attached to a suitable pipette. Transfer toxic or irritating materials in a well-ventilated area, preferably under a hood. Avoid storing incompatible chemicals together. Label all laboratory reagents. Attach poison stickers where indicated. Have available in the immediate area suitable neutralizers for acids and alkalies (sodium bicarbonate vs. mineral acids, dilute acetic acid vs. alkalies). Make available in the immediate area of volatile, flammable and explosive ~ ® Now; 32 GENERAL PROCEDURES materials suitable fire-fighting equipment (fire extinguishers, fire blankets and respirators). 9. Make periodic inventory of dangerous chemicals to avoid stock-piling of hazardous materials. 10. Familiarize other laboratory personnel with the precautions necessary in handling new hazardous materials. Following are specific suggestions for the more common hazardous chemicals: a. Acetone, ether. Highly flammable. Keep in a cool place and remote from sparking apparatus and open flames, including pilot flames. Keep containers tightly closed. Use with adequate ventilation. Avoid prolonged or repeated contact with the skin. b. Acids. Avoid breathing vapor and contact with the skin or eyes. c. Alkalies, caustics. Avoid contact with the skin, eyes and clothing. Wear goggles or shield when handling. Add crystals slowly to water to avoid spattering. d. Bichloride of mercury. Avoid contact with skin and eyes. e. Chloroform. Nonflammable, but keep vapors away from open flame, electric hot plates or any hot metals to guard against formation of the toxic gas phosgene. Use only with adequate ventilation. Avoid breathing vapor. Avoid prolonged or repeated contact with the skin. f. Denatured alcohol, methyl alcohol, etc. Use only with adequate ventilation. Avoid breathing vapor. g. Formaldehyde. Use with adequate ventilation. Avoid breathing vapor. Avoid prolonged contact with the skin. h. Hypochlorites. Store powders in cool dry place. Dust can be damaging to the skin and injurious internally, if inhaled. The calcium salt of hypochlorous acid is explosive when heated suddenly to above 100° C; if mixed with com- bustible substances, deflagration occurs. Wear chemical safety glasses and a respirator. i. Iodine. Use crystals with adequate ventilation; transfer under hood. Avoid contact with the skin and eyes. j. Phenol. Rapidly absorbed through the skin. Causes severe burns. Avoid contact with the skin, eyes and clothing. Avoid breathing vapor. k. Potassium dichromate Harmful dust. May cause rash or external ulcers. Avoid breathing dust or solution spray. Avoid contact with the skin or eyes. 1. Xylene. Rapidly absorbed through the skin. Flammable. Keep in a cool place and use with adequate ventilation. Avoid breathing vapor. Avoid con- tact with the skin and eyes. This list covers most of the hazardous chemicals commonly used in the public health laboratory. It is not exhaustive, however, and appro- priate caution must be exercised when any other chemicals are used in the laboratory. C. Treatment of Accidental Poisoning from Hazardous Chemicals For immediate advice call the nearest poison information center. 1. Volatile substances and gases— Whether the victim is over- come by ether, chloroform, carbon monoxide or any other volatile substance or gas, the principles of immediate treatment are the same: First—At once remove the patient well out of the area of the toxic agent into air as fresh as possible. GENERAL PROCEDURES 33 Second—If the patient is not breathing, see that the air passages are open and then apply artificial respiration by the mouth-to-mouth method, using oxygen inhalator if available. Keep the artificial respiration going until a physician sees the patient. Third—Seek medical help, but institute the first two measures immediately. 2. Substances dangerous internally—Whatever the toxic agent ingested, with the important exception noted below, the cardinal prin- ciples are immediate dilution of the agent and prompt emptying of the stomach. Both dilution and emptying can be effectively accom- plished by having the patient drink at once huge quantities of luke- warm water until vomiting takes place. Repeat the process again and again until thorough stomach lavage has taken place. If a stomach tube is available, use it. The special emetics and antidotes that are recommended in texts are rarely at hand for quick use when needed, nor are they always effective. Instead, if the drinking of copious amounts of water has not induced the expected vomiting, simply tickle the back of the patient’s throat with your finger or try soapsuds in warm water or two tablespoonfuls of table salt in a half pint of warm water as simple emetics. To lessen danger of aspiration, keep the subject’s head as low as possible and turned to one side. Time is of the essence because the dilution with water and the vomiting which you effect, if done at once, can prevent absorption of most of the poison which has been ingested. Prompt action may save a life that might be lost waiting for the doctor or a hospital ambulance. A simple universal antidote should be available to use in poisoning after as much of the poison as possible has been removed by the methods discussed in the foregoing. This antidote consists of pul- verized charcoal, two parts; tannic acid, one part; and magnesium oxide, one part. Give one heaping teaspoonful in water and induce vomiting or pump out the stomach with a tube. Medical aid should be called as soon as possible, but only after these immediate first-aid measures are under way. In the meantime, keep the patient warm and avoid excessive or too vigorous handling. Exception—If large quantities of concentrated acid or alkali have been ingested, do not force water or attempt to provoke vomiting, because there may be danger of rupture where the corrosive agent has already weakened the walls of the stomach or esophagus. Instead, dilute the poison with the prompt drinking of moderate quantities (say, 1 to 2 glassfuls) of water; for acid poisoning, large (5 to 1 glassful) doses of milk of magnesia, aluminum hydroxide, milk or raw eggs. For alkali poisoning give vinegar, lemon juice or grape- fruit juice. 34 GENERAL PROCEDURES 3. Substances damaging on contact.— Toxic agents that injure the skin, eyes or mucous membranes are caustic acids and alkalies, strong formaldehyde, hypochlorites, phenol, potassium dichromate, xylene and other irritating chemicals. One essential rule applies to all. Dilute the agent by thoroughly rinsing the affected part at once with copious quantities of water—for as long as 15 min in the case of caustic burns. Call for medical advice. V. INOCULATION OF MEDIA AND FISHING OF COLONIES A. Inoculation and Fishing of Culture Plates Most specimens received in the bacteriology laboratory contain a mixture of organisms. Therefore, the initial inoculation must be made on a solid medium in such a manner as to obtain isolated colonies of all the microorganisms present. It is important that the original inoculum constitute a representative sample of the specimen. To insure this, a swab should be rotated on a small section of an agar plate; all the growth on an agar slant should be thoroughly mixed and a small amount of the mixture transferred to an agar plate; a sputum must be emulsified, etc. The agar plate should then be streaked from the original inoculum by some method of cross-hatching which will insure (a) that some of each microorganism present are carried along, and (b) that isolated colonies are obtained (see Fig 4). Since satis- factory streaking requires considerable exposure of the plate, care must be taken to avoid extraneous contamination from talking; from air currents due to open windows, heating systems and unnecessary traffic in the room; from careless movement of the hands and arms over the exposed plate, etc. Furthermore, in the inoculation of media and the fishing and transferring of cultures, the microbiologist must always be aware of the possible hazard of laboratory-acquired infec- tions. For further discussion of this important subject, the reader is referred to Chapter 3. If the initial streaking has been done well, there will usually be no difficulty in fishing isolated colonies for further study. Nevertheless, care should be taken in choosing the colonies to be fished, selecting only those that are well isolated. When fishing from pour plates, special care must be taken to observe small colonies directly over or under those tentatively chosen for fishing. The use of a hand lens or a colony counter—stereoscopic microscopic or Quebec—is often help- ful in selecting colonies to be fished. The actual fishing should be done with a straight needle, care being taken to touch only the center of the selected colony. One should avoid touching the surface of the agar. If GENERAL PROCEDURES 35 Figure 4—Method of agar plate inoculation. A good distribution of colonies may be obtained by streaking at right angles on four sections of the plate, beginning at 1 and adjusting the amount of overlap at 2, 3 and 4, depend- ing on the nature of the medium and the inoculum. If properly done, discrete colonies will appear in at least one quadrant of the plate. possible, the plate should be left on the table so that the cover may be kept partially over it to prevent dust particles from falling on the agar surface. However, it is often necessary to hold the plate off the table to get satisfactory light for fishing. In this case the afore- mentioned precautions to avoid contamination must be observed. B. Inoculation of Culture Tubes The same precautions apply for work with culture tubes. Although it is a common practice, it is not necessary routinely to flame the open end of a tube when the cotton plug has been removed unless bits of cotton adhere to the tube. The flame may be sufficient only to warm the mouth of the tube, not to sterilize it. Passage to and from the flame may cause contamination by the introduction of dust particles. 36 GENERAL PROCEDURES On the other hand, if the contents of the tube are to be poured (as in the preparation of pour plates), the lip of the tube must be held in the flame long enough actually to sterilize it. In inoculating culture tubes the solid medium, when so tubed that it has a deep butt and a short slant, is first stabbed to the bottom of the butt and the needle then drawn over the slant so as to produce sufficient surface growth with which to work. Other types of media may require only stabbing or streaking. Swabs may be used also in inoculating the slant, taking the same precautions to insure satisfactory deposition of the inoculum on the surface of the medium. A straight needle should be used in inoculating fermentation tubes, rubbing the needle with the inoculum on it against the side of the tube, which should be held in such a manner that air bubbles cannot enter the inner tube. C. Recognition of Blood Culture Contamination A useful device for recognizing contamination when it occurs after the taking of a blood culture is the planting of two portions of the specimen in the following manner: First inoculate 10 to 15 ml of the blood in the syringe into a flask containing 75 ml of liquid medium. Then inoculate 1 ml of the remaining blood in the syringe into a little citrate in a petri dish and add 15 ml of warm melted agar to make a pour plate. If no or too few colonies appear in the pour plate, the possibility that any growth appearing in the flask is the result of a contaminating microorganism should be considered. Usually any microorganism originally present in the blood will grow in the pour plate as well as in the flask. D. Incubation in Increased Carbon Dioxide It has proved easy to maintain an atmosphere of increased carbon dioxide, which stimulates the growth of a number of bacteria con- sidered to be microaerophilic, including such pathogens as Neisseria gonorrheae and Brucella abortus. The candle jar affords the simplest means of obtaining increased carbon dioxide tension and is widely used. However, the difficulty of handling, the time consumed, the increase in moisture due to the burning of the candle, and the relatively low concentration of carbon dioxide produced make this method less desirable than others for continuous use with large numbers of specimens. Some laboratories have used large airtight cans or jars to which outlets have been attached making it possible to remove some of the air and replace it with carbon dioxide. But this method is unsuitable for large numbers of specimens because it is difficult, and filling the jar or can and adding the carbon dioxide are GENERAL PROCEDURES 37 time-consuming. Another method employs a standard incubator which is sealed as completely as possible and into which a small amount of carbon dioxide is allowed to flow constantly. This method has many advantages but gives variable results, because the difficulty of meter- ing the flow of carbon dioxide causes variations in the carbon dioxide tension within the incubator. A practical method of maintaining adequate carbon dioxide in an ordinary incubator has been described by Reese, et al.’® By this method a measured quantity of carbon dioxide is permitted to flow into the incubator; then the carbon dioxide is shut off and the in- cubator is allowed to remain unopened during the incubation period. Anaerobic incubators adaptable to anaerobiosis or carbon dioxide of varying tensions are now available from commercial sources.* E. Incubation under Anaerobic Conditions Early bacteriologists believed that to culture obligatory anaerobic organisms successfully all free oxygen must be excluded from them. Newer knowledge has established that it is necessary to remove free oxygen only from the immediate environment of the bacteria or to use some method that will maintain a low oxidation-reduction potential in the media. Various methods are followed to secure adequate anaerobic condi- tions. One method makes use of potassium hydroxide and pyrogallol to absorb the oxygen from the atmosphere in the sealed container in which the organisms are being incubated. Another method admits hydrogen or a mixture of gases to the jar. By catalytic action the hydrogen combines with the oxygen in the jar to form water. The catalytic agent commonly employed is platinized asbestos, which acts rapidly when heated. In the Brewer anaerobic jar the heating element is enclosed in a gastight tube within the catalytic mass. A drying agent may be used to remove the water that is formed. When a method involving the use of hydrogen or a mixture of gases is employed, appropriate precautions should be taken to prevent an explosion. Such precautions may include the use of a micromanipu- lating valve which will maintain less than 2 Ib pressure and the usual circumspection desirable when handling a highly inflammable gas. Anaerobiosis may be produced if the oxygen or air in the jar is replaced by flushing with nitrogen or some other inert gas to remove the air and leave the atmosphere as nearly pure inert gas as possible. A method of obtaining anaerobic conditions which does not require evacuation apparatus, gas cylinders or electricity has been described * National Appliance Co., distributed by Scientific Products. 38 GENERAL PROCEDURES by Parker.” Oxygen is removed from sealed containers by the oxida- tion of activated iron wool. In the use of broth cultures for anaerobic microorganisms, the oxygen is driven off by boiling the medium immediately before use and cooling rapidly. Cooked meat medium has been used in this manner ; the chopped tissue acts as a reducing agent and in addition as a nutrient for the bacteria. Thioglycolate broth is one of the most commonly used liquid media for obtaining growth of anaerobes. The combination of a small amount of agar, 0.1 per cent, and sodium thioglycolic acid in an enriched-base broth provides satisfactory con- ditions for the growth of such microorganisms as Actinomyces bovis, anaerobic streptococci, and members of the Clostridium group. It is important that this medium be stored in the dark at room tempera- ture. Detailed information will be found in the chapters on anaerobes. A special petri dish cover devised by Brewer makes use of the chemical absorption of air trapped in a very shallow space between the surface of the agar and the top of the dish. The oxygen is ab- sorbed by sodium thioglycolate or a similar substance in the agar which will maintain a low oxidation-reduction potential in the medium. Also used are the so-called “shake tubes”, which are deep tubes of glucose agar. The agar is melted and cooled to below 50° C, the inoculum added and mixed thoroughly, and the medium permitted to solidify rapidly. Strict anaerobes grow only in the depth of the medium, facultative anaerobes grow throughout the medium, micro- aerophilic organisms grow in the area between the surface and the deep portion, and of course aerobic organisms grow at the surface. This method permits determination of the oxygen requirements of the organism. F. Biochemical Tests The classification of bacteria is a complex process involving the use of many types of tests. Some of the general methods may be listed as follows: morphological, cultural, physiological, serological and pathological. Biochemical tests are based on measurements of chemical changes brought about by the physiological activities of bacterial cells or substances derived from them. Since enzymes mediate all chemical changes produced by bacteria, either intracel- lularly or extracellularly, it follows that biochemical changes are dependent on these substances. The results of such tests when cor- related with those obtained by other methods of study are very helpful in ascertaining the true nature of an organism. Biochemical tests frequently delineate satisfactorily a group of related organisms pend- GENERAL PROCEDURES 39 ing the application of more specific tests for recognition of individual members. Hundreds of biochemical tests are being used today in bacteriology. Some of the more commonly used are the test for the reduction of nitrates, the fermentation of carbohydrates, the hydro- lysis of urea, hydrogen sulfide production, gelatin liquefaction, bile solubility, sodium chloride sensitivity and catalase production. VI. STAINS A. Blood Film Stains Wright's stain (methylene blue, eosin) Wright's stain (certified, powder form) ......... cet iiiinninnnnnnn 03g Geert] (CP) ..cocisvsammmms sams nm sing tabs mabe Bast Ramat sts 3.0 ml Methyl alcohol (absolute, 20et0ne TYEE) uvsuve sos vasinen « sniss vamesiss 97.0 ml Place the powder in a dry mortar, grind with pestle. Add the glycerol, grind. Add the methyl alcohol, mix. Store overnight in a tightly stoppered bottle. Filter. Store 1 week before using. Since this stain contains 97 per cent methyl alcohol, it also acts as a fixative. Prefixing can be carried out, but it is not necessary. Staining procedure—Cover the air-dried preparation for 1 min with about 10 drops of the stain solution. Add an equal volume of Sorensen’s phosphate buffer, pH 6.4, and mix. A film may form when the buffer is mixed with the stain. The pH is important. If results are inferior, try a buffer of pH 6.6, 6.8 or 7.0. Let stand 3 to 4 min. The best staining time may vary for stains with different certification numbers. Wash by #boding the slide with water before pouring off the stain, followed by washing for 30 sec. This procedure helps remove any formed precipitate or scum. Some recommend washing with the same buffer, as used above, in place of water. The preparation should have a tan or brick red color. Air-dry (blotting should be avoided; however, blotting has been done to help remove formed scum when it threatens the quality of the preparation). May-Gruenwald stain (Jenner’s methylene blue, eosin) Certified Jenner's eosinate of methylene blue (powder form) ......... 05 ¢g Methyl alcohol (absolute, acetone free) ...........ccviiiiiivnnnn nn. 100.0 ml Carefully heat the methyl alcohol to 50° C. Slowly add the pow- dered dye. Stir until dissolved. Cool. Store overnight in a dark stop- pered bottle. Filter. Store in the dark. Staining procedure—ZFlood the unfixed film for 3 to 5 min. Wash with water, air dry. 40 GENERAL PROCEDURES Giemsa stain (azure, methylene blue, eosin) Stock solution: Certified Giemsa stain (powder form) ........coviieeniiinennnnnnnn. 05g GITEREOL SCLC BLY oii vivisilviinmmmedoitin asens Sibissssamininrs sutasemivs w wisssaiasvimimioral she 33.0 ml Methyl alcohol (absolute, acetone free) ..........c.cceviiiinnnnnn. «33.0 ml Heat the glycerol to 55° C. Slowly add the dye powder with stirring. Hold at this temperature for 17% to 2 hr. Cool and add the methyl alcohol. Mix. Allow to sediment overnight in a desiccator to prevent absorption of moisture. Pour into small (30 ml) bottles and stopper tightly. Store 2 weeks, then filter before use. Staining procedure—Use Coplin jars for staining. Add 2 ml of stock solution to 98 ml of Sorenson’s buffer, pH 7.0. Thin films usually are first fixed in methyl alcohol. These should be stained 20 to 30 min. Unfixed thin films can be stained by increasing the stock solution in the buffer to 3 per cent and decreasing the staining time to 15 to 20 min. Thick unfixed films should be stained about 45 min. The staining time may vary for stains with different certification numbers. Wash by standing in distilled water for 3 to 5 min. Some recom- mend washing in Sorenson’s buffer, pH 7.0, in place of distilled water. Unfixed films will need a reduced wash treatment and should merely be dipped into the water or buffer. Drain, dry. Do not use heat, do not blot. An ordinary electric fan is useful for air drying. Wright’s-Giemsa stain 2 Stock solution: Giemsa stain (certified, powder form) ...................... LL. 20 g GIVCRTOL LC. PLY i: nvwmsionn ood cmmniosin on 4 2.0 mosishsiesncn s+ 3S aitin no's sraceimemmimien o 100.0 ml Wright's stain, stock solution (see Wright's stain) ................. 900.0 ml Heat the glycerol to 55° C. Slowly add the Giemsa powder with stirring. Hold at this temperature for 2 hr. Keep covered to prevent absorption of moisture. Cool. Add 100 ml of aged Wright's stock solution. Mix. Let stand overnight, then add 800 ml of aged Wright's stock solution. Filter. Staining procedure—Use as for Giemsa stain. B. Acid-Fast Stains Ziehl-Neelsen stain Carbolfuchsin: Basic fuchsin (certified, 3% by weight in 95% ethyl alcohol) .......... 10.0 ml Phenol (5% in distilled WaLEr) .....civonvvrsisterermnirsennsnmnnnes 90.0 ml GENERAL PROCEDURES 41 Mix. Let stand several days before use. Acid alcohol: Bitiyl Bleohol {O596) emma i an stmumeies so enim sme wo sixonirmndt 54.5 ¥ 8 4 59 97.0 ml Hydrochloric acid (concentrated) ......covivvvrnrnenrervnnnnnnersens 3.0 ml Loeffler’s methylene blue (see Section D following). Staining procedure—Stain with carbolfuchsin by flooding slide with stain and gently heat to steaming point. Hold at this temperature for 3 to 5 min, cool. Wash in water for as short a time as practical. Decolorize with acid alcohol. The amount of decolorization will have to be determined by experience. Usually this will be 1 min or less. The film should have a light pink color remaining. Wash in water for as short a time as practical. Counterstain with Loeffler’s methylene blue for 5 to 30 sec. Wash, dry, examine. Acid-fast organisms are red, the background and nonacid-fast organisms should be blue. Do not allow the carbolfuchsin to dry on the slide while heating. If several slides are processed together through staining jars, cross- contamination may occur. This could result in the presence of acid- fast organisms on slides which originally were free of them. Only new, unused, clean slides should be used. Richards and Miller’s fluorescence stain Fluorescent stain,'® Solution A: Fill aleohol (OBYGT . ..ovieemiinios i 5 saints ss seimmmisis od a s masters 10.0 ml Auramine 0 (90% dye content) .........cievviinnnnntnnnnanninnenan 01g Solution B: Distilled Wallr . ..onenmns suvsnsussseenmens sis sess erie ines se seeys 87.0 ml Liquefied phenol ....comsesmvemnmsmmmmsis se vonims core eum sbesss sae 3.0 ml After the dye has dissolved in solution A, add solution A to solu- tion B. Acid alcohol: Ethyl 2leohol (F096) ui: sasmmnmnt iss snmmamas s 3 sabiine 4.5 4 5.5 Snes » 100.0 ml Hydrochloric acid (concentrated) .:svenmiveves «vniimsmins sas os puanmoss 0.5 ml Sodium CHIDEIAE . ..: oc ssumns ¥ 55 SER AUREL £ 5.5 FRR aTS Sian 2e TF Aik isis 05 g Staining procedure—Flood the slide with stain for 2 to 3 min. Wash briefly in water. Decolorize for 3 to 5 min with acid alcohol. Wash briefly in water. Dry. Examine, using a light source emitting ultraviolet light, with the usual microscope assembly for fluorescent microscopy. Only acid-fast organisms should be fluorescent. 42 GENERAL PROCEDURES Kinyoun’s stain (modified) Carbolfuchsin.: Basic fuchsin (Certified) . ...ocommmens issssmmoninms osnpmenivm vrs sob 40 g PHENOL snnumes 5s 53 mmimivies os semen £285 SVERTLE 8 SLATE ws bo 5 oh 80g Fihyl 2lcohol (9392) .uuvess + samnvainn sis suaismvens 245s vem oa oy 20.0 ml DSHHEd "'WEIEE: asinvmmmivie 35 visit Memes stellen atin s &nwncuimlate s 438 to 100.0 ml DEETHOL NB.: 7 ction iw oocnirumetaieie v0.5 Ar STREET fo Sunset iresus wut winsome SHER EG on hand Acid alcohol: BIL Gleolol (7070) + cvs sin ssnanlones vibls anes su bimsivas ns shoves 99.0 ml Hydrochloric acid (concentrated) .... ouvseeroiervnssissressnsnseess 1.0 ml Loeffler’s methylene blue (see Section D following). Methylene blue for Kinyoun stain of tissues (stock solution): Methylene DINE scones +i + smiferm sds § 8 SERUTS § 5 54 mBlimmd aliising « § » s so 14 g Ethy) 'aloohiol COBEY uss wsmusns« dakimmnnly vs 45 s bumbaids oo 55 »¢ bowion » 100.0 ml For use, add 10 ml of this stock solution to 90 ml of tap water. Staining procedures—For staining slides in the cold, flood the slide with Kinyoun’s carbolfuchsin. Add 1 or 2 drops of Tergitol No. 7, mix. Let stand for from 1 to 10 min. Wash briefly with water. Decolorize with acid alcohol until the film is a light pink. Wash with water. Counterstain with Loeffler’s methylene blue for 5 to 30 sec. For staining tissue sections, deparaffinize, then rehydrate in dis- tilled water. Place in Kinyoun’s carbolfuchsin for 1 hr at 56-58° C (some use room temperature). Wash briefly in water. Differentiate in acid alcohol until tissue is pale pink. Wash with water. Counter- stain with the methylene blue for Kinyoun’s staining of tissues as presented above. Do not use Loeffler’s methylene blue. Stain for 30 to 60 sec. Wash with water. Dehydrate, clear, and mount as usual. C. Gram Stain Hucker’s crystal violet, solution A: Crystal violet (CRIBARH) ......uvnvssss va s@oimm somo ovine vm mnines sv ues 20 g Eihyl aleohol (9595) sainsesissmsoninssess SAnimiasss ivan vae sos bag 20.0 ml Solution B: Ammon OZBIE ..cvmnen +1 versdnmns 28% SEARO ITEIAES § ATA § 08 g DisBled ‘War “uous vs smmmmess § + dswmeinas ss sranwmeines s 3 reminder LVS 80.0 ml Mix solutions A and B. Store 24 hr before use, The resulting stain is very stable. Burke's iodine: Potastimn Iodide . dos sensmviveamas Suniel snes un eaies sama mes siz 20 g LL ee 10g GENERAL PROCEDURES 43 Place the KI into a mortar, add the iodine and grind with a pestle for 5 to 10 sec. Add 1 ml of water and grind, then add 5 ml of water and grind, then 10 ml and grind. The KI and iodine should now be in solution. Pour into the reagent bottle. Rinse the mortar and pestle with the water needed to bring the total volume in the reagent bottle to 100 ml. Hucker’s counterstain (stock solution): Satranin 0 (CREHREd) .....0 vonmmiioinin vi sdnimmn © 29 8nEbiomhien Foss base 25g Tih] BICOROL (0800) . ow wmnsinsrn s snioniin.sinin 4 49800 ebmias wes eames 44 100.0 ml For use, add 10 ml of stock solution to 90 ml of distilled water. Staining procedure—Stain 1 min with Hucker’s crystal violet. Wash briefly in water. This wash step greatly influences the allowable decolorization time, A good wash procedure is to dip for 5 sec in a 250 ml beaker into which tap water is running at a rate of about 30 ml per sec. Rinse off the water with iodine solution, let Burke's iodine solution stand on the slide for 1 min. Wash with water, do not blot dry. It is recommended that the slides be wet when they are subjected to decolorization. Use three Coplin dishes containing 95 per cent ethyl alcohol for decolorization. Decolorize for 30 sec in each dish. Wash 5 sec, then counterstain for 10 sec with safranin. Wash 5 sec, dry, examine. After 10 slides, the alcohol in the first dish should be discarded. Refill with 95 per cent ethyl alcohol and place last in the decolorization sequence. Other decolorizers can be used if a proper correction is made in the decolorization time. For example, n-propyl alcohol would require about 1 min, 95 per cent #n-propyl alcohol 30 sec, and acetone 15 sec in each Coplin dish. Glanders bacillus (Actinobacillus mallei) may stain poorly with safranin, For these organisms use the carbolfuchsin of the Ziehl-Neelsen stain as counter- stain. Wash with water, blot dry, and examine. Since safranin washes rapidly from bacterial cells, keep this wash step to a minimum. Gram-positive organisms are blue, Gram-negative red. D. Loeffler's Methylene Blue Solution A: Methylene blue (90% dye content) .......cceviiirerniiiinneeennnnnn 03 g Ey] aleohol (0598) . ciovmimsnts 2+ wuiimtin vale wa: sisoieie v's y unanlii +s soak 30.0 ml Solution B: Dilute potassium hydroxide (0.01% by weight) .........coovvvnn.n. 100.0 ml Mix solutions A and B. 44 GENERAL PROCEDURES Staining procedure—Stain 1 to 2 min, Wash lightly with water. Blot dry. Examine. E. Malachite Green Stock solution (saturated aqueous): Malachite green (0XBIa18) ; concmvnis s3amRmE Aiki 5 5.8 FHITITE 3 § 5 200 0 80g Distilled WoLer .oswss sss vemmnniv ss ss womans 555 Sunswas so ¥ os vevmd ve 100.0 ml For use, dilute 1 part of stock solution with 2 parts of distilled water. F. Wayson's Stain (For staining diphtheria organisms) Solution A: Basie FUCHS. « vs viv + # « ummiinioe & # $5 SHEARS £ EOE TAH 1545 ARPES SRF a 02 g Methylene BIE « u. iui vies svnmmenies ss ume seme srs 3's 3 Memeo 1s para 075 g Ethyl alcohol (absolute) ................. coon... crnTRra ee Sis 20.0 ml Solution B: 5% phenol in distilled Water ........c.viiviiiiieirreeennernnrnneenns 200.0 ml Dissolve the fuchsin and methylene blue in the absolute alcohol (solution A). Add the phenol to the distilled water (solution B). Add solution A to B. Staining procedure—Stain for a few seconds. Wash with water. Blot dry. Examine. G. Dienes Stain (For colonies of pleuropneumonia-like organisms, PPLO) MEIhVICHE DIUE «voi issnmnmeis bas sinaaeiond 47 somnssiemot uot sim a's 46 mmrkssnce) 4k 250 g Ae TL; ommnnicorsvesmmnins od ves assiaiiess § 3 Senior GL 3 £5 samakans 125 ¢g MAliOms . . doummmion ss smears s 3 5 babes § 4 PYRENEES MEET § 35 REE 100 ¢g Sodium CHIOTIAE, wows 29 » smepsdios » 5 4 + snsmons + + + 4 Poors wees ¢ 3 3 srmEed 025 g DISNGA WEEE suns 32 simanhin + £3 ain ssaibininm + + sosipsasacaon: 0% + + msmsogoaensee « & 8 100.0 ml Preparation of cover slips—By means of a cotton swab, make a thin film of the stain on clean cover slips. The film should be uniform, and light. Dry. These cover slips may be stored almost indefinitely. Staining procedure (for colonies, suspected of being PPLO, growing on agar)—Cut a 1 cm square agar block from an area con- taining suspected colonies. Transfer this block to a microscope slide, colony side up. Place a treated cover slip, stain side down, on the agar block. The staining reaction is complete within a few minutes. All colonies stain, but in about 15 min the bacterial colonies decolorize, while the PPL.O colonies retain their color. GENERAL PROCEDURES 45 Examine at a low magnification (100) and with transmitted light. The preparation can be preserved by sealing the cover slip to the slide with melted paraffin. It is also possible to stain PPLO colonies in situ by dropping a 1:100 dilution of the Dienes stain directly onto the colony. Examine with the 16 mm objective. H. Macchiavello Stain (Rickettsia stain)?1® Reagent A: Basic fuchsin (0.25% in distilled water). Reagent B: Citric acid (0.5% in distilled water). Reagent C: Methylene blue (1.0% in distilled water). Staining procedure—Fix the film lightly with heat. Stain 3 to 5 min with freshly filtered reagent A. Pour off the stain, then dip the slide in fresh reagent B. Immediately remove the slide. Wash in a dish containing running tap water. Flood the slide with reagent C for a few seconds. Wash in running tap water. Blot dry with filter paper. Rickettsiae are a bright pink or red against a bluish background. VII. TEST PROCEDURES A. Hydrogen Sulfide Production This test is for the release of HoS from sulfur containing amino acids in the medium. Lead acetate is used to form a dark sulfide, thus indicating H,S production. Test procedure—Inoculate with a needle on the surface of the slant (CM No. 38), and stab into the deep agar layer. Incubate at the optimum temperature for growth of the test organism. Observe at 18-24 and 44-48 hr. A darkening indicates HS production, If the organism produces gas from glucose, this may cause the agar to break into pieces. Only darkening is significant. B. Indole Production This is a test for the ability of bacteria to produce indole from tryptophane. The use of hydrolyzed casein (U.S.P. XV) or tryptone assures adequate amounts of tryptophane in the medium. Kovacs’ reagent: Para-dimethyl-amino benzaldehyde .....occsissvvnesessns smvnansesnss 50g Aryl or butyl Blealiol . cum siws sss summiae vas ss 2 swarms v1.4 Hy iw vain +o sain 75.0 ml Hydrochloric acid (concentrated) ..........cccveveiririnsnepeveensan 25.0 ml Mix the amyl alcohol and benzaldehyde and heat to 50°-60° C in a water bath until dissolved. Cool. Then slowly add the hydrochloric 46 GENERAL PROCEDURES acid. Store in the dark, in a brown bottle with a glass stopper. If the final reagent is dark in color (light brown is permissible), it should not be used. In fresh reagent this dark color could result from the use of old benzaldehyde, amyl alcohol or hydrochloric acid. Test procedure—Incubate the inoculated medium (CM No. 2) for 4 days at the optimum temperature for the organism being tested. Add 0.2 ml Kovacs’ reagent to 5 ml of culture. Shake. A positive test consists of a red color appearing in the Kovacs’ reagent which layers itself over the culture medium. Indole can be tested for as soon as good growth appears. Usually 24 to 28 hr is sufficient, If the organism does not grow in this medium, ordinary tryptophane broth may be tried. C. Voges-Proskauer Test Some bacteria form 2-3 butylene glycol from glucose. This can then be oxidized to diacetyl. Diacetyl in the presence of arginine and potassium hydroxide gives a reddish color. This constitutes a positive Voges-Proskauer test. Proteose peptone is used in the medium, since it is rich in arginine. Reagents: 5 per cent a-naphthol in absolute ethyl alcohol (best re- sults are obtained with the use of a high grade of g-naphthol such as Eastman No. 170) ; 40 per cent potassium hydroxide. Test procedure—Inoculate the medium (CM No. 112). Incubate for 18-48 hr at the optimum temperature for growth of the organism. Add 1 ml of the culture to a clean test tube. Add 0.6 ml of the 5 per cent a-naphthol reagent. Shake for 5 sec. Add 0.2 ml of the 40 per cent potassium hydroxide solution. Shake for 5 sec. A crimson to ruby color should develop within 2 to 4 hr for a positive test. Do not read after 4 hr. Negative test may develop a copper or faint brown color. D. Methyl Red Test Methyl red is used in this test solely as a pH indicator. Some organisms, such as Escherichia coli, produce sufficient acidity from the glucose in this medium to stop growth and to give a red color with methyl red. This constitutes a positive test. Other organisms, such as the Klebsiella-aerogenes group, do not produce sufficient acidity to stop growth, and subsequent attack on the proteose peptone results in a rise in the pH, with the result that a yellow color is obtained with methyl red. This constitutes a negative test, Methyl red is red at pH 4.2 and yellow at pH 6.3. This test is significant only when the proper culture medium (CM No. 112) is used. GENERAL PROCEDURES 47 Methyl red indicator: Dissolve 0.1 g methyl red in 300 ml of 95 per cent ethyl alcohol. Dilute to 500 ml with distilled water. Test procedure—Inoculate the medium with the test organism, in duplicate. Incubate 5 days at the optimum temperature for the organism. Add 5 drops of methyl red indicator to each tube. Mix. A red color indicates a positive test. A yellow color indicates a nega- tive test. Intermediate colors indicate doubtful results. E. Reduction of Nitrates This should be a test for the disappearance of nitrates from a medium and the consequent appearance of nitrites, ammonia or nitrogen gas. There is no simple quantitative test by which one can follow the disappearance of nitrate. The appearance of nitrites, how- ever, can easily be tested for and this usually constitutes the basis of the nitrate reduction test. It is possible that the reduction process may have gone so far that all the nitrites formed have subsequently been reduced to ammonia or nitrogen gas. In this case a negative test for nitrite does not mean the absence of nitrate reduction. Culture medium: The usual formula for extract broth (CM No. 3) or extract agar (CM No. 4) is used, with the addition of 0.1 per cent potassium nitrate. Nitrite reagents—reagent A: 5 N acetic acid (1 part glacial acetic acid to 2.5 parts distilled water) .. 1000.0 ml Sulfanille 0d .cumsnvimmsnansiy eds amsrenesirreie peal dems hers 80g Reagent B: 5 N acelle aol Lio densi nmin dn olin they visi uete tems beds yebics 1000.0 ml Dimethyl-o-naplthylaming v..qcosisntioves sms ny sos wns webs sens 6.0 ml Test procedure (broth medium)—Inoculate three tubes of media, keep a fourth tube as an uninoculated control. Incubate at a tempera- ture favorable for the growth of the organism being tested. Test for the appearance of nitrites by using one tube on the 1st, 2nd and 3rd days following inoculation. Test for nitrite by adding 2 or 3 drops of reagent A, followed by an equal amount of reagent B. A red color indicates a positive test and should be recorded “nitrites present.” A negative test should be recorded “nitrites absent.” If a test is positive, check the uninoculated control. It is possible that nitrous acid has been absorbed from the air. If the control is also positive, the results are not significant. Nitrogen gas can be tested for by observing for gas bubbles prior to addition of the nitrite reagents. If gas bubbles are present but the nitrite test is negative, it is possible that nitrate has been reduced beyond the nitrite stage. 48 GENERAL PROCEDURES Test procedure (agar slant medium)—Inoculate three agar slants. Keep a fourth slant as an uninoculated control. Incubate at a temperature favorable for the growth of the organism being tested. Test for the appearance of nitrite by using one tube on the 1st, 2nd and 3rd days following inoculation. If the test is positive, test for nitrite in the uninoculated control (given in preceding test procedure). Test for nitrite by dropping equal amounts (2 or 3 drops) of reagents A then B onto the surface of the agar slant. A red color indicates a positive test. F. Catalase Test Test reagent: Fresh solution of 10 per cent hydrogen peroxide. Test procedure—The organism should be grown on a solid culture medium such as an extract agar slant (CM No. 4). Usually an 18-24 hr culture is used, grown at optimum temperature. Set the slant in an inclined position and add 1 ml of hydrogen peroxide solution so that it covers the growth. The appearance of bubbles indicates a posi- tive test. G. Coagulase Test This test is an aid in the identification of pathogenic staphylococci. Human or rabbit plasma can be used ; however, some authorities con- sider that rabbit plasma gives the more dependable results. Most of the usual forms of plasma are acceptable, including oxalated, citrated, rehydrated lyophilized or rehydrated desiccated (see also Chapter 6, “Staphylococcus Infections”). Small (Wassermann) test tubes which are clean, dry and sterilized are used. Place 0.5 ml of plasma into the test tube, For cultures grown on solid media, a loopful of the growth can be transferred into the plasma and suspended. For broth cultures, add 0.5 ml of an 18-24 hr culture to the plasma and mix. Incubate in a water bath at 37° C. Observe frequently for coagulation. Usually all positive results will appear within 3 hr, although incubation should be continued up to 4 hr. Longer incubation times cannot be recommended. Any degree of coagulation, however slight, is considered a positive test. H. Oxidase Test (for colonies of Neisseria) Dimethylparaphenylenediamine hydrochloride* ...................... 10 g DiISBNEA WALEE . ..ouvns iss sation £5 53 0MAEE 2 4 § 58 REE 3 £38 50 0mminios 100.0 ml Fresh reagent should be prepared each week and stored in the refrigerator. * Or the oxalate salt. If the oxalate is used, dissolve with gentle heating. 20 GENERAL PROCEDURES 49 Test procedure—For Neisseria, inoculations on chocolate agar should be incubated at 37° C in an atmosphere of 3 to 10% COs, and the incubation period should be from 18 to 48 hr. Since certain other organisms produce oxidase, Gram-negative diplococci should be observed from the oxidase-positive colonies to prove the presence of Neisseria. The oxidase reagent can be applied over specific colonies with a dropper or loop, or 2 ml of reagent can be run over the entire agar surface. Let stand 5 to 10 min before observing. Color changes are pink to maroon-black. If transfer of the organism is desired, pick from the colony while it is in the pink stage. Prolonged exposure to the reagent kills the bacteria. J. Sorensen's pH Buffers (pH 5.29 through 8.04) Buffer solutions added to culture media and to many types of reagents employed in diagnostic tests serve to prevent significant fluctuation in the hydrogen ion concentration. The reagents listed below can be purchased from almost any reliable chemical or biologi- cal supply company at a moderate cost or may be prepared from the anhydrous salts. The primary phosphate (potassium dihydrogen phosphate) may be dried at 110° C and the secondary phosphate (disodium hydrogen phosphate) at 130° C. The anhydrous salts readily absorb moisture from the air, so that prompt weighing of the dried salt is important. Reagents. M/15Na;HPO, (Solution A) is prepared by dissolving 9.464 g of anhydrous salt in distilled water to make 1 liter of solution ; M/15KH,PO, (Solution B) is prepared by dissolving 9.073 g of anhydrous salt in distilled water to make 1 liter of solution. Mix as indicated in the following tabulation: Solution A Solution B Solution A Solution B pH (ml) (ml) pH (ml) (ml) 5.29 0.25 9.75 6.81 5.0 5.0 5.59 0.50 9.50 6.98 6.0 4.0 5.91 1.0 9.0 7.17 7.0 3.0 6.24 2.0 8.0 7.38 8.0 2.0 6.47 3.0 7.0 7.73 9.0 1.0 6.64 4.0 6.0 8.04 9.5 0.5 K. Dichromate Cleaning Fluid (For removal of acid-fast bodies from glassware) Potassinn dichromate «usu s sassmnad oss anh wens ss sel muses» 2 50.0 g Sulfuric acid (CONTEMIBLEAY «cu vunnns s 22s nmit 5 2 & pen ainsnces oo 2 vibes 1,000.0 ml 50 GENERAL PROCEDURES VIII. LABORATORY ANIMALS?1:22 Guinea pigs, mice and rabbits are the animals commonly used in the diagnostic procedures described in this volume, and the com- ments of this section refer chiefly to these small animals. A. Care and Feeding The reader is referred to the companion handbook of the American Public Health Association.2 B. Identification of Test Animals Accurate results in tests are absolutely dependent on reliable identi- fication of the animals used. Otherwise, the report for one specimen may actually concern an entirely different specimen. For this reason each animal under test (or set of animals with identical inocula) must be kept in a separate cage. Mark both cage and animal with as secure a device as possible. When cages are cleaned, work with only one cage at a time to avoid any possible misplacing of animals. Remove the animal from its cage to a temporary box, clean the cage, return the animal at once, then proceed to the next cage. C. Injections of Guinea Pigs Lift the guinea pig with the hand over the back of the animal and the thumb and fingers under the forelegs. For an injection, have an assistant hold the animal by circling the shoulders with the left hand and holding the forelegs separately between the fingers. The heel of the hand helps support the animal’s back. The right hand circles the hindquarters from the back and holds the hind legs separately be- tween the fingers. The assistant extends the body of the animal, which leaves the hands of the operator free for the injection. It is best to remove the hair about the injection site with an electric clipper. For intracutaneous injections use a 4% in. 26 gauge needle. With bevel uppermost, insert the point of the needle gently into the skin so that it is barely visible. A raised bleb will appear as the material is pushed out of the syringe. For subcutaneous injections use a 22 to 24 gauge needle. For intraperitoneal injections use a similar needle, but have an assistant hold the pig with head down so that the intestines drop away toward the diaphragm. Then insert the needle posterior to the umbilicus. D. Injections of Mice Mice can be injected without an assistant, Grasp the tail of the mouse with the right hand ; it will immediately stretch away. Now use GENERAL PROCEDURES 51 the thumb and first finger of the left hand to grasp the skin of the neck and invert the mouse in the palm of the hand, holding the tail and left hind leg between the base of the thumb and the third or fourth fingers. Use a 22 to 24 gauge needle for subcutaneous and intraperitoneal injections as described under guinea pig injections immediately foregoing. Intravenous injection into the tail vein of a mouse is facilitated by the use of a square of 74 in. wire “hardware cloth” in a wooden frame as described by Crispen and Kaliss.? The mouse clings to one side of the screen while the tail is passed through one of the holes and then held firmly by the tip during injection using a 27 gauge needle. E. Injections of Rabbits Lift the rabbit by a gentle grasp of the ears and the skin over the shoulders, meanwhile supporting the weight of the animal with the other hand beneath the hindquarters. For intracutaneous and subcutaneous injections have an assistant hold the rabbit, which is a quite placid animal, and proceed as described under guinea pig injec- tions, except that the back should be chosen as the injection site rather than the abdomen. For intraperitoneal injections the animal will need to be restrained by being secured on a board. For intravenous in- jections, either use a special box with only the head protruding or have the animal held by an assistant. Use a 24 to 26 gauge needle for injections into the marginal ear vein. Always use the most distal end of the vein, in order to save the remainder for any subsequent injec- tions. Before injection of the needle rub the veins lightly with an alcohol-moistened pledget of cotton, if necessary to make the veins more prominent. F. Post-Mortem Examinations Place test animals that die in the refrigerator at once, and autopsy as soon as possible, exercising throughout the precautions against accidental laboratory infection described in Chapter 3. Fasten the animal on its back to a tray or board, then swab the thorax and abdomen with an effective germicide. Cut through the skin the full length of the abdomen and thorax, then cut transversely to right and left at either end of the primary incision. Free the skin from the underlying tissues, noting the condition of the abdominal wall where the inoculation was made and the various regional lymph nodes. Make a longitudinal incision along the middle of the peritoneum and the thorax, examining the organs for pathological changes as described 52 GENERAL PROCEDURES in the chapters of this volume on the various etiologic agents of disease. When the post-mortem examination is complete, cover the opened area with cotton soaked in disinfectant, wrap the animal in heavy paper, and complete disposal as described in Chapter 3. IX. FLUORESCENT ANTIBODY TECHNICS IN DIAGNOSTIC BACTERIOLOGY Fluorescent antibody technics have rapidly assumed importance in recent years. Their real value in the diagnostic laboratory is known for only very few diseases. However, as research and evaluation studies are completed, it must be admitted that many still unrealized applications will be forthcoming. Perhaps the most important factor that leads to the development of a successful fluorescent antibody diagnostic test is that a specific antigen-antibody relationship can be demonstrated. The early work of Coons and his co-workers has served through the years as a basis for the more successful developments in fluores- cent antibody microscopy. The number of publications now counted in the literature on this subject precludes a review of their applications in the many areas of medical immunology. Excellent reviews have been published by Coons,?#25 Liu,?® and Goldman.?? Recently, Cherry, et al.,?® made available a manual entitled Fluorescent Antibody Techniques in the Diagnosis of Communicable Diseases which serves as a guide for those interested in initiating fluorescent antibody tech- nics in the diagnostic bacteriology laboratory. It is the purpose of this section to acquaint the reader with the diagnostic and potentially diagnostic implications of the fluorescent antibody technic for bacterial infections. Basic information related to the various aspects of the technic will be given. Methods used in the more significant and advanced investigations will be described briefly. A. Fluorescence Microscopy and Photography Fluorescence microscopy is readily performed, but it is considerably more exacting than ordinary light microscopy. The critical alignment of the microscope in relationship to the light path is a basic pre- requisite to observing the fluorescent image in the microscope field. Fluorescent antibody dark-field microscopy requires the use of objectives of lower numerical aperture than that of the condenser. Fluorescent lenses or other substances must be eliminated from the optical system. Complementary or crossed filters are necessary in fluorescence microscopy. It is strongly recommended that standard GENERAL PROCEDURES 53 books, such as those by Shillaber?® and Needham,’ on basic micros- copy be studied in order to perform satisfactory fluorescence miscroscopy. 1. Microscopes—Standard microscopes fitted with a cardioid- type dark-field condenser are used by most workers. In order to take advantage of maximum light benefits, a monocular head is preferred for observing fluorescence reactions. Achromatic objectives appear to be satisfactory and perhaps preferable to apochromatic and fluorite objectives for fluorescence microscopy. A dark-field condenser permits easier visualization of the fluorescent image against a black back- ground. With a bright-field condenser, the excessive amount of white light in the background makes it difficult to discern some of the bacteria. 2. Light sources and filters—High-pressure mercury vapor lamps are available from commercial sources. The most common bulb is the Osram HBO 200, which provides light of sufficient intensity per unit of area and in the proper spectral range to make possible good visualization of the small quantities of fluorescein on the specimen. Filters are used (1) to pass the excitation light needed to cause the fluorescein to fluoresce and then (2) to eliminate the excitation light, making it possible to observe the fluorescent light of the image in the microscope. A pass filter is therefore placed between the light source and the specimen, and a barrier filter in the eyepiece between the specimen and the observer's eye. Brightest fluorescence reactions with dark-field condensers are ob- tained with a Schott BG 12, 3 mm pass filter and a Schott OG 1, 2 mm barrier filter, or equivalent Corning and Wratten filters. With such filters, wave lengths of 350-450 mu in the ultraviolet and blue- violet range are utilized. Some workers prefer to use ultraviolet irradiation only and find the Corning 5840 or 5970-Wratten 2A or 2B or Euphos filter combinations satisfactory. A good filter combina- tion should pass no observable light when the two are placed in tandem position to each other and held up to the light. Heat-absorbing filters, Schott BG-14 (4 mm) and Schott BG-22 (3 mm), should be placed between the light and the other filters to prevent their overheating and cracking. Fluorescence units which permit the easy visualization of specimens using tungsten light as well as blue-violet are especially helpful in examining clinical material. 3. Photography—In our experience the best color photomicro- graphs of fluorescent bacteria have been obtained with Super Ansco- 54 GENERAL PROCEDURES chrome, daylight-type film. Exposure times of 3-4 min are used commonly. Tri-X daylight-type film is desirable for black and white photographs, and exposure times of 2-4 sec usually are satisfactory. B. Labeling Antibody Solutions with Fluorescent Dyes Coons, et al.,3! used fluorescein isocyanate to label antibody globu- lin, Later, in a now classical publication, the method for synthesiz- ing this compound was described. Their methods were followed by most workers until Riggs, et al.,*® and Marshall, et al. ,** reported a method for synthesizing fluorescein isothiocyanate and rhodamine isothiocyanate. Fluorescein isothiocyanate offers the advantage of shelf stability not demonstrated for fluorescein isocyanate. The synthesis from fluorescein amine is simpler and less hazardous, utiliz- ing liquid thiophosgene, rather than phosgene gas, which is used to synthesize fluorescein isocyanate. Fluorescein isothiocyanate is used by most investigators now. It may be added directly as a dry powder to the buffered globulin or serum solution. Others prefer to add it as an acetone slurry which causes the entire amount to dissolve and enter into the coupling reaction at the beginning of the labeling period. As with fluorescein isocyanate, a satisfactory ratio of fluorescein isothiocyanate to protein is .05 mg per mg of protein. In the CDC laboratory, streptococcus and plague antibody globulins labeled with fluorescein isothiocyanate have remained stable for at least 2 years when stored frozen ( —40° C to —60° C), at 0° to 5° C, at room temperature or after lyophilization. Satisfactory preservation from contamination may be maintained with a 1:10,000 concentration of merthiolate or phenyl mercuric borate. Normally the stability of the conjugates can be maintained during shipment through the mails. Orange-red fluorescein dyes as protein labels used in combination with fluorescein isothiocyanate may permit differentiation of two antigens in a given preparation. Clayton?® reported the use of nuclear- fast red. Since no other work with this dye has been reported, its potentialities remain unknown. Isocyanate derivatives of rhodamine B®®¢ and tetramethyl rho- damine3” were used in the same manner as the isocyanate of fluores- cein, Presumably the stability of these compounds is no greater than that of fluorescein isocyanate, although no definite information is available. Commercially available rhodamine isothiocyanate? and Lissamine rhodamine RB 2003% perhaps are receiving the most in- terest. They are both stable compounds and simple to use. The former is used in the same manner as fluorescein isothiocyanate. Lissamine GENERAL PROCEDURES 55 rhodamine RB 200 is first converted to the sulfonyl chloride deriva- tive with phosphorus pentachloride and the dye is added slowly to the globulin or serum, which is buffered at pH 9.0. After dialysis for several days against buffered salt solution, the labeled protein is ready for use. Smith, et al.,?® used Lissamine rhodamine-labeled normal serum as a counterstain to eliminate nonspecific fluorescence of fluorescein dye in formalin-fixed tissues. A yellow fluorescing dye, 1-dimethyl aminonaphthalene-5-sulfonyl- chloride, was used by Weber,*® Clayton, Mayersbach,* and Redetzki.#? Although protein labeled with this dye retains its serological integrity, its brilliance is superseded by fluorescein. Its use- fulness for contrast staining, therefore, would not appear to equal that of the orange-red dyes described above. C. Use of Fluorescent Antibody Solutions The manner of using fluorescent antibody solutions will be de- termined partially by the information sought. For example, if bacterial cells or their antigens are to be detected, the “direct” staining technic would be the simplest and most rapid test to perform, although the more laborious “indirect” and “inhibition” tests could also be used. If antibodies are to be detected in serum, the inhibition or indirect test would be the method of choice. Regardless of the method selected, its value can be only as great as its specificity and its sensitivity. Therefore, before any assumptions are made that fluores- cent antibody tests are reliable for identifying a particular antigen or antibody, the test must be thoroughly investigated and evaluated under a variety of conditions. 1. Direct test—The direct fluorescent antibody test is the easiest to perform and involves the smallest number of factors that might interfere with specificity and sensitivity. Dried films of material containing the organism or antigenic substance are fixed on micro- scope slides and stained with the labeled antibody solution. After a relatively short reaction time, the excess reagent is rinsed from the slide and the films are mounted and examined. A positive reaction is indicated when brilliantly fluorescent organisms or areas are ob- served in the: fluorescence microscope. The test requires a different labeled antibody solution for each antigen to be detected. This is no longer a serious disadvantage, however, because of the ease with which antibody globulin can be labeled with the fluorescent dye. 2. Inhibition test—The staining obtained by using the direct fluorescent antibody test can be inhibited by using the unlabeled, 56 GENERAL PROCEDURES homologous antibody to block the reaction of the labeled antibody. With proper controls the test can be used advantageously to confirm the specificity of the direct test. It can also be used to detect antibody in serum both qualitatively and quantitatively. The test, as proposed by Coons, et al.,*! is performed in two principal steps. The antigen is treated with unlabeled antibody for a period of time and the excess rinsed off. Then, the film is treated with fluorescein-labeled antibody of the same type, and the excess rinsed away. Positive inhibition of fluorescence is demonstrated when the antigen no longer fluoresces or when fluorescence is reduced in comparison with control films. For bacterial systems somewhat better success has been experienced with modification of this inhibition test. Goldman*® and Moody, et al.,** treated films with mixtures of unlabeled and labeled homol- ogous antibody solutions and demonstrated inhibition of fluorescence. Reactions were sharper and more specific than those obtained using the two-step procedure. These observations were confirmed with streptococcus, Brucella, plague, and tularemia systems by Moody, et al.,*>*8 Biegeleisen, et al.,*” and Winter and Moody.*® The reasons for this are not clearly understood. Best results from the one-step modification of the inhibition test are obtained when appropriate dilutions of labeled globulin are used with an equal volume of diluted unlabeled antibody. Goldman*? reported the use of this test for detect- ing antibody for Toxoplasma gondii in human sera. The test also has been used to titrate Brucella antibody in human and animal sera.*6:47 Titers obtained by this method were approximately one-tenth as high as those obtained by agglutination tests but were of the same level of specificity. 3. Indirect test—The indirect fluorescent antibody test also may be used to localize antigen and to detect antibody in serum. The anti- gen on the slide is treated with varying dilutions of unlabeled homologous antibody for a short period and the excess washed off. The antigen, now coated with antibody, is then treated with antibody homologous for the serum globulin of the animal in which the un- labeled antibody was produced. In a positive reaction, the antigen be- comes fluorescent. The indirect test was originally described by Weller and Coons*® for staining viral antigens. Liu®® and Kaplan®! indicated that the effect of building up “layers” on the antigen may increase significantly the degree of fluorescence. The test has been found useful in titrating antibody in serum for atypical pneumonia,’® Herpes simplex virus,%? Treponema pallidum, Brucella, 647 and anthrax.5* The indirect test may be useful in screening sera for a variety of antibody types by varying the kinds of antigens used on the film. GENERAL PROCEDURES 57 Such an application would require one labeled globulin for testing serum from a single animal species. The additional controls and extra time required to perform the test may render it less advantageous, however, than the direct test. 4. Complement staining—Goldwasser and Shepard® introduced a modified indirect test designed to stain complement which has been fixed in a reaction between antigen and immune serum. Tissue culture cells were infected with polio virus and were exposed to a mixture of immune human serum and fresh, normal guinea pig serum as a source of complement. After a reaction period, the material was rinsed off and the film stained with labeled rabbit antibody prepared against guinea pig globulin. The virus stained brilliantly. Although this test has not been applied to bacterial systems, it might be expected to work in any system that will bind complement. Successful use of the test will depend upon very careful selection and analysis of control tests. Goldwasser and Shepard® indicated that some normal guinea pig and monkey sera, when used as complement sources, caused staining in the absence of immune sera. 5. Fixation methods—The method selected for fixing bacterial antigens to the slide depends somewhat upon the chemical nature of the molecules involved. In general, gentle heat fixation, as proposed in 1956,* has been suitable for most bacterial antigens. Chemical methods have been used successfully also. Winter and Moody*® used ethanol, methanol, formalin, and dioxane for fixing Pasteurella pestis. Greater numbers of group A streptococci can be retained on the slide by 95 per cent ethanol or absolute ethanol fixation than by gentle heat.*> The staining intensity of alcohol-fixed films was no greater than those which were heat fixed. In this case, alcohol fixation is par- ticularly advantageous when throat swab cultures contain small numbers of Group A streptococci. Bacterial cells and antigens are readily stained in impression films and tissue sections from infected animals or human beings.?456-59 For impression films, gentle heat fixation was satisfactory in most cases. Formalin was perhaps the most common fixative used for tissue sections. 8,54,59 6. Staining procedure—Fixed films or tissues on slides are covered with a small drop of appropriately diluted fluorescent anti- body. Care must be taken to cover the entire film to prevent partial or inadequate staining of fringe areas, which makes reading difficult. Staining at room temperature is satisfactory. Other temperatures have not enhanced the efficiency of the technic. Slides are stained in a 58 GENERAL PROCEDURES moist atmosphere provided by placing over the slides a large petri dish cover fitted with moist filter paper. To attain maximal staining in the direct test, 15 to 30 min is usually sufficient. The excess reagent is removed by rinsing 10 min in phosphate-buffered salt solution in the slightly alkaline range. Slides are carefully blotted dry and mounted with buffered glycerol salt solution and a cover slip. The same basic manipulations described above are used for the in- hibition and indirect methods, using appropriate staining and rinsing periods. 7. Examination of films—Methods of estimating fluorescence in- tensity of stained bacteria or their antigens are somewhat subjective, but with experience the readings appear to be sufficiently reliable to be useful. Most workers arbitrarily assign plus values to the level of fluorescence observed. For systems in which the only information desired is whether or not a reaction has occurred, very low levels of fluorescence may be accepted as significant. Caution should be exer- cised, however, in judging weak reactions as specific, because of the numerous “normal” antibodies present in sera and because of other factors unexplained. It is imperative also that the specimen be read only after it can be ascertained that the light source and microscope are in accurate alignment and that the field is properly focused. Read- ing of control films in comparison with the test is extremely important. 8. Diagnostic applications in bacteriology—The feasibility of using fluorescent antibody tests in diagnosing bacterial infections of public health significance has been tested with many systems. For the purpose of this section, only those studies will be described which included fairly complete comparisons between conventional and FA tests, even though other studies have reported what may be equally reliable information. a. Identification of streptococcal groups from throat swabs—TIt has been estimated that over 90 per cent of the throat infections caused by beta-hemolytic streptococci are in Group A. Therefore, the develop- ment of fluorescent antibody tests for detecting Group A streptococci has received the most emphasis. Moody, et al.,*> described methods for preparing fluorescent antibody reagents for grouping beta-hemolytic streptococci. The use of the reagents for identifying Group A streptococci from throat swabs was then evaluated extensively in 1958 and 1959 (to be published) to establish the validity of the test with direct films from throat swabs and with those made from young broth cultures of the throat swab. It was demonstrated that many more specimens positive for Group A streptococci could be demon- GENERAL PROCEDURES 59 strated with fluorescent antibody on young broth cultures than by conventional methods performed immediately after collecting the swab. It is also remarkable that by fluorescent antibody staining of films made from broth inoculated with the swab and incubated 2 to 3 hr the presence of Group A streptococci was demonstrated consid- erably more often and more readily than by staining films made di- rectly after collecting the throat swab. Positive films made directly from throat swabs usually contained very few cocci, which were seldom in well-defined chain formations and often enmeshed in pharyngeal secretions or other material which made the reading of such tests unreliable. Extensive retesting of specimens, from which results obtained with the two technics were divergent, established the specificity of the test. The test now recommended for routine use by Moody and co-workers is performed as follows: 1) Place throat swab into 1 ml Todd-Hewitt broth (Difco) and incubate 2 to 6 hr at 35°-37° C. 2) Centrifuge, resuspend sediment in physiological salt solution, and repeat centrifugation. 3) Prepare duplicate films of washed sediment, allow to air-dry and fix 1 min with 95 per cent absolute ethanol. Rinse briefly with salt solution. 4) Stain one film with fluorescein-labeled Group A streptococcus antiglobulin (absorbed with Group C streptococci) and one film with labeled normal rabbit globulin (absorbed with Group A streptococci) for 15 to 30 min in a moist atmosphere at room temperature. 5) Rinse 10 min in buffered salt solution, then briefly in distilled water. Blot dry gently. 6) Add a drop of buffered glycerol salt solution and a cover slip. 7) Examine on the fluorescence microscope, using a BG-12—OG-1 filter com-~ bination, a dark-field condenser, and oil-immersion objective. The results of fluorescent antibody tests for Group A streptococci must be interpreted with care. The Group A streptococcus anti- globulin is derived from antiserum which is specific when used in the Lancefield precipitin test. For use as a fluorescent antibody reagent, the conjugate must be absorbed with Group C streptococci to remove cross-reactions with Groups C and G streptococci. Such conjugates normally can be used if diluted 1:50 or 1:100. The control conjugate is derived from nonimmunized rabbits and the conjugate is absorbed with Group A streptococci. Both conjugates normally will stain Staphylococcus aureus. Therefore, extreme caution must be ex- ercised in reporting the presence of Group A streptococci if fluores- cent micrococci unchained or few in number are observed. A reliable Group A streptococcus identification can be reported only if brightly fluorescent chains of cocci are seen in films stained with Group A streptococcus conjugate and not in those stained with normal rabbit conjugate. The ubiquity of antibody in normal rabbit and human 60 GENERAL PROCEDURES sera constitutes a serious problem in fluorescent antibody tests, especially when the kind of specimen being examined may possibly contain Staph. aureus. Happily, experience so far indicates that the presence of the organism in young cultures of throat swabs is de- tected only rarely. The problem becomes more serious, however, with older cultures. Removal of the cross-staining reaction can be assured by absorption with certain strains of Staph. aureus, but the re- action for the homologous Group A streptococci is seriously dimin- ished. It was concluded that the fluorescent antibody test for rapid identification of Group A streptococci from throat swabs was more sensitive than and as specific as the conventional pour plate, cultural- precipitin grouping methods. The recommended procedures have been evaluated in more than 40 state public health laboratories covering nearly 30,000 patients. The agreement in results obtained with con- ventional and fluorescent antibody tests is approximately 96 per cent. Some workers reported somewhat better success in detecting Group A streptococci in films made directly from throat swabs.6%6! It was unclear, however, what controls were used and whether or not pres- ence of Staph. aureus was considered. Wolfe and Cameron®? reported success in staining films made directly from growth on trypticase soy agar which had been inoculated in the physician’s office and mailed to the state laboratory. A modification of the recommended technic was developed and evaluated by Redys, et al.®® They found that inhibition of common antigen fluorescence by preliminary treatment of films with Group C streptococcus antiserum eliminated problem cross- reactions of Group C and Group G streptococci. Some state public health laboratories have experienced difficulty in using fluorescent antibody tests for examining large numbers of routine throat swab specimens. Admittedly more time may be required to examine completely all specimens by the recommended procedure than would be required to screen cultures for the presence of beta- hemolytic streptococci and then determine the streptococcal group by performing fluorescent antibody tests with such colonies. The latter procedure has been used successfully by some laboratories, but it re- quires additional evaluation. b. Detection of enteropathogenic Escherichia coli types in fecal specimens— Whitaker, et al.,%* used flucrescent antibody tests to study an epidemic of infantile enteritis caused by E. coli 0127:B8. The study was made on 128 frozen stool specimens and rectal swabs which had been collected in 1954. Final comparisons of cultural and fluorescent antibody test results indicated that the latter test was highly specific and as sensitive as conventional methods. GENERAL PROCEDURES 61 Nelson, et al.,®® used antibody pools containing serotypic “O” antibody for the ten enteropathogenic E. coli types to screen films made from rectal swabs collected from 375 children under 2 years of age. When a positive reaction was obtained with one of the pools, additional films were stained with conjugates of the componential antibodies of the pool. The authors reported that greater numbers of enteropathogenic E. coli infections could be detected with fluorescent antibody than with cultural technics and that the method was highly specific. Thomason and co-workers®® reported on a comprehensive study of methods and reagents that contribute to successful use of fluorescent antibody for detecting enteropathogenic E. coli in films. A detailed report on the use of the technic in a survey of infants afflicted with enteritis is included. They prefer to use conjugates containing anti- body that will stain not only the “O” antigen, but also the “B”, based on the possibility that organisms might be encountered possessing large amounts of capsular antigen which might inhibit the staining of “O” antigen if only “O” antibody were used. It was also noted that “OB” conjugates can be used in higher dilutions than “O” con- jugates, which may reduce the chances of reporting cross-reactions by rare organisms stained with low dilutions. With the exception of minor differences in methods used for han- dling specimens and staining films both groups of workers have used similar technics. The results obtained show that by using fluorescent antibody methods more patients with enteropathogenic E. coli organ- isms are detected than by using conventional cultural methods. Thomason, et al., have repeatedly been successful in demonstrating E. coli by fluorescent antibody in all specimens that were culturally positive. Briefly, their method for preparing films and performing the fluorescent antibody test is as follows: 1) Prepare a 1:5 dilution of antibody pools I and II, using sterile 1:10,000 merthiolated salt solution. Pool I contains “OB” antibody for Types 026 :B6. 055:B5, 0111:B4, and 0127:B8. Pool II contains “OB” antibody for Types 086:B7, 0119:B14, 0125:B15, 0126:B16, and 0128:B12. Reagents must be refrigerated while not in actual use. 2) Dilute individual antibody conjugates 1:20 with merthiolated salt solution. 3) Prepare a suspension of feces or wash off rectal swab in 0.5 to 1.0 ml of sterile .85 per cent salt solution. 4) Prepare films approximately 1.0 cm in diameter immediately, air-dry, gently heat-fix, and stain 15 min with 1 drop of the diluted antibody pools. Rinse films 10 min in buffered salt solution. If fecal material is scanty, as is often the case with swabs, incubate the suspension 1 hr at 35° C before preparing films. 62 GENERAL PROCEDURES 5) If fluorescent bacilli are observed in films stained with liquid from either of the pools, stain additional films of the specimen with individual component antibodies of the pool. If only a few fluorescent organisms (10 or less) are seen per film report a negative result. 6) For isolation of the organisms, streak one loopful of fecal suspension on enteric isolation media suitable for growth of E. coli. 7) Perform slide agglutination tests with lactose-positive colonies, using multi- valent E. coli “OB” antisera. Transfer colonies that give positive agglutina- tion reactions with any of the pools to blood agar base slants for additional study with “O” or “OB” antiserum and fluorescent antibody reagents, since testing colonies directly from media containing bile salts may result in false- positive agglutination reactions. The authors note that the fluorescent antibody test is more sensitive than isolation procedures in that fewer organisms, whether viable or nonviable, are required to give a positive reaction. Detection of E. coli organisms from plates may require the testing of perhaps 20 to 25 colonies before a positive agglutination reaction is obtained. c. Identification of Neisseria gonorrheae in exudates—Deacon, et al.®"% have described fluorescent antibody technic for detecting N. gonorrheae in films of swab specimens collected from infected men and women. Although only 25 males were tested, brilliantly stained organisms were detected in urethral films. The reaction was dependent upon possession of a K-antigen by cells present in most freshly collected exudate from gonorrhea patients. From the study with males evolved the development of fluorescent antibody tests for detecting N. gonorrheae in females. The authors recommend the following procedure for rapid identification of N. gonorrheae in females by the direct and delayed FA methods.%8 1. The greatest yield of positive findings may be expected if specimens from the vagina, cervix, and urethra are included in the examination. The vagina and urethra, as a combination, will result in satisfactory findings and may be used where complete clinic facilities are not available, such as examination table and speculum. 2. Duplicate films from each site should be prepared. These are used for direct FA identification. At least one slant may also be prepared from each site and examined by the delayed FA procedure. 3. Direct FA slides demonstrating positive results constitute a complete ex- amination. Delayed FA testing needs completion only if the direct procedure fails to demonstrate gonococci. If desired, delayed FA testing may be used to confirm direct FA findings. 4. Fixation of air-dried films (direct or delayed) is best accomplished by 10 min in 3 per cent formalin in phosphate-buffered salt solution pH 7.2. This is followed by a thorough washing in distilled water, and finally blotting before application of fluorescent antibody. Positive findings from any site constitute a completed examination. d. Identification of Bordetella pertussis in nasopharyngeal films— Fluorescent antibody technics have been described for identification GENERAL PROCEDURES 63 of B. pertussis in films prepared from nasopharyngeal exudate collected from patients suspected of having whooping cough.8®-7 Kendrick et al.,”® examined 130 patients by complete fluorescent antibody and conventional cultural-serological tests,» They obtained agreement in the two procedures in 119 specimens (21 were positive by both and 98 negative by both). Of the remaining 11 specimens, 4 gave positive cultural and negative FA reactions, while 7 gave nega- tive cultural and positive FA reactions. Logical explanations were offered for divergent results. The use of normal bovine serum conjugated with Lissamine rhodamine RB 200 used in a proportion of 1:20 as a counterstain “aided considerably in visualizing the organisms against the back- ground of ciliated epithelial cells and other types of cells or debris which was sometimes present in direct smears.” Donaldson and Whitaker® reported that 31 of 36 patients showing clinical pertussis symptoms yielded positive FA films. Although no comparisons with cultural methods were described in this study, the authors were convinced that the reactions were specific and that the FA test was as sensitive in detecting pertussis patients as were cultural methods used in other studies. X. INTERPRETATION OF LABORATORY RESULTS In the past, there was a period of relative stability in the “disease etiology-laboratory detection” relationship which permitted the in- terpretation of laboratory findings with comparative ease. During recent years, however, the laboratory report has become increasingly difficult to interpret in view of the continued decline in the incidence of many bacterial, protozoan and spirochetal infections, the emergence of the viral and mycotic diseases to a relatively more important posi- tion, the identification of numerous “new” pathogens, the develop- ment and refinement of technical procedures for their detection, and the expansion of knowledge concerning the effects on laboratory find- ings of immunizing agents and therapy, particularly the use of drugs and antibiotics which may suppress, delay or modify the immune response, These developments necessitate a greater degree of coopera- tion between the laboratorian, the clinician and others who utilize the diagnostic services of the laboratory. The value of a laboratory test is dependent on the quality of the specimen submitted, the validity of the test performed, the proficiency of the laboratorian, and the intelligence with which the results are interpreted. 64 GENERAL PROCEDURES It has become axiomatic that the laboratory report should be in- terpreted in the light of the case history and clinical symptoms. How- ever, a correct interpretation of the findings frequently requires a more thorough understanding of the problem than can be obtained from the symptomatology alone. This broader knowledge may be developed more fully when the laboratory is made an equal partner with the clinical and epidemiological disciplines in obtaining the answers to questions pertaining to individual or group disease etiology. Generally, laboratory tests rely on two procedures: (1) demonstra- tion and identification of the causative organism and (2) demonstra- tion of substances, such as antibodies, produced by the host in re- sponse to the disease. In general, the recovery of a pathogenic organ- ism in the body fluids, tissues or exudates is more conclusive than the indirect evidence of infection provided by the presence of antibodies. However, as in some viral and rickettsial diseases, the serological test may be the most practical or the only test available. Demonstration of the causative microorganism requires knowl- edge of its occurrence and persistence in a given part of the body and, consequently, the type of specimen most suitable for examination. The proper interpretation of a laboratory report is also related to the manner in which the specimen has been collected and handled. Further, it is necessary to evaluate the possibility of obtaining growth and mutiplication of the suspected pathogen when the specimen has been collected following vaccination or the institution of treatment. For example, absence of growth may mean that the pathogen sought is not the cause of the infection or that growth has been suppressed by antibacterial therapy. Continuance of clinical infection and cultivation of the causative pathogen in the presence of appropriate dosage by antibacterial drugs may indicate bacterial resistance to the therapeutic agent or insufficient penetration to the site of bacterial multiplication. In some cultural procedures, such as the phage typing of staphylococci, it is advisable to type all cultures in a set from selected sources on the same day to reduce the chance of variations which might be introduced by test conditions. Otherwise, the results must be interpreted with some caution. Finally, the laboratory report, indicating the identifica- tion of a given microorganism, is of significance only when there is well-established evidence based on epidemiological and clinical con- siderations that the microorganism can actually cause a specific disease. GENERAL PROCEDURES 65 Satisfactory use of the laboratory for the demonstration of anti- body or other host products requires a knowledge of (1) the develop- ment and time of appearance of such products, (2) their persistence, and (3) the effect of vaccination or treatment on their development and persistence. Here again it should be emphasized that a quantitative serological report on only one specimen from a given patient is of decidedly limited value and that no single “diagnostic titer” can be cited. Of much greater significance is the rising or falling titer, which may be observed when specimens are collected in the acute and con- valescent stages of the disease. The later specimen should be ex- amined with a repeat aliquot from the first specimen in order to be sure that any change is real and not merely a laboratory variation. Furthermore, in reports on serological tests, consideration must be given to the possibility of cross-reactions, anamnestic reactions, non-~ specific reactions, and false-positive and -negative results. For ex- ample, in serological tests for syphilis, a reactive finding may be a biologic false positive in a nonsyphilitic person; a nonreactive find- ing may mean only that the specimen was collected too early in the course of the infection. There are values and limitations inherent in each type of examina- tion performed in the laboratory, and all are subject to technical errors on the part of the person performing them as well as to varia- tions in the sensitivity of the antigens, reagents and media employed. Consequently, the interpretation of laboratory findings, especially when the results appear to be incompatible with the clinical diagnosis, necessarily requires consideration of whether the test was performed by a well-qualified laboratory using accepted standard procedures, satisfactory equipment and reagents, and competent personnel. Con- stant review and evaluation of technical procedures and performance is necessary to insure reliable results. When the laboratory report involves a procedure with which the clinician is unfamiliar, consultation with the laboratory director to verify the value of the procedure and the significance of the results may be indicated. In conclusion, it is apparent that, while the interpretation of the laboratory report in terms of the presence and extent of the disease in the patient remains the exclusive responsibility of the attending physician, who should have the clinical judgment and experience needed to assign the proper significance to the facts reported by the laboratory, satisfactory interpretation of the laboratory report may ultimately require consultation with the laboratorian, health officer, 86 GENERAL PROCEDURES epidemiologist or a combination of these. Occasionally the situation may in addition require consultation with recognized authorities in a particular field of medicine. RoBert A. MAcCreaby, M.D., Chapter Chairman* James W. BarrHoroMEW, Pu.D. Cuarres C. Crort, Sc.D. C. A. LAwreNcg, Pu.D. Max D. Moopy, Pa.D. E. J. Sunkes, Dr.P.H. CarL WALTER, M.D. Lours WEINSTEIN, M.D. REFERENCES 1. Advisability of Routine Laboratory Examination. Committee on Advisability of Routine Laboratory Examination of Food Handlers, Laboratory Section, American Public Health Assn. A.J.P.H. (Suppl.) 26:98-100, 1936. 2. Technical Methods and Procedures, American Association of Blood Banks. Burgess, 1956. 3. Microscopy in Medicine. New York, N. Y.: American Optical Co., 1950. 4. The Theory of the Microscope. New York, N. Y.: Bausch & Lomb Optical Co., 1951. 5. International Centrifuges—Dealer Sales Manual. Boston, Mass.: Inter- national Equipment Co., 1954. 6. WALTER, CARL W. Aseptic Treatment of Wounds. New York, N. Y.: Mac- millan, 1948, Chapter XVI. 7. The Care and Handling of Glass Volumetric Apparatus. Cleveland, Ohio: Kimble Glass Co. 8. DeGowin, E. L., Haroin, R. C, and ALSEVER, J. B. Blood Transfusion. Philadelphia, Pa.: Saunders, 1949, p. 130. 9. McCurrocH, Ernest C. Disinfection and Sterilization. Philadelphia, Pa.: Lea & Febiger, 1945. 10. ReppisH, GrorGe F. Antiseptics, Disinfectants, Fungicides, and Chemical and Physical Sterilization. Philadelphia, Pa.: Lea & Febiger, 1954. 11. Sax, N. I. Dangerous Properties of Industrial Materials. New York, N. Y.: Reinhold, 1957. * Grateful acknowledgment is made for the assistance of the following persons : Dr. E. Kass of the Boston City Hospital J. P. BAKER, E. S. Gober and N. J. BLAIkLoCK of the American Optical Company W. R. Saunpers and J. R. BENForD of the Bausch and Lomb Optical Company J. F. LusseN of E. Leitz Company R. W. Lyons and A. G. Dixon of the International Equipment Company G. W. Tuomas of Fisher Scientific Company J. W. Korko of Corning Glass Works J. E. SmitH of the Long Beach, California, City Health Department Dr. P. B. DEws, Dr. D. G. Frienp, Dr. A. J. McBay of the Harvard Medical School Dr. E. Urpyke of the Communicable Disease Center W. BowmaN of the Georgia Department of Public Health Miss H. Girerte, Miss G. Stuart, Mrs. M. Jounson, Mrs. M. Hormes, T. Day, Miss M. MeveseriAN and Dr. J. NEweLL of the Massachusetts Department of Public Health Miss J. GATELY of the Cambridge City Hospital, Cambridge, Mass. Dr. H. M. Kurtz, University of Southern California Mzs. E. B. GooLpEN of the Childrens’ Hospital, Los Angeles GENERAL PROCEDURES 67 12. 13. 14. 15, 16. 18. 19. 20. 2i. 22. 23. 24. 25. 26. 27. 28. 29. 30. 3. 32. 33. HawmiLron, A., and Harpy, H. L. Industrial Toxicology. New York, N. Y.: Paul B. Hoeber, 1949. Toxic Eye Hazards. New York, N. Y.: National Society for the Preven- tion of Blindness, 1949. A Guide to Safety in the Chemical Laboratory. Manufacturing Chemists Assn. New York, N. Y.: Van Nostrand, 1954. The Origins and Prevention of Laboratory Accidents. Report No. 4. London: Royal Institute of Chemistry, 1949. Reese, A. M., Morris, JANIE F., and Sunkes, E. J. The Conversion of a Standard Incubator to a Carbon Dioxide Incubator. J. Lab. & Clin. Med. 34:865-872, 1949. Parker, C. A. Anaerobiosis with Iron Wool. Australian J. Exp. Biol. & M. Sc. 33:33-38, 1955. Ricuarps, O. W., and MiLLer, D. K. An Efficient Method for the Identifi- cation of M. tuberculosis, with a Simple Fluorescence Microscope. Am. J. Clin. Path. (Tech. Suppl.) 5:1-8, 1941. MaccHiaveLLo, A. Estudias sobre Tifus Exantematico. III, Un Nuevo Metodo para Tenir Rickettsia. Rev. chilena de hig. y med. prev. 1:101-106, 1937. CARPENTER, C. M., SunkLAND, L. G., and Morrison, M. The Oxalate Salt of p-Aminodimethylaniline—An Improved Reagent for the Oxidase Test. Science 105 :649-650, 1947. Sranerz, C. A. Care of Laboratory Animals. New York, N. Y.: American Public Health Assn., 1954. WabsworTH, A. B. Standard Methods of the Division of Laboratories and Research of the New York State Department of Health. Baltimore, Md.: Williams & Wilkins, 1947. CrispEN, CHARLES G., Jr, and Kariss, NATHAN. A Simple Device for Facilitating Injections in the Tail Veins of Mice. Am. J. Clin. Path. 35:387-388, 1961. Coons, A. H. Antigens and Antibodies Labeled with Fluorescein. Schweiz. Z. Path. Bakt. 22:693-699, 1959. ——————— The Diagnostic Application of Fluorescent Antibodies. Schweiz. Z. Path. Bakt. 22:700-723, 1959. Liu, C. The Use of Fluorescent Antibody in the Diagnosis and Study of Viral and Rickettsial Infections. Ergeb. Mikrobiol. Immunitdt. Exper. Ther. 33:242-258, 1960. GoLpMmAN, M. Immunochemical Staining with Fluorescent Antibody. Int. Rev. Trop. Med. 1:215-245, 1960. Cuerry, W. B.; GorpmaN, M.; Carski, T. E.; and Mooby, M. D. Fluores- cent Antibody Techniques in the Diagnosis of Communicable Diseases. PHS Pub. No. 729. Washington, D. C.: Gov. Ptg. Ofc., 1960. SHILLABER, C. P. Photomicrography in Theory and Practice. New York, N. Y.: John Wiley & Sons, 1949. NeepuAM, G. H. The Practical Use of the Microscope. Springfield, IIL: Charles C Thomas, 1958. Coons, A. H.; CreecH, H. J.; Jones, R. N.; and BEerrLINER, E. The Demonstration of Pneumococcal Antigen in Tissues by the Use of Fluores- cent Antibody. J. Immunol. 45:159-170, 1942. Coons, A. H., and Karran, M. H. Localization of Antigen in Tissue Cells. II. Improvements in a Method for the Detection of Antigen by Means of Fluorescent Antibody. J. Exper. Med. 91:1-13, 1950. Rices, J. L., et al. Isothiocyanate Compounds as Fluorescent Labeling Agents for Immune Serum. Am. J. Clin. Path. 34:1081-1097, 1958. 68 34. 35. 36. 37 38. 39. 40. 41. 42. 43. 44, 45. 46. 47. 48. 49. 50. 51. GENERAL PROCEDURES MarsHALL, J. D., Jr, Evetann, W. C, and SmrrH, C. W. Superiority of Fluorescein Isothiocyanate (Riggs) for Fluorescent Antibody Technique with a Modification of Its Application. Proc. Soc. Exper. Biol. & Med. 98:898-900, 1958. Crayton, R. M. Localization of Embryonic Antigens by Antisera Labelled with Fluorescent Dyes. Nature 174 :1059-1060, 1954. SiLversTEIN, A. M. Contrasting Fluorescent Labels for Two Antibodies. J. Histochem. 5:94-95, 1957. Hiramoro, R.,, Enger, K., and Pressman, O. Tetramethyl Rhodamine as Immunohistochemical Fluorescent Label in the Study of Chronic Thyroidi- tis. Proc. Soc. Exper. Biol. & Med. 97:611-614, 1958. Cuapwick, C. S., McEntecart, M. C, and Namn, R. C. Fluorescent Protein Tracers: A Trial of New Fluorochromes and the Development of an Alternative to Fluorescein. Immunology 1:315-327, 1958. Smita, C. W.,, MarsHALL, J. D., Jr, and Everanp, W. C. Use of Con- trasting Fluorescent Dye as Counterstain in Fixed Tissue Preparation. Proc. Soc. Exper. Biol. & Med. 102:179-181, 1959. WEBER, G. Polarization of the Fluorescence of Macromolecules. 2. Fluores- cent Conjugates of Ovalbumin and Bovine Serum Albumin. Biochem. J. 51:155-167, 1952. MAversBACH, H. Immunhistologische Methoden. II. Ein Weiteren Markierungfarbstoff: Dimethyl-1-1 Naphthyl-amino-sulfonsdure-5. Acta Histochem. 5:351-368, 1958. ReperzK1, H. M. Labelling of Antibodies by 5-Dimethylamine-1-naphthalene Sulfonyl Chloride—Its Effect on Antigen-Antibody Reactions. Proc. Soc. Exper. Biol. & Med. 38:120-122, 1958. GoLpMAN, M. Staining Toxoplasma gondii with Fluorescein-labelled Anti- body. II. A New Serologic Test for Antibodies to Toxoplasma Based upon Inhibition of Specific Staining. J. Exper. Med. 105:557-573, 1957. Moony, M. D., Goroman, M., and TrHomAsoN, B. M. Staining Bacterial Smears with Fluorescent Antibody. I. General Methods for Malleomyces pseudomallei. J. Bact. 72:357-361, 1956. Moony, M. D., Eruis, E. C., and Uppyke, E. L. Staining Bacterial Smears with Fluorescent Antibody. IV. Grouping Streptococci with Fluorescent Antibody. J. Bact. 75:553-560, 1958. Moony, M. D., BieGeLEISEN, J. Z., Jr, and TAvior, G. C. Detection of Brucellae and Their Antibody by Fluorescent Antibody and Agglutination Tests. J. Bact. 81,6:990-995, 1961. BIEGELEISEN, J. Z., Jr, Brabpsuaw, B. R., and Moony, M. D. Demonstra- tion of Brucella Antibodies in Human Serum. A Comparison of the Fluorescent Antibody and Agglutination Technics. J. Immunol. 88,1:109- 112, 1962. Winter, C. C., and Moooy, M. D. Rapid Identification of Pasteurella pestis with Fluorescent Antibody. II. Specific Identification of Pasteurella pestis in Dried Smears. J. Infect. Dis. 104 :281-287, 1959. WELLER, T. H., and Coons, A. H. Fluorescent Antibody Studies with Agents of Varicella and Herpes zoster Propagated in witro. Proc. Soc. Exper. Biol. & Med. 86:789-794, 1954. Liu, C. Studies on Primary Atypical Pneumonia. I. Localization, Isolation, and Cultivation of a Virus in Chick Embryos. J. Exper. Med. 106 :455-466, 1957. KarLan, M. H. Localization of Streptococcal Antigens in Tissues. I. Histologic Distribution and Persistence of M Protein, Types 1, 5, 12, and 19, in the Tissues of the Mouse. J. Exper. Med. 107:341-352, 1958. GENERAL PROCEDURES 69 52, 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. O’DEa, J. F., and Dineen, J. X. Fluorescent Antibody Studies with Herpes simplex Virus in Unfixed Preparations of Trypsinized Tissue Culture. J. Gen. Microbiol. 17 :19-24, 1957. Deacon, W. B., FaLcong, V. H,, and HArrLS, A. A Fluorescent Test for Treponemal Antibodies. Proc. Soc. Exper. Biol. & Med. 96:477-480, 1957. Currry, W. B., and Freeman, E. M. Staining Bacterial Smears with Fluorescent Antibody. V. The Rapid Identification of Bacillus anthracis in Culture and in Human and Murine Tissues. Zbl. Bakt. 1 Abt. Orig. 175: 582-604, 1959. GoLpwAsseEr, R. A., and Sueparp, C. C. Staining of Complement and Modification of Fluorescent Antibody Procedures. J. Immunol. 80:122-131, 1958. Karan, M. H., Coons, A. H., and Deane, H. W. Localization of Antigen in Tissue Cells. IIT. Cellular Distribution of Pneumococcal Polysaccharides Types II and III in the Mouse. J. Exper. Med. 91:15-30, 1950. TroMASON, B. M., Moony, M. D., and GoLbmAN, M. Staining Bacterial Smears with Fluorescent Antibody. 1I. Rapid Detection of Varying Numbers of Mallcomyces pseudomallei in Contaminated Materials and In- fected Animals. J. Bact. 72:362-367, 1956. Moony, M. D., and Winter, C. C. Rapid Identification of Pasteurella pestis with Fluorescent Antibody. 111. Staining Pasteurella pestis in Tissue Impression Smears. J. Infect. Dis. 104 :288-294, 1959. WHITE, J. D,, and BrunpeLr, G. P. The Use of the Fluorescent Antibody Technique for Demonstration of Pasteurella tularensis in Formalin-fixed Tissue. Bact. Proc. S.A.B., 1959, p. 136. HavpersoN, S., DonaLpson, P., and SuLkin, S. E. Identification of Strep- tococci in Bacterial Mixtures and Clinical Specimens with Fluorescent Antibody. J. Bact. 76:223-224, 1958. WaRrIeLp, M. A.; Pace, R. H.; ZuerLzer, W. W.; and Sturserg, C. S. Immunofluorescence in Diagnostic Bacteriology. I. Identification of Group A Streptococcus in Throat Smears. Am. J. Dis. Child. 101:160-163, 1961. Worrg, M. D., and CAMERON, G. M. Fluorescent Antibody Technique versus Cultural Methods as a Routine Procedure for Finding Beta-hemolytic Streptococci in Throat Cultures. Pub. Health Lab. 17:76-82, 1959. Repys, J. J., Ross, M. R,, and Borman, E. K. Inhibition of Common- Antigen Fluorescence in Grouping Streptococci by the Fluorescent Anti- body Method. J. Bact. 80:823-829, 1960. WHITAKER, J.; Pace, R. H.; StuLBerg, C. S.; and ZurLzer, W. W. Rapid Identification of Enteropathogenic Escherichia coli 0127 :B8 by the Fluores- cent Antibody Technique. Am. J. Dis. Child. 95:1-8, 1958. NeLson, J. D., WHITAKER, J., and HeEmpstEAD, B. Diagnosis of Entero- pathogenic Escherichia coli Diarrhea by Fluorescescein-labeled Antibodies. J. Pediat. 57 :684-688, 1960. (a) Tuomason, B. M.; Cuerry, W. B.; Davis, B. R.; and PoMALES- LesroN, A. Rapid Presumptive Identification of Enteropathogenic Escherichia coli in Faecal Smears by Means of Fluorescent Antibody. I. Preparation and Testing of Reagents. WHO Bull. 25:137-152, 1961. (b) —————— Ibid. 2. Use of Various Types of Swabs for Collection and Preservation of Faecal Specimens. WHO Bull. 25:153-158, 1961. (c) CuEerry, W. B.; THOMASON, B. M.; PoMALES-LEBRON, A.; and EwINg, W. H. Ibid. 3. Field Evaluation. WHO Bull. 25:159-171, 1961. Deacon, W. E.; Peacock, W. L., Jr.; FrReemaN, E. M.; and Harris, A. Identification of Neisseria gonorrhoeae by Means of Fluorescent Antibodies. Proc. Soc. Exper. Biol. & Med. 101 :322-325, 1959. 70 68. 69. 70. 71. 72. 73 GENERAL PROCEDURES -, et al. Fluorescent Antibody Tests for Detection of the Gonococcus in Women. Pub. Health Rep. 75 :125-129, 1960. DonavrpsoN, P., and WHITAKER, J. Diagnosis of Pertussis by Fluorescent Antibody Staining of Nasopharyngeal Smears. Am. J. Dis. Child. 99: 423-427, 1960. WHITAKER, J., DonaLpsoN, P., and Nelson, J. D., Diagnosis of Pertussis by the Fluorescent-Antibody Method. New England J. Med. 263 :850-851, 1960. KenprIick, P. L., ELbErRING, G., and EveLanp, W. C. Fluorescent Antibody Techniques. Methods for Identification of Bordetella pertussis. Am. J. Dis. Child. 101 :149-154, 1961. Kenprick, P. L., Lawson, G. McL, and Miiier, J. J. “Hemophilus pertussis” in Diagnostic Procedures and Reagents (3rd ed.). New York, N. Y.: American Public Health Assn., 1950. ELpErING, G., HorNBECK, C., and BAKER, J. Serological Study of Bordetella pertussis and Related Species. J. Bact. 74:133-136, 1957. CHAPTER 2 MAILING, RECEIVING AND PROCESSING SPECIMENS I. Specimen Mailing IT. Specimen Receiving III. Specimen Processing IV. Specimen Reporting V. Referral of Specimens References Appendix Since the methods of mailing and processing specimens have a profound effect on the ultimate findings of public health laboratories, it is obvious that they may be almost as important as the teclinical procedures used in examining the specimens. I. SPECIMEN MAILING If the specimen is to be sent through the mail, it must be packaged and shipped according to postal regulations. These regulations can be considered in two parts, one dealing with the kind and size of con- tainer that may be used, the other with the kind of information that may accompany the specimen. Postal regulations governing the kind and size of containers were specifically outlined in the U. S. Official Postal Guide, 1953 edition.! Later editions have not included this information but state only that material which is ordinarily nonmail- able, such as all “disease germs and scabs . . . may be sent through the mails if they conform to special regulations as to preparation and packaging . ..” (see Parts 124 and 125 of the Postal Manual). Regulations stipulating what information may accompany a third or fourth class package are ambiguous and are not easy to interpret. In general it may be stated that only such information can be given on a case history slip or descriptive blank as is required for identify- ing the patient, the specimen, the test desired, and the physician or other person to whom a report of the findings is to be sent. Other information not descriptive of the patient or specimen, such as whether the physician desires a phone call or report by telegram, can- not be sent at postal rates for third and fourth class mail. This is n 72 PROCESSING SPECIMENS true whether the instructions are handwritten, are typewritten, or have been indicated by check marks opposite printed items. Briefly the sender may give the following types of information: —Name, age, sex, color, occupation, and address of patient —Provisional diagnosis, provided written matter is descriptive of patient or specimen —Purpose for which the specimen was taken —Type and source of specimen —Clinical symptoms —Therapy and dates given —Physician’s name, address and telephone number The Post Office Department has ruled that under the title, “Purpose for Which Specimen Was Taken,” the physician may indicate the tests desired. The outside wrapper may carry only the name and address of the addressee, the return address and name of the sender, and a notation that a “Specimen for Bacteriological Examination” is enclosed. All request cards or slips should be designed to conform to the rules and regulations of the U. S. Post Office Department. Since these rules are subject to interpretation by officials currently in authority, the wording of a form may be declared unacceptable even though approved by a high authority in an earlier régime. A. Types of Containers (Specimen Mailing Outfits) There are many containers suitable for mailing various types of specimens to the laboratory. These are of two general types, (1) cylindrical with a metal bottom and a metal top that screws on, and (2) cardboard boxes, of which there is a great variety. Cylindrical containers are stronger than those which are square or oblong and therefore offer greater protection to their contents than do box-type containers. They are ideally suited for use with test tubes and round bottles and are more likely to retain any fluid which may escape from a broken specimen bottle or tube. On the other hand they are hard to stack and take up more space than the box type. 1. Double containers—Double containers are necessary when- ever the nature of the specimen is such that pathogenic organisms may be present, capable of infecting post office or laboratory em- ployees if breakage occurs in transit. Examples of such specimens are feces, sputa, spinal fluids, body exudates, bacterial and fungus cultures. Specimens such as water, milk, or clotted blood for serological testing for syphilis should be sent in single containers with sufficient internal packing to absorb all liquid in the event the inner glass jar or tube should be broken in transit. PROCESSING SPECIMENS 73 Blood specimens intended for cultural examination or febrile agglutination testing must be mailed in double containers, as must any other specimen that may contain pathogenic bacteria, viruses or fungi. Double containers should be used when in the judgment of the mailer the specimen for mailing is of an extremely hazardous nature and in the event of breakage there is definite danger to persons han- dling the container. Definite is interpreted to mean that the micro- organism involved has a high rate of survival in any temperature range or is capable of being airborne. This would include specimens to be examined for tubercle bacilli, diphtheria bacilli, typhoid bacilli, etc. When the nature of the specimen is such that definite danger of infection does not exist, a single mailing case will suffice, providing that there is sufficient packing to absorb any leakage. 2. Survey containers—When large numbers of specimens are submitted for the same type of examination, it is usually not necessary to employ an individual specimen kit for each specimen. Some manu- facturers are able to supply small but sturdy cardboard boxes furnished with two decks perforated to receive up to 25 blood tubes and hold them rigidly in place. A list of the names of the patients and examinations requested, numbered to correspond with the number- ing of the specimens, should accompany the package. 3. Containers for refrigerated specimens—Several types of con- tainers are available or may be improvised for shipping frozen specimens, An expendable container, made from insulated paper bags and cardboard boxes, suffices when small numbers of specimens are to be sent at one time. Enough dry ice to keep the specimen frozen 24 to 48 hr longer than the time estimated for it to arrive at its destination is placed in the bag with the frozen specimen. This bag containing the specimens and dry ice is put into a strong cardboard carton for shipment. A satisfactory insulated shipping container can also be made by placing a small, strong cardboard carton inside another strong card- board carton of at least four times its volume, packing the intervening space on all six sides with shredded paper, sawdust, shavings, shredded cork or other insulating material. Specimens should then be packed with an adequate amount of dry ice (as above) in the interior carton. Where specimens are shipped routinely or on a schedule, an in- sulated picnic ice chest makes an excellent shipping case and racks may be devised which will hold specimens in an upright position inside the chest. 74 PROCESSING SPECIMENS B. Directions for Collecting and Submitting Specimen Physicians, health officers, nurses and laboratory workers should exercise every precaution to see that the proper type of specimen is taken to yield the desired information, and that it is properly pro- tected during shipment to the laboratory. 1. Precautions (a) Specimens which must be kept warm until ex- amined—Very few types of specimens need be kept warm until examined. Cough plates for Hemophilus pertussis* are best kept at body temperature until received at the laboratory, where they should be placed immediately in the incubator. Specimens to be cultured for Neisseria gonorrheae and N. meningitidis should, without exception, be inoculated at once on suitable culture media and the culture thus made should be kept warm until placed in the incubator. Stool specimens which are to be examined for Entameba histolytica should also be kept at body temperature and examined within 2 hr in order to permit recovery of trophozoites. 1. (b) Specimens which must be kept frozen or chilled until examined—The majority of specimens sent to laboratories for bac- terial and serological examinations benefit from being kept at tempera- tures around 5° C. The exceptions to this procedure are listed in the preceding paragraphs. Clotted blood specimens, however, should not be refrigerated until the clot has formed, as quick chilling will in some instances delay clotting long enough to permit the cells to settle out before the clot forms. The result will be an almost clear plasma clot above, but firmly united with the lower clot containing most of the red cells. Efforts to obtain the serum from such a specimen are time-consuming and sometimes end in failure. Water, milk and food specimens to be examined for food-poisoning bacteria should be shipped and stored at 5° C until examined. Blood specimens for serological examinations should also be kept at this temperature in the laboratory until examined. Whole blood, clotted or unclotted, should not be allowed to freeze, since this will cause hemolysis. 1. (c) Use and misuse of preservatives—Preservatives are of value in the submission of only a few kinds of specimens. Buffered glycerol-saline solution has been used successfully for many years for preserving fecal specimens to be examined for enteric pathogens. Recent studies by Shipe et al? indicate that EDTA solution is * Currently designated Bordetella pertussis. PROCESSING SPECIMENS 75 superior to buffered glycerol-saline solution as a preservative for fecal pathogens. Polyvinyl alcohol is useful for treatment of fecal films to be stained and examined for protozoan cysts or trophozoites and helminth ova. A few preservatives may also be used for other special purposes. In most instances, however, preservatives, because of misuse, are more of a hindrance than an aid to the public health laboratory. Too often specimens, especially tissues, are submitted in formalin or other tissue-fixing fluids, which of course renders them unsuitable for bacteriologic or virologic study. Constant education of those who send specimens to the public health laboratory seems to be the only feasible method for eliminating this problem. 1. (d) Age of specimen in relation to validity of examination results—All specimens should be examined as soon as possible after being taken. A study of the death curve of most pathogens will show that the longer the time elapsed, the greater the possibility of obtain- ing false-negative results. Overgrowth by contaminants is often troublesome in specimens which are not examined promptly. While no thorough studies have been made on the viability of dysentery bacilli in fecal specimens, some of the more delicate strains, at least, are unable to survive mail shipment in detectable numbers. Messenger delivery for laboratory examination within 5-6 hr of collection of the specimen is advisable, particularly when release from quarantine is at issue. On the other hand, where the number of bacteria present is to be determined, as in milk or water, distorted figures may result either from a multiplication of the bacteria present or from the death of some of them. Since the accuracy and reliability of laboratory findings are prob- ably not increased by delay in transit, all specimens should be delivered to the laboratory by messenger, whenever practicable. 1. (e) Other causes of unsatisfactory specimens (1) Loose-fitting corks and lids—Many specimens are lost or rendered unfit for examination because cork stoppers come loose in shipment. Taping a cork after inserting it snugly is a good preventive measure. When container lids come off in transit it is not uncommon that a specimen will arrive in a container different from the one in which it was mailed. “Helpful” mail clerks sometimes attempt to replace specimens in containers from which they have escaped, but in most cases they have no way of knowing whether they have replaced such specimens in the right containers. Any specimen should be regarded 76 PROCESSING SPECIMENS as of uncertain identity if the container lid has been lost, even though a mail clerk has subsequently covered the container with gummed paper. (2) Insufficient identification—When the patient's name or other positive means of identification is not furnished, it is advisable to pro- ceed with the examination while the specimen is as fresh as possible but to withhold the report while asking the physician to identify the specimen if he can do so with certainty. (3) Weekend shipments—The sender of specimens should be advised to become acquainted insofar as possible with transporta- tion schedules so that specimens do not lie for long periods of time en route to the laboratory. On weekends or holidays, it is best that whenever possible the sender hold them at the point of origin, main- taining the proper temperatures until they can be shipped so that they will arrive during laboratory work hours on work days. The laboratory should, for its part, make arrangements to receive speci- mens that do arrive on weekends or holidays and place them under proper temperatures again until they can be examined. 2. Instruction booklets or pamphlets (a) Types of examinations and appropriate specimens—A booklet or pamphlet should be pro- vided by all laboratories outlining exactly what tests are available and noting the specimens appropriate for these tests. The booklet should give instructions for taking specimens, how to ship them, where to ship them and how long before results can be expected in each case. Knowledge of these matters will substantially reduce the number of specimens which are improperly taken or are otherwise unsuitable for the examination desired. Public relations also will be less likely to suffer when it proves necessary to reject specimens that cannot be examined. 2. (b) Interpretation of results—While it should always be re- membered that a laboratory does not make diagnoses, an explanation of test results is often helpful. This explanation should include the name and type of test performed, an interpretation of the results, and a note of any limitations which may affect the validity of the results reported. C. Forms, Printed or Mimeographed 1. Specimen cards or slips—FEach specimen container should be accompanied by a blank identification form. These forms should be designed to elicit all the information allowable under postal regulations PROCESSING SPECIMENS 77 described at the beginning of this chapter. One is more likely to get a form filled out if the information requested is kept to an absolute minimum and requires little writing. (a) Combination specimen and report forms—Some laboratories have found it desirable to employ combination specimen and report forms which reduce the amount of clerical work required of the laboratory in rendering reports of its findings. The advantages of this system and some of the problems which it presents are discussed elsewhere in this chapter. (b) Vehicle for instruction—The back of the identification form affords a convenient place to impart instructions for the taking of specimens, the kind of specimen needed, where to send it, and when to expect results. Similar instructions can be printed on the back of report forms, although an even better use of this space provides information on interpretation of results. 2. Labels—A return label bearing the name and address of the laboratory should be affixed to each specimen container. This label should have a space for the name and address of the sender. Many laboratories use labels of a special color for each kind of specimen or type of examination. Among other advantages color facilitates rapid sorting of specimens when they are received in the laboratory. 3. Special direction tags and inserts—When necessary, special handling tags or inserts may be supplied with the container. Tags should be so designed as to attract attention. Special instruction slips that will help to educate users of laboratory services are often worth- while if employed judiciously. Postal regulations must be kept in mind in designing all labels. Il. SPECIMEN RECEIVING The method of receiving and processing specimens in the laboratory will be influenced by such factors as: time of heaviest receipt, fre- quency of receipt, most efficient use of diagnostic staff, and type of clerical records maintained, as well as other factors which may be peculiar to a given laboratory. A. Receiving Room As a rule the receiving area of the laboratory is most conveniently located adjacent to the container and shipping rooms and the clerical office. Depending on the volume of specimens received, it may be 78 PROCESSING SPECIMENS necessary in some laboratories to designate a specific room for the receiving and routing of specimens which is staffed by full-time receiving clerks. In other laboratories it may be feasible to dispense with a special receiving room. This would apply to laboratories where volume is small or where specimens are brought in mostly by special messengers, the patient, or the physician. The average laboratory will receive its largest volume of specimens in the morning, consisting of specimens received at the post office or collection stations during the late afternoon and night. If arrange- ments can be made for early delivery, it may be advisable to have those employees who are responsible for receiving and opening the mail start at an earlier time than the technicians so that specimens will be available to the diagnostic staff on arrival. B. Routing Specimens to Laboratory Sections Upon arrival of specimens in the receiving room those employees assigned to this duty will sort and group the mailing cases in canvas laundry baskets or other receptacles designated to receive the speci- mens for each of the laboratory divisions or sections. The procedure of grouping specimens is simplified in large laboratories by the use of appropriately colored address labels and/or symbols which indicate the type of specimen within the mailing case. C. Opening, Numbering and Recording Procedures will vary from one laboratory to another but the follow- ing might be considered representative : 1. Opening and numbering—The specimen is carefully with- drawn from the mailing case and the data card is examined for completeness of information. Identical numbers (in serial sequence) are placed on the data card and the specimen. At this time the speci- men may be separated from the data card, processed, and examined. The numbered specimens are placed in sequence in suitable racks (which are best constructed in rows of ten if the daily specimen volume is large). A variation may be to place the specimens in centrifuge baskets or carriers which accommodate five tubes per row. The data cards are numbered and dated in sequence, using a serial numbering machine or numbered gummed labels, or by hand with ink or pencil. In laboratories where the work load is large and diversi- fied it may be considered advisable to have several numbering series, one for each major division. In other laboratories it may be necessary PROCESSING SPECIMENS 79 to place a letter prefix to the number to identify a particular service or type of specimen. The numbers may be written on small strips of tape or gummed labels at the time that the data card is being numbered, and placed on the specimen container. Glass-marking pencils also may be used but there is the risk of inadvertent blurring due to handling of the containers, If the specimen container has a gummed label or paper cover, ink or pencil may be used. Regardless of the numbering procedure, it is imperative, first, that extreme care be exercised to avoid duplication of numbers or omis- sion of numbers from the sequence ; and—most important of all—that the numbers on the specimen and its data card are identical. 2. Recording—The specimens and their data cards or slips pursue different courses for a time after the serial numbers have been applied. While the specimen is being processed in the laboratory the data cards are taken to the records office, where a work (or accession) sheet is prepared containing the serial numbers of the specimens and space for recording results on each specimen. The data cards and work sheets are returned to the laboratory and the results may be entered on both by a laboratory worker. The data cards on those specimens requiring further examination are held in the laboratory until final completion of the results. Data cards, when results have been recorded on them, travel back to the records office, where they supply all the information needed for typing reports to the physician. When multicopy combination specimen and report forms are used, the method of handling is somewhat different. When results are entered on such a form by the laboratory worker and the date is stamped or inserted by a clerical worker it becomes a completed report, one copy of which may be sent to the physician. The work sheets may be retained for the compilation of laboratory statistics and for quick reference so long as needed. Post binders of proper size are a convenient means of keeping these sheets. D. Problem Specimens Despite diligent attempts by laboratories to provide physicians and others with proper and safe mailing cases for each type of specimen, as well as suggested precautionary measures to take in collecting and submitting specimens, laboratories are continually plagued by “prob- lem specimens.” Specimens are often received with incomplete or no identification, mislabeled, in loose-lidded or otherwise unsatisfactory containers, too old for satisfactory testing, and so on. Each laboratory has its own procedures for the processing of these problem specimens. 80 PROCESSING SPECIMENS E. Multiple Requests Not infrequently several tests are requested on a single specimen, sometimes involving two or more sections of a laboratory. In such cases the several sections of the laboratory may need to confer, especially if there is insufficient material for all of the tests requested. If multiple requests on single specimens occur frequently enough, it may be desirable to designate one person or group to handle their routing through appropriate sections of the laboratory and issuance of the report(s). F. Specimen Rejection Specimen containers from which the contents have leaked out, or which have been damaged, should be handled cautiously. Whether an examination would be practical and valid must be weighed against the potential hazards involved in further handling. The information on the data card will most likely need to be transferred to a new card and the damaged container properly decontaminated (see Section ITI, “Specimen Processing,” which appears subsequently in this chapter). Specimens that are rejected for such reasons or for other causes, such as hemolysis or insufficient amount, should be placed in proper sequence, numbered, entered, and reported as rejected for the pertinent reason. G. Variations in Methods These procedures for receiving and routing specimens from arrival at the laboratory to the preparation of the specimens for examination are meant to serve only as a guide to indicate the basic principles that should be observed. There will of course be variations relating to the specific needs of each laboratory. Serial numbering—The type of serial numbering system used is variable from one laboratory to another and may vary within a labora- tory. Starting serial numbers with Number 1 on January first and continuing throughout the calendar year gives a ready index of the number of specimens received in a category over a certain period of time. Many laboratories number their specimens beginning with Number 1 each day. This reduces the number of digits which must be hand- written and also permits processing of specimens in prenumbered positions in racks, centrifuges, etc. Processing in prenumbered posi- tions affords one means of recognizing any confusion of specimens, PROCESSING SPECIMENS 81 since work sheets also can be prenumbered. The risk of confusing reports of specimens assigned the same number on different dates can be obviated by using a double numbering system—the first (daily serial) being applied to the tube (or other container) and card by a glass-marking pencil when the mailing case is first opened, the other (yearly serial) immediately before the work sheets are prepared. Dating and numbering machines are available at moderate cost which will speed up this numbering procedure and will, if desired, imprint a signature and name of the laboratory in the same operation. H. Receiving after Regular Hours 1. Weekend and holiday receiving—In those laboratories where specimens are accepted on every day of the year, the individual(s) responsible for receiving specimens on weekends and holidays should be thoroughly instructed in the proper storage of the different types of specimens, particularly differentiating between specimens which must be refrigerated and those which must be frozen. As a rule, bacteriologists are given such assignments, since there are usually cultures or reactions which must be observed and recorded and some- times urgent examinations must be made. If reports are to be issued, a typist may be needed. 2. Off-hours receiving—Although most small specimens brought to a laboratory during off-hours may be placed in a depository main- tained for that purpose, larger specimens including animal heads for rabies may not be so accommodated. Receiving and handling of such specimens should be geared to the facilities and personnel available to the laboratory. Such personnel may include night watchmen, janitors, hospital personnel and police. It may also be necessary to have a person on call to receive such specimens. Special written in- structions for the handling of these specimens should be posted in all areas where they could be delivered, and the individuals who might be involved in handling them should be informed of the posted in- structions. J. Personnel Handling Specimens The person in charge of receiving, opening and numbering speci- mens should have laboratory experience broad enough to recognize the nature of the examination requested and which section(s) of the laboratory, if any, can render the desired service. Any person meeting these requirements will usually have a bachelor’s degree with training in bacteriology and serology and certainly a year or two of experience in one specialty or the other at the laboratory where he is assuming 82 PROCESSING SPECIMENS this responsibility. With less academic background, a longer period of experience would be desirable. Clerical employees, after careful training, can assist in this work. Technical personnel may also share in this activity, depending on whether their work is so scheduled as to leave them with free time while the specimen mail is being opened. K. Written Orders and Work Procedures Many laboratories have manuals setting forth the orders and work procedures for the entire laboratory. Some laboratories, however, have not, and orders in these work procedures have been developed as problems have arisen through a period of years. The written manual, of course, is preferable. In either case, when orders are given or procedures are changed, these should be issued in written form to those responsible for and involved in the change. lll. SPECIMEN PROCESSING All specimens for biological studies are potentially infectious ma- terials and personnel must be trained to handle them as such. Leak- proof trays should be available to receive damaged and leaking containers and questionable specimens should be separated from the processing until personnel can protect themselves. Rubber gloves, face shields, disinfectant solution and protective clothing should be readily available. Specimens obviously damaged and contaminated which cannot be salvaged should be promptly processed for disposal, usually by autoclave sterilization at 120° C for not less than 20 min. Material, after examination is completed, can be processed for dis- posal by the same means. Some materials can be incinerated if facili- ties are available. The proper labeling or identification of specimens for disposal is a responsibility important to all personnel. Advances in the development of single-service containers have been made—for example, evacuated tubes for collecting blood specimens. Nevertheless, most laboratories reuse containers. After sterilization of the used containers they are cleaned by one of two methods, acid dichromate to remove all organic material and/or detergents com- bined with mechanical dishwashing. Care must be exercised to insure complete removal of detergent or other cleaning agent, whether the operation is by hand or machine. Covers and stoppers for containers can be reused if precautions are taken to prevent carry-over of speci- men material. Metal threaded or snap-on covers are available with replaceable liners of rubber or of a pulp backing with polyvinyl plastic facing. It should be noted, however, that after autoclaving, some plastic materials will yield substances toxic to bacteria. PROCESSING SPECIMENS 83 If the mailing case is contaminated, it can be sterilized by auto- claving. However, this process eventually destroys the paper or causes rust of metal components, which limits reuse, IV. SPECIMEN REPORTING A. Routine Reports Communication of the results of a laboratory examination to the physician or other person responsible for submitting the specimen is, of course, the primary aim of the report. Other purposes are served, however, which are important to the health of the public. Copies of positive laboratory reports are regularly furnished by most public health laboratories to local or state health officials who are responsible for carrying out any control measures required for the disease in- volved. Additional copies are often needed, also, for other administra- tive reasons and multicopy report forms have become the rule rather than the exception in public health laboratories. The amount of clerical work involved in the issuance of more than one copy of each report has encouraged the employment of a variety of labor-saving devices. Some of the most useful may be noted here: 1. Electric typewriters with adjustable tensions permit more legible copies to be produced at a faster speed and at the same time reduce the physical effort put forth by the typist. 2. Continuous-fold forms with interleaved, single-use carbon sheets and mar- ginal perforations fit sprockets on the typewriter platen and keep all copies in perfect alignment. 3. Folding and stuffing machines, especially if they have an automatic feeding mechanism, can save a great deal of labor at what is often the busiest time of the day—when reports are being prepared for mailing. 4. Other machines combine two or more operations into one, for example: dating and/or time-stamping a form while at the same time imprinting a serial number of as many as 5 or 6 digits and the signature of the laboratory director. 5. Some forms have several possible results of the examination printed on them, with a blank box alongside each result for checking the proper one. 6. Combined specimen and report forms are more fully discussed below. Many laboratories have report forms for different types of tests printed on paper stocks of different colors or with different inks. This practice may be considered labor-saving since it facilitates checking and filing. Color schemes for this purpose are most effective if ex- tended also to include specimen cards and mailing container labels. The elimination of unnecessary data and verbiage from reports is an obvious means of economizing space and labor. Combined specimen card (history blank) and report forms are employed in some laboratories. These forms save work for the labora- 84 PROCESSING SPECIMENS tory personnel, since the name and address of the physician, the name and other information about the patient, the character of the specimen and the type of examination desired are all inserted before submission to the laboratory, leaving only the results of the examination to be added at the laboratory. While considerable labor is saved by the use of these combined forms, some disadvantages should be mentioned. Physicians and others who fill out the greater part of the form are not uniformly careful about pressing the pencil firmly enough to make legible copies. Unless a laboratory clerk goes to the trouble of rewriting the copies before the original is separated, they constitute a dubious record for the files of the laboratory and the follow-up work of the health depart- ment. The original, which is mailed to the physician after the results have been inserted, is often miscarried because the sender’s hasty scrawl cannot be deciphered by the mailman. Another fault is potentially, at least, more serious than any of those already mentioned. Specimen mailing containers are purposely made easy to obtain and the official report blanks become equally easy to secure when the combined forms are used. If, as is sometimes the case, the blank forms carry the printed name or signature of the laboratory director, the forging of reports becomes a relatively simple matter. This objection can be obviated if an endorsing machine is employed to add the director’s signature after the form returned with the speci- men to the laboratory has been completed and is ready for mailing to the submitting clinician. When combined specimen and report forms are used in two, three or more copies, they have until recently been supplied with interleaved carbon. The considerable amount of handling which they receive, especially while being rolled up and placed in the mailing container, inevitably causes a good deal of smudging on one or both sides of each copy. Two recent innovations, however, overcome this difficulty : One is a newly developed paper, known as NCR, produced by the National Cash Register Company and is available to any printer. On NCR paper four or five legible copies can be obtained without the use of carbon paper. No special writing inks or instruments are required, although a sharp point, like that on the ubiquitous ballpoint pen, makes the best copies. A second development is a dry photographic process which is both economical and fast. A single-copy combined specimen and report form is used and after the laboratory findings have been added, copies can be made on a low-cost sensitized paper at a rate of several hundred per hour by a single machine. The original is retained by the labora- PROCESSING SPECIMENS 85 tory as its file copy. A shortcoming in this process is its failure to reproduce blue markings well, so that illegible copies result when blue ink is used in filling out the originals and even blue-black ink some- times fails to yield good copies. Thin-line writing, as with ballpoint pens, also reduces the legibility of copies. Nevertheless this method is being used in their reporting procedures by a number of labora- tories, where it is apparently considered satisfactory. B. Special Reports While clerical economy makes it desirable that reporting be routin- ized as much as possible, special laboratory reports are at times necessary to give a physician the benefit of observations not commonly encountered or not always significant. Preliminary or progress reports may even become routine in some types of examinations and are par- ticularly helpful where a final report cannot be issued for several weeks, as is the case when specimens are to be cultured for tubercle bacilli or fungi. Explanatory aids to the use of the laboratory—Probably few physicians are fully acquainted with all of the services which laboratories are able to furnish them. To remedy this situation some laboratories prepare informational pamphlets outlining the types of tests performed; the appropriate specimen(s) to submit in each case and the most favorable time(s) for collecting them; factors which render specimens unsatisfactory; definitions of terms and in- terpretation of laboratory findings and other helpful data. Some laboratories are able to place their pamphlets in the hands of medical students, by whom they are nearly always gratefully received. Use is also made of the backs of specimen cards and report forms to furnish similar information on a more limited scale. Inserts into envelopes carrying laboratory reports to physicians may be employed for the same purpose. V. REFERRAL OF SPECIMENS Many state and some city health department laboratories are able to provide a referral service for local laboratories within their jurisdic- tions which are unable to perform certain tests. For example, some agglutination tests require antigens which are unobtainable com- mercially ; the typing of some species of bacteria calls for the use of sera or phages which are not readily available. Not many private or hospital laboratories are as yet equipped to do much virus diagnos- tic work and necessarily they transmit their specimens to their own 86 PROCESSING SPECIMENS state laboratory. There is hardly any field of diagnostic laboratory work in which help is not sought by local laboratories at least oc- casionally. A still broader referral service is furnished by several laboratories of the U. S. Public Health Service. Their Communicable Disease Center Laboratories in Atlanta, Ga., will accept a great variety of bacteriological, parasitological, mycological and virological specimens when submitted through state health department laboratories for diagnostic examinations which the latter are not able to make. The Venereal Disease Research Laboratories, also located in Atlanta, will make tests with treponemal antigens as well as perform standard tests with lipoidal antigens in cases which have exhibited puzzling serological reactions. The Rocky Mountain Laboratories of the U. S. Public Health Service in Hamilton, Mont., provide expert diagnostic service in rickettsial infections and will also accept some types of specimens for virological and bacteriological examinations. HowaArp J. SHAUGHNESSY, PH.D., Chapter Chairman Evan T. Bynog, Pu.D. H. Giueert CreceLius, PH.D. Rosert E. Evans, Pu.D. Grorce F. Forster, PH.D. Joun Hawmirton, M.D. WirLiaM J. HAuscer, PH.D. Frienp Lee Mickie, Sc.D. Morris ScHERAGO, D.V.M. REFERENCES 1. U. S. Official Postal Guide (1953 ed.). Washington, D. C.: U. S. Post Office Dept., Chapter IV. 2. Surpg, E. L., Jr, Fierps, ApeLADE, and SHEA, Janice R. Comparison of Three Preservatives for Bacterial Pathogens in Fecal Specimens. Pub. Health Lab. 18:95-103 (Sept.) 1960. APPENDIX Postal Regulations on Shipment of Diseased or Contaminated Materials According to the 1953 edition of the U. S. Official Postal Guide, Chapter IV, “Mailability and Packaging,” are quoted for your information only, since they are not included in the new Postal Manual. Diseased Tissues and Other Specimens A. Specimens of diseased tissues, blood, serum, and cultures of pathogenic micro- organisms may be admitted to the mail for transmission to United States, state, municipal, or other laboratories in possession of permits issued by the Solicitor, certifying that said laboratory has been found to be entitled to PROCESSING SPECIMENS 87 receive such specimens only when enclosed in mailing cases as prescribed in this article. However, bacteriologic or unfixed pathologic specimens of plague and cholera shall not be admitted to the mails except when prepared specifically as follows: 1. Pathologic specimens of plague and cholera which have been immersed for at least 72 hr in four times their volume of 4 per cent formaldehyde gas in water or other fluid of equal or superior disinfecting power, for a period sufficient to fix or harden the central portions of the specimen, may be admitted to the mails if packed in the same manner as herein prescribed for other unfixed pathologic tissues in paragraphs 3, 4, and 5 of this article. Cultures and infectious material of plague, cholera, anthrax, undulant fever, and tularemia may be admitted to the mails if enclosed in stout glass tubes sealed by fusion of the glass and packed in a large, stout glass container with a layer of absorbent cotton soaked in 4 per cent formalde- hyde surrounding the inner container. The outer glass container shall be closed with a rubber stopper or cork of good quality or by fusion of the glass. This double glass container shall then be packed in accordance with the provisions of paragraphs 4 and 5 of this article. Specimens of sputum, feces, pus, unfixed diseased tissue, or other in- fectious material fluid in nature or shipped with nondisinfecting fluid shall be placed in stout glass containers of suitable size (but not more than 3 in. in diameter) closed with a metal cover having a rubber, cork, or paraffined paper leakproof washer or with a cork or rubber stopper of good quality, or by fusing the glass. Large fixed specimens of diseased tissue may be prepared for shipment outside mail bags when packed in accordance with the provisions of the following paragraph. The aforesaid glass container shall then be placed in any of the following described containers: (1) a cylindrical sheet-metal box with soldered joints, closed by a metal screw cover; (2) a paraffin-impregnated heavy cardboard container, with ends made of metal or a suitable substitute. A sleeve type of closure may be employed provided that the overlap is at least one-third the length of the cylinder and in any case at least 2 in. The closure shall be sealed with tape; or (3) a one-piece bored wooden cylinder at least three-sixteenths of an inch thick in its thinnest part, with a threaded screwtop. The screwtop covers shall be provided with rubber or felt washers and shall be threaded with sufficient screw threads to require at least one and one-half full turns before they will come off. The glass tubes in the above containers shall be completely and evenly surrounded by absorbent cotton or other suitable absorbent in quantity sufficient to absorb the contents of the glass container if broken. The sheet-metal box with its contents shall then be enclosed in a closely fitting wooden or papier-mache box or tube, at least three-sixteenths of an inch thick at its thinnest part, or in a sheet-metal box or tube of sufficient strength to resist rough handling and support the weight of the mails piled in bags. The tube shall be tightly closed with a screwtop cover with sufficient screw threads to require at least one and one-half full turns before it will come off. (a) Cultures in solid media, blood, serum, spinal fluid, fixed and com- pletely disinfected diseased tissue, and infectious materials on swabs shall be transmitted in a stout glass container of suitable size (but not more than 3 in. in diameter), closed with a plastic or metal cover having a rubber, cork, or paraffined paper washer, or with a stopper of rubber, PROCESSING SPECIMENS paraffined cork, or cotton, the last sealed with paraffin or covered with a tightly fitting rubber cap. The tube shall then be packed in a single wooden or papier-mache cylindrical box or tube, at least three-sixteenths of an inch thick in its thinnest part, or in a sheet-metal box or tube of sufficient strength to resist rough handling and support the weight of the mails piled in bags. The glass container in this box or tube shall be completely and evenly surrounded by absorbent cotton or other suit- able absorbent packing material. Cultures in media that are fluid at the ordinary temperature (below 45° C or 113° F) may be mailed if packed in stout glass vials closed by fusing the glass and enclosed as in paragraphs 4 and 5 of this article. (b) Large specimens of fixed diseased tissue shall be placed in securely sealed glass containers, or in securely closed (hermetically sealed or screwtop or approved patent-top) metal containers with the necessary preservative fluid. The container shall be surrounded by sawdust or other suitable absorbent material to protect against breakage or leakage. Specimens of blood dried on glass microscopic slides for the diagnosis of malaria or typhoid fever by the Widal test or of other conditions shall be sent in any strong mailing case which is not liable to breakage or loss of the specimen in transit. . Large pathological specimens of fixed diseased tissue and shipments of large numbers of small specimens may be prepared for shipment outside mail bags. Small specimens of sputum, blood, serum, spinal fluid, pus, feces, fixed or unfixed diseased tissue, or other material fluid in nature or shipped with fluid, forming part of such a shipment shall be placed in stout glass containers as in paragraph 3 of this article and individually, evenly wrapped in absorbent cotton or other suitable absorbent material in sufficient quantity to absorb all the fluid in case of breakage. Small and large specimens so prepared shall be shipped in a strong, securely closed box endorsed in accordance with article 59 (f), and marked “Specimen for Bacteriological Examination,” and be transported outside mail bags. . Upon the outside of every package of diseased tissue, blood, serum, or cultures of pathogenic microorganisms admitted to the mails shall be written or printed the words, “Specimen for bacteriological examination. This package shall be pouched with letter mail,”* except that when dis- patch is made to nonstop trains in catcher pouches or large specimens or shipments are prepared as prescribed in paragraph 8 of this article, they shall be marked “Specimen for Bacteriological Examination.” * Information supplied by officials of the Post Office indicates that the statement regarding pouching of specimen with letter mail is no longer officially in effect unless first class postage is affixed. CHAPTER 3 LABORATORY INFECTIONS AND ACCIDENTS I. Extent of the Problem A. Agents Involved B. Personnel C. Type of Work Involved D. Sources of Laboratory-Acquired Infections II. Recommended Safety Procedures . Handling Clinical Specimens . Transferring Cultures Special Precautions When Working with Coccidioides . Pipetting . Centrifuging Homogenizing . Use of Syringe and Needle . Working with Animals . Lyophilization Ventilated Cabinets . Disposal of Contaminated Material AOZoEEHDO Wp ITT. Prophylactic Procedures IV. Reporting of Laboratory Infections and Accidents References The continued reporting of instances of laboratory-acquired infec- tion, together with the accumulating knowledge of the potential sources of laboratory contamination, have increased awareness of the hazards of working with infectious agents. Technical personnel should understand the nature and extent of the risks involved, both for their own protection and for the protection of their associates. Those work- ing in a supervisory or administrative position should likewise be acquainted with the problem in order that they may provide the necessary safeguards for the healthful operation of the laboratory. This chapter is written with the hope of pointing out the areas of greatest potential danger and the measures that have been recom- mended for the protection of the laboratory worker. I. EXTENT OF THE PROBLEM Accurate figures regarding the incidence of laboratory infection are not available, The nearest approximation to such ideal data is obtained 89 90 LABORATORY INFECTIONS by a tabulation of recorded instances. Some insight into the kinds of agents most frequently involved, the most important sources of infec- tion, and the kinds of accidents most often resulting in infection can be gained by an appropriate classification of these reported infections. The data in Tables 1 and 2 include cases reported in the literature, cases occurring in the United States between 1930 and 1950 and collected in a survey of 5,000 laboratories! and a few published and unpublished cases which have come to the attention of the authors since 1950. A. Agents Involved Table 1 shows that all classes of infectious agents have caused in- fections in the laboratory. Bacteria, with which this volume is primarily concerned, have been incriminated more frequently than any other kind of agent. Significant numbers of infections have also been caused by the parasites and the fungi. The viruses and rickettsiae are recognized sources of accidental infection? and are included here for comparative purposes. Table 1—Distribution of Cases of Laboratory-Acquired Infections According to Type of Personnel and Work Performed Type of Infection Total No. of Rick- Para- Distribution Infections Bacterial Viral ettsial sitic ~~ Fungal No. of cases 2,262 1,303 519 293 62 85 No. of deaths 96 53 31 8 2 2 Personnel: Trained scientific personnel* 1,534 856 358 206 57 57 Students 82 80 — i 1 — Animal caretakers, janitors, etc. 221 137 39 40 i 4 Othert 87 48 — 15 1 23 Types of Work: Diagnostic 525 393 96 3 16 17 Research 726 289 230 155 31 21 Production of biologicals 51 19 31 1 —— — Classwork 37 37 es — ee — Combination of activities 599 335 81 133 11 39 * Including research assistants, professional and technical workers, and graduate students. + Including clerks, maintenance workers, occasional visitors, and others. LABORATORY INFECTIONS 91 B. Personnel A classification of the personnel involved in laboratory-acquired in- fections shows that trained scientific personnel far outnumber all others. These figures would have more significance, however, if they could be referred to the numbers of persons at risk in each category.? On the other hand, a review of individual instances shows that persons not directly associated with the laboratory work, even visitors, have not escaped. C. Type of Work Involved An attempt to analyze the available data with respect to the type of laboratory work involved (Table 1) emphasizes the importance of diagnostic work as a source of laboratory infection as compared to research and other types of activity. Although these figures should not be interpreted to indicate the incidence of laboratory infection in the various categories, they show that routine diagnostic procedures involving the infectious agents considered in this volume have been important sources of infection. It is interesting to note that more viral infections have occurred among research workers than among those concerned with diagnosis. This ratio will undoubtedly change as more and more laboratories become concerned with diagnostic pro- cedures for virus diseases. At present many laboratories limit their diagnostic work in virus diseases to serological procedures. D. Sources of Laboratory-Acquired Infections The bacterial, parasitic and fungal infections are summarized in greater detail in Table 2, and an attempt has been made also to identify the sources of infection insofar as the available information will permit such a classification. Bacteria belonging to more than 25 different genera have been associated with laboratory-acquired infec- tion, including most of the known pathogens and some organisms which are ordinarily not thought to be pathogenic. Typhoid fever, brucellosis and tuberculosis, however, account for more than half of the bacterial infections. The majority of the cases of leptospirosis occurred outside the United States. Until recently these organisms have not been handled extensively in this country and those working with leptospira can profit by this experience. The four cases of infec- tion with Serratia marcescens* show that a microorganism which does not naturally infect man may not be entirely innocuous to a worker who is exposed to extremely large numbers. Table 2—Sources of Laboratory-Acquired Infections Bacterial Infections Typhoid Brucel- Tuber- Tula- Strepto- Salmo- Shi- Lepto- Diph- An- Source* Total Fever losis culosis remia coccus nellosis gellosis spirosis theria thrax Otherf No. of cases 1,303 292 274 161 86 67 54 54 45 40 36 194 No. of deaths 53 21 2 3 1 3 — 1 6 — 3 11 Clinical specimens 103 9 11 36 1 4 1 29 1 9 — 2 Autopsy, including known accidents 95 1 —_ 59 — 21 — — — 1 —_ 13 Aerosols 35 4 18 — 5 — — —_— —_ — 2 9 Contact with infected ani- mals and ectoparasites 126 i 22 4 37 5 1 —_— 13 —_ 2 39 Worked with agent 412 162 95 24 14 17 35 12 4 19 1 29 Handled discarded glass- ware, etc. 21 7 4 3 — 1 — 1 —_ — 4 1 Known accidents involving— Needle and syringe 66 — 10 4 5 5 — 2 7 4 — 29 Pipetting 66 39 4 — 3 2 3 5 3 1 2 4 Spilling and splatter- ing 41 10 9 3 4 1 1 — 3 2 5 3 Injury with broken glass, etc. 4 2 4 1 2 8 — — 1 — —— 26 Bite of animal or ectoparasite 28 — nr — 1 1 — — 9 —— — 17 Centrifuge 2 — 1 re — —_ — Re — — — 1 z6 AdolLviodavi SNOILD3IdANI Parasitic Infections Fungus Infections Amebi- Toxo- Coccidioi- ~~ Histo- Source Total asis plasmosis Malaria ~~ Other** Total domycosis plasmosis ~~ Other} No. of cases 62 20 15 9 18 85 52 18 15 No. of deaths 2 — 1 rn 1 2 1 — 1 Clinical specimens 17 11 — — 6 — — re — Autopsy, including known accidents — — — —_ — —_ — —_ — Aerosols 1 — — 1 39 35 4 — Contact with infected animals and ectoparasites 5 2 —_ 3 —_ — — — Worked with agent 16 5 y 1 3 27 7 13 7 Handled discarded glass- ware, etc. x — — —_ — i — — l Known accidents involving— Needle and syringe 3 _— 2 1 rs 2 — 1 1 Pipetting 1 re re — 1 — — — Spilling and splattering 3 1 1 — 3 2 2 — — Injury with broken glass, etc. — — —_ pe — 1 1 re — Bite of animal or ecto- parasite 8 — 1 6 1 2 — — 2 Centrifuge — — — on So 3 Fo — 3 * The source was not indicated for 288 cases. T Including relapsing fever (38), erysipeloid infection (32), agent not specified (26), staphylococcal infections (16), glanders (14), ratbite fever (13), Treponema pallidum (10), Hemophilus influenzae (7), tetanus (6), meningococcal meningitis (5), cholera (9), Neisseria gonorrheae (4), pneumococcal pneu- monia (4), Serratia marcescens (4), plague (3), Pseudomonas aeruginosa (1), leprosy (1), and Pasteurella multocida (1). ** Including leishmaniasis (4), ascariasis (2), strongyloidiasis (2), giardiasis (1), pinworm (1), Leucocytozoon infection (1), trypanosomiasis (1), Chilo- mastix mesnili (1), sarcosporidiosis (1), schistosomiasis (2), hookworm (1), and Fasciolopsis buski (1). Including ringworm (4), sporotrichosis (4), actinomycosis (3), moniliasis (2), and blastomycosis (2). Source: Sulkin & Pike. See Chapter references 1-3. Adolviodgvi SNOILD3dNI €6 94 LABORATORY INFECTIONS In considering the potential sources of infection, it is significant to note that in the majority of instances the exact source of infection is unknown. Only about 20 per cent of cases of bacterial infection are preceded by a known accident. In a large number of instances it is known that the infected individual had worked with the agent, but when and how infection took place is obscure. The sources of rela- tively few cases have been designated as aerosols, although it is recog- nized that in many of these individuals who “workedywith the agent,” the infection was probably airborne. All of the infections in which known accidents were involved could be included under the six headings indicated in Table 2. The data indicate that one should be particularly cautious when using a needle and syringe or when pipetting material containing pathogenic microorganisms. Although the potential hazard of a procedure involv- ing the use of a centrifuge is recognized, only two bacterial infections could be traced to this source. Il. RECOMMENDED SAFETY PROCEDURES The numerous cases of proved laboratory-acquired infection should impress upon laboratory directors the importance of protecting labora- tory workers while engaged in the handling of infectious materials. Safety measures are constantly being recommended and numerous devices are being invented to minimize the chances of accidental in- fection. Obviously the safety measures instituted and the devices employed in any given laboratory will depend upon the nature of the work performed, the size of the laboratory, and other circumstances. All laboratory personnel should be provided with an appropriate set of rules, which may take the form of a safety manual. Such a set of rules, which was compiled especially for those working with the tubercle bacillus, has been published.® The rules should emphasize caution against smoking or eating in areas where infectious agents are handled. Vigilance is required to see that rules are enforced and that workers do not become careless as they become more familiar with certain technics. New and inexperienced employees should be instructed regarding the risks involved and the safety precautions indicated in the par- ticular type of work they are expected to do. Crowded conditions and undue haste can increase the frequency of accidents. Laboratories should be so designed or arranged that wastes can be promptly dis- posed of. An autoclave should be conveniently located for the steriliza- tion of certain materials. All work surfaces should be swabbed with disinfectant at frequent intervals, Appropriately placed safety signs LABORATORY INFECTIONS 95 have been recommended in some instances, and first aid kits should be available for treatment of minor injuries. The least obvious sources of infection are often the most dangerous and laboratory workers should realize that some of the most common procedures regularly result in the production of aerosols. In instances where highly infec- tious materials are to be handled workers may protect themselves through the use of goggles, masks and gowns. These precautions, how- ever, do not psévent the production of aerosols which may contaminate the environment. Ventilated cabinets (see heading “J” which follows), if effectively used, can eliminate this potential hazard. Safety devices have an important place but are often cumbersome and inconvenient to use. The worker should remember that there is no substitute for skill and attention to technical details. A. Handling Clinical Specimens Various clinical specimens constitute important sources of labora- tory infection. Specimens should be submitted in proper containers, the worker should exercise care in processing the specimen, and any potentially infectious specimen should be autoclaved when discarded (see Chapter 2). Such materials as blood clots and sputum are some- times homogenized by being forced through a syringe, a procedure which may easily result in the formation of a dangerous aerosol. A disease which is of prime importance to those handling specimens of blood and those cleaning glassware soiled with blood or serum is viral hepatitis. Hepatitis heads the list of viral infections acquired in the laboratory. One serological laboratory reported that 50 per cent of those cleaning glassware for one year or more became infected. Wearing rubber gloves has consequently been recommended for the washing of glassware contaminated with blood. On the other hand, the use of gloves in the handling of needles and syringes contaminated with blood has been discouraged” on the ground that this measure provides only a false sense of security. Such needles and syringes should be boiled for at least 10 min. Following an injury with a needle or broken glassware, which might result in the introduction of hepatitis virus, the only available prophylactic measure is the admin- istration of gamma globulin (see Section III of this chapter, fol- lowing). B. Transferring Cultures There are a number of ways in which such a common procedure as transferring cultures may result in the introduction of bacteria into the environment. Studies on atmospheric contamination suggest 926 LABORATORY INFECTIONS that the most hazardous laboratory procedures are those in which bubbling or splashing occurs. Bubbles produced as a result of shaking release tiny droplets as they burst. These droplets or droplet nuclei can remain suspended in the air for some time. A ventilated hood should be used whenever it is necessary to shake suspensions of highly infectious agents.’ A heavily contaminated loop inserted into flame may cause the spattering of viable organisms. The Microbincinerator,* providing a shield for the needle as it is being flamed, was designed to overcome this difficulty. A hot loop plunged into a broth culture may also cause spattering. A suggestion for avoiding these errors is to use a sterile applicator for making transfers. The applicator is discarded into dis- infectant following use, thereby obviating the necessity of flaming.? These precautions are especially important in transferring cultures of tubercle bacilli. Shaking a broth culture may be desirable from a technical point of view but it may also wet the plug of the container or produce an aerosol which will be liberated when the plug is removed. Wet plugs have long been recognized not only as a source of culture contamina- tion but also as a hazard to the worker. The use of small or com- partmented baskets, which prevent the tubes from falling over, is help- ful in keeping plugs dry. More obvious errors are allowing infectious material to be flipped from the transfer needle or permitting the needle to touch objects while it is contaminated. Special precautions when working with Coccidioides—This fungus far surpassed others as a cause of laboratory-acquired infec- tion due to the fact that infectious arthrospores are readily liberated from the mycelia and distributed in the atmosphere.l® Sealed culture plates suspected of containing this fungus may be examined after autoclaving.!* Other workers have recommended moistening the culture on solid media before making transfers.’ Tween 80 in physiological salt solution has been used for this purpose.!? It has also been suggested that agar slant cultures can be moistened by plunging through the cotton plug a sterile needle attached to a syringe contain- ing physiological salt solution, thus reducing the chance of liberating a cloud of spores when the plug is removed. Another suggestion is based on the observation that arthrospores are not formed until after about 9 days of growth. Therefore, very young cultures on blood agar (2-4 days) may be examined with minimal hazard to the laboratory worker and if Coccidioides is suspected, mice should be inoculated.'* : Manufactured by National Instrument Co., 5005 Queensbury Ave., Baltimore 15, Md. LABORATORY INFECTIONS 97 C. Pipetting A large proportion of the known accidents resulting in infection have occurred in connection with the use of pipettes (Table 2). The most frequent mishap is to suck infectious fluid into the mouth. The mouth may also be contaminated by droplets from the pipette con- tents, even though the level of the fluid in the pipette does not reach the mouthpiece. In addition to the risk from the fluid manipulated by the pipette, contact of the finger with the proximal end of the pipette followed by placement of the pipette in the mouth affords an oppor- tunity for the spread of contamination from hand to mouth. The use of a cotton plug in the pipette and attaching a mouthpiece to a pipette by means of rubber tubing are well-known precautions but are not entirely successful in eliminating these dangers. Rubber bulbs to fill pipettes* and several nonautomatic pipetting devices'® will eliminate the chance of drawing infectious fluids into the mouth. Bubbling should be avoided in the process of pipetting. Another common error in pipetting is to permit a drop to fall from the pipette. Such a drop, particularly if it lands on a hard surface, will spatter and form an aerosol. Blowing out the last drop from a pipette containing infectious material may also create an aerosol.!® Receptacles for contaminated pipettes should be located con- veniently for the worker and should be large enough to allow for complete immersion in a suitable disinfectant without contaminating the edge of the container above the disinfectant. Flat pans or other shallow containers are generally more suitable, since they afford less chance of splashing and fit more conveniently into standard-size autoclaves for sterilization when this is desirable. Disinfectants can- not be depended upon to kill all types of microorganisms. D. Centrifuging Infectious aerosols are sometimes produced during centrifuging. Materials sealed in centrifuge tubes or bottles quickly become airborne when they are released by a leak or fracture of the container caused by high centrifugal force.’ The natural draft created by the fan action of the rotating head acts to distribute the materials through the laboratory. Accessories are now available which permit sealing of the centrifuge cup. To insure maximum safety, a ventilated cabinet is * A safety pipette filler (Propipette) is available through the Scientific Products Division, American Hospital Supply Corp., 1200 Leon Place, Evanston, 111. + Manufactured by the International Equipment Co., 1284 Soldiers Field Road, Boston 35, Mass. 98 LABORATORY INFECTIONS used for filling centrifuge cups and for opening the cups after cen- trifuging. In those instances where a leak or fracture has allowed the material to contaminate the container, immediate decontamination steps may be taken. E. Homogenizing Although homogenizers are used more extensively in virological than in bacteriological work, laboratories should be aware of the risks attendant upon their use. A homogenizer employing an open mixing bowl is obviously an excellent means of contaminating the atmosphere. A closed homogenizer, such as the Waring blendor, may also liberate infectious aerosols through a poorly fitting cover or a loose base assembly. When the cover is removed, particles may escape from the mixing bowl for some time after mixing. One laboratory has designed a hermetically sealed lid and a device for removing the contents with- out liberating infectious material.’® A new type of mixing bowl which eliminates many of the hazards has been devised and tested by workers at Fort Detrick. In any event the equipment should be care- fully checked for leaks before use, operated in a ventilated hood, and promptly autoclaved after use. F. The Use of Syringe and Needle In addition to the accidental puncturing of the skin with a con- taminated needle, there are several other ways in which the operator may infect himself.2® Syringes should be checked for defects before use, and Luer-Lok syringes are recommended for animal inocula- tions, Bursting bubbles at the tip of the needle while attempting to remove the air from a syringe should be prevented by shielding the needle with alcohol-soaked cotton or by holding the needle in a small tube containing cotton. As the needle is withdrawn from a vaccine bottle stopper, an aerosol is released unless the needle is surrounded with a cotton pledget soaked in alcohol?! Manipulations which cause the needle to vibrate even imperceptibly should be avoided. Under certain circumstances the use of a face mask is indicated, especially when inoculating animals with bacteria suspended in mucin. There is no way to prevent accidental self-inoculation except through ex- treme caution. G. Working with Animals Infected animals may be a source of infection not only to the trained scientist but also to the animal caretaker. Animal caretakers and others who must handle infected animals and cages should wear LABORATORY INFECTIONS 99 heavy-duty rubber gloves, rubber sleevelets, face masks and glasses (or goggles). The process of parenteral injection of animals requires the pre- cautions necessary for the handling of the syringe and needle men- tioned above. Although not a common procedure in the diagnostic laboratory, the intranasal inoculation of animals introduces a special hazard. Consequently, inoculation chambers have been constructed to protect the worker from the droplets invariably introduced into the air by this technic.2%?223 The chamber can be disinfected with steam formaldehyde after the inoculated animals have been removed. The danger of infection from animals after they are inoculated depends on the agent, the manner in which it is excreted, and the possible presence of microorganisms in exposed lesions. Infection may result from the simple manual examination of cutaneous lesions without gloves. If extreme precautions are indicated, heavy-duty gloves or cotton gloves over the surgical rubber gloves may be used. Since the time of Koch it has been known that tuberculous guinea pigs may infect normal pigs housed in the same room.?* Obviously such animals are also a potential source of infection to man. Laboratory workers should be aware that cross-infection among communally housed infected animals may invalidate experimental results and im- pose an additional safety hazard. In instances where the bedding in the cages may be contaminated, the debris should be disturbed as little as possible before being sterilized. Floors should be mopped with a disinfectant solution. The animal autopsy and disposal of the carcass require careful attention. If sufficient time is allowed for the blood to clot after the death of the animal, the chance of spattering droplets of infected blood is diminished. Small animals may be placed in a stainless steel tray, which is in turn placed on a towel soaked in Lysol. The carcass may be transferred to a waxed waterproof paper bag or a polyethylene plastic bag and then incinerated after autoclaving. It is the responsi- bility of the worker to see that the carcass is properly disposed of. Rubber gloves (surgeon’s) and operating room gowns with sleeves of sufficient length to permit placing the sleeve cuffs under the glove cuffs should be worn when autopsying animals. Cotton gauze face masks provide partial protection but a commercial respirator* is re- quired for complete protection when the technic produces a fine aerosol. If the examiner does not wear glasses, goggles are recom- mended. * American Optical Co., Buffalo, N. Y. 100 LABORATORY INFECTIONS Normal laboratory animals may be healthy carriers of agents infec- tious for man. Such inapparent infections may result from inadequate wild rodent control in the animal colony (see Chapter 19). H. Lyophilization There are two aspects of the process of lyophilization which call for particular attention from the point of view of safety—contamination of the apparatus and opening or breaking of ampuls containing dried material. It has been shown that the apparatus becomes heavily con- taminated by contact with the manifold outlets. Some have advised placing the lyophilizer in a ventilated cabinet. A simple cotton filter may prevent contamination of the vacuum gauge and pump. Accidental breakage of ampuls containing dried cultures may liberate a large amount of infectious dust. The intentional opening of ampuls may also release infectious material into the environment unless proper precautions are taken. One worthwhile practice is to surround the ampul with an alcohol-moistened pledget of cotton while the neck of the ampul is being broken.?® A simple device consisting of a sleeve into which the ampul is inserted while it is being broken has been recommended to prevent the liberation of infectious material. ?7 J. Ventilated Cabinets The ventilated cabinet which provides glass shielding, and ventila- tion to sweep contaminated air away from workers, have not been in wide use in the bacteriology laboratory because of the absence of proof that conventional technics are hazardous. Recent studies at Fort Detrick and elsewhere have shown that aerosolization of infec- tious agents may result during common laboratory procedures re- ferred to here and serve to emphasize the importance of using specially designed cabinets for carrying out certain laboratory procedures. Several different types of transfer cabinets, properly equipped with ventilating devices for the control of aerosols, have been designed®® and some are available commercially.* In some instances,® portable cabinets constructed from flexible plastic materials may be utilized. It should be noted that no safety hood is perfect in removing all in- fected air from the work area, since eddies and cross-currents occur by which a portion of the infected air may escape from the hood and be inhaled by the worker. Essential facilities within a protective cabinet are gas, illumination, ultraviolet lamp and receptacles for con- * Manufactured by Kewaunee Mfg. Co., 5107 S. Center St. Adrian, Mich., and S. Blickman, Inc., Weehawken, N. J. LABORATORY INFECTIONS 101 taminated pipettes or other glassware. The cabinet should be designed to accommodate electrical equipment. Water and a source of negative pressure are desirable. These cabinets are usually equipped with a blower which provides a minimum air flow of 50 linear feet a minute across the opening of the hood. Variable exhaust according to the re- quirements of the procedure may be obtained easily by an adjustable opening or damper between the exhaust fan and the hood. For eliminating infectious material from exhausted air, filtration units are perhaps the most practical. Spun glass air filters are recommended for this purpose.?® Gas or electric incineration may also be used. Ultra- violet radiation provides partial disinfection of the interior of the cabinet but complete sterilization may be accomplished with steam formaldehyde. It must be remembered that the efficiency of most ultraviolet sources deteriorates with use. The ability of the glass to transmit germicidal wave lengths diminishes with use (solarization), so that a lamp which still produces a few lumens of blue light may be transmitting relatively small quantities of effective radiation. Thin films of grease and dust cut down very markedly the transmission of the shorter wave lengths. Lamps must be sponged frequently to remove accumulating dust.3° An ultraviolet meter* should be used to determine when lamps should be replaced.®! K. Disposal of Contaminated Material The proper disposal of contaminated material is of vital importance in the prevention of laboratory infections. It is good practice to dis- card all such material into a receptacle provided with a cover which overhangs the side a half inch or more. All discards should be auto- claved promptly (see Chapter 1). A firm rule should be established that the laboratory worker must never leave a discarded tray contain- ing infected material. If he is to be gone longer than a few minutes the tray should be placed in an autoclave and sterilization started, or it should be placed on a table or bench marked “Contaminated.” In some laboratories a can or jar which is closed except for a funnel at the top is used for discarding contaminated fluids. One may also use a 2 liter Erlenmeyer flask containing 300 to 500 ml of 5 per cent phenol. A number of laboratory infections have resulted from the handling of glassware autoclaved but not sterilized due to some defect in the apparatus not reflected in the performance of the steam pressure gauges and thermometers. This experience emphasizes the necessity for frequently checking sterilization procedures. * Sm-600 ultraviolet meter, Westinghouse Electric Corp., Pittsburgh, Pa. 102 LABORATORY INFECTIONS lll. PROPHYLACTIC PROCEDURES All personnel should be vaccinated against those agents to which they are likely to be exposed in the diagnostic laboratory whenever effective immunization procedures are available. In many laboratories, personnel rotates in the various departments; consequently these workers should be immunized by all available immunization pro- cedures. Each laboratory should post a calendar schedule for primary and booster inoculations of the various immunizations that are to be carried out, with an official in the laboratory designated to see that laboratory workers actually report for the immunizations as due. Prophylactic immunization is available for some of the agents that have been responsible for laboratory infections. Immunization, how- ever, does not necessarily eliminate risk of infection, although there is evidence that infections which have occurred in immunized persons tend to be less serious. In one instance®2 no infection followed the accidental ingestion of approximately one billion virulent typhoid bacilli in a previously immunized person, who received sulfadiazine and chloramphenicol shortly after the accident had occurred. Other investigators have presented convincing evidence of the efficacy of vaccine prophylaxis in lessening the severity of tularemic infection among laboratory workers. Although the efficacy of gamma globulin, especially in the prevention of infections from serum hepatitis virus, has not been firmly established, some laboratories engaged in the processing of blood administer gamma globulin once a month to em- ployees with the objective of preventing hepatitis. A high percentage of laboratory workers begin their tuberculosis work in the tuberculin-negative state. BCG vaccination may be used for such workers on the ground that it is frequently recommended for tuberculin-negative medical and nursing personnel.?? Since the figures indicate that a high proportion of tuberculous infections probably acquired in the laboratory are of pulmonary origin * frequent chest X-ray examinations are imperative. In view of the insidious nature of early pulmonary tuberculosis, x-rays should be taken at intervals not to exceed 6 months. In those who have recently become tuberculin- positive, chest examinations should be made at 3-month intervals for at least 2 years. : It may be desirable to store serum specimens from each laboratory worker, as well as to perform appropriate skin tests and chest x-rays at regular intervals. * Most of the tuberculosis infections which could be proved as laboratory- acquired were cutaneous, that is, tuberculous chancres. LABORATORY INFECTIONS 103 IV. REPORTING OF LABORATORY INFECTIONS AND ACCIDENTS It is recommended that each laboratory set up a definite system within its own organization for reporting all accidents as they occur so that appropriate prophylactic measures may be instituted. A record of accidents without sequelae, together with those resulting in infec- tion, could be of considerable value. Many laboratory-acquired infections and accidents never come to the attention of anyone not associated with the incident, Unless the situation is unusual, it is not likely to be reported in the literature. Yet, in order to improve safety measures it is necessary for microbiologists to know the pathogens involved and the circumstances under which the infection or accident occurred. When specific diagnostic serologi- cal procedures are available, it is sometimes a revealing experience to determine the titer of antibodies in the blood serum of all the labora- tory workers as an indication of the number of unrecognized infec- tions. A high rate of subclinical infections will indicate safety de- ficiencies which may become serious if work is begun on a more virulent microorganism. The authors of this chapter feel that regular reporting of laboratory-acquired infections and accidents, together with pertinent information regarding the nature of work, probable source, causative agent involved and the outcome, is essential if appro- priate safety measures are to be instituted. Such information should be sent to the Chairman of the Committee on Laboratory Infections and Accidents of the American Public Health Association. S. Epwarp Surkin, Pu.D., Chapter Chairman Esmond R. Long, M.D., Sc.D. Roeert M. Pike, PH.D. M. MicHAEL SiGeL, Pu.D. Cuarces E. Smite, M.D, D.P.H. Arnotp G. Wepum, M.D. REFERENCES 1. Surkin, S. E, and Pike, R. M. Survey of Laboratory-Acquired Infec- tions. A.J.P.H. 41:769, 1951. 2. — Viral Infections Contracted in the Laboratory. New England «J. Med. 241:205, 1949. 3. —— Laboratory-Acquired Infections. J.A.M.A. 147 :1740, 1951. 4, Paring, T. F. Illness in Man Following Inhalation of Serratia marcescens. J. Infect. Dis. 79:226, 1946. 5. Fisu, C. H., and Seenprove, G. A. Safety Measures in a Tuberculosis Laboratory. Pub. Health Rep. 65 :466, 1950. 6. HintoN, W. A. Acute Infectious Hepatitis: A Hazard for Workers in Blood Testing Laboratories. Pub. Health Lab. 5(3) :2, 1947. 104 LABORATORY INFECTIONS 7. Geruis, S. S. Viral Hepatitis: An Occupational Hazard to Medical Tech- nologists. Am. J. Med. Tech. 17:293, 1951. 8. AnpersoN, R. E, Sten, L., Moss, M. L., and Gross, N. H. Potential Infectious Hazards of Common Bacteriological Techniques. J. Bact. 64:473, 1952. 9. GiBLIN, M., and BAUER, H. Personal communication to S. E. Sulkin. 10. SmrrH, C. E. Coccidioidomycosis. M. Clin. North America 27:790, 1943. 11. Looney, J. M.,, and SteIN, T. Coccidioidomycosis: The Hazard Involved in Diagnostic Procedures, with Report of a Case. New England J. Med. 242:77, 1950. 12. Conant, N. F. Personal communication to C. E. Smith. 13. WiLuELM, S. Isolation of Coccidioides tmmitis from Sputum. Bull. U. S. Army Med. Dept. 5:468, 1946. 14. Crerrz, J. R., and Pucker, T. F. A Method for Cultural Identification of Coccidioides immitis. Am. J. Clin. Path. 24:1318, 1954. 15. WepuM, A. G. Nonautomatic Pipetting Devices for the Microbiologic Laboratory. J. Lab. & Clin. Med. 35:648, 1950. 16. JomanssonN, K. R., and Ferris, D. H. Photography of Airborne Particles During Bacteriological Plating Operations. J. Infect. Dis. 78:238, 1946. 17. RerrmanN, M., and PHiLLips, G. B. Biological Hazards of Common Labora- tory Procedures. ITI. The Centrifuge. Am. J. Med. Tech. 22:14, 1956. 18. Smapkr, J. E. The Hazard of Acquiring Virus and Rickettsial Diseases in the Laboratory. A.J.P.H. 41:788, 1951. 19. Rerrman, M.,, Frank, M. A, Sr.,, AiG, R,, and Webpuwm, A. G. Infectious Hazards of the High Speed Blendor and Their Elimination by a New Design. Applied Microbiol. 1:14, 1953. 20. WepuM, A. G. Bacteriological Safety. A.J.P.H. 43:1428, 1953. 21. RerrMAN, M., Aig, R. L., MicLer, W. S,, and Gross, N. H. Potential In- fectious Hazards of Laboratory Techniques. ITI. Viral Techniques. J. Bact. 68 :549, 1954. 22. Vax pEN Expe, M.,, and Hussarp, A. J. G. An Apparatus for the Safe Inoculation of Animals with Dangerous Pathogens. J. Hyg. 43:189, 1943. 23. MEvEr, K. F., Quan, S. F,, and LArsoN, A. Prophylactic Immunization and Specific Therapy of Experimental Pneumonic Plague. Am. Rev. Tuberc. 57:312, 1948. 24. Lurie, M. B. Experimental Epidemiology of Tuberculosis. Airborne Con- tagion of Tuberculosis in an Animal Room. J. Exper. Med. 15:743, 1930. 25. REeIrMAN, M., et al. Potential Infectious Hazards of Laboratory Techniques. I. Lyophilization. J. Bact. 68:541, 1954. 26. —————— Ibid, II. Handling of Lyophilized Cultures. J. Bact. 68:545, 1954. 27. Heckvry, R. J. Personal communication to S. E. Sulkin. 28. Pures, G. B., Novak, F. E., and Arg, R. L. Portable Inexpensive Plastic Safety Hood for Bacteriologists. Applied Microbiol. 3:216, 1955. 29. Decker, H. M., Ger, F. A, Harstap, J. B.,, and Gross, N. H. Spun Glass Air Filters for Bacteriological Cabinets, Animal Cages and Shaking Machine Containers. J. Bact. 63:377, 1952. 30. Weobum, A. G.,, Hanger, E, Jr, and PriiLies, G. B. Ultraviolet Steriliza- tion in Microbiological Laboratories. Pub. Health Rep. 71:331, 1956. 31. Mier, O. T., Scumirr, R. F., and Pures, G. B. Applications of Germicidal Ultraviolet in Infectious Disease Laboratories. I. Sterilization of Small Volumes of Air by Ultraviolet Irradiation. A.J.P.H. 45:1420, 1955. 32. Cork, H. E, and Trrpp, J. T. Prophylaxis Following Accidental Ingestion of Salmonella typhi. Am. J. Clin. Path. 20:669, 1950. 33. Long, E. R. The Hazard of Acquiring Tuberculosis in the Laboratory. A.J.P.H. 41:782, 1951. CHAPTER 4 CULTURE MEDIA I. Introduction II. Sterilization of Culture Media ITI. Preparation of Culture Media—General Considerations IV. Tabulation of Culture Media V. Formulas and Preparation of Culture Media References I. INTRODUCTION Bacteriological culture media are designed to promote growth and reproduction of microorganisms. Concurrent with this requirement, growth substrates are formulated for the purpose of producing specific antigens, allowing selective growth of certain organisms, and demonstrating other biological characteristics, such as hemolysis, spore formation, pigment production, and certain enzymes or enzyme systems. In each case there is an attempt to supply the organism with materials essential for reproduction. Furnishing essential growth substrates for many bacteria is a simple matter, while other microorganisms have not as yet been propagated on artificial media. A working knowledge of the microorganism’s physiology affords an efficient approach to formulation of a culture medium best suited for the intended purpose. Often paucity of the etiologic agent in a specimen requires that the isolation medium be able to promote growth and reproduction from a small inoculum. Thus, the indi- vidual cell, through its own synthetic ability, must establish a micro- environment suitable for its growth and reproduction. Hinshelwood?! believes this phase of the growth cycle to be concerned with the pro- duction of intermediate metabolites essential for growth. The duration of this conditioning of the microenvironment, or lag phase, for establishment of a favorable oxidation-reduction potential and CO. microatmosphere about the cell is in large measure de- termined by the medium on which the cell is placed. Culture media and environmental conditions which are less than ideal for these 106 CULTURE MEDIA reactions prolong the lag phase or result in loss of viability of the cell. The bacteria pathogenic for man vary considerably in their nutritional needs for growth and reproduction. Many of the En- terobacteriaceae grow well in media containing a few simple organic carbon and nitrogen compounds, while the more fastidious gono- coccus, streptobacillus and Hemophilus require complex organic substrates as well as precisely controlled environmental conditions. Tremendous advances have been made in our concepts of bac- teriology since the pioneering work of Pasteur and of Koch, yet much of our present knowledge represents technical elucidation of the principles long ago recognized by these men and their con- temporaries. Lister’s unwieldy dilution technics for obtaining pure cultures were replaced by Koch's gelatin plate. In Koch's laboratory Frau Hesse made the unique contribution of incorporating relatively non-nutritive agar jelly in culture media, thereby allowing isolation and morphological studies at temperatures heretofore impossible with gelatin plates. Pasteur derived an appreciation of the effects of organic compounds on the growth of microorganisms from his studies on optical rotation of isomeric tartrates and subsequently laid the foundations of experimental design for study of the chemical nature of unicellular growth, The varied biological nature of bacteria becomes more evident when we examine their growth requirements in terms of nutrition, metabo- lism, respiration, temperature requirements and response to physical and chemical factors. For example, the type of nutrient required varies from bacteria classed as autotrophs, which are able to satisfy their nutritional needs from inorganic salts, and the heterotrophs, which require one or more organic carbon or nitrogen compounds, to the obligate parasites, which have not as yet been grown outside of the host. Metabolic activities, which constitute the “how” of nutrition, and the growth responses of bacteria to chemical and physi- cal factors are diverse enough to form a major part of the basis for their taxonomic classification. Obviously it is beyond the scope of this chapter to discuss adequately these topics; therefore, the in- terested reader is referred to recently published excellent books covering these subjects. Some of the above characteristics in terms of their influence on the formulation and use of bacteriological cul- ture media will be briefly discussed. A. Nutrition Nutritional requirements of bacteria are fulfilled by supplying the raw materials necessary for building cellular material (protoplasm) CULTURE MEDIA 107 and for furnishing the requisite energy for the cell to carry out these anabolic reactions. In general, materials used for heterotrophic cell synthesis consist mainly of water, CO,, inorganic ions, and organic and inorganic sources of carbon, nitrogen and sulfur. Indi- vidual needs for these compounds may differ not only between generic groups but within clones of a given species. Water is universally required by bacteria for growth and consti- tutes about 80 per cent of the cell mass. It not only serves to solubilize compounds which must be transported through the cell wall, but it actively enters into many of the chemical reactions oc- curring within the cell. Materials must be able to pass through the cell wall—hence solubility and permeability factors determine in part which compounds are suitable as bacterial food. The cell may be able to assist in solubilizing compounds through extracellular en- zymatic breakdown of large protein, carbohydrate and lipid molecules into smaller assimilable units. Energy is required for the active transport of solutes across the cell wall against concentration gradients, as well as to maintain con- centration gradients within the cell and to effect anabolic reactions of protoplasm synthesis. Therefore, if viability, growth and re- production are to be maintained, the sum of the energy-producing (exergonic) reactions must exceed the energy-requiring (ender- gonic) reactions. It is to their advantage that bacteria contain enzyme systems which convert low-entropy materials to higher levels of free energy with little effect on the entropy of the cell itself. Oxybiontic carbohydrate dissimilation, and, to a lesser degree, an- oxybiontic dissimilation are the most efficient energy sources for heterotrophic bacteria, the energy being supplied through phos- phorylation reactions and reactions involving Coenzyme A in the form of high-energy compounds. - Short-chain carbon compounds produced as a result of these reactions are readily utilizable in the synthesis of cellular protoplasm. Energy is also derived from hydrolysis of low energy-yielding organic phosphates and from coupled oxidation-reduction reactions involving pairs of amino acids (Stickland reaction). Carbon and nitrogen sources for heterotrophic growth are usually provided from hydrolyzed protein products derived from meat, blood, casein, lactalbumin, gelatin, yeast, cottonseed and soya meal. Many bacteria possess enzymes capable of reducing protein molecules to smaller utilizable compounds, but only if the protein medium initially contains sufficient nitrogenous compounds of small molecular size to insure vigorous growth of the inoculum.? Proteins are 108 CULTURE MEDIA rendered suitable for bacterial use by breakdown into smaller soluble, nonheat-coagulable moieties through (1) acid hydrolysis, (2) alka- line hydrolysis, (3) enzymatic (e.g., pancreatic, tryptic, papaic) hydrolysis, or (4) a combination of these methods. Protein mole- cules are degraded in order of decreasing molecular size to meta- proteins, proteoses, peptones, peptides and amino acids. Depending on the protein source and method of digestion, a “bacteriologic peptone” contains a mixture of products of hydrolysis which are characteristic of the method of production but still present a problem in accurately defining the preparation. Consistency be- tween lots of “peptone” is maintained in the digestion process, although this cannot be absolutely controlled. Many desired growth effects from a bacteriologic peptone may relate not so much to the peptone products as to other components present in the hydrolysate. Enzymatic hydrolysis is accompanied by less destruction of growth and substrate factors than acid or alkaline digestion. Acid hydrolysis destroys the amino acids tryptophane and cystine, as well as certain vitamins which are essential for the growth of many bacteria and must be replaced in these preparations. Limited quantitative analyses of several commercial peptones revealed considerable differences in the distribution of nitrogenous compounds and in the pH of 1 per cent solutions of these digests.®* Even these data provide little information concerning the supply of essential nutrients which can be compared with information derived from nutrition studies em- ploying synthetic media. Stokes, Gunness and Foster® determined the relative abundance of eight members of the B-complex vitamins present in several com- monly used hydrolysates and extracts. They were able to classify them into four groups based on the relative abundance of vitamins. Yeast extracts were classed as having the highest vitamin content, while meat extracts, brain-heart infusion and heart infusion con- stituted a group having the next highest vitamin activity. Next in order were neopeptone, proteose peptone and tryptose, while prepara- tions of some peptones and tryptone were lowest. More important, these authors conclude that “if the peptones, meat extracts, etc., are used singly or in some combinations in concentrations of 1 or 2 per cent, the resultant media may be deficient in thiamine, riboflavin, pantothenic acid, pyridoxine and p-aminobenzoic acid, but not in nicotinic acid, biotin or folic acid.” Brewer® reported that vegetable digests were more satisfactory than meat infusions in supporting adequate growth of several of the more fastidious organisms. Limited analyses on most Colab? products are available, as well as the vitamin, CULTURE MEDIA 109 amino acid and inorganic content of several Colab and Sheffield} casein hydrolysates. A number of the amino acids derived from protein hydrolysis serve as essential metabolites for many of the heterotrophs. Amino acids are incorporated directly into bacterial protein; act as precursors for the synthesis of other amino acids, vitamins or short-chain peptides (streptogenin, glutamine); provide energy for anabolic activities; and serve as a nitrogen source. Due to their amphoteric nature, amino acids also act as buffers in media, resisting rapid changes in pH. In most cases they serve multiple functions. Most heterotrophs require the naturally occurring amino acid (L-isomer), although some organisms exhibiting racemase or D-amino acid oxidase activity are able to utilize the D-isomers. Another considera- tion is the resultant effect of D-amino acids on growth as shown by Goodlow et al.,® who were able to accelerate smooth to rough variation in Brucella abortus in a synthetic medium by adding the “unnaturally occurring” amino acid, D-alanine. Indiscriminate addition of amino acids to synthetic culture media has been shown to induce toxic effects on bacterial growth because of metabolic antagonisms with other essential amino acids. Thus the unsuitability of a particular “peptone” may not be related so much to the absence of essential metabolites as to the presence of toxic compounds such as fatty acids, excessive concentrations of certain amino acids, or toxic reaction products evolved during preparation of the medium. More detailed information on the complex inter- relationships of amino acids in bacterial nutrition may be found in the report by Gladstone? and the monograph by Knight.1° B. Inorganic lons Heterotrophic bacteria need certain inorganic ions as components of cell protoplasm, as activators of enzymes, and in order to maintain osmotic pressure, concentration equilibria and electric balance between the cell and its environment. Inorganic ions in culture media may enhance, antagonize or replace each other in function. There is a universal requirement for phosphorus, iron and magnesium, and probably for potassium, manganous, calcium, and stannous and chloride ions. Phosphorus is required by all bacteria and is involved in metabolic reactions which produce energy through the synthesis and hydrolysis of high-energy phosphate bonds. Magnesium acti- vates phosphorylation enzymes. Iron-containing cytochromes par- + Technical data, Sheffield Chemical, Norwich, Conn. 110 CULTURE MEDIA ticipate with the flavins and dehydrogenases in maintaining biological oxidation-reduction potentials, while sodium and chloride ions are most likely associated with membrane permeability and osmotic and electrokinetic equilibria. An example of the importance of iron concentration on growth and specific metabolic activities was demon- strated by Pappenheimer,* who found an inverse relationship be- tween the concentration of iron necessary for optimal growth and that required for toxin production by Corynebacterium diphtheriae. There was an optimal but narrow range of iron concentrations above and below which there was no toxin production. Relatively little is known about the functions of other inorganic ions because the organism’s requirement for them is so small that most of them are supplied as contaminating “trace” elements with organic materials or other inorganic salts. For example, it is interest- ing to note that the requirement for cobalt was demonstrated only after the need for the cobalt-containing vitamin B-12 had been established.’?® Cobalt was required in such small amounts that its presence as trace-element contamination of the iron salts required for growth also satisfied the requirement for bacterial synthesis of vitamin B-12. Antagonism and enhancement effects of ions further complicate definition of the role of individual ions. However, the recent use of chelating agents for studying inorganic needs of bacteria is adding valuable information on the role of single inorganic ions. Chelating agents are compounds which combine with metals by both valence and coordinate bonds forming ring structures radically different in their chemical properties from the metal or ion. This binding can be so complete that qualitative tests for the presence of the ion are negative, Citrate is an example of a chelating agent produced by the bacterial cell. The cellular function of this type of compound in ion binding is as yet obscure. C. Oxygen The variability in oxygen requirements of bacteria crosses taxo- nomic lines. Obligate anaerobes cannot grow and often lose viability in the presence of oxygen, while another group of bacteria, classed as facultative anaerobes, are able to grow either in the presence or absence of molecular oxygen. The next logical group of micro- organisms are those which have been thought to require a reduced oxygen tension for growth and constitute the microaerophilic bacteria. Lamanna and Mallette,'* however, believe that bacterial growth in a microaerophilic environment may depend more on increased CULTURE MEDIA m carbon dioxide content than on reduced oxygen tension and actually appears to require both molecular oxygen and CO,. Finally, at the other extreme are those bacteria, classed as obligate aerobes, whose growth is adversely affected by reduction in oxygen tension. Molecular oxygen serves as a hydrogen and electron acceptor and participates in exergonic (energy-yielding) reactions in terminal aerobic respiration. It is the final electron acceptor in a series of oxidation reactions, involving enzyme systems (oxidases) active at oxidation-reduction (O-R) potentials lower than that of oxygen, which are generally mediated by dehydrogenases, flavins and cytochromes. Organic and inorganic peroxides formed in these reactions may be lethal for organisms not having the ability to dis- pose of them. Fuller and Maxted!® showed that the peroxides elaborated by viridans streptococci, while growing in mixed culture with a Lancefield Group A streptococcus, prevented beta-hemolysin production and inhibited growth of the Group A strain. One protective mechanism for removal of peroxides is based on the ability of many bacteria to produce enzymes such as catalase and peroxidases, which destroy peroxides. Catalase formation is a physiological characteristic often used in bacterial taxonomy. How- ever, survival of bacteria grown on artificial media does not rest solely on the ability to produce catalase, inasmuch as the streptococci and clostridia may produce peroxides but not catalase. Also, poison- ing the catalase system does not necessarily inhibit growth of aerobic bacteria which can produce peroxides. Catalase present in fresh blood added to culture media may aid in the disposal of metabolic peroxides. The clostridia possess the potential to produce peroxides, but the low O-R potential of the medium prevents their formation. Oxygen can be excluded from the microenvironment on solid media by using one of the many exclusion or replacement technics. Liquid media can be poised at low O-R potentials by the addition of strong reducing compounds such as sodium thioglycolate, cysteine, gluta- thione, ascorbic acid or the “glucose évolue” (prepared by heating a 10% glucose solution in 0.1 N NaOH for 10 min at 110° C), which react with and remove molecular oxygen. Lepper and Martin'® showed that broth media containing meat have two reducing systems—one due to auto-oxidation of unsaturated fatty acids of the meat and the other to glutathione and fixed thiol groups from denatured muscle protein. The use of strong reducing compounds or meat particles in liquid media allows anaerobes to be grown in tubes exposed to an atmosphere containing oxygen, permitting the organism to use its reducing powers for synthesis reactions. 112 CULTURE MEDIA The addition of 0.1 per cent agar to liquid media also aids the initiation of anaerobic growth and the growth of small inocula by reducing oxygen diffusion into the medium and retarding the dis- persion of reducing substances and COs formed in the microenviron- ment. In addition to retarding diffusion, the presence of a colloidal system such as agar, gelatin, blood proteins or starch may promote growth of organisms by adsorbing toxic compounds such as fatty acids from the environment and rendering them nontoxic, or by increasing the concentration of nutrients around the cell. D. Carbon Dioxide Bacterial requirements for CO. have been shown to be uni- versal.1™1® Wood and Werkman demonstrated the assimilation of CO,,2° and other studies?! utilizing C®*O, proved the Wood-Werk- man hypothesis. A logical extension of this hypothesis revealed that initial CO. requirements could be satisfied by the substitution of intermediate products of CO. fixation, such as are found in the Krebs cycle and in other reactions occurring during the cell's in- termediate metabolism. The COs produced as a product of inter- mediate metabolism may then suffice to meet the future growth needs of the cell. The practical implications of these findings have long been appreciated by the bacteriologist, who routinely alters the gaseous environment of cultures to provide an increased CO. tension for initial isolation of certain medically important bacteria. Adequate conditions are obtained by simply enclosing a lighted smokeless candle in an airtight container (candle jar) holding the cultures, the final CO. volume being 2 to 3 per cent.?> For ex- ample, growth of Group A streptococci and beta-hemolysin produc- tion are enhanced by incubation in a candle jar. However, initial isolation of certain bacteria such as Brucella abortus and Vibrio fetus sometimes requires higher concentrations of CO, which may be obtained by adding measured amounts of sodium or calcium car- bonate to dilute sulfuric acid.® This requirement for increased COq tension often provides a valuable clue to the identification of certain pathogenic bacteria. However, inasmuch as many of these organisms rapidly adapt to growth under normal atmospheric conditions, the presence or absence of this characteristic should be noted early. Il. STERILIZATION OF CULTURE MEDIA High-temperature sterilization employing moist heat is the most reliable means of destroying microorganisms. The bactericidal effect is probably related to the destruction of vital enzyme(s) CULTURE MEDIA 113 through protein denaturation resulting in coagulation. The latter effect of heat on proteins is used to prepare Loeffler’s serum medium and the various coagulated egg media. Tyndallization, the first reliable method for heat sterilization of liquid media,?® was described by John Tyndall in 1877. The medium is heated at 100° C for 30 min on 3 successive days. Vegetative cells present are readily killed at this temperature, and spores in the medium held at room tempera- ture between heating periods germinate to the vegetative state, be- coming susceptible to the subsequent heating. This method now has limited application and presents many disadvantages when compared with sterilization procedures employing steam under pressure. The prolonged heating of media containing polysaccharides and phosphates may depolymerize polysaccharides and caramelize sugars, as well as form other undesirable precipitates and reaction products. Agar and gelatin lose their ability to gel if subjected to prolonged or repeated heating, especially if the reaction of the medium is either very acid or very alkaline. Spores of obligate anaerobes, being unable to germinate aerobically, are not killed. Less apparent is the possible racemization of L-amino acids to the unusable D-isomer. For these reasons intermittent heating is not recommended for sterilization of media when it is important to preserve the integrity of components which are affected by prolonged heating. Steam under pressure obtained in autoclaves or pressure cookers is pre- ferred for sterilization of most media because the higher tempera- tures reduce the exposure time of media components to heat. The time-temperature relationship requirements for sterilizing culture media vary with the volume and type of container. Times as short as 2-3 min at 121° C (15 1b) are sufficient, although not prac- tical routinely, when containers are well-spaced and volumes are small. Liter volumes require at least 20 min at 121° C provided the liquid is preheated before autoclaving. Most media are sterilized by heating at 121° C for 15 min for volumes up to 500 ml and 20 min for liter quantities. Instructions for the preparation of many media indicate pres- sure rather than temperature for sterilization. As it is sometimes desirable to sterilize media at temperatures lower than 121° C (15 Ib), the pressure-temperature relationships for saturated steam most likely to be encountered are shown in Table 1. Best results are ob- tained from steam sterilization when certain principles are observed : 1. The autoclave must be efficient and should be equipped with a drain line thermometer. 2. The autoclave must not be overloaded. 3. Bulk media should be preheated to avoid lag time. 114 CULTURE MEDIA 4. Tubes should be packaged loosely and never placed in containers capable of entrapping air. 5. All air in the autoclave must be completely replaced by steam to maintain proper pressure-temperature-time relationships. Flasks and tubes should not be filled to more than two-thirds capacity. Culture media must be removed from the autoclave as soon as possible after sterilization. No Table 1—Temperature of Saturated Steam at Various Pressures Temperature Gauge Pressure Degrees Degrees (Ib per sq in.) Fahrenheit Centigrade 0 212.0 100.0 5 227.1 108.4 7 2323 111.3 10 239.4 115.2 1 241.5 116.4 12 243.7 117.6 13 245.8 118.8 14 247.8 119.9 15 249.8 121.0 20 258.8 126.0 Caution: Temperature-pressure relationships will vary with the atmospheric pressure and the efficiency of the autoclave; therefore temperature must be the criterion for sterilization. “For each 1,000 feet elevation above sea level the pressure in the sterilization chamber should be increased approximately 0.5 pound per square inch gauge.”*** A small autoclave in good working order may require between 3 and 15 min either to reach the desired temperature or to return to atmospheric pressure, depending on the type of autoclave. These intervals will vary somewhat with the liquid load in the chamber and with the temperature of the materials placed in the autoclave. Every precaution must be taken to avoid any prolonged heating of media which might result in alteration or destruction of essential com- ponents or might permit the formation of undesirable precipitates or toxic products from the myriad chemical reactions occurring in the milieu of peptides, amino acids, carbohydrates, growth factors and inorganic ions. Heating a complex medium may enhance, reduce or have no apparent effect on its growth-promoting properties. There- fore, strict adherence to instructions regarding method of steriliza- tion must be observed. CULTURE MEDIA 115 Efficient sterilization procedures require that everything in the autoclave be in contact with flowing steam. If no air is discharged before introduction of the steam under 15 Ib pressure, the tempera- ture will be 100° C. If two-thirds of the air is replaced with steam, the temperature will rise to 115° C. If all the air is discharged, the temperature will be that of saturated steam, namely 121° C.*** The gravity-flow system of air removal from the autoclave is reliable because steam weighs about half as much as air at these temperatures. Air entrapment within containers in the autoclave can result in faulty sterilization and is not reflected in temperature readings. Use of covering materials which prevent free exchange of steam and air within the autoclave must be avoided. Muslin and kraft-type paper are choice materials for wrapping items to be autoclaved. Heavy canvas or excessive wrapping with paper retards penetration of steam, and cellophane is unsuitable for this purpose as it is almost impervious to penetration. The most common causes of air en- trapment result from clogged exhaust lines and defective or im- properly adjusted thermostat valves. For a more complete discus- sion of the theory and principles of steam sterilization, the reader is referred to Chapter 1 of this book and to the excellent texts by Perkins,2* McCullouch,?® Reddish,?® and Sykes.2? The preparation of heat-coagulated protein media, such as Loeffler’s coagulated serum medium, requires an autoclave equipped with a cut-off valve in the exhaust line. Any sudden change in temperature or pressure during and after the coagulation process will result in a “scrambled egg” appearance of the serum or egg medium. Special care must be taken during preparation of the medium to avoid in- corporation of air bubbles. Screw-cap tubes are recommended con- tainers, as the increase in pressure within the closed tube assists in the maintenance of a smooth surface. After applying the caps, the tubes of media are slanted in layers not more than two or three deep and are covered with several layers of paper to prevent rapid heating. All outlets of the cool autoclave are closed and the pressure is rapidly raised to 15 Ib. After 10 min the exhaust valve is opened slightly and the temperature slowly raised to 121° C by gradually replacing the entrapped air with steam while at the same time main- taining a constant pressure. The sterilization process is continued for 15 min, then all inlet and exhaust valves are closed and the chamber is allowed to return slowly to atmospheric pressure. An additional cooling time is allowed for dissipation of heat from the covered tubes. Alternate methods of inspissation and sterilization provide for inspissation followed by Tyndallization in an Arnold 116 CULTURE MEDIA sterilizer, or by sterilization in an autoclave at 111° C (7 1b). Detailed methods for inspissation and sterilization of serum media using a single-chamber autoclave, pressure cooker or double-chamber auto- clave have been described by Levine.?® Caution: Media containing dyes (e.g., Lowenstein-Jensen or Petroff) deteriorate markedly at a temperature of 121° C. Heat-labile components—Heat sterilization of certain culture media may render them unsuitable for reasons discussed elsewhere. A broth medium containing 1 per cent of a heat-sensitive disaccharide may, after autoclaving, contain a sufficient concentration of mono- saccharides to produce a positive fermentation reaction from the monosaccharide alone. The time apparently saved by this method of preparation is more than offset by the time and effort wasted in puzzling over aberrant fermentation reactions. Therefore, heat- sensitive carbohydrates, polyhydric alcohols and other compounds used in media for biochemical tests should be sterilized by passage through a bacteria-retaining filter. Ordinarily, these carbohydrates are made up in 10% solutions (when solubility permits), are steri- lized by filtration, and are added to the sterile base medium to a final concentration of 0.5-1.0%. A less desirable means of sterilizing compounds subject to hydrolysis by heat consists of carefully auto- claving small volumes of 10 to 20% aqueous solutions at 118° C maintained for exactly 10 min. A number of carbohydrates and polyhydric alcohols commonly used in biochemical tests, together with methods for their sterilization, appear in Table 2. Sterility testing of these media is mandatory, and media for determination of biochemical reactions must be pretested, using cultures known to produce positive and negative reactions with the test compounds. Sterilization using the mechanical means of passing solutions of heat-labile components through bacteria-retaining filters overcomes many hazards to definitive biochemical determinations. Among the types of filters used for cold sterilization are: (1) asbestos pads (Seitz), (2) diatomaceous earth candles (Berkfeld, Mandler), (3) porcelain candles (Chamberland-Pasteur, Coors, Selas), (4) sintered (fritted) glass, and (5) cellulose nitrate membranes (“Gelman” or “Millipore” type). The advantage of one filter over others depends on the type and volume of material to be filtered. The essential characteristics of most of these filters are discussed by Porter,? and detailed information may be obtained from the manufacturers con- cerning porosities, capacities, cleaning and general uses of filtration apparatus. CULTURE MEDIA 117 Table 2—Sterilization of Carbohydrates and Polyhydric Alcohols Final Stock Method Concentration Solution of in Medium Compound (%) Sterilization (%) Arabinose 10 SF 0.5 Adonitol 10 A 0.5 Glucose 10 A 0.5 Dextrin 107 A 0.5 Dulcitol 25 A 0.5 Glycerol 10 A 0.5 Glycogen 10 A 0.5 Lactose 10 A 0.5 Maltose 10 SF 0.5 Mannitol 10 A 0.5 Rhamnose 10 A 0.5 Salicin 101 A 0.5% Sorbitol 5 A 0.5% Sucrose 10 A 0.5 Trehalose 10 A 0.5 Xylose 10 SF 0.5 SF = Seitz filter; A=autoclave at 118° C for 10 min. T Solution must be added at 30° C to prevent crystallization. I Store at room temperature. lll. PREPARATION OF CULTURE MEDIA—GENERAL CONSIDERATIONS Water—Always use distilled water for preparing culture media unless specifically directed otherwise. Tap water is an un- known quantity and varies considerably in ion content and pH. Substances such as chlorine or copper in tap water may adversely affect growth, and the hydrogen ion concentration of tap water may change from day to day. The use of deionized water is not con- traindicated provided that the dangers associated with failure to renew inactive resins, and the possibility that organic matter may pass through the resins, are recognized. Reagent chemicals—Highest-purity chemicals will be used unless otherwise stated. Inorganic chemicals should be of analytical- reagent grade; and saccharides, polyhydric alcohols and glucosides used for fermentation tests should be free, by bacteriological test, from chemical contamination by other fermentable compounds. 118 CULTURE MEDIA Agar—Suggested specifications for bacteriological-grade agar proposed by the Committee on Bacteriologic Technic of the American Society for Microbiologyf are more restrictive than those outlined in the U. S. Pharmacopoeia XVI. In general, agar suitable for bac- teriological procedures should be clear in solution (low sol. turbidity) and in plates (low debris count). Melting and gelation temperatures and the content of other substances such as chlorides, reducing com- pounds and protein nitrogen should be kept within prescribed limits.?® Agar which contains more than 25 viable spores per gram is excessively contaminated. To test for contamination dis- solve 2 g of agar in 100 ml of broth medium (CM No. 11) and auto- clave for 5 min at 121° C. Cool the medium to 45°-50° C, pour into four petri plates, and incubate at 32° or 35° C for 48 hr. The total count on four plates should not exceed 50 colonies.?® Semisolid agar used for motility studies should be tested for suitability, using a known, weakly motile culture each time a new lot of agar is used, as variations in the purity of agar from lot to lot may require readjustment in concentration. Gelatin—Specifications for bacteriological-grade gelatin have been outlined by the Committee on Bacteriologic Technic.?® TU. S. Pharmacopoeia standards require that bacteriological-grade gelatin contain no coliform bacteria and no more than 10,000 viable bacteria per g. In addition the A.S.M. committee recommended that the ma- terial be clear in solution, free of suspended particles, and contain no fermentable carbohydrate when tested under specified conditions. It is important to keep in mind that the solidifying power of gelatin is gradually destroyed by prolonged or repeated heating and that charring is likely to result when a flame is applied directly to the flask containing a gelatin solution. Bacteriological peptones—The descriptive term “peptone” is used for hydrolysates of proteins or proteinaceous materials. These contain varying amounts and kinds of amino acids, peptides, peptones, proteoses, vitamins and carbohydrates, depending upon the protein source and the method of hydrolysis. These have already been re- ferred to briefly. The U., S. Pharmacopoeia XVI refers to three classes of peptones: (1) “peptic digest of animal tissue,” (2) “pep- tone, dried (meat peptone),” and (3) “pancreatic digest of casein (tryptone).” In each case the specifications are general, sufficiently so that many peptones would fit into each of the three classes, Dif- + Formerly Society of American Bacteriologists. CULTURE MEDIA 119 ferences within a class depend on the degree of and treatment sub- sequent to digestion, and on the source protein used in preparing these peptone products. “Peptic digest of animal tissue” solutions should (a) produce no precipitate (complete digestion) when tested with glacial acetic acid; (b) show the presence of tryptophane by the bromine test; (c) con- tain not more than 50 bacteria or clumps of bacteria per 10 consecu- tive oil-immersion fields (see procedure below); (d) fail to produce acid when inoculated with Streptococcus liquefaciens but possibly produce acid when inoculated with Escherichia coli; and (e) produce indole, acetylmethylcarbinol, and hydrogen sulfide when tested with appropriate cultures. “Peptone, dried (meat peptone)” differs from the above in color, odor and Kjeldahl nitrogen content (14.2-15.5%). Growth-support- ing requirements, freedom from fermentable carbohydrates, and tests for indole, acetylmethylcarbinol and hydrogen sulfide are not specified for this peptone. This peptone is not considered suitable for bac- teriological work. “Pancreatic digest of casein (tryptone)” specifications are similar to those for “Peptic digest of animal tissue,” as outlined in the current U. S. Pharmacopoeia. U.S.P. XVI specifies that there be no undigested casein in a 10 per cent solution of the digested material, and that the digest contain not less than 10 per cent Kjeldahl nitrogen. At specified concentrations and employing specific test organisms, solutions of the digest should (1) be free from, or show only a trace of, fermentable carbohydrates, (2) allow production of indole; acetylmethylcarbinol and hydrogen sulfide, (3) be free from nitrite, and (4) support growth of the designated test organisms. The U.S.P. specifications afford little help toward direct applica- tion of information derived from nutrition and metabolic studies using synthetic media. Chemically defined media are usually im- practical for routine diagnostic procedures either because of their technical complexity in preparation or because of the requirement for special strains of bacteria and large inocula. At the present time only microbiological tests are reliable for determining the adequacy of media. However, chemical analyses of each of the various “basic” peptones used in culture media, such as the fairly extensive analyses published by Consolidated Laboratories and Sheffield Chemical Corp. for their products and supplementary data available from others, are also helpful to the microbiologist who is attempting to formulate media using these peptones. At the same time, addi- tional quality control of these products should be more firmly estab- 120 CULTURE MEDIA lished and expanded. Despite these considerations, dehydrated definitive media, or peptones, with added meat or yeast extract are usually sufficient for adequate growth of most bacteria and should be substituted whenever possible for fresh infusions, provided com- parison studies have shown the substitute medium to fulfill the necessary requirements. Generally, dehydrated media are con- siderably better and more reliable. Examination of peptones, infusions and extracts for microbial content?? a) Dissolve 1 g of dry or concentrated material in 10 ml of sterile distilled water; or use laboratory-prepared infusions or decoctions undiluted. b) Spread 0.01 ml on 1 sq cm of a glass slide. c¢) Stain by Gram’s method. d) Examine with an oil-immersion lens. No more than a total of 50 bacteria or clumps should be visible in 10 consecutive fields. This test rules out the use of highly contaminated materials or of materials in which growth has actually occurred. Determination of pH—Measurement of the final pH of a prepared culture medium is an essential part of the formula protocol, whether the medium is prepared from the dehydrated product or from individual components. This may be accomplished colorimetri- cally or preferably potentiometrically.2®3! Final pH readings should not deviate from the recommended reading by more than 0.2 of a pH unit. The reaction of many media can be adjusted prior to auto- claving after experience has determined the effect of heat on the final hydrogen ion concentration of the particular medium. For example, media containing sodium bicarbonate are likely to be more alkaline after autoclaving owing to the breakdown of the bicarbonate ion and evolution of COs. Distilled water exposed to air absorbs CO, with a resultant drop in pH which might cause errors in the final pH of the medium. The latter problem can be overcome either by using freshly distilled water or by boiling the water shortly before use. Preferably the final pH reading is taken after the medium has cooled to about 25° C. If the reaction is de- termined electrometrically, the standard buffer and the medium should be at the same temperature, for a change in the tempera- ture of distilled water from 25° to 40° C results in a change in the pH reading from 7.0 to 6.7, respectively. CULTURE MEDIA 121 Storage of media—If storage of prepared media is necessary, they should be placed in a cool atmosphere and in the dark, inasmuch as certain dyes, indicators and growth factors (for example— riboflavin, folic acid and pyridoxine) are adversely affected by light. Storage in the refrigerator usually prolongs the shelf life of most media; however, media containing small amounts of agar such as thioglycolate broth (CM Nos. 19 and 20) must be stored at room temperature unless sealed, or evacuated and sealed. Media should not be subjected to long periods of storage, and stored media to be used for the isolation of fastidious bacteria, as might be encountered in blood cultures, should first be heated to remove dissolved oxygen. Solid media not containing heat-labile or heat-coagulable in- gredients may be remelted once (no more) and reslanted or poured to plates in order to provide the necessary moist surface optimal for recovery of many fastidious pathogenic bacteria. Dehydration of tubed media can be prevented by the use of screw caps or autoclav- able plastic caps which retain moisture. Placing agar plates in air- tight plastic bags effectively reduces dehydration of these media. Therefore, unless adequate protection is assured, it is recommended that storage of prepared media be kept to a minimum. Prolonga- tion of the time available for chemical reactions to occur in a medium even at refrigerator temperatures may introduce additional variables in an already complex reagent. Modifying or formulating media for isolation of pathogenic bacteria—The efficacy of a new formulation or modification of a culture medium for the isolation of pathogenic bacteria from clinical sources must be determined by rigorous testing in parallel with the best media in current use. The use of old laboratory strains for this purpose constitutes, at most, only the preliminary step, for a medium developed and tested using only this criterion may be un- suitable under conditions of actual usage. Gutiérrez-Vasquez3? found that although H37Rv stock strains grew equally well on three media being compared for isolation of Mycobacterium tuberculosis, clinical application revealed that considerable discrepancies existed between these media in efficacy for isolation, and he concluded that results obtained from experiments carried out with laboratory strains could not be applied a priori to diagnostic work. Fig 1 demonstrates the differences encountered when two stock cultures of Vibrio comma having varied culture histories were plated on an alkaline starch-milk medium. 122 CULTURE MEDIA Figure 1—Surface colonies of Vibrio comma on alkaline starch-milk agar, pH 8.5, after 24 hr at 35° C: a was isolated in 1962; b in 1959. The initial isolates of strain b from the patient produced similar reactions to a. These differences again emphasize that selection of an adequate isolation medium must ultimately be based on its per- formance using clinical specimens, should the temptation arise to “improve” on an author’s formula. To be differentiated from the foregoing discussion is the use of carefully selected and maintained test cultures for quality control work, antibiotic testing, sterility testing, disinfectant testing and the like. Although these cultures are usually selected on the basis of their performance in specific measurements or tests, they may not be gen- erally representative of the type species or of organisms found in clinical materials. Dehydrated media—Many of the media listed in the following section, or media with comparable properties, can be obtained from commercial sources. These media are indicated by an asterisk (*) after the title. The use of dehydrated media is recommended when- ever possible, as these products afford the advantages of good con- CULTURE MEDIA 123 sistency from lot to lot, less labor in preparation and greater economy. Nomenclature used in Section V (formulas and preparation) is be- lieved to be sufficient for obtaining the dehydrated medium, if avail- able, from one of the several manufacturers, e.g., Albimi Laboratories (Brooklyn, N. Y.); Baltimore Biological Laboratories (Baltimore, Md.) ; Case Laboratories (Chicago, Ill.) ; Consolidated Laboratories (Chicago Heights, Ill.) ; Difco Laboratories (Detroit, Mich.). Of necessity many brand names have been used to designate pep- tone components in media formulas. However, in cases where the brand appears to be of no importance, a definitive name (usually the U. S. Pharmacopoeia nomenclature) or the general term “peptone component” has been used. Specific nomenclature of these compo- nents can be found in the various commercial media manuals. A knowledge of the various commercial names for similar products is helpful. Trypticase (BBL), Casitone (Difco), N-Z Case (Sheffield) and Peptone “C” (Albimi) are examples of peptones which con- form to the U. S. Pharmacopoeia standards for pancreatic digest of casein, and can be interchanged within prescribed limits. Neverthe- less caution must be exercised when interchanging peptones or other complex organic components of media, inasmuch as there are varia- tions among products of the same name from different manufacturers which might directly affect the growth response of certain bacteria. Moreover, the rather delicate balance between salts and dyes in many of the selective or enrichment media may be upset by the substitution of peptones in these media. Dehydrated culture media, useful as they are, provide no panacea for the practicing microbiologist, and the careful worker will check each lot of dehydrated medium as carefully as when a medium is prepared from the individual components to insure that it produces the desired growth response or reactions. All manufacturers recom- mend certain media for growth of specific organisms, and one®* lists suitable organisms for performing both positive and negative control tests on media used for determining biochemical reactions. This necessity for pretesting media was reported by Karlstrom, Brandon and Levin® in describing some of the difficulties experienced with SS agar, bismuth sulfite agar, and Loeffler’s serum medium. Their advice is basic in any pretesting procedure: “Our experience has shown that it is well to check the pH of all dehydrated media, and to run controls on many of these by using the appropriate micro- organisms.” Kendrick®® checked Bordet-Gengou media obtained from two commercial sources and found that colony size and hemolysis by 124 CULTURE MEDIA Bordetella pertussis on the medium from one source was definitely inferior to the reactions obtained on the other. Werner et al.37 using a dehydrated medium designed for the selective isolation of Group A streptococci, reported that 87.5 per cent of the Group A strains isolated in the study would have been missed had this medium been used alone. Schaub et al.*® for whom the medium was originally prepared, obtained excellent results, while the experience of Hunter, Blair and Rust®® with this medium paralleled that of Werner and his colleagues. These examples are not cited to discourage use of the many ex- cellent dehydrated products available, for the deficiencies noted are quickly rectified ; rather they are cited to emphasize the necessity for predetermining the suitability of each lot of medium for its intended use. It must be kept in mind, however, that culture media, whether prepared from dehydrated products or from fresh infusions, should be tested for performance before use. They cannot be expected to perform as described unless (1) sound technical methods are em- ployed, (2) media are prepared according to directions, and (3) media are used in the manner recommended. Dehydrated media, like other laboratory tools, must be properly cared for. They should be stored in a cool, dry place, or in the refrigerator if there is likelihood of exposure to heat. Bottles which are sealed and stored properly will keep for long periods of time. Material in bottles which have been opened may deteriorate rapidly, depending upon atmospheric temperature and humidity. There may or may not be visible evidence of deterioration, such as browning or darkening and hardening of the contents. Preferably bottles which have been opened should be used within a short time or they should be discarded. Sterile complete media—Completely prepared media sealed in tubes, bottles and plates should probably be employed in labora- tories not having sufficient personnel and facilities availabe for properly making and testing media formulated from ingredients or from dehydrated materials. A small number of prepared media can be selected which will provide even a small laboratory with the ma- terials necessary for processing a wide variety of specimens rapidly and efficiently. E. B. Braj, Pu.D., Chapter Chairman C. W. CHRISTENSEN, PH.D. E. I. PetrAN, PH.D. H. D. Vera, Pu.D. CULTURE MEDIA 125 31. 33. 34. 35. 36. 37. 38. 30. 40. 41. . Starch Agar for Gonococcus and Meningococcus, Mueller and Hinton 43. 45. TABULATION OF CULTURE MEDIA Broth for Fermentation Studies Peptone Solution for Indole Production Extract Broth Extract Agar Meat Infusion Broth, Plain Meat Infusion Agar, Plain Phenylethylalcohol Agar, Brewer and Lilley Phenolphthalein Phosphate Agar Mannitol Salt Agar, Chapman Agar for Staphylococcus Enterotoxin, Dolman et al. Casein-Soy Digest Broth Casein-Soy Digest Agar . Extract Gelatin Milk, with Indicator Semisolid Agar Blood Agar Heated Blood (Chocolate) Agar Bordet-Gengou Agar Thioglycolate Broth with Glucose Thioglycolate Broth without Glucose or Indicator . Brain-Heart Infusion Broth, Dehydrated . Brain-Heart Infusion Agar, Dehydrated . Beef Heart Infusion for Streptococcal Media Todd-Hewitt Broth . Todd-Hewitt Agar with Sheep Blood . Tryptose Extract Agar with Sheep Blood . Tryptose Infusion Agar with Sheep Blood Thallous Acetate-Tetrazolium-Glucose Agar Beef Heart Infusion Broth with Blood Pike’s Sodium Azide-Crystal Violet Enrichment Broth Blood Digest Enrichment, Fildes Yeast Extract, Thj6tta and Avery Levinthal’s Broth Levinthal’s Agar Tryptose Phosphate Agar, Semisolid Tryptose Dextrose Vitamin B Broth Tryptose Dextrose Vitamin B Agar Lead Acetate Agar Yeast Autolysate Broth for Brucella Yeast Autolysate Agar for Brucella GC Medium Base Agar with Hemoglobin, for Gonococcus McLeod's Agar with Plasma and Hemoglobin Peptone Agar with Plasma and Hemoglobin Gelatin-Blood Agar 126 46. 47. 48. 49. 50. 51. 52. 53a. 53b. 54. 55. 56. 57. 58a. 58b. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. CULTURE MEDIA Gelatin-Egg Albumin Agar Tryptic Digest Broth, Douglas Tryptic Digest Agar, Douglas Douglas’ Agar with Ascitic Fluid and Carbohydrates Transport Agar Medium for Gonococcus, Peizer ef al. Transport Agar Medium for Gonococcus, Stuart et al. Kligler’s Iron Agar Triple Sugar Iron Agar Triple Sugar Iron Agar Eosin-Methylene Blue Agar Endo’s Agar Sodium Desoxycholate Agar Sodium Desoxycholate Citrate Agar Bismuth Sulfite Agar, Wilson and Blair Bismuth Sulfite Agar, Hajna and Damon Salmonella-Shigella (SS) Agar MacConkey’s Agar Brilliant Green Agar Tetrathionate Enrichment Broth Selenite (F) Broth Broth for Nitrate-Reduction Test Agar for Nitrate-Reduction Test Citrate Agar, Simmons Urea Broth, Rustigian and Stuart Urea Agar, Christensen Semisolid Agar for Motility Test Agar Base for 10% Carbohydrate Medium Glycerol-Sodium Chloride Solution, Buffered Dorset’s Egg Medium OF Agar, Hugh and Leifson Hormone Agar for Plague Vaccine Crystal (Gentian) Violet-Hormone Agar, Meyer and Batchelder PPLO Broth PPLO Agar Pai’s Egg Medium Cystine-Tellurite-Blood Agar, Frobisher and Parsons McLeod's Agar for Type Determination of Corynebacterium diphtheriae. Loeffler’'s Coagulated Serum Raffinose-Serum-Tellurite Agar, Whitley and Damon Glucose-Serum-Tellurite Agar, Whitley and Damon Heated Blood-Tellurite Agar, Kellogg and Wende Agar for in vitro Virulence Test, Frobisher ef al. Thiol Broth with 0.1% Agar Liver Infusion Broth for Vibrio fetus, Plastridge Broth for Leptospira, Verwoort-Wolff Korthof’s Broth for Leptospira Stuart’s Broth for Leptospira CULTURE MEDIA 127 91. Chang’s Broth and Semisolid Agar for Leptospira 92. Fletcher's Semisolid Agar for Leptospira 93. Packer’s Extract Broth for Erysipelothrix 94. Packer’s Sodium Azide-Crystal Violet Extract Agar 95. Extract Gelatin for Erysipelothrix 96. Lead Acetate Agar for Hydrogen Sulfide Production by Erysipelothrix 97. Edwards’ Extract Agar with Crystal Violet for Erysipelothrix 98. Carbohydrate Base Broth for Erysipelothrix 99. Glycerol-Asparagine Medium, Proskauer and Beck 100. Egg Yolk-Potato Flour Medium, ATS 101. Egg-Potato Flour Medium, Lwenstein- Jensen 102. Tween-Albumin Medium 103. Egg-Potato Starch Medium, Petragnani 104. 7H-10 Medium, Middlebrook and Cohn 105a. Sabouraud’s Agar 105b. Sabouraud’s Agar with Chloramphenicol 105¢c. Sabouraud’s Agar with Cycloheximide 106. Cornmeal Agar 107. Potato Glucose Agar for Fungi 108. Mycophil or Mycological Agar 109. Littman’s Oxgall Agar 110. Rice-Tween 80 Agar 111. Glucose-Asparagine Broth for Histoplasmin and Tuberculin 112. Glucose-Peptone Broth for MR-VP Tests 113. Sabouraud’s Maltose Agar 114. Chopped Meat Medium 115. Blood-Glucose-Cystine Agar, Francis 116. Extract Broth for Antibiotic Assays V. FORMULAS AND PREPARATION OF CULTURE MEDIA All culture media formulas appearing in this section were sub- mitted by the contributors of the various chapters appearing in this book. Formulas are essentially as described by the contributing authors, altered only in the interest of uniform presentation. They are referenced to original work, as well as to pertinent modifications. There are many other media formulations which will be found useful in diagnostic procedures. Often diagnostic laboratories which receive a variety of specimens cannot effectively or conveniently employ large numbers of culture media. However, a few media carefully selected may serve many purposes, so that numerous highly specialized media are not necessary. Certain modern media have a broad coverage in their ability to promote growth of many kinds of microorganisms. CM Nos. 11 and 12 are two such media. CM No. 128 CULTURE MEDIA 11 can be used for a great variety of bacteria; blood for blood broth or Levinthal’s broth base (see CM No. 33) can be added. CM No. 12 can be used as is for many pathogens and it is recommended for the preparation of blood agar and chocolate agar media. Multiple- function preparations, that is, a single medium which can be used to determine both indole production and nitrate reduction, are available and serve to reduce the number of media necessary for diagnostic procedures. Semisolid fermentation media incorporating cystine, sulfite and pancreatic digest of casein can be used (a) for aerobic and anaerobic fermentation studies, (b) for motility determinations, and (c¢) for maintenance of stock cultures. The addition of glucose and papaic digest of soybean meal to this medium has been reported to result in luxuriant growth of many of the more fastidious patho- gens. Descriptive literature on these media may be obtained from the several manufacturers producing them. Many dehydrated media formulas in this section contain the nota- tion “peptone components” instead of the brand-name peptone. The appropriate peptone can be determined by consulting one of the media manuals supplied by manufacturers of culture media when such a medium is to be prepared from the individual components. Note that names of media or components followed by an asterisk (*) may be obtained in the dehydrated form from commercial sources. In all cases where the commercial medium is used, instructions appearing on the container will govern the method of preparation. All solutions are aqueous unless otherwise specified. I. Broth for Fermentation Studiest Pancreatic digest of casein, US.P. XVIf..... .......... 0g Sodium chloride ......cciiiiiinriniiiiiiiiiineaen 5 ¢g Bromcresol purple§ (10 ml of an 0.2% aqueous solution) 002 g WEEE cit iss Soviaiic £5 DUS EENAIETTs 2 5 Sanh we wkd 1,000 ml a. Dissolve the peptone and salt in water. Add the indicator and adjust the reaction to give a final pH of 6.8=+. b. Dissolve 0.5-1.0 g of the dry carbohydrate in each 100 ml of medium and dispense in tubes. c. Pack tubes loosely in baskets or in test tube racks and autoclave at 118° C for 10 min. + Semisolid fermentation media may be prepared by adding agar to give a final concentration of 0.3-0.35%. 1 Other peptones shown by bacteriological test to be free from carbohydrates may be used. § Other suitable indicators may be used, provided the final pH is appropriately afjused, i.e, media with phenol red as the indicator should have a final pH or74=%. CULTURE MEDIA 129 d. Remove from the autoclave promptly and incubate at 35° C for 24 hr to check sterility. e. If filtered carbohydrate solutions are used (see Table 2), add them to 100 ml amounts of the autoclaved broth, dispense aseptically in tubes, and incubate to check sterility. 2. Peptone Solution for Indole Production Pancreatic digest of casein, U.S.P. XVIf.....ccovvveinnnnn 10 g Sodium ChIOHAR .. «comers is « + smmmmens sss + shames sss oi 5g WVBLEE. . ovornomms won 0» 0 on orererwieins « © wwwstmnion be = § § SFWRAIHEIRE & § S80 FW 1,000 ml Dissolve peptone and salt in water and tube in desired amounts. Autoclave at 121° C for 15 min. Final reaction should be pH 7.2%. T Satisfactory positive results for indole production by bacteriologic test are required in the U.S.P. XVI specifications. Other peptones having a high available tryptophane content may be substituted for pancreatic digest of casein. 40 3. Extract Broth*t Pepione COMPONRL +... vumwsnivrs vanens stssis ans saions sos ve 5 Beef GRUTHOL ov contin son ab sam smteit oh RSET 3 WRLEE: &iirivsn rumen sim asmm mms dvi ess pee 1,000 a. Dissolve ingredients in water and, if necessary, filter through paper to clear. b. Dispense as desired and autoclave at 121° C for 15 min. Final reaction should be pH 6.9=+. + Several suitable dehydrated media are available as “nutrient” or “extract” broth. This medium is not suitable for fermentation studies.41 4. Extract Agar* Extract broth (CM No. 3) . ei senicvenssssssanmosessssrms 1,000 ml AGBE nse yo yormmpupss « 3 siaigsonares SOEHRTEEATS § § SOREN £5504 40 15¢ a. Dissolve the agar in the extract broth, with frequent agitation and heat. The temperature must be raised to the boiling point to obtain satisfactory solution. b. Dispense in tubes or flasks as desired and autoclave at 121° C for 15 min. The final reaction should be pH 6.9+. 5. Meat Infusion Broth, Plain* Lean beef muscle (or equivalent of dehydrated infusion) 375-500 g Tryptose, thiotone, or proteose peptone ................. 10 g Sedu. CRIOTHE vss sammie wrias evar Sas Eos 5g WWILET coivii os sisR aa dias aaa Sadan sna ais sons wis snms 1,000 ml 130 CULTURE MEDIA a. Prepare infusion from fresh meat as in CM No. 23, or b. Suspend dehydrated infusion in water, allow to stand for 5 min, mixing thoroughly until a clear solution has been obtained. Add other ingredients. c. Dispense as desired and autoclave at 121° C for 15 min. Final reaction should be pH 7.4. 6. Meat Infusion Agar, Plain* Meat infusion broth, plain (CM No. 5) .....cvvvvvnnnnn. 1,000 ml ATAT 40s 3 naamnm on £3 500s 88 A Gar ails Same $50E 15¢g a. Dissolve the agar in the broth with frequent agitation and heat. b. Dispense as desired and autoclave at 121° C for 15 min. The final reaction should be pH 7.4. 7. Phenylethylalcohol Agar, Brewer and Lilley#243* Pancreatic digest of casein, USP. XVI ................. 15¢g Papaic digest of soybean meal ......ccevvieiiiernvnnennnn 5g SOUT TCHIBTIAC.. wcvms a5 00 pimps ss osm emis wos Beinn = ds 5g Phenylathvlnleonol . os covivas sas smmannamons vas samme ea ss mn 25 ¢g AGEL: os asietimms 3 35 GEERT $3 FeRaiaeiars + s » Fa wiemogiie dommes 15¢ WVBIEE 0 wlidoe 35 ohm so + sa wmbionis + o's biaminisda bla ds cme 1,000 ml a. Suspend ingredients in water, mix thoroughly, and heat to dissolve the agar. b. Dispense as desired and autoclave at 118° C (12 1b) for 15 min. The final reaction should be pH 7.3=+. 8. Phenolphthalein Phosphate Agar,** Modified?’ Bxtract agar (CM No. 4) iiiivisiiissnines ansonnsns is 1,000 ml Phenolphthalein phosphate as sodium salt ................ 05g WalElr oianie vin saan Enns Teast ae esos G RRS EF 100 ml a. Dissolve the phenolphthalein phosphate salt in 100 ml of water and filter through a Seitz E. K. filter. The stock solution may be stored at 4° C. b. Add the phenolphthalein phosphate solution to sterile, melted and cooled extract agar, pH 7.4, in the proportion of 1 part of phenolphthalein solution to 49 parts of extract agar. Pour plates and incubate overnight at 35° C to free agar of surface moisture. The complete medium can be stored at 4° C and plates poured as needed. CULTURE MEDIA 131 9. Mannitol Salt Agar, Chapman** BEBE CRITABE . oovtvisinnin stviarsiniieliititme aia ootiniimraae: 410 Fin aim iare 1 g Peptonit. COmMpPOneHl ii ssnwddnnddidsnnemunine s swelon 10 g =Mamitol . commen isvssrarsrsisrsdosvees sass nue 10 g SON CHIOTIAR iar im se sn am aiming nt dik emit BAP smn 75 g Phenol red (10 ml of 0.25% aqueous solution) ......... 0.025 g ALOT lori vin Sennen SEs RR EVRA EGE ves reli 15 WHEE oni co Shs Sn mis SEs HAS PAAR SA Sass Sra 1,000 ml a. Dissolve components in water with frequent agitation and heat. b. Dispense as desired and autoclave at 121° C for 15 min. The final reaction should be pH 7.4=+. 10. Staphylococcus Enterotoxin Agar, Dolman ef al.*7-4? Proteose peptone or equivalent ..............eiiiiinneannn. 5g Soditiny HOPE: ii vunsnrs arsams vanes sss ine sess rsdesiss 5g BOE riviv isi ad SERRATE TS 8 AR RRA TERS FARR eSATA 3g BALE ici iost in siulgfnTinmemmasebmissesnst gomibiasasmtrispn sesmipasnintommsgieimin 500 ml a. Dissolve ingredients in water with heat and agitation. b. Add aqueous solutions of the following salts: ALHOONIIY JBCIBIE rina nm ss sivin sins asd smambittioin seman tise 5 g Dipotassium phosphate, BEpHPO,; wove vvvsvmnerrrsrssmmmenns Lg Monopotassium phosphate, KH,PO, .ocipivnrsrerrnmneres 1 gz Magnesium sulfate, MgSOL7H,0 ..ovvvviiiiniiiiinnnnnnn. 02g Colcivm chloride), ‘CaCl, . . « + vuwrimmmnns 2 ww MNRAS dwn 01g c. Make up solution to 1,000 ml, heat to boiling to melt agar, and adjust pH to 7.4. Autoclave at 121° C for 20 min, d. Pour medium into petri dishes while still hot to a depth of 0.5-1 cm. Evenly seed the surface of the cooled medium with a few drops of a young culture of the staphylococcus strain being tested. Il. Casein-Soy Digest Broth* Pancreatic digest of casein, US.P. XVI ........cc0vu0ss 17 ¢g Papaic digest of soybean meal ............covviiiiiinnnn 3 g Sodium, chloride v.oe1es svmvsn sf ss ramunnrsss srnnmamss es 5 g Dipotassium phosphate, K,HPO, i. snmnuevnessssnnmsness 25g GIUGOBE 2 05 vonmianins 5 5 530 Masao + 3 5 5a aii « 5 + 3 $F HEmw _ 25g Vater o.iriiitinenennrenenraneneesnnansnsssnnannannns 1,000 ml Dissolve ingredients in water, dispense as desired, and autoclave at 121° C for 15 min. The final reaction should be pH 7.3. 132 CULTURE MEDIA 12. Casein-Soy Digest Agar, General-Purpose Pancreatic digest of casein, US.P. XVI ........ccvvnn.... 15g Papaic digest of soybean meal .........covviiniiiininnnn.. 5g Solum CHIOEIAR i oo nniinei eosin nasiton dram 45 4 Sams 5g ARAL swims 653 SRETramEEE V8 30 SASTRY V5 3 SRE SISA 15 g NVBIRET vumarses So mmme ses 5% 20 RuEiinras SET OREEes by mea 1,000 ml Dissolve ingredients in water with frequent agitation and heat. Dispense as desired and autoclave at 121° C for 15 min. The final reaction should be pH 7.3=+. 13. Extract Gelatin®%*} Extract broth (CM No. 3) ..oviiiiiiiiiiiiiii iene 1,000 ml GEIL pei i samba $5.54 38 based 8.5 55 it wonemlilirns wo winrnBiile 120 g a. Soak gelatin in broth for 15 min before heating. Stir and gradually raise the temperature to 65° C to dissolve the gelatin. This procedure should be carried out in a double boiler to avoid burning the gelatin. b. Make up to original volume; if medium is not clear, heat to boiling point and filter while very hot. c. Dispense in tubes as desired and autoclave at 121° C for 15 min. Prolonged heating must be avoided or the power of the gelatin to solidify will be lost. The final reaction should be pH 6.8. + Dehydrated media are available as “nutrient gelatin.” 14. Milk with Indicator a. Add 1 ml of 1.6% alcoholic solution of bromcresol purple to 1,000 ml of skim milk. If desired, litmus may be substituted. b. Dispense in tubes and autoclave at 115° C (10 1b) for 15 min. OR* Slit. ITE POWABY . vik iouniit ae 20 iibiscnitinn & § 8:0 snl 5.2 ie win 100 g Bromcresol purplet (10 ml of 0.2% aqueous solution). .. 0.02 g WALEED icomeive i 45 5 Suuom ass ¢ § # SESH 35 § SARS S84 35 § 20 1,000 ml a. Dissolve powdered milk in water which has been preheated to 50° C and add the indicator. b. Dispense in tubes and autoclave at 115° C (10 Ib) for 15 min. The final reaction should be pH 6.8=+. + Litmus (0.75 g) may be substituted. 15. Semisolid Agar® Meat infusion broth (CM No. 5) ..oeviiviiiiininnnnnn. 1,000 ml BRIBE crainie » worpuaninerestieo® § Sg BioRad. 6 es oarnsd 57% A tysonens 1-15 ¢ CRICHEE wcocis ok anh wmuilnd £4 5 SABRE SR SRAIRE 002 5 2 smn 1 g CULTURE MEDIA 133 a. Dissolve agar and glucose in the infusion broth with frequent agitation and heat. b. Dispense in tubes or flasks as desired and autoclave at 121° C for 15 min. The final reaction should be pH 7.4=. For Neisseria meningitidis, add : Potassium chloride, BCL .::canisrsn sn vane smn vas spesnmes 02 g Calcium chloride, Cally svvsssicvmnsvnss swarms ssn 01g 16. Blood Agar Agar Dase ..uvser sir censr snes ssnsnpa sre yeaa wee ve 1,000 ml Detbrinated BIO ...vave ves vrssechbapass snersssmsvanssas 50-100 ml a. Melt sterile agar base and cool to 45° C. Add sterile, defibrinated blood, which has been preheated to 37°-45° C, in the amount desired. b. Dispense in tubes or plates and incubate at 35° C for 24 hr to check sterility. Surface bubbles in the medium can be removed by rapidly flaming the surface before the agar solidifies. However, adequate care in mixing the blood and agar medium will prevent the formation of bubbles. Choice of a blood agar base medium is predicated on the growth re- quirements of the organisms being studied. The medium should be isotonic for erythrocytes. The agar concentration (usually 1.5%) is so adjusted that the surface of the medium is of the desired soft- ness. Blood agar plates should be used soon after preparation in order to avoid loss of labile constituents and surface moisture. Base agar media used for isolation and for demonstration of hemolysis of beta-hemolytic streptococci should be free of reducing sugars, inas- much as these compounds interfere with hemolysis.1%52 Selection of species blood for blood agar is as important as choice of the base medium. Brown’s®® classic description of hemolytic reactions by streptococci in horse blood media has been valuable for classification of members, of this genus; however, subsequent studies have shown that sheep blood is the blood of choice for the selective isolation of ILancefield-groupable beta-hemolytic streptococci,38:54:55 and for Staphylococcus aureus.®®5" It must be kept in mind, how- ever, that sheep blood agar—while advantageously differentiating most nongroupable streptococci (by their inability to produce beta hemolysis on sheep blood) and eliminating troublesome Hemo philus hemolyticus (by its inability to grow on most media containing sheep blood)®® in pharyngeal cultures—also eliminates Hemophilus influenzae, which may be of medical importance. Rabbit and horse blood may allow better growth of many genera and widen the spectrum of bacteria producing beta hemolysis on 134 CULTURE MEDIA media incorporating these bloods; therefore, the purpose for which the medium is to be used must be considered when selecting the blood. The use of human blood or blood bank blood should be avoided because undesirable-antibodies, antibiotics or carbohydrates may be present. Many horse bloods have been reported to contain high titers of antistreptolysins. The concentration of blood should not be so great as to obscure reactions by weakly hemolytic organisms; likewise, the blood agar layer in plates should be of sufficient/depth for good growth but not_so thick as to obscure hemolytic reactions. A thin layer (4-5 ml) of base agar just covering the bottom surface of the plate effectively prevents spreaders from interfering with reading when blood agar pour plates are made for demonstrating subsurface hemolysis by streptococci. Sterile, defibrinated blood (sheep or horse) is collected by insert- ing a canula or needle, connected by rubber tubing to a stoppered flask containing glass beads, through the disinfected skin into the external jugular vein. A slight negative pressure in the collection flask facili- tates the flow of blood. The flask is shaken continuously as the blood flows over the glass beads, followed by an additional 5 min shaking period after all the blood has been collected. Rabbits and smaller animals are usually bled by syringe and needle from the heart and the blood is injected into a flask and shaken with glass beads. 17. Heated Blood Agar (Chocolate) Agar: Base our siisidilaahinsinss pelvatibu st {ove und sos 24 be 1,000 ml Defibrinated blood (see CM No. 16) .......coevrinnnnn.. 50-100 ml a. Melt sterile agar base. CM No. 12 is recommended, although CM No. 6 may be used for certain purposes. Cool to 50°-60° C. b. Add the sterile, defibrinated blood and heat at 75°-80° C just until the mixture turns chocolate brown. Keep well mixed during heating. c. Dispense as desired and incubate one or two representative con- tainers from each batch overnight at 35° C to check sterility. 18. Bordet-Gengou Agar, Modified??* Potato Infusion: Poeled, sliced DORIS , vuviitiunive soimnniis 2 + bnammds 500 g CHINEREDL. i, tiene oct wipitiphipmsean va bmsmpeonscinraim rae lene 40 ml WVILEE: oo nniivss in oh 50min s 5 Ann Esa R mnt § winery 1,000 ml a. Boil potatoes in the solution of glycerol and water until very soft. Strain through several layers of gauze and restore volume. CULTURE MEDIA 135 b. Pour infusion into tall cylinders and allow to stand until a clear supernate can be filtered off. If not used immediately, the infusion should be sterilized. Base Agar: Potato ITUSIOM. “Views sve saisinnibisiim sion aviies inn ssins 500 ml Sodium, SHIOTIE. oi vou rev rmmmmmnsins sriilumesbiavmsssm 11.25 g ANTAL |v cps mimarhms sim saa wb SAR ET Rs FS 50 g WALEE oso vets its sn esas s v5 nA Rn EP 1,500 ml a. For more luxuriant growth or for vaccine production, supple- ment with 1% of a peptone which cultural tests have shown will support the growth of Bordetella pertussis. b. Dissolve salt and thoroughly wet the agar with a portion of the water. Add the remainder of the water and dissolve the agar with heat. c. Add the potato infusion and dispense as desired. No adjustment of reaction is necessary. d. Autoclave at 121° C for 15 min. Complete Medium: BASE anaes ou immune vo 25 ram + 2 wel wiimmseis 5 miei 100 ml Defibrinated blood (see CM No. 16) ......cevvuvennnnn 15-20 ml a. Melt the base agar, cool to 45° C, and, for each 100 ml, add 15-20 ml of defibrinated sheep blood which is less than 72 hr old. b. Dispense the medium in plates or tubes and incubate overnight at 35° C to check sterility. Each lot of medium should be checked for growth-promoting properties, colony appearance, and characteris- tic hemolytic zone with a control culture of Bordetella pertussis. Satisfactory plates should have a moist surface and should be cherry red in color. 19. Thioglycolate Broth with Glucose,’ Modified*} Pancreatic digest of casein, USP. XVI ........c..... 15 g VCYRLINE sum cvs o rian 495 EaBRw TES SER i 05 g GUICOBE South. ads anton wos ss whines os vandals tas +4 5 g NCASE CULTARE re. 004% + woe SRSA GA Freire so on Reb wo 5 g Sodium ChIOTIAR: .. cvivninsinns restainsvnis SEES TS 555 25 g Sodium thioglycolate} ......ccvviiiniiiirinirnrnnenns 0.5 g Resazurin (1 ml of a 0.1% aqueous solution) ......... 0.001 g IATIE coiosiator aon 000 M3 BAT HR ak ig ease 46 ERE R wr0 075 g WVBLET «vom aan os sss onismp snes sss SREHs os ASSURE SER 00 a 1,000 ml a. Suspend ingredients in water and dissolve with heat. Boil for at least 1 min to insure solution of the agar. + Should conform to U. S. Pharmacopoeia specifications. i Or thioglycolic acid, 0.3 ml. 136 CULTURE MEDIA b. Dispense in tubes, filling them to a depth of at least 10 cm, autoclave at 121° C for 15 min, and store in the dark at room temperature. Do not refrigerate. The final reaction should be pH 7.1, c. If oxidation occurs, as will be indicated by the pink color of the resazurin, the tubes can be reheated once in flowing steam or in a boiling water bath to lower the oxidation-reduction potential. 20. Thioglycolate Broth without Glucose or Indicator* Several modifications of CM No. 19 are commercially available from which glucose, resazurin and, in some cases, yeast extract have been omitted. If the medium is to be used for fermentation tests, formulas without yeast extract are recommended. Fermentation reactions are determined by adding the test carbohydrate in a final concentration of 0.5 to 1.0%, checking the sterility, and inoculating with the test organism. Acid production as an index of fermenta- tion is detected by pipetting small portions of the culture from time to time to small volumes of 0.05% aqueous bromthymol blue in a spot plate. For use as a maintenance medium for stock cultures, add a small amount (0.1 g) of CaCO; to each tube prior to addition of the medium and sterilization. 21. Brain-Heart Infusion Broth, Dehydrated* Call Drang «ass sivsvasasmvvsnoni viene infusion from 200 g Beet heart «vvonmnsss vrennn svn sammie infusion from 250 g Depiohie COMPONBILE » vorvwe immed s summits ansne sins 10 g GIIBORE: .vviiiiomishivin ment nse lpr a rae TEa Th a8 2-2 Sodium CHIOTIAR wissvinscesiorvesigs babes wm sr 5 g Disodium phosphate, Na, HPO, ....:u0rmeansivassmmnnes 25g WERE oruneesvsmmnmismss Samm tet sR CEI r ee ame wes 1,000 ml a. Obtain in dehydrated form and rehydrate as per instructions on the container. b. Dispense as desired and autoclave at 121° C for 15 min. Final pH, 74+. 22. Brain-Heart Infusion Agar, Dehydrated* Calf Drains. wows se somo dss 2 Sranmes dt vi asan infusion from 200 g BEBE BATE voor wn wowing 3% woman d sows o shies infusion from 250 g Pepiont COMPOTBAL. simmnisinm sss wines wrasse sis s Wem 10 ¢g GEIUICOBE |, cuiminns 55 5 60 mum ann 5% thin (oo © adrinsmonst + 35ers 2 g Sodium: CHlorlde . «3 sommuninss snes s pe swbiwmming ie o si 5 g Disodium phosphate, Na,HPO, ......ovvvnvnvnnnnnnnn. 25g BIIE wis mnie anisms 3 Sapo $475 GORI Ak ¥ 8 0 15 ¢ CULTURE MEDIA 137 a. Obtain in dehydrated form and rehydrate as per instructions on the container. b. Dispense as desired and autoclave at 121° C for 15 min. Final pH, 74+. For Isolation of Clostridium botulinum: c. Plates should be dried, preferably for about 30 min at 35° C before use. Plates which have been poured and kept in the re- frigerator longer than 4 days are likely to be unsatisfactory. 23. Beef Heart Infusion for Streptococcal Media®? a. Trim fat and fascia from fresh beef heart and pass heart muscle through a meat grinder. b. Add 1,050 ml distilled water to each pound (453.6 g) of ground meat and stir. Skim off solid fat that rises to surface and place infusion in the refrigerator overnight. c. Bring slowly (3 or 4 hr) to 85° C and keep at that temperature for 20 min with frequent stirring. Skim off fat and strain through gauze in a colander; express juice from the meat. Filter through paper. 24. Todd-Hewitt Broth, Modified®4* Beef heart infusion (CM No. 23) .c.comiimiarsaomnraneis 1,000 ml NCODEDIONE «oun vos sama wie ede spas ERR ER 20 g Adjust reaction to pH 7.0 with N NaOH and add: Sodiuttl CHIOTIQE. cms +1 3500 sme + 3 « taswimariin seo wnmmmmm ese os we 2 gz Sodium bicarbonate, NaHCO, «.c:.ivaimsmsssnrsnisminsonrns 2 g Disodium phosphate, NaHPO, «oi conmmusris ssvmmmons ssoas 04 g GUISORE 4541 svammnrons » © 1 woman » $3 SERRE T ¥ NEWS prs 559 2 gz After adding chemicals, bring slowly to a boil. Boil for 15 min and filter through paper. Dispense as desired. Sterilization may be accomplished by filtration, flowing steam (Arnold sterilizer) for 1 hr on each of three successive days, or by autoclaving separated tubes at 115° C (10 1b) for 10 min. Final reaction should be pH 7.8. 25. Todd-Hewitt Agar with Sheep Blood, Modified? Todd-Hewitt broth, modified (CM No. 24)* .............. 100 ml BNO 0 003 oainiiatiss 33 5 RAY A $2 BATHS v0 chia meinen: iwi iwsnimae 15¢g a. Heat to dissolve agar and dispense in flasks. Autoclave at 121° C for 20 min. 138 CULTURE MEDIA b. Cool to 45° C and add 4 ml of sterile defibrinated sheep blood, mixing blood evenly into the medium. Pour approximately 20 ml per plate into sterile petri dishes having covers lined with filter paper. When solidified, seal dishes with parafilm. 26. Tryptose Extract Agar with Sheep Blood* PLYDIOBE viii re anitn n neni vrs Sree sw eR EE 10 g Beet extract oun tio bo duiiliiive vibe ainsi wns weiniiettet mime 3g Soin. cHIOEAR «usw veo Gomme denims pwsiiedniornd omm sin 5g AGEY sitters sitio midis SRI as 4 SE TR Sed oe 17 g GL Ty 1,000 ml a. Dissolve ingredients with heat, dispense as desired, and autoclave at 121° C for 15 min. The final reaction should be pH 7.2%. b. Cool to 45° C and add 4 ml of sterile, defibrinated sheep blood per 100 ml of medium. Pour plates with 20 ml of medium per plate and seal with parafilm strips if desired. 27. Tryptose Infusion Agar with Sheep Blood? Beef heart infusion (CM No. 23) ...cvviiiiiiiiinnnnennn. 1,000 ml TIYDIOSE. cei ios simaiiis x® ¢ Rivioioinins i x % » BSI RIRATTES + § SRBRBE LEE oF 10 g Sodium chlorkde « cauives + 3+ maining + « sh onmmis s +3 sown 3 5¢g AGA sins 23 $3000mEs £ 1 5 STARHINGS § ¢ 5 FRSARE $4 CRO, 17 g a. Add ingredients to beef heart infusion and heat to dissolve. Adjust reaction to pH 7.6-7.8 with N NaOH if necessary, dispense as desired, and autoclave at 121° C for 15 min. b. Cool to 45° C, add 4 ml sterile, defibrinated sheep blood per 100 ml of medium, and pour plates. When the agar hardens, invert plates and prop bottoms on lids for 30 min or longer to allow drying of surface. Store and incubate unsealed. 28. Thallous Acetate-Tetrazolium-Glucose Agar, Barnes® Pantone (CEVIHE)T + suvintunies somnmvaivssdiiivssnipmes son 10 ¢g ETT ER LN SL re, JO 10 g GIUCOBE" + i's bitint wav anit nes sasmasie vas shes sue ELE Seas 10 ¢g 2, 3, 5-triphenyltetrazolium chloride .. 0lg THalGUS BORIBIR ous sn biomass sole vane nova unions soe 1 g ABET aise sei aint ea Rae Wes I So a 14 g WVBLET wiih ssmsmiin s3asimesd ans sare sdviot so earanmes ioe 1,000 ml a. Dissolve peptone and Lab-Lemco in water and adjust pH to 6.0. Add agar and dissolve with heat, then autoclave at 121° C for 15 min, t1% tryptone (Difco) and 0.5% yeast extract may be substituted for the Evans peptone and Lab-Lemco, respectively.68 CULTURE MEDIA 139 b. Add sterile stock solutions of glucose, tetrazolium chloride and thallous acetate to the cooled basal medium to yield the appropriate concentrations. 29. Beef Heart Infusion Broth? with Blood Beef heart infusion (CM NO. 23) .vvvveiinieriererenenns 250 ml NEODEDIONE + co cvivmmesn ss vrmmnioinns + 45 pusans 4.65 23ABFERELS 5 g Sodium CHIOTIAE cen cas is enmmmmnns o 2s anammas ++ 5 oanwmes o 125 g a. Mix ingredients and boil to dissolve. Adjust reaction to pH 7.8 with N NaOH. Filter through paper until clear. b. Dispense in 5 ml amounts to tubes and autoclave at 121° C for 20 min. c. Add 1 drop of defibrinated sheep, horse or rabbit blood, as indicated, to each tube of the cool medium. 30. Pike's Sodium Azide-Crystal Violet Enrichment Broth,%” Modified®? Beef heart infusion (CM No. 23) :.esvesvmnnes connsnnss ee 100 ml TIVDIOSE + .onienivon sos vos smienni nes se sws sie ses sy vanesiee 1 = Glucose (1 ml of 2% aqueous solution) ........eeveuu.n.. 0.02 g a. Adjust reaction to pH 7.6-7.8 with N NaOH and sterilize at 121° C for 15 min. When cool, add 5 ml of sterile, defibrinated rabbit blood and dispense to tubes in 2 ml amounts. b. On the day of use add to each tube the following aqueous solu- tions, previously sterilized by autoclaving : Sodium azide, 0.195 ..cvversarssrssnnssennensnnenssssioseie 0.15 ml Crystal violet, 0.004% «ovensivs sc anamainsa iss oaniihine + 5 wo aam 0.10 ml 31. Blood Digest Enrichment, Fildes®®* Sodium chloride, 0.85% Solution ...cvveviinrnereenennnnnnns 150 ml Hydrochloric acid, concentrated .......ccovviiieveiinennnns 6 ml Defibrinated blood (see CM No. 16) .....c.covvivevnvnnnnn. 50 ml Granulated. PEPIN. vvvmvissnsimnmyses sr sor rms vs sessed: lg Sodium hydroxide, 20% SOIUtiON ....cvevreveerneensnenennns 12 ml a. Add salt solution, HCI, defibrinated blood and pepsin to a sterile 250 ml glass-stoppered bottle and shake well. b. Place mixture in water bath at 55° C until it is digested (between 2 and 24 hr). c. Add 12 ml NaOH to the digested blood and adjust final reac- tion to pH 7.0-7.2. d. Add chloroform to 0.25% concentration and store in the re- frigerator. 140 CULTURE MEDIA 32. Yeast Extract, Thjotta and Avery® Brewer's yeast, fresh yeast, or baker's yeastf ............... 100 g WBLEE «vs sinnembumn ss sramnman sss SrEame sens sowsmmmns es saws 400 ml a. Adjust reaction to pH 4.6 and boil over a free flame for about 10 min. b. Allow yeast to settle at room temperature and withdraw super- nate aseptically. c. Store in screw-cap bottles under refrigeration. T Use yeast free from starch and fillers. 33. Levinthal's Broth,’ Modified™ Broth Base: Meat infusion broth (CM No. 3) oui eevessummnonns 1,000 ml Defibrinated blood (see CM No. 16) .......cveenvuennn. 100 ml a. Adjust reaction of broth to pH 7.5. b. Add defibrinated sheep, rabbit or horse blood and heat rapidly to boiling, then remove from flame. c. Filter through paper to remove coagulum. d. Filter through a bacteria-retaining filter for sterilization and store at 4°-6° C. Broth Medium for Hemophilus influenzae: Broth DATE .wsivcens srsmmenmesy 0 semmameslnee » sob amie £5 1 part e. Add sterile broth base to sterile meat infusion in desired volumes. 34. Levinthal's Agar,” Modified Agar Base: Meat infusion broth (CM No. 5) ....ovvvvvnnnnennn.. 1,000 ml BIGOT 0 rv I ETRE Sok AeA S00 FF aR A Fe TAS 30 g a. Dissolve agar in the meat infusion broth with frequent agita- tion and heat. b. Dispense as desired and autoclave at 121° C for 15 min. Final reaction should be pH 7.5=+. Complete Medium: c. Melt the desired volume of meat infusion agar base, cool to approximately 60° C, add an equal volume of the modified Levinthal broth base (CM No. 33), and dispense in plates. CULTURE MEDIA 141 35. Tryptose Phosphate Semisolid Agart Tryptose or equivalent ..vessnemrmmesiruissnusEess wees 20 g GIIICOBE «oo vmvimais vmises vas vam Faas a kes So Fina i SRE ol 2 Z Sorry CHIOEIAR + .conmns sum nmes brandon sn ho mba Fa an aech an 5 g Disodium phosphate, NagHPOy v.uvvvvviiiiiniennnnnn.. 25g ITAL ootssitininmmmins mmaimnioimi akin anmon pra ssesmnse im secu cms mia asse 1-2 g WHEEL reins minimsiminsmmacmmomatie vera wines ia ce onrmsmonsiwra gm sen 1,000 ml a. Dissolve all ingredients in the water. The solution must be raised to the boiling point for satisfactory solution of the agar. b. Dispense in tubes or flasks as desired and autoclave at 121° C for 15 min. Final pH should be 7.3. 1 Commercially available without agar as tryptose phosphate broth. 36. Tryptose Dextrose Vitamin B Broth* Tryptose or eatiVAIBAL . cos inniis sins simsiie srs same 20 g GIICOBE: cov oi mmmsn shi hE Ribsns: Sikhs vaca arwsbsnsssklos achiais Wiansme 1 g SOIL CHOBE vv sors 0 wim niiw animes w wm wiwimceirmain scm mssmonn 5 g Thiamine hydrochloride .........ovviiieeineennnennnns 0.005 g IWVITHBE werner wn wcusmommatommiinnnss wonon reowinmemessaca awn ress seme ww sesmcpsezans 1,000 ml a. Dissolve all ingredients in water. b. Dispense in tubes or flasks as desired and autoclave at 121° C for 15 min. The final reaction should be pH 7.2. 37. Tryptose Dextrose Vitamin B Agar* Tryptose dextrose vitamin B broth (CM No. 36) ......... 1,000 ml ALOE oir iinit ws a WBE Eitri. iw SRI Ret ar ncn nmmari nck 8 mim chetmi mnie 15g a. Dissolve the agar in broth with heat. b. Dispense in flasks or tubes and autoclave at 121° C for 15 min. The final reaction should be pH 7.2%. 38. Lead Acetate Agar* Proteose pepione or ThiOtONe .....ovivvvivevevvessersns 5 ¢g Bacto pepfone or equivalent ....osvssssssvsnn srsrsinene 15 ¢ GIUCOSE sun esis ssummms is » Srvsanimues soesnmvnss Porn inns 1 g Lead inceinte, CPy CHPHO, cies sovimuions ns sansms 02 ¢g Sodium thiosulfate; Nag8,0p suwwvivs sve cimvinme vis ania 0.08 g ATUL ivinsn 090 4 SHRARRIL 85 2H ERASE + 33TH £855, wins Fim 15 g WWVBLEE icin » 4 3 5 Renitrtitnh.s 33 SUREINAEE E65 Bailie: os 2a wmsmmatn 1,000 ml a. Dissolve ingredients in water and tube in 7 ml amounts so as to give both a slant and a deep butt for stab inoculation. 142 CULTURE MEDIA b. Sterilize in autoclave at 121° C for 15 min, remove, and cool in a slanted position. The final reaction should be pH 6.6. 39. Yeast Autolysate Broth for Brucella* Peptone “MM” (AIM) .cvsesssrssvesmaves sasvsenssvess 20 g Yeast AUIGITERLE ov vevrrvrunenssswimene nen vsrmsse es 2 g CC ICTEE Lorine roetnsisiatersraneorasmusssianatate terse tarsnensns ero rasaimsasspssotm dard 1 g Sodinm CHIOTIE) «cvs sinssninmmssbsivnmensd S300 s SAFREEH 45 3 .%g Sodium bisulfite, NaHS0, «evi cv vrsssnmmvennsersvason ve 01g WHEE. cvevvpriiniivoltvmmdnnior se nuomreris sitet ak 1,000 ml a. Dissolve ingredients in water, dispense as desired, and auto- clave at 121° C for 20 min. The final reaction should be pH 7.0=. b. For Vibrio fetus, first add 0.1% agar, then proceed as above. 40. Yeast Autolysate Agar for Brucella* Brucella broth (CM No. 39) ..iiiiiiiiiiiiiiiineennnnnns 1,000 ml AEOT . acicviv 3 0a RS AA tina RRA Roh A TR RH SARA 15¢ a. Dissolve agar in broth with frequent agitation and heat. b. Dispense in flasks or tubes as desired and autoclave at 121° C for 20 min. The final reaction should be pH 7.0. 41. GC Medium Base Agar with Hemoglobin for Gonococcus™ GC Medium Base Agar :* Proteose peptone No. 3 or equivalent .......c.cvvunuens 15¢g Sodium Chloride . .:osnsmmsnss ss smnyesssssansins piss 5¢ Dipotassium: phosphate, HPO, ue rdissivonmmessvees 4g Monopotassium phosphate, KHo,POy ..vvvvvniivininnnn. lg COTTIATTN acini ov sin simmons ch nH Rimimctrminst, Bin men Be Abd Bib Aine lg 0g a. Suspend dry ingredients in the cold water and heat gently to the boiling point to dissolve the agar. b. Dispense 100 ml amounts and autoclave at 121° C for 15 min. The final reaction of the base agar should be pH 7.2. Hemoglobin Solution: Hemoglobin® i sss weno ns tsa sanmne iiss Samson Lae 10 g WVALEE ooismervnivies s mammniaieiem © 5 SHsmmehn 0 sosRmevia ees mk 500 ml a. Dissolve hemoglobin completely in cool water with frequent agitation and stirring. This may take 15-20 min. b. After solution of the hemoglobin is accomplished, dispense in 100 ml amounts. Autoclave at 121° C for 15 min. CULTURE MEDIA 143 Complete Medium :t DATE HOE 1 oa ars SA TIE Sw a A AA ST ara 100 ml Hemoglobin. Solution cos vreenssnvssessvrsnsmbaiioses 100 ml Supplement A. or supplement BX ...covesisivssssssmava 2 ml a. Melt agar base and cool to 50°-60° C. Warm hemoglobin solu- tion to the same temperature. b. Mix agar base and hemoglobin solution, then add the supple- ment. Mix thoroughly and pour plates. + This medium when completed has an agar concentration of only 1%. This will require extreme care in handling and inoculating the plates, because the surface will be very moist and soft. Supplement A is a fortified yeast con- centrate prepared to conserve both thermolabile and thermostable accessory growth factors, to which has been added crystal violet 1:6,000. Supplement B is the fortified concentrate without crystal violet. 42. Starch Agar for Gonococcus and Meningococcus, Mueller and Hinton3¥ A. Starch Paste: (1) Suspend 1.5 g of ordinary starch (cornstarch or laundry starch, not “soluble starch”) in 10 ml of cold water; (2) pour slowly into 90 ml of boiling water while stirring. B. Agar Solution: Dissolve 17 g of agar in 500 ml of water and autoclave for 15 min at 121° C. C. Base Medium: Meat infusion, double strength ..................... 300 ml Casamino acids or equivalent ...........ovvenenennnns 175 g Starch paste CA) iii. isnmnivees svnvnssinss Uasmvrne 100 ml WHIBE susie 009 soisonniistes ais smiinimns £s S/R sin we uhm 100 ml a. Add starch paste (A) to the meat infusion-casein hydrolysate solution while the agar is being autoclaved. Adjust the reaction to pH 7.4-7.6. b. While the sterile agar (B) is still hot, add the base medium (C) and mix. c. Dispense in test tubes (20 ml each for pours, 5 ml for slants) or flasks of 120-200 ml and autoclave not longer than 10 min at 115° C (10 1b). The final reaction should be pH 7.4. T Double-strength meat infusion is prepared as described in CM No. 23, except that 1 kg of ground meat is infused with 1,000 ml of water. 43. McLeod's Agar with Plasma and Hemoglobin™75 Base Agar: Fresh, ground beef heart muscle ..................... 600 g Proteose peptone No. 3 or equivalent ................ 10 g Disodini phosphate, Na HPO, eos vis svmmusoss i save 2g BUGHE Gare se a nnsaitsds Saami gas be SENTRA Ew PETIA 20 g WHEE count os ovnitonsinn 4 S Anime sss o Sanmmiame + § ¢ ssivenss 1,000 ml 144 CULTURE MEDIA a. Add peptone, ground heart muscle, and disodium phosphate to the water and bring to a boil. The infusion mixture should be stirred constantly to prevent burning unless the heating is done in a double boiler. b. Cool to 60° C and continue the extraction for 45 min, stirring mixture frequently. c. Place mixture in flowing steam so that temperature of the medium will be between 80° and 90° C. Continue the extraction for 30 min. d. Cool to 45° C and filter through two layers of cheesecloth, expressing all the liquid. Then filter through paper to clear. e. Restore to original volume, dissolve agar with heat, dispense in flasks as desired, and autoclave at 115° C for 10 min. Store in the refrigerator. The final reaction should be pH 7.4. Enrichment Mixture: Sterile horse DIAM. vouivvvas sss memmnns ss ss sgmasonsss ss 300 ml Hemoglobin solution} ......cceeeerriereneeeeeenenaenn 500 ml Glucose (10 ml of 20% aqueous solution) .............. 2g Nile blue A, 74% dye content, 0.04% aqueous solution... 24 ml Disodium phosphate, Na,HPO,.12H,0, 15% aqueous BOITEION ions « 3 5 vn nmmninivi » + § wo Wem + 3 3 3 SASHES 04 3 5.2590 100 ml f. Combine the ingredients (sterilized separately) in the order given and adjust reaction to pH 7.4, using the 15% Na,HPO, solution. Store mixture in the refrigerator for not longer than 8 weeks. The 15% disodium phosphate can be conveniently sterilized by autoclaving at 121° C for 15 min in amounts of 50 ml in 4 oz bottles. The Nile blue A can also be heat-sterilized, while the glucose solution should be filter-sterilized. Complete Medium: g. Melt base agar and cool to 60° C. For each 100 ml of melted base agar add 31.1 ml of the sterile stock enrichment solution. Mix thoroughly and pour plates. The final reaction should be pH 7.4. + Proportions: 25 ml settled horse red cells and 475 ml of water. 44, Peptone Agar with Plasma and Hemoglobin Base Agar: Proteose peptone No. 3 or equivalent ........ccouvunn. 20 g Sodium CHIOTAE . ovvmvms in ons srmmme nn s 2 woeaisions s « o8 Sg BGAT comnmis 00 20S RaT ens £35 AE 24 SUES 38 Spe 20 g CULTURE MEDIA 145 a. Suspend ingredients in water and dissolve the agar with heat and frequent agitation. No adjustment of reaction is necessary. b. Dispense in flasks in suitable amounts and autoclave at 121° C for 20 min. Enrichment Mixture: Horse plasm moves snysmewnse ss s semis oa oaaneasd ss 260 ml Horse hemoglobin, 0.5% solution} ..................... 430 ml Disodium phosphate, Na,HPO,.12 H,0 (15% aqueous SOMEONE) wii 4 + 3 samieliiuins 55 FREE 4 6 § $5 RAINES + § re 50 ml Glucose (10 ml of 20% aqueous solution) .......... ... 2g Nile blue A, 74% dye content, 0.04% aqueous solution ... 9 ml c. Combine ingredients, sterilized separately, and store in the refrigerator. The 15% disodium phosphate can be conveniently sterilized by autoclaving at 121° C for 15 min in amounts of 50 ml in 4 oz bottles. The Nile blue A can also be heat-sterilized, while the glucose solution should be filter-sterilized. Complete Medium: d. Melt the base agar and cool to 60° C. For each 100 ml of the agar base, add 50 ml of the enrichment mixture. Mix thoroughly with a swirling motion to prevent bubble formation and pour plates. The final reaction should be pH 7.4. 1 Proportions: 5 ml settled horse red blood cells and 995 ml water. 45. Gelatin-Blood Agar™ Proteose peptone No. 3 or equivalent ...........ouun.n.. 20 ¢g Sodintny chloTIde « : 3 mwaimss £4555 BEBat © 5 15 Sukwers § + awkiv 5 g Disodium phosphate, NagHPO, «vvueeermnvrninses canine 5 g GUICOBE “ain 5 1 1s sommmin: pi w 4 ewaipuonsin 49's 3 2imminns 4 4 § 3 Rhy 05g GEIB : vcore 0 4 Buncmievesn « 8 + § 35 ioe 0:3 + 3 FRIEIGEIE % + 3 on{sFwele 40 g BBE | iociininn wt $AWHES 3.3 8 SERRA ASF 4 8 BEETS + 3 » Wiwinioim 20 ¢g WBLEE win 5 5.0 vuummusn #4 3 sinensis £ ¢ 3 bawimnmisie 5 5 »R@ismE LE 1,000 ml Blood, sterile, defibrinated (see CM No. 16) ............. 250 ml Nile blue A, 74% dye content, 0.04% sterile aqueous solu- FIOM wos 23 sommes v5 oramsmen +» anamamasis + § 4 sme sus 7.5 ml Base Agar: a. Suspend the gelatin, salts, peptone and glucose in half the water and soak for 20 min so that the gelatin is thoroughly wetted. b. Dissolve the agar in the remainder of the water. c. Combine the two solutions and make up weight to 1 kg. Adjust the reaction to pH 7.4-7.6. d. Dispense in flasks in definite volumes and autoclave at 121° C for 15 min. Store in the refrigerator if not to be used immediately. 146 CULTURE MEDIA Complete Medium: e. Melt the agar base. For each 1,000 ml add 250 ml of blood, 7.5 ml of Nile blue A solution, and mix thoroughly. The agar base should be at 85°-90° C when the additions are made. f. Dispense rapidly, as the mixture is thick and hardens quickly; 6 ml volumes of the medium can be dispensed either in 16X133 mm culture tubes stoppered with paraffined corks, or in suitable screw- cap vials having phenol-formalin-free plastic caps. The final reac- tion should be pH 7.4==. Keep tightly closed prior to use. 46. Gelatin-Egg Albumin Agar™ GIVERIOL: . .unmniininn « sianniin' sds oss HURAREES oo 0 AR etiS Hews mere 4 ml Eg albumin, Tresh ...vepuivns dp sememhive's © s 4 5 smuanias s +5 15 ml OX SETUMT .vvwvns « 2 sniminims was » « vTmmeiees £4 PEPPESEEE © § 8 58 3 ml GEISER 37, dhntatiins vs yeionmitnes 35 Siummion ¢ x » www xis veh 10 ¢ BEE om 4 53 Fommionniun § 2 Wio Biwleloiol § Bie 3 ipaerhie rash Sle 0b Mdent le olen 04 g WHEE is svnninmenion ss smmmaninios 1 5 Ssmans + # + LAVAS. 43 £45 100 ml a. Soak agar and gelatin in cold water for 20 min. Dissolve with heat and frequent agitation, taking care not to burn the gelatin. Add the glycerol, with thorough mixing. b. Adjust the reaction to pH 6.5 with 1% potassium dihydrogen phosphate (approximately 30 ml) and autoclave at 121° C for 12 min. c. Cool in a water bath at 45° C; add the albumin and serum supple- ments. The temperature must not exceed 45° C after these supple- ments have been added. Preparation of Serum: Sterilize serum by Seitz filtration. The filter must be preliminarily corrected by filtering 50 ml of 1% KH,PO,. After filtration, the serum is transferred to sterile serum bottles, inactivated on two successive days at 56° C for 30 min, and then stored at 4° C until used. Preparation of Egg Albumin: Fresh eggs are scrubbed with “Emulsept” and soaked for 1 hr in a solution of “Roccal.” Other wetting agents may be used if satis- factory disinfection of the shell can be demonstrated. After disinfec- tion of the egg is completed, the larger (butt) end of the egg is opened, using sterile technics, the albumin removed with a pipette or syringe and transferred to sterile bottles. Care must be taken to + Instead of ox serum, 0.25 ml of supplement A or B (Bacto) may be used. CULTURE MEDIA 147 avoid rupture of the egg yolk sac, as any admixture of yolk makes the albumin unsuitable for use. Egg albumin must be used fresh, as it deteriorates rapidly under storage. Complete Medium: After addition of the supplements, 3 ml of the finished medium is rapidly dispensed to vials closed with phenol-formalin-free plastic screw caps which have a well to hold a cotton swab applicator. The applicator should be long enough to reach the bottom of the vial and, in use, it is virtually embedded in the medium. The vials are filled with CO,, tightly closed to prevent loss of the added CO. by diffusion, and stored at room temperature for use within 8 days of preparation. The vials must be kept upright at the time of use to prevent excessive loss of COs. 47. Tryptic Digest Broth, Douglas™ a. Mix 150 g of minced lean beef or horse muscle with 250 ml of water. b. Heat to 80° C and add 250 ml of 0.8% solution of anhydrous calcium carbonate. ¢c. Cool to 45° C, add 5 ml of Cole and Onslow’s pancreatic ex- tract and 5 ml of chloroform. Pancreatic Extract, Cole and Onslow: Fresh, fat-free pig pancreas, minced ................. 500 g WARE ..oivoiiins art ein sus SRuss Rrasbame Re sie ssse same 1,500 ml FL ry TY 500 ml 1) Mix pancreas, water and alcohol throughly, place mixture in a large stoppered bottle, and allow it to stand for 3 days at room temperature. Shake repeatedly. 2) Strain the extract through gauze and filter through paper. 3) Add 0.1% of concentrated HCl. A precipitate will be formed which soon settles out. 4) Filter the extract and store in a stoppered bottle at 4° C. d. Incubate the meat-pancreatic extract mixture at 35° C for 6 hr, stirring frequently. e. Add 40 ml of N HCl and boil the mixture for 1 hr. f. Cool and filter through paper. g. Adjust the reaction to pH 7.6-7.9, boil for 1 hr, and filter through paper after cooling to 45° C. h. Autoclave at 121° C for 15 min. 48. Tryptic Digest Agar, Douglas Douglas Broth (CM Noi 47) +uvvvcvsimmuons snvmpsminse 1,000 ml BIT onssmmmrm pes ees emir frie eS SAE 20 g 148 CULTURE MEDIA a. Combine agar and broth, and heat to boiling to dissolve agar. b. Filter while hot through absorbent cotton, if necessary, and dispense in tubes or flasks. Autoclave at 121° C for 15 min. 49. Douglas’ Agar with Ascitic Fluid and Carbohydrates®® a. Melt 100 ml of sterile Douglas’ agar (CM No. 48), pH 7.4-7.8, in a flask and cool to 48°-50° C. b. Add 20 ml of sterile, sugar-antibiotic-bile-free ascitic fluid, pH 7.2 to 8.0; 5 ml of a sterile 20% aqueous solution of the carbohy- drate; and 1 ml of a sterile solution of Andrade’s indicator. Andrade’s Indicator :81 WWOLEE areas 25:00 Huimin +a 8/00 ealSTthles $a Tosaimssoraiesmnt i sit rome 100 ml Sodium hydroxide, N/1 solution ............cocvuenn. 16 ml Acid Fuchsia} ...ovivvvssnrsnosee svssvsessorssvsves 01-05 g Each lot of acid fuchsin must be standardized. Make up a small amount of indicator, using 0.1-0.5 g of acid fuchsin per 100 ml. Incorporate this indi- cator in a small amount of medium in which it is to be employed and test with appropriate cultures. c. Dispense into tubes, replace cotton plugs with rubber stoppers, and allow to harden in a slanted position. d. Check sterility by incubating at 35° C for 24 hr. t Acid fuchsin (certified), Allied Chemical and Dye Corp. The concentra- tion of acid fuchsin to be used and instructions for preparation of Andrade’s indicator appear on the label. 50. Transport Agar Medium for Gonococcus, Peizer, Steffen, Klein? Agar Base: Bacto Peptone or equivalent ..«: vevswes sx sssmmas sas 5s ¢ 10 g Cornstaroh sume » vo vesmmmnns so vamos » 3 pwimaswre ss 53 5g Sodium chloride ....vvsvvriermvermrrrevcnersmeenses en 3g ATHY © conrnmr os vs REE SR RTE $F Fwcnswrtoeninern wie 10 g WOLEE cvisnivss sonmmmum eis 55 50s ine ows £088 SERSRAES 5 1,000 ml a. Add ingredients to water, stir well, and heat in an Arnold sterilizer or flowing steam to dissolve the agar. Distribute in con- venient amounts and autoclave at 121° C for 15 min. b. To each 30 ml of the melted, cooled agar base add the following sterile solutions in the order listed : Disodium phosphate, Na,HPO,.12 H,0O, 15% aqueous SOUMION nme vu snmp rs sms +s SERVEITOTE 5 € § SESE 2.1 ml Cysteine monohydrochloride 10% aqueous solution ........... 0.2 ml Calcium chloride, CaCl,, 1% aqueous solution ............... 1 ml Bovine albumin, Armour’s Fraction V, 20% in saline solution 5 ml Crystal violet, 0.08% aqueous solution ...................... 1 ml CULTURE MEDIA 149 c. Tube in 2 ml amounts in sterile vials having an inside diameter of approximately 12 mm. After the medium hardens, replace cotton plugs with sterile rubber stoppers. 51. Transport Agar Medium for Gonococcus, Stuart, ef al.83* REEL ERT 100 + 5 Sree we» » Wlashincurmivnyis ou ooimsentiiaied’l Fuss winivarers 6g Thioglyeolic SCT cunmens +5 35 srummn iss se ameminsis ve shmmie 2 ml Sodium glycerophosphate, 20% w/v in water .............. 100 ml Calcium chloride, CaCl, 1% aqueous solution ............ 20 ml Methylene blue, 0.1% aqueous solution ............ocueu... 4 ml VALET svnuiiinm iss 0amiie ve 4 5n 8 wirbross sia: 3 4 4respoensare: + § ¢ § sovsresssne 1,900 ml a. Mix agar with 1,900 ml of sterile distilled water and dissolve with heat. b. Add thioglycolic acid (or sodium thioglycolate) and adjust the reaction to pH 7.2 with N NaOH. c. Add sodium glycerophosphate and calcium chloride solutions and again adjust reaction to pH 7.4. d. Add methylene blue and dispense in small screw-cap vials, filling vial almost to the top. Heat at 85° C for 1 hr, afterward screwing caps down tightly. The final reaction should be pH 7.3. 1 Sodium thioglycolate, 3.3 g, may be substituted for thioglycolic acid. 52. Kligler's Iron Agar84¥ Originally, Kligler included lead acetate in the double sugar medium of Russell® to detect the formation of HoS. This medium has been modified by incorporating more sensitive indicators of HsS production and acidity as in Modifications 1, 2 and 3. Peptone component ........oeeirieeiiinininnenannnnn 20 g LBEOEE viviicsn wammesininismnton Solid Homi snrs Smit Peers hes. iim 10 g GIICOSE Livers sraanmanad sos Tabs mas pane smmen sss HERES 1 g Seditim. 'ChIorlde .voevdusaivers srr ormyprsasess eres 5 g Phenol red (10 ml of a 0.25% aqueous solution) ...... 0.025 g DUBE © iin di ihi nd mn wm PAR wR memset We eth hres oe 12-15 g WBE © covnivss srs sone an Bs seus aa eas sas SAATATS 230 1,000 mi Modification No. 13%: Add Beef extract ....ovveviiiniinenieeennnineisennenennes 3 g YRUSE GRLIBEE iis ovis sna lilnimiison ie Emons Saisie sscsns itis 3 g Ferrous sulfafe ..usevsvsmnvsnmasnsonsoress sess LE 02. g Sodium thiosulfate 03 =g Or, Modification No. 234; Add Ferric ammonium, CHIBI vevsvi svt toseminns wasn sn sis 03+ .g Sodium thiosulfate ..usmvmmaerismnrrnssssns rims 05 . ¢ Or. Modification No. 3, Hardy86: Add Modification No. 1 or No. 2, plus 1% sucrose. 150 CULTURE MEDIA a. Dissolve all ingredients in water, with frequent agitation and heat. b. Dispense in tubes and autoclave at 121° C for 15 min. The final reaction should be pH 7.4=. c. Cool in a slanted position so that the agar will form a deep butt. Tighten caps on screw-cap tubes during storage, or otherwise prevent dehydration. If screw caps are used, be sure they are loose after inoculating the medium; otherwise aberrant reactions due to the accumulation of CO. may occur. 53a. Triple Sugar Iron Agar, Modified?** Hajna®" modified the medium of Krumwiede®® by including an indicator of H,S production and substituting phenol red for Andrade’s indicator. POlypeptonie o.oo: srsnimyvss vs sibs way i vo Ean aabnss »8 uri 20 g Sodium. ehlotide . coon sis samme Rk mnie Be SE 5 g NACITEE Cs tterrros salsosssmtimn so sale soskumesm vastaia monsoons: i048 rane 10 g SUCIONE. monte s va onilmnes ves reasmnme os yews Cos EFEe 10 g GIUCOBE +.ovivsisvnonsns 155s os semaines s Ses awans si sesve 1 g Ferrous ammonium sulfate ........coiiiiiiiiiinennnnn 02 .g Sodium thiosulfate .........c.oiiitiiriiiiiiinneennnn. 02 g Phenol red (10 ml of a 0.25% aqueous solution) ...... 0.025 g ABUL wtnnato +4 # rnEEIT S32 BARBED ES 5 3 3 SAFRHCERE 45 050 13 g VBI. uraisivivias B enibinionssni 3 5 + bis gowbmmncton 438 #.n ibabintrsit 2 40a thas ition 1,000 ml Prepared and used as is Kligler’s iron agar (CM No. 52). The final reaction should be pH 7.4. 53b. Triple Sugar Iron Agar, Modified®3* Beef GXIrBct ..osvnunivnnsssson nn rsdsssnsmmonss ss ons 3 g Yeast EXITah +o: nmvnans os ht vauibons sees samunnss sss 46% 3 g Bacto Peplofi® ..uuveveresvniommnsshsnsmmmsimmns sms 15 g Proteose PePLONE «event vere iiiiineeeienanneenenen 5 g Lactose cues vomemn 08 sss samemie a6 vs 3 sanmeswess « Same 10 g SHCEOSR. i +05 4% 3 2bicincdin 4 3 3 bEBRA0ES 4 300 Birmwimntion’s 3 3 BRI 10 g TRECOSR rirsciv + 2 siaiisaresiodis Co waiomaiulonssin sds remapsarameids © aoarasess 1 g Forrong stlfale . somrorss os srumin ve v1 3 vepmsivens s move 02 ¢ Sodium thiosulfale cevs«rovammmmness s vamssoes os sues 03. xg Sodium CHIOTIIE omens tsi sammnsodns be 2otmainss £1 meinen 5 g Phenol red (10 ml of a 0.25% aqueous solution) ...... 0.025 g AGEL Sivan » S0aname £9 rr eae RET 1 § SEAT ¢ ¥ 30 aR 12 g WBE sis aa saver oneness 5 SARSWID EL RIOT AVEE LE $3 IGA 1,000 ml Prepared and used as is Kligler’s iron agar (CM No. 52). CULTURE MEDIA 151 54. Eosin-Methylene Blue Agar,t Levine's, Modified* Several modifications of the original formula proposed by Holt- Harris and Teague? have been developed. Levine® evaluated the influence of variation of the ratio of eosin Y to methylene blue. He reported that the optimum ratio was eosin 6 : methylene blue 1 when based on actual dye content. Peplone. component wuss ss snmmevs vas so smss sos «stm 10 g Dipotassium phosphate, KogHPO, .....ooviviiiiiit, 2 g LAGIORE vovws » svoeummnivies ov Spree aig 4+ SANTEE 44 ¢ Siokmas 10 g Eosin Y, certified (20 ml of a 2% aqueous solution) ... 04 g Methylene blue, certified (13 ml of a 0.5% aqueous SOHEEIIRY: vx vo oemcwsnmsnin = + win sommminindiidin = Hoobs Woramwsein ss + 3 2H 0.065 g AGE mm 103 433 van v2 3 0 8% SETS £4 8 5 REI £8 & of 13 g Wer civics ss 5.9 SOREN £5 3 § BORRRGAE © 5s Huma « & § § piss 1,000 ml a. Dissolve all ingredients in water with heat. b. Dispense in flasks as desired and autoclave at 121° C for 15 min. For use, melt agar medium and pour plates. The final reaction should be pH 7.1. 1 Certified dyes must be used if the volumes listed in this formula are to be followed. Any variation in dye content must be checked by assay to be certain that the complex formed by the eosin and methylene blue is correctly pro- portioned. The carbohydrate component of the original Holt-Harris and Teague formula was 0.5% lactose and 0.5% sucrose. This combination can be used if desired for the detection of intestinal pathogens, but it should not be used in water bacteriology. 55. Endo's Agar, Modified* Peplone component sever sss vos sumnss «ss somone +54 sani 10 g LaclOBl commis os 5a ammmaiin £ + 5 swam on £5 SEdsimen ets vo 10 g Dipotassium phosphate, KegHPO, ..........coooiin.... 358 Sodium: bisulfite, NalISO; «.vvvvsirmvnnnisins vumones mrs 25g Bogie Lfuchsing wuissvsommn nosis smpwmessssssonsmscorssn 04-05 g AGA - 125500510000 5 2 5 ma ERIS © & » § HENS £23 33 WRITE 5 553 15 g WVBIER: . oooviirisnacnn + + « wonimanmesesen ¢ + 3 ORRERWE £ 8 83 RRNTEIN 25 3 3 1,000 ml a. Dissolve ingredients in water with frequent agitation and heat. b. Dispense in flasks and autoclave at 121° C for 15 min. c. Cool to 45° C, evenly distribute the flocculent precipitate by gentle rotation of the flask, and pour plates. Prepared plates should be kept in the dark and used within 4 days of preparation. The final reaction should be pH 7.4. + If medium is prepared from individual components, the required concen- tration of basic fuchsin must be determined by standardization. 152 CULTURE MEDIA 56. Sodium Desoxycholate Agar? Modified®34* Peplone 'COMPONBILE 5 eviviun vis he tbteeile 555 5 witivin nein in 10 ¢g Lia0E008. silanes vite suaisiniinivlv simi tins +5 Shad ctloms + 10 g Dipotassium phosphate, KogHPO, ..vvvvinvniennnnnn... 2 ig Ferric citrate (or ferric ammonium citrate, 2 g) ...... = SOCINNGEUTALE. vo o'ss nmeive dB os San mmeiales & 3 Sumbmae vas lg Sodium desoxYCHOIAe ove vss sas snrninins ss savonmmrins ss 1, 2 Sodium ChIOTIAR +... : sasvue a4 ss saammnwn « vs wxpissens sss 5 g AGBY Sienna ase sw amamwing 3 55 SAREEAE § 5 4 SESE VES 15-16 ¢ Neutral red (3 ml of a 1% aqueous solution) ......... 003 g WNARIRE svnnnnns « 3 sannnnieine + 30 ommmmntiie + ARG AEES ba 3 1,000 ml a. Dissolve all soluble ingredients in water and add agar. Allow to soak at least 10 min to wet agar thoroughly. b. Heat gently with constant agitation and boil for 1 min to dis- solve agar. Do not overheat, as excessive heating increases the in- hibitory effect. The final reaction should be pH 7.2. c. Pour in porous-top plates or dispense in tubes containing 20 ml of medium for later preparation of plates. Larger volumes should not be used, or there will be too much exposure to heat in melting the agar. Do not autoclave. Brodie®® investigated the mechanism of inhibition of bile salt media and found that sodium taurocholate could be substituted for sodium desoxycholate if the latter is not available. He demonstrated that the inhibitory effect is the result of the combined action of the electro- lytes and the bile salt. Any variation in the bile salt concentration or the bile salt itself must be controlled by variation of the electrolyte concentration. The exact ratio can only be controlled by titration. 57. Sodium Desoxycholate Citrate Agar,®? Modified®34* Lean beef muscle (or equivalent of dehydrated IOSUSIONY. - +0 venisnavns sr amuiplesoins soe smuses + suamasion 330-375 g Peptone COMPONENE .u..osvsvissvssssssmms esis savsve 10g Lactose! |. suns enern ass ornnmmsei te sibmas sis insosmenis 10g Sod CIAIR ovis or wna aamanis.s mame sss GIFT 20 g Ferric citrate (or ferric ammonium citrate,f 2g) ..... 1g Soi ACSORVONOIARE . ovivenssmonny, mrtatmsindsnm 5.48 Neutral red (2 ml of a 1% aqueous solution) ........ 002 g ASHE 2, hh cleat hii tee dai ve ate vr hs ai gd aie vere ey 17% .g WBE ocodvassniomend cnn sms Daitaia dives os Shien ee Buivconion 1,000 ml Prepare and use exactly as sodium desoxycholate agar (CM No. 56). T Green scales. CULTURE MEDIA 153 58a. Bismuth Sulfite Agar, Wilson and Blair®%95*} Agar Base: Peptone component coves esssrssissresrevesssesersors 10 g Beth CXITROL «vores ne revamp A EE a 5g AAT camnesrsvsneraneres sheaves bees seine te ian 20 g WRIST unnsva sve svomsh webs amme Eas 3 Sasaaan ess suas 1,000 ml a. Dissolve all ingredients in water with heat and adjust reaction topH 7.2. b. Dispense in flasks in 100 ml quantities or other desired volumes and autoclave at 121° C for 15 min. Bismuth Sulfite Solution: Bismuth ammonium citrate powder, U.S.P. IX} ...... 6g SOtium SUES: senwrs vos summers ses PEEREEEE EE RERERS 10-20 g GIUCOBE. iiivii snnnvmass ss eun@aaes i vannvsie nes nes 10 g Disodium phosphate, Na,dHPO, ....ccvvinvniinnen. 10 g VATHEBE. ovo 3 S00 SAAR 9 SARTRE a boobasashotin tang st orion 200 ml a. Dissolve the bismuth ammonium citrate in 50 ml of boiling water, the glucose in 50 ml of boiling water, and the sodium sulfite in 100 ml of boiling water. (Caution—Sulfur dioxide is evolved rapidly from hot solutions.) b. Mix the bismuth ammonium citrate and the sodium sulfite solutions, boil, and while boiling, add the disodium phosphate. c. Cool and add the glucose solution. Restore to 200 ml volume, d. Store at room temperature in a well-stoppered pyrex flask in the dark. Iron Citrate-Brilliant Green Solution: Perric cifrate, U.S. P. wc: ur vumnminrss sniows SE ERE 1 g Brilliant green, 1% aqueous solution .................. 12.5 ml WHEE. coin: sampilisss sapaitonma sn & pat Runn bd WF SAMS 100 ml Dissolve ferric citrate in water with heat and add brilliant green solution. Store in a well-stoppered pyrex bottle at room temperature in the dark. Complete Medium: ATor Base ..: i onmsaviias s Orman s 5 05 3 FuaRART § 600000 1,000 ml Bismuth. sulfite solufion ...sesewsis is sssmsnmines +200 200 ml Iron citrate-brilliant green solution ............ccce.... 45 ml + Do not autoclave commercial dehydrated bismuth sulfite agar. + Amend Drug and Chemical Co., Inc, 117 East 24th Street, New York 10, N. Y. § The weight of the sodium sulfite may be varied within limits as a means of standardizing other ingredients (various peptones, agar, etc.) in the medium. 154 CULTURE MEDIA a. Melt the agar base and add the bismuth sulfite solution and iron citrate-brilliant green solution with thorough mixing. b. Pour in porous-top plates at least 74 in. deep and use within 4 days of preparation. Store plates in the refrigerator in the dark. The final reaction should be pH 7.7 =. 58b. Bismuth Sulfite Agar, Modified, Hajna and Damon?%:97%} Agar Base: Boal eRIract’ vores ve sin cnmniie s » + srmrnionnt « § Sevlemenies x 5 Thiotone or thiOPEPIONE «uve ix svemeivn iss sesmmne ses 10 GIUCOBE. . wnsmwn + + Form RERLIS 53 SHEMET & £8 WEREEESS E04 5 5 a. Dissolve all ingredients in water with heat. Adjustment of the reaction is unnecessary. b. If the agar base is to be stored, dispense in flasks in 100 ml quantities or other desired volumes and autoclave at 121° C for 15 min. Bismuth Sulfite Solution: Bismuth ammonium citrate, 10% aqueous solution ... 500 ml Ammonium hydroxide, NH,OH, concentrated BAUEOUS GOMILION «is i «x ntrerniainiinin os 0 dieieinss sd don 14 ml Sodium sulfite, anhydrous, Na,SOg, 20% aqueous SOIT su BE brut rwiain: eaTiin mimomanconns Sta.» = wiglmamizecnins & oie on 1,000 ml Dibasic sodium phosphate, Na,2HPOy .....cuv...... 100 g Ferric ammonium sulfate, Fe,NH,(SO,),.12 H,O 10% aqueous solution ........coevvevvnniinennn eens. 100 ml a. Add the ammonium hydroxide to the bismuth ammonium citrate solution and allow to stand until clear. Several additional milliliters of ammonium hydroxide may be required. b. Add the sodium sulfite solution and mix, then add the dibasic sodium phosphate and mix. c. Add the ferric ammonium citrate and boil for 2 or 3 min. d. Stopper the flask or bottle with a rubber stopper and store in the dark at room temperature. Do not refrigerate. Complete Medium: Agar DASE sunrises sssrnonnsss sa areas se RES. 1,000 ml Bismuth sulfite solulion .oossmses ssvsamsnses eevenmnoss 70 ml Brilliant green, 1% aqueous solution ................. 4 ml + Do not autoclave commercial dehydrated bismuth sulfite agar. CULTURE MEDIA 155 Melt the agar base, add the bismuth sulfite solution and the brilliant green solution. Cool to 50°-60° C and pour in porous-top plates. 59. Salmonella-Shigella (SS) Agar* Agar Base: Beek emIratl «vos rosmndivins ss savmmunres wahvmteses 58 5 ¢ Peptone cOMPONTAL unas ex ss semmuning sss avsmvres sass 5 g Bil SE cpoetio: fopmmem oi os Sewtitud le sy siammemsiniam ae 85g ADOT crm ok 1 Reet rs Neils uh « sed SAR asd os 135 ¢g Wall cviwves ionmmnins bn § samnavedios. sod OEE v3 So 1,000 ml a. Dissolve the agar in 500 ml of the water with heat. b. Dissolve the other ingredients in the remainder of the water, combine, and restore to original volume. c. Dispense as desired and autoclave at 121° C for 15 min. Complete Medium: AZar DASE «vis ist sani st Sramnnssesst EARTHS 1,000 ml LACtOR cussbeionion conens aime ssn ane yaee se 10 g Sodium citrate, Nag C¢H;0,2H,0 ............ 85 g Sodium thiosulfate, Na,S,05.5H,0 ............ 85 g Ferric citrate pearls, USP. ..ccvivevsnissenvesss 1 g Neutral red (2.5 ml of a 1% aqueous solution) .. 0025 g Brilliant green, certified (0.33 ml of a 0.1% Aeon SOMMON) cmv san ommmmmeriece sawn 0.00033 g a. Melt agar base and add all ingredients except the dye solutions. b. Adjust the reaction to pH 7.0 and add the neutral red and brilliant green. c. Mix thoroughly and pour about 20 ml per plate. Do mot autoclave. Final reaction should be pH 7.0. 60. MacConkey's Agar,*® Modified®34* Peptone COMPONEHL vss « buonibinsin + 4 + moaning as s oRe 20 g Ly 10 g Bile Bal o 21 aimumae 5 5.05 Bighorn £8 3 bwtemmmeissom * o wnowisaeie 1.5 ¢g Sodium CHIOTIAE wows «2 e325 mamma's 7s saikumanes « 3 Semis 3 g AGATE uw sso stvmst bs + sammsnins 8 5 ehmwmsnsg 2 Gsm 13.5-15 g Neutral red (3 ml of a 1% aqueous solution) ....... 003 g Crystal violet (1 ml of 0.1% aqueous solution) ...... 0.001 g VWALEE 00: 5 sa smimiowa 56 & snsonimeii § § snsiemiains « Forni 1,000 ml a. Add all dry ingredients to water and dissolve with heat. b. Adjust the reaction to pH 7.2 and add dye solutions. c. Dispense in flasks or tubes as desired and autoclave at 121° C for 15 min. Pour in deep plates and allow surface to dry. Final reaction should be pH 7.1. 156 CULTURE MEDIA 61. Brilliant Green Agar®-101% Veoost CRUEL vi von vivenun uss dabumsanss hiss weapons 3 g PEOIONE COMPONGIEL: ov eieluin's nie wei sninmisiotes wale samiaiie wp 10 g Sodium chloride . ....oiivs sass sminbe es isssmabiiee 5 g Lacione ..ciuisden susrnsmad vasa sespaaismvevenaessy 10 g SUCEOLE vvwruivmmendenurimvdiins neh ew yess 10 g PHEADL TO... co vcoinimibin emma sins sp baiine sii pismo 008 ¢ Brilliant green (5 ml of a 0.25% aqueous solution) .... 0.0125 ¢ AGU v0 vin POF RIERA ERE SARE RGR SAAS Fe 20 g I LC 1,000 ml a. Suspend ingredients in water and let stand for 5 min. Mix thoroughly and heat with occasional agitation. Boil for 2 to 3 min to insure solution of the agar and autoclave at 121° C for 15 min, no longer. b. Cool the sterile medium to 45°-50° C, pour plates, and allow them to dry about 2 hr with lids slightly ajar. The final reaction should be pH 6.9. 62. Tetrathionate Enrichment Broth,1°2 Modified3:34* Peptone component ....... pre PRR Ie 5g Bie SHS ores mos mmm on sa EE EH Ae lg Colciumncarhonate, ‘Cally «ovovnnvns sevins isn agosmmvemns 10 g Sodium thiosulfate, NasS,0, 5H, 0 voc: virernnnnrmnnen 30g lodine-lodide SOIUHION vovsvisvrmsrmvsssrmenronsed severe ss 20 ml WRIEE cures vonsnes savins shoassms spas vsins snsasnsst srs 1,000 ml a. Dissolve the peptone, bile salts and sodium thiosulfate in the water and dispense 500 ml amounts in liter flasks. Add 5 g calcium carbonate to each flask and autoclave. b. Cool to less than 45° C and, if broth is to be used that day, add 20 ml of the iodine-iodide solution. Dispense to sterile tubes in 10 ml amounts, taking care to maintain a uniform suspension of the calcium carbonate. Or: Dispense broth base without iodine-iodide solution to sterile tubes in 10 ml amounts and add 0.2 ml of the iodine-iodide solution to each tube on the day it is to be used. The broth base can be stored indefinitely after sterilization. Do not heat after the iodine solution has been added. Todine-Iodide Solution: Yodine Cresublimed) =. 1. canons sommes sls des 2 6g Potassium jodide; BT Lo etiam ing srnememmen’s sumimnmnis messy s 5% B15 et I er rN NN RS RE 20 ml Dissolve the potassium iodide and iodine in part of the water and make up to volume. CULTURE MEDIA 157 63. Selenite (F) Broth103* Pepione COMPONENE «+x snwwwnin § # so onaimanions s a8 WaEwEe +8 5g TACIT. 1h uo vow ein bo 38 RR Eres WR ETD 5 AEE § 4g Sodium phosphates, anhydroust .......cevvvvineinunennnn. 10 g Sodium acid selenite, NaHSeO, «iv ipnrivnonsrorgrneeses 4g WHET: ons nsvmmaisvn sha ssa nemesis sad mnnitiien Dat re sana tee 1,000 ml a. Dissolve the dry ingredients in the water with gentle heat. b. Dispense in tubes to a depth of at least 2 in. and sterilize by ex- posure to flowing steam for not more than 30 min. Do not autoclave. Tubed media may be stored in the refrigerator. c. Final reaction should be pH 7.0=. Alternatively, a proportionate amount of the dry ingredients can be added to a sewage effluent, water sample, or other large specimen and dissolved without heat for the enrichment of suspected enteric pathogens. + The ratio of monosodium phosphate to disodium phosphate must be de- termined with each lot of sodium acid selenite. The total phosphate concen- tration will be 1%, adjusted so that the solution containing the sodium acid selenite is buffered at pH 7.0. Uninoculated media exhibiting large amounts of the red precipitate of oxidized selenite should not be used. 64. Broth for Nitrate-Reduction Test*} Extract broth (CM NO: 3) ceiver ssrimimuuns avvnnmnvnens 1,000 ml Potassium nitrate (nitrite-free) KNOz ....cvivevninnn... lg Dissolve the nitrate in the broth, dispense in tubes as desired, and autoclave at 121° C for 15 min. No color should appear when an uninoculated control is tested with the e-naphthylamine-sulfanilic acid reagents for the presence of nitrite (pink to red color). See Chapter 1 for method of performing the test. Final reaction should bepH 72x. For obligate anaerobes, add 1 g each of glucose and agar to 1,000 ml of the broth medium and tube in deep columns, For Erysipelothriz insidiosa, use CM No. 3 prepared with 1% proteose peptone and proceed as in CM No. 64 above. + Commercially available dehydrated products of slightly varying formulas are suitable. . . Co. . % Poorly rinsed tubes may contain sufficient nitrites to give a positive reaction. 65. Agar for Nitrate-Reduction Test* Broth for nitrate reduction test (CM No. 64) ............. 1,000 ml AGE siaiiieNusg » 1 FRNEERA EL OE § SURE. SB SPIRO 27 ip #oripieaiiie 12 g a. Dissolve the agar in broth by heating to boiling and dispense in 3-5 ml amounts to tubes. 158 CULTURE MEDIA b. Autoclave at 121° C for 15 min. Place tubes in position to make a short slant and allow to cool. The final reaction should be pH 6.8=+. 66. Citrate Agar, Simmons04* Magnesium sulfate, MgSO, «cvvovcrivrnnrisisrraaniine 02 g Monoammonium phosphate, NH,H,PO, ............... 1:g Dipotassium phosphate, KoZHPO, ...ovvvnivninnenn..... 1 g Sodium citrate, NagCaH;O0;.5H,0 ..ouvvvnnnvnnnnnnnn. 2 g Sodium CHIOTIAE uuwas oa summsmns s 54 + piaiianm + 5 vavvnies 5.2 Bromthymol blue (20 ml of a 0.4% aqueous solution) ... 0.08 g BLBL. oes ohveivesivnhunmmeee crane tor CIEE 15 go VIBE Ls vine omit oda an Siam lass dm a alo wea are me mei shuts 1,000 ml a. Dissolve ingredients with heat, dispense in tubes and autoclave at 121° C for 15 min. Allow medium to cool in slanted position. Final reaction should be pH 6.8. b. Inoculate by stab and streak. Growth accompanied by alkali production changes the medium from green to a deep Prussian blue color. 67. Urea Broth, Rustigian and Stuart105* TECH. : niniian's § § $TMATH STF 03 3 GADRGEES 3 5 LAPGHGH A 5 5 Fie 20 ¢ Monopotassium phosphate, KHoPO, v.vvvvnennnieinnnn. 91 g Disodintn phosphate, Na HPO, ..«..... cuciesirosvene 95 g Yash SXiract on ovivisns imme cosas sa ginevns dive vives 01 ¢g Phenol red (4 ml of 0.25% aqueous solution) ........... 0.01 g WWOlRr 5 ooesseiisnnestnsrsrstinnhiohas seemsnionss sensory 1,000 ml a. Dissolve dry ingredients in the water and add the phenol red solution. Do not heat. b. Sterilize by filtration, preferably through a glass or porcelain filter, and tube in at least 2 ml amounts. Final reaction should be pH 6.8+. 68. Urea Agar, Christensen!®%*} TITER 2 irilons danni E Bs a ERO 3.5 3 3 ERIE & 3 niorus 20 g GLUCOSE: outils ot + Sininiif § nd MPRA AE © SRR 6.8.0 4 mii 1 g Pepione cOmpOnBHt u...: its rtmrin sss ssssvnsoinsss sve 1 g Soditim chloride .....cocs 0s cnrvicsnnesn sndipaipsniois danse 5 g Monopotassium phosphate, KHyPOy ..covvvvnnnnen.... 2 g Phenol red (5 ml of 0.25% aqueous solution) .......... 0012 g WARE oon tials nes Stim os Chaabames us an odnte 1,000 ml BAAR 250 Seg vamos baad SERA 08 SHAS ais ererrp 15 g + Medium without agar is also commercially available. CULTURE MEDIA 159 a. Dissolve all ingredients except agar in 100 ml of water and sterilize by filtration. Do not heat. b. Dissolve the agar in 900 ml of water with heat and autoclave at 121° C for 15 min. c. Cool agar to 50° C and add the filter-sterilized solution of chemicals. Mix thoroughly. Tube and slant so that a deep butt results. Final reaction should be pH 6.8. 69. Semisolid Agar for Motility Test, Edwards and Bruner,” Modified* Pancreatic digest of casein, USP. XVI} coun vivvvnninss 10 g Bef @GRIMANE . covinminn s+ + sbveminis oo PSRERR © © 53 SPREE 3g GRIBIIIE vv vo + morons § 414 sp ominsies © v's SRRREIE Ss § § $m os 80 g Sodium chloride ......vviniiiieierieeiiiiieeennnennanann 5g BEAL samme s+ 3 vammamiv va 1 #2 iaRE Ses 4 5 RRSEOES 5 08 SPSVETHG 4g WVEBIEE coved » wnmmmipes 3 + § TRETAES £7 5 3 Sidemins § § § SEEEETGA. 1,000 ml a. Dissolve dry ingredients in the water with gentle heat. Temp- erature must be raised to the boiling point to insure adequate solu- tion and even distribution of the agar. b. Adjust the reaction to pH 7.4 and dispense in tubes or flasks as desired. c. Autoclave at 121° C for 15 min. Final reaction should be pH 722. In any subsequent reheating, particular care must be taken to see that the agar is liquefied. The medium becomes liquid at about 50° C, but the agar will not melt under 95° C. + Other peptones may be used. . I For detection of motility at 22° C, reduce the gelatin concentration to 4%. 70. Agar Base for 10 Per Cent Carbohydrate Medium108* Pancreatic digest of casein, USP. XVI ............... 10 g Sodiom chIOTIAE ..cuumemsins ins ssmniEhuipms oosainsns goss 5 g BEBE oiuivniovinton se is mamas So a Eis Fa PW 15 g Phenol red (10 ml of 0.18% aqueous solution) ........ 0.018 g WBIET vos imuuimn s bout Rua ve Ss Se ped he RTA 6 1,000 ml a. Suspend ingredients in water and add 100 g of the carbo- hydrate (glucose or lactose). Allow to stand for about 5 min and mix thoroughly. b. Heat with occasional stirring and boil for 2 min. c. Dispense and sterilize at 115° C (10 Ib) for 15 min. Slant tubes for cooling. The final reaction should be pH 7.4=+. 160 CULTURE MEDIA 71. Glycerol-Sodium Chloride Solution, Buffered0%:110} Soditin, Chloride oo 2 4 +s avviogn + » srs Twmss 5 » 5amamess iad 42 ¢ Dipotassium phosphate, TPO, (ovveiniss vs svmmnnmn sass 31g Monopotassium phosphate, KHoPO, .....coovviiniii.in, 1 g GIVeerol uanises tn stags hitniums olds wns sal sea bieiniole + 2a 300 ml NVALBE of 4+ opin + 2 + stibisprioiainis » oes Spiwininaie § # FRATHIAE £3 82% 700 ml a. Dissolve ingredients in water and add sufficient phenol red (0.25% aqueous solution) to impart a pink color to the solution. b. Dispense in bottles and autoclave at 121° C for 15 min. Final reaction should be pH 7.2=+. Solutions which become acid (yellow) upon standing should be discarded. + Base without glycerol is commercially available. 72. Dorset's Egg Medium Basic Formula: ER RO. cairo Crmaguatipsnees mun dvnivtn es spits 750 ml Water or other QHURHE ...evesiyenesontme suinrons denis ns 250 ml Mix thoroughly, dispense in previously sterilized tubes or screw- capped vials. Sterilize as directed for the preparation of Loeffler’s coagulated serum medium (CM No. 81). Use sterile technic throughout to minimize contamination. There are many variations of this basic formula, apparently de- pending upon the availability of various diluents and the preference of the bacteriologist. As early as 1902, Dorset!!! suggested the use of whole egg without diluent, egg yolk without diluent, and egg yolk with water as a diluent for the cultivation of Mycobacterium tuber- culosis. Since that time, the variations suggested in the literature have been very numerous. Levine and Schoenlein!!? compiled five modifications labeled Dorset’s medium; and all five had been sub- jected to further variation. Francis!*® used McCoy and Chapin’s!!* egg yolk medium in which the egg yolk was diluted with two volumes of physiological salt solution. Many of these variations were de- veloped after unsatisfactory results were obtained with the original formulas. It is safe to say that the primary reason for dissatisfaction with an egg medium is failure to sterilize the medium properly. The im- portant principles to be followed as a guide in the preparation of any egg medium are: (1) The eggs must be fresh; (2) the procedure should be carried out using all possible precautions to avoid any and all unnecessary contamination, including preliminary disinfection of the shell; and (3) the exposure of the medium to heat should not be CULTURE MEDIA 161 greater than the minimum time period required to secure satisfactory coagulation of the egg protein and effective sterilization. A good medium has a moist, soft, glistening surface. 73. OF Agar, Hugh and Leifson!t5* Pancreatic digest of casein, U.S.P. XVI ..ovvvinirinnnes 2g Dipotassium phosphate, K,HPO, ...vvvvvnvnnivnnenn, 03:.¢ Sodium CHIOTIAE «.cuvvvr eres crmnn ss sinsanionons es snmn 5-'g ALBEE finuatione an i Brhmiip sans Sn Satatai bss hapiarin rs 56 wots 3 vg Bromthymol blue (3 ml of a 1% aqueous solution) ..... 003 g Carbohydrate (100 ml of 10% aqueous solution) ....... 10 g Water, 18 BIBRE .unacmnrs sompnmns ve suspense Es SR 1,000 ml a. Dissolve all ingredients except the carbohydrate in water, add the bromthymol blue solution, and adjust the reaction to pH 7.1=. Bring to boiling to dissolve agar and autoclave at 121° C for 15 min. b. Add the filter-sterilized carbohydrate to the cooled medium to a final concentration of 1%. c. Dispense in sterile tubes to a depth of about 124 in. + Do not use alcoholic solution. 74. Hormone Agar for Plague Vaccine, ''® Modified!17* Ground lean Teel DATE «cv wus duwimsinss ss ph wipe Sein s.5 5 5000s 500 ¢ WRIT civnaistod saints § 7 3 walbhbmis 10.8 5 mmr 4 § Sawin 1,000 ml Protecse peptone No. 2 ve. iiosvmuins sss subivee Ct 2s omnes 20g Sodium chloride uve eiiiie ie ciiee cena 5 ¢g Cysteine monohydrochloride .............covivvnvrnnnne 01 g Sodium sulfite, Na,SO4 (2.5 ml of 10% aqueous solution) 025 g ABUL copmnmis oss smu » 1 4 SROSREE 4 1» § SAT ob § 500000 23 .g a. Trim excess fat and fascia from beef heart and grind. b. Boil ground heart, peptone and water for 1 hr, drain and filter broth, and restore volume to 1,000 ml. c. Add sodium chloride, cysteine (dissolved in 10 ml of water), and freshly prepared sodium sulfite solution to the filtered broth. d. Heat to boiling, add the agar, and boil until agar is completely dissolved. Adjust reaction to pH 7.3== with NaOH and restore to original volume if necessary. e. Dispense in Roux bottles and autoclave at 121° C for 25 min. Cool in slanted position. 75. Crystal (Gentian) Violet-Hormone Agar, Meyer & Batchelder!8 Hormone agar (CM No. 74) ....oviviiiiiininiieenenvn.. 690 ml Crystal violet,7 0.1% aqueous solution ........coeevveuenn... 10 m! T “Gentian Violet, Improved,” by Coleman and Bell. 162 CULTURE MEDIA a. Melt sterile hormone agar with heat, add crystal violet solution, and mix uniformly. b. Dispense in petri plates. 76. PPLO Broth!19¥ Beef heart for infusion (DCO) «..:snmmmnive vives suv os 50g Pepione {DI0Y svi rimimmemsin sos rami dmt os Sa L155 10 g Sodiam. ChIOFIAR! ..ame ss vin vsmmTines emt ss vos BORE 5¢g WHEE vnuins snmemnon sae 8 0eomsniaiias 2 so ami oes sainhm uss 1,000 ml a. Combine the beef heart for infusion with the water and hold for 1 hr at 50° C, then heat to boiling for a few minutes to coagulate proteins. Strain through three or four layers of gauze and make up to 1,000 ml. b. Dissolve peptone and salt in infusion broth with heat, adjust reaction to pH 8.04, and boil for 5 min. (This excess alkalinity is helpful in producing a clear medium, which is very important.) c. Filter through filter paper and readjust pH to 8.0 with HCI (for best growth of PPLO, the medium should not be filtered more than once). d. Dispense as desired and autoclave at 121° C for 15 min. Final reaction should be pH 7.8. e. Before use, enrich broth with either 1% PPLO serum fraction (Difco), 25% ascitic fluid, or 10-20% serum.20 77. PPLO Agar'19¥¢ PPLO broth (CM No! 70). conics ssta'sos mame vs saimamsns 1,000 ml ATRIE win ois stmmmnntis #5 5 aE 1 famelumuve na 43am 14g a. To the PPLO broth (CM No. 76), following filtration in step ¢, and while the pH is still greater than 8.0, add the agar and heat to boiling until agar is dissolved. b. Readjust reaction to pH 8.0 with HCL c. The medium while boiling hot is filtered through a thin layer of cotton contained between two layers of gauze. d. Dispense as desired and autoclave at 121° C for 15 min. e. Before use this agar medium must be enriched with either 1% PPLO serum fraction (Difco), 25% ascitic fluid, or 10-20% serum.12° + Difco PPLO agar is equally suitable for growth of these organisms (see Chapter 22). % The agar must be known to be satisfactory for cultivating PPLO, as not all lots of bacteriological agar are satisfactory for growth of these organisms.121 CULTURE MEDIA 163 78. Pai's Egg Medium,'?? Modified!2? Meat infusion broth (CM NO. 5) ..suisismcssssvsassnnes ss 300 ml GIHICOBE © vvnvnins syns vi vies i Tai PEs eo AES VES BIT ATES Sz Whole, mixed hen’s eggs .....oviiiiiirininnennnnnesensnnns 700 ml GIYeetol, ‘CP. conics vrumomismains vc vnumssin we xis 80 ml a. Dissolve glucose in broth, then mix in the hen’s eggs and glycerol, taking care to avoid bubbles. b. Dispense and sterilize in the same manner as for Loeffler’s medium (CM No. 81). 79. Cystine-Tellurite-Blood Agar, Frobisher and Parsons!?* Meat infusion agar (CM No. 8) ..ucivisvsmvrmirsnsss 1,000 ml Sterile, defibrinated or citrated blood (see CM No. 16) 50 ml Potassium telluritet (150 ml of 0.3% aqueous solution) 045 g Dry SYNE . ossmsviinnss sninmatiimns aah sommes ream 0.035-0.05 g a. Melt sterile meat infusion agar and cool to 50° C. Preheat blood and sterile potassium tellurite solution to 37° C and add to the melted agar, then add the dry cystine. b. Mix thoroughly and dispense in pour plates approximately 15 ml of the medium per 9 cm petri plate. + Certified potassium tellurite is available from the A. H. Thomas Co. i Need not be sterilized if taken directly from the reagent bottle. 80. McLeod's Agar for Type Determination of Corynebacterium diphtheriae, Modified? Meat infusion agar (CM No.6) ....coeviiiiiiiiinnnnnn. 1,000 ml Sterile, defibrinated blood (see CM No. 16) ............ 50 ml Potassium tellurite (150 ml of a 0.3% aqueous solution) 045 g a. Melt agar and cool to 90° C. b. Add sterile blood and hold at 90° C for 10 min. c. Preheat the filter-sterilized potassium tellurite solution to 37°- 40° C, and add it to the cooled (42° C) blood agar. Mix well and pour about 12 ml per plate, 81. Loeffler's Coagulated Serum*t Meat infusion broth (CM No. 5) +vivivsivenmennrsensmmmen 300 ml GIUCOSE .onwissvvasmmnsss ors souienm esos 33 SanBams ses 5 ims Bakes 3 Fresh, clear mammalian serum ..........coouiveininennnnnn.. 700 ml a. Dissolve glucose in broth and add serum. b. Mix well, taking care to avoid entrapment of air bubbles, and tube in slants with shallow butts. + Each lot of commercial product should be tested for suitability.35 164 CULTURE MEDIA c. Sterilize, using an autoclave with a manually operated air-escape valve. To avoid foaming during autoclaving, close the air-escape valve and raise pressure to 15 lb immediately. After 10 min, open the valve very slightly so that steam enters and air escapes slowly while full pressure is maintained. When the temperature reaches 121° C, close the valve and hold for 15 min. Turn off steam but do not open autoclave until the pressure has slowly reduced to zero and the autoclave has cooled. The vacuum may be broken by admit- ting air slowly to the cooled autoclave. 82. Raffinose-Serum-Tellurite Agar, Whitley and Damon!2¢ Proteose No. 3 agar™ or equivalent ... oo siiuerdenatneen ss 45 g Phosphate-buffered water, pH*7.2 .. ci vives reinnnynans 100 ml a. Dissolve proteose No. 3 agar by heating at 100° C in the buffered water. Autoclave at 121° C for 15 min. Phosphate-Buffered Water, pH 7.2: Na, BPO, (M/13=947 g per liter) viv oddities. 72 ml Nabtl,PO, (M/15==8.00.2 per Her) =. v..viiie waivvizhitni vi 28 ml AVELEE viv ivi toroste doi iiss ars Tnsps pion analogy ns tis Selb lock 900 ml b. Cool to 50° C in a water bath and add 10 ml of stock raffinose- serum-tellurite solution prepared as follows: Raffinose-Serum-Tellurite Solution: Mammalian serum (preferably human) ................. 30 ml Raffinose, 10% aqueous SOON .....vvvevinrnrinennnnnnns 24 ml Potassium tellurite,f 0.5% aqueous solution ............. 6 ml 1) Sterilize human serum (can be obtained from serological speci- mens) and the 10% raffinose solution by Seitz filtration. 2) Prepare the 0.5% solution of potassium tellurite by grinding 0.5 g of C.P. dry potassium tellurite to a very fine powder in a small dry mortar. Add 10 ml of buffered distilled water gradually, with stirring. After grinding, and when particles have settled, remove clear supernatant to a 100 ml graduate. Repeat process until all the tellurite appears to be dissolved. Add 0.33 ml of 10% KOH to the mortar, rinse sides of mortar with 10 ml of distilled water, and add rinse to graduate. Make up volume in graduate to 100 ml and Seitz-filter. - The final reaction will be about pH 9.6. ¢. Add 10 ml of stock raffinose-serum-tellurite solution aseptically and mix thoroughly. + Keep C.P. potassium tellurite powder in a desiccator. If powdered tellurite is used, add 0.33 ml of 10% KOH to the solution. CULTURE MEDIA 165 d. Tube so as to obtain long slants, and check sterility by incuba- tion at 35° C. These slants will remain satisfactory indefinitely in the refrigerator if dehydration is prevented. 83. Glucose-Serum-Tellurite Agar, Whitley and Damon'?? Base Agar: Proteose No. 3 agar* or equivalent ...........c0vunnnn 45 g AGAT, SrARUIAL vs 5 viiilnk pve o's § Hakicvass so vimiitimismes vs 05¢g Buffered water (see CM No. 82) ....c.vovvvnnnnnnnns 100 ml Glucose-serum-tellurite solution} .........ecevvevnen.. 8 ml a. Dissolve the Proteose No. 3 Agar and the granular agar by heating in M/15 phosphate-buffered water, pH 7.2. b. Autoclave at 121° C for 15 min, then cool to 50° C in a water bath. c. Add 8.0 ml of a sterile stock glucose-serum-tellurite solution and mix thoroughly. d. Pour plates of not less than 5 mm thickness and incubate over- night at 35° C to test sterility. Stored in the refrigerator, this medium is good for at least 1 month. T The glucose-serum-tellurite stock solution is prepared as described for the raffinose-serum-tellurite solution in CM No. 82 by substituting 12 ml of a 20% glucose solution for the raffinose solution. 84. Heated Blood-Tellurite Agar, Kellogg and Wende128 Dextrose proteose No. 3 agar* or equivalent .............. 42 g WIEBE 41 cous sniines s1 sa sannuaes sas ansians vos summa soe 100 ml a. Autoclave at 121° C for 20 min. Cool to 75° C and add 5 ml of a sterile 0.8% aqueous potassium tellurite solution (autoclaved at 121° C for 20 min). b. Immediately add 7 ml of fresh rabbit blood and heat at 75° C for 15 min. Cool to about 45°-50° C and pour plates. 85. Agar for in vitro Virulence Test, Frobisher ef af.120.130% Base Agar: Proteose peptone (Difco) ......vvvirivireninvnennnn. 2 g Grated QE le 35 Den stvniniid sab aed Rie 28 5 a bainirid 175 Sodium chloride, CP. i scumeriiusasionmmms sg rama 025 g WABLOT ios ons Sma $52 ame Snes Gasset sa a wise 100 ml a. Dissolve proteose peptone (use only lots which have been shown by preliminary testing to be satisfactory), sodium chloride, and agar in distilled water by steaming. Adjust the reaction to pH 7.82 and dispense in 100 ml amounts in bottles or 12 ml in tubes and autoclave at 121° C for 12 min. Store at room temperature. 166 CULTURE MEDIA b. For use, melt the agar, cool to 45° C, and add 20% of sterile serum or serum substitute and mix just before pouring into petri plate. The serum substitute is prepared as follows : Serum Substitute: t Casamine acids (DIC) :..couvvvvivinssvrvavises sevens lg TWEE BO 5s arora 0a gots ives heim morose Samioimawon loss 1 ml GIICRIOl, TP. ooinnnn cum domi anemia n Sadiis hse ad be 1 ml WVRLEE oon vn vnin wis smi wn $06 Swi ve 4's SEA sw JE 100 ml Add the components to the distilled water, shaking gently after each addition. Warm to 50° C to aid solution of the solids. Autoclave at 121° Cor 12 min. 1 Both agar and serum substitute are commercially available. Each lot should be tested for toxin production with known toxigenic intermedius strains of Corynebacterium diphtheriae. See Chapter 8 for special precautions to be followed. 86. Thiol Broth with 0.19, Agar!3* Protecse peptone No. 3 iv. ou cunvimmns sss mmo sss a 10 WERE CRILACE «2: + 55s mmmins a0 swam: + » sbinimistvssin oie s wn 5 CHITOSE Sumario 4 1 23 5500s 2 #55 mise Aiols » » #0 mpmswidsionst 8 + 38 in 1 Sodittn. ChlOTIAe: . . «vues ct 55 + summing » 05 so ewuaE Ess sos 5 Thiol ‘COMPO +. os coniims bs 23 5 biainls £2 055 eames 3 o's i 8 ABE. alulohun's § a5 sanhom Fits Sn uhaimins ¢ s 3 5 + Bowseniie esti arene 1 p-Aminobenzoic acid (10 ml of 0.5% aqueous solution) ... 005 g Water, freshly distilled ......cweimn ssisnvmvnnss rss smes 1,000 ml a. To dissolve completely, suspend ingredients in freshly distilled water and heat to boiling. b. Distribute in tubes or flasks and sterilize by autoclaving at 121° C for 15 min. The final reaction of the medium should be pH 72x. 87. Liver Infusion Broth for Vibro fetus, Plastridge!32* PEO ATRUSION (mers stern eiie nt FA eema ie Bein Be peared SR man oo 500 ml PRDIONE vs suman ss samdesimes ou samba sano 34% Snes 256 5 5. .g AGRE invasive Xmen o sas Busnes SEAT ERTS LS SER TEEE RA 15g a. Prepare liver infusion as in beef heart infusion broth (CM No. 23), using 1 kg fresh liver instead of ground meat. The infusion is not sterilized before incorporation of the other ingredients. b. Add peptone and heat in flowing steam for 20 min. Cool and adjust pH to 7.2. c. Heat in flowing steam 30 min and filter through coarse paper. d. Add agar and heat to boiling to dissolve. Dispense in tubes to a depth of more than 4 cm and autoclave at 121° C for 15 min. The final reaction should be pH 7.2+. CULTURE MEDIA 167 88. Verwoort-Wolff Broth for Leptospiral3s:134 Pepione, Witle's, ‘Or L0YDIOR ..oocmmammens sb ammemnssses 15g MWALBE 050mm niin © 55 SRa@went 1 § SER LoE SS PHUTHGETE LES 6 1,500 ml a. Bring to a boil and add 300 ml of Ringer’s solution. Ringer's Solution: Sodium CHOTIAR «im soins 3 samme sss 3 5 hamnnas ss 335 85 g Crlcium chloride, Cally? HD 5 vvessenmmsmnvssssas 02 ¢ Potassium chlonide, ICI . os commun ys ss pugmomes sas 02 g Sodium carbonate, NasCOp cuvnvsvnremmmnrvavivains 001 g WBEEE inc 55 ome 2 5.5 ob SRB © 3 & mdr Getler § § 5 2000 1,000 ml b. Continue boiling for 5 min, remove from heat, and add 150 ml of M/15 Serensen’s phosphate solution, pH 7.2. Sorensen’s M/15 Phosphate Solution: Disodium phosphate, Na,HPO, (M/15=947 g per liter) 108 ml Monopotassium phosphate, KH,PO, (M/15=9.08 g per THEY os uo wwii 00 28 3 3 SRUSERT £ 55 9 FAREEES § 57 SOFIE > 42 ml c. Boil mixture until precipitation is complete (about 30 min). Place in refrigerator overnight, than filter or siphon off the clear supernatant. Leave 150-200 ml in the bottle, if necessary, so as not to disturb the precipitate. d. Autoclave at 121° C for 20 min. Cool to room temperature and add sterile, inactivated (56° C for 30 min) rabbit serum to a final 10% concentration. The final reaction should be pH 6.8-7.2. 89. Korthof's Broth for Leptospira, Modified!35} Tryptose. Solution: TOVDIOBET. vrourn «£55 5 itenpsndi 6 £ 401 HARIER 5235 SUH E £2 08 1 g WVBLET © ivi cis + # 5 moans waie 5 3 % ARE Sains + & 8 saranmmse 100 ml Salts Solution: Sodium chloride, 2.5% aqueous solution ............. 11.2 ml Sodium bicarbonate, NaHCO, 0.1% aqueous SOUIEOI writ i 7 inn ns + § SHWE 5 35 5 HEAR 8 4 ml Potassium chloride, KCl, 0.1% aqueous solution .... 8 ml Calcium chloride, CaCl,.2 H,O, 0.1% aqueous BOGEN sions vor oibmimnng & 5 2 nsw Ewmy vt bs wim e « 0) 8 ml Monopotassium phosphate, KHo,PO,, 2.5% aqueous SOHIIGIL writs 5 Sinica s wank © arid 3c 5 5 Sri £ 4 3 1.44 ml Disodium phosphate, Na,HPO,.2 H,0O, 2.5% aqueous SOWIAON. vps ns mmsmmpns est Sandy pws 4+ 3 bawaras 3 $53 7.68 ml WHE cimres b sh mwmmmns £4 £4 Frame 4 + Fammsmmems +4 160 ml + Modified by the Department of Veterinary Medicine, Walter Reed Army Institute of Research, Walter Reed Army Medical Center, Washington, D. C. # Other peptones may be used.136 168 CULTURE MEDIA a. Adjust reaction of salts solution to pH 7.4 with N NaOH and autoclave at 121° C for 15 min. Filter through two thicknesses of filter paper to remove precipitate and recheck the pH. b. Combine 8 parts of salts solution with 1 part tryptose solution (1%) and sterilize at 121° C for 15 to 20 min. c. Before using, add sufficient sterile, inactivated (56° C for 30 min), slightly hemolyzed rabbit serum to give a final concentration of 10%. 90. Stuart's Broth for Leptospira, Modified!37*} Glycerol-Asparagine-Salt Solution: Glycartl lL (opIonal) “Lh oa vies seis eri vnvas ses son se 10 ml Sodium clorlde S.. ohn bvsbsinnvnmsd dos vase £oee 3.85 g AoASDBIBEING ovens ems woe bs bss bis was ows ss wamnsr ss 026 g Ammonium chloride, NH, Cl . coun cvisvmisivi ony innsh 0.54 g Magnesium chloride, MgCl,.6 HO .............. arr 08 g WORIEE . cos namms wed Dosim sing stm mbm tf nares oh 330 ml a. Add 340 ml of glycerol-asparagine-salt solution to 1,500 ml of distilled water. Then add 160 ml of M/15 Serensen’s phosphate buffer, pH 7.6. Sorensen’s M/15 Phosphate Buffer, pH 7.6 Disodium phosphate, Na,HPO, (M/15=9.47 g per BEE). ssn wrmmaleninsvi iv pop oils Sana Sone be sms 139.2 ml Monopotassium phosphate, KH,PO, (M/15=9.08 g per THEE) | i snnnvumvmiinvssvnis vi sts domninsiienhiosvns ons 20.8 ml TOE] comms shim Doki IE Pe Pe Ee gir Ritts 160.0 ml b. Mix solutions thoroughly and filter to remove precipitate. c. Dispense in desired amounts in tubes or flasks and autoclave at 121° C for 20 min. d. Just prior to inoculation, add sterile, inactivated (56° C for 30 min), slightly hemolyzed rabbit serum to a final concentration of 8-10%. + Modified by the Department of Veterinary Medicine, Walter Reed Army Institute of Research, Walter Reed Army Medical Center, Washington, D. C. 91. Chang's Broth and Semisolid Agar for Leptospiral®® Broth Medium Semisolid Medium Torplose Juounvernnsdirahmmp se sws Lg 08 g Liver extract (Lilly 343) ......... 07 g 05g Disodium phosphate, Na,HPO, ... 2 g 2g Monopotassium phosphate, KH,PO, 04 g 04 g Sodinny chloride ....iomaemmerinmy 4 g 3% AGRE wtih spine sitive smr anew es — 2 g WHEE o.vvunrvun vrs ssmsvaanss bus 1,000 ml 1,000 ml CULTURE MEDIA 169 a. Dissolve components in water with heat and autoclave at 121° C for 20 min. b. Cool and add 150 ml of sterile, inactivated horse serum and 15 ml of hemoglobin solution prepared by mixing 1 part horse red blood cells with 3 parts distilled water. Final reaction should be pH 7.3-7.5. c. Dispense as desired to sterile flasks or tubes. 92. Fletcher's Semisolid Agar for Leptospiral®* Peptone Component .. cuvies is os samisom ox + » wnmiaiees 1 2 » sivwinn 03 g Beef extract ............ 8 § § SERS Sh SEE 5 § EE 02 g Sodium chloride ......... on ee Eo ARI 05 g BIBI parts a to aman a a ee ats 15g WHEE tt saan do ra Sr dam ew a ae eid 920 ml a. Suspend ingredients in cold water and heat to boiling to dissolve, Autoclave at 121° C for 15 min. b. Cool the medium to 56° C and add 8-10% of sterile rabbit serum. c. Dispense in sterile screw-capped tubes in 5-7 ml amounts. Inactivate the whole medium the day following its preparation by placing the tubes in a water bath at 56° C for 1 hr. The final reac- tion should be pH 7.8-8.0. 93. Packer's Extract Broth for Erysipelothrix,14® Modified!4! Proteose peptone NO. 2 ...ivuiiiniiienrinennreinennnnnns 5g BEE CRITACE vvivninn sv 1 nainppenivie » slminmigiio ads Sai Mrstaim:s oni 3g TEVDIONE + ssinnisin + £4 24-5 AMNTES SE3 SRRSERIHE C3 53 Rolin 4 4 woe 5¢ Sodium chloride .. qs sawmamans 21s genase v's» same anes ve ssy 5g 1g GIUE0SE ;snmmrrs th ewer es os gma Th 1 Suen oF SPATE VVBLEE iciasnonivrivos viptesuns se iguitorein: pied age onsets Rie 4 oar STINE 8/0 80 Sa oToT 1,000 ml a. Dissolve all ingredients in water with heat, dispense in 0.75 ml amounts in screw-cap tubes and autoclave at 121° C for 15 min. Final reaction should be pH 7.6. 94. Packer's Sodium Azide-Crystal Violet Extract Agar for Erysipelothrix,'*! Modified!4? Proteose peptone No, 2 ....cvui sess ssspone eave syshopnns 5 ¢g Beef extract ..oenon iiss venammames v1 vanan es § § ores. 3. z TIVOIone oisrssmmeacs os 2 somemmues s + « suming » « « sesevdees 5 g Sodium chloride .......oieniiieri iia 5 g GIACOBE. ais inition sat 30nd ls 3 0A 0 E 43 033 Sarin s 5 ¢g Botlift’ oo. :ii0itniii sds Hm 150000 anddd al i snatlnis 1 g Crystal violet (10 ml of 0.1% aqueous solution) ........ 001 g Sodiuta azide, Nay ais hia viii vindionsn ids sain o4 g BEAT Tocovn svamisisn sists sake iul os 1 COSRERIATS 2 3 data 25 ig LE 1,000 ml 170 CULTURE MEDIA a. Dissolve all ingredients in water with heat, dispense in 100 ml amounts, and autoclave at 121° C for 15 min. b. Before inoculation, melt and cool to 50° C, add 10% citrated blood (bovine blood is preferable, as it is less readily hemolyzed in the presence of sodium azide), and pour plates. Final reaction should be pH 7.5=+. 95. Extract Gelatin for Erysipelothrix!4 TIVDIONE cons somsmurninss Semen iios ve FEE mRuEos Lh sm Senda 10 g BEET BHITACL! 5.0 summimns +58 0s swimmer vomimioie sn womens 3g GEILIN sltns oo i aan as sa smismmireisive waa ee ws PEE 120 g GUICOSE 4ivv sts suman sss spmmpuvns s 6s ssmves ve vam awaemss 5g Soditm ChIOTIAE «cuwvn ss ve svmsnmmnes s SrRRinvomes bt avsmkin 5g WVALEE aie ta ahmeronmma vss » SINAN e 0 kmisrelorn pris sldinislirwrasate 1,000 ml a. Dissolve all ingredients in water with gentle heating to avoid destruction of gelatin. b. Dispense in screw-cap tubes and autoclave at 121° C for 15 min. Final reaction should be pH 7.24. ¢. Stand tubes upright and allow to harden. Inoculate by stabbing medium. Incubate at 20° C. 96. Lead Acetate Agar for Hydrogen Sulfide Production by Erysipelothrix'4! Beef infusion agar (CM No. 6, pH 7.6) ....ccouvunn.... 1,000 ml Lead aceiate, C.P., Ph (C,H0,),3H,0 ..vevensrnesnin 05 ¢g Sodium thiosulfate, NagSoOg..euvrerueiiiiienniniinnnn. 25g a. Dissolve all ingredients with heat. Tube in amounts to give a 2 in. butt, autoclave at 121° C for 15 min, and stand tubes nearly upright for medium to solidify. b. Inoculate by stabbing medium. 97. Edwards' Extract Agar with Crystal Violet for Erysipelothrix,43 Modified44 Proteose peplone NO. 2 ...ueoniminnss sovmmnonn ss sna BEBE CRITATE hci s senatian ss s+ # mbisimins sven imiememin nnn ware TTIVDIOHE © wus 5 ov obiommman.sis o vam: v4 v4 rewnrovmey § Gi Sodium Chloflde. ...cosmvrsns sammmss ess Ss vummet is 6a CHITOSE unis vos 3 § 10RWREEE S 3 BRPRRRHERE $3 BEART wine BOUIN waves 3 sRBiit 35 Simei vine e o Sivinimersios yh Sw Crystal violet (2 ml of 0.1% aqueous solution)....... AGEL oovsmminmms s »anamany s ¥ 5 SePRR@RE 1 4 § GOREHES E506 00 EE Sem nnnwn 3 03.03 0% 09108 10% 0309 MN —* I] - . . . . — 3 CULTURE MEDIA 171 a. Dissolve all ingredients with heat, dispense in 100 ml amounts and autoclave at 121° C for 15 min. Final reaction should be pH 7.5%, b. Before use, melt and cool to 50° C, add 10% citrated blood, and pour plates. 98. Carbohydrate Base Broth for Erysipelothrix!4! Proteose peptone .......eveveiriiinrreessrecnensnnnes 10 g Sodium CHlotide .svsansmnsersves snus memes 25 g Potassing. chlotide, KCL .....eosmmnnvene sss onesie vroes 01 g Bromcresol purple (16 ml of 0.1% ethanolic solution). . 0.016 g WRLEE. cums on 200 shmop EGER Smows, sn fd smite s 4m 1,000 ml a. Dissolve ingredients with heat, adjust to pH 7.0, and tube in 10 ml amounts in tubes containing gas tubes. Autoclave at 121° C for 15 min and allow to cool. b. Add filter-sterilized carbohydrate solution to give a final con- centration of 0.5%. Final reaction should be pH 7.0. c. Incubate inoculated medium for 10 days. 99. Glycerol-Asparagine Medium, Proskauer and Beck, Modified!45* Monopotassium phosphate, KHoPOy «..vvvvvvenninnnn... 5 ¢ ATDOIOGINE muse ponmmw anv rns bs SE A 5 g Potassium sulfate, KoBO, wns snr anne swvis anisms spon 05g BIVREIBL. risswrmciore: « ncnssimimmmitinsis® Bio. METSIEEN E8 20s SETA ss § wiwite 20 ml WOLBE sunmmme s9s were es swum § 4S RuRBAET § 5 RES 1,000 ml a. Dissolve ingredients in the order given in water, care being taken that each ingredient is completely dissolved before the next is added. Adjust reaction to pH 7.0 with 40% NaOH, then add: Moagnesiom citrate, Mga (CaHz O00) vvvnnnnnnnivninvennnnns 15 ¢ b. Dispense in 9.6 ml amounts to tubes. Autoclave at 115° C (10 Ib) for 20 min. 100. Egg Yolk-Potato Flour Medium, American Trudeau Society (ATS) 146,147a Bod oll simnns 1 erenemsen wa wransens ot soem aimng wos va mami 500 ml Potato flour water containing 2% glycerol ................ 500 ml Malachite green, 1% in 50% alcohol .............ccouvunnnn. 20 ml a. Make potato flour water by adding 20 g of potato flour to 500 ml of water, to which 10 ml of glycerol has been added. Heat to boiling with constant stirring. Cool to 50° C. 172 CULTURE MEDIA b. Carefully clean fresh hen’s eggs with wet gauze, rinse them in alcohol, and separate the yolks from the whites. Add 1 whole egg to each 11 yolks until 500 ml of egg yolk-whole egg mixture is obtained (approximately 24 eggs). c. Add 500 ml of the egg yolk mixture to 500 ml of potato flour water, then add the 20 ml of malachite green. Mix thoroughly and dispense into sterile screw-cap tubes or vials. d. Sterilize and coagulate the medium by inspissation at 90° C for 1 hr. Incubate 48 hr to check sterility. Before use remove excess water of condensation. 101. Egg-Potato Flour Medium, Lowenstein-Jensen, Modified!48:14%% Potato Flour-Salt Solution: Monopotassium phosphate, KHyPO, ...covvvvni..t. 24 ¢g Magnesium sulfate, MgSO, 7H,O ...vvvernevrsien. 024 g Magnesium citrate, Mgg(CgHgOp)g.uuveeininnannnn. 06 g ABPITEEING 2 snsnnns s svsnmensss aps eames esas sameness 36 g GITCEIOL 00 eh viens sia snailbhin i vil namineisa dss wana via's 12° ‘ml Water .ivniavecnissbadbieaintorssie nade saihio sans 600 ml Potato Flour cos evssismis@onesas aves anenss seen sve 30 g Dissolve salts and asparagine in water, add glycerol and potato flour, and autoclave the mixture at 121° C for 30 min. Final Medium: Potato flour-salt solution (sterile) .........c...ccoen.. 612 ml Homogenized whole egg mass .......eivveveeernennss 1,000 ml Malachite green, 2% aqueous solution ................ 20 ml a. Use eggs not more than 1 week old. Clean by vigorous scrub- bing in a 5% soap and soda solution. Leave the eggs in the soap- soda solution for 30 min, then run cold water over them until the water becomes perfectly clear. b. Break eggs into a sterile flask and homogenize completely by shaking (a Waring blendor or sterilized kitchen egg beater is an excellent tool for this purpose). Filter through four layers of sterile gauze. c. Add 1 liter of homogenized whole eggs to the flask of the potato flour-salt solution, which has been cooled to room temperature, and to this add 20 ml of malachite green solution. Mix thoroughly and let stand for 1 hr at room temperature. d. Tube this medium by means of a sterile aspirator bottle, funnel with bell attachment, or similar device, using 5-6 ml for each 150 mm pyrex test tube. Solidify by inspissation at 85° C for 50 min. Check for sterility by incubation at 35° C for 48 hr. CULTURE MEDIA 173 e. Store medium in refrigerator. Storage at 5° C for as long as 11 months has no appreciable effect on time of appearance of growth or on the number of typical colonies of tubercle bacilli that will develop. 102. Tween-Albumin Medium50% Monopotassium phosphate, KHyPO,.....coovvivnnnnn. 1.5 g Disodium phosphate, NagdHPO, «..evvvvnnnnvnnnnn.n. 1.5 g 1-Glutamic acid (as sodium Salt) v..iisvvmvanrisonsns 0.5 g Ammonium sulfate, (NH) 550, ...cvvvviiviviiinnan 0.5 g Sodium citrate, NazCeH 0,2 HO ................. 04 ¢g Ferric ammonium citrate (green scales) ............. 004 ¢g Magnesium sulfate, MgSO.7 Hy0 .ovvvvnnnnnnnnnen. 005 g Pyridoxine-HCl (1 ml of 0.1% aqueous solution) .... 0.001 g Biotin (0.5 ml of 0.1% aqueous solution) ............ 0.0005 g Tween 80 (5 ml of 10% aqueous solution stored under refrigeration less than 4 weeks)t ........c.vuvnn... 0.05 % VWALEE «cov vos vnimdimsin s sn aivindinn 3 wabinaelnns soo iiss 1,000 ml a. Dissolve ingredients, in the order given, in the water. Adjust the reaction to pH 6.6, if necessary, with HCI. b. Autoclave at 121° C for 15 min, cool to 45° C, then add: Albumin (Plasma Fraction V, Armour Laboratories) : 100 ml of a sterile 5% solution in 0.85% NaCl solution, neu- tralized with sodium hydroxide solution. If used in media with Tween 80, inactivate lipase by heating in a water bath at 56° C for 30 min. Sterilize by filtration and test sterility in thioglycolate medium (CM No. 19) 5 g Glucose: 10 ml of an autoclaved 50% solution in 0.005 M CUTIE ARIA oiiis hanno waiininriis sons snsnitis wie i shnia doles 5g Catalase}: 3 ml of 0.1% solution in 0.85% NaCl solution steri- lized by filtration through a sintered-glass filter ...... 3 ug/ml c. Dispense in sterile flasks or screw-cap tubes and incubate to check sterility. Polysorbate 80, U.S.P., product of Atlas Chemical Industries, Inc., Wil- mington, Del. $Obtainable as Catalase “Crude” from Nutritional Biochemicals Corp., Cleve- land 28, Ohio. 103. Egg-Potato Starch Medium, Petragnani, Modified**™* Mixture A ME rvs cnaurenmivins sn simmmnms 3 5 § svmsie sm + s SSEIENRES $58 5 225 ml Potato SBICH wwuvmast v4 0 stsitn 5+ so ton vaon 4s +s manimtinsish 4 9..& BOPIOIR .. . commits oo bikiomempyiiie: 4 » Kener, Toe TATE fas 15g Diced DOIAtO , wmmemns 14 5 enninans $43 550a0 08 s 10 wreesivsas 3 150 g 174 CULTURE MEDIA Heat in a double boiler for 1 hr, stirring constantly. Mixture B Pods (yolks andi Whites) oie doivriivrinnivanins bern sins sents 8 eggs GUVCRTOL, CPi vvvmimmiin sanisni sg wenn sums sms sm vmmmsin e 18 ml Malachite green, 2% aqueous SOMItION +.vvevnvrrnennnsonnn 15 ml Mix well. Mixture C GIICOBE vss ils nmaimin ns 3.55 wnwininm as os wile wove sias + one imma tas 15g ASDSIEITING Lh s vniaitems + sas wmmie s 5 Spweieiia vevinidutesse vs 15 ¢ WWHIHE ois hv ole booiama Wams a3. RIAITE® 4 4 ww baiiachin:s vo mniivreriraliy 50 ml Add ingredients to the water and warm until all are dissolved. Complete Medium: Combine mixtures A, B and C in a sterile flask. Pass through one layer of gauze. Dispense in sterile tubes and sterilize in a slanted position. Sterilize by inspissation for 1 hr at 80° C. 104. 7H-10 Medium, Middlebrook and Cohn**"* Base Solids: Magnesium sulfate, MgSO,7 HyO ...evvvvrvnnnnnnn.. 05g Ferric ammonium citrate (green scales) .............. 04 g Sodium citrate, NagCqHz072 HO ..unvvnnnnnnn..... 40 g Ammonium sulfate, (NH )580, ..vovrvrrrernnrnrssen 50g 1-Glutamic acid (as sodium salt) ....civivevuivvrsvenes 50 g Disodium phosphate, Na,HPO, .....covvvviivnrenennnn 150 g Monopotassium phosphate, KHo,PO, ..........coontn. 150 g Store at room temperature after mixing and indicate on label that 0.45 g will make up to 100 ml with distilled water at pH 6.6 if 1.5 g of Baltimore Biological Laboratories agar is used per 100 ml. Oleic Acid-Albumin Complex (5%) :* a. Dissolve 0.12 ml of oleic acid in 10 ml of N/20 NaOH by rotat- ing in a 25 ml flask. b. Prepare a 5% albumin solution by adding 5 g of bovine albumin Fraction V to 95 ml of 0.85% NaCl solution. c. Add 5 ml of the oleic acid solution to 95 ml of the 5% albumin solution and adjust the reaction to pH 6.8. d. Sterilize by filtration through a Seitz filter and dispense in sterile tubes. e. Incubate at 35° C overnight. Inactivate in a 56° C water bath for 30 min, then store at 4° C. CULTURE MEDIA 175 Glucose Solution (50%): Dissolve 50 g of glucose in 60 ml of distilled water, then add 1 ml of a 10% solution of citric acid. Autoclave for 10 min at 121° C. Heat-Labile Constituents: Oleic acid-albumin complex (5%) ......ccvvnvennnennn 10 ml Glycerol, CP. init iii ii ieee 0.5 ml Glucose (5096 SOIIBIONY . vio vovnines sins emving sss os smn 04 ml Pyridoxine HCl (100 pg per ml) ....v.nuvvvssessnnnss 1.0 ml Biotin (50 pg per-ml) cosvsvvniviisissomavssnsss arms 1.0 ml Malachite green (100 pg per ml) .....ovvvineennnn.n. 1.0 ml Catalase, technical (100 ug per ml) ......c.ovvvninnnnn.. 0.3 ml Filter to sterilize and store in sterile tubes each containing 13.5 ml. Preparation of Complete Medium: a. Weigh out 0.45 g of base solids into a 250 ml Erlenmeyer flask; dissolve in 100 ml of warm distilled water. The reaction should be pH 6.6. b. Add 1.5 g of agar (Baltimore Biological Laboratories) to each 100 ml of the dissolved base solids. c. Autoclave this solution at 115°-121° C (10-15 1b) for 10 min. d. Add the contents of one tube (13.5 ml) of heat-labile con- stituents to each flask containing 100 ml of sterile, cooled (less than 50° C) agar-base solids mixture. Mix without forming excess bubbles. e. Dispense into sterile tubes or plates and allow to solidify. 105a. Sabouraud's Agar, Modified!5! NEOPEPLONE + vette ieee ee teat ce eeeeeeeaeneeenenns 10 g GLUIGORE | , wonton 25.4 3 $5000 2:0 5.2 5.0 5 SERIE 2 5 # 2.00 mfisin e § 5 2.0 wn 20 g ALAN iiramamvm aes San p45 5843 ROR E 156 FRRTE LETT AEB 20 g er rrr iss nero sn emer ae frre ted ov 7 ee de 1,000 ml a. Add ingredients to the water and dissolve the agar with heat. b. Autoclave at 118° C (12 1b) for 15 min. Do not overheat. Dispense as desired. The final reaction should be pH 6.5-7.0. 105b. Modified Sabouraud's Agar with Chloramphenicol'52 Add 0.05 mg per ml (0.05 g per liter) of chloramphenicol to CM No. 105a prior to autoclaving. 105¢c. Modified Sabouraud's Agar with Cycloheximide!®3 Add 0.5 mg per ml (0.5 g per liter) of cycloheximide (Acti-dione) to CM No. 105a prior to autoclaving. 176 CULTURE MEDIA 106. Cornmeal Agar®* CortimEnl’ came dues iin tne Sh anh mn a ASR Banh ee Ea ck #4 40 g AGEr [Ll eke bens sr ida rei hiue aes vit sen hess vie 20 g LS I he J cn NE ow oT. I a 1,000 ml a. Simmer 40 g of yellow cornmeal (enriched or unenriched) in 1 liter of water for 1 hr. b. Filter through eight layers of gauze and then through coarse filter paper (E & D folded filter paper No. 192). Restore to volume. c. Add the agar and dissolve by heating to boiling point. d. Dispense in tubes or flasks as desired and autoclave at 121° C for 15 min. The final reaction is about pH 6.6. 107. Potato Glucose Agar for Fungi* SUCed DOLBIOEE ' John nnmusvscdpiavin sien snn vom earumnes 300 g GHICOER 10iitis sir vivod omimcnipin Simin Slo mit ondieie 4 pi de Reve Silncnst 10 g BEAT saidirisrtss tsi iss sss shaiads sede mares d is manne 15¢g EL a. 1,000 ml a. Boil potatoes in water for 15 min, filter through cotton, and make up volume to 1,000 ml. b. Add dry ingredients and dissolve agar with heat. No adjustment of pH is necessary. c. Dispense in tubes or flasks as desired and autoclave at 121° C for 15 min. 108. Mycophil or Mycological Agar®*®* Papaic digest of soy bean meal ... Lill cabenaeade sini 10g GIICOBE un vad aaasas sx vvas ses Wala RES va A RREEATS STEWS 10 g AAT orevs iene duaiienrrcos vs panies ive w rie sale syns 15-16 g WVREBE | rid ties wininiiin sed mois wna dois oan wmf wwe mind 1,000 ml Dissolve ingredients in water with heat and autoclave at 121° C for 15 min. Final reaction should be pH 7.0. 109. Littman's Oxgall Agar!55* Pepione COMPBONEHL wovvvesrssrpssserss wrsnrmwsny es vans 10 g CHUB vive vim Hogmstimincom momar a bib iapuinss: iain wis owsbinspronmswiesn wwisinie 10 g Oxgall, dehydrated (... 0. viii vines si assis inns Pr esvns 15 g Crystal violet (1 ml of 1% aqueous solution) .......... 0.001 g CL I PA EEL LA SR NE SEE AEM 20 g WVBHBE ii ue vin mvminmimmmiinioinmaimmmin mins wd mi ible et acs 1,000 ml a. Add dry ingredients to water and dissolve with heat. Add the crystal violet solution. b. Dispense measured volumes to flasks or screw-cap bottles and autoclave at 121° C for 15 min. Final reaction should be pH 7.0. CULTURE MEDIA 177 c. For use, melt the agar base, cool to 45° C, and add 30 units of streptomycin per ml of medium. Dispense 30 ml of the complete medium per petri plate and allow plates to stand at room temperature for 6-8 hr before inoculation. 110. Rice-Tween 80 Agar!s® “Cream of Rice” or vitamin-free rice .....c.veveevennennns 10g 1 pe 10 g TED WHILE hus sunmmitimemes ss svmmtimmns 4 AvEsRARTos so SOOCmE § 1,000 ml Tween 80 (Polysorbate 80, USP.) ...cvviviviivnnennn... 10 ml a. Heat water to boiling, sprinkle in Cream of Rice, boil for 30 sec, then let stand for 30 sec. b. Filter through cotton and restore volume of the filtrate to 1,000 ml. ¢. Add the agar and Tween 80, dissolve by heating to boiling, then autoclave at 121° C for 15 min and pour plates. See reference for “Slit” method of inoculation. I'11. Glucose-Asparagine Broth for Histoplasmin and Tuberculin 157-159 1-ASPRTAGUIE «tn ov visinioints 05.4 50s bie atEss ns mm wmioin ox » wrk bial 14 g Dipotassium phosphate, KogHPO, .....ovviuniniinnnen. 131 ¢g Sodium citrate, NagCqHz07.5% Hy0 ..vvnvvvvnnnnnn... 0.90 g Magnesium sulfate, MgSO,.7 HO ...oovnvunvicessrnies 1.50 g Ferric citrate, U.S.P. VIII, scales .........oevvvvnnenn. 0.30 g Glycerol, U.S.P. (for histoplasmin) ............eevn.n. 25 ml (for coecidioldind «.5. seamen is aname 25 ml (for tuberctllin). «oui idnmiiine sd vrmuse 100 ml GlICO8E: covrersmmmmmmr ratios amiss testo 10 g Witter, 10 SHORE. co navn im ivram pret vam desmsimymmmhy 1,000 ml a. Dissolve asparagine in 300 ml of water heated to 50° C. Dis- solve each of the other salts separately in 25 ml of water (ferric citrate in hot water) and add each to the hot asparagine solution in the order listed, mixing well after each addition. Add glucose and glycerol and make up to 1,000 ml. b. Dispense in 3 liter Fernbach culture flasks, 1,500 ml per flask, and autoclave at 115° C (10 1b) for 25 min. 112. Glucose-Peptone Broth for MR-VP Tests* Proteose peptone or polypeptone .............ceeeeeeenn.n. 7g CRICOSE .ivfina mein eam gmat dval fo wins tomo wn whivmeipin 5g Dipotassium phosphate, KgHPO, .....covvvivninnnnnnn... 5g WBLET nvunsvmviysmmses soem shad dummies sss sieaesen nhs 1,000 ml Dissolve in water, tube in 5 ml amounts, and autoclave at 121° C for 15 min. The final reaction should be pH 6.9%. 178 CULTURE MEDIA 113. Sabouraud's Maltose Agar* Neopeptone, proteose peptone, or polypeptone.............. 10 g Maltose ...uunmens res srsamsnes +» 2 vodmmbenony s seaunwses ss 40 g AUT ww ov's svwnmninies 3 SEOBRETe #5 Muse m pr 4 wibby 15¢g MVELEE ve vuon womminivrasns & 2 8 SISTeslert § + & MRGIRIRE o4 5 5 Sisdosmonses wwe » 1,000 ml a. Dissolve ingredients in water with heat and frequent agitation. Dispense in tubes and autoclave at 121° C for 15 min. The final reaction will be pH 5.6. b. Cool tubes in a slanted position. This medium is used for en- hancement of pyocyanin production by Pseudomonas aeruginosa. 114. Chopped Meat Medium? a. Grind finely 1 1b of fat-free beef heart or muscle. Add meat to 500 ml of boiling N/20 NaOH (XN /15 for muscle) and boil for 20 min. b. Allow to cool, remove fat, and strain through muslin. Allow the meat to dry partially. c. Adjust pH of the fluid to 7.5 and add 1% peptone. d. Tube the meat, about 2 in. deep, in each tube and add the pep- tone broth until the level is about 1 in. above the level of the meat. e. Heat in a boiling water bath for 30 min and then autoclave at 121° C for 15 min. 115. Blood-Glucose-Cystine Agar, Francis, ’* Modified62 Veal infusion, double strength ...cvvivsesrsvamimes cisasns 1,000 ml Pancreatic digest of cagelnn, U.S. P. XVI ...screvencssnsins 10 g Sodium chloride .....eovvnvsirvsssnsrenscrsvreveners nse 5g Cystine, or cystine hydrochloride ............ccoevvvnnn... lg BTAE Saint Si linelesns wins Sesnivenenioss 8 wi HOARE BA SR SARE Dice it 20 g GIUCOBE ov uiimmvn is Drm smmt ibis Basking bask» PaLsE PEE S050 10 g Rabbit or horse DIood uueius sisvunsvnves wos snmnse or snnvns 80 ml a. Proceed as for beef heart infusion (CM No. 23), except that 1 kg of ground veal is infused with 1,000 ml of water. b. Dissolve the peptone, sodium chloride, and agar in 900 ml of the veal infusion with heat and frequent agitation. Add the cystine dissolved in a small amount of 0.1 M NaOH. c. Dissolve the glucose in 100 ml of the veal infusion and sterilize by filtration. d. Adjust the reaction of the base agar (b) to pH 7.6-7.8 and autoclave at 121° C for 20 min. Cool to 60° C and add the blood and glucose solution. e. Maintain the complete medium at 60° C in a water bath for 3 hr with frequent mixing to insure complete solution of the cystine. CULTURE MEDIA 179 f. Dispense in tubes and plates as desired and incubate one or two representative plates or tubes at 35° C overnight to check sterility. 116. Extract Broth for Antibiotic Assays* Boel extrant oorvinsimnnit poss seme suomi smsae vines 15 ¢ Peplone (Gelysate or Bacto) .uesvivrsensvnnssanavnns 5 9 Sodium chloride ....vviriiiriieiinienrineeenannennns 35 g GIBEEER. cvrvcrasovimmimmimnsistadl gies BURT 4 2 So SERIE +5: HAT 1 g Yeast CRIACE «vos wniviies 25:0 355 EHEL00E $0.5 sn minies +55 Twn 15 ¢ Monopotassium phosphate, KH,POy ...ovvnvvivnenin, 132 ¢ Dipotassium phosphate, MoHPO, cuts ves canviosne ss annin 3.68 g BVBIBE vires vost 0 wnemisintusons sin win Bininii00 & 4 40 4 HBRIGEI £3 2 SRE 1,000 ml a. Dissolve components in water with frequent agitation and heat. b. Dispense as desired and autoclave at 121° C for 15 min. The final reaction should be pH 7.0. REFERENCES 1. 10. 1, 12. 13, 14. 15, HinsaeLwoop, C. N. The Chemical Kinetics of the Bacterial Cell. Ox- ford: Clarendon Press, 1946. Porter, J. R. Bacterial Chemistry and Physiology. New York: John Wiley & Sons, 1946. Difco Manual (9th ed.). Detroit, Mich.: Difco Laboratories, Inc., 1953. Hook, A. E., and FaBian, F. W. Chemical and Biological Studies on Peptones. Mich. Agr. Exp. Sta. Tech. Bull. No. 185, 1943, 31 pp. Stokes, J. L., GunnEess, M., and Foster, J. W. The Vitamin Content of Ingredients of Microbiological Culture Media. J. Bact. 47:293-299, 1944. Brewer, J. H. Vegetable Bacteriological Media as Substitutes for Meat Infusion Media. J. Bact. 46:395-396, 1943. Products for the Clinical Laboratory. Culture Media and Laboratory Preparations. Chicago Heights, Ill.: Consolidated Laboratories, Inc., 1961. Goonrow, R. J., Braun, W,, and Mika, L. A. The Role of D-alanine in the Growth and Variation of Brucella abortus. Arch. Biochem. 30: 402-406, 1951. GLADSTONE, G. P. Inter-relationships Between Amino Acids in the Nutri- tion of B. anthracis. Brit. J. Exper. Path. 20:189-200, 1939. KnicuT, B. C. J. G. Bacterial Nutrition. Special Report Series No. 210. Brit. Med. Res. Council, 1936, 182 pp. PapPENHEIMER, A. M,, Jr. Diphtheria Toxin. III. A Reinvestigation of the Effect of Iron on Toxin and Porphyrin Production. J. Biol. Chem. 167 :251-259, 1947. Rickes, E. L., et al. Vitamin By, a Cobalt Complex. Science 108: 134, 1948. —— Comparative Data on Vitamin B;, from Liver and from a New Source, Streptomyces griseus. Science 108 :634-635, 1948. Lamanna, C., and Marrerre, M. F. Basic Bacteriology: Its Biological and Chemical Background (2nd ed.). Baltimore, Md.: Williams & Wil- kins, 1959. Furiter, A. T., and Maxtep, W. R. The Production of Haemolysin and Peroxide by Haemolytic Streptococci in Relation to the Non-Haemolytic Variants of Group A. J. Path. & Bact. 49:83-94, 1939. 180 16. 17. 18. 19. 20. 21. 22. 23. 24. 2s, 26. 27. 28. 29. 30. 3% 32. 33. 35. 36. 37. 38. CULTURE MEDIA Lepper, E.,, and Martin, C. J. The Chemical Mechanisms Exploited in the Use of Meat Media for the Cultivation of Anaerobes. Brit. J. Exper. Path. 10:327-334, 1929. VALLEY, G., and Rerrcer, L. F. The Influence of Carbon Dioxide on Bacteria. J. Bact. 14:101-137, 1927. GLADSTONE, G. P., FiLpes, P., and RicaHARDSON, G. M. Carbon Dioxide as Essential Factor in the Growth of Bacteria. Brit. J. Exper. Path. 16:335- 348, 1935. RamN, O. Notes on the CO,-Requirement of Bacteria. Growth 5:113- 118, 1941. Woop, H. G., and WerkmAN, C. H. The Utilization of CO, by the Propionic Acid Bacteria in the Dissimilation of Glycerol. J. Bact. 30: 332, 1935. Woon, H. G., WerkMAN, C. H., Hemingway, A. and Nir, A. O. Fixation of Carbon Dioxide by Pigeon Liver in the Dissimilation of Pyruvic Acid. J. Biol. Chem. 142:31-45, 1942. Frrcuson, W. Optimal Carbon-Dioxide Tensions for Primary Isolation of the Gonococcus: Response of the Organism to Other Gaseous En- vironments. Am. J. Syph. 29:19-55, 1945. BuLrocH, W., in A System of Bacteriology in Relation to Medicine. Vol. 1. London: H. M. Stationery Office, 1930, pp. 38-41. Perkins, J. J. Principles and Methods of Sterilization. Springfield, IIL: Chas. C Thomas, 1956. (a) pp. 64-65; (b) pp. 54-57. McCurrocH, E. C. Disinfection and Sterilization (2nd ed.). Philadelphia, Pa.: Lea & Febiger, 1945. ReopisH, G. F. Antiseptics, Disinfectants, Fungicides, and Chemical and Physical Sterilization (2nd ed.). Philadelphia, Pa.: Lea & Febiger, 1957. Sykes, G. Disinfection and Sterilization. New York: D. Van Nostrand, 1958. Levine, M. Two Agar-less Media for the Rapid Isolation of Coryne- bacterium and Neisseria. J. Bact. 46:233-237, 1943. Manual of Microbiological Methods. Society of American Bacteriologists, Committee on Bacteriological Technic. New York: McGraw-Hill, 1957. Standard Methods for the Examination of Dairy Products (11th ed.). New York: American Public Health Association, 1960. Crark, W. M. The Determination of Hydrogen Ions (3rd ed.). Balti- more, Md. : Williams & Wilkins, 1928. Horan, W. A. Bacteriology: Storage of Media. Am. J. M. Technol. 25:413, 1959. GUTIERREZ-VASQUEZ, J. M. Los Medios de Agar-Carbon, Agar-Sangre, y Lowenstein-Jensen-Holm en el Cultivo de Mycobacterium tuberculosis. II. Resultados Comparativos por el Método de las Diluciones Seriadas. Rev. Latinoamer. Microbiol. 1:125-136, 1958. Products for the Microbiological Laboratory. Baltimore, Md.: Baltimore Biological Laboratory, Inc. KarLstrROM, A., BRANDON, G. R., and Levin, W. Some Observations on the Use of Dehydrated Culture Media. Pub. Health Lab. 15:175-177, 1957. KENDRICK, P. Personal communication, 1960. WERNER, G., CorNFELD, D., HuBBArD, J. P., and Rakg, G. A Study of Streptococcal Infection in a School Population: Laboratory Methodology. Ann. Int. Med. 49:1320-1331, 1958. ScHAUB, I. G., ef al. Ecologic Studies of Rheumatic Fever and Rheumatic Heart Disease. Procedure for Isolating Beta-Hemolytic Streptococci. Am. J. Hyg. 67 :46-56, 1958. CULTURE MEDIA 181 39. 40. 41. 42. 43. 4. 45. 46. 47. 48. 49. 50. 51. 52, 53. 54. 58. 56. 57. 58. 59. 61. 62. Hunter, D. H.,, Brak, E. B, and Rust, J. H, Jr. A Comparison of Culture Technics for the Isolation of Beta-Hemolytic Streptococci from Acute Respiratory Infections. Bact. Proc. 1962, p. 81. RucuHort, C. C, KaLLas, J. G., CHINN, BEN, and Courter, E. W. Coli- Aerogenes Differentiation in Water Analysis. II. The Biochemical Dif- ferential Tests and Their Interpretations. J. Bact. 22:125-181, 1931. Vera, H. D. Relation of Peptones and Other Culture Media Ingredients to the Accuracy of Fermentation Tests. A.J.P.H. 40:1267-1272, 1950. Brewer, J. H., and LiLey, B. D. Paper presented before a meeting of the Maryland Association of Medical and Public Health Laboratories Dec. 2, 1049. Liey, B. D.,, and Brewer, J. H. The Selective Antimicrobial Action of Phenylethyl Alcohol. J. Am. Pharm. A. (Scient. Ed.) 42:6-8, 1953. Barser, M., and Kuper, S. W. A. Identification of Staphylococcus pyogenes by the Phosphatase Reaction. J. Path. & Bact. 63 :65-68, 1951. BLAIR, J. E. Personal communication, 1960. CaapMAN, G. H. The Significance of Sodium Chloride in Studies of Staphylococci. J. Bact. 50:201-203, 1945. DoLMAN, C. E.,, WiLson, R. J., and Cockrorr, W. H. A New Method of Detecting Staphylococcus Enterotoxin. Canad. Pub. Health J. 27:489- 493, 1936. Dorman, C. E., and WiLsoN, R. J. Experiments with Staphylococcal Enterotoxin. J. Immunol. 35:13-30, 1938. ———— The Kitten Test for Staphylococcus Enterotoxin. Canad. Pub. Health J. 31:68-71, 1940. Standard Methods for the Examination of Water and Sewage (9th ed.). New York: American Public Health Association, 1946. HircHENs, A. P. Advantages of Culture Mediums Containing Small Per- centages of Agar. J. Infect. Dis, 29:390-407, 1921. CasMmaN, E. P. A Noninfusion Blood Agar Base for Neisseriae, Pneu- mococci, and Streptococci. Am. J. Clin. Path. 17:281-289, 1947. Brown, J. H. The Use of Blood Agar for the Study of Streptococci. Monograph No. 9, The Rockefeller Institute of Medical Research, 1919, 350 pp. Ferrer, A. E, and Stevens, D. A. Sheep Blood Agar for the Isolation of Lancefield Groups of Beta-Hemolytic Streptococci. J. Lab. & Clin. Med. 39:484-491, 1952. WANNAMAKER, L. W., A Method for Culturing Beta-Hemolytic Strep- tococci from the Throat. New York: American Heart Assn., 1956. Hospital-Acquired Staphylococcal Disease. Recommended Procedures for Laboratory Investigation. Atlanta, Ga.: Communicable Disease Center, U. S. Dept. Health, Education, and Welfare, 1958 page viii. MirLianN, S. J, Batowin, J. N., Ruemms, M. S., and Weiser, H. H. Studies of the Incidence of Coagulase-Positive Staphylococci in a Normal Unconfined Population. A.J.P.H. 50:791-798, 1960. Krumwiepg, E,, and KuTTNER, A. G. A Growth-Inhibitory Substance for the Influenza Group of Organisms in the Blood of Various Animal Species. The Use of the Blood of Various Animals as a Selective Medium for the Detection of Hemolytic Streptococci in Throat Cultures, J. Exper. Med. 67 :429-441, 1938. Kenprick, P. L. Whooping Cough. Sixth Annual Yearbook, American Public Health Association. New York: The Association, 1936. ‘ Brewer, J. H. A Clear Liquid Medium for the “Aerobic” Cultivation of Anaerobes. J. Bact. 39:10, 1940. Dorman, C. E, Personal communication, 1959. WiLson, A. T. Personal communication, 1960. 182 63. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 85. 86. CULTURE MEDIA Toop, E. W, and Hewirr, L. F. A New Culture Medium for the Production of Antigenic Streptococcal Haemolysin. J. Path. & Bact. 35: 973-974, 1932. Swrrr, H. F. “The Streptococci,” in Bacterial and Mycotic Infections of Man. Philadelphia, Pa.: Lippincott, 1948. Barnes, E. M. Methods for the Isolation of Faecal Streptococci (Lance- field Group D) from Bacon Factories. J. Appl. Bact. 19:193-203, 1956. Niven, C. F. Personal communication, 1960. Pike, R. M. Isolation of Hemolytic Streptococci from Throat Swabs. Experiments with Sodium Azide and Crystal Violet in Enrichment Broth. Am. J. Hyg. 41:211-220, 1945. FiLpes, P. New Medium for the Growth of B. influenzae. Brit. J. Exper. Path. 1:129-130, 1920. Tujy6rrA, T. and Avery, O. T. Studies on Bacterial Nutrition. II. Growth Accessory Substances in Cultivation of Hemophilic Bacilli. J. Exper. Med. 34:97-114, 1921. LevintHAL, W., and FernBacH, H. Morphologische Studien an In- fluenza-bacillen und das 4tiologische Grippeproblem. Ztschr. Hyg. 96: 456-519, 1922. PirtMAN, M. Variation and Type Specificity in the Bacterial Species Hemophilus influenzae. J. Exper. Med. 53:471-492, 1931. Diagnosis of Gonococcal Infections. Circular 121. Detroit, Mich.: Difco Laboratories, Sept. 1947. MUELLER, J. H., and HintoN, J. A. A Protein-Free Medium for Primary Isolation of the Gonococcus and Meningococcus. Proc. Soc. Exper. Biol. & Med. 48:330-333, 1941. Prizer, L. R.,, and SterreN, G. I. Modification of the Horse Plasma Hemoglobin Agar for Primary Culture of the Gonococcus. Usefulness of Nile Blue A in This Medium. J. Ven. Dis. Inform. 23:224-226, 1942. THAYER, J. D., Scuusert, J. H, and Bucca, M. A. The Evaluation of Culture Mediums for the Routine Isolation of the Gonococcus. J. Ven. Dis. Inform. 28:37-40, 1947. CARPENTER, C. M,, in Diagnostic Procedures and Reagents (3rd ed.). New York: American Public Health Association, 1950, pp. 32-33. HirscHBERG, N. Use of Solid Medium for Transportation of Specimens for Gonococcus Culture. Am. J. Syph. 29:64-70, 1945. Buck, T. C, Jr. A Transport Medium for Neisseria gonorrhoeae. J. Ven. Dis. Inform. 28:6-9, 1947. Doucras, S. R. A New Medium for the Isolation of B. diphtheriae. Brit. J. Exper. Path. 3:263-267, 1922-23. CARPENTER, C. M,, in Diagnostic Procedures and Reagents (2nd ed.). New York: American Public Health Association, 1945. ANDRADE, E. Influence of Glycerine in Differentiating Certain Bacteria. J. Med. Res. 14:551-556, 1905-06. Prizer, L. R., STEFFEN, G. I, and Krein, S. Simple and Efficient Trans- port Method for Gonorrheal Specimens. Pub. Health Rep. 64:599- 603, 1949. Stuart, R. D., TosuacH, S. R, and Parsura, T. M. The Problem of Transport of Specimens for Culture of Gonococci. Canad. J. Pub. Health 45:73-83, 1954. KLiGLER, I. J. A Simple Medium for the Differentiation of Members of the Typhoid-Paratyphoid Group. A.J.P.H. 7:1042-1044, 1917. Russell, F. F. The Isolation of Typhoid Bacilli from Urine and Feces, with the Description of a New Double Sugar Tube Medium. J. Med. Res. 25:217-229, 1911-12. Harpy, A. J. Personal communication, 1960. CULTURE MEDIA 183 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. HajNa, A. A. Triple Sugar Iron Agar Medium for the Identification of the Intestinal Group of Bacteria. J. Bact. 49:516-517, 1945. Krumwiepg, C. J. and Korn, L. A. A Triple-Sugar Modification of the Russell Double-Sugar Medium. J. Med. Res. 37:225-227, 1917. SurkiN, S. E, and Witerr, J. C. A Triple Sugar Ferrous Sulfate Medium for Use in Identification of Enteric Organisms. J. Lab. & Clin. Med. 25 :649-653, 1940. Horr-Harris, J. E., and Tracug, O. A New Culture Medium for the Isolation of Bacillus typhosus from Stools. J. Infect. Dis. 18:596-600, 1916. Levine, M. The Effect of Concentration of Dyes on Differentiation of Enteric Bacteria on Eosin-Methylene Blue Agar. J. Bact. 45:471-476, 1943. LerrsoN, E. New Culture Media Based on Sodium Desoxycholate for the Isolation of Intestinal Pathogens and for the Enumeration of Colon Bacilli in Milk and Water. J. Path. & Bact. 40:581-599, 1935. Bropik, J. Observations on the Differential Inhibition of Coliform Bacilli and Rough Variants of Intestinal Pathogens. J. Gen. Microbiol. 2:1-7, 1948. Wirson, W. J., and Bramwr, E. M. McV. Further Experience of Bismuth Sulfite Media in the Isolation of Bacillus typhosus and Bacillus para- typhosus from Feces, Sewage and Water. J. Hyg. 31:138-161, 1931. Mackie, T. J., and McCartney, J. E. Handbook of Practical Bacteriology (9th ed.). Baltimore, Md.: Williams & Wilkins, 1953, p. 183. Haj~a, A. A. Preparation and Application of Wilson and Blair's Bis- muth Sulfite Agar Medium. Pub. Health Lab. 9:48-50, 1951. —— and Damon, S. R. New Enrichment and Plating Media for the Isolation of Salmonella and Shigella Organisms. Applied Microbiol. 4:341-345, 1956. MacConkey, A. T. Lactose-Fermenting Bacteria in Faeces. J. Hyg. 5:333-379, 1905. LesTER, V., Jurcens, A., and KristEnseN, M. The Use of Trypsinized Casein, Bromthymol Blue, Bromcresol Purple, Phenol Red, and Brilliant Green for Bacteriological Nutrient Media. Brit. J. Exper. Path. 6:291- 299, 1925. KavurrmMaNN, F. Weitere Erfahrungen mit dem Kombinierten An- reichrungsverfahren fiir Salmonella-bacillen. Ztschr. Hyg. 117:26-32, 1935. Garton, M. M,, and Quan, M. S. Salmonella Isolated in Florida during 1943 with the Combined Enrichment Method of Kauffmann. A.J.P.H. 34:1071-1075, 1944. KaurrMANN, F. Die Bakteriologie der Salmonella-Gruppe. Copenhagen: Einar Munksgaard, 1941. LerrsoN, E. New Selenite Enrichment Media for the Isolation of Typhoid and Paratyphoid (Salmonella) Bacilli. Am. J. Hyg. 24:423-432, 1936. Simmons, J. S. A Culture Medium for Differentiating Organisms of Typhoid-Colon-Aerogenes Groups and for Isolation of Certain Fungi. J. Infect. Dis. 39:209-214, 1926. RusticiaN, R., and Stuart, C. A. Decomposition of Urea by Proteus. Proc. Soc. Exper. Biol. & Med. 47:108-112, 1941. CHrisTENSEN, W. B. Urea Decomposition as a Means of Differentiating Proteus and Paracolon Cultures from Each Other and from Salmonella and Shigella Types. J. Bact. 52:461-466, 1947. Ebpwarps, P. R., and Bruner, D. W. Station Circular No. 54, Lexington (Ky.) Agric. Exp. Sta., 1942. 184 108. 109. 110. 111. 112. 113. 114. 115. 116. 117. 118. 119. 120. 121. 122, 123, 124. 125. 126. 177. 128. CULTURE MEDIA Carton, M. L., and Furron, M. Presumptive Media for Differ- entiating Paracolon and Salmonella Cultures. J. Lab. & Clin. Med. 31: 824-826, 1946. Teacug, O. and CrurMaN, A. W. A Method of Preserving Typhoid Stools for Delayed Examination and a Comparative Study of the Efficacy of Eosin-Brilliant Green Agar, Eosin-Methylene Blue Agar, and Endo Agar for the Isolation of Typhoid Bacilli from Stools. J. Infect. Dis. 18:653-671, 1916. GreeNFIELD, M. Laboratory Aids in Diagnosis of Enteric Infections. Southwestern Med. 20:385-388, 1936. Dorset, M. The Use of Eggs as a Medium for the Cultivation of Bacillus tuberculosis. Am. J. Med. (American Medicine) 3:555-556, 1902. Leving, M., and ScuoenrEIN, H. W. A Compilation of Culture Media. Baltimore, Md. : Williams & Wilkins, 1930, 969 pp. Francis, E. Cultivation of Bacterium tularense on Mediums New to This Organism. Pub. Health Rep. 37:102-115, 1922. McCoy, G. W., and CrmariN, C. W. Studies of Plague and Tuberculosis Among Rodents in California. Pub. Health Bull. No. 53 (USPHS). Washington, D. C.: Gov. Ptg. Off., 1912. HucH, R., and LerrsoN, E. The Taxonomic Significance of Fermentative versus Oxidative Metabolism of Carbohydrates by Various Gram-Nega- tive Bacteria. J. Bact. 66:24-26, 1953. Huntoon, F. M. “Hormone Medium,” a Simple Medium Employable as a Substitute for Serum Medium. J. Infect. Dis. 23:169-172, 1918. MEYER, K. F. Personal communication, 1960. — and BATCHELDER, A. Plague Studies. Selective Mediums in the Diagnosis of Rodent Plague. J. Infect. Dis. 39:370-385, 1926. Morton, H. E., Smrra, P. F., and LeBerMAN, P. R. Investigation of the Cultivation of Pleuropneumonia-Like Organisms from Man. Am. J. Syph. 35:361-369, 1951. LyxN, R. J, and Morton, H. E. The Inhibitory Action of Agar on Certain Strains of Pleuropneumonia-Like Organisms. Applied Microbiol. 4:339-341, 1956. SmrtH, P. F., and Morton, H. E. The Separation and Characterization of the Growth Factor in Serum and Ascitic Fluid Which is Required by Certain Pleuropneumonialike Organisms. J. Bact. 61:395-405, 1951. Par, S.-eEn. A Simple Egg Medium for the Cultivation of Bacillus diphtheriae. Chinese M. J. 46:1203, 1932. Brerz, G. B., and FroBisuer, M. Enrichment of Loeffler’s Medium with Glycerol. CDC Bull. X, No. 5. Atlanta, Ga.: Communicable Disease Center, May 1951. FroBisHER, M., and Parsons, E. I. Further Studies of Tellurite Plating Media for Corynebacterium diphtheriae. A.J.P.H. 43:1441-1442, 1953. McGuican, M. K, and FroBisHER, M. Mediums for the Study of Diphtheria. J. Infect. Dis. 59 :22-29, 1936. WarrLey, O. R.,, and Damon, S. R. Raffinose Serum Tellurite Agar Slants as a Replacement for Loeffler’s Medium in Diphtheria Diagnosis. Pub. Health Rep. 64 :457-460, 1949. . A Transparent Dextrose Serum Tellurite Plating Medium. Its Use as an Adjunct to Microscopic Examination of Smears Made from Loeffler Slants in Routine Diphtheria Diagnosis. Pub. Health Rep. 64: 201-212, 1949. Kerrocs, D. K., and WENDE, R. D. Use of a Potassium Tellurite Medium in the Detection of Corynebacterium diphtheriae. A.J. P.H. 36:739-745, 1946. CULTURE MEDIA 185 129. 130. 131. 132, 133. 134. 135. 136. 137. 138. 130. 140. 141. 142. 143. 144. 145. 146. 147. 148. 149. FroBisHER, M., King, E. O., and Parsons, E. I. A Test in vitro for Virulence of Corynebacterium diphtheriae. Am, J. Clin. Path. 21:282- 285, 1951. HerMmaNN, G. J, Moore, M. S., and Parsons, E. I. A Substitute for Serum in the Diphtheria in vitro Toxigenicity Test. Am. J. Clin. Path, 29:181-183, 1958. HuppLesoN, F. A Satisfactory Medium for the Isolation, Cultivation and Maintenance of Viability of Vibrio fetus (Bovine). J. Bact. 56:508, 1948. Prastringe, W. N. Cultural and Serological Observations on Vibrio fetus. J. Bact. 42:816, 1941. VAN THieL, P. H. The Leptospiroses. Leiden: Univ. of Leiden Press, 1948. Worrr, J. W. The Laboratory Diagnosis of Leptospirosis. Springfield, I11.: Chas. C Thomas, 1954. KorraOF, G. Experimentelles Schlammfieber beim Menschen. Zbl. Bakt. I Abt. Orig. 125:429-434, 1932. Aviston, J. M. and Broom, J. C. Leptospirosis in Man and Animals. Edinburgh: E & S Livingstone, Ltd., 1958. Stuart, R. D. The Preparation and Use of a Simple Culture Medium for Leptospirae. J. Path. & Bact. 58:343-349, 1946. CHANG, S. L. Studies on Leptospira icterohaemorrhagiae. 1. Two New Mediums for Growing L. icterohaemorrhagiae, L. canicola, and L. biflexor and a Method for Maintaining the Virulence of L. icterohaemorrhagiae in Culture. J. Infect. Dis. 81:28-34, 1947. Frercuer, W. Recent Work on Leptospirosis, Tsutsugamushi Disease, and Tropical Typhus in the Federated Malay States. Tr. Roy. Soc. Trop. Med. & Hyg. 21:265-288, 1927-28. Packer, R. A. The Use of a Sodium Azide(NaNy) and Crystal Violet in a Selective Medium for Streptococci and Erysipelothrixz rhusiopathiae. J. Bact. 46:343-349, 1943. ConNELL, R. Personal communication, 1960. —— and Lancrorp, E. V. Studies on Swine Erysipelas. V. Presence of Erysipelothrix rhusiopathiae in Apparently Healthy Pigs. Canad. J. Comp. Med. 17:448-453, 1953. Epwarps, S. J. Studies on Bovine Mastitis. A Selective Medium for the Diagnosis of Streptococcus Mastitis. J. Comp. Path. & Therap. 46: 211-217, 1933. ByrNE, J. L.; ConNELL, R.; FRANK, J. F.; and Moy~N1HAN, I. W. Studies of Swine Erysipelas. II. Cultural Characteristics and Virulence of Strains of Erysipelothrix rhusiopathiae Isolated in Different Regions of Canada. Canad. J. Comp. Med. 16:129-148, 1952. Youmans, G. P. A Method for the Determination of the Culture Cycle and the Growth Rate of Virulent Human Type Tubercle Bacilli, J. Bact. 51:703-710, 1946. National Tuberculosis Association. Diagnostic Standards. New York: The Association, 1955, p. 55. Tuberculosis Laboratory Methods. VA-Armed Forces Cooperative Study on the Chemotherapy of Tuberculosis. Washington, D. C.: VA Depart- ment of Medicine & Surgery, Central Office, July 1960: (a) p. 31; (b) pp. 32-33; (c) pp. 33-34. LoéwensteIN, E. Die Methodik der Reinkulture Tuberkelbazillen, aus dem Blute. Deutsche med. Wchnschr. 56 :1010, 1930. Horm, J., and Lester, V. Diagnostic Demonstration of Tubercle Bacilli. Acta tuberc. scandinav. XVI fasc, pp. 3-4, (1941). Abstracted in Pub. Health Rep. 62:847-854, 1947. 155. 156. 157. 158. CULTURE MEDIA . Dusos, R., and MmprLeBrook, G. Media for Tubercle Bacilli. Am. Rev. Tuberc. 56:334-345, 1947. . Emmons, C. W. Phialophora jeanselmei comb. n. from Mycetoma of the Hand. Arch. Path. 39:364-368, 1945. . Ajerro, L. Cultural Methods for Human Pathogenic Fungi. J. Chronic Dis. 5:545-551, 1957. . Georg, L. K. Use of Cycloheximide Medium for Isolation of Derma- tophytes from Clinical Materials. A.M.A. Arch. Dermat. Syph. 67:355- 361, 1953. . GorooN, M. A, Braprey, E. G.,, and Grant, V. Q. The Influence of Different Types of Corn Meal Agar upon Chlamydospore Production by Candida albicans. J. Lab. & Clin. Med. 40:316-320, 1952. Littman, M. L. A Culture Medium for the Primary Isolation of Fungi. Science 106:109-111, 1947. TascapyiaN, C. L. Routine Identification of Candida albicans: Current Methods and a New Medium. Mycologia 49 :332-338, 1957. SmrrH, C. E, et al. The Use of Coccidioidin. Am. Rev. Tuberc. 57:330- 360, 1948. Emmons, C. W., Orsen, B. J., and Eroringe, W. W. Studies of the Role of Fungi in Pulmonary Diseases. I. Cross Reactions of Histoplasmin. Pub. Health Rep. 60:1383-1394, 1945. . Long, E. R,, Semert, F. B., and Aronson, J. D. A Standardized Tuber- culin (Purified Protein Derivative) for Uniformity in Diagnosis and Epidemiology. Tubercle 16:304-322, 1935. . SmirH, L. D. Personal communication, 1960. Francis, E. Tularemia. X. The Amino Acid Cystine in the Cultivation of Bacterium tularense. Pub. Health Rep. 38:1396-1404, 1923. . CampBerL, C. C. Reverting Histoplasma capsulatum in the Yeast Phase. J. Bact. 54:263-264, 1947. CHAPTER 5 STREPTOCOCCUS INFECTIONS I. Introduction Cultivation of Streptococci Fluid Media Blood Agar II. Collection and Preparation of Specimens A. Blood B. Anterior Nasal, Nasopharyngeal, and Throat Specimens C. Pus, Sputum, Cerebrospinal Fluid, Discharges, Exudates, Urine D. Milk III. Bacteriological Examination Blood Agar Plates IV. Serological Identification Capillary Pipette Grouping Capillary Pipette Typing of Group A Streptococci V. Evaluation and Reporting of Results References I. INTRODUCTION Certain species of streptococci have acquired binary designations (Strep. fecalis, Strep. agalactiae,* and the like) and these have achieved general currency among workers with particular interests, such as veterinary and dairy bacteriologists. Bergey? lists 19 species with fairly well-defined characteristics. Sherman? has divided the streptococci (exclusive of the obligate anaerobes) into four main groups, chiefly on the basis of physiological characteristics but with some consideration of serological constitution (Table 1). Workers concerned with disease-producing streptococci find the type of hemol- ysis produced by a strain to be one of its most useful characteristics, since most strains producing acute disease are beta hemolytic, while strains that produce chronic disease or are nonpathogenic are generally of the viridans or nonhemolytic types. This association between hemolysis and pathogenicity is so useful that cultivation on blood agar plates has come to be the method of choice in primary isolation. * To prevent confusion with Salmonellae, staphylococci or Shigellae species, streptococcal species will be abbreviated as shown. 187 Division and Group or Species Pyogenic: Strep. pyogenes Strep. agalactiae Strep. equi Strep. equisimilis (“Human” C) Strep. zooepidemicus (“Animal” C or “animal pyogenes’’) Strep. dysgalactiae “Minute hemolytic” Group G streptococci Group E streptococci Group H streptococci Viridans:} Strep. sanguis Strep. salivarius Strep. mitis Strep. bovis Strep. thermophilus Lactic: i Strep. lactis Strep. cremoris Enterococcus: Strep. fecalis Strep. fecalis var. liquefaciens Strep. fecalis var. zymogenes Strep. durans Table 1—Divisions and Some Differential Characters of the Streptococci* Lancefield Group a ow» TEQEan gf uu 22 Beta | Hemolysis + H+ Ht + * + + Growth in Presence of 0.1% 6.5% Methylene NaCly | rsa ++ Vila: vy ++ + + ++ + + Blue in Milk Survives 60° C for 30 min dtl) * * HH $4 & + Gelatin + + Trehalose + +H H+ + + Sorbitol 1 * * Ld Ce J Xo 4 Colony Pro- duction on 5% Sucrose Agar§ J {1h * Modified by E. L. Updike, from Sherman.? 1 6.5% NaCl added to a simple glucose-free heart infusion broth. § Produces large succulent or gelatinous colonies on 5% sucrose agar. ... Data not available or not pertinent. ** Occasional variations from type reactions. . } Many other poorly defined viridans streptecocci have been described. 88lL SNO220201Ld3YULS SNOILD3dNI STREPTOCOCCUS INFECTIONS 189 The classification of streptococci into serological groups and types has been of the greatest importance in differentiating streptococci, and much of our knowledge of streptococcal disease and epidemiology has arisen from studies which could not have been carried out with- out serological identification of strains.® A summary of the main host associations of the several serological groups is given in Table 2. Table 2—Summary of Recognized Serologic Groups of Streptococci Group Usual Habitat Usual Pathogenicity A Man Many human diseases B Cattle Mastitis C Many animals Many animal diseases Man (human strains) Mild respiratory infections D Dairy products; in- testinal contents of Genitourinary tract infections, endo- man and animals carditis, wound infections (enterococci) E Normal milk None known Swine Pharyngeal abscesses of swine F Man Questionable; found in respiratory tract G Man Mild respiratory infections—rare Dogs Genital tract infections H Man Questionable; found in respiratory tract K Man Questionable; found in respiratory tract L Dogs Genital tract infections M Dogs Genital tract infections N Dairy products None 0 Man Occur in upper respiratory tract but not associated with disease; endo- carditis The chief pathogen for human beings, Group A, is subdivided into some 46 types on the basis of a precipitin reaction involving the M protein, which is related to virulence. Acquired immunity to Group A streptococci is largely type specific, being dependent upon the develop- ment of antibodies to the M protein. Specific streptococcal diseases, such as scarlet fever, erysipelas, etc., are not generally associated with particular types. An exception is acute hemorrhagic nephritis, which occurs following infection with certain types (1, 4, 6, 12, 25 and 49) much more frequently than with other types. The serological differentiation of streptococci in milk is of im- portance. Even though the presence of Group B, C, D or E organ- 190 STREPTOCOCCUS INFECTIONS isms is of little danger to the consumer, Group A streptococci in milk may cause epidemics of septic sore throat and scarlet fever. Most Group A streptococci are beta hemolytic, but nonhemolytic strains have been encountered. Group A streptococci are sensitive to peni- cillin, which is the drug of choice in the treatment of infection by those organisms. Most of the hemolytic streptococci are sensitive to the sulfonamides, penicillin, aureomycin, terramycin, chloromycetin and erythromycin, but the Group D organisms are as a rule highly resistant to the sulfonamides, penicillin and streptomycin. Viridans and nonhemolytic strains are variable in drug sensitivity. Recently Group A streptococci resistant to tetracycline have been encountered here and abroad. Cultivation of Streptococci Various streptococcal strains, even within Group A, differ con- siderably in growth requirements, in the conditions required for pro- duction and maintenance of cellular and diffusible antigens and enzymes, and in resistance to injurious agents. The media used in routine streptococcal bacteriology necessarily represent a compromise among the many media available, each of which may have superiority from a particular point of view. Considerable variation exists in the media preferred by various laboratories for streptococcal study. It is perhaps advisable to select a set of media, to become thoroughly familiar with their use and how streptococci appear on them, and then to stay with them. In general it may be said that if blood is added, streptococci can be grown in most media used for cultivation of other fastidious microorganisms, Fluid media—There is no synthetic medium that will give maximal growth of all streptococci, but some of the defined media used for cultivating mammalian cells, such as Holmes’s medium A,* have given good growth of all Group A streptococci tested. However, the media generally used for routine purposes are compounded from peptone and meat extract or infusion. For sterilizing such media, filtration is superior to flowing steam, which in turn is superior to autoclaving, although autoclaving may be used for many purposes. Adding to such media supplements of serum, blood or fluid of ascites improves strep- tococcal growth. The optimal pH for initiating the growth of streptococci is about 7.8. Streptococci grown in glucose—containing media produce lactic acid, and the resultant lowering of pH is the chief factor limiting the amount of growth that occurs. Phosphate and bicarbonate buffers may STREPTOCOCCUS INFECTIONS 91 be used to retard the pH fall. When stock cultures are needed for short-term storage, the medium of choice (CM No. 29)* contains no added glucose, because without it the pH does not fall so low and the cultures will remain viable for several months at refrigerator tempera- tures (2°-6° C). On the other hand, when maximal bacterial yield is important, as in preparing extracts for grouping and typing, a buffered glucose-containing medium is used (CM No. 24). In the presence of certain peptones some Group A streptococci produce an active proteolytic enzyme that digests the M substance. Since successful typing depends on the M content of the cocci, it is essential to use a peptone (for example neopeptone), which prevents the formation of the active enzyme. When grouping only is to be performed, any medium that will give adequate growth is satisfactory, since the group carbohydrate is always produced. Blood agar—The species of blood used in blood agar plates is important to the outcome of the test. The preferred blood for primary isolation is sheep blood, because it contains a factor that inhibits the growth of Hemophilus hemolyticus, an organism that may cause confusion in nose and throat cultures. Human blood, either freshly defibrinated or discarded from blood bank reserves, should be used only when sheep blood cannot be obtained, as its content of the hemophilus-inhibiting factor is irregular, Rabbit and horse blood also lack the factor but are useful for subculture in the study of colony form and hemolysis. The blood agar medium recommended for primary isolation contains no added glucose (CM No. 26) because hemolysis is favored by this omission. Special blood agar plates for the study of colony forms are discussed in the following paragraphs. Blood agar in plastic petri dishes is now available commercially and may be welcomed by laboratories that do not care to pour their own plates. A highly important factor, and one that is often neglected, is the moisture content of blood agar plates. The size and appearance of streptococcal colonies are influenced by the moistness of the agar. Moist plates allow the growth to spread out and favor hydration of the streptococcal capsule. Methods for insuring moist plates vary accord- ing to the work load of the laboratory. When only a few plates are used daily, a convenient method is to pour a supply that will last a week or two, seal the plates with parafilm strips, and store in the refrigerator until used. A filter paper disk (10 cm in diameter) placed in the top of the petri dish before sterilizing serves to absorb excess * CM = culture medium as presented in Chapter 4. 192 STREPTOCOCCUS INFECTIONS moisture. As an alternative method, a group of plates may be stored in polyethylene freezer bags. In laboratories doing work on a large scale, where plates are poured daily and are used while strictly fresh, the plates are moist enough without taking special precautions to avoid drying. Hemolysis by Group A streptococci growing as surface colonies is due to streptolysin S alone, because streptolysin O is inactivated by atmospheric oxygen. There are some strains that produce only streptolysin O, and these strains will appear nonhemolytic when grown on the surface of blood agar plates. To recognize such strains, anaerobic conditions must be supplied—by incubating the plates in an anaerobic jar, by making pour plates (in which sufficiently anaerobic conditions exist deep in the agar), or by making a stab with the inoculating loop into the agar. Anaerobic conditions are also necessary for development of the pigment produced by certain Group B strains. Some streptococcal strains grow best in an atmosphere containing about 5 per cent COs, and hemolysis is sometimes favored. A candle jar is a simple device for this purpose. Il. COLLECTION AND PREPARATION OF SPECIMENS A. Blood Sterilize the skin by swabbing with 4 per cent iodine followed by wiping with 70 per cent alcohol. Withdraw approximately 10 ml of blood into a sterile syringe and deliver to a flask containing 2.0 ml of 3 per cent sodium citrate solution or 1 mg heparin. In the labora- tory, transfer the blood with aseptic technic to a flask containing approximately 100 ml of Todd-Hewitt broth (CM No. 24). If it is also desired to know the number of organisms per unit of blood, transfer 1.0 ml and 2.0 ml of blood to two sterile petri dishes. Add 20 ml of melted blood agar base (CM No. 25) to each dish and rock to insure uniform distribution of the blood. Incubate the cultures at 35° C and read daily for 2 weeks. In general, it is not necessary to add antagonists to media for the blood culture of patients receiving chemotherapy. Most media made with peptones or meat infusion contain sufficient sulfonamide antago- nists to prevent sulfonamide inhibition of streptococcal growth, but if desired, 0.25 ml of 1 per cent solution of para-aminobenzoic acid may be added for each 5.0 ml of blood. It has also been shown that penicillinase is not needed when blood culture is performed on patients receiving penicillin.” However, if the quantity of blood or other fluid STREPTOCOCCUS INFECTIONS 193 added to the medium is relatively large, penicillinase may be added in the proportion of 10 units to 15 ml of medium. B. Anterior Nasal, Nasopharyngeal and Throat Specimens Swabs from the upper respiratory passages are the commonest types of culture for hemolytic streptococci. The technic of swabbing the area to be cultured is as important in streptococcal isolation as is cultivation of the specimen obtained. Swabs for throat cultures are made from small sticks (about 2 mm in diameter) around one end of which just enough cotton is wrapped to cover the wood. Individual swabs are introduced into test tubes, held in place by a cotton plug. They are sterilized in the autoclave at 121° C for 20 min, With the patient’s tongue depressed and the throat adequately exposed and illuminated, the swab is passed across the tonsils, if present, and the pharynx, care being taken to avoid touching the tongue or lips, which would con- taminate the swab. If exudate is present, it should be touched with the swab. The swab is then returned to the test tube from which it was taken and the tube is suitably labeled. Anterior nasal cultures are taken by introducing the swab into the nares about an inch. Nasopharyngeal cultures are more readily tolerated when the swab is made of wire to which a small amount of cotton has been securely attached at one end by twisting, with a loop formed at the other end of the wire to facilitate manipulation. Elevate the tip of the patient’s nose with one hand and gently introduce the swab along the floor of the nasal cavity, under the middle turbinate, until the pharyngeal wall is reached. If obstruction is encountered, force should not be used to overcome it and the nasopharyngeal culture cannot be taken on that side. The swabs should be spread onto blood agar within an hour or two of taking the cultures. When this cannot be done, it is common prac- tice to place the swab in a tube containing 0.5 ml of broth (CM No. 29), without blood, for transfer to the laboratory. However, the numerical balance between streptococci and other microorganisms may be upset by this procedure. The survival time of streptococci on dry cotton swabs appears to be variable and is subject to influences that are not thoroughly under- stood. Dacron wool has been proposed as a substitute for cotton, but more work is needed to justify recommending a change from cotton. Hollinger ef al.® have described a method in which throat swabs are inoculated onto filter paper strips. The strips are then air- dried and may be held for several days or sent through the mail to a central laboratory. The strips are used to inoculate blood agar plates 194 STREPTOCOCCUS INFECTIONS by direct contact for several hours. The method gives promise of use- fulness in streptococcal studies, but the survival of other microorgan- isms on the strips is unknown. In streptococcal upper respiratory infections, streptococci may be recovered from throat cultures or nose cultures or both. When only one type of culture can be taken, it should be a throat culture. Naso- pharyngeal cultures often yield almost pure culture of streptococci, even though throat cultures may, at the same time, give highly mixed floras; and particularly in convalescent patients nose cultures may re- main positive long after the throat has become negative. Anterior nasal cultures are of interest in view of Hamburger’s demonstration that carriers of streptococci in that site are often important sources of dissemination of streptococci to the environment.? C. Pus, Sputum, Cerebrospinal Fluid, Discharge, Exudates, Urine Depending on the worker’s judgment as to the number of strepto- cocci in the material, blood agar plates (CM No. 26) are streaked so as to give well-isolated colonies, and Todd-Hewitt broth (CM No. 24) is inoculated. A Gram stain of the original material may indicate roughly the number of organisms present and if necessary the fluid may be centrifuged and the sediment cultured. If heavily contaminated with other bacteria, inoculate Pike's medium (CM No. 30) and streak from it after overnight incubation. D. Milk Keep the specimen at refrigerator temperature as much of the time as possible prior to culturing, or mix with one-third volume glycerol if the milk is to be stored at room temperature for longer than a few hours. Add 0.1 and 1.0 ml or suitable dilutions of the milk, which has been mixed by shaking, to sterile petri dishes in duplicate. Add to the petri dishes blood agar base (CM No. 26) which has been melted and cooled to 45° C and has had 5 per cent defibrinated blood added to it; mix by tilting to distribute the milk uniformly in the blood agar, allow to harden, and incubate at 35° C under aerobic and anaerobic conditions. It sometimes happens that aerobically incubated plates re- ceiving 0.1 ml of milk will show growth of hemolytic streptococci, whereas plates receiving 1.0 ml of milk will not. This is because milk contains lactenin, which in sufficient concentration inhibits the growth of Group A and some other streptococci. With the smaller inoculum of milk the lactenin is added in insufficient quantity to inhibit growth. Lactenin is not active under anaerobic conditions? STREPTOCOCCUS INFECTIONS 195 Ill. BACTERIOLOGICAL EXAMINATION Blood Agar Plates Streak the material to be cultured on the surface of moist sheep blood agar plates (CM No. 26) and with the sterile platinum loop distribute the inoculum over the plate so that well-isolated colonies will be present in some area when full growth is achieved. There are numerous methods of accomplishing this dilution. In the case of throat cultures a satisfactory method is to rub the throat swab over about one-sixth the area of the plate. Pass a sterile inoculating loop through the primary inoculum onto about half of the remaining uninoculated area of the plate, using about 20 to-and-fro strokes. Then pass the loop through this just-inoculated area onto the remaining uninoculated area of the plate without reentering the site of primary inoculation. Then stab the platinum loop into the agar at one or two points. There are many other systems for spreading the inoculum, and each worker develops a technic of his own by which the desired dilution is accomplished. Swabs from the two sides of the nose may be cultured on a single blood agar plate by dividing the plate in two with a china-marking pencil. Inoculate each swab on a small segment of one-half the plate and spread the inoculum with the platinum loop to achieve well-isolated colonies, making a stab into the agar. When laboratory materials must be conserved, nose and throat cultures may be plated on a single plate, using two-thirds of the plate for the throat specimen. When parafilm-sealed plates are used, it is advantageous to reapply the parafilm strip during incubation. Incubate the plates at 35° C and examine at 18 to 24 hr for colonies of streptococci, observing hemolysis and colony form. Oc- casionally in carriers more than one streptococcal strain may be present in a single culture. Therefore in carrier surveys it may be desirable to look for variation in colony form and to pick multiple colonies when variations are observed. This procedure is of no value unless serological grouping is to be performed. Colonies suspected of being streptococci are examined by making a slide for Gram stain. If Gram-positive cocci are found, and if further studies of the strain are to be undertaken, inoculate a tube of blood broth (CM No. 29) with material from a single well-isolated colony, and, after 18 hr incubation, streak on a blood agar plate to determine that the growth is in pure culture. Store the blood broth culture in the refrigerator to serve as a stock culture. 196 STREPTOCOCCUS INFECTIONS Another method of plating throat cultures is a combination pour- streak plate. Wash the throat swab off in 1 ml of broth (CM No. 29 without blood), drain the swab against the inside of the tube, and re- place in its original tube. Transfer a loopful of the inoculated broth to 15 ml of melted blood agar base (CM No. 26) which has been cooled to 45° C. Add 0.75 ml blood and make a pour plate. When the agar has solidified, rotate the swab over a small area of the surface and spread inoculum over one-half the plate as described above. Incubate at 35° C and read at 24 hr. If streptococci are at all numerous and other bacteria are not too numerous, this method allows the worker to observe colony morphology of surface colonies and hemolysis of both surface and deep colonies on a single plate with the material of primary culture. In carrier surveys, where the number of streptococci may be small compared to other bacteria, the number of positive cultures can be increased if the swab, after being rubbed off on the blood agar plate, is placed in 2.0 ml of Pike’s enrichment broth (CM No. 30). After overnight incubation, a small loop of this broth is plated on a new blood agar plate. In the recognition of streptococcal colonies the most important features are hemolysis and colony form. Hemolytic zones occasionally are limited to the area underlying the colony and can best be in- spected when the streptococcal growth is pushed aside. There are three main types and one subtype of hemolysis which were named, defined and thoroughly studied by Brown.!! His classification is based primarily on microscopical appearance of zones around colonies grow- ing deep in blood agar plates. Alpha hemolysis—The colony is immediately surrounded by a zone, which may be very narrow, of intact but discolored erythrocytes that have a green or brownish green color. Outside the zone of dis- coloration a zone of clearer hemolysis may be seen; this zone may be so narrow as to be almost invisible, or it may attain considerable width; continued incubation or refrigeration will usually widen this zone. Alpha-type colonies may easily be mistaken for beta-type colonies if the hemolytic zone is wide and the inner zone of greening is narrow. In the latter case, microscopical examination of colonies growing deep in the agar may be necessary for exact identification. Some strains which produce alpha-hemolytic colonies in atmos- pheric air produce beta-hemolytic colonies when grown under anaerobic conditions or under increased CO; tension. Strains char- acterized by alpha-hemolytic colonies in blood agar are commonly STREPTOCOCCUS INFECTIONS 197 referred to as the viridans group, or green streptococci. The alpha- prime type of colony is surrounded by a slightly hazy zone of hemol- ysis that is less sharply defined than in beta hemolysis. By using the microscope one can see that the zone of hemolysis contains a moderate number of unaltered corpuscles, and these are most numer- ous in the immediate neighborhood of the colony. No visible dis- coloration or greening occurs. Continued incubation or refrigeration for another 24 hr causes considerable widening of the zone of hemol- ysis and may cause these strains to be confused with beta-hemolytic streptococci, Some strains which produce alpha or alpha-prime hemolysis on sheep blood agar produce typical beta hemolysis on rabbit blood agar, and are, in fact, beta-hemolytic streptococci. Beta hemolysis—The colonies of beta-hemolytic streptococci on blood agar plates are surrounded by a clear zone in which few or no intact erythrocytes are visible. A subdivision of this type is the double- zone beta-hemolytic type. This colonial type, after producing a zone of hemolysis like that of other beta-type strains forms, on standing at room temperature or on refrigeration, a second ring of hemolysis separated from the first ring by a zone of intact erythrocytes, All the double-zone beta-hemolytic strains which have been recorded are members of Group B, but not all Group B strains produce double zones. Alternate zones of hemolysis and unhemolyzed erythrocytes may be produced with these strains by alternating incubation and re- frigeration. Some Group C strains produce a particularly large and brilliant zone of hemolysis. This is also true of some Group D strains, the hemolytic areas of which have a tendency to coalesce in a characteris- tic manner. Alpha and nonhemolytic strains occur in most of the groups, particularly Groups D, H and K, but also occasionally in Group A, especially in Type 12. Gamma streptococci—The third type of reaction of streptococci to blood agar is seen in those strains, colonies of which produce no detectable change in the blood surrounding the colony. These are commonly referred to as indifferent or nonhemolytic types. Colony form is a useful feature in the recognition of streptococci and some strains have colony forms (mucoid and postmucoid) which are so distinctive that they may be selected for further study on this basis regardless of hemolysis. Furthermore, colony form, when the strains are grown on appropriate media, may indicate whether or not the strain is encapsulated and may allow selection of variants that differ in M production. 198 STREPTOCOCCUS INFECTIONS Figures 1 through 6—Colony forms on moist Todd-Hewitt sheep blood agar: (1) mucoid; (2-4) postmucoid; (5) mixture of postmucoid and non- mucoid colonies on crowded area of plate; (6) nonmucoid. Figures 7 through 9—Colony forms on dry tryptose infusion sheep blood agar: (7-8) matt; (9) glossy. STREPTOCOCCUS INFECTIONS 199 The appearance of streptococcal colonies depends strikingly on the medium used. Unfortunately, there is no known single medium on which all the colonial characteristics of streptococci are manifested. On the medium recommended for primary isolation (CM No. 26) colonies are small and surface characteristics are not fully developed, so that dependence rests on hemolysis rather than on colony form. Two other media are recommended for colony form study because they particularly favor the development of colonial characteristics. Colony forms on moist Todd-Hewitt blood agar (CM No. 25)— On this medium the streptococcal capsule is well developed and is the predominant factor in determining the appearance of colonies. The glucose in this medium is important in capsular synthesis, and the preservation of moisture ensures capsular hydration. Strains pro- ducing a capsule on this medium grow as mucoid or postmucoid colonies, Strains that do not produce a capsule grow as nonmucoid colonies. The development of these characteristics is best observed in well-isolated colonies. The mucoid colony (Fig 1) is large (3-7 mm in diameter) and watery. It has a smooth, mirror-like surface, is rather flat, and is round, oval or somewhat irregular in outline. Contiguous colonies coalesce when they touch, and in crowded zones the growth appears like a pool of glycerol. Mucoid colonies become converted to postmucoid colonies on standing. With many strains the conversion of some or all of the colonies has already occurred when the plate is first examined following the usual incubation period. The postmucoid colony is characterized by marked surface irregu- larities which have developed as a result of the spontaneous collapse of the colony into folds, ridges or papillae as the capsular material has disintegrated. The conversion of mucoid to postmucoid colonies can also be effected by applying hyaluronidase solution directly to the colony. Postmucoid colonies do not all look alike, and a variety of appearances is illustrated (Figs 2-5). The nonmucoid colony (Fig 6) is smaller (1.0-2.5 mm) than the postmucoid. It has a well-defined, round or slightly irregular edge and is usually hemispherical or dome- shaped, although it may be rather flat. Its surface is usually smooth and glistening but may be dull and stippled. Its shape and surface do not change on standing or after application of hyaluronidase. When India ink preparations are made from material taken directly from the colony, mucoid colonies are found always to contain cocci with capsules. Postmucoid colonies usually contain encapsulated cocci, although many unencapsulated ones may also be found. Nonmucoid colonies consist of unencapsulated cocci only. Although strains that grow in mucoid or postmucoid colonies often are M+, some M— 200 STREPTOCOCCUS INFECTIONS strains are encountered. Similarly, nonmucoid colonies may be M+ or M—. Colony forms on dry tryptose infusion blood agar (CM No. 27 )— On this medium, the effect of the streptococcal capsule on colony form is minimized and other features are brought out. The medium should be allowed to dry after pouring by propping the plates on their lids for an hour or so, or by storing unsealed for a day or longer. The colonies on this medium are much smaller than on Todd-Hewitt blood agar; it is therefore advisable to use a colony microscope for differentiating them. The matt colony (Figs 7-8) is small (0.4 to 1.0 mm in diameter), has an entire or irregular edge, and is dome-shaped or hemispherical, sometimes with a peak in the center. The surface is finely or coarsely stippled, and occasionally corrugations are present around the edges. The matt characteristic is best shown in crowded areas or in zones of confluent growth. The glossy colony (Fig 9) is about the same size as the matt colony. It has an entire edge and hemispherical contour and its surface is perfectly smooth and mirror-like. India ink preparations made directly from the colonies of either form usually reveal no capsules, although a small capsule may be seen. Incorporation of hyaluronidase in the medium does not alter the mattness or glossiness of colonies on this medium. Matt colonies are usually M+ but occasionally may be M —. Glossy colonies are usually M —. Cultures sometimes show a mixture of matt and glossy colonies or a mixture of highly matt and less matt colonies. A useful procedure is to study the variants separately because often one variety will be M + and the other M —. A few strains on this medium will appear as mucoid or post- mucoid colonies (although they are much smaller than corresponding colonies on Todd-Hewitt sheep blood agar) and judgment cannot be made as to their mattness. Incorporation of hyaluronidase in the blood agar plates suppresses the capsular function and it can then be de- termined whether the colonies are matt or glossy. Some strains of Group B streptococci have red-pigmented colonies when grown anaerobically, as in poured plates. All Group F strains and some Group G strains form “minute” colonies. These colonies are no larger than a pinpoint, with zones of hemolysis very large com- pared to the size of the colony. They grow slowly both on solid and in liquid media. Their growth is favored by the use of a candle jar for CO2 during incubation. Distinguishing beta-hemolytic streptococci from other micro- STREPTOCOCCUS INFECTIONS 201 organisms— On sheep blood agar the confusing organisms are hemo- lytic staphylococci, Gram-negative cocci, and green streptococci. The Gram-negative cocci are differentiated by Gram-stained films. A simple, convenient and usually reliable method of differentiating streptococci from staphylococci is the catalase test, in which 0.5 ml of 3 per cent hydrogen peroxide solution is added to some bacterial growth from a blood agar plate (care being taken not to transfer any of the catalase-containing red blood cells) ; or 0.5 ml of a broth culture (CM No. 29) without blood is added. The evolution of gas bubbles is characteristic of staphylococci but does not occur with streptococci. The addition of a few drops of saturated sodium dodecyl sulfate solution to the reaction mixture causes the bubbles to persist as a foam and facilitates reading of the test. The green streptococci are sometimes difficult to distinguish from beta-hemolytic streptococci, particularly when plates are incubated more than 24 hr. Repeated subculture often reveals their true nature. On horse, rabbit or human blood agar, colonies of H. hemolyticus may resemble very closely those of hemolytic streptococcus, although the former are more translucent than the latter usually are. Gram- stained films are necessary for final differentiation. Occasionally the Gram stain will show a mixture of Gram-positive cocci along with Gram-negative rods and thus may be confusing. Subculture of such a colony on sheep blood agar fails to produce growth. If sheep blood is not available, transfer to a tube of broth containing no blood. Hemophilus will not grow. IV. SEROLOGICAL IDENTIFICATION The final identification of a streptococcal strain properly rests on determination of its specific serological group and type. Serological identification has played such a large role in streptococcal bacteriology and epidemiology that a description of the methods used is given here, even though typing sera are currently available only from the Com- municable Disease Center of the U. S. Public Health Service, Atlanta, Ga., and they are issued by that organization only for research and epidemiological studies. More detailed directions may be obtained else- where in the literature. Grouping and typing are accomplished by testing acid extracts of streptococcal cells against absorbed sera of known antibody content. Approximately 40 ml quantities of modified Todd-Hewitt broth (CM No. 24) without blood in 50 ml centrifuge tubes are inoculated from the pure stock cultures and are incubated at least 18 hr or until a heavy growth is obtained. The broth culture is centrifuged and the 202 STREPTOCOCCUS INFECTIONS clear supernatant fluid is pipetted off or decanted. The bacterial sedi- ment is mixed with 0.4 ml of N/5 HCI. A loopful of the suspension should give an orange-red color with a drop of 0.01 per cent thymol blue on a spot plate; that is, extractions should be carried out at pH 2.0 to 2.4. If necessary, more N/5 HCI is addded to obtain this range. The mixture is transferred to a pointed 15 ml centrifuge tube, heated in a boiling water bath, shaken at intervals of 3 min for 10 min, and cooled. A small drop of 0.01 per cent solution of phenol red is added, which colors the solution a distinct yellow. Then 0.03 to 0.33 ml of N/5 NaOH is added drop by drop until a faint pink color appears. The first faint pink color indicates a pH of 7.0 and a good extract will have a pH between 7.0 and 7.8. If too alkaline, the extract should be readjusted with N/20 HCI, since nonspecific pre- cipitation of the immune serum may occur when the pH of the extract is over 7.8. The tube is then centrifuged and the supernatant fluid, which should be crystal clear, is pipetted or decanted to a small test tube. The extracts may be stored for several days in the re- frigerator. Refrigeration of the neutralized extracts sometimes facilitates clarification. Capillary Pipette Grouping Grouping may be performed either for final identification or as a necessary step in identifying members of Group A. In the latter case, it is necessary only to differentiate Group A streptococci from the other groups. The sterile end of a 1.5 mm capillary pipette is dipped into the Group A serum until a column of serum between 1.0 and 1.5 cm long has been slowly drawn in by capillary action. This end of the pipette is then wiped with paper tissue and dipped into a drop of extract until an equal amount of extract has been drawn into the pipette. Air bubbles must not separate serum and extract. The pipette is again wiped, inverted and plunged 1 or 2 mm into plasticine in the pipette stand so that the extract is on top of the serum and a column of air lies between the plasticine and the fluid in the pipette. Within 5 to 10 min a positive reaction is shown by the formation of very fine precipitate at the junction of serum and extract. With weak sera or extracts, a longer time may be required. If the pipettes are placed in the incubator at 35° C for 1 hr, weak cross-reactions with sera of other groups may occasionally occur; hence readings made within 5 to 10 min probably indicate more specifically the group to which the streptococci under examination belong. Upon standing, the precipitate formed early may redissolve, or it may clump and fall to the bottom of the serum. The acid extracts STREPTOCOCCUS INFECTIONS 203 are made for typing as well as for grouping and contain a relatively large amount of group carbohydrate. For this reason difficulties from antigen excess may arise, especially when weak sera are used. If an extract fails to group with full-strength extracts, precipitation may follow using extracts that have been diluted 1:4 or 1:16 with 0.85 per cent NaCl solution. Alternative methods involve the use of conical capillaries or pointed 7 mm tubes. The latter require 0.05 ml of serum for each of the groups, but when adequate serum is available, it is the best method. Extracts for grouping (but not for typing) may also be made by the hot formamide method,'* by using an enzyme preparation obtained from Streptomyces albus® or by autoclaving suspensions by the method of Rantz and Randall,'® all of which have the advantage of not requiring acidification and subsequent neutralization. A procedure of value chiefly in epidemiological studies and not recommended for use in laboratories that do not routinely check results by serological methods is the presumptive identification of Group A streptococci introduced by Maxted.!™ This procedure is based on the selective inhibition of Group A streptococci by bacitracin- sensitivity disks. Most strains of the other groups are able to grow in the presence of a concentration of bacitracin that inhibits the growth of Group A strains. The useful concentration of bacitracin is critical and the disks should be assayed with known sensitive and resistant strains. Another method that has recently been proposed is identification of Group A streptococci by the fluorescent antibody technic. This technic has the advantage of rapidity in that results may be available within 2 or 3 hr of taking a throat culture. Satisfactory results with the fluorescent antibody technic depend on: (1) meticulous adherence to prescribed procedures; (2) specialized training; (3) high-quality lighting and optical equipment; (4) carefully prepared, tested and evaluated fluorescent antisera. At present the procedure can be recommended for use only by individuals who have had thorough training in the handling of the equipment and the testing and control of the reagents, and in laboratories that have facilities for checking results by precipitin grouping. (See Chapter 1 for technic.) Capillary Pipette Typing of Group A Streptococci The typing sera should be sufficiently strong and specific that a type may be identified on the basis of a positive reaction in one serum without the necessity of testing for negative reactions in all the others. Tt is thus possible to save time and materials by testing extracts against 204 STREPTOCOCCUS INFECTIONS the sera for the six or eight types prevalent in a given locality. Test- ing with all the typing sera is necessary only when the strains under examination are not the types commonly encountered in a given area. Only those extracts are tested which have reacted with Group A serum. The sterile end of a 1.0 mm capillary pipette is placed in the serum until a column 1.5 to 2.0 cm long has been drawn in by capillary action, This end of the capillary pipette is wiped clean with paper tissue, then dipped into the extract, and an equal column is run in after the serum. If an air bubble separates serum and extract, the pipette is discarded and another one set up. The column is allowed to run to the middle of the pipette and the pipette is carefully wiped with soft paper tissue, then inserted into the plasticine of the pipette stand so that the serum is on top of the extract. Similar preparations are made with each serum to be tested. As soon as an extract has been set up, the tubes are examined for cloudiness with a hand lens. If foreign particles are present, the pipettes are discarded and the test is repeated. The tubes are incubated for 2 hr at 35° C and preliminary reading is made. A final reading is made after the pipettes have stood overnight in the refrigerator. Most positive reactions will appear at the 2 hr reading. When extracts set up only against the common types are negative at that time, they may be set up immediately with the rest of the sera. The following scale is used: plus minus (=), just visible; plus (+), a few fine masses visible with the lens; 2 plus (+ +), usually beaded through- out and visible to the naked eye; 3 plus (++ +), and 4 plus (++ + +), column filled with masses of precipitate. Each positive test must be interpreted in the light of the known reaction of the serum with homologous extract. Readings of plus or weaker should not be accepted. V. EVALUATION AND REPORTING OF RESULTS A complete report on a streptococcal culture gives such informa- tion as: a. Type of hemolysis (alpha, alpha prime, beta or gamma). b. Counts (the number of colonies developing per ml of blood or other fluid will be given if determined). c. Proportion of colonies in the culture that are streptococci. If nose and throat swabs have been streaked directly on blood agar plates, these figures may be very helpful to the clinician, since his evaluation of a culture containing only two or three streptococcal colonies may be quite different from that of a culture in which 90 per cent or more of the colonies are streptococci. If the original material has had a preliminary incubation in broth in the laboratory or in transit to the laboratory before plating, this figure may be misleading due to the differential growth and survival rates of various bacterial species. STREPTOCOCCUS INFECTIONS 205 d. Serological group and type. These designations may be offered only when the serological determinations have actually been made. Attempts to arrive at presumptive grouping on the basis of physiological tests, bacitracin sensi- tivity, and the like may be reported as such but should not convey the impres- sion that serological studies have been done. e. Physiological characteristics. Certain non-Group A streptococci with binary designations may be identified on the basis of the characteristics indicated in Table 1 of this chapter, preceding. Such cultures should be reported giving the names of their species but not the name of the group with which they may be associated (such as Strep. agalactiae, which belongs to Group B) unless grouping has been done. f. Antibiotic sensitivity. This may be the characteristic of chief interest to the person submitting the culture and is usually specifically requested. Sensi- tivity testing is described in Chapter 27. g. Anaerobic streptococci should be reported as such. In many cases it is impossible or unnecessary to do a complete study of the streptococcal strain. Many reports will consist only of some statement like “60 per cent beta-hemolytic streptococci.” When grouping and typing are done, that information, of course, is of primary importance and, except with group D streptococci, little sig- nificant information would be added by supplying physiological characteristics. Grouping is a procedure which should perhaps be done by all major public health and clinical laboratories because the information ob- tained thereby is essential in evaluating the significance of a strep- tococcal culture, Typing, on the other hand, is of interest chiefly to the epidemiologist and the laboratory research worker. ArRMINE T. WirsoN, M.D., Chapter Chairman PauL P. FRANK NEeLL F. HoLLINGER, PH.D. ANN G. Kutrner, M.D., Pa.D. ReBecca C. LancerFieLp, Pu.D. MacLyn McCarty, M.D. CuarrLes H. RaMMeELkAMP, M.D. LoweLL A. Rantz, M.D. Evang L. Uppyke, Sc.D. REFERENCES 1. Breen, R. S.,, Murray, E. G. D,, and SmitH, NATHAN R. In Bergey's Manual of Determinative Bacteriology (7th ed.). Baltimore, Md.: Williams & Wilkins, 1957, pp. 506-529. 2. SHERMAN, J. M. The Streptococci. Bact. Rev. 1:3-97, 1937. 3. LancerieLp, R. C. Specific Relationship of Cell Composition to Biological Activity of Hemolytic Streptococci. Harvey Lect. Series 36, 1941, pp. 251-290. : 4, RammeLkamp, C. H. Jr, and WEAver, R. S. Acute Glomerulonephritis, The Significance of the Variations in the Incidence of the Disease. J. Clin. Invest. 32:345-358, 1953. 206 5 10. 11. 12. 13. 14. 15. 16. 17. STREPTOCOCCUS INFECTIONS HoLmeEs, R. Long-Term Cultivation of Human Cells (Chang) in Chemically Defined Medium and Effect of Added Peptone Fractions. J. Biophys. & Biochem, Cytol. 6:535-536, 1959. Krumwiepg, E, and KurrNer, A. G. A Growth-Inhibitory Substance for the Influenza Group of Organisms in the Blood of Various Animal Species. The Use of the Blood of Various Animals as a Selective Medium for the Detection of Hemolytic Streptococci in Throat Cultures. J. Exper. Med. 67 :429-441, 1938. KirBy, W. M. M. Addition of Penicillin Tnactivator to Routine Culture Media. Stanford M. Bull. 2:158-161, 1944. HoLLINGER, N. F., et al. Transport of Streptococci on Filter Paper Strips. Pub. Health Rep. 75:251-259, 1960. HAMBURGER, M., Jr., GREEN, M. J., and HAMBURGER, V. G. The Problem of the “Dangerous Carrier” of Hemolytic Streptococci. II. Spread of Infec- tion by Individuals with Strongly Positive Nose Cultures Who Expelled Large Numbers of Hemolytic Streptococci. J. Infect. Dis. 77:96-108, 1945. WiLsoN, A. T., and RosensLum, H. The Antistreptococcal Property of Milk. II. The Effects of Anaerobiasis, Reducing Agents, Thiamine and Other Chemicals on Lactenin Action. J. Exper. Med. 95:39-50, 1952. Brown, J. H. The Use of Blood Agar for the Study of Streptococci. Rockefeller Institute Med. Res. Monograph No. 9, 1919. WiLsoN, A. T. The Relative Importance of the Capsule and the M-Antigen in Determining Colony Form of Group A Streptococci. J. Exper. Med. 109 :257-270, 1959. Swirr, H. F. “The Streptococci.” In Bacterial and Mycotic Infections of Man (R. J. Dubos, Ed.). Philadelphia, Pa.: J. B. Lippincott, 1948, pp. 286-290. FuLLer, A. T. The Formamide Method for the Extraction of Poly- saccharides from Hemolytic Streptococci. Brit. J. Exper. Path. 19:130-139, 1938. Maxtep, W. R. Preparation of Streptococcal Extracts for Lancefield Grouping. Lancet 255 (2) :255-256, 1948. Rantz, L. A., and RanpaLL, E. Use of Autoclaved Extracts of Hemolytic Streptococci for Serological Grouping. Stanford M. Bull. 13:290-291, 1955. MaxteEp, W. R. The Use of Bacitracin for Identifying Group A Hemolytic Streptococci. J. Clin. Path. 6:224-226, 1953. CHAPTER 6 STAPHYLOCOCCUS INFECTIONS I. Introduction A. Microscopical Appearance B. Cultural Appearance C. Classification II. Collection and Handling of Specimens ITI. Bacteriological Examination A. Microscopical B. Isolation and Cultivation C. Identification 1. Coagulase tests 2. Bacteriophage typing D. Sensitivity to Antibiotics IV. Serological Examination V. Evaluation and Reporting of Results References I. INTRODUCTION The staphylococci are responsible for a number of pathological conditions, chiefly of a suppurative nature, which affect various tissues and organs of the body. They are the usual cause of furuncles, car- buncles and osteomyelitis; they are commonly associated with infec- tions of wounds or burns, either as the primary infectious agent or in association with other microorganisms; and they are responsible for some cases of pneumonia, meningitis, bacteremia, endocarditis, pyarthrosis, conjunctivitis, renal abscesses and miscellaneous other conditions. Hospital-acquired staphylococcal infections have assumed considerable importance in recent years; in particular, staphylococcal infections in newborn infants, often occurring in epidemic form, and breast abscesses in nursing mothers now present serious problems of prevention and control. They have also been incriminated as a cause of enterocolitis, which sometimes follows the oral administration of antibiotics. Staphylococcus enterotoxin is responsible for many out- breaks of food poisoning (see Chapter 11). 207 208 STAPHYLOCOCCUS INFECTIONS Pathogenic staphylococci are normally parasitic and are found on the skin and mucous membranes, where they occur either tran- siently or as part of the permanent bacterial flora of these regions. Potentially pathogenic staphylococci can be isolated in cultures of the skin of about 20 per cent of all individuals, and in cultures of the nose of approximately 50 per cent. Staphylococci sometimes are found in the environment, as in dust, air or on articles of daily use. They are especially numerous in the immediate vicinity of heavily infected persons. The pathogenic staphylococci are represented by a few species of a large group of micrococci essentially universal in distribution. Although the majority of micrococcal species are nonpathogenic, several species present an appearance which closely resembles that of the pathogenic staphylococci, both microscopically and in culture. Some are commonly present on the human skin and mucous mem- branes, where they represent a potential, and in some cases an almost unavoidable, source of contamination. In the laboratory diagnosis of staphylococcal infection it therefore becomes highly important to differentiate between the pathogenic varieties and the nonpathogenic environmental forms. A. Microscopical Appearance Microscopically the pathogenic staphylococci appear as spherical cells and typically occur in irregular clusters. This grouping is par- ticularly characteristic in stained preparations made from an agar culture. In broth the clusters are usually small, and single cocci, pairs or short chains are seen; long chains are never found. The cocci average about 0.8 to 1 p in diameter; and generally the staphylococci in any given culture tend to be fairly uniform in size and arrange- ment. In cultures 24 to 48 hr old the cocci are Gram-positive, while occasional Gram-negative cocci may be observed in older cultures. In comparison with the pathogenic staphylococci, many nonpathogenic micrococci also occur in irregular clusters; however, the individual cells often appear as diplococci and tend to be somewhat larger and less uniform in size than staphylococci. B. Cultural Appearance On agar the colonies are round, raised, opaque, smooth and glisten- ing and exhibit characteristic pigmentation which ranges from deep gold to yellow, cream or white. The development of pigment is de- pendent upon several factors, It is produced only in the presence of STAPHYLOCOCCUS INFECTIONS 209 oxygén and is best developed on a solid medium containing a carbo- hydrate or blood. While the color usually is distinct after incubation at 35° C for 24 hr, it is often intensified when the culture is held at room temperature for another day or two. Milk agar, to be men- tioned again, distinctly enhances the production of pigment. Cultures on extract agar containing no blood or carbohydrate may appear white or nearly white. Cultures in broth are nonpigmented and usually uniformly turbid, with an amorphous, occasionally stringy sediment. When cultured on blood agar the colonies of a majority of freshly isolated strains of staphylococci are surrounded by a zone of hemol- ysis; a few strains are nonhemolytic. Staphylococci may produce at least three antigenically distinct hemolysins, designated as alpha (a), beta (B), and delta (2) hemolysin, respectively. They differ in their activity against the erythrocytes of various species of ‘animals. The alpha hemolysin is active against the red blood cells of rabbits and sheep but its action on human cells is negligible. It produces a wide, clear zone of hemolysis with blurred edges. The beta hemolysin lyses the erythrocytes of sheep and cattle; human red blood cells are lysed only occasionally, and those of rabbits rarely, if ever. Charac- teristically this lysin produces a zone of discoloration, or “partial hemolysis,” after incubation at 35° C for 24 hr which becomes clearer when the plate is subsequently held at room or refrigerator temperature. The delta hemolysin is especially active against human and rabbit erythrocytes and also lyses the red blood cells of sheep and some other species. Tt produces a narrow, sharply defined zone of complete hemolysis. The alpha hemolysin is produced principally by strains that are pathogenic for man. The beta hemolysin is as- sociated chiefly with strains of animal origin. The delta hemol- ysin may be formed by strains which produce either the alpha or beta lysin or both. The simultaneous presence of two hemolysins may affect the ap- pearance of the culture on blood agar. When a strain produces both alpha and delta hemolysin, the narrow zone produced by the latter may be masked by the relatively wider zone of the former on either sheep or rabbit blood agar. Strains that produce both alpha and beta hemolysins, or beta and delta hemolysins, may exhibit a zone of clear lysis surrounded by a zone of partial hemolysis on sheep blood agar, although on rabbit blood agar only a zone of clear lysis would be seen, for beta hemolysin does not affect rabbit cells. The final differentia- tion of the hemolysins requires tests for neutralization by specific antisera. A scheme for such tests was described in 1959 by Elek in his volume, Staphylococcus pyogenes. 210 STAPHYLOCOCCUS INFECTIONS The pathogenic staphylococci are aerobic, and facultatively an- aerobic, microorganisms. Strictly anaerobic micrococci are encountered very rarely, being represented by species distinct from the pyogenic staphylococci, with which this chapter deals; one species is in- frequently associated with puerperal fever. The colonial appearance of the staphylococci readily differentiates them from such other pathogenic cocci as the meningococcus, gonococcus, pneumococcus and streptococcus. Microscopical examination of stained preparations further differentiates these microorganisms from the staphylococci. C. Classification Although the staphylococci are members of the family Micrococca- ceae, they have undergone a varied taxonomic history and there is no general agreement on their generic or specific classification. The term “staphylococcus” has long been used by the bacteriologist and the clinician, often as a generic name, to denote this group of pathogenic cocci with characteristic morphology and cultural properties. Species have been identified according to differences in pigmentation—for example, Staph. aureus* (golden), Staph. albus (white, nonpig- mented), Staph. citreus (lemon-yellow). When they are freshly isolated and grown on a suitable solid culture medium, the majority of pathogenic staphylococci tend to produce a deep golden pigment, although the shade of color may vary from deep gold to pale cream and fully pathogenic white variants are known to occur. Almost invariably these strains, regardless of the presence or absence of pigmentation, are coagulase-positive. Some staphylococci conform in biological and biochemical properties to the pigmented strains but remain white and are regarded by some in- vestigators as a separate species, Staph. albus. In the most recent edition of Bergey's Manual of Determinative Bacteriology! only two species are recognized: Staph. aureus and Staph. epidermidis. The former is comprised of coagulase-positive, pathogenic or potentially pathogenic, staphylococci which usually exhibit a golden pigment but may occur as white variants. When used in this sense, the term Staph. aureus is essentially synonymous with Staph. pyogenes or Micrococcus pyogenes, terms which are not in- frequently encountered in the literature. Staph. epidermidis is coagu- lase-negative and its growth on agar is porcelain-white. It is parasitic on the skin. This microorganism is often found in stitch abscesses, where mild suppuration appears to result from its ability to become * To prevent confusion with streptococcus, Salmonella and Shigella species, Staphylococcus species will be abbreviated as shown. STAPHYLOCOCCUS INFECTIONS 211 established in traumatized tissue rather than from any inherent patho- genic capacity. The staphylococci may be separated serologically into Type A (pathogenic) and Type B (nonpathogenic) by means of precipitin reactions with specific polysaccharides? Among the pathogenic staphylococci, three serological groups were established by Cowan,? based on agglutination with specifically absorbed immune sera. This classification has been confirmed by others,*5 who have recognized several additional serological groups. Il. COLLECTION AND HANDLING OF SPECIMENS The wide distribution of micrococci presents special problems in the collection of certain specimens that are to be examined for staphy- lococci. When the nature of the infection is such that material for culture must be obtained by aspiration, particular precautions must be taken in sterilizing the skin. When drawing blood for culture or when aspirating fluids from the body cavities, the skin should be painted with tincture of iodine and washed with 70 per cent alcohol. This procedure must not be perfunctory, nor may it be performed in haste. Adequate precautions should be taken to avoid surface con- tamination in the collection of pus from boils, carbuncles or superficial abscesses and draining sinuses. Pustules in the region of the nose and mouth must never be squeezed to express pus, for the highly vascular nature of this area renders it especially vulnerable to extension of the infection. Aspirated fluids are transferred aseptically from the syringe to a sterile tube. Preferably, and especially for transport over any great distance, the tube should be tightly closed with a cork or rubber stopper, or a screw-capped tube may be used. Purulent discharges from wounds, sinuses and similar lesions are collected on sterile swabs which are placed in sterile, cotton-plugged tubes for transport to the laboratory. Care should always be taken to avoid touching the swabs to the skin at the edges of the lesions. In the experience of one author (R. I. W.) the chance of contamination of a specimen from a draining sinus is reduced by the use of a sterile glass pipette, which is inserted into the sinus tract after suitable sterilization of the skin surfaces. When only a small amount of pus is available, 2 ml of extract broth (CM No. 3) may be added to the tube to prevent drying. It sometimes is the custom to break off the portion of the applicator that has been handled while collecting the specimen and to drop the 212 STAPHYLOCOCCUS INFECTIONS swab into the tube. Manipulation of such a swab in the laboratory is awkward and often increases the possibility of contamination. The authors prefer that about one-half inch of the handle of the swab be allowed to project through the plug at the top of the tube. This can be passed quickly through a flame if necessary and permits easier han- dling when the specimen is planted on culture media. Urine is collected and delivered into a sterile rubber-stoppered or screw-capped tube for transport to the laboratory. In both males and females the specimen may be collected by catheter when there is good indication for its use. However, an increasing body of opinion holds that catheterization for the collection of specimens for culture should not be the routine practice, for the procedure itself may be the source of an infection of the urinary tract. In both sexes, therefore, a voided midstream specimen which is collected after carefully cleansing the area of the urethral orifice is acceptable and generally proves satis- factory. Specimens of sputum or feces are collected in a sterile, wide-mouth, cork-stoppered or screw-capped 2 oz bottle. Instructions for the collection and handling of specimens in out- breaks of suspected staphylococcal food poisoning are outlined in Chapter 11. Although the staphylococci are relatively hardy microorganisms and are not readily susceptible to adverse environmental conditions, specimens should nonetheless be delivered to the laboratory promptly after collection. Should any delay be expected, the specimens should be refrigerated. Whenever it is anticipated that antibiotic therapy will be used, it is highly advantageous to obtain at least one culture from the lesion or from the blood before therapy is started. Tests for sensitivity of this culture to antibiotics not only provide information which may guide the clinician in planning therapy, but they serve as a point of refer- ence for subsequent cultures in the event that antibiotic-resistant strains emerge during the course of treatment. Ill. BACTERIOLOGICAL EXAMINATION A. Microscopical Gram-stained preparations of the specimen furnish only presump- tive evidence of the presence and relative numbers of staphylococci. Pus and purulent fluids should be spread in thin films over the slide. Stained films of urine and body fluids are made after centrifugation of the specimen. In stained preparations the cocci are usually grouped STAPHYLOCOCCUS INFECTIONS 213 in small clusters and pairs ; single cocci may also occur, while in fluid specimens short chains of 3 or 4 cocci occasionally are seen. In any event, the evidence supplied by the Gram stain must be confirmed by culture and by a coagulase test, as described in the following. B. Isolation and Cultivation The choice of a culture medium for the isolation of staphylococci is governed to some extent by the purpose for which the culture is made. In the diagnostic laboratory, blood agar prepared on a base of beef heart infusion or casein soy peptones is recommended (CM No. 16). Good growth of staphylococci occurs on this medium, which also per- mits the growth of most of the other microorganisms that might com- monly be encountered in pathological material. Rabbit or sheep blood in a concentration of 5 per cent is to be preferred; human blood is generally less satisfactory. When the objective is the ready isolation of pathogenic staphylo- cocci without regard to the possible presence of other microorganisms, as might be the case in certain epidemiological investigations, a differential or selective medium may be employed. Agar (CM No. 9) containing about 7 per cent sodium chloride provides the required selectivity, for it permits the growth of pathogenic staphylococci while inhibiting the growth of most other microorganisms. With mannitol and a suitable indicator, “high salt” agar is available commercially. A disadvantage of a high salt agar is that incubation for 48 hr usually is necessary for the development of good growth. One of the authors (L. R. K.) routinely uses milk agar, composed of 20 per cent canned evaporated milk in a trypticase soy base containing 4 per cent agar and about 7 per cent sodium chloride. While growth may be delayed for a day, pigmented colonies of staphylococci are readily identified. A differential medium is provided in the form of phenolphthalein phosphate agar (CM No. 8).6 The usefulness of the medium is based on the formation of phosphatase by the pathogenic staphylococci. After incubation for 24 hr at 35° C, the plate is exposed briefly to the fumes of concentrated ammonia. Colonies of pathogenic staphylococci assume a bright pink color, a result of the liberation of free phenolphthalein by the phosphatase. Streak pus, purulent fluids or sputum directly on a blood agar plate and inoculate a tube of thioglycolate broth (CM No. 19). Deposit the material near one edge of the plate and streak widely from this area to obtain well-isolated colonies. One loopful, or a small drop delivered from a capillary pipette, of the sediment from centrifuged specimens of body fluids is placed on the plate and streaked in a 214 STAPHYLOCOCCUS INFECTIONS similar manner, and a tube of thioglycolate broth is inoculated. Incubate at 35° C. For culturing feces, emulsify about 1 g of the solid specimen in 5 ml of extract broth, and streak a loopful of the solid suspension on a blood agar plate and on a plate of phenylethyl alcohol agar (CM No. 7) or high salt agar. Streak fluid fecal specimens directly on both media. Incubate at 35° C. Phenylethyl alcohol agar inhibits the Gram- negative bacilli and permits the ready isolation of staphylococci.” It may be noted that when a culture of staphylococci is grossly over- grown with Proteus, successive subcultures onto phenylethyl alcohol agar or salt agar usually allow isolation of the cocci without difficulty. Staphylococcal diarrhea is sometimes encountered in patients who are undergoing treatment with antibiotics. In a suspected case the presence of large numbers of Gram-positive cocci in clusters in a Gram-stained preparation of the stool specimen is highly suggestive that staphylococci are responsible. This presumptive evidence is then confirmed by culturing the specimen for staphylococci, as described above, in addition to plating on the differential media usually em- ployed for isolation of the Gram-negative enteric bacilli. For blood cultures it is preferable that both the collection of blood and preparation of the cultures should be done by members of the laboratory staff who are experienced in bacteriological technics. In some hospitals, blood cultures are delegated to relatively less ex- perienced members of the house staff. Consequently, in addition to the recommended procedure, an alternate method for use by the house staff is described herein which requires minimum manipula- tion and reduces the possibility of contamination. I. Recommended procedure for blocd cultures 1) Collect 10 ml of venous blood as described in Chapter 1 and transfer aseptically to a tube containing sufficient sterile sodium citrate, potassium oxalate or heparin to prevent clotting. Mix the blood and anticoagulant immediately and thoroughly by rotating the tube gently. In the laboratory : 2) Pipette 4 ml of blood into a flask containing 45 ml of extract broth. 3) Pipette 6 ml of blood into a flask containing 45 ml of extract agar (CM No. 4) which has been melted and cooled to about 45° C (comfortably warm to the skin of the wrist). Mix the blood and agar by gentle rotation of the flask and pour approximately equal amounts into each of three petri dishes. This permits a rough estimate STAPHYLOCOCCUS INFECTIONS 215 of the number of colonies per milliliter, on the basis of about 2 ml of blood per plate. 4) Incubate the broth culture and the plates at 35° C. 2. Alternate procedure for blood culture 1) Collect 5 ml of venous blood aseptically as described in Chapter 1. 2) Remove the needle from the syringe and, with sterile precau- tions, transfer the blood directly from the syringe to a flask containing 45 ml of extract broth. Mix the blood and broth by gentle rotation of the flask. 3) Incubate the blood culture at 35° C. Instructions for culturing samples of food and other specimens obtained in outbreaks of suspected staphylococcal food poisoning are outlined in Chapter 11. C. Identification The identification of pathogenic staphylococci is based upon the gross appearance of the culture, microscopical examination, and the performance of a coagulase test. Cultures which present characteristic colonies of typical pigmentation that hemolyze blood agar and exhibit Gram-positive cocci of typical morphology and grouping may be regarded as staphylococci. When freshly isolated, staphylococci usually exhibit some degree of pigmentation and hemolyze blood agar. How- ever, complete reliance should not be placed upon either of these properties. Occasional strains do not produce hemolysis, and the pigment may vary from a deep gold to a very pale cream. If desired, the color of a weakly pigmented culture can be determined by scrap- ing a loopful of growth from the plate and examining it against a white background. Incubation for at least 48 hr may be needed to bring out the paler shades of color. Further differentiation into pathogenic and nonpathogenic varieties must be made. This is ac- complished by the coagulase test, which is performed on every culture that exhibits the characteristic morphological and cultural properties. I. Coagulase test The ability to clot blood plasma is a characteristic and, for practical purposes, a specific property of the pathogenic staphylococci, including the enterotoxigenic strains.® While an occasional coagulase-negative 216 STAPHYLOCOCCUS INFECTIONS strain has been incriminated as the etiologic agent of an infection, the opinion of the majority of investigators seems to be that the coagulation of plasma by staphylococci represents the most reliable laboratory test for the identification of pathogenic strains currently available. The test is performed as follows :° a. Tube test: 1. In 0.5 ml of fresh citrated rabbit plasma, undiluted or diluted 1:10 in extract broth (CM No. 3), suspend 1 loopful of the growth from an agar culture 18 to 24 hr old. Or, to 0.5 ml of plasma add 0.1 ml of a culture in nutrient broth. Cultures on high salt agar should not be used for the test. 2. Set up controls in the same way with known coagulase-positive and coagulase-negative cultures. 3. Incubate in the water bath at 37° C and examine for clotting at intervals of 30 min for 3 hr and if no clot is observed at the end of this time, again at 6 and 24 hr. With the majority of coagulase-posi- tive cultures a clot is formed within 3 hr, and frequently within one- half to 1 hr. A positive test reaction is represented by any degree of clotting— from a loose clot suspended in the plasma to a solid clot that is im- movable when the tube is inverted. Note: Fresh rabbit plasma is definitely to be preferred for the coagulase test. For the small laboratory that does not have access to fresh rabbit plasma, a dehydrated product is available commercially which is reconstituted for use with distilled water according to directions which accompany the material. Human plasma may also be used for the coagulase test if it is not over 3 or 4 days old, but the reactions are often weaker than with rabbit plasma. It is debatable whether human plasma from the blood bank should be employed, although it is reported to give satisfactory results by some laboratories. Individual samples of blood plasma vary considerably in the length of time they remain satisfactory for the coagulase test. Therefore, if bank plasma is used, it should first be care- fully checked with known coagulase-positive and coagulase-negative cultures. b. Slide test: The use of the slide test as an alternate procedure is based on the fact that essentially all coagulase-positive staphylococci are clumped by human plasma, while coagulase-negative cocci are not.!® The slide test is particularly useful in surveys which involve the examination of a large number of cultures or of many colonies on a single plate. Some skill is required in performing and reading the test, so that it should be performed only after familiarity with it has been gained by a fairly extensive series in parallel with the tube coagulase test. Since the reaction depends upon the presence of clumping, only fresh STAPHYLOCOCCUS INFECTIONS 217 human plasma can be employed, and each lot of plasma must be checked beforehand by tests with known coagulase-positive and coagu- lase-negative cultures. The test is performed as follows: 1. Place a small drop of water on each of two glass slides. 2. With an inoculating needle, emulsify the growth from a typical colony in the drop on one slide and transfer the material remaining on the needle to the second slide for subsequent staining by the Gram method. Thorough emulsification of the bacterial growth is essential— a preparation which exhibits autoagglutination before the plasma is added is unsatisfactory for the test. 3. Add 1 loopful of fresh human plasma to the suspension on the first slide and mix with the needle by a continuous circular motion for 5 sec. 4. Easily visible, white clumps usually appear immediately or within 5 sec and represent a positive reaction. A uniformly turbid suspen- sion with no clumping after 5 sec represents a negative reaction. 5. If the test is negative and if the Gram-stained preparation shows cocci of typical morphology and grouping, set up a coagulase test by the tube method described above. 2. Bacteriophage typing The bacteriophage typing of staphylococci provides a method by which individual cultures can be differentiated with a reasonable degree of accuracy.’ While the method is comparatively new, its value has been well established and it promises to be of increasing usefulness in the future. Phage typing is particularly useful in the study of sets of cultures of staphylococci that have been isolated from related sources; and in certain epidemiologic investigations. The maintenance and control of stocks of typing phages is time consum- ing, and proper interpretation of the lytic patterns requires consider- able experience. This would appear to preclude the use of phage typing by the small laboratory and suggests that at present its use should be limited to those laboratories that are in a position to estab- lish a continuing phage-typing service. Some 24 regional laboratories for staphylococcal phage typing have been established in various public health laboratories in the United States.!® There are two national reference laboratories in the United States: Department of Bac- teriology, Hospital for Joint Diseases, New York City, under the direction of Dr. J. E. Blair; and the Staphylococcus and Strepto- coccus Unit, Communicable Disease Center, U. S. Public Health Service, Atlanta, Ga., under the direction of Dr. E. L. Updyke. The 218 STAPHYLOCOCCUS INFECTIONS former provides phages and their specific propagating strains of staphylococci to those hospitals and medical centers where phage typing appears to be feasible, and the latter supplies phages for typing to certain public health laboratories. Canada has a national reference laboratory for staphylococcal phage typing; Bacteriologi- cal Laboratories, Laboratory of Hygiene, Department of National Health and Welfare, Ottawa, under the direction of Dr. E. T. Bynoe. This laboratory provides phages for typing and their propagating strains to the provincial public health laboratories and to other interested hospital or university laboratories (in Canada). Standardized methods are essential if phage typing of staphylococci is to be reliable and if the results obtained in different laboratories are to be compared intelligently. Of special importance are the control methods used to determine the suitability of the phages for typing. The procedures recommended by the International Committee on Phage Typing of Staphylococci were outlined in a recent paper? which describes in some detail the methods that have been found satisfactory for propagating the phages and defines a standard testing routine whereby stability of the phage preparations can be verified. These tests comprise (1) titration to establish the appropriate dilu- tion of phage for typing; (2) determination of the lytic spectrum, that is, the lytic activity of a phage on a selected set of test strains of staphylococci; (3) periodic checks on the activity of the phages dur- ing use. Technical details are given of the methods recommended for determining the phage type of staphylococci. Also discussed in this paper is interpretation of the differences that may be observed be- tween the phage patterns of strains during routine typing. It is im- portant that close collaboration be maintained between the laboratory and the epidemiologist, for only by mutual exchange of information can proper evaluation and interpretation of the observed phage patterns be reached. Typing phages in lyophilized form are now being commercially prepared. It is essential that all phages to be used for typing, whether prepared by the individual laboratory or by a commercial manu- facturer, meet certain established criteria of potency and host range as set forth in the recommendations in the above-mentioned paper.” There is reason to believe that commercial manufacturers of the typing phages will endeavor to produce preparations which con- form to the established standards. However, those who use the commercial preparations should become familiar with the recom- mended criteria and should, for their part, faithfully perform those control tests that are required at the laboratory level. STAPHYLOCOCCUS INFECTIONS 219 D. Sensitivity to Antibiotics Several laboratory procedures are available for estimating the inhibition of bacterial growth by antibiotics. Results may be defined in terms of the concentration per milliliter of the agent which com- pletely inhibits growth or in general terms, such as “sensitive,” “moderately resistant” and “resistant” (strains), according to arbi- trary standards often set at 1, 10 and 100 micrograms, respectively. The results obtained by different methods vary considerably!® and the selection of a method will depend upon the precision desired, the availability of materials, and the number of tests to be performed. Two basic methods are in general use—the tube dilution method, which is the more precise, and the disk method, which is qualitative but serves in many diagnostic laboratories to provide information about the relative susceptibility or resistance of cultures to the anti- biotics. Certain properties of staphylococci and staphylococcal infec- tions often make a precise method almost essential. The tube dilution test and the disk method are described in Chapter 27. IV. SEROLOGICAL EXAMINATION Because of the universal distribution of staphylococci and the frequency of exposure to these microorganisms, the majority of human sera often agglutinate staphylococci in vitro in varying degrees, although agglutination tests per se have no special diagnostic signifi- cance. While the pathogenic and nonpathogenic staphylococci have been shown to fall into distinct serological groups, the serological differen- tiation of individual cultures of pathogenic staphylococci has been restricted to those few laboratories where an intensive study of the problem has been made.!® In these laboratories and under certain conditions, the correlation between serological identification and phage typing has been found to be fairly close. In general, however, this correlation is less exact. Both methods have their advantages, but a serological testing routine such as is used in phage typing has not yet been defined. It would appear that the technics of serological identification of staphylococci are not now readily adaptable to the diagnostic laboratory. V. EVALUATION AND REPORTING OF RESULTS In the identification of staphylococci few difficulties are presented by their morphology and cultural appearance. However, because of the wide distribution of micrococci, it is of considerable importance 220 STAPHYLOCOCCUS INFECTIONS to know whether the organisms isolated in cultures of pathogenic ma- terial are pathogenic or whether they represent nonpathogenic, environmental forms. Fortunately, a simply performed in vitro test that differentiates pathogenic and nonpathogenic strains is available in the coagulase reaction. Pigmentation and hemolysis of blood agar are useful supplementary criteria and often are exhibited by freshly isolated strains. In the final analysis, however, a positive coagulase reaction provides the most acceptable evidence of pathogenicity, regardless of the presence or absence of these cultural properties. The identification of staphylococci is not complete until a coagulase test has been performed. Occasional strains of typical staphylococci may be coagulase- negative on first isolation but become positive after one or two trans- fers. When such a strain is morphologically and culturally typical but coagulase-negative, subculturing once or twice in broth or on blood agar is often advisable before it can be regarded as coagulase-negative. In the bacteriological examination of foods that are incriminated in food poisoning, occasional strains of enterococci may be en- countered that give a “false positive” coagulase reaction.?’ The morphology of these microorganisms and the performance of a test for heat stability should differentiate them readily from the staphylo- cocci. The individual laboratory must decide which of the methods of determining sensitivity to the antibiotics will best suit its needs. Inhibition in the tube dilution test should be reported as the concentra- tion in micrograms or units per milliliter that completely inhibits gross turbidity after incubation for 16 to 24 hr (Minimal Inhibitory Con- centration). The minimal bactericidal concentration, if desired, may be determined by transferring a standard quantity from each of the clear tubes to antibiotic-free media and observing for growth after incubation. Sometimes it is requested that the laboratory interpret the results of tests for sensitivity to the antibiotics in terms of “sensitive,” “moderately resistant” or “resistant.” This is usually the case when the disk method is used, and the interpretation may be qualified by reporting the amount of antibiotic in the disk(s) which inhibited or failed to inhibit growth of the culture. It is recommended that the results of positive cultures of pathogenic staphylococci be reported as “Staphylococcus aureus, coagulase- positive.” Joun E. Brawr, Pu.D., Chapter Chairman GEORGE GEE JAcksoN, M.D. L. Roranp Kunn, Pa.D. Rosert I. Wise, M.D., Pu.D. STAPHYLOCOCCUS INFECTIONS 221 REFERENCES 1 2 3 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. Bergey's Manual of Determinative Bacteriology (7th ed.). Baltimore, Md.: Williams & Wilkins, 1957. JurianerL, L. A., and WiecaArp, C. W. The Immunological Specificity of Staphylococci. J. Exper. Med. 62:11-21; 23-30, 1935. Cowan, S. T. The Classification of Staphylococci by Slide Agglutination, J. Path. & Bact. 48:169-173, 1939. Hoses, B. C. A Study of the Serological Type Differentiation of Staphylo- coccus pyogenes. J. Hyg. 46:222-238, 1948. . OepiNG, P. Serological Typing of Staphylococci, Acta path. et microbiol. scandinav. (Suppl.) 93:356-365, 1952. BArser, M., and Kuper, S. W. A. Identification of Staphylococcus pyogenes by the Phosphatase Reaction. J. Path. & Bact. 63:65-68, 1951. Lictey, B. D., and Brewer, J. H. The Selective Antibacterical Action of Phenylethyl Alcohol. J. Am. Pharm. A. (Scient. Ed.) 42:6-8, 1953. CrUIKSHANK, R. Staphylocoagulase. J. Path. & Bact. 45:295-303, 1937. Fisk, A. The Technique of the Coagulase Test for Staphylococci. Brit. J. Exper. Path. 21:311-314, 1940. CapNESS-GrRAVES, B.; WiLLiams, R.; Harper, G. J.; and MiLes, A. A. Slide Test for Coagulase-Positive Staphylococci. Lancet 1:736-738, 1943. WiLLiams, R. E. O,, and Rippon, J. E. Bacteriophage Typing of Staphylo- coccus aureus. J. Hyg. 50:320-353, 1952. Bram, J. E, and Carr, M. The Techniques and Interpretation of Phage Typing of Staphylococci. J. Lab. & Clin. Med. 55 :650-662, 1960. Jackson, G. G., Dowring, H. F., and Lepper, M. H. Bacteriophage Typing of Staphylococci. J. Lab. & Clin. Med. 44:14-28; 29-40; 41-50, 1954. Wisk, R. I, Cranny, C. and Srink, W. W. Epidemiological Studies on Antibiotic-Resistant Staphylococci. Bull. Univ. of Minn. Hosp. 26:174-190, 1954. AnpersoN, E. S., and Wirriams, R. E. O. Bacteriophage Typing of Enteric Pathogens and Staphylococci and Its Use in Epidemiology. J. Clin. Path. 9:94-127, 1956. BorMAN, E. K.,, Chairman. Statement on Availability and Uses of Staphylo- coccal Phage Typing. Committee on Staphylococcal Phage Typing, Labora- tory Section, American Public Health Assn. A.J.P.H. 49:1184-1188, 1959. Brag, J. E, and Wirriams, R. E. O. Phage Typing of Staphylococci. A statement prepared in behalf of the International Committee on Phage Typing of Staphylococci. WHO Bull. 24:771-784, 1961. Jackson, G. G., and Finranp, M. Comparison of Methods for Determining Sensitivity of Bacteria to Antibiotics in vitro. Arch. Int. Med. 88:446-460, 1951. OEepiNG, P., and WiLriams, R. E. O. The Type Classification of Staphylo- coccus aureus: A Comparison of Phage Typing with Serological Typing. J. Hyg. 56:445-454, 1958. Evans, J. B.,, Buerrner, L. G., and Niven, C. F, Jr. Occurrence of Streptococci That Give a False-Positive Coagulase Test. J. Bact. 64: 433-434, 1952. CHAPTER 7 PNEUMOCOCCUS INFECTIONS I. Morphology II. Type Differentiation ITI. Isolation from Mixed Cultures by Mouse Inoculation IV. Differentiation from Other Cocci A. Bile Solubility B. Inhibition by Optochin C. Immunological Specificity of C Carbohydrate V. Examination of Specimens A. Sputum B. Cerebrospinal Fluid and Pus C. Blood VI. Media The availability of effective antimicrobial drugs for treatment of pneumococcal infections has resulted in a general abandonment of procedures for making a specific etiological diagnosis. This situation is unfortunate for a variety of reasons, not the least of which is the obligation of the physician (which he can no longer discharge) to make accurate diagnosis of the patient’s disease. In addition to this ethical and scientific injunction there are several practical reasons for specific diagnosis, including proper treatment of the individual patient and accurate prognosis. Pneumococcus causes a variety of pyogenic infections such as sinusitis, otitis media, menin- gitis and occasionally empyemas, peritonitis, pyarthrosis, endocarditis and pericarditis. As has long been known, it is by far the commonest and most important cause of acute pneumonia in persons of all ages. Because this is the case and because pneumococcus in nature is highly susceptible to so many of the antimicrobial drugs, the general tendency in medical practice is to treat acute bacterial pnuemonias as though all were caused by pneumococcus. If a “satisfactory” response is not obtained with antipneumococcal therapy, the physician com- monly shifts to other drugs, singly or in combination. By this time it may be impossible to be sure of the cause of the patient’s illness, so that therapy can no longer be properly guided; or else the disease 222 PNEUMOCOCCUS INFECTIONS 223 process may have become so far advanced that the changes are irreversible and no therapy can help. Acute bacterial pneumonia is not uncommonly caused by Klebsiella pneumonia (Friedlinder’s bacillus) and by Staphylococcus pyogenes. Both species cause very severe disease associated with considerable mortality. Appropriate treatment of these diseases should be started at the earliest moment, which necessitates etiological diagnosis. Although differentiation of pneumococcus, Klebsiella and staphylo- coccus offers no difficulty, regardless of the source in the patient from which the material containing them derives, the situation is more complicated than this statement indicates because all three organisms are commonly carried in the normal human pharynx. To determine which is responsible for disease may be difficult without adherence to certain principles in the examination of sputum. I. MORPHOLOGY In sputum or exudates pneumococcus exists in diplococcal form. In the test tube it is often found in chains of variable length. The cocci are usually described as lancet-shaped, although they are sometimes difficult to differentiate from various streptococci. In addition, some strains of pneumococcus may lack the typical lanceolate form. For this reason, although the shape of the cocci is helpful, it is by no means diagnostic. In fresh sputum and exudates and in young cultures, pneumococcus is strongly Gram-positive. However, it undergoes autolysis readily so that preparations which have been allowed to sit at room tempera- ture or in the 37° C incubator may become partially or wholly con- verted to Gram negativity. Along with the change in staining reaction, there occurs loss of the ability to effect a capsular swelling or “quellung” reaction in the presence of specific antipneumococcal serum. On the surface of fresh, moist blood agar plates of proper compo- sition, young cultures of pneumococcus (18 to 24 hr) give rise to smooth, glistening colonies from 1 to 2 mm in diameter, except for Type 3, which forms much larger, juicy colonies that are easily recognizable. As the culture ages and autolytic changes begin, the center of the pneumococcal colony may collapse, giving it a checker- like appearance. The rate and extent of autolysis are highly variable from strain to strain. Identification of pneumococcal colonies in pure or in mixed culture on the surface of blood agar plates is facilitated greatly by the use of oblique reflected illumination and of low-power magnification (45X) provided by a dissecting microscope. 224 PNEUMOCOCCUS INFECTIONS The colonies on blood agar are surrounded by a zone of alpha hemol- ysis which increases in size as the culture ages. Under ideal circumstances it may be possible to differentiate pneumococcus from viridans streptococci on morphological grounds alone. However, in most cases except for the highly characteristic colonies of Type 3 pneumococcus, differentiation cannot be made with assurance. Il. TYPE DIFFERENTIATION Immunological Considerations Pneumococcus is subdivisible into about 80 specific immunological types, depending upon differences in the chemical and immunological properties of the complex polysaccharides which compose the capsules that surround the virulent cells. Type differentiation depends upon the availability of specific diagnostic antipneumococcal serum for each of the types. A variety of reactions can be used for type differentiation—for ex- ample, agglutination, precipitation, mouse protection and capsular swelling. Of these, capsular swelling or the quellung reaction is by far the most useful for practical diagnostic purposes because it can be applied directly to bacteria in sputum or exudates as well as to pneumococci from cultures on solid or in fluid laboratory media, and the reaction can be examined immediately. A loopful of material presumed to contain pneumococcus is spread on a glass slide. A loopful of antipneumococcal serum and a loopful of 1 per cent aqueous alkaline methylene blue to stain the bacterial bodies are mixed on the surface of a cover slip and the inverted cover slip is applied to the area of the slide bearing the bacterial suspension. The wet preparation is then examined immediately under the oil- immersion lens with reduced and slightly oblique illumination. The reaction of specific antibody with the polysaccharide capsule causes the capsule to swell so that it appears as a sharply demarcated re- fractile zone of variable width surrounding the coccus. The size of the swollen capsule depends both upon the amount of polysaccharide in the capsule and upon the potency of the anti- capsular serum. For this reason it is important not to use bacterial suspensions containing excessive numbers of pneumococci and it is well to limit the number of organisms to less than 50 per oil-immer- sion field to avoid inhibition of capsular swelling by the presence of excess antigen. When the amount of capsular antibody is insufficient to cause capsular swelling, agglutination of the pneumococci may still PNEUMOCOCCUS INFECTIONS 225 be observed under the microscope. Type 3 pneumococcus, which pro- duces the most polysaccharide, gives a much larger capsular swelling reaction than that of any other capsular type. Although there are about 80 pneumococcal types, use of the quellung reaction for practical clinical diagnosis is not as difficult as this large number of different types would imply. It is not necessary to examine separately for quellung with each antiserum, since several different sera can be combined in a pool, which is then applied to the unknown pneumococcal suspension. If a quellung occurs with a particular pool, the suspension is then tested with each of the indi- vidual sera making up the pool. A further simplification arises from the fact that most pneumococcal infections, whether in children or adults, are caused by only a few out of the many immunological types. In adults, Types 1, 2, 3,4, 5, 7 and 8 are responsible for about two-thirds of all cases of pneumococcal pneumonia. Types 6, 14, 18 and 19, in addition, are important in children. Despite the great convenience and elegant specificity of the quellung reaction, its use unfortunately becomes more limited with each passing year, as supplies of specific antisera, whether singly or in pools, have been proving more and more difficult to obtain. Pooled sera for recog- nition of the first 33 capsular types are now commercially available and it is to be hoped that steps will be taken to provide individual type-specific antisera as well—not only for use in clinical diagnosis but also for study of the epidemiology of pneumococcal infections. Ill. ISOLATION FROM MIXED CULTURES BY MOUSE INOCULATION Most of the types of pneumococci that cause human disease are highly virulent for the laboratory mouse (Type 14 is an exception). Mouse virulence is a useful property, especially in the identification of pneumococci in sputum specimens because the other Gram-positive species of cocci that are present are usually of very low virulence and so are eliminated. The mouse acts, so to speak, as a “filter” which permits the outgrowth of pneumococcus while destroying most of the other Gram-positive species that are present. In similar fashion, it may facilitate isolation of microorganisms of the genus Klebsiella from the sputum of patients with Friedldnder’s pneumonia. Inocula- tion is carried out by the intraperitoneal route. A small amount of freshly obtained mucopurulent sputum is emulsi- fied in an equal volume of isotonic sodium chloride solution by al- ternately aspirating and expelling it in a syringe without attached 226 PNEUMOCOCCUS INFECTIONS needle. Between 0.25 and 1 ml of the emulsion is then injected intra- peritoneally. The mouse will die, often in less than 24 hr, and usually within a period of 4 days following inoculation. Animals surviving 4 days should then be sacrificed and examined because approximately 5 per cent will be found to harbor pneumococci. Pneumococci will generally be found in pure culture in the peritoneal exudate and heart blood. Following confirmation of the presence of Gram-positive cocci in the peritoneal exudate by stain, the fluid is cultured on blood agar in the presence of a bile salt or optochin disk as described below. If specific antisera are available, type diagnosis can be made by quellung reaction with the peritoneal fluid. Before use in the test for quellung reaction, the fluid should be diluted with sufficient isotonic sodium chloride solution to give a density approximating that of an over- night culture of pneumococcus. It should be noted that mouse inoculation for the isolation of pneu- mococcus is of advantage only when a mixed bacterial flora is pres- ent, as in the case of sputum. No advantage is to be gained by the inoculation of mice with cerebrospinal fluid from patients with meningitis. Spinal fluid or pus should be cultured directly on blood agar or in broth containing blood. IV. DIFFERENTIATION FROM OTHER COCCI A. Bile Solubility A characteristic of pneumococcus that distinguishes it as a species from other members of the genus Streptococcus is its susceptibility to the action of bile salts such as desoxycholate or taurocholate. Growth of pneumococcus is entirely inhibited in the presence of very low concentrations of whole bile or individual bile salts. Furthermore, suspensions of pneumococci are quickly lysed by bile due to activation of the autolytic enzymes of the cell. Streptococci, on the other hand, are not lysed by bile salts and inhibition of their growth requires much higher concentrations than in the case of pneumococcus. As a practical diagnostic reagent, bile may be used in two ways: (1) by the application of a bile-containing disk to a blood agar plate on which a culture of pneumococci has been streaked (if pneumo- coccus is present, inhibition of its growth will occur in a zone sur- rounding the disk) and (2) by determining whether the bacterial sus- pension is bile soluble. A saline-washed suspension of Gram-positive cocci from growth in broth or on the surface of an agar plate is mixed with an equal volume of sterile ox bile or of 0.1 per cent PNEUMOCOCCUS INFECTIONS 227 sodium desoxycholate (or sodium taurocholate). The mixture is placed in a water bath at 37° C and examined for clearing at intervals up to 1 hr. If pneumococcus is present, clearing will usually occur within a few min. In tests for bile solubility it is necessary to wash the bacterial suspension before adding bile, especially when serum is present. Serum inhibits the reaction. B. Inhibition by Optochin Growth of pneumococcus is inhibited by optochin (ethylhydro- cupreine hydrochloride), whereas the growth of streptococci is not affected unless very large concentrations of the compound are used. Because of the selectivity of growth inhibition by optochin, this property can be used to distinguish pneumococcus from streptococci. The most convenient method is the application of optochin-containing disks to plates upon which unknown cultures are streaked. As in the case of bile disks, growth of pneumococcus is inhibited in a zone surrounding the disk, whereas streptococci are unaffected. Optochin disks do not appear to possess any advantage over bile disks in pneumococcal identification. C. Immunological Specificity of C Carbohydrate Pneumococcus possesses as a constituent of its cell wall a C carbohydrate which distinguishes it with reference to its species just as the C carbohydrates of hemolytic streptococci can be used to define the various groups of these organisms. The C carbohydrate of pneumococcus is prepared by acid extractions—as in the case of streptococci—and is tested with antiserum in precipitin reactions, Although the C precipitin reaction may be of use in identifying pneumococcus under certain undefined circumstances, as a practical measure it is superfluous in clinical diagnosis. V. EXAMINATION OF SPECIMENS A. Sputum The greatest success in identifying pneumococcus in sputum is obtained with fresh specimens. The sputum of patients with pneumo- coccal pneumonia is often rusty or blood-streaked, but these prop- erties are not diagnostic. Select a small mass of mucopurulent sputum and wash it free of saliva by gently manipulating it with a sterile wire loop or pipette in a petri dish containing isotonic saline soluton. Remove to a clean petri dish and add a small amount of saline solution. Employing a 228 PNEUMOCOCCUS INFECTIONS sterile syringe without needle attached, emulsify the sputum and saline mixture, When well emulsified, prepare a film and stain immediately by Gram’s method. If Gram-positive cocci are present and specific typing sera are available, carry out direct typing by quellung reaction (commonly an answer can be obtained by this method within a few minutes). Streak a loopful of emulsified sputum on the surface of a blood agar plate and then apply one or more bile disks. Examine after 16 to 24 hr incubation at 37° C for colonial morphology and for inhibition of growth by bile. Single colonies may be selected for typing by quellung reaction, or for restreaking on the surface of a second blood agar plate to which a bile disk may then be applied. Inoculate a mouse intraperitoneally with 0.25 to 1.0 ml of the sputum emulsion. Examine after 12 to 14 hr. At this time the mouse is often obviously ill; if necessary, it may be sacrificed and the peritoneal contents removed for morphological, cultural and im- munological diagnosis. As noted above, death of the mouse commonly occurs in less than 24 hr. The contents of the peritoneal cavity are washed out with saline solution, stained by Gram’s method and cultured on blood agar in the presence of a bile disk. Typing by quellung reaction can be carried out with a high probability of success if the sputum inoculated into the mouse contained even small numbers of pneumococci. At times mice injected with large amounts of sputum will show a mixed peritoneal culture at death. Usually, however, the heart blood yields a pure culture. Whether or not specific type diagnosis is contemplated, mouse inoculation of sputum is one of the most useful technics available for the etiological diagnosis of acute bacterial pneumonia, facilitating the recovery of Klebsiella as well as of pneumococcus. When cultural methods alone are used, failure to make a diagnosis occurs frequently even when carried out by experts. It is therefore recommended that mouse inoculation be employed routinely in the examination of sputum. Occasionally two and rarely three or more types of pneumococcus may be present in a single specimen of sputum. When sputum or mouse peritoneal fluid is examined by the quellung technic and capsular swelling is observed to have affected only a fraction of those bacteria manifesting typical pneumococcal morphology, it is worth- while to continue the study with the remaining available anticapsular sera. Such a procedure may effect the identification of additional pneumococcal types. When pneumococci are present in cultures of sputum, blood and/or other body fluids from a patient, capsular PNEUMOCOCCUS INFECTIONS 229 typing should be carried out with each culture to determine whether or not more than one type of pneumococcus is present. B. Cerebrospinal Fluid and Pus As noted above, when it is anticipated on clinical grounds that a pure culture of pneumococcus is likely, mouse inoculation is super- fluous and should be omitted. The material (pus or spinal fluid) should first be smeared on a slide and examined after Gram-staining. If pneumococcus is suspected from the results of microscopical ex- amination, direct typing by quellung reaction should be carried out if serum is available. The fluid should also be cultivated on blood agar plates with bile disks and in 0.1 to 1 ml amounts in a tube of blood broth. C. Blood Because of the prognostic importance of bacteremia in pneumo- coccal infection, a blood culture should always be obtained when this diagnosis is entertained. Taking sterile precautions, 20 ml of venous blood should be withdrawn, 15 ml of which are inoculated into a flask containing 80 ml of suitable meat infusion-peptone broth. The re- maining 5 ml are added to a sterile tube containing a small amount of anticoagulant; a blood agar pour plate is then made following the addition of 1 or 2 ml of the patient’s blood in anticoagulant to a tube containing meat infusion-peptone agar at 46° C. Pneumococci are identified after 24 to 48 hr incubation at 37° C by the technics of staining, capsular swelling and/or sensitivity to bile described in the foregoing. VI. MEDIA Commercially prepared, dehydrated media are generally used in diagnostic laboratories for the cultivation of pneumococcus. Most batches of these dried media contain components which are inhibitory to pneumococcal growth in greater or lesser degree. The inhibitory properties can be neutralized in part by the addition of 1 to 2 per cent of human or animal blood; or by the addition of sterile serum in final concentration of 5 to 10 per cent. The source of the blood or serum is not important in the case of pneumococcus. Besides reduc- ing the inhibitory properties of culture media, the addition of blood or serum retards the rate of pneumococcal autolysis. The bacteria therefore retain their viability for a much longer time, whether at 37° C or in the icebox. 230 PNEUMOCOCCUS INFECTIONS Uniformly satisfactory media for pneumococcus are best prepared from fresh meat infusion to which is added 1 per cent of any good brand of peptone. The final pH should be in the neighborhood of 7.4. Sterilization is carried out in the autoclave, but it must be .em- phasized that overheating is to be avoided. Overheating causes the medium to become inhibitory, possibly because of the formation of humins. Glucose need not be added to a medium prepared from fresh meat infusion; in fact, added glucose may exert a detrimental influence on pneumococcal recovery, since cultures containing excess glucose quickly become acid and cause the death of pneumococcus. For this reason, if the medium contains an excess of glucose, cultures sus- pected of containing pneumococcus should be examined after 4 to 8 hr of incubation at 37° C. Corin M. MacLeon, M.D., Chapter Chairman RoBeErr AUSTRIAN, M.D. MaxweLL FiNvcanp, M.D. CHAPTER 8 DIPHTHERIA I. Collection of Specimens II. Preparation of Original Cultures and Films A. Inoculation of Slants B. Detection of Other Pathogens C. Preparation of Direct-Swab Films D. Reporting on Direct-Swab Films E. Inoculation of Tellurite Plates 111. Incubation of Original Cultures IV. Examination of Original Cultures V. Staining and Microscopy VI. Examination of Growth on Plates VII. Pure Culture Studies A. Preparation of Pure Cultures B. Examination of Pure Cultures 1. Morphology 2. Toxigenicity (virulence) 3. Determination of type 4. Methods References It is the purpose of this chapter to present, first, methods by which the bacteriological laboratory may furnish physicians with reliable data relative to the diagnosis of possible cases of diphtheria and, second, means of isolating pure cultures of Corynebacterium diph- theriae and of determining their types and toxigenicity. In clinically recognizable cases of diphtheria of the respiratory tract, C. diphtheriae is usually present in large numbers in the lesions and membranes. Experience has shown that in these cases, as well as in subclinical infections, the diphtheria bacilli may readily be cultivated on an appropriate medium provided chemotherapy has not too recently been administered. When cultivated, spread and stained in a suitable manner, the organisms in such cultures can usually be recognized by a person properly trained in such work. Further, experience has shown that the presence, in diphtheria-like lesions, of organisms 231 232 DIPHTHERIA morphologically resembling C. diphtheriae may be accepted as pre- sumptive evidence, not only of the identity but also of the toxigenicity of the organisms. On the basis of such morphological evidence alone physicians usually administer antitoxin if they have not previously done so on the grounds of clinical observations. Completed evidence of the identity and type of the microorganism in question may be obtained only after isolation of a pure culture of C. diphtheriae from the patient.!-® However, using procedures de- scribed below*-7 it is possible to demonstrate the presence of toxigenic C. diphtheriae in most cultures from cases and contacts within 24 hr of swabbing the patient’s throat. In contrast with cultures from clinically probable cases of diph- theria, difficulty often arises in examining cultures from doubtful cases or from presumably healthy persons made during surveys. Toxigenic C. diphtheriae in original cultures from many such persons occurs in morphologically unrecognizable (coccoid) form or forms poorly differentiated from diphtheroids. The recognizable form de- velops from the coccoid readily on subculture. Even when clearly identifiable as C. diphtheriae by morphological, tinctorial and cultural properties, such strains are often totally atoxigenic. Less trouble is experienced with cultures from known convalescent carriers or case- contact carriers.® For these reasons the isolation and study, in pure culture, of all microorganisms which may be C. diphtheriae are essential parts of the work of the diagnostic laboratory, since they are the only known means of identifying them with certainty. The procedures described in this chapter are not the only ones available for the purpose.®-*® They have been selected because they are known, on the basis of comparative studies, to yield satisfactory results if carefully followed. A choice of methods is given in some places, since one method may be more applicable in some laboratories than another. (See flow chart.) I. COLLECTION OF SPECIMENS The physician is fully justified in expecting accurate diagnostic data from the laboratory. He must remember, however, that the accuracy which he demands depends in large part on his own in- telligence and care in collecting and submitting specimens. In making a throat culture, illuminate the pharyngeal region prop- erly and, if necessary, gently depress the tongue. Routinely collect material from throat and/or nose on wooden or aluminum applicators padded with a small amount of absorbent cotton. It is sometimes con- FIRST | DIPHTHERIA 233 = SWAB DAY [2 Gime, Lod Flood == Fp a gar es DAY RIVTT THIRD DAY FOURTH DAY FIFTH DAY SIXTH DAY 0000 = i ] McL. Agar TYPING Flow chart of a typical scheme showing sequence of culturing, isolating, and identifying C. diphtheriae, in the laboratory, venient, in preparing swabs, to pad some of them with a minimum of cotton for use in the nose. Sterilize and keep the swabs in sterile tubes until used. For clinical diagnostic purposes keep the swabs separate. Use one or two sterile swabs for each person cultured. Use the first swab to obtain material from the throat lesions or tonsillar crypts; the other may be passed to the nasopharynx through one nostril—this is optional. Rub a sterile swab over any white spots, ulcerations or in- flamed areas and immediately withdraw it. Whether the swab is im- mediately used to inoculate media or is itself shipped directly to the laboratory must depend on local circumstances. In general, inoculate culture media as soon as possible after collecting the specimen. Long delays in transporting swabs to the laboratory probably cause some reduction in numbers of viable organ- 234 DIPHTHERIA isms, especially swabs from convalescents and contact carriers.’ Do not leave a swab in any culture. Return it to the laboratory in a separate tube to be used for making films and other cultures. Il. PREPARATION OF ORIGINAL CULTURES AND FILMS Since no single procedure is applicable under all conditions and since several procedures now in use have been found valuable in differ- ent laboratories, they are mentioned here. It is desirable to have various laboratories conduct comparative studies with these methods. A. Inoculation of Slants Rub the swab gently over the entire surface of a slant of moist Loeffler’s medium (CM No. 81), modified Pai medium!® (CM No. 78), or raffinose serum tellurite agar* (CM No. 82). Rotate the applicator between the thumb and forefinger while the swab moves over the medium. Use the same swab to inoculate a serum antitoxin plate for the RIVTT (see Section VII B 2(e), which appears later in this chapter). B. Detection of Other Pathogens It is important to detect pathogens other than C. diphtheriae in any infection of the upper respiratory tract. For this purpose, after inoculating the media especially intended for C. diphtheriae, rub the swab on the surface of a plain, unheated blood agar plate and dis- tribute the inoculum to obtain well-isolated colonies. Other plating media such as heated blood agar or Bordet-Gengou agar may be similarly inoculated at this time if thought desirable. For further details of the study of various other pathogens, see appropriate chapters in this book. C. Preparation of Direct-Swab Films After inoculating culture media, prepare films by rubbing each swab in a drop of water on a clean slide over an area about 1 sq cm. Stain with alkaline methylene blue and examine for C. diphtheriae. D. Reporting on Direct-Swab Films If the film appears to contain C. diphtheriae, make a tentative report immediately by telephone to the physician. State clearly in the report to the physician that any identification of diphtheria bacilli based on examination of such films is tentative and is subject to correction when cultures are examined. DIPHTHERIA 235 The examination of swab films is extremely important, not only for the early specific treatment it makes possible in the otherwise missed case, but also because early administration of antibiotics may prevent or greatly retard the growth of diphtheria bacilli in culture, though not their toxin production in the patient. Do not make final identification of diphtheria bacilli (or report their absence) in direct-swab films unsupported by bacteriological evidence from cultures, since some species of Nocardia, Actinomyces and related organisms frequently present in the normal throat closely simulate C. diphtheriae morphologically. Most of these organisms do not grow rapidly on slants of the media recommended for C. diphtheriae in this chapter, but there are some species that do. Further, C. diphtheriae sometimes occurs in temporarily unrecogniz- able coccoid forms. In such cases it is not detected until it is isolated in pure culture or until it reacts in the RIVTT. E. Inoculation of Tellurite Plates a. Use the same swab to inoculate the surface of plates of cystine tellurite blood agar'® (CM No. 79), heated blood tellurite agar?® (CM No. 84) or dextrose serum tellurite agar® (CM No. 83). Use the first two media when Loeffler or modified Pai slants are used for initial cultures; the last when the raffinose serum tellurite agar slants are used for initial throat cultures. b. Whatever plating medium is chosen, distribute the inoculum over the entire surface in graded amounts in such a manner as to obtain numerous well-isolated colonies. Ill. INCUBATION OF ORIGINAL CULTURES 1. Temperature—Incubate cultures at 35° C. 39°C 2. Arrangement—Incubate inoculated plates in an inverted position. Do not place more than two plates to a pile during the first 18 to 24 hr, since large stacks of plates interfere with the even and rapid penetration of heat throughout the incubator, IV. EXAMINATION OF ORIGINAL CULTURES A. Time Examine original Loeffler, modified Pai, and raffinose serum tellurite slants at various intervals during the first 18 to 24 hr of incubation. On diagnostic cultures, make at least one preliminary 236 DIPHTHERIA examination 2 to 8 hr after inoculation and in any case late in the same day on which the culture is inoculated. These early examinations are often positive, and overgrowth by contaminating microorganisms later in such cultures is largely avoided. Reincubation of Loeffler and/or modified Pai slants negative after 18 to 24 hr and reexamination after another 24 hr are recommended. In release and contact cultures, diphtheria bacilli are found frequently on the second day and not on the first. B. Method 1. Prepare a film from each Loeffler, modified Pai, or raffinose serum tellurite agar slant culture, using a selected portion, or after thoroughly mixing the entire growth on the surface of the slant with a sterile wire. Emulsify a small portion of the growth in a drop of broth on one end of a slide marked into three spaces (see Iig 1). Do not stain until after other films are placed on the same slide as directed in Section VI E (1) of this chapter. OOO" Figure 1—Method of marking slide into three spaces for making diagnostic films: (1) film from raffinose slant; (2A) and (2B), films from dextrose serum tellurite plate. (Courtesy Dr. S. R. Damon, formerly Director, Bureau of Laboratories, Indiana State Board of Health.) 2. If individual colonies from slants or tellurite plates are to be examined, films from them are made separately in space 2A of the slide (Fig 1). VY. STAINING AND MICROSCOPY A. Stain 1. Stain all films prepared for diagnostic purposes with Loeffler’s alkaline methylene blue. DIPHTHERIA 237 2. The nature of the staining solution is of importance. Prepare it in accordance with directions given in Chapter 1. Granule stains such as Albert’s, Ljubinsky’s or Ponder’s, while useful for special studies, are not recommended for diagnostic purposes. Too great an emphasis on granules stainable by these methods leads to error and causes the more fundamental morphological characteristics to be overlooked. Further, many diphtheria bacilli do not form granules. B. Cellular Morphology 1. Diphtheria bacilli are Gram-positive, markedly pleomorphic rods which are nonmotile, lack capsules, and do not form spores. They vary greatly in size and shape, depending on the type of C. diphtheriae, the age of the culture, and the medium upon which they are grown. There is wide variation even in a single field, where the straight or slightly curved rods may vary from 1 to 8 u in length. Chains are not formed. 2. In the early (1-8 hr) growth of a throat culture on Loeffler’s blood serum medium or raffinose serum tellurite medium, the diph- theria bacilli often outgrow the other throat flora and appear in almost pure culture. At this age, morphology is distinguishing, with the similarity of all types most striking. Presumptive diagnosis at this early stage is frequently possible, therefore, and is of inestimable value if serum therapy has not yet commenced.?! 3. In these young cultures the bacilli stain intensely with Loeffler’s alkaline methylene blue. Characteristically the septum is not centrally located but occurs in the thicker end of the bacillus in such a way as frequently to produce a distinguishing wedge-shaped segment; the other segment is longer, is slightly curved and tapers to a point. The wedge form as a rule is seen only in cultures of true C. diphtheriae. In general, diphtheria bacilli are not of even thickness throughout. Some may be swollen at one end, some at both ends, while still others are pointed at both ends, with a swollen portion between. The indi- viduals lie at angles with one another, rather than in palisades, and open V and Y formations are frequent (Fig 2). 4. As the culture grows older, the pleomorphism of the diphtheria bacilli increases and the barring produced by unstained portions of the cells becomes more marked. In some strains, at about 12 hr deeply staining, refractive, metachromatic granules begin to appear which may be round or oval and thicker than the rest of the bacillus or thinner and surrounded by a less deeply staining area. By 18 to 24 hr the culture may show all degrees of staining and all possible forms, 238 DIPHTHERIA Figure 2—Drawing of a young culture of C. diphtheriae on Loeffler’s medium, magnification about 1000x. Note the angled grouping and the characteristic wedge shape of several cells; also cocci in the lower part of the field. This culture was almost completely overgrown by the cocci in 24 hr. (Courtesy Dr. R. A. MacCready, Director, Division of Diagnostic Laboratories, Massachusetts Department of Public Health, published by permission of the Canadian Journal of Public Health.) including the characteristic club forms, short “tear drop” forms (Fig 3), and even coccoid forms, as well as overgrowth by the normal throat flora. The microscopic identification of diphtheria bacilli is admittedly a difficult art that can be mastered only by considerable experience, but the careful study of the stained film is still an exceedingly useful and rewarding laboratory procedure. 5. Cellular morphology of the types of diphtheria bacilli may be distinguished on dextrose serum tellurite agar or unheated blood agar as follows :5.9.22-24 DIPHTHERIA 239 Figure 3—Microphotograph of C. diphtheriae (mitis type) in a frequently seen form, cultivated on Loeffler’'s medium 18 hr, stained with alkaline methylene blue, magnification 1500 x. (Photo courtesy Dr. R. A. MacCready.) a. Intermedius type—Pleomorphic, with club-shaped forms com- monly seen; few or no metachromatic granules; many solid-stain- ing forms. Barred cells are deeply stained, with pale blue areas between the bars (Fig 4A). b. Mitis type—Pleomorphic, with well-developed metachromatic granules in most strains; barred forms infrequent. Typical, rather slender rods, as compared to intermedius type (Fig 4B). c. Gravis type—Uniform, short, stout, heavily staining. Occasional tear-, club- and wedge-shaped forms seen. Some barred and shadow forms may be present (Fig 4C). C. Report If microorganisms thought to be C. diphtheriae are seen, report to the physician immediately by telephone. VI. EXAMINATION OF GROWTH ON PLATES Examine tellurite agar plates after 20 to 24 hr of incubation. By means of a low-power lens (about 8X) but preferably with a binocu- lar dissecting microscope, search for colonies likely to be those of C. diphtheriae. It must be remembered that tellurite media are not absolutely selec- tive for C. diphtheriae, especially with carrier cultures. Colonies of certain diphtheroids and micrococci are particularly likely to be con- fused with those of C. diphtheriae. 240 DIPHTHERIA Figure 4—Microphotographs of C. diphtheriae: A =intermedius type; B =mitis type; C=gravis type. Cultivated on unheated blood agar, stained with alkaline methylene blue, magnification 1000 x. (Courtesy Dr. E. T. Bynoe, Chief, Bacteriological Laboratories, Department of National Health and Welfare, Ottawa, Canada.) Since the appearance of the various colonies differs considerably on the plating media recommended in this chapter, a description of colonies on each medium is given here. A. Colonies on Cystine-Tellurite-Blood (CTB) Agar (CM No. 79) Descriptions are based on an incubation period of between 24 and 48 hr. 1. Mitis type—Circular, soft and butyrous in consistency, smooth and polished or glistening, usually rounded or domed but sometimes slightly conical; from 0.5 to 2.5 mm in diameter; of very dark slate color, almost black but not jet black. They are usually opaque from rim to rim; no zones of hemolysis (Fig 5). 2. Intermedius type—Flatter than mitis-type colonies; usually dry and opaque; very small and uniform in size; often have very narrow translucent zone at edge. 3. Gravis type—Larger than mitis-type colonies, ranging up to 4 mm in diameter; surface dull, matt or rough, with radial ridges; form, low and conical ; margins irregular. Often somewhat friable in consistency. Sometimes all of these characteristics are diminished and the colonies approach the mitis type in appearance. 4. Minimus type*—Exceedingly minute on all media (0.1 to 0.5 mm) ; usually flat with a black center and a brownish, translucent periphery. The surface may be rough or smooth. * The minimus type is not recognized by some members of this committee but is considered by them to be identical with the intermedius type. It is described here because it is recommended by Frobisher as a control in the in vitro toxi- genicity test. DIPHTHERIA 241 Figure 5—Appearance of colonies of C. diphtheriae (mitis type) on CTB agar. (Photo courtesy Dr. Martin Frobisher, Formerly Consultant, CDC, USPHS, Chamblee, Ga.; published by permission of W. B. Saunders Co., Philadelphia, Pa.) 5. Diphtheroid colonies are usually light greenish gray in color, dark in the center, with a lighter, translucent periphery. They are more distinctive when well isolated; when crowded, they often have an appearance indistinguishable from colonies of mitis-type C. diph- theriae. 6. Micrococcus colonies of some species are usually flatter and thinner than those of C. diphtheriae and are of an intense, glistening, jet black color. Staphylococcus aureus colonies tend to a slaty tint and often closely resemble those of C. diphtheriae. Usually, Staph. aureus colonies are larger and show a center or concentric rings of lighter gray color. 7. Yeast colonies are white. 8. Spore formers, etc., sometimes appear but their colonies are readily distinguishable by their brown or greenish color, mucoid or dry texture, hardness, surface membrane, etc. A little experience soon serves as a guide to selection of colonies. When there is doubt, fish the colonies. 242 DIPHTHERIA B. Colonies on Heated Blood Tellurite (HBT) Agar (CM No. 84) Descriptions are based on an incubation period from 24 to 48 hr. 1. Mitis type—Similar to mitis-type colonies on CTB agar but may be somewhat larger, flatter and less shiny, with slightly irregular margins. 2. Intermedius type—Similar to intermedius-type colonies on CTB agar but may be less shiny and have slightly irregular margins. 3. Gravis type—Like gravis-type colonies on CTB agar but larger and rougher. 4. Minimus type—Like minimus-type colonies on CTB agar. 5. Diphtheroids, micrococci, etc.—Same as on CTB agar. C. Colonies on Dextrose Serum Tellurite (DST) Agar (CM No. 83) The most significant characteristics are those observed at the end of 18 to 24 hr of incubation because it is at this time that parallel film preparations from Loeffler, Pai and/or raffinose serum tellurite slants are generally examined. Descriptions given here are of 24 hr colonies. 1. Mitis type— Variation in colony size is common, 0.3 to 1.25 mm (average, 1.0 mm) ; gray, sometimes a dull gray with a darker gray center, smooth, round, convex and glistening. Entire edge is of butyrous consistency. 2. Intermedius type—Very small gray colony, 0.25 to 0.5 mm (average, 0.3 mm); very slightly raised, but sometimes flat with a tiny black dot or dark gray center. Edge is serrated or entire, with surface somewhat matt-like in some strains; uniform in size. 3. Gravis type—Size 1.0 to 1.5 mm (average, 1.25 mm), gray with dark grayish black center, translucent periphery, raised and round. In some strains edge is crenated to a variable extent, is brittle and tends to fracture radially when touched with a needle. 4. Staphylococcus aureus—Colony varies in size in the differ- ent strains, somewhat flat with elevated center, glistening, round, black or bluish black, with a very thin, translucent periphery. 5. Other micrococci—Round, raised, grayish, glistening and resemble the mitis-type diphtheria colony somewhat. Often show some blackening in center of colony which in that event helps to differ- entiate them. DIPHTHERIA 243 6. Diphtheroids—Very small, dead white and pinpoint in size; a few strains have brown tint and are large. 7. Yeasts—White, moist to dry, round and varying in size according to species. 8. Spore formers—Usually light brown, usually mucoid, of good size but not spreading unless the medium is quite moist. Generally entirely suppressed. 9. Streptococci—Vary in size, texture and elevation; majority of strains are small, flat and round, with glistening black centers; periphery is thin, translucent. Some strains are light brown in color, while others are black—these may have to be picked to be certain of their identity. 10. Pneumococci—Round, flat, dull, dark greenish colony with lighter greenish edge ; matt-like texture. D. Transfer of Colonies from CTB and HBT Agar 1. Transfer suspected colonies directly to slants of Loeffler or modified Pai medium or to plates for the in vitro toxigenicity test (IVTT) (see Section VII B 2(d) of this chapter). After overnight incubation of the slants, examine the growth by means of methylene blue stained films. As a rule, C. diphtheriae is readily recognizable in such preparations. Use those slants showing, on microscopic examina- tion of the growth, pure cultures of C. diphtheriae for tests of toxi- genicity and for type determination as described in Section VII of this chapter. 2. To facilitate diagnosis based on the film from the original cul- ture (Section IV B) portions of two or three suspected colonies may be suspended in drops of water on space 2A of the same slide as used for the initial culture (see Fig 1). However, since the morphology of C. diphtheriae is often distorted on CTB or HBT agar, it is usually more satisfactory to await growth on the Loeffler or modified Pai slants. These must in any event be inoculated to obtain pure cultures for typing and for toxigenicity tests in animals, and often for the IVTT. 3. Whether or not colonies thought to be C. diphtheriae are seen, streak another plate of CTB agar or HBT agar with mixed growth from the initial Loeffler or modified Pai culture and proceed with these as with the first CTB or HBT agar plates. These second plates 244 DIPHTHERIA frequently yield pure cultures of C. diphtheriae, even though the first plates failed to do so. 4. Mixed growth from the CTB or HBT agar plates or from the original Loeffler or modified Pai slants may also be streaked on antitoxin plates for the RIVTT at this time (see Section VII B 2(e) of this chapter). E. Transfer of Colonies from DST Agar 1. Emulsify up to three suspected colonies in drops of water on space 2A of the slide used for the initial raffinose serum tellurite slant (see Fig 1). In space 2B of the slide make a thin film repre- sentative of the mixed growth on the DST plate. 2. Stain the films with methylene blue and examine with the microscope. 3. On the basis of this examination render a report. 1f positive, tele- phone the report immediately to the physician. 4. Mixed growth from the raffinose serum tellurite slant or from the DST agar plate may also be streaked on antitoxin plates for the RIVTT at this time (see Section VII B 2(e) of this chapter). VII. PURE CULTURE STUDIES Perform toxigenicity tests on all suspected organisms isolated from specimens submitted for original diagnosis. A. Preparation of Pure Cultures Broth subcultures of pure cultures are preferable as inoculum in toxigenicity tests and in typing procedures, although broth (or even salt solution if used promptly) suspensions of pure cultures on a freshly prepared, moist Loeffler slant or other solid medium may be used. B. Examination of Pure Cultures 1. Morphology—By methods previously described (Sections V A, B and C of this chapter), stain and examine microscopically films of the growth from isolated cclonies transferred to Loeffler or modified Pai slants (Section VI D). The morphology of C. diphtheriae growing in broth is not always distinctive; it frequently resembles diphtheroids. Do not discard cultures showing mixtures of C. diphtheriae with other organisms unless other cultures from the same patient are found to be pure. If such pure cultures are not DIPHTHERIA 245 present, replate and purify the mixed culture. Test all cultures which are morphologically typical or suggestive of C. diphtheriae for toxi- genicity as directed in the following section. 2 (a). Toxigenicity (virulence): Intradermal method using rab- bits or guinea pigs.2® Use only pure cultures. 1. Principle of test—This method permits the same animal to be used for both the test and the control injections. The suspension or culture to be tested is injected intradermally into the nonimmune animal, About 5 hr later, diphtheria antitoxin is given intravenously for the rabbit, intraperitoneally or intracardially for the guinea pig, and the same suspension is reinjected as a control into a different area of the skin of the same animal. The antitoxin does not obliterate the characteristic reaction of the skin produced in response to the first injection of virulent bacilli. This is because damage done during the 5 hr interval between first and second injections, when the animal was not immune, is not repaired. However, the animal is rendered immune by the antitoxin and the tissues are specifically protected against the second (control) injection. 2. Preparation of culture—A heavy suspension in broth may best be obtained by 24 hr cultivation in a meat infusion broth (CM No. 5) or a hydrolyzed casein broth (CM No. 11) containing about 0.1 per cent serum. Alternatively, prepare a heavy, even suspension of microorganisms by adding an appropriate amount of broth (pH about 7.2) to an 18-24 hr growth on a Loeffler or modified Pai slant or other satis- factory medium. 3. Selection and preparation of animal—Do not use pregnant ani- mals. Use rabbits when more than 10 cultures are to be tested at one time; guinea pigs for smaller numbers of cultures. The skin of the back of the guinea pig is much tougher to inject than that of most rabbits. The abdominal skin of the guinea pig is softer but the area available is limited and reactions are more likely to become confluent and confusing. Select white or light-colored animals—it is difficult to read reactions in heavily pigmented skin. Prepare rabbit or guinea pig by clipping the hair of the back as short as possible, first with a 000-head clipper and then with a 0000 head. Hand clippers are not satisfactory and depilatory is undesir- able. Satisfactory clipping is essential. Moisten the clipped area and with an indelible pencil, mark two areas not less than 2 cm square for each culture upon the area to be injected. Draw a line along the 246 DIPHTHERIA backbone and a parallel line at least 2 cm laterally on each side. Draw lines at right angles to enclose the desired number of squares. Avoid making squares over the vertebrae, sacral region or shoulder girdle (Fig 6). Figure 6—Intradermal toxigenicity tests on a rabbit. (Photo courtesy Com- municable Disease Center, Atlanta, Ga.; published by permission of W. B. Saunders Co., Philadelphia, Pa.) 4. Making the test injections—With a syringe graduated in 0.1 ml and a 5 in. needle of 26 gauge, inject 0.2 ml of the suspensions being tested into (not under) appropriate areas of skin. A small bleb or “button” should appear at the site of each injection. Save the space below each test injection to make the control injection, The relation of control to test should habitually be the same. Store the syringes containing the test suspensions at 4° C to 10° C until they are to be reinjected into the animal. 5. Injection of diphtheria antitoxin and reinfection of suspen- sion—DBetween 5 and 7 hr after the primary injections, inject 500 to 1000 units of diphtheria antitoxin intravenously into the rabbit; intraperitoneally or intracardially into the guinea pig. Immediately thereafter, in the case of the intravenously or intracardially injected animals, inject 0.2 ml of the test suspension of C. diphtheriae intra- dermally into fresh areas of skin. In the case of the intraperitoneally injected animal, allow 1% hr to elapse before reinjection. DIPHTHERIA 247 6. Control cultures—Include a toxigenic strain which is known by previous test to elicit characteristic reactions (Park 8 strains are not satisfactory) in the series of test suspensions to serve as a “positive” control. Similarly, a suspension of C. pseudodiphtheriticum or C. xerosis may be used as a “negative” control but is not required. 7. Readings and inter pretations—Make preliminary readings in the rabbit after 48 hr; in the guinea pig after 24 hr. Report any definitely positive readings at this time by telephone to the physician. Since the lesions become fully developed in the rabbit only after 72 hr and in the guinea pig after 48 hr, make final readings only after these intervals. The lesion produced by the suspension of known toxigenic C. diphtheriae injected approximately 5 hr before giving the antitoxin is characterized by a central necrotic area 5 to 10 mm in diameter, surrounded by a zone of redness 10 to 15 mm in dian - ter. A central, indurated, hemorrhagic area is usually present. I.esion; are usually larger, more intense, and more easily read in the rabbit (Fig 6). The same control suspension injected after the antitoxin elicits a pinkish nodule 5 to 10 mm in diameter in the guinea pig and a similar, though often larger, reaction in the rabbit. There is no necrosis beyond an occasional very small, central, pimple-like suppuration easily distinguished from the true diphtheritic necrosis. If, in the areas injected before and after the administration of antitoxin, a tested “unknown” culture elicits reactions similar to the corresponding injections of the positive control, the interpretation is that the tested culture contains toxigenic C. diphtheriae. If the tested culture does not elicit the response characteristic of toxigenic strains, the interpretation is that the culture does not contain toxigenic diphtheria bacilli. If both test and control injections of the organisms under investigation show necrotic reactions, the following possibilities should be considered: (a) the organism is not C. diphtheriae; (b) it is a mixed culture; (c¢) antitoxin was not given prior to the control injection; or (d) the wrong antitoxin was given and the culture may be C. ulcerans, requiring tests with a filtered broth culture.?® 8. Discussion—The margin of safety, insofar as the density of sus- pension is concerned, is large. Although satisfactory results may be obtained with suspensions more dense than McFarland Suspension 3, do not use suspensions less turbid than this. A 3-hr interval between first injection of test culture and antitoxin allows for an interpretation of the test, but the lesions are relatively small and may lack the characteristic necrosis; the reactions obtained at the 4, 5 and 248 DIPHTHERIA 6 hr intervals are much more distinct. Animals may be killed by the infection if intervals much over 6 hr are allowed. There is a wide margin of safetv with respect to the quantity of diphtheria antitoxin allowed. As many as 7000 units may be ad- ministered at no sacrifice to validity of the test, but so large an amount is wasteful. 2(b). Tests of single cultures subcutaneously—Cultures are oc- casionally encountered which give doubtful readings when tested in the skin of the rabbit or guinea pig as described above, Some cultures give a result at variance with that expected on the basis of morphology and other tests; other cultures exhibit very slight toxigenicity in test animals, although perhaps causing fatal diphtheria in human beings. For these and other reasons, recourse may be had to injecting a large dose of the living culture subcutaneously (not intraperitoneally) into a test guinea pig and an antitoxin-protected control, A maximal dose of the culture is used because cultures vary greatly in pathogenicity. A 1-2 ml dose may produce at most a doubtful, local inflammation, while 5 ml of the same culture will produce a fatal intoxication with diagnostic pathological changes. For this test use a heavy broth culture 48 to 72 hr old, or the entire growth from a Loeffler or modified Pai or RST slant inoculated 18 to 24 hr previously. Prepare two 6-ml broth cultures or suspend the organisms from the solid medium in about 12 ml of sterile broth. Shave or clip areas about 3 cm in diameter on the guinea pig abdomens and clean with alcohol. Into the control pig inject 1000 units of diphtheria antitoxin intra- peritoneally. After 1 to 2 hr inject into each pig about 5 ml of the test material subcutaneously in the previously prepared areas. If the culture is wholly atoxigenic, neither pig will show any evidence of local necrosis or intoxication within 10 days, or paralysis later, If the culture has any toxigenicity, the unprotected pig will die in one to several days or will show an area of necrosis or inflam- matory reaction or both. The protected pig will exhibit no effect of the injection. Autopsy guinea pigs that die within 10 days of the test injection. Look for extraneous causes of death such as pneumonia, enteritis or trauma. Specificity of death should be corroborated by finding sub- cutaneous gelatinous exudate, or one or both adrenal glands enlarged and hemorrhagic or excess fluid in peritoneal and/or chest cavity, or all of these. These specific findings are usually best observed when pigs die quickly (within 24 to 48 hr) and are promptly autopsied. Characteristic post-mortem findings are not invariably present. DIPHTHERIA 249 2(c). Method using chicks?7-30 1. Selection and maintenance of stock—Use Plymouth Rock or Rhode Island Red chicks 7 to 15 days old. Leghorn chicks have been found not so well adapted to laboratory handling as the larger breeds. Sound, guaranteed pullorum-tested stock is usually available at poultrymen’s supply houses. Avoid chilling chicks by open windows or cold rooms. It is absolutely essential that the chicks have warm, dry quarters (85° F) at all times. A $50 mail order brooder will house about 300 chicks and occupies little more space than a household refrigerator. Never use chicks when the stock is showing evidence of epizootic disease or numerous deaths from an unknown cause. Error may arise if chicks arrive from the dealer without food or water, late in the day, and remain overnight with neither—consequently are in a weakened condition when desired for use. It is preferable to buy day-old chicks, since these have not usually eaten commercial food containing anti- biotics. They are shipped by mail or express. Give newly arrived chicks, even when several days old, a 24 to 48 hr rest and feeding period before using them in an experiment. Day-old chicks are held until they are at least 7 days old. Commercial feeds free from anti- biotics are recommended. The feeding of antibiotics to chicks by dealers could introduce error, although there is no clear evidence on this point. 2. Method of testing toxigenicity—About half an hour before in- jecting test culture into chicks, prepare an antitoxin control chick for each culture to be tested by injecting 50 to 100 units of diphtheria antitoxin intraperitoneally. A few drops of alcohol should be used to moisten and control the feathers. A method of holding and in- jecting the birds properly is shown in Fig 7. The serum is quickly absorbed. Occasionally a chick dies suddenly after the antitoxin injection. The exact reason for this is not clear. Mark each protected chick by a spot of eosin on top of the head. Use dyes of different colors on head, tail, wings, etc., to identify test control pairs. Large numbers of combinations are thus available. Leg bands or web punches may be used but are not necessary. A half hour after preparing the controls inject intraperitoneally 4 to 5 ml of each well-grown 48 hr broth culture (CM No. 5 or CM No. 11) to be tested into one control chick and one test chick, making careful note of the identifying marks of each chick for each culture. As with the subcutaneous test in guinea pigs, dosage is of maximal size because of the great variation in pathogenicity of cultures Chicks 250 DIPHTHERIA Figure 7—Intraperitoneal injection into a chick. Note method of holding feet. The injection is given fairly low in the abdomen to avoid the gizzard, which can easily be palpated. (Photo courtesy Communicable Disease Center, Atlanta, Ga.; published by permission of W. B. Saunders Co., Philadelphia, Pa.) appear to be wholly unaffected by a volume of 5 ml of fluid in the peritoneal cavity. 3. Results—When the tested strains are toxigenic, about 80 per cent of the unprotected chicks die within 24 hr; 12 per cent during the next 24 hr; relatively few are found alive 72 hr after inoculation. When they survive for 48 hr or longer, leg or wing paralysis is well advanced by this time, which leaves no doubt as to toxigenicity of the culture. The controls should remain perfectly well. If the cultures are atoxigenic, all chicks will remain perfectly well. 2(d). The in vitro method (IVTT)3-35 This test depends on the occurrence of a precipitin reaction, in agar, between diphtheria toxin produced by the test culture and diphtheria antitoxin in the agar. This permits determination of toxigenicity by cultivation of the organism to be tested on the surface of serum agar in a petri plate. No test animal is required. Diphtheria antitoxin diffuses into the agar in a gradient of concentrations from a centrally placed strip of DIPHTHERIA 251 antitoxin-impregnated filter paper. When toxin diffuses (also in a gradient of concentrations) from the growing organisms on the plate, it encounters the antitoxin. Where the two coexist in the proper proportions, a precipitate occurs which is visible as a faint white line in the agar (Fig 8). The test is rapid, inexpensive and sensitive. It Figure 8—The in vitro test for toxigenicity. (Photo courtesy Communicable Disease Center, Atlanta, Ga.; published by permission of W. B. Saunders Co., Philadelphia, Pa.) is highly reliable but only if directions are followed exactly, and certain specific precautions are taken in performing it, as follows: 1. Composition of the basal medium (CM No. 85) Proteose peptone (DIFOY, ..vvuvervssvvvss sav nvmess ss save sor vas 200 g Granulated agar (Difco or equivalent) ...........coiviiuinnennnn. 1.75 g NACL (CCT vous suv vuns insures sin mwssicims ivoms s imasbastormneinFomm mine 025 g DisliNed Water «uuninssmamvmmminiinmm aes hinm simmer d Sass GEE 050 100 ml Heat in steam to melt agar. Adjust to pH 7.8. Autoclave for 15 min at 121° C. The medium is most conveniently dispensed in 10 ml tubes or in 100 ml bottles. Store at room temperature. When needed for a test, melt the basal agar, restoring volume with sterile distilled water if necessary, and cool to 45° C. Add 20 per cent of the sterile serum or serum substitute (described in the following paragraph) aseptically to the plate or agar just before pouring in the agar. Mix serum (or substitute) and agar well. * Variable. Use only batches shown by preliminary test to give satisfactory results. 252 DIPHTHERIA 2. Serum and serum substitute for the basal medium—Rabbit serum is satisfactory. Probably some sheep and horse sera are equally satisfactory and some human sera may serve. Whatever serum is used, collect it as soon as possible after the clot is formed. It must be virtually free from erythrocytes and hemoglobin. The serum keeps well in the refrigerator and, for purposes of this test, appears to improve with storage if sterile. Whatever the source, select only samples of serum which give good positive reactions in careful tests, using intermedius- or minimus-type strains as positive controls. Regardless of type, use strains which regularly give positive but minimal reactions in this test. No minimus- type strain has been found atoxigenic by this method. A known atoxigenic strain should also be included as a negative control in these tests. Serum substitute: For serum, the following substitute has been found convenient and in some instances superior: Distilled [a0 Foi ciara s + 4S iniintos vain idm siains 3 u soups 100 ml Casaming agids Sm in . ok ubibiuiin sd 1's sleleii states slates ties somioliin vin + lg TWEEA. BO oh rt cotinine +5 Cuba te area 4. WR eee 1 ml Glycerine LC PD Yt tvs snvnaind os sioner + 8 seattle vs + 3 Sma 1 ml Shake gently and warm at 50° C to dissolve. Autoclave at 121° C for 12 min. Use in place of serum in the IVTT and RIVTT.3* 3. Antitoxin—For reasons not yet clear, some samples of com- mercial antitoxin are unsatisfactory for this test, since they produce excessive precipitate in the agar and/or nonspecific lines and/or fail to react with toxigenic cultures. This has no relation whatever to the quality of these antitoxins for prophylactic or therapeutic purposes. Each lot of antitoxin, regardless of its source, must be tested for suitability in this procedure. Antitoxins found to be satisfactory for this test have been obtained from Connaught Laboratories, Lederle, Squibb and Wyeth. 4. Preparation and inoculation of plates—Dilute concentrated diph- theria antitoxin so as to contain about 500 units per ml. Into this dip a strip of sterile filter paper about 1.5X7 cm. Drain off the excess fluid and place the strip, with as little agitation as possible, in the center of a dish of fluid, basal serum agar at about 45° C. Allow the strip to settle to the bottom. After the agar has solidified, place the dish in the incubator for about 1 hr with cover partly open to dry the surface. Inoculate the cultures to be tested with a loop, each in a single line entirely across the plate at an angle of about 90° to the paper strip, DIPHTHERIA 253 about 1 cm apart. Crowding of growth streaks may be avoided by not testing more than four to seven cultures (five to eight including positive control) on one 10 cm petri plate. Always include a positive control as noted above. Use as inocula for this test well-grown broth cultures, growth from solid media, or pure colonies fished directly from tellurite plates used for initial isolation. 5. Incubation and reading of plates—Incubate plates at 35° C, not more than two to a pile. A positive result will show one or more white lines of precipitate in the agar, about 5 to 10 mm distant from the paper, beginning at and extending out from the line of inoculation at an angle of 45° and away from the filter paper (Fig 8). The lines are often small and faint, so that they can be seen only with strong oblique light against a dark background. To identify them, a hand lens (about 2X) and some practice are usually required. Often the lines are visible in 24 hr at 35° C, but 72 hr may be required for full development. Lines appearing after 72 hr or in locations differ- ent from those mentioned, or in multiple locations, are of doubtful significance. Lines may also be produced by C. ulcerans2® Verify doubtful reactions in animals as described in Sections VII B 2(a), (b) and (c¢) of this chapter. 2(e). The rapid in vitro method (RIVTT)—It is possible, by means of the RIVTT, to demonstrate the presence of toxigenic C. diphtheriae in about two-thirds of original positive diagnostic speci- mens within 24 hr after receipt of the specimen. The special medium for the standard in vitro toxigenicity test is made somewhat selective by adding to it 0.033 per cent of potassium tellurite, which retards growth of many of the contaminating microorganisms in the original swabs or original Loeffler, Pai or raffinose serum tellurite cultures but has little inhibitory effect on corynebacteria. 1. Medium—The same agar base, sterile normal serum or serum substitute, sterile filter paper strip and diphtheria antitoxin diluted to 500 units per ml are used as in the standard in vitro toxigenicity test. 2. Method of preparation—Into a new or unscratched petri dish, put: Sterile normal serum or serum substitute .................0. iia... 2.0 ml Sterile 0.3 per cent solution potassium tellurite ...................... 1.5 ml Melted agar base ............. SE 48 3 SORRBRREE Be ARETE FRE REP Siti 10.0 ml Mix by rotating gently. A smooth surface is essential for satisfactory results. 254 DIPHTHERIA Saturate a strip of sterile filter paper with diluted diphtheria anti- toxin and press to the bottom of the dish before the agar hardens. After the agar hardens place the plate in the incubator, with cover partly open, to dry the surface. 3. Making the test—Use swab directly from the patient to inoculate the plate by drawing it across the plate at a right angle to the filter paper. Rotate the swab in so doing. or Thoroughly mix with a loop the growth on original diagnostic cultures and streak a small portion of the mixed growth across the plate as above. Similarly inoculate a known toxigenic minimus or intermedius strain on each plate as a positive control. Include also a known atoxi- genic strain as a negative control on each plate. 4. Reading the test—Reduction of the potassium tellurite by growth of the various organisms will cause the lines of inoculum to turn grey. The reading is made in the same manner and at the same intervals as with the standard IVTT test. 3. Determination of type—In 1931 certain starch- and glycogen- fermenting rough colony strains of C. diphtheriae were isolated dur- ing an epidemic at Leeds, England. They were designated as the gravis type, since they seemed to be related to malignant, antitoxin- refractory (grave) cases of diphtheria. Other strains, not fermenting starch and glycogen and forming smooth colonies, were called the mitis type, since they were found at that time in milder cases. Many were atoxigenic. A type described as having some properties of gravis and mitis types but with smaller, flatter colonies, was called the intermedius type. These types have been described by many sub- sequent workers.36-37 In 1944, during an epidemic in Baltimore, Md., a type was observed which differed from the others in that it fermented glucose slowly or not at all under certain cultural conditions and formed very minute colonies. This type was designated as the minimus type,3® although it is regarded by some workers as identical with the intermedius!239-41 type. It is included here because certain strains of it have been desig- nated as standard positive controls for in vitro methods of testing toxigenicity. Distinctive characteristics of these types of diphtheria bacilli are shown in Table 1. DIPHTHERIA 255 Table 1—Differentiation of Types of Diphtheria Bacilli . : Hemol- Call Fermentation of Pelli- ysin Colony Morphol- Glu- Su- Gly- cleon Produc- Types Form ogy cose crose Starch cogen Broth tion Gravis Rough Fig 4C + oe + 4 fe == Mitis Smooth Fig 4B + — — — — + Intermedius Small Fig 4A + —_— — — — & Minimus Minute Intermedius mitis-like, or barred o% — — — — — * Slowly if at all. Several other varieties of C. diphtheriae have since been found which differ from those most commonly observed. For example, some virulent strains ferment sucrose.*2:43 Extensive investigations indicate that the exotoxins formed by all types are immunologically homogeneous. ** 4. Methods—Several investigators have found serological typing to be of some epidemiological significance. As this method is still in the investigative stage and as there is no general agreement on the definition of types, no standard reference strains and no national or international reference typing-center methods for this procedure are given here. Those interested are referred to the literature cited.*5-48 The cultural methods of determining types as described by the original Leeds workers are given here with as little modification of the original procedures as possible. In determining types of diph- theria bacilli, adhere to these methods in the interest of uniformity and comparability of results. Use only pure cultures as inocula for the tests required in type determination. Determine type as soon as possible after isolation of the culture, since type characteristics tend to change upon subculture in the laboratory. a. Fermentation of glycogen—Prepare glycogen (Eastman “high- est purity” or equivalent) in a 5 per cent aqueous solution and auto- clave at 121° C for not over 12 min. Add, aseptically, 0.3 ml of this to 3 ml of broth (CM No. 5 or CM No. 11) dispensed in 13100 mm culture (serological) tubes and containing just enough bromecresol purple to give a definite purple color. The inoculum must not contain 256 DIPHTHERIA serum or fermentable substance, Incubate the culture at 35° C for at least 1 week unless fermentation occurs sooner, Observe daily for change in pH. b. Fermentation of starch—Determine this in the same manner as fermentation of glycogen except that “soluble starch according to Lintner” of highest purity obtainable is prepared in a 2 per cent aqueous solution. Use only freshly prepared starch solutions, as starch is apt to hydrolyze spontaneously. c. Fermentation of glucose—For purposes of differentiating mini- mus strains, it is important that the method given be followed in exact detail.*® Use Difco heart infusion broth (CM No. 29) without blood. Adjust the pH to 8.0 so that it will be 7.8 after autoclaving. Add indicator and dispense as directed for glycogen. Autoclave and check pH. To each tube containing 3 ml of broth aseptically add 0.3 ml of 10 per cent aqueous-solution C. P. glucose sterilized by filtration. Use the same precautions regarding inoculum as are indicated under glycogen. Incubate at 35° C. Freshly isolated, true minimus strains will not ferment glucose under these conditions in less than 6 days if at all. Other strains ferment in 24-48 hr. Do not report the result of any fermentation test in which growth is not satisfactory. Growth is usually not as heavy in unfermented media as it is when fermentation occurs. d. Colony form—TFor differentiation of types, use the heated blood agar of McLeod (CM No. 80). This is melted and poured into petri plates. When the agar is firm and the surface dry, streak one culture on each plate, taking care to use very small inocula and spreading widely over the surface of the plate so as to secure well-isolated colonies. Incubate the plates at 35° C for 48 hr (Fig 5). In evaluating and recording results bear in mind that colonies of several different types are often observed on McLeod’s medium, even though inoculated with one pure culture. Only experience and judg- ment can enable the investigator to arrive at a reasonable estimate of the tendency of the culture as a whole, Usually it is best to examine only well-isolated colonies and to form a sort of mental average of the group. e. Pellicle formation—Inoculate broth (CM No. 5 or CM No. 11) in the usual manner; watch for the development of a scum or a definite surface membrane (pellicle). Examine also any other broth cultures of this organism, since only one of several cultures may show a pellicle. Incubate at 35° C for at least 6 days. DIPHTHERIA 257 Avoidance of errors—Pellicles often break up and sink quite early in the age of the culture. Slight shaking will often cause the precipi- tation of a heavy pellicle to the bottom of the tube, resulting in an erroneously negative notation. A thick growth of smoothly suspended organisms just under the surface of broth, or a fine veil of growth over the surface, may be mistaken for a pellicle. The true nature of these developments becomes apparent upon the least agitation of the tube, when they disperse as a smooth, cloudy suspension. It is essential that well-buffered, carbohydrate-free and serum- free broth, such as is used for fermentation tests, be selected for these investigations, since even a slight acidity tends to lower the surface tension and pellicles may not then be so well supported upon the surface. f. Hemolysin production—Inoculate a tube of infusion broth (CM No. 5 or CM No. 11) similar to that used for fermentation tests and pellicle studies and incubate for 48 hr at 35° C. Place 1 ml of the culture in a clean 13100 mm tube. Add 1 ml of a 2 per cent suspension of thrice-washed human erythrocytes in 0.85 per cent salt solution. Place in a 35° or 37° C water bath for 1 hr and then in the icebox overnight. Reading—Avoid agitating the tubes. Read hemolysis as present or absent, depending on the appearance of a clear, ruby red color above the cells in the bottom of the tube. The test is wholly qualitative, the amount of hemolysis having no recognized significance with regard to type. A control tube prepared with sterile broth instead of broth culture and washed erythrocytes of the same lot must show absolutely no hemolysis. ; For the primary isolation of C. diphtheriae, a medium not yet so widely tried as those described in detail in this chapter, but of recog- nized value and worth further comparative study, may be made by the modified formula of Tinsdale®® as an alternative. ACKNOWLEDGMENT—The Subcommittee for the Chapter on Diphtheria takes pleasure in acknowledging with thanks the assistance of the following in the preparation of this chapter: Dr. Elizabeth I. Parsons (deceased), Communicable Disease Center, U. S. Public Health Service, Chamblee, Ga. ; Miss Helen Gillette and other members of the staff, Massachusetts State Health Department, Boston, Mass.; Miss Ona R. Whitley, Indiana State Health Department, Indianapolis, Ind.; Miss Dorothy E. Helmer, Laboratory of Hygiene, Department of Na- tional Health and Welfare, Ottawa, Ontario, Canada. MarTIN Frowisuer, Sc.D., Chapter Chairman E. T. Bywox, Pu.D. Samuel R. Damon, Par.D. Roser A. MAcCreaDy, M.D. 258 DIPHTHERIA REFERENCES 1. Nurrar, G. H. F, and GramaM-SmrrH, G. S. The Bacteriology of Diphtheria. Boston, Mass. : Cambridge Univ. Press, 1908. 2. Denny, F. P. Observations on the Morphology of B. diphtheriae, B. pseudodiphtheriae and B. xerosis. J. Med. Res. 9 N. S. 4:117, 1903. 3. BECkLER, E. A. A Plea for a Quick Bacteriological Diagnosis of Diphtheria. Boston Med. & Surg. J. 178:531, 1918. 4. WarrLey, O. R,, and Damon, S. R. Raffinose-Serum-Tellurite Agar Slants as a Replacement for Loeffler’s Medium in Diphtheria Diag osis. Pub. Health Rep. 64 :457, 1949. 5. ————————— A Transparent Dextrose-Serum-Tellurite Plating Medium. Its Use as an Adjunct to Microscopic Examination of Smears Made from Loeffler Slants in Routine Diphtheria Diagnosis. Pub. Health Rep. 64:201, 1949. 6. SantoPADRE, G. Sulla Prova di Tossinogenisi in vitro del C. diphtheriae., Riv. Ist. sieroterap. Ital. 28:213, 1953. 7. Parsons, E. I; Froeisuer, M.; Moore, M.; and Aiken, M. A. Rapid © Virulence Test in Diagnosis of Diphtheria. Proc. Soc. Exper. Biol. & Med. 88:368, 1955. 8. FromrisHER, M. and VAN VOLKENBURGH, V. A. Increased Numbers of C. diphtheriae Demonstrable by Extensions of Bacteriological Procedures. Am. J. Hyg. 22:292, 1935. 9. Bywog, E. T,, and HeLMER, D, E. Bacteriological Observations on Diphtheria in Halifax. Canad. J. Pub. Health 36:135, 1945. 10. Cooper, R. E, et al. Laboratory Diagnosis of Diphtheria. Lancet 1:865, 1940. 11. Doucras, S. R. A New Medium for the Isolation of B. diphtheriae. Brit. J. Exper. Path. $:263, 1922. 12. MueLLer, H., and MiLier, P. A. A New Tellurite Plating Medium and Some Comments on Laboratory Diagnosis of Diphtheria. J. Bact. 51:743, 1946. 13. MuEtLLER, J. H. Nutrition of the Diphtheria Bacillus. Bact. Rev. 4:97, 1940. 14. Knox, R. Comparison of Loeffler’s Inspissated Serum and Tellurite Blood Medium in the Laboratory Diagnosis of Diphtheria. Month. Bull. Min. Health 3:34, 1944. 15. Wricut, H. A. The Laboratory Diagnosis of Diphtheria. A Note on Some Present Day Methods. Edinburgh M. J. 50:737, 1943. 16. CruriksHANEK, R. Diphtheria. 3. Laboratory Aspects. Pub. Health 17:57, 1943. 17. Serregrs, T. F. Personal communication. 18. Brerz, G. B., and FroBisHEr, M. Enrichment of Loeffler’s Medium with Glycerol. CDC Bull. May 1951. 19. FromisHEr, M., Jr.; Parsons, E. I.; Yeates, E. L.; and Gay, K. L. A Comparative Study of Tellurite Plating Media for Corynebacterium diphtheriae. Am. J. Hyg. 48:1, 1948, 20. FromisuEir, M., and Parsons, E. I. Further Studies of Tellurite Plating Media for C. diphtheriae. A.J.P.H. 43:1441, 1943. 21, Dawiews, J. B, Jomnson, M. P. and MacCreapy, R. A. Laboratory Diagnosis of Corynebacterium diphtheriae: Early Report of Positive Cul- tures in 0-8 Hours, Canad. J. Pub. Health 42:185, 1951. 22. Perry, C. A.,, WHITLEY, O. R,, and PetrAN, E. Types of C. diphtheriae in Maryland. Am. J. Hyg. 23:580, 1936. 23. WaIitLey, O. R. A Study of Corynebacterium diphtheriae and Related Organisms in Maryland. J. Lab. & Clin. Med. 20:1024, 1935. DIPHTHERIA 259 24. 2s. 26. 27. 28. 29. 30. 31. 32 33. 34. 33. 36. 37. 38. 39. 40. 41. 42. 43. 45. ————— Corynebacterium diphtheriae gravis Found in Maryland J. Lab. & Clin. Med. 19:943, 1934. Fraser, D. T., and WEeLp, C. B. The Intracutaneous “Virulence Test” for Corynebacterium diphtheriae. Tr. Roy. Soc. Canada 20 (Sec. V): 343, 1926. HerMANN, G. J., and Parsons, E. I. Recognition of C. diphtheriae-like Corynebacteria (Corynebacterium ulcerans) in the Laboratory. Pub. Health Lab. 15:34, 1957. FroBisHER, M., Parsons, E. I, and T'ung, T. The Use of Chicks in Testing the Virulence of Corynebacterium diphtheriae. Am. J. Hyg. 35:381, 1942. BranuAM, S. E.,, and WormALDp, M. F. The Use of the Chick in Titration of Diphtheria Antitoxin. J. Immunol. 72:478, 1954. Kor, R. W., Branuam, S. E., and Rices, D. B. Comparison of the Guinea Pig and Chick in Evaluation of Diphtheria Toxin for the Schick Test. Applied Microbiol. 4:242, 1955. BranuaM, S. E, GraBowski, M. W,, and Rices, D. B. Evaluation of the Chick as a Test Animal in the Assay of Diphtheria Toxoids. Applied Microbiol. 5:286, 1957. ErLek, S. D. The Recognition of Toxicogenic Bacterial Strains in vitro. Brit. M. J. 1:493, 1948. ———————— The Plate Virulence Test for Diphtheria. J. Clin. Path. 2:250, 1949. OucutErLONY, O. In vitro Method for Testing the Toxin-producing Capacity of Diphtheria Bacteria. Acta path. et microbiol. scandinav. 26:516, 1949, HerMANN, G. J., Moore, M., and Parsons, E. I. A Substitute for Serum in the Diphtheria in vitro Toxigenicity Test. Am. J. Clin. Path. 29:181, 1958. Fropisaer, M., King, E. O., and Parsons, E. I. A Test im vitro for Virulence of Corynebacterium diphtheriae. Am. J. Clin. Path. 21:282, 1951. Anperson, J. S., Harrop, F. C., McLeop, J. W., and Truomson, J. G. On the Existence of Two forms of Diphtheria Bacillus—B. diphtheriae gravis and B. diphtheriae mitis—and a New Medium for Their Differentia- tion and for the Bacteriological Diagnosis of Diphtheria. J. Path. & Bact. 34:667, 1931. McLeon, J. W. The Types mitis, intermedius and gravis of Corynebacterium diphtheriae. Bact. Rev. 7:1, 1943. FroBisHER, M., Apams, M. L., and Kunns, W. J. Characteristics of Diphtheria Bacilli Found in Baltimore since November 1942. Proc. Soc. Exper. Biol. & Med. 58:330, 1945. Hewitt, L. F, Serological Typing of C. diphtheriae. Brit. J. Exper. Path. 28:338, 1947. Orping, P. On the minimus Type of Diphtheria Bacillus. Acta. path. et microbiol. scandinav. 27 :907, 1950. Jouwnstong, K. I, and McLeop, J. W. Nomenclature of Strains of C. diphtheriae. Pub. Health Rep. 64:1181, 1949, Erier, C. H., and Frosrsaer, M. An Outbreak of Diphtheria in Baltimore in 1944. Am. J. Hyg. 42:179, 1945. Mauss, E. A, and Keown, M. J. Saccharose-Fermenting Diphtheria Bacilli. Science 104:252, 1946. ZiNNEMANN, K. Neutralisation of C. diphtheriae-Type Toxins with Standard Antitoxin as Determined by Skin Reactions in Guinea Pigs. J. Path. & Bact. 58:43, 1946. Rominson, D. T. and PeENey, A. L. P. Serologic Types among gravis Strains of C. diphtheriae and Their Distribution. J. Path. & Bact. 43:403, 1936. 260 DIPHTHERIA 46. Ferris, A. A. Serological Types of Corynebacterium diphtheriae in Australia. J. Path. & Bact. 62:165, 1950. 47. FreeMmAN, V. J. and MinzeL, G. H. Serologic Studies of Corynebacterium diphtheriae. III. Cultural Grouping and Serologic Typing of Diphtheria Field Cultures from the Pacific Coast Region of the United States. Am. J. Hyg. 55:74, 1952. 48. HeErMANN, G. and Parsons, E. I. A Study of Antigenic Relationships in Some Strains of Corynebacterium diphtheriae. Am. J. Hyg. 61:64, 1955. 49. Parsons, E. I, and FroBisHer, M. Differentiation of minimus Type C. diphtheriae by Slow Fermentation of Dextrose. Science 113:317, 1951. 50. KarLstrOM, A., BARGER, R. H., and BranpboN, G. R. A Simplification of Modified Tinsdale’s Medium. Pub. Health Lab. 20:44, 1962. II. III. Iv. VIL VIL VIIL CHAPTER ¢ TUBERCULOSIS AND LEPROSY . Introduction Microscopical Examination of Stained Material A. Sputum B. Other Clinical Material C. Staining D. Examining the Film E. Sources and Prevention of Positive Error F. Interpretation Demonstration of Tubercle Bacilli in Sputum and Other Body Exudates by Culture A. Collection of Specimens B. Preparation of Containers C. Preparation of the Specimen D. Technic for the Culture of Tubercle Bacilli Methods of Testing Tubercle Bacilli for Drug Susceptibility A. Direct Tests on Solid Medium B. Method of Reading and Recording Results of Sensitivity Tests C. Subculture in Tween-albumin Liquid Medium D. Subculture Para-aminosalicylic Acid Sensitivity Tests E. Subculture Isoniazid Sensitivity Tests F. Catalase Tests . Demonstration of Tubercle Bacilli by Inoculation of Animals Special Problems in the Demonstration of Tubercle Bacilli in Tuberculous Lesions . A. Chemotherapy B. Limitations of Sputum Examinations C. Tuberculous Lesions D. Tissue Sections E. Necropsies Laboratory Diagnosis of Leprosy A. Scraped Skin Incisions B. Nasal Samples Discussion References 261 262 TUBERCULOSIS AND LEPROSY I. INTRODUCTION Tuberculosis—Although the acquisition of knowledge about tuberculosis has been enormous since Robert Koch made his epochal announcement in 1882 describing the tubercle bacillus as the etiologic agent of tuberculosis, this disease still claims a tremendous toll of human life. The tubercle bacillus is a member of the family Mycobacteriaceae— a group which includes pathogenic and nonpathogenic acid-fast bacilli. The term “tubercle bacillus” includes only those acid-fast micro- organisms that cause human, bovine or avian and murine tuberculosis. Mycobacteria possess the unusual characteristic of retaining dyes which resist decolorization by acids and alcohol. It is because of this characteristic that they have been called “acid-fast” bacilli. Mycobacteria tend to be long, narrow rods varying in length from 2 to 7 pu and measuring about 0.2 p in thickness. Beaded forms are often seen. Pleomorphic and nonmotile, they may appear singly or in small irregular clumps in stained preparations. They may be seen within the phagocytic cells of the lesion or may appear free in the surrounding milieu. Although tuberculosis can be recognized clinically and radio- graphically, the demonstration of tubercle bacilli in the sputum or tissues of a patient remains the surest method of establishing a diagnosis. The finding of the tubercle bacillus in pathological material will to a large extent indicate the activity of the disease process and often dictates whether or not the patient will be sent to a sanatorium or tuberculosis hospital for prolonged treatment. Conversely, the absence of tubercle bacilli in specimens obtained from the patient after a careful search for these microorganisms has been made should turn the clinician’s attention to a search for nontuberculous infection and other possible causes for the lesions. Since so much depends upon the bacteriological examination of specimens obtained from tuberculosis suspects, it is most important that the laboratory procedures be as reliable as possible. The pro- cedures most widely employed to demonstrate tubercle bacilli in pathological material are (1) microscopical search for the bacilli in stained preparations; (2) cultivation of the bacilli on artificial media; and (3) demonstration of tuberculosis lesions after animal inocula- tions. Leprosy is one of the oldest known chronic infectious diseases. In spite of the common impression that leprosy is rare because of the low incidence in this country, millions of persons throughout the TUBERCULOSIS AND LEPROSY 263 world suffer from it. If all cases were recognized and reported, it might rank as one of the more frequent infectious diseases. Since thousands of American troops have served and are serving in the Southwest Pacific and the Orient, cases of leprosy must be expected in parts of the country where the disease is not endemic. Microscopically, Mycobacterium leprae is indistinguishable from the tubercle bacillus. New, effective methods of treatment make it most important for the physician to recognize the disease clinically and, more particularly, to know the role which the laboratory can play in establishing a diagnosis. The etiological agent is M. leprae, an acid-fast bacillus first recog- nized by G. Armauer Hansen in 1874. Although the organism was described before Robert Koch succeeded in isolating and identifying the causative agent of tuberculosis, the leprosy bacillus has never been cultivated on artificial media or in tissue culture. It has been im- possible to transmit the disease to normal human beings or to ex- perimental animals (except for the foot pads of mice). Il. MICROSCOPICAL EXAMINATION OF STAINED MATERIAL The demonstration of acid-fast bacilli in sputum or other pathologi- cal material is only presumptive evidence of tuberculosis since it does not identify M. tuberculosis. The examination of stained material is the least sensitive diagnostic bacteriological procedure for tuberculosis, but because of its simplicity and, above all, its speed, it is an important and useful test. A. Sputum Direct film—Pour the specimen into a petri dish and examine under a good light against a black background. Pick out the most necrotic or purulent portions and spread on a new microslide. Wooden applicator sticks are satisfactory for this purpose. In the case of clear, mucoid or saliva specimens, it is essential to select yellow or opaque “likely” particles. Spread the material fairly evenly with the sticks, but leaving thick areas and thin areas. Allow to dry; fix by passing through a low flame two or three times. The film may be dried and fixed simultaneously on a surface warmed to about 60° C. Higher temperatures are to be avoided because of possible alteration in the morphology of M. tuberculosis. A covered water bath over a bunsen flame is satisfactory. The film is now ready for staining. Concentrated film—Concentration procedures, carefully carried out, will generally but not always increase the chances of finding some 264 TUBERCULOSIS AND LEPROSY acid-fast bacilli. The object is to gather all the mycobacteria from the specimen into a few drops that may be spread on a slide, It is distinctly advantageous to use the entire specimen, but where this is not possible, select the more likely portions by fishing with applicator sticks. Proceed as described in Section III of this chapter, which follows. Centrifugate all or a liberal part of the treated specimen. Tubes hold- ing 50 or 100 ml are often required. After neutralizing, prepare films from the sediment with a Pasteur pipette or wire loop. It is often advantageous to spread the material in a small area about 1 cm in diameter. Where the sediment is thin, prepare a film of two or more layers, allowing one layer to dry before the next is added. Serum or egg albumin may be used as a fixative. Heat gently, as above, before staining. Sodium hypochlorite, 5 per cent, can be used in place of sodium hydroxide.! (See comment in Section III.) The sediment need not be neutralized. This compound is a superior digestant, yielding small, clean sediments, It is powerfully tuberculocidal and therefore unsuit- able for the preparation of material for culture or animal inoculation. It is available as a household bleaching agent in such brand-name products as Clorox or Purex. Numerous other concentration procedures have been proposed.? Because of the relatively low specific gravity of the tubercle bacillus, it is not as readily collected by centrifugation as most other micro- organisms. Acceptable procedures designed to avoid this difficulty are chemical flocculation (see Section III) and hydrocarbon flotation. Hydrocarbon flotation®—The lipophilic mycobacteria attach to hydrocarbon droplets and are carried by them to the surface. Shake the specimen with an equal amount of 0.5 per cent sodium hydroxide. Dilute with water 10 to 20 times. Shake by machine with 1 ml of xylol (or other lightweight hydrocarbon). Allow to stand 15 to 20 min for flotation. Skim off the cream-like layer which has formed at the surface with a Pasteur pipette and prepare a multi- layered film. B. Other Clinical Material Handle the more purulent specimens such as sinus or fistula exudate, the contents of cold abscesses or pleural exudate as you would sputum. Always concentrate thin fluids, such as urine, pleural transudates or cerebrospinal fluid. Methods available are: gravity sedimentation TUBERCULOSIS AND LEPROSY 265 (see below) as a preliminary procedure for large volumes; centrifuga- tion; flocculation (see Section IIT); and hydrocarbon flotation (see above). Digestion is unnecessary except where one is also performing culture or animal inoculation of fluids likely to contain contaminating microorganisms, as in the case of urine. Fibrin clots involving a large proportion of a fluid specimen may enmesh many of the mycobacteria present. Therefore, either they should be examined or their formation should be prevented. Spread small clots on microslides for staining. Grind up large clots, as in the case of tissues (see Section VI), and prepare films from the resultant suspension. To prevent clotting, place in the container 1 mg of potassium oxalate for each ml of fluid to be collected. Acid-fast bacilli demonstrated microscopically in material from enclosed spaces (pleural, cerebrospinal, joint, peritoneal fluids) and in aseptically removed tissues are almost certain to be pathogens and most likely to be M. tuberculosis. Urine—Microscopical examination of urine is notably suspect because of the frequent presence of Mycobacterium smegmatis, a saprophyte derived from the recesses of the external genitalia. For this reason, clean, voided samples obtained after washing or aseptically catheterized specimens are to be preferred. Repeated (daily) single specimens are preferable to 24 hr collections for microscopical examination as well as for culture, because of the multi- plication of contaminating microorganisms, both acid-fast and other, during standing and the possible toxicity of the urine for tubercle bacilli. Always centrifuge the entire single specimen. If a 24 hr specimen must be examined, place the bottle in the refrigerator in a tipped position overnight for gravity sedimentation. Remove the sedi- ment to two 100 ml centrifuge tubes by means of a large pipette equipped with a rubber bulb. Centrifugate and proceed as usual. Flocculation (see Section IIT) is a useful concentrating procedure and hydrocarbon flotation is applicable. Cerebrospinal fluid—Because of the crucial need for prompt treatment, a microscopical examination of cerebrospinal fluid is more valid than other methods for the demonstration of tubercle bacilli. If there is a pellicle or clot, spread it on a slide for fixation and staining, but also concentrate the remaining fluid for examination. Centrifugate all of the cerebrospinal fluid obtainable at 3,000 rpm or faster, for 30 min, Carefully remove the supernatant, mix the sediment, and prepare a multilayered film of small diameter (5-10 mm). Plant on culture media or inject into animals any remaining sediment. 266 TUBERCULOSIS AND LEPROSY Hydrocarbon flotation (see above) is an advantageous alternative. Carry out the procedure in the collection tube, omitting the dilution step. Use approximately 0.2 ml xylol and remove the entire resultant surface emulsion for film preparation. Tissues (see Section VI for a comprehensive discussion)—An additional procedure worth mentioning is concentration of myco- bacteria from fresh or formalin-fixed tissues. Reduce specimen to a pulp in a hand tissue grinder. Mix with 5 to 10 parts of 0.5 per cent sodium hydroxide or 5 per cent sodium hypochlorite so as to render fluid. Transfer to a wide-mouth bottle containing a few glass beads; stopper. Shake by machine for 10 min. Incubate at 35° C overnight in the case of NaOH, 2 to 4 hr in the case of NaClO. Concentrate by centrifugation or, better, by hydrocarbon flotation (see above). Prepare films by the use of albumin or serum fixative. Where digestion is inadequate, add additional amounts of reagent, shake, and incubate longer. C. Staining The Ziehl-Neelsen technic—While numerous variations have been recommended, none appears to have superseded the Ziehl- Neelsen technic in simplicity and dependability. The following version is recommended (Ziehl-Neelsen method) :# Cover film with carbolfuchsin stain Steam gently 3 to 5 min Wash with tap water Add acid alcohol until film is colorless Wash with tap water Counterstain with methylene blue 5 to 20 sec Wash and allow to dry Caution: Do not boil or dry the carbolfuchsin on the slide. Maintain adequate steaming by intermittent passes of the bunsen flame. Cover- ing the stain with a strip of filter paper helps to control drying at the edges. Decolorize adequately: at least 174 min, not over 10 min. Counterstain very lightly. Heavy cellular counterstaining will reduce the chance of finding acid-fast bacilli by obscuration. Staining several film preparations in the same jar creates doubt of cross-contamina- tion. Placing slides too closely on the rack makes it possible for con- tamination to be carried across by staining reagents. Other methods—A higher content of basic fuchsin, 1 per cent Pottenger,® and a longer period of steaming, up to 20 min, will give deeper stain penetration. Cold staining may give good results after 20 min but is more reliable if prolonged to between 12 and 24 hr. This TUBERCULOSIS AND LEPROSY 267 may be done, without danger of contamination, by Steenken’s® method. Inclusion of a wetting agent in the formula is said to reduce cold staining time to about 3 min.” Malachite green or picric acid (in saturated solution) may be used as counterstains. The latter has two advantages: It is pale and it does not selectively stain cellular material. Fluorescent staining causes the bacillus to glow so that it is visible under lower magnifications of the microscope. Thus one may examine a film in considerably less time than is ordinarily required. A pro- cedure used by Thompson® is satisfactory. Heat-stain with 0.1 per cent auramine-0 in 3 per cent phenol and decolorize with acid alcohol. Examine under a binocular microscope equipped with yellow ocular filters* using a 21X objective and 20X wide-view eyepieces. Use an ultraviolet light source equipped with a violet filter.** While faster, this procedure is more complex and some believe there is a slightly increased incidence of false-positive findings. Before adopting a new staining technic the bacteriologist is advised to try it out parallel to the Ziehl-Neelsen procedure in a large number of specimens so that he may determine whether the method is satis- factory where only rare bacilli are present. Tissue Sections—Kinyon’s method, as adopted by the Armed Forces Institute of Pathology,® is recommended. Paraffin sections of 4 to 6 p prepared from tissues fixed (preferably) in formalin are processed as follows: Deparaffinize and rehydrate as usual. Stain in Kinyon’s carbolfuchsin solution for 1 hr at room temperature. Wash well in tap water. Differentiate in two changes of 1 per cent acid alcohol until tissue is pale pink. Wash well in tap water. Counterstain in a working methylene blue solution for a few sec. Dehydrate, clear, and mount as usual. An alternative is the method of Putt'® recommended in Section VI of this chapter. D. Examining the Film Examine sputum films for a period of 5-10 min before recording as negative. Examine fluids from enclosed spaces and tissues for as * Corning HR yellow shade No. 3486. + General Electric 100 watt AH4 mercury lamp in a Spencer No. 370 lamp housing ; transformer is required. ** Corning No. 5113. 268 TUBERCULOSIS AND LEPROSY long as 30 min to 1 hr, if necessary. Cross the preparation in both axes so as to sample representative portions. Distinguish between typical acid-fast rods (which are slender, with parallel borders, long or short, beaded or granular) and atypical forms (which are unusually broad or diphtheroid, unusually large in breadth and/or length, or broadly coccoid). A positive finding should be based only on “typical” rods but the presence of atypical forms should be recorded. M. tuberculosis may be somewhat pleomorphic, appearing in all men- tioned atypical shapes. Nevertheless, typical acid-fast bacilli are most likely to be M. tuberculosis, while atypical microorganisms are most likely to belong to other mycobacterial species. Tubercle bacilli from patients under chemotherapy occasionally show increased granularity (as opposed to beading) and a tendency toward long forms, a varia- tion within the limits of “typical” morphology. An extraordinary in vitro result of contact with isoniazid is loss of acid-fastness. This has been described in vivo,!* but it is not generally detected in routine examinations. A rough estimate of the number of bacilli found is sufficient, for example, the average number per oil-immersion field, or the number on the entire slide in a given period of search. The code recommended in Diagnostic Standards of the National Tuberculosis Association is satisfactory and is given here: —= No acid-fast bacilli found. 1 or 2=acid-fast bacilli in entire film, to be so recorded. + =3 to 9 acid-fast bacilli in entire film. + +4 =10 or more acid-fast bacilli in entire film. + + +4 =10 or more acid-fast bacilli in most oil-immersion fields. E. Sources and Prevention of Positive Error False-positive microscopic findings may result from inadequate decolorization in the staining process or from the presence of con- taminating tubercle bacilli or other acid-fast microorganisms. Tubercle bacilli present on previously used glassware may be destroyed by heating at 250° C for 2 hr or by exposure for 12 hr to an especially strong cleaning fluid such as dichromate in sulfuric acid. The efficiency of these measures may be checked by including glass slides, using films of M. tuberculosis as controls. Water supplies and solu- tions may be checked for contamination with acid-fast bacilli by oc- casionally making stained films from centrifuged sediments of these liquids. The oil-immersion microscope objective should be carefully cleaned after each use in order to destroy any adhering stained TUBERCULOSIS AND LEPROSY 269 mycobacteria, which could be transferred to the next film examined. This is particularly important where there is a strong tendency for the stained film to become detached from the slide—a situation obtaining when films have been prepared from cultures. As a general rule it is not advisable to examine stomach contents microscopically, since contaminating acid-fast saprophytes may be present in this material. However, where this is to be done, a new stomach tube and a dichromatic acid-cleaned syringe should be used. Should material be used for microscopic examination that could be contaminated with acid-fast bacilli from previously used instruments (bronchoscopes, cystoscopes, stomach tubes, catheters), positive find- ings must be viewed with suspicion and a warning should accompany the report, since these instruments cannot be rendered completely sterile with certainty. F. Interpretation When large numbers of acid-fast bacilli are found, it is probable that they are pathogens. When they are of typical morphology they are probably M. tuberculosis. When they are “atypical,” they may be other pathogenic mycobacteria, which include the unclassified strains such as Group I, light sensitive ; Group II, pigmented ; and Group III, nonpigmented.'? There is serious question whether final diagnosis of tuberculosis should ever rest on microscopic examination alone, Confirmation by culture should always be made, and subsequent animal inoculation if deemed necessary. In a minority of cases under chemotherapy, microscopical ex- amination may reveal tubercle bacilli where cultures and animal inoculation are unsuccessful. This occurs most commonly when the patient’s bacillary output is diminishing rapidly toward zero, usually in the first few months of treatment. However, failure to demonstrate tubercle bacilli due to loss of acid-fastness as a result of contact with isoniazid has not been shown to be of clinical importance. Ill. DEMONSTRATION OF TUBERCLE BACILLI IN SPUTUM AND OTHER BODY EXUDATES BY CULTURE Tt is well known that the demonstration of unmistakable tubercle bacilli furnishes the most important criterion we have in the diagnosis of tuberculosis. Twenty-five years ago no one would seriously have questioned the infallibility of a diagnosis based on the discovery of acid-fast bacilli in a sputum film. However, in the light of current 270 TUBERCULOSIS AND LEPROSY knowledge that other acid-fast organisms may be present in the sputum, it is necessary to employ more critical and sensitive methods for demonstrating true tubercle bacilli, that is, culture and guinea pig inoculation. Many negative results of examination of sputum films for tubercle bacilli gave the patient a false sense of security, but unless repeated cultures of sputum or gastric contents have been performed, such apparently negative results often come back from the laboratory to plague the physician. In other words, to the bacteriologist a negative sputum has little significance unless many successive specimens, sedi- mented and inoculated into animals and cultures, have revealed nothing. The value of one positive finding is absolute only when all the conditions of control have been fulfilled. Repeatedly negative specimens are reliable only when related to other confirmatory evidence. A. Collection of Specimens The laboratory should be furnished with adequate sputum (24 or 48 hr collection) or a gastric lavage, which is then concentrated and cultured for tubercle bacilli. If these initial examinations are negative by culture of concentrates, three consecutive gastrics or 48 hr sputa are requested for culture. The practice of such procedures is especially important in diagnosis and in determining bacillary negativity on discharge after treatment. B. Preparation of Containers All containers, preferably glass or made of a material that will resist cleaning solutions, are washed, dried and allowed to stand in the cleaning solution (saturate commercial sulfuric acid with com- mercial potassium bichromate) overnight. These containers are then rinsed thoroughly with tap water and dried. Since the morphology and acid-fast properties of tubercle bacilli are only partially destroyed by boiling and autoclaving, sterilize (2 hr at 130° C) in a dry-air sterilizer. Dead and intact bacilli may adhere to containers or slides and become a source of error in staining methods unless containers or slides have been treated with cleaning solution, C. Preparation of the Specimen To obtain adequate digestion of sputum with either dilute mild alkali (3% NaOH) or acid (4% H2SO.), forceful agitation of the specimen is most important. In this connection the use of paint- TUBERCULOSIS AND LEPROSY 27 conditioning machines for shaking sputum specimens was suggested in 1942.33 These sturdy machines subject specimens to much more violent agitation (760 three-dimensional shakes per min) than is possible by manual shaking. The Committee on Evaluation of Laboratory Procedures of the American Trudeau Society, as well as the Laboratory Committee of the Veterans Administration, has recommended more widespread adoption of shaking machines of this type. Besides preparing the sputum specimens for culture or films more efficiently, their use will save time and effort on the part of technicians. In recent years, several articles have appeared on the use of detergent sodium hydroxide mixtures and of the brand-name product Clorox to promote the digestion of sputum and body exudates before concentrating such material as a preliminary to microscopical ex- amination for tubercle bacilli. The addition of Tergitol to sodium hydroxide does enhance the action of the latter upon the sputum or other tenacious material to be digested. Clorox likewise has even greater digesting properties than the Tergitol-sodium hydroxide mixture. Such technics are not applicable to specimens to be cultured since most of the tubercle bacilli are killed. Unfortunately, any agent in this category, whether used alone or added to sodium hydroxide, intensifies the destructive action upon the tubercle bacillus. In material with few bacilli, this deleterious action is particularly objectionable. From our experience it would then seem preferable to use digesting agents suitable for cultivation purposes, preferably 2 to 3 per cent NaOH. Clorox or NaOH-Tergitol mixtures should be used only when the materials to be examined microscopically cannot be digested by ordinary means, that is, using 2 to 3 per cent NaOH or 3 to 4 per cent HoSOy4 or HCI, plus good mechanical agitation. Thorough shaking of the mixture cannot be overemphasized, since it minimizes prolonged chemical treatment and contact of the tubercle bacilli with such digesting agents. D. Technic for the Culture of Tubercle Bacilli Sputum—DMethod: The sputum specimen is collected in a 100 ml wide-mouth bottle stoppered with a good grade of rubber or cork, or a similar appropriate container. An equal volume of 3 per cent NaOH is added and the mixture homogenized for 10 min with a shaking machine, followed by 30 min incubation at 35° C. It is then centri- fuged for 15 min at high speed in sterile pyrex tubes which have been 272 TUBERCULOSIS AND LEPROSY sealed with sterile rubber caps. The sediment is neutralized with normal hydrochloric acid containing 0.04 per cent by volume phenol red indicator and adjusted on the acid side to a pH of about 6.4-6.8. It is best to approach the final pH with either N/10 HCl or N/10 NaOH, as may be required. The technic used for concentration and culture of sputum specimens is the standard procedure to which all diagnostic material is subjected. In some cases a few preliminary steps are necessary before this basic technic can be applied. Wayne has demonstrated that it is possible to recognize micro- colonies of tubercle bacilli as early as 3 to 5 days after incubation by using molecular filter membranes. Such membranes were employed over Lowenstein-Jensen egg medium. The microcolonies are stained with neutral red and are visualized by a slit of light from a standard microscope lamp.'® McKinney has described a similar technic for culture of tubercle bacilli on membrane filter after sputum has been digested with enzymes such as Pangestin.1® Gastric lavage—Tubercle bacilli often can be demonstrated in the gastric contents of patients whose sputum is consistently negative. Method: Levine tubes are boiled, rinsed and autoclaved at 20 1b pressure for 1% hr. The tubes are introduced through a nostril or the mouth, using a lubricant (glycerol) that is noninhibitory to the growth of the tubercle bacillus. With each swallow the tube is encouraged to progress until it reaches the stomach, when gastric contents are aspirated with a sterile 20 ml Luer syringe. It should be emphasized that nonpathogenic acid-fast bacilli are more likely to be found in the gastric contents than in the sputum. For this reason the laboratory technician should not depend solely upon a gastric film but should use culture or guinea pig inoculation for identifying acid-fast organisms. Urine culture—Glassware for collection of urine is sterilized, as stated previously. To each 100 ml of urine add 1 ml protein (serum or albumin) solution. Mix well and place in the refrigerator overnight. Carefully siphon off the supernatant fluid and centrifuge the sediment at high speed for 10 min. Transfer this precipitate to a sterile sputum bottle. Pleural fluid—Centrifuge all clear and slightly turbid specimens at 3,000 rpm for 10 min. Transfer resultant precipitate to a sterile sputum bottle. Nothing will be gained by a preliminary centrifugation TUBERCULOSIS AND LEPROSY 273 of specimens containing large amounts of pus. A 50 ml portion of this type of fluid will be ample for examination. Feces—The specimen is collected in a wide-mouth jar and diluted with 2 to 3 volumes of a saturated solution of sodium chloride. After thorough mixing, this is filtered through gauze to rid it of coarse particles and the filtrate allowed to stand undisturbed for 2 to 3 hr. The surface layer, or scum, is then removed with a sterile spoon and placed in a sterile sputum bottle. Spinal fluid—The spinal fluid is collected in a sterile tube and centrifuged for 15 min at 3,000 rpm. The supernatant fluid is de- canted and the sediment inoculated directly on culture media. If the sediment appears contaminated or contains a large amount of debris, it is digested with NaOH prior to cultivation. Another procedure that can be used is to add 0.1 ml of protein (serum or egg albumin) suspension for every 10 ml of spinal fluid. Mix until uniformly cloudy and centrifuge at high speed for 10 min, Decant the supernatant fluid. To guard against loss of this small amount of precipitate, the diges- tion technic is carried out in the centrifuge tube if necessary. Tissues—Finely minced tissue is ground in a mortar with a small amount of sterile sand. At least an equal volume of 2 per cent sodium hydroxide is added and well mixed with the specimen. Transfer to a sterile sputum bottle and then shake for 10 min in a shaking machine. Centrifuge at low speed for a few min and discard the precipitated clumps of undigested tissue. Centrifuge the supernatant fluid at high speed for 10 min and neutralize the precipitate as outlined in the procedure for sputum culture. Caseous material may be cultured directly if free from secondary organisms. Puncture is made through a seared area and the material removed and inoculated directly on culture media; or the material may be suspended in physiological salt solution or in 0.1 per cent albumin solution and then inoculated on culture media. Isolation and Identification—The choice of culture media for isolation of tubercle bacilli from pathological material is usually decided by personal preference. However, sufficient evaluation studies have been performed to indicate that certain media are more desirable than others for primary isolation of tubercle bacilli. When practicable, two different media should be employed. Modified Lowenstein- Jensen medium (CM No. 101) and the American Trudeau Society medium (CM No. 100) are most widely employed and are recom- mended for use in diagnostic laboratories. Liquid media (CM No. 274 TUBERCULOSIS AND LEPROSY 99 and CM No. 102) are primarily used for subcultures, drug sus- ceptibility testing, and research. Cultures are incubated at 35° C and should be examined at weekly intervals for a total of 8 weeks. Films should be made of all positive cultures to determine acid-fastness. Whereas acid-fast saprophytes usually appear in a few days and are chromogenic, human-type tubercle bacilli generally appear in 2 to 3 weeks and are rough buff-colored colonies. More recently, so-called unclassified mycobacteria have been described which may or may not be pigmented. These may be isolated from individuals showing clinical evidence of disease but they differ in cultural and pathological characteristics from the human and bovine types of tubercle bacilli, Most of them are drug-resistant, which provides a useful means of distinguishing these strains from more typical mycobacteria isolated prior to chemotherapy. These un- classified acid-fast mycobacteria may be characterized as follows: Classification—The following simplified terminology is sug- gested until a taxonomic classification has been adopted. The classifica- tion is based on pigment formation and rapidity of growth. These groups are not entirely pure and overlapping does occur.!? Group I: Pigment appears only when grown in the presence of light. Group II: Pigment appears when grown in the absence of light. Group ITI: Nonpigmented regardless of light exposure. Group IV: Rapid growers (3 to 4 days and generally pigmented). Pathogenicity—Only rarely do unclassified strains produce disease in guinea pigs. The light-sensitive (Group I) strains as a rule, when first isolated, produce disease in mice. The kidneys usually are highly involved. Biochemical Tests—The following biochemical tests may be used in differentiating the unclassified mycobacteria from pathogenic human and bovine tubercle bacilli : 1. Catalase test: Highly active, even though they are highly resistant to INH. This is in contrast to the human and bovine organisms, since they lose their catalase activity as they become resistant to INH (isoniazid). 2. Neutral red test: A negative test will ordinarily indicate an organ- ism other than true tubercle bacilli. 3. Niacin test: Human tubercle bacilli give a positive test, whereas bovine, avian and unclassified mycobacteria give a negative test. TUBERCULOSIS AND LEPROSY 275 IV. METHODS OF TESTING TUBERCLE BACILLI FOR DRUG SUSCEPTIBILITY Today, the laboratory assumes a more significant role in the treat- ment of tuberculosis than it has in the past. In years gone by, the therapy employed was bed rest, supplemented by pneumothorax, phrenic nerve operation and thoracoplasty. The sole purpose of these therapies was to alter the pathologic process, which in turn affected the etiological agent responsible for the disease. However, the method of attack has been reversed. This is largely due to the use of antituberculosis drugs which attack the etiological agent directly. It becomes of prime importance to determine whether or not the organism harbored by the host is susceptible to the drug or to combinations of drugs to be, or being, used in therapy. The method of ascertaining this information is to culture the tubercle bacilli on drug-containing media. The necessity for sensitivity tests prior to chemotherapy will become increasingly important as more and more individuals become infected with drug-resistant tubercle bacilli. These tests also furnish the clinician with the data he requires regard- ing the time of emergency and the degree of drug resistance of the organisms during treatment. Thus they offer guidance in the selec- tion of the drug regimen to be followed or in determining when it must be modified. A. Direct Tests on Solid Medium The drugs are added to the solid medium (CM No. 100 or No. 101) before inspissation. For streptomycin, concentrations of 10, 50 and 100 pg per ml are used; for para-aminosalicylic acid (PAS) concen- trations of 1, 10 and 100 pg per ml; for INH, concentrations of 0.2, 1 and 10 pg per ml. Fresh aqueous stock solutions of drugs are pre- viously sterilized by passage through sintered-glass filters. After thorough mixing, the drug-containing medium, as well as a batch of medium free of the drug for control tubes, is tubed, slanted and in- spissated for 1 hr at 90° C. After preparation of the specimen as described, aliquots of the sediment to be cultured are planted in two tubes of plain medium and in as many “drug” tubes as desired, the only limitation being the amount of sediment. With a small sediment, as few tubes as possible should be planted. Care must be taken that equal aliquots be used for each tube. The tubes are incubated at 35° C for 6 to 8 weeks and the sensitivity is estimated by comparing the amount of growth and the rapidity 276 TUBERCULOSIS AND LEPROSY of appearance of growth in the drug tubes with that in the control tubes, If growth in the control tubes is scant (less than 50 colonies) a subculture for indirect testing is made in Tween-albumin medium (CM No. 102) from a representative sample of the colonies. (An alternative method for indirect sensitivity tests is to make a suspen- sion of a representative sample of the colonies in sterile salt solution by either grinding in a mortar or by triturating against the side of a tube with a platinum spade.) The subculture or suspension is then tested by planting in the same series of solid-medium tubes, or in liquid medium as described in the next section. The direct sensitivity tests should be used whenever possible, because of the rapidity with which results can be obtained. Further- more, intervening subcultures may show altered susceptibilities. B. Method of Reading and Recording Results of Sensitivity Tests Growth is observed once a week for 35 days and recorded as follows: 44 =Surface of medium entirely covered 34- =Growth not quite confluent 2-4 =Innumerable colonies 14 =50 to 200 colonies Actual colony counts are made of less than 50 Form for recording results of SM, PAS and INH sensitivity tests: Results of Sensitivity Tests to Patient Date of Culture cei Lo8horatory No, of Culture Drug (in pg/ml of medium) TSM INH* PAS | l4days | 21 days | 28 days | 35 days 0 0 0.0 10 02 1.0 50 1.0 10 100 50 100 * A 10 pug tube should be used when cultures start to show resistance. For clinical records the tests may be reported as follows: When no growth is present in the drug tubes and there is good growth in the TUBERCULOSIS AND LEPROSY 277 control tubes, the culture is considered to be completely sensitive or nonresistant. When similar growth is obtained in the drug-containing tubes as compared with the control tubes, the culture is considered completely resistant, or nonsensitive. When there is definitely less growth in the drug-containing tubes as compared with the controls, the culture is considered partially resistant to the concentration of drug present. C. Subculture in Tween-Albumin Liquid Medium Streptomycin tests—The tubercle bacilli to be tested are first transferred to Tween-albumin medium (CM No. 102) from the original isolation medium by triturating some of the growth with a platinum spade on the side of a test tube containing 5 ml of Tween- albumin medium. Following incubation at 35° C for 3 to 5 days, evidence of growth in the form of a diffuse turbidity should be seen in the Dubos tubes. If the growth of this first generation in liquid medium is granular, it is agitated vigorously by pipetting back and forth and a second transfer is made by pipetting 0.1 ml into a tube of 5 ml of fresh Dubos medium (CM No. 102). A diffuse 7-12 day growth is desirable for the sensitivity tests. A series of test tubes containing Dubos medium and varying con- centrations of streptomycin is arranged. For fresh isolations ex- pected to be sensitive, concentrations of 0.5, 1.0, 2.5 and 5.0 ug of streptomycin per ml of medium are used. Each tube in the series (always including a control without streptomycin) is inoculated with 0.1 ml of the culture to be tested. A tenfold increase or decrease in the size of the inoculum does not appreciably affect the results of the sensitivity test. The tubes are incubated at 35° C. Readings are made on the 4th day and on every 3rd or 4th day thereafter until the 14th or 16th day. The tubes are shaken and the turbidity is assigned a value from 0 to 4 plus. The control tubes should show definite growth by the 4th day. Sensitive strains will usually show no growth in concentrations of streptomycin of 0.5 or more pug per ml when incubated for 14 days. Resistant strains found may then be tested with higher concentrations of streptomycin. The sensitivity may be expressed as the lowest concentration of streptomycin which prevented growth for a period of 14 days. D. Subculture Para—aminosalicylic Acid Sensitivity Tests Liquid media have been found unsuitable for testing the sensitivity of cultures of tubercle bacilli to PAS for two reasons: (1) The end 278 TUBERCULOSIS AND LEPROSY point between growth and inhibition is usually not distinct; (2) the inhibitory concentration is markedly influenced by the size of the inoculum. Therefore, sensitivity tests to PAS are done on solid media, utilizing either the modified American Trudeau Society (ATS) or the modified Lowenstein’s medium. E. Subculture Isoniazid Sensitivity Tests Solid media are more desirable than liquid media for these tests because of the variation observed in the latter with size of inoculum and length of incubation period. Therefore, either the American Trudeau Society (CM No. 100) or the modified Lowenstein’s medium (CM No. 101) may be used, adding sufficient isoniazid before inspissation to give concentrations of 0.2, 1.0, 5.0 and 10 pg per ml. Inspissation has no effect on the activity of the drug. F. Catalase Tests Catalase activity of tubercle bacilli has been shown to be related to INH susceptibility and virulence for guinea pigs. A negative catalase test is usually associated with resistance to INH and with attenuated virulence for guinea pigs. The colonies to be tested are covered with the reagent, equal volumes of 30 per cent hydrogen peroxide and 10 per cent Tween 80 in distilled water, at room temperature. Grossly visible gas bubbles will arise from the flooded colonies within 2 min. One may roughly quantitate the total amount of gas formed in 5 min. A strongly positive test suggests contamination or an acid-fast saprophyte. Atypical tubercle bacilli will also yield a strongly positive test. V. DEMONSTRATION OF TUBERCLE BACILLI BY INOCULATION OF ANIMALS Animal inoculation is often necessary for the detection of tubercle bacilli; for the differentiation of pathogenic from nonpathogenic organisms; and for the separation of human from bovine strains. Body fluids such as spinal and pleural fluid, exudates, and certain tissues which have been collected aseptically may be injected directly into guinea pigs (intraperitoneally or intramuscularly) to demonstrate the presence of pathogenic tubercle bacilli. Contaminated materials such as urine and feces should be treated by digestion procedures (as in the preparation for culture) when used for this purpose. The guinea pig is used for virulence testing, while the rabbit is the animal of choice when it is desirable to differentiate human from bovine tubercle bacilli. The bovine type of organism is invariably TUBERCULOSIS AND LEPROSY 279 lethal or rapidly progressive in the rabbit, whereas human-type tubercle bacilli cause only a localized infection in this animal. A. Guinea Pig Inoculation Since tuberculosis in man is nearly always a result of infection with human or bovine types of tubercle bacilli, the guinea pig is the animal of choice in routine laboratory diagnosis. If one is employing pure cultures, a dosage of 0.1 mg (moist weight) of culture is the usual inoculum. This produces a generalized infection by the 6th week. Acid-fast saprophytes and attenuated or avirulent strains of mycobacteria seldom progress beyond the site of inoculation. If possible, two guinea pigs are used for each determination, the animals having been tested with 1 mg of Old Tuberculin intra- cutaneously prior to their use. Only those found to be negative should be used. In guinea pigs, injection is made subcutaneously in the groin, always using the same side. The site of injection is palpated at 10 day intervals for developing local lesions, which give an early indica- tion of the result to be expected. Contaminated material may cause local inflammatory lesions, which appear at an earlier date (7-10 days). The animal is tested with 1 mg of Old Tuberculin intracutaneously after 4 weeks. If the test is positive, it is presumptive evidence of tuberculous infection. If paired animals are used, one of them is sacrificed at this time and the other is allowed to live 6 weeks. If only one animal is used, it is sacrificed after 6 weeks. If the skin test is negative in the only animal used, it is watched for another 2-3 weeks and necropsy is performed at that time. If the skin reaction is nega- tive in the case of paired animals, one is sacrificed after 2-3 weeks, the other after 2-3 months. At autopsy, the local lymph nodes, spleen, tracheobronchial lymph nodes and liver are searched for necrotic, caseous foci. The disease affects these organs in this order of sequence. The presence of such necrotic foci constitutes proof of tuberculous infection. However, films must be made from these lesions and acid-fast bacilli demon- strated before this can be considered final. Lesions from animals inoculated with negative culture material must be further cultured, and if there is any doubt, histological examination is helpful. B. Inoculation of Rabbits and Fowl When bovine or avian tuberculosis is suspected the laboratory technician should resort to the inoculation of rabbits or fowl. Rabbits 280 TUBERCULOSIS AND LEPROSY inoculated intravenously with bovine tubercle bacilli usually develop fatal tuberculosis within 3 or 4 weeks, whereas those which receive human bacilli usually survive the challenge infection. A few tubercles may be found in the lungs and kidneys. Avian tubercle bacilli inoculated intravenously into rabbits give rise to the Yersin type of disease, that is, a fulminating disease with- out macroscopic tubercles. Fowl (chickens are generally preferred) injected with avian tubercle bacilli die in a variable time, usually greatly emaciated. The disease is seen most prominently in the spleen, liver, kidneys and mesenteric lymph nodes.?18 VI. SPECIAL PROBLEMS IN THE DEMONSTRATION OF TUBERCLE BACILLI IN TUBERCULOUS LESIONS The demonstraton of tubercle bacilli in sputum or in fasting gastric contents indicates that a necrotic pulmonary lesion or lesions are liquefying and sloughing. The ease with which bacilli are found depends upon the amount of necrotic debris that is raised and upon the numbers of bacilli in the sloughing debris. On the other hand, not infrequently it is most difficult to find tubercle bacilli in the sputum or fasting gastric contents of persons with a minimal amount of tuberculosis, as revealed roentgenologically, even though sloughing and local dissemination of the disease may be occurring. A. Chemotherapy Before the introduction of chemotherapy it was not unusual for tuberculous patients to become “sputum negative.” Since chemo- therapy has been used, this situation is much more common. Before the days of chemotherapy little work was done to determine the numbers and distribution of bacilli in lesions. But now the examina- tion of material from necrotic lesions to determine the status of bacilli in tuberculous foci still persisting after a substantial course of drug therapy has led to a considerable amount of culturing and animal inoculation. Interest at first was on the reactions of the bacilli to the chemotherapeutic agents which had been used. However, it has been found that in a high percentage of “closed” lesions (i.e. filled-in cavities and necrotic areas of tuberculous pneumonia that had not liquefied and sloughed), the bacilli failed to grow on culture media or to produce progressive disease in guinea pigs. On the other hand, it was unusual to fail to obtain growth of virulent organisms from open cavities even when chemotherapy had been in use continuously for as long as two or three years and bacilli could not be demonstrated TUBERCULOSIS AND LEPROSY 281 in sputum or fasting gastric contents for many months preceding resection of the tuberculous disease. There remains to be made a thorough bacteriological study of “closed” lesions in tuberculous persons who have not received any chemotherapy. It is necessary to determine what difference there may be between the condition of the bacilli within untreated and treated “closed” lesions. B. Limitations of Sputum Examinations Since so much stress has been placed upon the presence or absence of bacilli in sputum, it is now essential to draw attention to dis- crepancies between sputum examinations and examinations of material directly from the pulmonary lesions. At best, sputum ex- amination must be regarded as a crude method of testing cases in which bacilli in relatively small numbers are being sloughed from the lung. It is common to find bacilli, sometimes in large numbers, in material from necrotic lesions of cases that had become “sputum negative” for months prior to resection of tuberculous pulmonary disease and also in cases in which no bacilli had ever been demon- strated in sputum. From such findings it is apparent that sputum studies frequently do not indicate either the presence or the number of bacilli within the pulmonary lesions, It has long been known that pulmonary tuberculosis is a chronic disease with a pronounced tendency to exhibit phases of remission and of relapse. It is equally clear that relapse may result from the persistence of bacilli in necrotic lesions that become free to be disseminated to new areas of lung parenchyma. This is due to the liquefaction and sloughing of necrotic lesions that may have been dormant for a long time. C. Tuberculous Lesions In the examination of tuberculous lesions certain features need attention. In the first place, tubercle bacilli commonly are most irregularly distributed within lesions, especially in the older necrotic ones. Even in cavity walls there often is a very irregular distribution. Bacilli seldom are found except in the innermost portion, the “lining,” of a cavity. Even in this location large areas may fail to show bacilli. In local and unpredictable foci, bacilli may be present in tremendous numbers. There is an even more unpredictable situation in “closed” necrotic lesions. Sometimes no bacilli can be found in sizable necrotic foci, whereas on occasion a small necrotic focus may show numerous organisms. One portion of a sizable old necrotic focus may contain 282 TUBERCULOSIS AND LEPROSY no bacilli, while in another area masses of bacilli in colony formation may be found. It is evident that examination of a single section of a cavity or of a necrotic lesion may or may not show the bacilli which may be present. Such incomplete examination may or may not reveal the probable proportions of the bacillary population. D. Tissue Sections In the examination of tissue sections it has become an accepted procedure to search carefully for bacilli in the outer fringe of the cavity wall and in “epithelioid” tubercles and giant cells, In most in- stances examinations of these areas of tuberculous tissue yield nega- tive results. For practical purposes such searches are a waste of time. Bacilli may perchance be present but, if so, they are so few in number that they seldom are found. E. Necropsies In necropsies, in which an open tuberculous cavity is found and in which there also is evidence of an acute lobular pneumonia, the acute pneumonic process may prove to be tuberculous with bacilli present in large numbers in lesions that show a preponderance of neutrophils. This is not a constant finding, but an acid-fast stain of such lesions will readily clarify the situation. From these remarks it is clear that tubercle bacilli are most irregularly distributed in the diseased areas and it is this situation which requires a realistic approach to the determination of the presence or absence of bacilli in tuberculous lesions, regardless of the organ in which they are found. When fresh tissue is to be examined the best results will be obtained if sizable areas of necrotic lesions are smeared on slides. The film should be thoroughly dried in an open flame before applying the usual acid-fast technic used in stain- ing films of sputum. It is preferable to crush the material in a hand tissue grinder before making the films in order that a more even distribution of bacilli may be obtained. Equally good results can be obtained by homogenizing the necrotic lesions after the tissue is fixed thoroughly in 10 per cent formaldehyde. Here it will be necessary to mix thoroughly a drop of blood serum or Mayer's egg albumin mixture with the necrotic material to be smeared thinly on a slide. Also it will be necessary to fix and dry the film more thoroughly in an open flame than in the case of fresh unfixed tissue lesions. Excellent results can be obtained by using an acid-fast staining technic on microscopic sections of tissue which has been thoroughly fixed in 10 per cent formaldehyde. Here it is preferable to use one of TUBERCULOSIS AND LEPROSY 283 the methods of cold staining, The whole process of staining can be completed within ten minutes.’ Since tubercle bacilli are so un- predictable in their distribution in necrotic lesions it often requires the examination of several slides before any bacilli can be found. This situation has led to questioning the dependability of the staining process. As a result, it is common to stain a known positive slide as a check. This precaution is unnecessary if reliable technical assistance is available and if the unpredictable location of bacilli within lesions is appreciated. Search of properly stained tissue sections is of value to show where and in what numbers bacilli may be present within lesions. However, when it is desired only to demonstrate the presence or absence of bacilli, the smear technic outlined above is more rapid and will give a much higher proportion of positive results with less labor. VII. LABORATORY DIAGNOSIS OF LEPROSY Due to the uncertainty which surrounds the successful inoculation of guinea pigs or culture media for the cultivation of mycobacteria from skin lesions, these methods are not useful in the diagnosis of cutaneous tuberculosis or leprosy, and negative results do not provide presumptive evidence of leprosy. Demonstration of mycobacteria by staining, however, provides the clinician and public health adminis- trator with essential information concerning the clinical type of leprosy and the probable prognosis. It is also the basis of administra- tive classification into “closed” (so-called noninfectious) or “open” (bacteriologically positive) cases. The organisms are not found in the epidermis but in the corium. The important sites to sample are infiltrations and the margins of suspected or definite lesions. Samples are also taken routinely from the ear lobes, the forehead or elbows, and the nasal mucosa. The intended pattern of samples must be carefully mapped on clean, labeled glass slides. A. Scraped Skin Incisions Scrub the site vigorously with a small pledget of cotton soaked in alcohol. Compress a fold of the skin tightly between a thumb and forefinger ; make a short incision 2-3 mm deep into the corium with a small scalpel or single-edge razor blade; use the blade to obtain scrapings from the walls of the incision, transferring the material to a small circular spot on a clean glass slide. Wipe the blade thoroughly with fresh cotton soaked in alcohol before sampling the next skin sites. 284 TUBERCULOSIS AND LEPROSY B. Nasal Samples Nasal samples are less important than thorough sampling of the skin, Hammer the tip of a short nichrome inoculating wire to form a sharp blade and bend it at right angles. After anesthetizing with cocaine and cleaning the surface of the septum, remove scrapings from the posterior portion of the septum by abrading suspected areas (found by speculum) or normal mucosa until slight bleeding points appear. Samples are transferred to small circular areas on the test slide. The wire is flamed and cooled between samples. The air-dried films are preserved in a dustproof slide box until stained, as follows: 1) Lay the slides on a leveled glass plate over a bath of boiling water for 30 sec heat fixation. 2) Cover with carbolfuchsin for 30 sec; rinse gently in water. 3) Counterstain for 30-90 sec in 0.2 per cent methylene blue in 4 per cent sulfuric acid by volume; rinse gently in water. 4) Shake off water excess, air-dry, and examine under oil. Since in tuberculoid and undifferentiated (indeterminate) lesions of leprosy, bacilli are very rare and may not be discovered, methods of high sensitivity must be combined with careful and critical ex- amination of the films. Differentiation time should be adjusted so that a red color is exhibited by epithelial and mucosal cells and paler tints by red blood cells. The essential observations in a useful report are: the numbers of fields searched, or the time expended before arriving at a negative decision; an estimate of the numbers of bacilli per field when modest numbers are found; and, when bacilli are numerous, an indication of the frequency and size of globi (clumped bacilli). VIII. DISCUSSION The methods described in this section are those commonly employed which for the most part have withstood the test of time. It is recog- nized, however, that many laboratory workers will prefer modifica- tions of the technics presented and scme may even prefer certain procedures not referred to here. - Modern technics of searching for the tubercle bacillus can greatly assist in making the diagnosis of tuberculosis. The advent of chemo- therapy has made it necessary to perform antibiotic sensitivity tests which, however, may aid the clinician in his appraisal of the course of the disease and in his choice of effective drugs as well. As with most other laboratory tests, the results must always be correlated with the clinical findings. TUBERCULOSIS AND LEPROSY 285 Safety is a major consideration for laboratories handling tubercle bacilli. Therefore, it is important that all personnel be thoroughly trained in the use of pipettes, transfer loops and other special equip- ment used in the laboratory. Wherever possible a bacteriological safety hood should be available for handling cultures. Ultraviolet irradiation of work areas and the liberal use of germicidal agents for cleaning are to be encouraged. Sanitary precautions are equally im- portant for procedures carried out in animal quarters and in the morgue. (See Chapter 3, “Laboratory Infections and Accidents,” for other suggestions.) MartIN M. Cummings, M.D., Chapter Chairman Oscar AUERBACH, M.D. WiLriaM FeLpman, D.V.M. Jou~x H. Hanks, Pu.D. Ebpcar M. MEepLAr, M.D. C. Ricuarp Smith, M.D. WILLIAM STEENKEN, Jr. Sc.D. REFERENCES 1. Ovriver, J. and Rrusser, T. R. Rapid Method for the Concentration of Tubercle Bacilli. Am. Rev. Tuberc. 45:450, 1942. 2. Wiis, H. S,, and Cummings, M. M. Diagnostic and Experimental Methods in Tuberculosis (2nd ed.). Springfield, Ill.: Charles C Thomas 1952, Chapter IV. 3. SmitH, C. R. The Flotation Method of Sputum Examination. Am. Rev. Tuberc. 37:525, 1938. 4. NEeeLseN, Fortschr. der med. 3:200, 1885 (a footnote to an article by Johne). 5. PortENGER, J. E. Controlled Staining of Mycobacterium tuberculosis. Am. Rev. Tuberc. 45:549, 1942. 6. STEENKEN, W., Jr. Tray for Staining Tubercle Bacilli. Am. Rev. Tuberc. 44:115, 1941. 7. Ausert, E. “Cold” Stain for Acid-Fast Bacteria. Canad. J. Pub. Health 41:31, 1950. 8. Tuompson, L. Comparison of Carbolfuchsin with the Fluorescent Dye Auramine for the Demonstration of Acid-Fast Bacteria. Proc. Staff Meet. Mayo Clin. 16:673, 1941. 9. Manual of Histologic and Special Staining Techniques. Armed Forces In- stitute of Pathology. Washington, D. C.: The Institute, 1957, page 175. 10. Purr, F. A. A Modified Ziehl-Neelsen Method. Am. J. Clin. Path. 21:92- 95, 1951. 11. Atri, L. Variazioni tintoriali e morfologiche del bacillo di Koch durante il trattamento con l'idrazide dell’acido isonicotinico. Osped. Maggiore 41 :209- 214, 1953. 12. Runvon, Ernest H. Anonymous Mycobacteria in Pulmonary Disease. M. Clin. North America 43:273, 1959. 13. SteeNnkeN, W., Jr, and Smite, M. M. A Shaking Machine and Con- tainers for Shaking Sputums and Other Body Fluids During the Process of Homogenization. J. Lab. & Clin. Med. 27:1582-1585, 1942. 286 TUBERCULOSIS AND LEPROSY 14. WaynNE, LAWRENCE G. Cultivation and Visualization of Mycobacteria on Molecular Filter Membranes. J. Bact. 69:92-96, 1955. 15. — Quantitative Aspects of Neutral Red Reactions of Typical and “Atypical” Mycobacteria. Am. Rev. Tuberc. 79:526-530, 1959. 16. McKinney, Ruta A. The Preparation of Tuberculous Sputum for Mem- brane Filter Filtration. Am. Rev. Tuberc. 77:1019-1022, 1958. 17. Torrey, W. W. C., and WitsoN, G. S. Principles of Bacteriology and Immunology (4th ed., revised by G. S. Wilson and A. A. Miles). Baltimore, Md. : Williams & Wilkins, 1955, pp. 517-524. 18. Wirrts, H. S., and Cummings, M. M. Diagnostic and Experimental Methods in Tuberculosis. Springfield, I1l.: Charles C Thomas, 1952, p. 56. CHAPTER 10 BACTERIAL INFECTIONS OF THE GASTROINTESTINAL TRACT I. Shigella and Salmonella A. Examination B. Specimens Feces and Other Intestinal Discharge Urine Blood Bile C. Isolation and Presumptive Identification Media Selection Inoculation of Media Picking Colonies Reactions on Triple Sugar Iron Agar Screening Presumptive Serological Tests Reporting Presumptive Findings D. Identification and Typing Purification Cultural and Biochemical Tests Serological Typing Reporting of Findings E. Reference Diagnostic Services 1. Phage Typing of S. typhosa 2. Salmonella Typing 3. Identification of Problem Organisms F. Serological Examination Agglutination Tests Sera for Control Test Procedure II. Enteropathogenic E. coli in Infantile Diarrheal Disease A. Classification and Description B. Nomenclature C. Preparation of Diagnostic Antisera and Technics of Agglutination Tests OB Antisera Polyvalent OB Sera O Antisera H Antisera D. Collection and Handling of Specimens 1. Feces 2. Throat and Nasopharyngeal Swabs 3. Blood E. Bacteriological Examination 1. Isolation Media 2. Isolation and Identification F. Serological Diagnosis G. Evaluation and Reporting of Results 287 288 GASTROINTESTINAL INFECTIONS ITT. The Cholera Vibrio A. Description and Classification B. Laboratory Procedures Isolation and Identification References I. SHIGELLA AND SALMONELLA A. Examination Control of enteric infections is an acknowledged major responsi- bility of public health authorities. The isolation and identification of enteric pathogens and the serological examination for evidence of typhoid fever have been basic tasks of the public health laboratory. The earlier and simpler procedures seemed adequate to aid in the clinical diagnosis of typhoid fever, but now more exacting procedures are required in the search for carriers and in attempts to obtain etiological diagnosis of the prevalent but ordinarily brief diarrheal disorders which cannot be differentiated by clinical findings alone. The required bacteriological studies commonly involve three steps. These may be done in one laboratory or in different laboratories. Isolation and presumptive identification, the first step, is designed to provide an early report useful as an aid in clinical diagnosis or as a guide in control. In general, these examinations are best performed in laboratories located near the patients, since some bacteria, notably Shigella, may remain viable for a relatively short time in fecal specimens. The second step entails the tests necessary for identifica- tion of species and especially the study of microorganisms that suggest but do not exactly correspond to the recognized bacillary incitants of enteric disease. Central laboratories are equipped to cover this phase of the work when it cannot be undertaken at the local level. The third step is the differentiation of serogroups, serotypes and phage types, which often requires final checking in a reference laboratory. Enteric infections are caused by a wide variety of bacteria with differing cultural requirements (see also Chapter 11, “Bacterial Food Poisoning”). The isolation of the pathogenic types of Escherichia coli (see Section II of this chapter) or the cholera Vibrio (see Section III) requires technics which diverge widely from those used in the study of salmonellosis and shigellosis. Even the optimum technics for isolation of Shigella and Salmonella differ. Pertinent clinical and epidemiological data are therefore essential to guide the bacteriologist in the selection of appropriate procedures, GASTROINTESTINAL INFECTIONS 289 It is true that in the hands of experienced workers alternative technics may provide equally dependable findings. Nevertheless, the procedures outlined here are those believed preferable for the great majority of public health laboratories. These technics diverge from the multiple tests which are an accepted part of studies in descriptive bacteriology. The preferable public health laboratory practice is to arrive at an accurate conclusion in the shortest possible time with every practicable economy in glassware, media and labor. Only in this way can there be maximum service from funds available. B. Specimens Feces and Other Intestinal Discharge Specimens for culture may be obtained as follows: 1. Passed specimens—In hospitals, the patient may be provided with a recently sterilized bedpan and the whole specimen or a portion thereof should be taken to the laboratory without delay. In public health laboratory practice, these specimens ordinarily are submitted by mail, using 1 oz screw-cap bottles or jars half filled with buffered (pH 8.4-8.6) 30 per cent glycerol in physiological salt solution (CM No. 71) with phenol red indicator. Approximately 1 g of solid or 2-3 ml of fluid feces should be added. The mailing container employed must meet the requirements of the postal regulations for the ship- ment of infectious material. Passed specimens are preferred in ex- amining for Salmonella, particularly in the search for carriers, since large inocula are indicated. 2. Rectal swabs—If the individuals to be examined are con- veniently available, the specimen for culture may be collected by rectal swab,! especially in examinations for Shigella and for the incitants of acute diarrheal disease in infants. In young children the ordinary cotton-tipped applicator may be readily inserted beyond the anal sphincter and a suitable inoculum collected by rotating the swab and swinging it gently in a circular motion. In adults, the use of a lubricated rubber tube with the applicator inside facilitates insertion of the swab. The major advantages of the rectal swab are that the specimens may be obtained as desired, assuredly fresh material is available for the inoculation of culture media, and the handling and disposal of fecal specimens is avoided. When the inoculum is collected by rectal swab, it is recommended that the media be taken to the bedside and planted immediately; otherwise the swab must be taken at once to the laboratory. 290 GASTROINTESTINAL INFECTIONS 3. Sigmoidoscopic swabs—Specimens collected on swabs dur- ing sigmoidoscopic examination from areas of maximum pathology in the lower bowel are significantly but not markedly superior for the isolation of Shigella. Urine Urine specimens have some value in determination of the rare urinary carrier of S. typhosa. Passed specimens collected with precau- tions to limit extraneous contamination are satisfactory. These may be mailed after adding equal amounts of urine to the glycerol salt solu- tion preservative. Blood Clotted venous blood collected under aseptic conditions is accept- able. The separated serum may be used for serological tests in possible S. typhosa infections and the macerated blood clot may be cultured for Salmonella and certain other microorganisms, More reliable results are obtained when 100 ml of an appropriate medium is inoculated directly with 10 ml of freshly drawn blood. Bile The examination of duodenal drainage is recommended (1) in the event typhoid bacilli or other Salmonella are not found in feces or urine from an individual whom serological tests or epidemiological data indicate to be a carrier; (2) to confirm the assumption that the infection is localized in the gall bladder when cholecystectomy is con- templated; and (3) for possible release of a carrier after chole- cystectomy. The criteria for judging the suitability of duodenal drainage for bacteriological examination have been outlined by Forsbeck and Hollon.? Gall bladders removed surgically also may be submitted for examination for S. typhosa. C. Isolation and Presumptive Identification Media Selection In examining for Shigella and Salmonella, the following culture media are of major importance : Shigella-Salmonella (SS) Agar (CM No. 59)—This is used for initial plating and/or for plating from enrichment broth. It inhibits most coliforms but permits the growth of virtually all Salmonella and most Shigella encountered in public health laboratory practice in the United States. GASTROINTESTINAL INFECTIONS 291 Wilson and Blair’s Bismuth Sulfite (WB) Agar (CM No. 23)— This is primarily for isolation of S. typhosa and other Salmonellae, both for initial plating and for plating from enrichment broth. Brilliant Green (BG) Agar (CM No. 61)—This is recom- mended for plating from the enrichment broth and is useful primarily for Salmonellae other than S. typhosa and S. paratyphi A. Sodium Desoxycholate Citrate (DC) Agar (CM No. 57)—This is helpful in examinations for Shigellae, since these are occasionally inhibited on SS agar. Enrichment Broth, Tetrathionate Brilliant Green (CM No. 58) or Selenite F (CM No. 63), is useful primarily for Salmonella. The latter may aid in the isolation of Shigella. The former without brilliant green is sometimes recommended. Different combinations of media are indicated for varying purposes. For examination of specimens from acute diarrheal disease for Shigella and Salmonella, one or two plates of SS or DC agar and enrichment broth subcultures to one or two plates of BG, WB or SS agar make an effective combination. Some recommend that this be supplemented by at least one plate of a less inhibitive enteric medium, such as Endo’s (CM No. 55), eosin-methylene blue (CM No. 54), MacConkey (CM No. 60), or desoxycholate agar (CM No. 56). In the examination of specimens from food handlers for the detec- tion of chronic carriers of S. typhosa, one heavily inoculated streak plate of WB agar is of the greatest importance. This may be supple- mented by two pour plates. Examination of specimens from food handlers is recommended only when clinical or epidemiological data suggest the presence of a carrier. When searching specifically for Shigella, as in epidemic situations, one or two well-inoculated plates of SS or DC agar will give excellent results. This permits the ex- amination of large numbers of specimens. Enrichment broth and BG agar are particularly effective for the isolation of Salmonellae other than S. typhosa and S. paratyphi A. Where no information is at hand, an acceptable procedure makes use of SS agar, WB agar (streak), one of the less inhibitive media, and enrichment broth, plus BG or SS and WB agar for subculturing—thus utilizing three or more plates and one tube for the initial isolation. Inoculation of Media Employ a technic that will, if possible, provide a good distribution of isolated colonies over a major portion of each plate. The following procedures are suggested : 292 GASTROINTESTINAL INFECTIONS For inoculation of plating media, use sterile cotton-tipped swabs. If the specimen is collected by rectal swab, streak this directly on the plates and then inoculate the fluid enrichment medium. If material is submitted in glycerol salt solution, streak the plates with a swab dipped into the fecal mixture. After streaking a quarter to a half of each plate, inoculate the fluid enrichment medium. Using a second sterile swab for each plate, cross-streak half the plate (inoculating one-half of the uninoculated area by streaking through one-half of the heavily inoculated area). Streak the remaining uninoculated area, using the same swab, without touching the other inoculated areas. One must learn by experience the maximum amount of material which can be inoculated onto the various plating media in order to provide a desirable number of well-isolated colonies over a substantial portion of the medium. Very little or no growth may occur on WB agar even with a very heavy inoculation. The amount of inoculum will vary materially, depending on the population under study. When examining specimens for carriers of S. typhosa, two pour plates of WB may be used also, one inoculated with 5 ml and another with 0.1 ml. The medium, melted and cooled to 45° C, is poured into the petri plates in which the inoculum has been placed. The plates are then rotated gently to mix and distribute the inoculum evenly. S. typhosa does not produce typical blackening of bismuth sulfite agar when the colonies are very numerous and crowded. Inoculate brilliant green agar and/or other plates from the enrich- ment broth after incubation for 18-24 hr with a sterile wire loop approximately 5 to 6 mm in diameter. Cross-streak to obtain areas of heavy, medium and light inoculation. Examine plates after approximately 20 hr incubation and again after 44-48 hr when the results of the first examination are not significant. The WB agar ordinarily requires the longer incubation. Fluid or semifluid medium (CM No. 5 or No. 15) inoculated with blood should be plated after incubation for 24 hr, 48 hr, 1 week, 2 weeks, and 1 month, Picking Colonies The most important and the most difficult step in isolation of enteric pathogens is the picking of colonies. For this purpose a suitable source of light is essential. The Quebec Colony Counter provides a good quality of artificial light, a dark background, and desirable magnifica- tion, but the preference of the individual experienced worker should be considered. In picking, time and energy may be saved by arranging conveniently the materials needed. It should be an unbroken rule to Table 1—Character of Growth of Bacilliary Incitants of Enteric Disease on Selective Plating Media Microorganism Shigella S. typhosa Salmonella group (other than S. fphsa) and rizona group Alkalescens-dispar group E. coli and Klebsiella- Aerobacter group Proteus group E. freundii, Provi- dence and Arizona groups Pseudomonas group SS Agar (CM No. 59) Colorless or pale pink; transparent to mod- erately opaque; round; raised; 3+ mm. Sh sonnei may be large, flat and irregular. Similar to Shigella, Similar to Shigella; occasionally with darker center, Similar to Shigella; tend to be more opaque. Usually inhibited; pink to red; opaque, some mucoid. Colorless; some with black centers; trans- parent; irregular; dis- crete. Mey Suzgest Shigella colt. Usuall colorless, often rownish. Desox: Sills Agar CM 56) Mac- Eo "Agar (CM No. 60) EMB Agar (CM No. 54) Colorless; transparent; 2-7 mm; usually round. Sh. sonnei may be large, flat and ir- regular. Occasionally with dark centers. Similar to Shigella, Similar to Shigella. Similar to Shigella. Red gpaque on CM No. 56 and CM No. 60; dark center and metallic sheen on CM No. 54; some mucoid. Usually Spreading on CM Nos, 56 and 60; yuly 9 dserate on 0. May sug- gest Shigatta or Sal- nk Mex suggest Shigella colt. May suggest Proteus group. * These characteristics may vary on different lots and brands of media. Plating Media* Desoxycholate Citrate Agar (CM No. 57) Colorless or pale pink; translucent or nearly transparent; 2-3 mm. h. sonnei may be irregular in size and shape. Similar to Shigella. Similar to Shigella. Similar to Shigella. Usually inhibited pink to red; opaque, some mucoid. Colorless or pale pink, transparent, discrete. May Supgest Shigella colt. Usually colorless; sometimes brownish. WB Agar (CM No. 58) Inhibited; occasionally small, greenish colo- nies with depressed centers. Black surrounded by black or brown zone with metallic sheen; 3+ mm; subsurface colonies jet black, well defined, no sheen. Variable. Few similar to S. typhosa; some green to brown; some markedly inhibited. Light to dark green, smooth; glistening. Usually inhibited; some strains have dark brown or green colonies. Discrete green, some with dark centers. Some strains inhibited. Similar to E. coli. Variable. BG Agar (CM No. 61) Inhibited. Usually inhibited. Pink to fuchsia sur- rounded by red zone; occasionally brown with little change in medium, Usually inhibited; rare strains similar to Salmonella. Usually inhibited; some strains have vel lowish green coloring. Usually inhibited; . some small reddish. Similar to E. coli. Pink to purplish; ir- regular edges; may suggest Salmonella. TVNILSILNIOYLSVD SNOILD34ANI €62 294 GASTROINTESTINAL INFECTIONS pick plates in serial order, as this is the best assurance against errors in numbering. The character of the colonies on the different media is given in Table 1.* There is no substitute for experience in learning the selection of suspicious colonies. Workers must be encouraged to pick freely, preferably two or more of each type of suspicious colony. Even those who are very experienced must finally rely on differential tube medium to separate the “negative” from the possibly “positive” colonies. It should be emphasized that on the surface of all highly selective enteric media there may be viable microorganisms which have not grown. To assure picking pure cultures, only the elevated center surface or the extreme edge of a colony should be touched. A scooping motion, with the needle sweeping the surface of the agar, is to be avoided. Pick all suspicious colonies to triple sugar iron (TSI) agar (CM Nos. 53a and 53b) or Kliger’s iron agar with 1 per cent sucrose added (CM No. 52). First, stab the center of the butt to the bottom and then streak the slanted surface. Suspicious colonies from pour plates of bismuth sulfite agar should be picked and streaked for purity on SS or similar medium and colonies picked the next day from this to the TSI agar. Reactions on Triple Sugar Iron Agar In Table 2, the reactions of 18-24 hr cultures in TSI agar of various groups of Gram-negative bacilli isolated from feces or urine are indi- cated. In examining for Shigella and Salmonella, cultures are dis- carded as indicated. Screening The rapid urease test is the most useful screening procedure.’ With a small loop transfer a generous portion of the growth to a tube con- taining 0.2 ml urea medium (CM No. 67). Read the results after 30 min in a 37° C water bath. A change in color to pink or fuchsia indi- cates hydrolysis of urea and constitutes a positive test. Urease-positive cultures isolated from feces may be discarded. Examine all urease- negative gas formers, regardless of HS production, by the rapid indole test.® Incubate heavily inoculated tryptone broth (CM No. 2) 2 hr in a 37° C water bath. Add Kovacs’ reagent (see Chapter 1) and shake gently. The formation of indole is indicated by a red color in the layer of the reagent on the surface of the broth. Organisms that are indole-positive by this method are usually not Salmonella and may GASTROINTESTINAL INFECTIONS 295 be discarded. In laboratories handling small numbers of specimens it is convenient to perform the rapid urease and indole tests simul- taneously, reading both after 2 hr incubation. The procedure at this point will vary. When examining specimens from patients with undiagnosed enteric disease, particularly when Shigella or Salmonella is indicated, proceed at once to presumptive serological tests. However, in the examination of specimens for release Table 2—Reactions of 18-24 Hour Cultures in Triple Sugar Iron Agar (CM No. 53) . HS Indicated Procedure Reaction on Pro- Microcrganisms for Organisms Slant Butt duction Suggested Isolated from Feces* Alkaline Acid — Shigella, S. typhosa, Screen and identify as Proteus, Alkalescens- indicated. dispar group Alkaline Acid Be S. typhosa, Proteus, Screen and identify as anaerogenic Sal- indicated monella Alkaline Acid and + Salmonella, Proteus, Screen and identify as gas Arizona indicated (ordinarily many pathogens). Alkaline Acid and — Proteus, occasion- Screen and identify as gas ally Salmonella indicated (ordinarily very few pathogens). Acid Acid — Streptococci, staphy- Screen and identify as lococci, occasionally indicated, if a Gram- S. typhosa, other negative rod ; discard Gram-negative rods others. Alkaline (spread- Acid and + or — Proteus Discard. ing growth) gas Acid Acid and i E. coli; Klebsiella- Examine serologically gas Aerobacter group, for enteropathogenic Arizona E. coli when indicated; otherwise discard. Alkaline Alkaline — Alcaligenes, Pseudo- Discard. monas, Mimeae Purplish Alkaline — Pseudomonas species Discard. * Applicable only to pure cultures. Note: Alkaline slant indicates lactose and sucrose not fermented. Acid slant indicates lactose and/or sucrose are fermented. Alkaline butt indicates glucose not fermented. Acid butt indicates glucose fermented. 296 GASTROINTESTINAL INFECTIONS or from suspected carriers, the biochemical tests outlined below may be performed before presumptive serology to conserve antisera, which are often in short supply. Presumptive Serological Tests Microorganisms selected on the basis of biochemical screening or clinical history are checked with multivalent and other selected agglutinating sera. The following sera obtained either commercially or from a central laboratory are required : Multivalent Shigella dysenteriae (Group A) Multivalent Shigella flexneri (Group B) Multivalent Shigella boydii (Group C) Sh. sonnet (Group D) Group D Salmonella Multivalent Salmonella Vi Mark a 1 by 3 in. glass slide with a glass-marking pencil into 10 or fewer squares approximately 75 by 1% in. In the first row of squares place a drop of agglutinating serum, diluted as indicated, in each square. In the second row of squares, place a drop of the corres- ponding dilution of normal serum in each square (from an unim- munized animal of the same species as that in which the agglutinating serum was produced). Using a sterile needle, take up a portion of the growth from a TSI slant and emulsify first in the normal serum and then in the agglutinating serum. The mixture should be heavily turbid. In practice, the growth from at least five cultures may be suspended in succession in as many drops of serum, then observed for agglutina- tion by rocking the slide gently and examining with a bright light and a dark background. Definite agglutination in one of these sera and not in a corresponding dilution of normal serum indicates the probable serogroup of the microorganism. Cross-reactions are not uncommon among Shigellae, especially of Groups B and C. The desirable sequence in presumptive serology is to proceed from the most probable to the least probable. Ordinarily Group B and Group D comprise the most prevalent Shigellae in the United States, but past experience in the individual laboratory may indicate the common occurrence of other groups. Reporting Presumptive Findings In undiagnosed illness an early report of presumptive findings is highly useful and much appreciated by the health officer or clinician. It is desirable to report by telephone when possible, since the presump- GASTROINTESTINAL INFECTIONS 297 tive nature of the report should be emphasized. The final report after further study usually but not always will be in agreement. D. Identification and Typing The publications of Kauffmann” and Edwards and Ewing® provide detailed procedures and classifications for the differentiation and identification of Salmonella and Shigella and other Gram-negative bacilli found in the intestinal tract of man. It must be kept in mind that strains of bacteria are occasionally encountered that fail to fulfill the accepted criteria for identification of any given species. Salmonellae that ferment lactose or sucrose or that produce indole as well as anaerogenic strains have been reported.*™1° Divergence in one or more properties does not necessarily exclude a given species. Purification—The plating of cultures to establish purity is always indicated in systematic bacteriology. However, in diagnostic bacteriology, with careful picking of single isolated colonies from the plates and critical inspection of slants it is necessary to plate only those cultures upon which the report is based and those in which the possible presence of more than one species is indicated. Cultural and biochemical tests—When the reaction in TSI agar (CM No. 53) suggests either Salmonella or Shigella, study the following properties (see Table 3 and Edwards and Ewing®). 1. Motility may best be observed either in semisolid motility medium (CM No. 15 or No. 69) or by microscopic examination of a hanging drop of broth culture. 2. Hydrolyzation of urea—Inoculate the surface of a slant of urea agar (CM No. 68) or a tube of urea broth (CM No. 67), and incubate, Decomposition of urea is indicated by a change in the color of the medium from yellow to violet red that is usually apparent within a few hours. Some strains produce urease very slowly, how- ever, and failure to obtain the characteristic color change should not be considered final until the cultures have been incubated for 4 days. 3. Utilization of citrate—Inoculate lightly the slant of sodium citrate agar (CM No. 66) and incubate. Record growth and change in color of indicator after 18-20 hr. No change in color occurs unless there is growth. 4, Indole production—Test for indole production by adding 0.2-0.3 ml of Kovacs’ reagent to approximately 5 ml of a 1 to 4 day culture in tryptone broth (CM No. 2) and shake gently. The formation of indole is indicated by a red color in the layer of reagent on the surface Table 3—Biochemical Tests for Identification of Shigella and Salmonella Acid From Group or Type Motility Indole Urease Citrate KCN Mannitol Xylose Rhamnose Suggested a V* = oe -— + — — Sh. flexneri or Sh. boydit — il ss — — + — RX Sh. sonnet id #5 m= dred — = — pe Sh. dysenteriae Type 2 (Sh. ambigua) — Vv so -_ — — — — Sh. dysenteriae other than Type 2 (Shiga or Sachs) — + — P= fon + + + (—)t Alkalescens-dispar group (Sh. alkales- cens) + —_ — Vv = =f pin) = S. typhosa ot = = I — + + (=) + (—) Salmonella other than S. typhosa * V =variable. + (—)=occasionally negative. 862 TVNILSILNIOCYLSVSD SNOILD3ANI GASTROINTESTINAL INFECTIONS 299 of the medium. As a control, always incubate and test at the same time one tube of uninoculated medium ; one inoculated with an indologenic culture such as E. coli; and one with a nonindologenic culture such as S. typhosa. Record no indole formed only when this is indicated in a culture incubated for at least 4 days. 5. Test for fermentation of carbohydrates—Inoculate culture medium (CM No. 1 or CM No. 3) containing an indicator and carbohydrates, preferably in tightly stoppered tubes, and observe for at least 3 weeks unless acid is produced sooner, Tests for production of acid from mannitol, xylose and rhamnose are most helpful (see Table 3). Slow fermentation of lactose and sucrose may have to be determined, as in the case of Sh. sommei and certain strains of Escherichia and other lactose-fermenting species. Determination of acid production in medium containing salicin, sorbitol and dulcitol may also be helpful. If gas production is demonstrated in triple sugar iron agar, no further tests for this property are necessary. When this is question- able, however, inoculate glucose broth in a fermentation tube. Tests for gas production from other carbohydrates are seldom necessary. 6. Liquefaction of gelatin—Inoculate extract gelatin (CM No. 13) by stabbing to the bottom of the tube. Incubate at room temperature or at 35° C for at least 2 weeks unless liquefaction is demonstrated sooner. Refrigerate for 30 min before recording liquefaction. 7. Other tests—Consult Edwards and Ewing® on the criteria for identification of E. coli and the group designated as Klebsiella- Aerobacter, Arizona, Bethesda-Ballerup, or Providence. Tests that may be helpful are (1) growth in KCN medium,’* (2) methyl red,’? (3) Voges-Proskauer,'? (4) ninhydrin,'® and (5) phenylala- nine.14 Serological typing—The sera required for identifying and typing the most commonly encountered Salmonella and Shigella are available commercially. It may be possible to obtain others from a central laboratory. Directions for the preparation and standardization of such sera are outlined in detail by Edwards and Ewing.® Slide agglutination test: Follow the directions that accompany the sera; also see the section in this chapter under Heading C (Isolation and Presumptive Identification), “Presumptive Serological Tests.” Include with each test controls consisting of a known culture with known serum and the unknown culture with serum from a normal animal of the same species as that in which the agglutinating serum was produced. 300 GASTROINTESTINAL INFECTIONS Tube agglutination test: Follow the directions that accompany the serum. If directions are not available, determine the specific dilutions of each serum by testing with known strains of homologous and heterologous species of microorganisms. Adjust the turbidity of sus- pensions of the microorganisms to that of McFarland Turbidity Standard No. 3'® or its equivalent. Combine in small tubes (10-11 mmX78-80 mm) equal volumes (0.3-0.5 ml) of each serum dilution and a suspension of the microorganisms in salt solution. For purposes of control, combine the suspension being tested with (1) salt solution and (2) a low dilution of serum from a normal animal of the same species as that in which the agglutinating serum was produced. Also test the agglutinating serum with a known strain of the homologous species. Incubate the tests at from 48° to 52° C for 15-18 hr and record the reactions. If no agglutination occurs, repeat the test with a heated suspension of the unknown culture (100° C for 30 min), since the presence of thermolabile K antigen may interfere with agglutination of the living microorganisms. On the other hand, heating may result in spontaneous clumping, thus rendering the test unsatisfactory. Interpret results as outlined in the directions accompanying the sera. Reporting of findings—Report the isolation of a species or serotype of Salmonella or Shigella when this has been determined. Report the isolation of a microorganism having properties of Shigella (or Salmonella) that requires further study when this is the case. Considerable time may be required for identification and typing, especially if cultures are sent to a reference laboratory. Report the isolation of species other than Shigella or Salmonella only when the circumstances under which they are found suggest etiological significance. Members of the Alkalescens-dispar group (formerly designated Shigella), for example, are seldom the incitants of infectious processes. Incorporate in the report explanatory information that may be helpful to the physician or health officer. Changes in nomenclature may be especially confusing. Thus in some instances it is advisable to give the older, more familiar names in parenthesis—for example, Sh. dysenteriae Type 1 (Shiga dysentery bacilli) and S. paratyphi (S. paratyphi A, or paratyphoid A bacilli). E. Reference Diagnostic Services Certain tests rarely needed and tests requiring reagents which are difficult to keep are available from a reference laboratory. This service has three major purposes: GASTROINTESTINAL INFECTIONS 301 1. Phage typing of S. typhosa—This examination which has important epidemiological implications is available to public health laboratories through Regional Centers and the National Typing Center at the Communicable Disease Center, USPHS. It is important for each bacteriologist using this service to familiarize himself with the instructions set forth in the report of the Subcommittee on Enteric Phage Typing of the Coordinating Committee on Laboratory Methods, American Public Health Association. This committee report lists all Regional Typing Centers.'® Briefly, it is suggested that colony fishings which yield significant reactions be subcultured immediately upon egg medium (CM No. 72) and the subcultures, after overnight incubation, stored in the refrigerator until the organism has been identified. If it proves to be S. typhosa, then one or more egg medium cultures should be sent without delay to the Regional Center most con- veniently located. For each culture, the following information should be supplied: case or carrier and name; source (feces, blood, bile, gall bladder) ; related previous phage typings; duration of carrier state if known; and epidemiological relationships among cultures in relation to outbreak. The more complete this information, the more meaning- ful will be the annual reports of the Reference Laboratory as to phage types in the United States. 2. Salmonella typing is available in some state public health laboratories and through others may be obtained from the Communi- cable Disease Center Laboratories at Atlanta, Ga. 3. Identification of problem organisms—A major purpose of the Communicable Disease Center Laboratories at Atlanta, Ga., is to receive and examine rare species of bacteria which have been identified presumptively and those presumed to be of significance but which have not been definitely classified. This service is of great value in helping to maintain high standards in enteric bacteriology. Cultures are accepted only when submitted by or through the various state public health laboratories. These state laboratories share the re- sponsibility of assuring the CDC that a critical selection of micro- organisms is being submitted. The central laboratories of the larger states maintain a comparable service for the laboratories within their jurisdiction. F. Serological Examination Agglutination tests—The examination of patients’ blood serum for agglutination with S. typhosa is one of the oldest laboratory pro- cedures. Its value as an aid in diagnosis, however, has declined with 302 GASTROINTESTINAL INFECTIONS (1) general use of the vaccine in certain segments of the population, (2) the decrease in the incidence of typhoid fever, and (3) the recognition of ever-increasing numbers of antigenically related sero- types of Salmonella. As in most serological reactions with patients’ serum, little significance can be attached to the result of an agglutina- tion test with a single specimen. A rise in titer (fourfold or greater) for a specific antigen in a second serum specimen collected 7 to 10 days following the first is usually considered significant. Sera should be tested for both somatic or granular (“O”) and flagellar or floccular (“H”) agglutinative properties. As an aid in diagnosis the former are more significant than the latter, since they generally develop earlier in typhoid fever and disappear more rapidly following administration of typhoid vaccine. Suspensions for the slide test are available commercially, while those for tube tests are not. The former, however, does not provide a satisfactory differentiation of floccular and granular clumping, which can be determined with the tube tests. The testing of patients’ sera for agglutination with Salmonella other than S. typhosa has been limited by the multiplicity of serotypes and their antigenic relationships. Under special circumstances, as in an epidemic, it may be practicable to test for agglutinative properties for the strain of Salmonella isolated in that particular outbreak. Agglutina- tion tests with Shigella have usually been considered impracticable. Recent reports!™1? indicate that the hemagglutination test is a sensi- tive and reliable procedure by which to demonstrate antibodies for Salmonella and Shigella in patients’ sera. The practical indication for purposes of diagnosis remains to be determined. Central and reference laboratories may plan to include these tests in their services. The demonstration of antibodies for the Vi antigenic factor affords a practical and reasonably reliable method of screening large groups of individuals for possible typhoid carriers?’ The hemagglutination test?-24 has proved more reliable and sensitive for this purpose than the procedures recommended earlier.202> Serum specimens should be sent to central or reference laboratories for this test. When Vi agglutinative properties are demonstrated, even in very low dilutions of the serum, a series of fecal specimens should be examined before ruling out the carrier state. The possibility must be kept in mind of a hidden chronic infection from which the typhoid bacilli are rarely excreted. 1. Preparation of specimen—Allow blood drawn aseptically to clot at room temperature. Separate the serum from the cells by centrifuga- tion and refrigerate until used in the test. The clot may be used for GASTROINTESTINAL INFECTIONS 303 culture if the specimen is taken during the acute phase of the disease. Chylous or markedly hemolyzed sera should be considered unsatis- factory, as these interfere with the reading of agglutination. (See Chapter 1, “General Procedures.”) 2. Suspensions—Satisfactory suspensions for the rapid slide- agglutination tests are available commercially. These are usually pre- pared from killed suspensions of smooth cultures by the methods of Welch or Diamond. 26:27 For the tube-agglutination test, employ carefully standardized sus- pensions for demonstrating granular and floccular agglutination.?® Sera for control—Use human or rabbit sera of known ag- glutination titers for purposes of control. Test each new lot of sus- pension for sensitivity. Suspensions may be standardized for the slide test by the method of Diamond?” and Huddleson.2® Test procedure—Slide-agglutination test: The equipment includes 3X2 in. microscopic slides with six paraffin wax rings about 1 cm in diameter ; slide holders that will accommodate three of these slides and a mechanical rotator that can be set at approximately 175 rpm (this equipment is commonly used in the microflocculation tests for syphilis). Pipette 0.04 ml of serum No. 1 into the first wax ring on each of two slides. Pipette serum No. 2 into the second depressions and continue until each slide bears 0.04 ml of serum of 10 different specimens. Add 0.03 ml of S. typhosa somatic (“O”) antigen to each specimen on slide No. 1 and 0.03 ml of flagellar (“H”) antigen to each specimen on slide No. 2. If suspensions of other Salmonellae are tested, follow the same procedure. Place the slides on the mechanical rotator and rotate for 4 min. Read over a lighted viewing box. By using 0.04 ml of serum with 0.03 ml of antigen, the test is comparable to a 1:40 dilution by the tube method. Any specimen giving a 2 plus or stronger reaction is then checked for possible higher titer. This is done by using smaller amounts of serum with 0.03 ml of antigen, as described by Huddleson.?® For example, 0.02 ml of serum is equivalent to a 1:80 dilution, 0.01 ml to a 1:160 dilution, and 0.005 ml of serum to a 1:320 dilution. Higher dilutions are obtained by diluting the specimen with serum known to contain no agglutinative properties and repeating the test as above. Tube agglutination test: A tube test, using appropriate antigen, is an alternative elective procedure. Combine equal volumes (0.3-0.5 ml) of serum dilutions (1:10 and greater in geometric progression) and somatic and flagellar suspensions standardized for this purpose in small tubes (10-12 mm<75-80 mm). Incubate at 50° to 55° C over- 304 GASTROINTESTINAL INFECTIONS night and record the agglutination. Always include one tube of known agglutinating serum and one of nonagglutinating serum with each sus- pension. Il. ENTEROPATHOGENIC E. COLI IN INFANTILE DIARRHEAL DISEASE It is now widely accepted that certain serogroups of E. coli (entero- pathogenic*) are among the etiologic agents of infantile diarrhea, particularly that of the newborn. The outstanding serogroups are E. coli 111:B4, 55:B5 and 127 :B8F which have been isolated from sporadic cases and institutional outbreaks of this disease in many countries throughout the world. These serogroups have been par- ticularly prevalent in epidemics involving newborn infants in nurseries and are but rarely encountered in healthy infants, children, and adults who have not been exposed to the disease. Corroborative evidence of the pathogenicity of these organisms has been provided by a series of feeding experiments in adults®’-3* and in a single observa- tion in an infant.3%:36 As the search for additional etiological agents has progressed, sero- groups of E. coli other than 111, 55, and 127 have been found associated with infantile diarrhea. E. coli 26 :B67 has been isolated in a sufficient number of sporadic cases of diarrheal disease to be reckoned the fourth etiologically important serogroup of E. coli. The proved and suspected enteropathogenic E. coli serotypes are listed in Table 4. Appraisal of the significance of additional serogroups as causative agents of infantile diarrhea must await further study. The above-mentioned organisms represent but a small fraction of the more than 140 serogroups of E. coli known thus far. The procedures outlined below, which aid in the etiological diagnosis of E. coli diarrheal disease of infants, are concerned solely with E. coli serogroups 111, 55, 26, and 127. A. Classification and Description On the basis of morphological, cultural and physiological charac- teristics, enteropathogenic coliforms are members of the species Escherichia coli. Like the other members of this species, they are Gram-negative rods; they rapidly ferment lactose and glucose, with * The term “enteropathogenic” is suggested for those serogroups of E. coli which are known to be responsible for diarrheal disease of infants, in order to differentiate them from others responsible for appendicitis, peritonitis, urinary tract infections, etc. For the sake of simplicity, abbreviated here as E. coli 111, E. coli 55, E. coli 127, and E. coli 26. GASTROINTESTINAL INFECTIONS 305 Table 4—Serotypes of Enteropathogenic E. Coli Antigens Designation Synonyms 0 Old New H E. coli 026 26 B6 60 (-) 026 :B6 Type E893 11 E. coli 026:B6 32 055:B5 B. coli, beta type 55 BS 59 (— 086:B7 Type E990 86 B7 61 (— B. colt neapolitanum 111 B4 58 (=) E. coli-gomez 2 B. coli, alpha type 4 Type D433 6 0111:B4 0112:B11 S. guanabara 112a B11 66 (=) 112¢ 0119:B14 { a 537-52 119 B14 69 4 306 GASTROINTESTINAL INFECTIONS Table 4 (continued) Antigens K Designation Synonyms 0 Old New H 0124:B17 411 124 B17 72 (=) 16 19 30 32 HX1 0125:B15 Canioni 125a B15 70 6 125b 11 19 An 25 0126 :B16 Type Eoll 126 B16 71 (=) 2 27 0127 :B8 Holcomb 127 B8 63 (=) 0128:B12 Cigleris 128 B12 67 (=) 2 8 9 10 12 Source: Adapted from Ewing et al.37 by permission of author and publisher. production of acid and visible gas; they produce indole; they are methyl red-positive, do not produce acetylmethylcarbinol, and do not utilize citrate as the sole source of carbon. On the basis of differences in other biochemical activities, fermentative types (biotypes) are found within serogroup 111 (see Table 5). Most enteropathogenic serogroups are nonhemolytic on sheep or horse blood agar, while strains of serogroup 26 may or may not hemo- lyze blood. Colonies of E. coli 111 on blood agar are pearl gray, have an even margin, are slightly raised, frequently of smaller diameter than those of “normal” E. coli strains, and usually are slightly tena- cious when touched with a wire. Characteristically these colonies re- semble Shigella cultures on blood agar, inasmuch as they are an even gray color and often have an odor which has been described as semi- nal. Colonies of serogroups 55, 26 and 127 have little to distinguish them from “normal” E. coli colonies. GASTROINTESTINAL INFECTIONS 307 Table 5—Biotypes of E. Coli Serogroup 111:B4 Authors Charter and Type Kauffmann? Le Minor38 Taylor? E. coli 111:B4 Sucrose + == Salicin +d — Sorbitol }- + Sorbose — on E. coli 111:B4:2 Sucrose + Salicin +d +d Sorbitol + Sorbose — E. coli 111:B4:12 Sucrose er Salicin he Sorbitol ge Sorbose pu | ++) E. coli 111:B4:21 Sucrose Salicin Sorbitol Sorbose +41 + d=delayed. Note: Complete identification of serogroups 111, 55, 26 and 127 of E. coli requires full biochemical tests together with serology with OB and O antisera. It will be found that, with practice, quite accurate results can be obtained with OB sera by slide agglutination. As discussed in the following under the heading, “Nomenclature,” enteropathogenic E. coli strains contain somatic or O antigens as well as surface or B antigens. Furthermore, motile strains contain flagellar antigens. For detailed information on the antigens and serology of E. coli, the reader is referred to Kauffmann.” B. Nomenclature The antigenic formulas of the Kauffmann-Knipschildt-Vahlne schema for E. coli are now generally accepted as the designations of choice. In this schema the first arabic number refers to the particular thermostable somatic (O) antigen (111, 55, 26, etc.). The letter B, designating the kind of surface or envelope antigen, is given a specific number (B4, B5, B6). If an H or a thermolabile flagellar antigen 308 GASTROINTESTINAL INFECTIONS exists, it is designated by an arabic numeral. The known combinations of O, B, and H antigens of enteropathogenic E. coli are listed in Table 4. C. Preparation of Diagnostic Antisera and Technics of Agglutination Tests Diagnostic E. coli antisera are available from commercial and other sources. The following brief outline of methods is offered as a practi- cal guide for those who wish to prepare their own sera. For a detailed discussion of antigens and methods the reader is referred to Edwards and Ewing.® OB antisera—Antigens for production of OB antisera should preferably be living, nonmotile organisms*® grown for 18-24 hr in broth or on infusion (veal, heart, beef) agar plates. Suspension of the latter antigen is made in formalinized (0.5%) physiological (0.85%) salt solution and is standardized to an approximate turbidity of a No. 3 barium sulfate or equivalent standard. A suspension of freshly grown organisms should be prepared for each inoculation. The following dosages in the order given are injected intravenously into a rabbit over a period of 21-24 days: 0.25 ml, 0.25 ml, 0.5 ml, 0.5 ml, 0.5 ml, 1.0 ml, 1.0 ml, 2.0 ml, 2 ml. The animal is bled 3 to 5 days after the final inoculation. OB antiserum should be titrated with an agar-grown homologous culture by using both tube- and slide-agglutination technics. In the former method, formalinized suspensions of the organisms are com- bined with serial dilutions of serum and incubation is carried out at 35° C for 2 hr. The tubes are allowed to stand overnight in the re- frigerator (+5° to +10° C) before reading. Complete agglutination is characteristically in the form of a web. A serum titer of 1:320 in the tube is a satisfactory potency for an OB serum. An antiserum found satisfactory by this method should be diluted 1:10 and tested by slide-agglutination technic with living organisms of serogroups 111, 55, etc. Agglutination should be rapid and complete using the homologous culture, with no apparent reaction greater than a weak agglutination using heterologous culture. Strongly cross-reacting anti- bodies should be removed by absorption. Polyvalent OB sera—It is convenient to combine single OB sera into two or three pools of polyvalent OB sera. On the basis of incidence of the particular organisms in disease, OB sera for sero- groups 26, 55, 111 and 127 are usually combined into a single pool. OB sera for the remaining serogroups may be combined into one or GASTROINTESTINAL INFECTIONS 309 two additional pools, according to the strength of the sera, as de- termined by slide titrations with living homologous cultures. Usually a mixture of four specific antisera, each in a final dilution of 1:10, will provide a satisfactory polyvalent serum. O antisera—Antigens for O antiserum production are either heated agar-grown or heated broth-culture organisms. A convenient method is to grow the culture for approximately 16 hr in infusion broth. The broth suspension is heated in water or steam at 100° C for 2.5 hr to destroy the B antigen, then cooled, and formalin added to a final concentration of 0.5 per cent. Such a suspension is stable for at least one month. The schedule of immunization given for production of OB antisera will be satisfactory also for O antiserum production. No adjustment of antigen turbidity is necessary. O antisera can be titrated with the same antigen suspension as that used for immunization. In the event of excessive turbidity, the antigen should be adjusted against a nephelometric standard equivalent to a No. 3 barium sulfate standard. A considerable range of antigen turbidity will give approximately the same end point in titration of an O antiserum. Titrations should be made by the tube-dilution method, and incubation should be carried out for 18-20 hr at approxi- mately 50° C. A satisfactory titer for an O antiserum is 1:5120. H antisera—Unless extensive investigations are planned, it is not advisable to attempt identification of H antigens with specific anti- sera. E. coli strains are often sluggishly motile and require consider- able manipulation before they become suitable H antigens, either for immunization or identification. For details, see Kauffmann? and Edwards and Ewing.® D. Collection and Handling of Specimens 1. Feces—Bacteriological examination of the feces is the most important of the laboratory procedures. For optimal results the specimen should be cultured shortly after it has been passed; alternatively, it may be procured by means of a rectal swab and cultured immediately. Serogroups 111, 55, 26 and 127 survive in buffered 30 per cent glycerol solution (CM No. 71) for several days during shipment,** but exact data are not available as to percentile survival in this medium. It must be emphasized that this bacteriologi- cal examination should be undertaken as early as possible during the illness, since in the early phase of the disease the serogroups of E. coli under discussion are frequently present as the predominant or even sole coliform organism. : 310 GASTROINTESTINAL INFECTIONS 2. Throat and nasopharyngeal swabs—It has been demon- strated that the enteropathogenic E. coli strains are rather frequently found in the throat and/or nasopharynx of patients in the presence or absence of vomiting.® No information is available concerning survival of these pathogens on swabs under conditions of shipment. 3. Blood—There is no evidence that the four serogroups invade the bloodstream during the diarrheal disease; for this reason blood cultures are not indicated as a routine procedure for the recovery of enteropathogenic E. coli. Secondary invasion of the bloodstream with bacteria other than E. coli, however, may occur as a complica- tion of the disease. E. Bacteriological Examination 1. Isolation media—Blood agar plates (infusion or extract) and a differential culture medium—Endo( CM No. 55), MacConkey (CM No. 60), or eosin-methylene blue agar (CM No. 54)—are the media of choice for recovery of the serogroups of E. coli under discussion. Selective culture media, such as SS agar (CM No. 59), or desoxycholate citrate agar (CM No. 57), suppress in varying degree the pathogenic E. coli strains. Note that such a selective culture medium as SS agar should always be included in the bacteriological examination of fecal specimens for the demonstration of Salmonella or Shigella and it is useful also for the isolation of some strains of the E. coli under consideration. Every effort must be made not to overlook other pathogens in the search for enteropathogenic E. coli. 2. Isolation and identification—(a) Seeding of culture media: Regardless of the type of specimen—rectal swab, feces recently collected or feces in glycerol solution—inoculation of blood agar, differential plating media and selective media should be made as soon as the specimen reaches the laboratory. If the specimen has been collected by swab, streak approximately one-fifth the area of each of two blood plates—one MacConkey plate and the whole of an SS or desoxycholate plate—with the swab. With a sterile straight wire distribute the material from inoculated areas to obtain isolated colonies. If the specimen is feces, transfer material to the plates with a sterile cotton swab and distribute with an inoculating needle. Incubate the seeded culture media for 18 to 24 hr at 35° C. 2(b). Presumptive identification of E. coli: For the presumptive identification, an agglutination test with appropriate antisera and isolated colonies is carried out. The following antisera are used: GASTROINTESTINAL INFECTIONS 311 (1) polyvalent OB antisera A, B, etc., covering all serogroups; (2) group-specific OB antisera. Examination of colonies on blood agar usually yields the best results. If, however, the blood plate is overgrown by a spreader, efforts should be concentrated on the MacConkey agar plate. As a preliminary, pick at least 5 isolated coliform colonies and rub each into approximately 0.03 ml of each polyvalent OB antiserum deposited either on a microscopic slide or in a well slide.* Optimal turbidity can be regulated only with practice. If a microscopic slide is used, the serum drop should be distributed in a film approximately 5 mm wide by 15 mm long, placed parallel to the ends of the slide. Rock the slide to facilitate mixing and observe against suitable illumination, such as a slit lamp or a fluorescent lamp with frosted glass. In case of agglutination, reexamine the colony, if possible, giving this reaction by means of the indicated individual OB antisera. A rapid, complete agglutination is provisional evidence of group identity. In the event that isolated colonies do not agglutinate with poly- valent OB sera, material from the mass growth should be tested. When any degree of agglutination occurs, transfer from the serum- bacterial mixture an agglutinated mass of organisms, by loop, to a fresh blood or MacConkey plate. Streak out to obtain isolated colonies and reexamine after 18-24 hr. 2(c). Final identification of E. coli: Single colonies which ag- glutinate in group-specific OB antiserum have to be identified further; also, such colonies are not necessarily pure. Fish selected colonies from the primary isolation medium to new agar plates. Then select from the purification plate one or more single colonies for final identification on the basis of morphological characteristics, biochemical activities, and antigenic structure, Test these colonies in group-specific OB antiserum. If strong agglutination occurs, transfer some of the particular colony to (1) a double (or triple) sugar or a plain agar slant, (2) Endo or MacConkey agar, (3) 20 ml of broth in a tube or small flask, and (4) an agar stab or other suitable medium. The growth on (1) and (2) will serve to identify the isolate as E. coli and also for inoculation of carbohydrates and other test media. Gram stain examination should also be carried out. The growth in broth (3) should be heated in boiling water or steam for 30 min, cooled, and formalin added to a concentration of 0.5 per cent. This suspension serves as antigen in tests with group-specific O antisera. Most strains are agglutinated to approximately the same end point of * Perma-slides, Certified Blood Donor Service, 146-16 Hillside Avenue, Jamaica 35, N. Y. 312 GASTROINTESTINAL INFECTIONS the homologous antiserum as the strain used for immunization; an occasional motile culture will fail to agglutinate to more than partial titer of the antiserum. In the latter case, only absorption tests will give complete evidence of strain identity. Coons’ fluorescent antibody technic has been employed recently for the rapid identification of enteropathogenic E. coli in fecal speci- mens.*? The method deserves further exploration in order to establish its value and limitations for routine purposes. In research laboratories strains of E. coli of the four serogroups under discussion may be divided into phage types by means of suit- able bacteriophages.*® Both the determination of H antigens and phage types may have epidemiological significance. Serogroup 111 may be divided also into biotypes on the basis of fermentation of sucrose, salicin, sorbitol and sorbose. The correlation of biotypes with H antigens, according to different workers, is given in Table 5. F. Serological Diagnosis At the present time a sensitive serological test for the demonstration of group-specific E. coli antibodies and of a rise in titer of these antibodies in the sera of patients with diarrheal disease is not yet available for routine purposes. G. Evaluation and Reporting of Results When an enteropathogenic E. coli organism has been identified, first on a presumptive basis and later after final examination, reports should be submitted to the clinician at once. The finding of an entero- pathogenic E. coli does not necessarily indicate that this micro- organism is directly related to the disease of the patient ; the individual may be a carrier. It cannot be too strongly emphasized that in the search for special serogroups of E. coli, every effort must be made simultaneously to determine whether other pathogens, such as Salmonella and Shigella, are present in the specimen. The possibility must also be kept in mind that a mixed infection, including viral agents, may exist. : Information on the presence of enteropathogenic E. coli is im- portant not only for the management of the individual patient but also from an epidemiological point of view. So far as the individual patient is concerned, specific therapy may be considered: Sulfona- mides, aureomycin, terramycin, chloramphenicol and neomycin have been shown to be effective in vitro against enteropathogenic E. coli; neomycin and other antibacterial drugs have been used with success both therapeutically and prophylactically. The laboratory may aid GASTROINTESTINAL INFECTIONS 313 the clinician by determining the in witro sensitivity of the isolated strain to antibacterial agents, including antibiotics. From an epi- demiological point of view, the clinician must be aware of the fact that enteropathogenic E. coli and the associated disease may be spread to other infants in a nursery or ward, and every effort must be made to prevent the spread of this infection. For a discussion of the present status and the unsolved problems of enteropathogenic E. coli enteritis and a list of references, the interested reader is referred to a recent review article. lll. THE CHOLERA VIBRIO* (Vibrio comma, Vibrio cholerae) The problem of isolating and identifying the cholera vibrio is presented very infrequently to laboratories in the continental United States. However, with increasing rapidity of transportation the possibility of the infection bypassing quarantine measures which have hitherto been successful should be considered, particularly in connec- tion with modern air transport. Cholera is constantly active in India and China; from these foci it is always capable of spread to the rest of the world. In 1947 the disease extended to Egypt and Syria. Spread may be by contact with cases and convalescent carriers or their belong- ings, and contaminated water supplies may be a means of transmis- sion of epidemic disease. It is probable that recent contamination of water is necessary for spread of the disease, since the vibrios do not persist for extended periods. There is no chronic carrier state which maintains the disease in endemic. form, but some evidence suggests that individuals may carry the vibrios during an epidemic period for 1 to 2 weeks before the appearance of symptoms, or the disease may be aborted. The maximum period of excretion of vibrios by convalescents has been found to be 3 to 4 weeks in the occasional individual, but 90 per cent of convalescents are free of infection in a week to 10 days. The presentation to be given here is largely for the use of labora- tories which are only rarely called upon for the isolation and identifi- cation of Vibrio comma and which must act promptly with the materials at hand. A. Description and Classification The classification of I. comma is based on morphological, bio- chemical and serological criteria, all of which are of importance, * The disease and the causative microorganism are exhaustively reviewed by Pollitzer.45 314 GASTROINTESTINAL INFECTIONS with the biochemical criteria exhibiting anomalous behavior somewhat more frequently than the other attributes of the organism. The vibrios are Gram-negative slightly curved rods 2-4 x in length and about 0.5 u in width; roughly in the same size range as the Salmonellae, Many forms will appear perfectly straight; occasionally two or more curved forms will be attached end to end, forming an S or spiral shape. They are motile, with a single polar flagellum. No spores are formed and capsules have not been described. I. comma is much more susceptible to chemical disinfectants than are the other enteric pathogens. Sus- pensions of the organism undergo lysis in the presence of phenol or chloroform, and as a result of heating or repeated freezing and thaw- ing. However, growth occurs in the presence of an alkalinity (pH 9.5) which is inhibitory to most of the enteric organisms. V. comma is predominantly aerobic although a small amount of anaerobic proliferation occurs. On the surface of nutrient agar, growth is luxuriant; in nutrient broth it is delicate and a pellicle is usually developed in 24 hr. Lactose fermentation is usually late but may not occur at all. With few exceptions, glucose, maltose, mannitol and sucrose are fermented without gas formation; dulcitol and arabinose are not acted upon. Gelatin is liquefied. Indole is formed and nitrate is reduced to nitrite; these two simultaneous activities being responsible for the “cholera red” reaction which is elicited by the addition of a small amount of concentrated sulfuric acid to a pep- tone water culture. The cholera red test is of limited diagnostic value, since some nonpathogenic vibrios show it, Moreover, certain batches of peptone are unsuitable, and a negative reaction is therefore un- reliable unless the particular medium gives a positive reaction with known strains of the vibrios. Much attention has been paid to the hemolytic activity of V. comma. Most strains produce clearing of the medium around colonies on sheep or horse blood agar, but this is said to be due to hemodigestion rather than to the effect of a soluble toxin. The Greig test, performed by mixing equal volumes of a broth culture and a suspension of goat or sheep erythrocytes (5%), is usually negative (no hemolysis after 2 to 4 hr at 35° C) with the true cholera vibrios. Greig-positive strains, which appear to be identical with true cholera vibrios in every other respect, including antigenic composition, have been observed in Celebes and once or twice in Bengal, in apparently causative relation to epidemic diarrheal disease indistinguishable from cholera. The status of these hemolytic (El Tor and Celebes) vibrios is not yet fully established. GASTROINTESTINAL INFECTIONS 315 The serological classification of the cholera and cholera-like vibrios is based upon the specificity of the heat-stable O antigens; the H antigenic complex is nonspecific in that common factors are shared by a wide variety of vibrios. The cholera vibrio, and some of the El Tor types including the Celebes vibrios, are designated O group I, while water and other vibrios fall into other distinct and apparently unrelated O groups. O group I vibrios contain a common antigen designated A and are subdivided into the Ogawa and Inaba serotypes represented by antigenic structures AB and AC respectively.*® A more complex antigenic structure has been described but is not relevant to serological identification and typing. An AB (Ogawa) antiserum or an AC (Inaba) antiserum, or a mixture of the two, may be used for identification ; the serotypes cross-react to within one or two dilutions. Strains may be typed, using an Inaba antiserum absorbed with Ogawa antigen, and vice versa, but serotype apparently has no epidemiological or other significance. By the 3rd day of the disease, R forms may be encountered which are substantially identical in colonial appearance, though not necessarily in consistency and which are spontaneously ag- glutinable in salt solution; it is therefore essential that saline controls be run in serological identification of the vibrios. B. Laboratory Procedures* Isolation and identification—In acute cases of cholera the direct microscopic examination of carbolfuchsin-stained films of the in- testinal dejecta is of little or no value, since the line-up of curved organisms in parallel arrangement as described in textbooks is seldom observed. Because of the delicacy of the organism, cultivation pro- cedures should be carried out as promptly as possible, although storage of stool specimens overnight in the refrigerator is permissible. Specimens taken with a rectal swab or catheter are preferred. In the rice water stool the vibrios occur in enormous numbers and in prac- tically pure culture. VV. comma ferments lactose slowly or not at all. Its growth is inhibited on Salmonella-Shigella (CM No. 59) and eosin- methylene blue (CM No. 54) plating media. The usual plating medium for isolation consists of extract agar (CM No. 4) containing 0.5 per cent NaCl and 0.5 per cent sodium taurocholate and adjusted to pH 8. Colonies of VV. comma on this bile salt medium resemble those of Salmonella or Shigella in form and size except that they are almost completely transparent; colonies showing a faint haze usually fail to agglutinate in anticholera sera. Portions of suspected colonies are * For the internationally accepted procedures see Burrows and Pollitzer.47 316 GASTROINTESTINAL INFECTIONS rubbed up on a slide in small pools of specific serum of the proper dilution (see Section A preceding) and agglutination is demonstrated after rocking the slide. Normal serum and salt solution controls must be included. Colonies showing agglutination are examined micro- scopically for vibrio morphology and staining, transferred to nutrient agar slants, and incubated for 24 hr at 35° C; the resulting growth is transferred to alkaline extract broth (pH 9.2) and to the proper carbohydrate media, namely, sucrose and mannitol (acid without gas), and arabinose (negative). After inoculating the carbohydrate media, the 24 hr agar slant culture is washed down with 4 to 5 ml of salt solution; the resulting suspension is used to determine the highest reactive dilution of the specific agglutinating serum against this organism. The 24 hr alkaline broth culture is used to determine hemolytic activity of the organism by the following procedure : One ml of the broth culture is mixed with 1 ml of a 5 per cent suspension of sheep erythrocytes (sheep erythrocytes are equal or superior to goat erythrocytes), incubated for 2 hr at 35° C and stored overnight in the refrigerator. The tubes are examined for hemolysis after 2 hr and again the following morning. The test should not be reported as nega- tive before the second reading, although both readings commonly agree. In summary, positive identification of VV. comma requires the demonstration of Gram-negative, curved or slightly curved rods which ferment sucrose and mannitol but not arabinose, fail to hemolyze sheep red blood cells, and agglutinate in specific group I, O antiserum. Retrospective diagnosis on the basis of agglutinative properties in convalescent serum is almost completely unreliable. AvLsert V. Haroy, M.D., Dr.P.H., Chapter Chairman Arcor S. BrowNE, M.D. WiLLiaAM Burrows, PH.D. Marion B. CoLEMAN WiLriam W. Fercuson, Pu.D. Pear. KENDRICK, Sc.D. Erwin NEeTER, M.D. C. B. Seastong, M.D. REFERENCES 1. Harpy, A. V., WATT, J., and DeCArrr0, T. Studies of the Acute Diarrheal Diseases. VI. New Procedures in Laboratory Diagnosis. Pub. Health Rep. 57:521-524, 1942. 2. Harpy, A. V.; MackeL, D.; Frazier, D.; and HaMmEerick, D. The Relative Efficacy of Cultures for Shigella. U. S. Armed Forces M. J. 4:393-394, 1953. 3. ForsBeck, F. G., and Horron, G. C. Standards for Determining Suitability of Bile Specimens for Detection or Release of Typhoid Carriers. A.J.P.H. 27 :253-260, 1937. GASTROINTESTINAL INFECTIONS 317 4. 10. 11 2 13. 14. 15 16. 17. 18. 19. 20. 21. 22. 23, 24. 25. Garton, M. M., Haroy, A. V., and MrrcueLL, R. B. The Public Health Laboratory Diagnosis of Enteric Infections. Am. J. Trop. Med. 30:77-90, 1950. Medical and Public Health Laboratory Methods (James Stevens Simmons and Cleon J. Gentzkow, Eds.). Philadelphia: Lea & Febiger, 1955, pp. 625-626. Arnorp, W. M,, Jr, and WEAVER, R. H. Quick Microtechniques for the Identification of Cultures. J. Lab. & Clin. Med. 33:1334-1341, 1948. KaurrMaNN, F. Enterobacteriaceae (3rd ed.). Copenhagen: Munksgaard, 1961. Epwarps, P. R. and Ewing, W. H. Identification of Enterobacteriaceae (2nd ed.). Minneapolis, Minn. : Burgess Publishing Co., 1962. CoLEMAN, M. B., WiLson, M. W., and SickinGer, C. M. Serotypes of Salmonella in New York State Outside New York City. J. Infect. Dis. 104 :207-212, 1959. CoLEMAN, M. B,, et al. Annual Report of the Division of Laboratories and Research, New York State Department of Health, 1958, p. 91. MoELLER, V. Diagnostic Use of the Braun KCN Test within Entero- bacteriaceae. Acta. path. et microbiol. scandinav. 34:115-126, 1954. Standard Methods for the Examination of Water, Sewage, and Industrial Wastes (10th ed.). American Public Health Assn., American Water Works Assn, and the Federation of Sewage and Industrial Wastes Assn. New York: APHA, 1955, p. 392. Carrquist, P. A Biochemical Test for Separating Paracolon Groups. J. Bact. 71:339, 1956. Ewing, W. H., Davis, B. R, and Reavis, R. W. Phenylalanine and Malonate Media and Their Use in Bacteriology. Pub. Health Lab. 15(6) :153-167, 1957. McFarLaND, J. The Nephelometer: An Instrument for Estimating the Number of Bacteria in Suspensions Used for Calculating the Opsonic Index for Vaccines. J.A.M.A. 49:1176-1178, 1907. Regional Laboratories for Bacteriophage Typing of Salmonella typhosa. Pub. Health Lab. 8:91-93, 1950. NETER, E, et al. Enterobacterial Hemagglutination Test and Its Diagnostic Potentialities. Canad. J. Microbiol. 2:232-244, 1956. NETER, E., and WALKER, J. Hemagglutination Test for Specific Antibodies in Dysentery Caused by Shigella sonnei. Am. J. Clin. Path. 24:1424-1429, 1954. NETER, E.; DrRISLANE, A. M.; Harris, A. H.; and Jansen, G. T. Diagnosis of Clinical and Subclinical Salmonellosis by Means of a Serologic Hemagglutination Test. New England J. Med. 261:1162-1165, 1959. FeLix, A. Laboratory Control of Enteric Fevers. Brit. M. Bull. 7(3) :153- 162, 1951. SpauN, J. On the Determination of Vi-Antibodies by Hemagglutination. Acta path. et microbiol. scandinav. 29:416-418, 1951. Staak, H. H, and Sraun, J. Serological Diagnosis of Chronic Typhoid Carriers by Vi-Hemagglutination. Acta path. et microbiol. scandinav. 32:420-423, 1953. Suusert, J. H.,, Epwarps, P. R., and Ramsey, C. H. Detection of Typhoid Carriers by Agglutination Tests. J. Bact. 77 :648, 1959. CoLemMAN, M. B.; WiLson, M. S.; SickINGer, C. M.; and ALLISON, E. In Annual Report of the Division of Laboratories and Research, New York State Dept. Health, 1955, p. 88. SAINT-MARTIN, M. and DesraNLEAU, J. M. Results Obtained with a Glycerolated Vi-Antigen in the Detection of Chronic Typhoid Carriers. A.]J.P.H. 41:687-692, 1951. 318 GASTROINTESTINAL INFECTIONS 26. WeLcH, H., and Stuart, C. A. A Rapid Slide Test for the Serological Diagnosis of Typhoid and Paratyphoid Fevers. J. Lab. & Clin. Med. 21:411, 1936. 27. Diamonp, B. E. The Preparation of Rapid Antigens for the Diagnosis of Typhoid and Paratyphoid Fevers. Pub. Health Lab. 6(4) :74-77, 1948. 28. Diagnostic Procedures and Reagents (3rd ed.). New York: American Public Health Assn., 1950, pp. 220-222. 29. Huppreson, I. F. Brucellosis in Man and Animals. New York: Common- wealth Fund, 1943. 30. Kimry, A. C., Harr, E. G.,, and CoackrLey, W. Neonatal Diarrhea and Vomiting. Outbreaks in the Same Maternity Unit. Lancet 2:201-207, 1950. 31. Braun, O. H. and Hencker, H. Sauglingsenteritis durch Pathogene Colitypen Insebesondere E. colt 55/Bs. Ztschr. Kinderh. 70:273-285, 1952. 32. Ferguson, W. W, and June, R. C. Experiments on Feeding Adult Volunteers with Escherichia coli 111, Bs, a Coliform Organism Associated with Infant Diarrhea. Am. J. Hyg. 55:155-169, 1952. 33. Jung, R. C,, Fercuson, W. W., and WorreL, M. T. Experiments in Feed- ing Adult Volunteers with Escherichia coli 55, Bs, a Coliform Organism Associated with Infant Diarrhrea. Am. J. Hyg. 57:222-236, 1953. 34. WentworTH, F. H.; Brock, D. W.; Stureerg, C. S.; and Pace, R. H. Clinical, Bacteriological and Serological Observations of Two Human Volunteers Following Ingestion of Escherichia coli 0127:B8. Proc. Soc. Exper. Biol. & Med. 91 :586-588, 1956. 35. Nerer, E, and SHumMwaAy, C. N. E. coli Serotype D,g4: Occurrence in Intestinal and Respiratory Tract, Cultural Characteristics, Pathogenicity, Sensitivity to Antibiotics. Proc. Soc. Exper. Biol. & Med. 75 :504-507, 1950. 36. NETER, E., and Wess, C. R. Study on Etiological Role of Certain Serotypes of Escherichia coli and Effects of Antibiotic Therapy in Infantile Diarrhea. Exper. Med. & Surg. 9:385-388, 1951. 37. Ewing, W. H., Tatum, H. W, and Davis, B. R. The Occurrence of Escherichia coli Serotypes Associated with Diarrheal Disease in the United States. Pub. Health Lab. 15:118-138, 1957. 38. LE Minor, S. Etude bacteriologique d’Escherichia coli Isolés au Cours de Gastroenterites Infantiles.” Doctoral thesis, University of Paris, 1953. 39. CHARTER, R. E., and Tavior, J. Cultural and Serological Reactions of Strains of Bact. coli Isolated from Babies. J. Path. & Bact. 64:729-734, 1952. 40. WricHT, J., and VILLANUEVA, R. The Presence of H Antigen in Escherichia coli K Suspensions. J. Immunol. 72:389-392, 1954. 41. Fercuson, W. W. Unpublished data. 42. WHITAKER, J.; Pace, R. H.; StuLserg, C. S.; and ZurLzer, W. W. Rapid Identification of Enteropathogenic Escherichia coli 0127 :B8 by the Fluores- cent Antibody Technique. Am. J. Dis. Child. 95:1-8, 1958. 43. NicorLg, P.; LEMiNor, L.; Burriaux, R.; and Ducrest, P. Lysotypie des “Escherichia coli” Isole dans les Gastro-Enterites infantiles. II. Fréquence Relative des Types dans Differents Foyers et Valeur Epidemiologique de la Methode. Bull. Acad. nat. med. 26 :483-485, 1952. 44. NEter, E. Enteritis Due to Enteropathogenic Escherichia coli. Present- Day Status and Unsolved Problems. J. Pediat. 55:223-239, 1959. 45. PoLLITZER, R. Cholera. WHO Monograph No. 43, 1959. 46. ————, and Burrows, W. Cholera Studies. 4. Problems in Im- munology. WHO Bull. 12:945-1107, 1955. 47. Burrows, W., and PoLLitzer, R. Laboratory Diagnosis of Cholera. WHO Bull. 18:275-290, 1958. CHAPTER 11 BACTERIAL FOOD POISONING I. Introduction II. Collection of Specimens 111. Botulism A. Test for Toxin in Food B. Culture Tests for Cl. botulinum 1V. Staphylococci V. Streptococci VI. B. cereus VII. Cl. perfringens References I. INTRODUCTION Food poisoning outbreaks may be caused by one of a variety of etiologic agents. Certain bacteria or their metabolic products are the most important of these agents.! A number of metallic compounds may produce poisoning. A few foods are inherently poisonous due to the presence of alkaloids, or to more obscure but perhaps related sub- stances. The phenomenon of food allergy is also encountered. Finally, there are always a number of outbreaks in which no definite causative agent can be ascertained. At times the epidemiological evidence and clinical symptoms may afford important clues, but at other times this information is of little etiological assistance. Since the methods to be presented here deal only with bacteriologi- cal examination, no attempt will be made to outline procedures for those instances in which food is poisonous from other causes. There is ample evidence, however, that at times other agents cause very striking cases or outbreaks which may simulate bacterial food poison- ing. In this category are poisonings due to mushrooms or other fungi, the “milk sickness” following consumption of milk from cattle which have eaten white snakeroot or richweed, some forms of shellfish poisoning due to the presence of a toxic dinoflagellate in the shell- 319 320 BACTERIAL FOOD POISONING fish,2 and other animal or plant poisons conveyed directly or in- directly through food.! When bacterial food poisoning is suspected, the laboratory worker should bear in mind those microorganisms which have been clearly implicated as the cause of previous outbreaks, The bacteria against which the evidence seems definite are the following: 1. Clostridium botulinum, the toxin of which gives rise to clinical symptoms distinct from those produced by the other food poisoning microorganisms. . Members of the Salmonella group. . Enterotoxin-producing strains of staphylococcus. . Streptococcus. * . Clostridium perfringens. . Bacillus cereus. ALN The chief concern must be to detect these species, but the possibility of finding some other significant microorganisms should not be over- looked. The examination of suspected foodstuffs may be complicated by the greatly diversified nature of all the various bacteria which might possibly be involved; yet the laboratory worker is often spared much time when epidemiological and clinical data are available, since such information may narrow the search for the causal agent. Some salient characteristics of various types of bacterial food poisoning are given in Table 1. Questionnaires such as those distributed by the International As- sociation of Milk and Food Sanitarians, Box 437, Shelbyville, Ind., may be helpful. The questionnaire cited is entitled “Procedure for the Investigation of Food-borne Disease Outbreaks” (1957). Il. COLLECTION OF SPECIMENS Samples for laboratory examination must not be collected indis- criminately. Preliminary epidemiological investigation will reduce to a minimum the number of suspected foods in a given outbreak. Evidence concerning the particular foods most likely to be responsible should be sought by listing foods consumed by those made ill. While this does not always give clear-cut evidence, at least a number of foods can usually be eliminated. Some judgment may be called for in deciding which of the foods listed are most likely involved. The time when symptoms first appeared should also be recorded, along with other pertinent facts. * Fecal streptococcus strains belonging to Lancefield’s serological Group D have been implicated in bacterial food poisoning. For more extensive discussion, the reader is referred to Section V of this chapter (pp. 330 through 332). BACTERIAL FOOD POISONING 321 Table 1—Features of Food Poisoning Caused by Bacteria Onset of Symptoms Type of Food Commonly Involved Symptoms and Other Characteristics Botulism Staphylococcus food poisoning Salmonellosis* Group D strep- tococcus food poisoning Cl. perfringens food poisoning B. cereus food poisoning 6 hr to 8 days; average, 12-30 hr 1-6 hr; average, 214-3 hr © 5-72 hr 2-18 hr; usually + 11-15 hr do do Home-canned, low- acid vegetables Processed meat, potato salad, cream-filled bak- ery products, dairy products Poultry and poul- try products, processed meat Ground meats, dressing Reheated meats, meat pies and pastries, cold meats, stews and made-up dishes Foods containing cereal products, e.g., vanilla pudding Difficulty in swallowing, speech and respiration ; double vision. Death from paralysis of muscles of respiration Nausea, vomiting, abdominal cramps, diarrhea and acute prostration, and circulatory collapse in occasional severe cases. Usually no fever. No secondary cases Abdominal pain, diarrhea, chills, fever, frequent vomit- ing and prostration. Secondary cases may occur. Leukocytosis Nausea, seldom vomiting, usually abdominal cramps and diarrhea. Symptoms seldom persist longer r than 8-12 hr. No secondary cases. Fever and prostration absent * Covered in Chapter 10. When food from sealed containers is under suspicion, obtain the original container together with the complete label of the product, whenever possible. Make a record of code marks and any other identifying marks on the container itself. When it is necessary to transfer a representative sample of a suspected food to a smaller container, a sterilized sample bottle should be used. If the samples are of such a nature that they cannot be put into the common sizes of sterile containers, they should be given every possible protection from additional contamination in transit, _ Fecal specimens are often of value if secured early in the acute stage of the disease. Blood samiples are of lesser importance but may 322 BACTERIAL FOOD POISONING be useful in cases of botulism or occasionally in Salmonella infections, particularly where it has been impossible to obtain samples of sus- pected foodstuffs. If necropsy material is available from fatal cases, samples of the stomach contents, as well as portions of the colon, spleen and mesenteric lymph nodes, should be taken. It is important that all samples be collected promptly and that laboratory examination be started without delay. Where specimens must be shipped, arrangements should be made for their refrigeration before and during transit. lll. BOTULISM Caution: Botulinum toxin is extremely dangerous and the greatest precautions should be taken in handling suspected samples. Minute amounts of the appropriate types are lethal to man when ingested. Hence, in handling botulinic food specimens, cultures or filtrates, pipetting must never be done by mouth. Moreover, the possible absorption of toxin through the conjunctiva or broken skin should not be overlooked. Prepare a Gram-stained film directly from the liquid portion of the suspected foodstuff. If the food is solid (e.g., meat or fish) macerate a representative portion in sterile physiological salt solu- tion and examine the suspension after the larger particles have settled out, If the only sample submitted is an empty jar or can, the interior should be thoroughly washed out with a few ml of sterile physiological salt solution or nutrient broth. The washings then can be used for the direct microscopical and cultural examinations, Unused remainders of food samples should be kept refrigerated in case additional tests are needed. A. Test for Toxin in Food The liquor from the foodstuff or original container, or a physiologi- cal salt solution extract of the food, should also be tested for botuli- num toxin, as described below. Where a sufficient quantity of sample and the requisite facilities are at hand, bacterial filtration may be desir- able to eliminate any cells. Otherwise, to lessen contamination and avoid possible loss of toxin from adsorption on the filter, centrifuge the material and use the supernatant for animal inoculation. To avoid accidental breakage, plastic centrifuge tubes are recommended. After preparing the material for animal tests by filtration or centri- fuging, remove a small portion to a sterile test tube and heat for 10 min in a boiling water bath. This serves as a heat-labile toxin control. BACTERIAL FOOD POISONING 323 Portions of the unheated sample and heated control should now be administered intraperitoneally to guinea pigs or white mice. An alter- nate procedure, suitable for guinea pigs only, is to feed portions of the sample from a pipette. This requires a somewhat larger sample and is not as delicate a test for minute amounts of toxin, although it has the advantage of eliminating the occasional infections caused by bacteria in badly spoiled samples which have not previously been filtered. Inject two or more mice intraperitoneally with 0.1 to 0.5 ml of the filtered or centrifuged food sample and inject either one or two mice similarly with equivalent volumes of the heated control sample. Where guinea pigs are used, inject intraperitoneally 0.5 to 2.0 ml of the test material ; or feed 2 to 5 ml amounts. The doses given will be largely governed by the volume of sample available. If none of the test animals die, proceed at once with enrichment cultures. If the animals receiving heated controls survive while those given unheated food sample die, specific neutralization tests should be carried out, using types A, B and E botulinum antitoxins.* When these antitoxic sera are not on hand or cannot be obtained promptly, the material should be sent to a laboratory having the required facili- ties. When antitoxic sera are available, inoculate a group of mice or guinea pigs with mixtures of the food sample and of Types A, B and E antitoxins, respectively. Use the same dose of food sample as in the foregoing test and mix thoroughly in a syringe with 0.2 ml of the antitoxin. Hold each mixture for about 20 min at room temperature before injecting intraperitoneally, to insure full combination. Occa- sionally, when the serum is of low antitoxic potency while the food sample contains much toxin, larger amounts of serum may be needed to insure neutralization. However, if the serum has been preserved by addition of 50 per cent glycerol or 0.5 per cent phenol, confusion may arise from fatal reactions caused by the preservative, in the case of mice injected intraperitoneally with 0.5 ml or more of the serum. The condition of the animals injected with food specimens will afford evidence of the presence and type of botulinum toxin in the food. Given a toxin of high potency, test animals often succumb within a few hours, death being preceded by a bellows-like respira- * Types C and D may be disregarded, since they have not been implicated in human botulism. Types A and B toxins (the former more frequently in North America) are liable to be involved in outbreaks due to home-canned vegetables and fruits, less often to meat products. The reverse holds in certain European countries, notably Germany and France. Type E toxin should be suspected when the vehicle is pickled or smoked fish or marine mammal.3-5 324 BACTERIAL FOOD POISONING tion, constricted abdomen, and limb paralyses. Low-potency toxin may cause flaccidity of the abdominal muscles, with some dragging of the limbs. Then death may not occur for 3 or 4 days, or even longer ; and sometimes obviously sick animals will completely recover. It must be borne in mind that when animals, especially white mice, have been injected with unfiltered material from food samples, death may be due to infection by contaminating bacteria rather than to botulinum toxin. This possibility makes it all the more important that control animals be given heated material and antitoxin mixtures. Again, mice occasionally die in convulsions within a comparatively few minutes after being injected with a food extract. This should not be a source of confusion, since mice do not die in less than 3to5 hr after injection of large doses of botulinum toxin. In the event that equivocal results are obtained—for example, if all mice receiving suitably proportioned toxin-antitoxin mixtures die of typical botulism—consideration should be given to the remote possi- bility that Type C toxin, or a toxin of an entirely new type, may be involved. Such problems cannot be investigated satisfactorily in the average small laboratory. B. Culture Tests for Cl. botulinum Plating—W ith a loopful of the fluid sample, streak in succes- sion two agar plates, preferably using one of blood agar prepared from meat infusion (CM No. 16) and one of brain heart agar (CM No. 22). If blood agar and brain heart agar are not available, meat infusion agar without blood may be used (CM No. 6). Incubate the plates under anaerobic conditions at 35° C for 24 to 48 hr, after which examine them for colonies of Cl. botulinum. * Pick suspected colonies to large tubes of ground meat medium (CM No. 114) which have been heated and cooled before inoculation, add 0.5 per cent glucose to the tubes, and then incubate for 4 days at 30° C. At 35° C, some strains produce very little toxin under these conditions. Inject mice with centrifuged supernatant fluid from the meat cultures as a test for toxin and, if death follows, type with specific antitoxins as described in the following paragraphs. If ground meat medium is not available, thioglycolate broth (CM No. 20) may be substituted. . * These colonies may assume many forms, and long experience is needed for selecting them. On dry blood agar plates they are usually slightly hemolytic, small, flat, somewhat irregular in outline, fairly smooth in texture, and trans- lucent. On moist plates colonies tend to develop frond-like outgrowths, or only a thin continuous film of growth may be apparent. On brain heart agar, colonies tend to grow larger and often exhibit a mosaic pattern when viewed by trans- mitted light.4 BACTERIAL FOOD POISONING 325 Enrichment cultures—Botulinum toxin may not be demon- strated in the foodstuff, especially when the sample submitted is scanty or otherwise unsatisfactory, and it is often difficult to detect Cl. botulinum colonies in direct platings from the food sample. In such cases presence of the organism may be detected by inoculating appropriate enrichment media with the sample or with washings from the container promptly following receipt at the laboratory. Some- times very small scraps of the implicated food (e.g., a fragment of herring backbone retrieved from a garbage pail) repay cultural ex- amination. When sufficient food sample is available, inoculate 1 to 4 g (or ml) into each of three large tubes of ground meat medium (CM No. 114). Before inoculation this medium should have been held in a boiling water bath to expel air and then cooled. The addition of 1 per cent glucose will facilitate growth of the anaerobe and usually will promote toxin production by Cl. botulinum. The final pH should be 7.2 to 7.6 after autoclaving. Immediately after inoculation of the meat medium, heat two of the three tubes in a water bath at 80° C for 20 min to destroy vegetative bacterial cells. Leave the third tube unheated. These exposures to heat are arbitrary in degree and duration. If Type E botulism is suspected, it should be borne in mind that Type E spores are relatively heat- labile. On the other hand, advantage may have to be taken of the high thermal resistance of Type A spores when attempting to isolate a suspected Type A strain from material badly contaminated with other anaerobes, in which event incubate all three tubes anaerobically at 35° C for 3 to 4 days. If an anaerobic jar is not available, layer the medium in the tubes with sterile vaseline or agar to form a seal. Examination of these cultures should supplement the foregoing direct test for toxin in the food sample. Note any macroscopical evidence of growth and prepare Gram stains from each tube. Note whether Gram-positive bacilli, with or without subterminal spores, are present. Select one or more tubes for a toxicity test similar to that carried out with the original sample. The result may confirm the direct test with the food sample, or it may afford additional evidence when toxin cannot.be demonstrated directly in the food- stuff. In the event of a positive test, the meat medium cultures may be used for further purification and isolation of CI. botulinum if this is desired. Blood and bowel contents—In cases of botulism, toxin can sometimes be demonstrated in the blood or in the gastrointestinal con- tents by animal injection together with the use of specific antitoxins. 326 BACTERIAL FOOD POISONING It is necessary to dilute the bowel contents with sterile salt solution, centrifugalize, and filter through a Berkefeld or Seitz filter to eliminate the numerous contaminating bacteria which are present, before using the specimen for toxin tests. In cases of clinically typical botulism, demonstration by the above methods of toxin in a foodstuff, or the procurement of toxic cultures therefrom, usually serves to designate that food responsible for the episode. Use of type-specific antitoxic sera may furnish additional information about the type of Cl. botulinum involved. However, a reservation applies here to instances in which a culture of CL botulinum has been isolated from an empty discarded container and none of the suspected foodstuff remains for direct test of the presence of toxin. Such a finding may be very significant but it should be re- called that botulinum spores occur in soil and might conceivably have gained entrance to the empty container after it was discarded. Any conclusions drawn from such findings must therefore take into ac- count the conditions to which the container was exposed after first being opened. Similar arguments would apply to isolation of a culture of Cl. botulinum from the gastrointestinal tract in the absence of demonstrable botulinum toxin of homologous type in the stomach or lower bowel contents. IV. STAPHYLOCOCCI The ability to produce enterotoxin apparently is limited to coagu- lase-positive staphylococci.®? These organisms represent a homoge- neous, readily identifiable taxonomic entity.®® However, not all coagulase-positive strains produce enterotoxin.®!! Thus considerable caution must be used in interpreting the results of bacteriological examination of a food suspected of being the vehicle in a food poison- ing outbreak. Careful correlation with the epidemiological and clinical features of the episode is especially important. The significance of laboratory findings is also dependent on the history of the food samples examined! The laboratory examination should involve quantitative plating in suitable media and a direct microscopical examination of Gram-stained preparations from each sample. Emulsify a 10 g representative sample in 90 ml of sterile water in a sterile metal laboratory blender cup. A. Gram stain and further dilutions for plating should be made from the emulsified specimen. Streak plates of blood agar (CM No. 16) and inoculate the surface of salt mannitol agar? with 0.05 ml decimal dilutions of the food. BACTERIAL FOOD POISONING 327 There is no completely satisfactory selective medium for the enumeration of coagulase-positive staphylococci. However, Staphylo- coccus Medium No. 110 (Difco) and the Chapman-Stone medium are useful!3-1% regardless of the fact that coagulase-negative micrococci and occasional strains of genus Bacillus may grow luxuriantly on these media. Pick representative colonies and perform the coagulase test as directed in Chapter 6. From a practical standpoint it would seem desirable to consider any food found to contain large numbers of coagulase-positive staphylococci (5X 10° per g) unfit for human consumption. Although such findings prove nothing in a medicolegal sense, they would represent good presumptive evidence that the food had been grossly mishandled at some time prior to laboratory examination and might well contain enterotoxin. The finding of small numbers of coagulase-positive staphylococci in a food should not be considered particularly significant, but if such a food were inadequately refrigerated it might become dangerous. Fortunately, these organisms cannot grow appreciably or produce enterotoxin at temperatures below 50° F. The presence of large numbers of coagulase-negative micrococci in a food sample may be indicative of mishandling at some previous time, but there is no evidence that consuming the food would have any effect on the well- being of the consumer. There is, however, the complicating fact that in a heated food the staphylococci may have been killed without destruction of their enterotoxin, In such a situation the appearance of large numbers of Gram-positive cocci in the stained preparations might be expected. This also serves to reemphasize the importance of carefully consider- ing the epidemiological and clinical features of the episode under investigation. The only completely reliable test for the responsibility of a suspected food is to feed it to a group of human volunteers. This proceeding is not recommended. Therefore, when large numbers of coagulase-positive staphylococci are found in the food, for the further evidence that they are implicated it will be necessary to examine some of the isolated pure cultures for their ability to produce entero- toxin. Several methods for detecting enterotoxin in culture filtrates have been proposed. Feeding tests on monkeys are subject to fewer errors than are tests involving intraperitoneal or intravenous injection into monkeys or kittens, but monkeys are known to be more resistant to enterotoxin than man and may develop a resistance to enterotoxin 328 BACTERIAL FOOD POISONING if used repeatedly.’® In addition monkeys are difficult to work with and are relatively expensive. For these reasons injection of suitably prepared filtrates into kittens or cats may be the method of choice in the routine diagnostic laboratory. Care must be exercised to remove or neutralize other staphylococcal toxins that interfere with injection tests for enterotoxin.!”-20 When properly applied these tests are sensitive and reliable. Preparation of filtrates—Dolman and Wilson!® found a semi- synthetic soft agar medium (CM No. 10) satisfactory for the pro- duction of enterotoxin. Distribute the medium in shallow layers in petri dishes; flasks or bottles and, when cool, seed the surface evenly with a few drops of a young culture of the staphylococcus under study. Incubate at 35° C for 40 hr under an atmosphere of 30 per cent carbon dioxide and 70 per cent oxygen. Squeeze the cultures through cheesecloth and centrifuge at high speed. Decant the super- natant fluid and sterilize where necessary by passing through a Seitz filter. Monkey feeding tests—If available, Macaca mulatta may be used for feeding tests. The supernatant fluid from cultures prepared as outlined above may be fed without heating or filtration. Give 50 ml by stomach tube to each of three monkeys. Observe the animals for vomiting over a period of 6 hr. If enterotoxin is present, vomiting usually occurs in 2% to 3 hr. Monkeys may develop a tolerance upon repeated feeding and it is necessary to test with known enterotoxin those animals which fail to react in order to eliminate from future tests naturally resistant animals or those that have developed a tolerance. Preparation of filtrates for intravenous or intraperitoneal in- jection—Before a staphylococcus culture can be subjected to the kitten or cat test, it must first be Seitz-filtered and then treated for elimination of the alpha and beta toxins. For routine diagnostic laboratory procedure, heat the filtrate, adjusted to pH 7 by the addi- tion of dilute acetic acid, in a boiling water bath for 30 min. Remove any precipitate by centrifugation and use the supernatant for injec- tions. In most instances this heat treatment will inactivate the alpha and beta hemolysins, while the relatively heat-resistant enterotoxin will survive in sufficient proportion to affect the test animals. How- ever, considerable losses of enterotoxin occur when filtrates are heated to boiling for 30 min, and although the majority of food poisoning strains produce ‘enterotoxin of sufficient potency and heat stability BACTERIAL FOOD POISONING 329 to give positive reactions on injection, a negative reaction under such conditions would not necessarily indicate the absence of enterotoxin from the filtrate before boiling. Hemolysins may be inactivated also by formalinization, or by neutralization with specific antitoxins. Where the latter procedure is followed, the antitoxin employed must be free of enterotoxin- neutralizing antibody. Although these procedures are less simple and more time-consuming than boiling, they are to be preferred when filtrates weak in enterotoxin content are being studied. Treated filtrates should be warmed to 35° C before injection and should be administered slowly by either route. Dolman?! noted that a strain of staphylococcus produced potent enterotoxin, but only negligible amounts of alpha toxin, when grown on the above medium for 37% days in air at room temperature. Filtrates of cultures thus grown could be injected into cats without further treatment. This simplified method is not necessarily applicable to all enterotoxigenic strains, but it is worth trying. Intravenous injection of cats—Only healthy adult cats should be used. A moderate-sized meal given shortly before inoculation of the enterotoxin has been found to increase effectiveness of the vomit- ing stimulus, while refusal of an offered meal aids in the elimination of sick animals. Inject treated culture filtrates slowly into the saphenous vein at about the level of the knee. Depending on the potency of the toxin, 0.5 to 5 ml may be required. Intraperitoneal injection of cats—Kittens from 6 weeks to 3 months of age and weighing 350 to 700 g are most satisfactory for the test, but young cats weighing up to 1 kg may be used. Inject intraperitoneally 3 ml of the treated filtrate. If no reaction occurs, 5 ml of the filtrate should be given. Results of injection tests—The characteristic syndrome pro- duced by injection of enterotoxic filtrates is marked lassitude and apathy, followed by one or more bouts of retching and vomiting often associated with diarrhea, over a period of 30 min to 4 hr after administration. Repeated use of test animals is not desirable, as an increased tolerance to the enterotoxin may develop.22-23 If large numbers of coagulase-positive staphylococci were found in a food epidemiologically implicated in an outbreak of food poisoning, the source of the contamination of food might be sought in nose and throat cultures from food handlers or from cutaneous lesions in food handlers. Where a coagulase-positive staphylococcus is recovered 330 BACTERIAL FOOD POISONING from a food handler and the strain is available from the implicated food, it is recommended that the strains be typed for their bacterio- phage pattern as recommended in Chapter 6. Where the phage pattern of the strain from the food and that from the handler are identical, the probable source of the contamination is apparent. V. STREPTOCOCCI Streptococci have been implicated as the etiological agent in a number of food poisoning outbreaks.?4-%7 Cultures from these out- breaks that have been carefully studied have been found to be con- fined to the enterococcus group, specifically Lancefield’s serological Group D.27-80 In the literature only a few instances are recorded in which the streptococcus isolated from the incriminated food has been fed to human volunteers in pure cultures to confirm its toxigenic capacity. Human volunteers provide the only known acceptable means of test- ing the ability of a streptococcus strain to induce food poisoning symptoms in man. Consequently, for the great majority of the re- ported food poisoning outbreaks in which enterococci have been im- plicated, the evidence is largely circumstantial, being limited to the alleged symptoms of the illness, the time of onset, the presence of large numbers of enterococci in one or more of the foods consumed, and the apparent absence of other known food poisoning micro- organisms in the samples examined. The symptoms ascribed to enterococcus food poisoning are similar to those of staphylococcus food poisoning, although usually less severe’? While diarrhea occurs, neither uncontrollable vomiting nor acute prostration is characteristically present. The time of onset may vary from 2 to 18 hr after eating the food. Since enterococci are normally found in large numbers in the intes- tine of man and warm-blooded animals, it must be concluded that only rare strains which have grown under special conditions are capable of causing food poisoning. Thus far no distinctive physiologi- cal or serological characteristics among the streptococci implicated in food poisoning have been detected. Obviously the examination of stool specimens from afflicted persons for the presence of enterococci would yield no helpful information. If enterococci are suspected as the causative agent in a food poison- ing outbreak, the available evidence indicates that very large numbers of viable streptococci (10 billion or more) must be ingested at one time to produce symptoms. In contrast to staphylococcal food poison- BACTERIAL FOOD POISONING 331 ing, culture filtrates or heat-killed cultures do not give rise to symp- toms. It is therefore unnecessary and may be misleading to employ enrichment procedures for the detection of enterococci in suspected foods. Such procedures do not indicate the total numbers of en- terococci present in the food. For examination of suspected foods, stain by Gram’s method films made directly from several portions of the food and examine for the presence of large numbers of streptococcus-like microorganisms. Plate aliquots of the food quantitatively on blood agar or on a suitable extract agar medium containing glucose. In most instances the direct microscopic examination will suggest the appropriate dilutions to be prepared. The finding of large numbers of cocci in pairs or short chains in the films made directly from the foodstuff, or large numbers of strep- tococcus-like colonies on the agar plates, is indicative of a high enterococcus population. However, considerable caution is necessary in interpreting these findings. For example, if the food material had been subjected to conditions which favored salivary contamination, one should expect alpha-type streptococci to develop on the agar plates. Also, some fermented foods such as cheeses, certain varieties of sausages and fermented milk drinks normally contain indigenous streptococci and other lactic acid bacteria that may cause confusion. In such instances no significance should be attached to their presence. If an enterococcus food poisoning outbreak is to be suspected, no difficulty should be encountered in the detection and isolation of streptococci from the suspected food by use of nonselective plating technics. However, in some instances a selective method of isolating or quantitatively enumerating the enterococci in the food may be desired. No entirely satisfactory selective plating medium for quan- titatively enumerating enterococci in food specimens exists, but one which has met with partial success was proposed by White and Sher- man®? and modified by Dack et al.3° This medium consists of 0.5 per cent glucose, 0.5 per cent tryptone, 0.5 per cent yeast extract, 1.5 per cent agar, 0.01 per cent sodium azide, and penicillin in a concentra- tion of 100 units per liter (added to the melted and cooled medium just prior to use). Most enterococci grow quantitatively in this medium after an incubation period of 48 hr at 35° C. Some lacto- bacilli also grow on this medium. Barnes® has proposed a peptone-yeast extract-glucose agar medium containing 0.1 per cent thallous acetate and 0.01 per cent 2,3,5- triphenyltetrazolium chloride, pH 6.0, for the purpose of quan- 332 BACTERIAL FOOD POISONING titatively enumerating enterococci in foods. Although other strepto- cocci such as Strep. lactis and Strep. bovis are able to grow in this medium, the color of the colonies is somewhat distinctive from that of enterococci. This medium deserves further study. Several selective broth media (e.g., SF medium, buffered azide broth, enterococcus-presumptive broth) are commercially available for the detection of enterococci in foods. The selective agent in these media is sodium azide, and advantage may be taken of the rather high temperature limit for growth of the enterococci (incubate at 45° C) to provide further selectivity for these media. The Most Probable Number (MPN) technic may be employed with these media to estimate quantitatively the total numbers of enterococci present.34:35 Because these media are not entirely selective for enterococci, it is desirable to conduct confirming tests. Commercially available con- firmatory media can be employed (e.g., enterococci-confirmatory broth, ethyl violet azide broth). Positive presumptive tests also can be confirmed by streaking the broth cultures on either the penicillin azide agar or thallous acetate tetrazolium agar, as described above. Where positive identity is desired, isolate several colonies from one of the plates and culture in a suitable broth containing glucose. Determine their enterococcal identity by conducting the Lancefield precipitin test with Group D serum of known potency and specificity (see Chapter 5). If Group D serum is not available, inoculate tubes of glucose broth and incubate at 10° C and 45° C. Temper the tubes in water baths at the respective temperatures prior to incubation. Observe for turbidity development after 2 days at 45° C and after 1 week at 10° C. Addi- tionally inoculate tubes of glucose broth containing 6.5 per cent sodium chloride with the isolated cultures and incubate at 35° C. Observe for turbidity after 2 days. Growth in each of these environ- ments is reasonable assurance that the cultures are enterococci. For positive identity, caution must be exercised to avoid confusing the enterococci with certain Staphylococcus and Leuconostoc species. The enterococci are catalase-negative and fail to produce large amounts of carbon dioxide from glucose. To determine carbon dioxide production, layer inoculated glucose broth cultures with a paraffin-petroleum jelly seal and incubate at 35° C for 2 days. As an alternative, use the method of Williams and Campbell.2® Other tests proved of value as practical and convenient aids in identifying enterococci with respect to species have been summarized by Sherman. 37 BACTERIAL FOOD POISONING 333 VI. B. CEREUS Hauge®® reviewed the literature with reference to the occurrence of aerobic spore-forming, Gram-positive bacilli in foods implicated in food poisoning outbreaks. Over a period between 1947 and 1950, Hauge studied four large food-poisoning outbreaks caused by B. cereus which involved about 600 people. Christiansen, Koch and Madelung®® described an outbreak, caused by pudding, involving 15 of 18 adults and 106 of 136 children. B. cereus food poisoning is characterized by an incubation period varying from 8 to 16 hr but more often from 12 to 13 hr. The symptoms are nausea (seldom vomiting), abdominal cramps about the umbilicus, tenesmus, and frequently diarrhea. After 4-6 bowel movements the symptoms subside, usually within 6-12 hr. Generally, fever is not present. The foods involved in B. cereus outbreaks have been vanilla sauce powder and similar foods, prepared a day in advance and generally stored under conditions which permitted growth of B. cereus. B. cereus is often found in corn or potato starch, which are ingredients of vanilla sauce powder. Laboratory procedure—The Gram stain of the suspected food should show large numbers of Gram-positive, rod-shaped organisms. Where quantitative blood agar plates are prepared, there should be a preponderance of strongly hemolytic colonies of an aerobic, spore- forming bacillus appearing after 24 hr incubation at 35° C. Stool examinations may show few colonies of B. cereus and are not recom- mended. Tn implicated foods B. cereus has usually been found numbering millions of organisms per gram.*0 VII. CL. PERFRINGENS Cl. perfringens has caused outbreaks of mild illness characterized by abdominal cramps and diarrhea, usually without vomiting, com- mencing 8-20 hr (average 10-12 hr) after eating contaminated food.#1-4* Recovery from the illness is usually complete within 24 hr from the onset. Hobbs et al.,** in a study of outbreaks of CL per- fringens food poisoning occurring in Great Britain, found that the strains of the causative organism are feebly toxigenic, produce heat- resistant spores, and fit into the type A group. Some differences are reported in colonial characteristics** of the strains of Cl. perfringens isolated from food poisoning outbreaks. In the outbreaks reported by McClung,*2 Osterling,*3 and Hobbs et al.#* the meat involved had 334 BACTERIAL FOOD POISONING been insufficiently cooked to destroy the spores and was kept long enough after cooking to permit growth of the organisms. When meat cultures of living microorganisms were fed to human volunteers, McClung,*? Osterling,** and Hobbs et al.%* observed symptoms similar to those occurring in food poisoning outbreaks where Cl. perfringens appears involved. On the other hand, Dack et al.*® failed to cause illness in human volunteers fed strains (from food poisoning outbreaks) of Cl. perfringens grown in veal infusion broth containing 0.1 per cent agar and 0.25 per cent glucose, or in autoclaved chicken broth. It may be that the whole meat cultures are necessary to cause illness. All investigators agree that filtrates do not cause illness when fed to human volunteers. Laboratory procedure—Cl. perfringens should be sought by laboratory examination where epidemiological study of an outbreak points to a specific meat product as the possible causative agent. The incubation period from the time of ingesting the food to the onset of illness will not differentiate Cl. perfringens food poisoning from that caused by Strep. fecalis, B. cereus or from some Salmonella in- fections. A Gram stain of the implicated food specimen may prove helpful in showing a preponderance of Gram-positive rod-shaped organisms. Prepare quantitative blood agar plates with the food specimen unheated. Incubate the plates anaerobically at 35° C for 24 hr. If large numbers of nonhemolytic colonies resembling CI. per- fringens are found in the absence of significant numbers of Strep. fecalis, B. cereus or Salmonella, then significance may be attached to Cl. perfringens as the causative agent. According to Hobbs et al.,** if the anaerobic plates after examination are left aerobically at room temperature for 24 hr the aerobic colonies will develop, making it easier to recognize the anaerobic Cl. perfringens colonies. The cul- tures may be tentatively identified as Cl. perfringens by biochemical reactions. Acid and gas are produced from glucose, maltose, lactose and sucrose, but not from mannitol or salicin. Acid and a clot are formed in litmus milk incubated at 35° C for 24 hr. Anaerobic con- ditions may be obtained by boiling and cooling these differential media before inoculation and incubating the cultures in an anaerobic jar, or by sealing the cultures with sterile vaseline immediately after inoculation. Gam. M. Dack, M.D., Pu.D., Chapter Chairman Craupe E. Dorman, Pua.D.,, D.P.H. Harry E. GoresLINE, PH.D. CuArres F. Niven, Jr, PH.D. GLENN G. SrocumMm, PH.D. BACTERIAL FOOD POISONING 335 REFERENCES 1. 2 10. 11. 12. 13. 14. 16. vw. 18. 19. 21. 22. 23. 24. Dack, G. M. Food Poisoning (3rd ed.). Chicago, Ill.: Univ. of Chicago Press, 1956. SomMER, H.; WHEboN, W. F.; Korom, C. A.; and StoHLER, R. Relation of Paralytic Shellfish Poison to Certain Plankton Organisms of the Genus Gonyaulax. Arch. Path. 24:537-59, 1937. Dorman, C. E.,, and CuaNnG, H. The Epidemiology and Pathogenesis of Type E and Fish-Borne Botulism. Canad. J. Pub. Health 44:231-44, 1953. Dorman, C. E. Recent Observations on Type E Botulism. Canad. J. Pub. Health 48:187-198, 1957. ————— Type E (Fish-Borne) Botulism: A Review. Jap. J. Sci. & Biol. 10:383-395, 1957. CuapmaN, G. H, Lie, C. W., and Curcio, L. G. Isolation and Cultural Differentiation of Food Poisoning Staphylococci. Food Research 2:349-367, 1937. HussemanN, D. L, and TANNER, F. W. A Comparison of Strains of Staphylococci Isolated from Foods. Food Research 14:91-97, 1949. Evans, J. B,, and New, C. F., Jr. A Comparative Study of Known Food Poisoning Staphylococci and Related Varieties. J. Bact. 59:545-50, 1950. Evans, J. B., Buerrner, L. G., and Niven, C. F., Jr. Evaluation of the Coagulase Test in the Study of Staphylococci Associated with Food Poisoning. J. Bact. 60:481-84, 1950. Suaw, C, Stitt, J. M., and Cowan, S. T. Staphylococci and Their Classification. J. Gen. Microbiol. 5:1010-1023, 1951. Dorman, C. E. Ingestion of Staphylococcus Exotoxin by Human Volun- teers with Special Reference to Staphylococcic Food Poisoning. J. Infect. Dis. 55:172-32, 1934. CuaprMaN, G. H. The Significance of NaCl in Studies of Staphylococci. J. Bact. 50:201-203, 1945. ——————— An Improved Stone Medium for the Isolation and Testing of Food Poisoning Staphylococci. Food Research 13:100-05, 1948. ———— Comparison of Ludlam’s Medium with Staphylococcus Medium Number 110 for the Isolation of Staphylococci That Clot Blood. J. Bact. 58:823-824, 1949. McDivirr, M. E., and HussemANN, D. L. Comparison of Three Media for the Isolation of Enterotoxigenic Micrococci, A.J.P.H. 44:1455-1459, 1954. SuURGALLA, M. J., and Dack, G. M. Unpublished data. Davison, E., Dack, G. M,, and Cary, W. E. Attempts to Assay the Enterotoxic Substance Produced by Staphylococci by Parenteral Injection of Monkeys and Kittens. J. Infect. Dis. 62:219-223, 1938. Dorman, C. E., and WiLson, R. J. Experiments with Staphylococcal Enterotoxin. J. Immunol. 35:13-30, 1938. Dorman, C. E. Bacterial Food Poisoning. Canad. J. Pub. Health 34:97- 111, 205-35, 1943. ———— and Wison, R. J. The Kitten Test for Staphylococcus Enterotoxin. Canad. J. Pub. Health 31:68-71, 1940. DoLmAN, C. E. Antigenic Properties of Staphylococcus Enterotoxin. Canad. J. Pub. Health 35:337-51, 1944. MatHEsoN, B. H., and TuaTcHER, F. S. Studies with Staphylococcal Toxins. Canad. J. Microbiol. 1:372-381, 1955. OrteL, S. Lysotypie von Staph. aureus zur Klirung Epidemiologischer Zusammenhange bei durch Fischkonserven Hervorgerufenen Staphylo- kokken-Nahrungsmittelvergiftungen. Ztschr. f. Hyg. 144:407-424, 1958. Linpen, B. A, Turner, W. R,, and THoM, CHARLES. Food Poisoning from a Streptococcus in Cheese. Pub. Health Rep. 41:1647-52, 1926. 336 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. BACTERIAL FOOD POISONING Cary, W. E,, Dack, G. M,, and Meyers, E. An Institutional Outbreak of Food Poisoning Possibly Due to a Streptococcus. Proc. Soc. Exper. Biol. & Med. 29:214-15, 1931. Cary, W. E,, Dack, G. M., and Davison, E. Alpha Type Streptococci in Food Poisoning. J. Infect. Dis. 62:88-91, 1938. BUCHBINDER, L., OSLER, A. G., and SterreEN, G. I. Studies in Enterococcal Food Poisoning. I. The Isolation of Enterococci from Foods Implicated in Several Outbreaks of Food Poisoning. Pub. Health Rep. 63:109-18, 1948. SuerMAN, J. M, Smitey, K. L., and Niven, C. F., Jr. The Identity of a Streptococcus Associated with Food Poisoning from Cheese. J. Dairy Sci. 26:321-23, 1943. SHERMAN, J. M., Gunsarus, I. C, and Berramy, W. D. Streptococcus Food Poisoning. 57th Annual Report, New York State College of Agri- culture, Bull. Cornell Univ. Agr. Exp. Sta. 1944, p. 116. Dack, G. M.; Niven, C. F,, Jr; Kimrsner, J. B.; and MArsHALL, H. Feeding Tests on Human Volunteers with Enterococci and Tyramine. J. Infect. Dis. 85:131-8, 1949. Dack, G. M. Food Poisoning (3rd ed.). Chicago, Ill.: Univ. of Chicago Press, 1956, p. 203. Warr, J. C., and SuerMAN, J. M. Occurrence of Enterococci in Milk, J. Bact. 48:262, 1944. Barnes, Erra M. Methods for the Isolation of Faecal Streptococci (Lancefield Group D) from Bacon Factories. J. Appl. Bact. 19:193-203, 1956. ZaBorowsKkI, HELEN, Huser, D. A., and Rayman, M. M. Evaluation of Microbiological Methods Used for the Examination of Precooked Frozen Foods. Applied Microbiol. 6 :97-104, 1958. Fanernr, M. J.,, and Avres, J. C. Methods of Detection and Effect of Freezing on the Microflora of Chicken Pies. Food Tech. 13:294-300, 1959. WiLriams, O. B., and Cameserr, L. Leon. The Detection of Hetero- fermentation by Lactic Acid Bacteria. Food Tech. 5:306, 1951. SHERMAN, J. M. The Streptococci. Bact. Rev. 1:1-97, 1937. Hauce, StEINAR. Matforgiftninger fremkalt av Bacillus cereus. Nord. Hyg. Tidskr. 31:189-205, 1950. CuristiIaANSEN, O., Koc, Svenp O., and MaperLung, P. Et Udbrud af Levnedsmiddelforgiftning Forédrsaget af Bacillus cereus. Saertryk af Ugeskriftfor Laeger 113, 703-706, 1951. Smita, N. R, Goroon, R. E, and Crark, F. E. Aerobic Mesophilic Sporeforming Bacteria. U. S. Dept. Agr. Misc. Pub. No. 559, May 1946. NeLsoN, C. I. Flatulent Diarrhea Due to Clostridium welchii. J. Infect. Dis. 52:89-93, 1933. McCrunNG, L. S. Human Food Poisoning Due to Growth of Clostridium perfringens in Freshly Cooked Chicken. J. Bact. 50:229-31, 1945. OSTERLING, S. Matforgiftninger Orsakade av Clostridium perfringens. Nord. Hyg. Tidskr, 33:173-79, 1952. Hogss, Berry C., et al. Clostridium welchii Food Poisoning. J. Hyg. (Great Britain) 51:75-101, 1953. . Dack, G. M.; Suctyama, H.; Owens, F. J.; and KrsnEer, J. B. Failure to Produce Illness in Human Volunteers Fed Bacillus cereus and Clos- tridium perfringens. J. Infect. Dis. 94 :34-38, 1954. CHAPTER 12 BRUCELLOSIS I. Collection and Handling of Specimens A. Blood Serum B. Blood and Clot Cultures 1. Collecting outfits for whole blood culture 2. For clot culture C. Miscellaneous Cultures (Body Fluids and Tissues Other Than Blood) 11. Bacteriological Examination A. Blood Culture 1. Culture of whole blood 2. Clot cultures B. Miscellaneous Cultures (Body Fluids and Tissues Other Than Blood) C. Demonstration of Brucella by Animal Inoculation D. Differentiation of Brucella Species Methods of choice Procedure for culture media Tests for CO, Requirement and H,S production Dye-plate method Classification of cultures PRL pot III. Serological Examination A. Blood Serum 1. Rapid plate (Slide) method 2. Test tube method 3. The standard tube agglutination test for human brucellosis B. Fluids Other Than Serum IV. Evaluation and Reporting of Results A. Cultures B. Animal Inoculation C. Agglutination Tests References Brucellosis is a disease of animals transmissible to man through contact with infected animals, the ingestion of unpasteurized dairy products, inhalation, and accidents in the laboratory. All three Brucella species, Br. abortus, Br. melitensis and Br. suis, have been isolated in the United States from human as well as animal reservoirs. A new species of Brucella isolated from the wood rat, Neotoma lepida, reported by Stoenner and Lackman,! so far as is known has not been isolated from human infections. Since brucellosis may simulate 337 338 BRUCELLOSIS many diseases, the physician is dependent upon the laboratory for aid in confirming his clinical suspicions. Three procedures are of special value in diagnosis: (1) the isolation of the specific etiological agent from the blood or other tissues, (2) the agglutination test, and (3) animal inoculation. According to McCullough? and Spink et al. sternal marrow cul- tures have yielded positive results when blood cultures have been negative. With rare exceptions organisms of the Brucella group are slow-growing, require special culture media for growth, and are either dependent on or enhanced in an atmosphere of increased COs. The isolation of the etiological agent leaves no question as to the diagnosis, whereas the agglutination test is open to interpretive errors. Too much emphasis is placed on the agglutination test to the exclusion of culture methods in attempting to establish a diagnosis. The isolation and typing of the organism as to species gives very valuable epidemiological information. The determination of species is also important from the therapeutic point of view. Br. abortus infections usually respond to a single (broad-spectrum) antibiotic, whereas Br. suis and Br. melitensis in- fections frequently relapse or recur. Thus in suis and melitensis infections, in addition to a broad-spectrum antibiotic, streptomycin® and sulfadiazine’ may be required as well to effect a permanent cure. For these reasons every effort should be made to get cultures in suspected cases and correlate them with the agglutination findings. Brucella may remain dormant in reticuloendothelial cells for years following apparent clinical recovery. Activation may occur following injury or a debilitating illness with symptoms, as in the primary attack, together with the development of abscesses at the site of injury. Granulomatous lesions simulating tuberculosis®? mycosis” and/or cancer” may occur in the lung, brain, spleen, liver, kidney, bone marrow, etc. In such instances Brucellae are usually isolated, whereas the agglutination test is most commonly negative or too low in titer to be of diagnostic significance. The laboratory must be prepared to offer the physician adequate laboratory service to aid him in confirming his provisional diagnosis. The National Research Council Committee on Public Health Aspects of Brucellosis® has recommended two measures to insure consistent and reproducible results in agglutination tests the country over. They recommend “(1) that a standard antigen, prepared by a prescribed method from a designated strain of Brucella abortus be used in all instances; and (2) that a uniform procedure of conducting the tube agglutination test be utilized in all laboratories.” BRUCELLOSIS 339 For the preparation of antigen great care must be exercised in the selection of satisfactory smooth, agglutinable cultures to avoid erro- neous end results. Stock cultures frequently mutate, which renders them unsuitable for use. Antigens prepared in the laboratory should be standardized by reference to a standard tube test.’ Modifications of the flocculation test as devised by Hunter and Colbert! are satisfactory for testing human sera and are being widely adopted. Since there are no specific, uniform standards for conducting cultural tests at this writing, the authors of this chapter have detailed the procedures which in their opinion appear to be reliable and appli- cable for the average and smaller than average laboratories. Methods for examining dairy products will be found detailed in Standard Methods for the Examination of Dairy Products. See Chapter 3 regarding the handling of highly infectious micro- organisms, I. COLLECTION AND HANDLING OF SPECIMENSt A. Blood Serum After blood has thoroughly clotted in the test tube, properly identify the specimen and transmit it to the laboratory. Centrifuge the blood for 15 min at 3,000-4,000 rpm. Transfer clear serum aseptically to another sterile stoppered tube and retain under refrigeration for the agglutination test. Retain the clot under refrigeration for cultural studies. B. Blood and Clot Cultures 1. Collecting outfits for the culture of whole blood—Preferably use culture bottle or flask provided by the laboratory. The following types of outfits are suitable: a) Delayed culture (mailed specimens): Into a narrow-mouth bottle of 100 ml or 4 fl oz capacity, equipped with diaphragm stopper, dispense 50 ml of a suitable culture medium such as tryptose dextrose vitamin B broth (CM No. 36). Commercially dehydrated culture media, trypticase soy broth (CM No. 11), Bacto tryptose broth (CM No. 36) or Albimi broth (CM No. 39), are also satisfactory. These * Eleventh edition, prepared by committees of the American Public Health Association. New York, N. Y.: The Association, 1960. 1 See Chapter 1 for procedures in collecting specimens for cultural and serological tests. 340 BRUCELLOSIS broth media may be modified by the addition of 0.5-1.0 g of agar per liter prior to sterilization.? When these media are used in field collec- tion outfits for direct inoculation of uncitrated blood, further modify by the addition of 10 g sodium citrate per liter prior to sterilization.? After sterilization, using aseptic precautions replace about 10 per cent of the air above the medium with CO: in a suitable manner as follows: Sterilize the diaphragm with 70 per cent ethanol or with acetone, Provide, completely sterilized, a tuberculin syringe barrel containing absorbent cotton and fitted to a 23 gauge hypodermic needle, Plunge needle through the diaphragm. With rubber tubing attach open end of syringe barrel to one lead of a three-way stopcock, the other leads of which are used for attachment respectively to a vacuum pump and to a vessel containing a measured amount of CO collected over water by displacement. Exhaust approximately 10 per cent of air above the medium. Reverse stopcock and allow CO: to replace it. (A Y or a T tube with rubber tubing and pinchcocks may be used instead of the three-way stopcock.) This is essentially the method and apparatus pictured by Huddleson.!® Sterilize the dia- phragm of the culture container with 70 per cent ethanol or with acetone. Force needle through diaphragm and inject 5-10 ml of whole blood and mix thoroughly. Identify and transmit culture to laboratory. b) Immediate culture only: Use any suitable flask with cotton plug containing 50 ml of a suitable culture medium as above and sterilize after assembly. The use of this type of collection flask is predicated upon early incubation of the culture under suitable CO: tension. Inject 5 to 10 ml of whole blood from syringe under aseptic condi- tions into flask, mix thoroughly, identify, and place in incubator. c) Emergency use only (outfit with culture medium not available) : Use vial or tube (about 1 fl oz capacity) with suitable closure, con- taining 1 ml of 20 per cent sodium citrate, sterilized after assembly. Inject 5-10 ml of blood aseptically from syringe, mix, and transmit to the laboratory. At the laboratory transfer 1 ml quantities to several flasks as in (b) above, mix, and incubate under 10 per cent COo. d) The medium and method recommended by Castaneda’? may be used with good results for blood or blood clot cultures, 2. For clot culture—Dispense 15-20 ml of suitable culture medium as above in cotton-stoppered tube, flask or bottle, and sterilize, Ha BRUCELLOSIS 341 C. Miscellaneous Cultures Body fluids and tissues other than blood—Sternal bone mar- row, pleural fluid, cerebrospinal fluid, peritoneal fluid, urine and abscess material should be transferred to sterile, chemically clean cork-stoppered test tubes, properly identified, and promptly trans- mitted to the laboratory. Il. BACTERIOLOGICAL EXAMINATION A. Blood Culture 1. Culture of whole blood a) Incubation of primary culture: Incubate at 35° C under 2-10 per cent CO. tension. When a field outfit containing CO. similar to that described (see Section IB1(a) of this chapter) has been used, no additional CO. need be furnished but other cultures should be placed in sealed incubating chambers or jars. In the latter case, means should be provided for the introduction of CO-, either by exhaust and replacement of air or by the burning of a candle in the sealed space. Exactly 10 per cent CO: tension need not be attained; atmospheres of 2-3 per cent have been shown to yield good results’? Huddleson'* recommends the use of NasCOs in the medium as a source of CO. The methods devised by Damon et al.’ may be followed. Incubate for 4-7 days before making first transfer described below. Reincubate primary culture for 7 days, renewing COs: if it has been dissipated, and make second transfer unless growth has been obtained on first. Repeat for an additional 7 days and make third and final transfer at that time if growth has not been obtained. The high humid- ity which develops in sealed chambers or jars under these conditions promotes rapid growth of molds which will contaminate and render valueless many cultures if not controlled. A layer or tray of dry CaCl; (anhydrous, porous, 4 mesh) placed in the bottom of the chamber or jar will be found helpful in reducing the humidity. b) Transfers to solid medium: Use freshly poured slants or solidi- fied plates of tryptose dextrose vitamin B agar (CM No. 37). Com- mercially dehydrated culture media, trypticase soy agar (CM No. 12), Bacto tryptose agar (CM No. 37), or Albimi agar (CM No. 40), are also satisfactory. Mix primary culture, aseptically remove 0.5 ml, and streak over the entire surface of the medium in the plate, or transfer several loopfuls to an agar slant. Incubate under 2-10 per cent CO: tension for 4 days at 35°C. Methods of obtaining a suit- 342 BRUCELLOSIS able CO. tension are numerous. It is practicable to use large glass jars (of 1, 3 or 5 gal capacity with gasketed screw-caps) into which the inverted plates are placed. A lighted candle secured to a small tray is then placed in the jar near the top and the cap is screwed tightly in place. A candle allowed to burn undisturbed in the jar until it goes out will yield a CO: ten- sion between 2 and 3 per cent.!® The development of a high humidity within the chamber or jar not only is favorable to rapid growth of contaminating molds but also constitutes a hazard to the worker, since condensate on the plates may serve as a vehicle for the spread of Brucella to the hands of the person examining plates for growth. Hence, a drying agent (used as in Paragraph 1 (a) above) is recommended. c) Examination of culture plates: After the 4 day incubation period, examine plates for signs of growth. Characteristic Brucella colonies are 2-7 mm in diameter, spheroidal in shape, moist and slightly opalescent in appearance, and translucent. These character- istics may vary somewhat with available moisture and with pH. Do not consider a blood culture negative for Brucella until the third streaking has failed to yield the organism. d) Study of suspected colonies: Fish isolated colonies to several tryptose agar slants (CM No. 37) or slants prepared from commercial dehydrated media (see Section IT A 1 (b)) and incubate under COs tension at 35° C for 48 hr or for an additional 24 hr if sufficient growth has not resulted. Brucella species yield a fine, clear, trans- lucent growth with a slight amber tinge. Make a Gram stain and examine for Gram-negative pleomorphic coccobacilli. Brucella takes the counterstain poorly; therefore apply counterstain 1 to 3 min instead of the usual 30 sec. Make a spot slide-agglutination test with Brucella antiserum in suitable dilution (usually not less than 1:10). Control for pseudo agglutination by noting smoothness of emulsion of the growth in a drop of isotonic salt solution on glass slide. Con- firm positive slide test by tube-agglutination test. Identify species of Brucella as outlined in D below. 2. Clot cultures a) Inoculation of primary medium: Subject clot to as little handling as practicable in separating and removing serum. Observe aseptic precautions, preferably leaving clot in original container. Macerate clot by forcing through an autoclaved 10 or 20 ml glass syringe or with open end of a sterile straight-sided pipette about 9 in. long with a 2 ml capacity rubber bulb attached. Such pipettes, or “thieves,” BRUCELLOSIS 343 can be made from glass tubing 7 mm in diameter, with ends polished and slightly flattened by heating in a flame. When broken up, the clot is drawn into the pipette with the aid of the rubber bulb and transferred aseptically to tryptose dextrose vitamin B broth (CM No. 36) or to broth prepared from commercially dehydrated media (see Section I B 1 (a)), dispensed in screw-capped vials or cotton-plugged test tubes in 8-10 ml quantities before sterilization. The addition of 0.5-1.0 g of agar per liter to the broth before sterilization is recom- mended. In laboratories which handle specimens taken under varying condi- tions and shipped through the mail there will be many contaminated cultures which can be eliminated to some extent by adding 1.4 ml of 0.1 per cent aqueous crystal violet (certified) per liter of broth before sterilization. However, according to Huddleson'* this concentration of crystal violet may inhibit small numbers of Br. suis. Some workers have also noted that Br. melitensis cultures may be similarly inhibited. The medium recommended by Weed!” of the Mayo Clinic for poten- tially contaminated specimens is also satisfactory. b) Isolation of the organism: Proceed as directed above under 1 (b), (c¢) and (d). When using screw-capped vials do not close cap tightly during incubation under CO. tension. Identify species iso- lated in pure culture as directed in Section D below. B. Miscellaneous Cultures Body fluids and tissues other than blood—Inoculate primary medium, subculture, and identify as for blood cultures. Since Bru- cella may be few in numbers in pleural and cerebrospinal fluid, inocu- late guinea pigs with centrifugated sediment as in Section C. a) Urine—Using sterile equipment, collect 50 to 100 ml of catheter- ized urine. Centrifuge at a sedimenting force comparable to that produced by 3,000 rpm in a No. 2 centrifuge maintained for 30 min. Spread sediment over the surface of two tryptose dextrose vitamin B agar plates (CM No. 37), or agar plates prepared from com- mercially dehydrated media (see Section IT A 1 (b)) above: either a medium containing 1.4 ml of 0.1 per cent aqueous crystal violet (certi- fied) per liter added before sterilization or the medium recommended by Weed.!” Incubate plates under 2-10 per cent COs tension at 35° C for 48 hr. Reincubate an additional 24 hr if no growth occurs. Identify suspected colonies as directed in the section on blood cultures. b) Cerebrospinal, peritoneal and joint fluids—If a fibrinous clot or pellicle is present, remove it aseptically and grind in a mortar. Add 344 BRUCELLOSIS this ground material to the fluid portion, centrifuge, and inoculate as in Paragraph (a) above and Section C below. c) Abscess material and bone marrow—Spread material over solid media as in Section IT A 1 (b) above and inoculate animals as in Section C immediately following. C. Demonstration of Brucella By Animal Inoculation Use healthy male guinea pigs of 300-600 g body weight. Secure 4-5 ml blood from each prior to inoculation and test for Brucella agglutinative properties. Prepare specimens or samples in the man- ner suggested for cultural procedure (see Section B above). If material is likely to contain many contaminating organisms, inoculate animals subcutaneously—otherwise, intraperitoneally. Inject about 2 ml into each animal, at least two animals per specimen. Six weeks after inoculation procure 4-5 ml blood from each animal and test for Brucella agglutinative properties. Kill and autopsy those animals that react, examining each for characteristic lesions such as: 1) Spleen—Enlarged, sometimes 5-6X; usually with nodules that are at first hemorrhagic, later becoming encapsulated, gray and discrete, and occasionally having necrotic centers; occasionally ab- scesses (usually Br. suis). 2) Liver—Small (0.5-2.0 mm in diameter), gray, glistening, discrete nodules just below surface on the capsule; occasionally ab- scesses (usually Br. suis). 3) Genitalia—Sometimes abscesses in testes and epididymes (Sublumbar lymph nodes may also be involved). Make cultures from suspected lesions by rubbing the cut surface of the tissue over the surface of crystal violet tryptose agar. Incu- bate, fish colonies, and identify organisms as directed under the section on blood cultures. Kill and autopsy at 8 weeks those animals with negative agglutina- tion tests at 6 weeks. D. Differentiation of Brucella Species 1. Methods of choice—For a complete discussion of methods available for differentiation of Brucella species, see Huddleson.1® Many such methods are not readily carried out in the routine ex- amination of cultures. It is recommended that each culture isolated be identified by the dye plate method of Huddleson,'® supplemented by tests for ability to grow in the absence of CO. and by H.S pro- BRUCELLOSIS 345 duction. Cruickshank!® has recently reported on a filter strip method of typing cultures. The urease test'® is useful in the differentiation of Br. suis from other Brucella species but does not permit differentia- tion of Br. abortus from Br. melitensis. Serological differentiation of Brucella species is impracticable. 2. Procedure for culture media a) Tryptose agar slants (CM No. 37) or slants prepared from commercial dehydrated media (Section II A 1 (b) above) are the media of choice. b) Thionin tryptose agar: To prepare, melt previously sterilized tryptose agar (CM No. 37). Adjust medium while hot to pH 6.6- 6.8. Heat a small amount of 1 per cent aqueous thionin (certified) in a boiling water bath for 20 min and then add 0.1 ml* while hot to each 100 ml medium. Mix, pour plates or slants and use within 48 hr. The surface of the plates or slants must be dry before inoculation, which may be accomplished by placing them in the incubator 24 to 48 hr. ¢) Basic fuchsin tryptose agar: To prepare, melt previously steril- ized tryptose agar (CM No. 37). Heat a small amount of 1 per cent aqueous basic fuchsin (certified) in a boiling water bath for 20 min and then add 0.1 ml* while hot to each 100 ml medium. Mix, pour plates, and use within 48 hr. These plates should be dark rose red in color; 1 per cent basic fuchsin solution older than 2 months may deteriorate, yielding lighter plates not suitable for use. Always store basic fuchsin solution in the dark. 3. Tests for CO: requirement and H-S production—From each pure culture on tryptose agar or other medium inoculate two tryptose agar slants. Suspend a lead acetate strip about 1 in. below the cotton plug within one of the tubes for the detection of H2S. Incubate the plain slant aerobically and the one for H>S production under 2-10 per cent COz at 35° C for 4 days. Examine daily for both growth and H.S production, replacing the lead acetate strip with a fresh strip each day. Record HS produced as “None” (0), “Trace” (=), and “Moderate to marked” (+). 4. Dye-plate method—From each pure culture, streak one thionin tryptose agar plate or slant and one basic fuchsin tryptose agar plate or slant. Four cultures may be tested on each plate, one in * The amount is dependent upon the bacteriostatic action and dye content of each lot of dye. 346 BRUCELLOSIS each quadrant. Use moderately heavy inoculum, streaking one small area at the top of each plate heavily and streaking off onto the rest of the plate or quadrant. Incubate plates under 2-10 per cent CO. at 35° C for 72 hr. Examine and record the presence or absence of distinct growth. Do not confuse heavy inoculum with growth but look for distinct growth on portions of streaked plates after rubbing off the excess on the loop. 5. Classification of cultures*—The dye plate results constitute the most reliable criterion for species identification ; results of other tests are informative. The following schema illustrates the manner of interpretation: Basic Thionin Fuchsin CO, Plates Plates Needed H,S Production Classification Growth Growth No 0 to + throughout 4 days Br. melitensis Growth No growth No + throughout 4 days Br. suis No growth Growth Usually + for first 2 days only Br. abortus The urease reaction may be helpful in differentiation but is not clean- cut in all instances. Ill. SEROLOGICAL EXAMINATION A. Blood Serum 1. Rapid plate (slide) method'®2? (a) Special apparatus and materials: 1) Glass plates (slides)—Use rectangular plates of double-thick- ness window glass cut to suitable size, preferably with polished edges. Before use, these should be thoroughly clean and greaseless. Exact dimensions will vary from one laboratory to another, depending upon the size of viewing box (if used) and the usual methods of procedure, but it is recommended that the plate be no larger than necessary to accommodate six rows of compartments for tests, five or six to the TOW. * Occasional cultures do not conform to the above characteristics in all respects. This is usually the result of dissociation from smooth to rough variants. BRUCELLOSIS 347 Compartments may be ruled on the plate with a diamond-point or wax pencil. Alkyd resin paint or wax rings may be constructed in any convenient manner which permits thorough cleaning of the plate after use. Square compartments measuring 1 in. on each side, or circular compartments 1 in. in diameter, are suitable, Circular com- partments are readily made with a ring-maker and a hot (130°- 140° C) mixture of 90 per cent paraffin (m.p. 48° F) and 10 per cent petrolatum. When this mixture is used, the bulk of the wax is readily removed if the plates are immersed in cool water for a short time immediately after use. The ring-maker may be constructed by shaping a circle from 28 gauge wire around a 1 in. test tube, leaving 4 in. to ¥% in. of both ends of the wire projecting side by side. The circle is then wound carefully with one layer of No. 12 linen thread, which is secured by tying at each end. The projection wires are then bent at an angle of 60°-75° to the plane of the circle and are forced into any convenient handle. Multiple ring-makers may be constructed entirely of metal. Ordinary curtain rings of about 1 in, diameter can be welded to strips of metal for a multiple ring-maker so that an entire plate may be pre- pared at one time. 2) Illuminating device—Although an illuminating device is not necessary, some workers find that it contributes markedly to the ease of reading reactions. A simple wooden box, 14 to 17 in. long by 9% to 11 in. wide by 5 to 6 in. deep, with the top partly covered by a strip of wood 27% in. wide running lengthwise, serves the purpose very nicely. A 10 in. showcase bulb mounted in a socket at one end of the box under the strip of wood, or two 50 watt electric light bulbs (one at each end), will supply sufficient illumination. The strip of wood on top of the box protects the eyes of the worker reading the test and allows for viewing the test by indirect lighting. Some workers prefer to insert a metal mirror behind the light source, 3) Antigen dropper pipette—Prepare antigen bottles, 15-30 ml capacity, fitted with dropper pipettes having an outside diameter at the extreme tip of 0.07 in. and an opening of 0.06 in. The size cor- responds to gauge 15 by U. S. Standard Gauge No. 283. These dropper pipettes, if held vertically, will deliver 30-35 drops per ml. 4) Antigen suspension—Use antigen prepared specifically for the rapid plate method, ordinarily known as Antigen-Huddleson.® Al- though this antigen is available through supply houses, a method of preparation is described below. 348 BRUCELLOSIS (b) Preparation of antigen: 1) Selection of Brucella culture—For routine use, select an aviru- lent smooth, agglutinable strain of Br. abortus (N.I.H. Strain 456) obtainable from the National Institutes of Health at Bethesda, Md. ; or Br. abortus Strain 1119 obtainable from the Bureau of Animal Industry, U. S. Department of Agriculture. Streak a series of tryp- tose agar (CM No. 37) plates with a single light inoculum of the culture and incubate aerobically at 35° C for 3-4 days until one of the plates shows well-isolated colonies 2-7 mm in diameter. Examine colonies by both reflected and transmitted light. Typical smooth colonies are distinctly spheroidal in shape, slightly opalescent in color and translucent. When viewed from above by re- flected light the colonies have a moist, almost greasy appearance. Using a small lens (274-5X), scan the isolated colonies and select for use only those with the described characteristics which have circular edges and are not mucoid, opaque or wrinkled on the surface. If evidence of dissociation from the smooth state is apparent by any criteria, obtain another culture. 2) Seed cultures—From a smooth colony prepare one heavily seeded tryptose agar (CM No. 37) slant ina 34 in. (18 mm) diameter test tube for six pint-size Blake bottles to be used for antigen produc- tion. Incubate aerobically at 35° C for 24 hr. At this point make a Gram stain of each culture and test the growth for uniform emulsi- fiability in isotonic salt solution and for rapid slide agglutination in Brucella antiserum. (see III A 1 (c¢) above). If pure and sero- logically specific, emulsify the growth from each seed culture slant in 12-15 ml of sterile isotonic salt solution. Inoculate each bottle of tryptose agar (CM No. 37) with 2-2.5 ml of the resulting suspension and, by tilting the bottles, cause the suspension to flow over the entire agar surface. Incubate aerobically with the agar surface uppermost and on a slight slant (neck of bottle higher than bottom) for 48 hrat 35°C. 3) Harvesting growth—Add to each bottle 15-20 ml of isotonic* salt solution containing 0.5 per cent phenol or 0.3 per cent formalin. Phenolized salt solution is preferred by most authorities. Place the bottles on a flat surface with the agar surface uppermost so that the bacterial growth is moistened. Suspend the growth by tilting each * Some prefer to use hypertonic salt solution (12%) for this and further steps involving dilution of the antigen suspension for the rapid plate method. A recommended formula is: NaCl (A.C.S.) 12 g, glycerol (neutral) 20 ml, phenol (U.S.P.) 0.5 g, distilled water q.s. 100 ml. BRUCELLOSIS 349 bottle back and forth. This is readily accomplished without scraping. Stand each bottle on end until the operation has been completed. Pool the suspensions and filter through a layer of absorbent cotton to remove particles of agar.* 4) Preparation of crude antigen suspension—Sediment the organ- isms, preferably in an angle centrifuge, until firmly packed and dis- card the supernatant fluid. Take up the sediment in a minimal quantity of the phenolized or formalinized salt solution (about 5 ml for the growth from six pint-size Blake bottles). Insure even suspension by mixing with a tongue depressor, wooden applicator, or similar instru- ment, Filter by suction through a thin layer of absorbent cotton on a Buchner-type filter. 5) Titration of antigen suspension—Prepare minimal quantities of several dilutions of the crude suspension in the phenolized or formal- inized salt solution as follows: 1:2, 1:4, 1:6 and 1:8. Test each of these diluted antigens for sensitivity to an immune animal serum of known tube test titer (usually necessary to dilute antiserum so that an end point occurs at 1:160 or 1:320) according to the technic de- scribed under Paragraph (c¢) below. Select that dilution of antigen which gives results with 0.08, 0.04, 0.02, 0.01, and 0.005 ml of serum that are nearest to those obtained respectively with 1:20, 1:40, 1:80, 1:160 and 1:320 final dilutionsT in the tube test. Further test the selected dilution against two or more sera of varying titers from patients and against at least one known negative serum. If the selected dilution gives results comparable to those obtained with standard tube test, consider the titration adequate. In some cases it may be necessary to test interpolated dilutions, such as 1:3 or 1:5. Workers using isotonic salt solution as a diluent will find that on rare occasions such a suspension is insensitive ; sensitivity may be increased by using hypertonic salt solution (see first footnote to Paragraph (3) above). 6) Final antigen for test—Dilute the unused crude antigen suspen- sion with the phenolized or formalinized salt solution to the point indicated by the above titration. To each 20 ml add 0.01 ml of 1 per cent aqueous brilliant green and 0.005 ml of 1 per cent aqueous crystal violet and mix. Test the bulk of standardized antigen to check ac- curacy of dilution against the patients’ sera used above. If satis- * Where a virulent culture must be used for antigen, the suspension should be heated in a water bath for 1 hr at 60° C after filtration. 1 Some prefer to standardize plate test antigens in terms of a series of dilu- tions such as 1:25, 1:50, 1:100, etc. 350 BRUCELLOSIS factory, dispense in 15-20 ml quantities in bottles with dropper pip- ettes. When not in use, store in the refrigerator, (¢) The test: 1) Be sure samples of serum and antigen are at room temperature. 2) Shake the antigen gently but thoroughly and repeat at 2 hr in- tervals when in use. 3) Place 0.08, 0.04, 0.02, 0.01 and 0.005 ml amounts of each serum to be tested in a row of squares or circles. Test not more than five or six sera at one time. Attempts to determine end point or titer using smaller amounts of serum or diluted serum are not recom- mended. 4) Place a drop of antigen on each quantity of serum. 5) Mix the serum and antigen in each row with a fresh toothpick or wooden applicator, working from the smallest quantity of serum to the largest. 6) Lift the plate and tilt it back and forth no more than 3 min. 7) Read in bright indirect light against a dark background, that is, place gooseneck desk lamp over a black background with shade slightly tilted and read plate while held above the rim. When using an illum- inating box, place the plate on the box, turn on the light, and read the results. Avoid excessive drying of tests. The results may be read and recorded in serum amounts or in serum dilutions. Immediately after reading the tests, rinse plate with cold water or place in a tray containing cool water for subsequent thorough cleansing. 2. Test tube method — (a) Preparation of antigen: Use antigen suspension prepared from a smooth, avirulent Br. abortus culture (N.I.H. 456 or B.A.I. 1119) selected, grown in pint- size Blake bottles, and harvested as in Paragraph III A 1 (b) 3 above. Since a common agglutinin is demonstrated by smooth agglutin- able strains of Br. abortus, Br. suis and Br. melitensis, patients’ sera are tested with suspensions of only one species. The pooled suspension may be kept in the refrigerator and diluted as needed. Antigen for use in the test may be standardized by dilution of the crude suspen- sion with phenolized or formalinized salt solution to a density corres- ponding to 0.04 per cent bacteria by the centrifuge method of Fitch et al.,?*2 to 200 ppm silica (fuller’s earth) standard,?® to tube No. 1 of the McFarland nephelometer,?* or to 7 cm on the Gates apparatus?® BRUCELLOSIS 351 or comparable density on a photometer, After adjustment of density, check each antigen by making comparative tests with it and with a control antigen of known satisfactory sensitivity against sera of dif- ferent titers, including negatives (see Section 2 (b) below). Satis- factory antigens should give results identical with or very closely approximating the control antigen. (b) The test: 1) Prepare dilutions of the serum in isotonic salt solution, 0.5 ml final volume in each tube of the series. Preferably use a series of dilutions which require only simple manipulation, such as 1:10, 1:20, 1:40, etc., or 1:25, 1:50, 1:100, 1:200, etc. Make the initial dilution in sufficient quantity to permit serial transfer of 0.5 ml to the second and succeeding tubes, each containing 0.5 ml isotonic salt solution. Mix the contents of each tube thoroughly by drawing back and forth in a pipette five times before removing portion to next tube. Avoid frothing. Routinely make dilutions at least to 1:320 (or 1:400); these dilutions are doubled when antigen is added. 2) Prepare controls for each run of unknown sera. Include either a series of dilutions of a serum of known titer or a dilution of anti- serum known to yield complete agglutination. Include also a tube containing 0.5 ml isotonic salt solution only. 3) Add 0.5 ml antigen to each tube, including controls. Mix by shaking. 4) Incubate in a water bath at 37° C for 18-20 hr. Incubation at other temperatures and for other periods of time may yield titers that cannot be compared. 5) Read and record reactions after examining controls. Results may be read either as complete agglutination (+ ), partial agglutina- tion (P or=) and negative (—), or as 4+, 3+, 2+, 1+ and —, indicating approximate percentage of complete agglutination. Do not overlook zone reactions, particularly those which show slight or no agglutination in the lower dilutions. 3. The standard tube agglutination test for human brucellosis®?® (a) Serum dilutions: Place 0.9 ml of isotonic sodium chloride solu- tion in the first tube of a series of 10 tubes (13X100 mm) and 0.5 ml of the sodium chloride solution in each of the nine remaining tubes. With a 1.0 ml pipette (graduated to the tip), place 0.1 ml of serum in tube No. 1. Mix by aspiration, remove 0.5 ml of the mixture, and transfer it to tube No. 2. Mix and transfer 0.5 ml to tube No. 3. Continue this procedure until the contents of tube No. 10 have been 352 BRUCELLOSIS mixed. Discard 0.5 ml of the contents of tube No. 10. The series of 10 tubes now contains 0.5 ml each of serum dilutions ranging as fol- lows: 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, 1:2560 and 1:5120. 3 (b). Dilution and addition of standard antigen: Shake the antigen vial thoroughly. Remove aseptically a quantity of stock antigen suffi- cient for the proposed number of tests. The stock antigen is to be diluted 1:100 with isotonic sodium chloride solution containing 0.5 per cent phenol. Add 0.5 ml of diluted antigen to each tube con- taining the serum dilutions previously prepared. The final serum dilutions now range as follows: 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, 1:2560, 1:5120 and 1:10,240. 3 (c). Test controls: Prepare an antigen control (preferably in duplicate) containing 0.5 ml of isotonic sodium chloride solution and 0.5 ml of diluted standard antigen. Prepare two serum controls. For the positive serum control use a serum possessing a known Br. abortus titer. In addition, select a serum that possesses no known Br. abortus agglutinins for a negative serum control. Prepare both serum controls in serial dilutions and add diluted antigen as noted above. 3 (d). Incubation period: Shake all tubes thoroughly and place them at 35° C (water bath preferred for optimum results) for 48 hr. A period of time less than this (24 hr, for instance) frequently ex- hibits inconsistent results from day to day as well as a twofold or fourfold decrease in titer value. 3 (e). Reading and evaluating test results: Remove the racks of tubes from the water bath and allow them to stand at room tempera- ture while the results are being read. Read the results of all control tubes first. Remove two or three tubes at a time from the racks, hold them in front of a suitable source of light, and estimate the degree of agglutination as follows: 4 plus (+++ +) = complete agglutination and sedimentation, supernatant fluid clear 3plus (+++) = nearly complete agglutination and sedimentation, super- natant fluid about 75 per cent clear 2 plus (++) = marked sedimentation, supernatant fluid about 50 per cent clear 1 plus (+) = distinct sedimentation, supernatant fluid about 25 per cent clear Trace (x) = slight sedimentation on tube wall, supernatant fluid practically as dense as the antigen control Negative (—) = no evidence of agglutination, supernatant of same density as antigen control BRUCELLOSIS 353 The end-point titer of the serum is designated as the highest dilu- tion of serum (the smallest quantity of undiluted serum) that exhibits complete agglutination ( + + + + ) of the antigen. Degrees of partial agglutination, including those exhibited in prozones, are also recorded. B. Fluids Other Than Serum Occasionally there is some justification for making agglutination tests for Brucella on cerebrospinal, peritoneal or pleural fluid. The antibody titers in these specimens are generally very low and a nega- tive agglutination test does not rule out the possibility of a Brucella infection. Technics described for blood serum (see Section A above) are suitable. IV. EVALUATION AND REPORTING OF RESULTS A. Cultures The isolation of any species of Brucella from any fluid, tissue, secretion or excretion derived from the human or animal body con- stitutes conclusive evidence of infection. Exceptions may occur only under very unusual circumstances—for example, the isolation of Brucella from swabbings of tonsils after the patient has drunk raw milk from infected cattle. Negative cultures do not exclude brucel- losis; repeated negative blood cultures combined with negative agglu- tination tests are convincing but not conclusive evidence for the absence of brucellosis. B. Animal Inoculation Results on animal inoculation are subject to the same limitations as those on cultures. C. Agglutination Tests Blood serum— (a) In acute brucellosis little or no titer may de- velop during the first 10 days of illness. Successive specimens taken as the disease progresses will ordinarily show a significant titer some time during the 3rd to 6th week which may afterward decline. The test is usually positive at the time the physician is consulted. Titers up to 1:160 may persist for years after apparent clinical recovery. b) In chronic brucellosis there is no definite criterion whereby the significance of an agglutination test titer may be judged. No diag- nostic question is likely to be raised about persons yielding high titers (complete agglutination in titers higher than 1:160) with clinical find- 354 BRUCELLOSIS ings that are compatible with modern knowledge of brucellosis and showing no symptoms of tularemia. So long as any degree of agglu- tination is obtained in dilution of 1:20, infection with Brucella can- not be ruled out on the basis of agglutination tests alone. As a matter of fact, some individuals apparently do not develop detectable agglu- tinative properties (or have lost them), although infected. This is small comfort to the clinician but nevertheless true in the light of present knowledge. Laboratories making clot cultures routinely will automatically re- solve some of these problems by isolation of the organism from the nonreacting blood. Repeated blood cultures are helpful. Persons who have recovered clinically from primary brucellosis may years later develop complicating lesions, with Brucella being isolated from such lesions. Agglutinative properties in such instances are commonly absent, or are present in low dilution only, rather than in titers of diagnostic significance (1:160 or higher).2¢ Brucella agglutination reactions as high as 1:1280 may occur in subclinical or asymptomatic cases.?” Incubation periods as long as 7 months?® may occur; hence repeated agglutination tests are indicated to demonstrate rising titers. A recent epidemic (1960) in Iowa of over 150 cases emphasizes the prolonged incubation period that might occur in association with low-titered agglutination reactions and fol- lowed by progressively rising titers. Titers do not reflect severity of illness. c) Cross-reactions with Brucella antigen are ordinarily found only in sera from tularemia patients or from persons vaccinated against cholera. d) Anammnestic reactions: Persons who have recovered from brucel- losis may have Brucella antibodies restimulated nonspecifically by any subsequent febrile illness. In such instances agglutination-reaction titers may rise to 1:160 or sometimes higher in a few days and drop to negative or 1:20 within 10 days. Such reactions are confusing to interpret. I. H. Borts, M.D., Chapter Chairman Henry Bauer, PH.D. EarLe K. BormMAN CuARLES M. CARPENTER, M.D., D.V.M., Pu.D. SamueL R. Damon, Pu.D. CHARLES A. HunTER, PH.D. BRUCELLOSIS 355 REFERENCES 5 ® N 10. 11, 12, 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. StoENNER, H. G., and LackmaN, D. B. A New Species of Brucella Isolated from the Desert Wood Rat, Neotoma lepida Thomas. Am. J. Vet. Res. 18:947, 1957. McCurroucH, N. B. Laboratory Tests in the Diagnosis of Brucellosis. A.J.P.H. 39:866-869 (July) 1949. } Spink, W. W.; McCurroucH, N. B.; HurcHInGs, L. M.; and MINGLE, C. K. Diagnostic Criteria for Human Brucellosis. Report No. 2 of the National Research Council Committee on Public Health Aspects of Brucellosis. J.A.M.A. 149:805, 1952. Conn, H. F. “Brucellosis (Undulant Fever),” in Current Therapy. Phila- delphia: Saunders, 1960, pp. 4-5. EiseLe, C. W., and McCurroucH, N. B. The Treatment of Brucellosis. Illinois Med. J. 96 :241-244, 1949. Borts, I. H. Brucellosis in the United States and Iowa with Special Refer- ence to Incidence and Complications Noted in Iowa. Pub. Health Lab. 7:74-76 (July) 1949. WEED, L. A. Personal communication. Mayo Clinic, 1959. Spink, W. W.; McCurwucH, N. B.; Hurcaings, L. M.; and MINGLE, C. K. A Standardized Antigen and Agglutination Technic for Human Brucellosis. Report No. 3 of the National Research Council Committee on Public Health Aspects of Brucellosis. Am. J. Clin. Path. 24:496, 1954. Brucella abortus Tube Antigen for the Laboratory Diagnosis of Human Brucellosis. (Prepared and Tested According to the Recommendations of the Committee on Brucellosis of the National Research Council.) New York: Lederle Laboratories Division, American Cyanamid Co., 1954. HupbLesoN, I. F. Brucellosis in Man and Animals. New York: Common- wealth Fund, 1943. Hunter, C. A. and CorBert, B. L. Flocculation Tests for Brucellosis. J. Immunol. 77 :232-241, 1956. CasTANEDA, M. Ruiz. A Practical Method for Routine Blood Cultures in Brucellosis. Proc. Soc. Exper. Biol. & Med. 64:114-115, 1947. West, D. E., and Borman, E. K. The Culturing of Blood Clots for Brucella Organisms. J. Infect. Dis. 77 :187-192, 1945. HuppresoN, I. F. Personal communication. Damon, S. R.; BunneLr, Doris; Gay, KataLEeN ; and HurcHiNGs, L. M. Primary Isolation of Brucella from Human Blood Clots. Pub. Health Rep. 67 :883-887 (Sept.) 1952. FercusoN, W. Optimal Carbon-Dioxide Tension for Primary Isolation of the Gonococcus. Am. J. Syph. 29:19-55, 1945. WEED, L. A. The Use of a Selective Medium for Isolation of Brucella from Contaminated Surgical Specimens. Am. J. Clin. Path. 27:482-485, 1957. CruicksHANK, J. C. A Simple Methed for Testing Dye Sensitivity of Brucella Species. J. Path. & Bact. 60:328-329, 1948. Pacuro, G., and pEMELLo, M. TH1AGo. A Urease Test for the Differentia- tion of Brucella suis, J. Bact. 59:680—-691, 1950. Hupbreson, I. F. The Diagnosis of Brucella Infection in Animals and Man by Rapid Macroscopic Agglutination. Tech. Bull. No. 123, Michigan Agr. Exp. Sta, 1932. FrrcH, C. P.; DoxuaM, C. R.; Bissor, L. M.; and Boyp, W. L. Studies of the Test-Tube Agglutination Test for the Diagnosis of Bang’s Disease (Contagious Abortion). Tech. Bull. No. 73, Univ. of Minnesota Agr. Exp. Sta., 1930. FrrcH, C. P., DonuaMm, C. R,, and Boyp, W. L. Further Studies of the Test-Tube Agglutination Test for the Diagnosis of Bang’s Disease (Con- 356 BRUCELLOSIS tagious Abortion). Tech. Bull. No. 77, Univ. of Minnesota Agr. Exp. Sta., 1931. 23. Standard Methods for the Examination of Water and Sewage (10th ed.). New York, N. Y.: American Public Health Assn., 1955. 24. McFarranp, J. The Nephelometer. J.A.M.A. 49:1176, 1907. 25. Gates, F. L. A Method of Standardizing Bacterial Suspensions. J. Exper. Med. 31:105, 1920. 26. SCHIRGER, A., et al. Brucellosis: Experiences with 224 Patients. Ann. Int. Med. 52:827-837, 1960. 27. Jowroan, C. F., and Boris, I. H. Brucellosis and Infection Caused by Three Species of Brucella. Clinical, Laboratory and Epidemiological Observa- tions. Am. J. Med. 2:156-167 (Feb.) 1947. 28. Borts, I. H., et al. A Milk-Borne Epidemic of Brucellosis, Caused by the Porcine Type of Brucella (Brucella suis) In a Raw Milk Supply. J.A.M.A. 121:319-322, 1943. CHAPTER 13 PASTEURELLA INFECTIONS 1. Pasteurella pseudotuberculosis A. The Agglutination Test B. Characteristics of P. pseudotuberculosis 1. Morphology and Staining 2. Cultural Characteristics 3. Biochemical Reactions 4. Phage Tests 5. Animal Inoculations C. Evaluation of Findings 11. Pasteurella multocida A. Methods of Isolation 1. Culture 2. Animal Inoculation B. Identification of Isolates 1. Morphology 2. Growth and Biochemical Activities 3. Serological Identification 4. Pathogenicity Tests C. Serological Tests D. Allergic Skin Tests E. Interpretation of Results 111. Pasteurella tularensis A. Collection and Handling of Specimens B. Bacteriological Examination 1. Culture 2. Animal Inoculation 3. Culture of Animal Inoculation Material 4. Characteristics of P. tularensis C. Serological Examination 1. The Agglutination Test 2. The Precipitin Test (Modified Ascoli) D. Evaluation and Reporting of Results IV. Pasteurella pestis A. General Characteristics of Human Plague B. Principles of Identification of Plague 1. In Arthropods and Rodents 2. In Man C. Collection and Handling of Specimens 1. Precautions 2. Collection of Specimens and Preparation of Films 3. Collection of Fleas and Inoculation of Flea Material 357 358 PASTEURELLA INFECTIONS D. Identification of P. pestis and Its Antigens 1. Procedures for the Isolation of P. pestis a. Cultivation b. Animal Tnoculation 2. Storage and Disposal of Specimens and Cultures 3. Serological Examination E. Report and Its Evaluation References I. PASTEURELLA PSEUDOTUBERCULOSIS Since discovery by Pfeiffer in 1889, the microorganism Pasteurella pseudotuberculosis has been isolated from a wide range of animal species. In addition, up to 1948 about a score of human infections, probably of gastrointestinal origin, were recorded.! Since that year the organism has been isolated with increasing frequency from human cases of abscess-forming lymphadenitis.>® In experimentally in- fected rabbits acute or chronic manifest infection or merely a rise of the specific agglutination titer without corresponding pathological lesions has been observed. Apparently, therefore, depending on the virulence of the invaders and the resistance of the hosts, pseudotuberculosis can lead to mani- festations ranging from latent infection to a fulminant acute form.” There can be no doubt that in certain countries a vast reservoir of pseudotuberculosis exists in many species of wild animals. Moreover, the infection has been found in animals bred or kept by man—for example, in mink, turkeys, cats and canaries. Under these circum- stances suspicious cases, for instance those with symptoms suggesting gastroenteritis, typhoid fever, or chiefly appendicitis, ought to be looked for by the clinician as well as by the laboratory worker.2-%? In the case of specimens such as pus, feces or autopsy material, cultivation must be resorted to and the organisms isolated must be subjected to biochemical and serological identification tests, and/or animal inoculation to the extent necessary. Until Daniels cultivated P. pseudotuberculosis from the stool of one patient on Leifson’s medium, cultivation from the feces had proved successful only in heavily infected animals. In human pseudotuberculosis, which may masquerade as acute or subacute appendicitis, main reliance has to be placed on isolation of the organism from mesenteral lymph nodes or on agglutination tests. A. The Agglutination Test H, O and R antigens!®-!* and in certain strains also an exo- toxin®®1% have been defined. Five specific O types have been classi- PASTEURELLA INFECTIONS 359 fied.”1% The O antigen of Type II is related to the somatic antigen 4 of Salmonella Group B%-'* and the O antigen of Type IV to the somatic antigen 9 of Salmonella Group D.* Serological cross-reactions occur with plague bacilli,***® but these should be avoidable if pure O sera are used for the agglutination tests. Production of O sera—Cultures of the five O types are used for antigen and serum production.* Forty-eight hour cultures grown at 22° to 30° C are washed off with physiological salt solution and the suspensions adjusted to the turbidity of Wellcome 4 or McFarland 8 standard. The bacterial suspensions are then heated at 100° C for 2% hr. After it has been proved that the suspensions are sterile and agglutinable only in known sera developed in rabbits against the corresponding types, the antigens are ready for use. The salt sensitivity of the strains (in 0.85% and 3.5% NaCl solution) should be tested by incubating 1 ml amounts of the antigen at 35° or 52° C (incubator or water bath) overnight. Spontaneous agglutination should not occur and the turbidity should be uniform. However, should pseudoagglutination be present, it often can be overcome by serial passages in semisolid agar at room temperature or in mice. Increasing doses (0.25, 0.5, 1.0, 1.5, 2.0 and 2.5 ml) are used for the intravenous injection of rabbits at 4 day intervals. As a rule, satisfactory agglutinin titers can be obtained in this manner. The test animals are bled between 7 and 10 days after the last injec- tion and their serum is inactivated by heating it for 30 min at 56° C in a water bath. After addition of merthiolate (1:10,000) the serum is stored in the refrigerator, preferably in vaccine bottles with diaphragm stoppers. Tube agglutination—For the detection of O and H agglutinins, use 48 hr cultures of the five type strains grown at 22° C on infusion or proteose agar, either living or killed by washing off with 0.25-0.5 per cent phenol or 0.2-0.25 per cent commercial formalin solution. Adjust density of the suspensions to that of Wellcome 2 or Mec- Farland 4 standard. The phenol- or formaldehyde-killed antigens, if stored in the refrigerator, keep for around 3 to 6 months but must be tested now and then for spontaneous agglutination. Combine 0.5 ml amounts of the type antigens with 0.5 ml of suitable serum dilu- tions and incubate the tubes for about 20 hr at 52° C in a water bath or at 35° C in an incubator. * Can be obtained from the National Collection of Type Cultures, Colindale, London, or from the Central de Collections des Types Microbiens, Lausanne, Switzerland. 360 PASTEURELLA INFECTIONS For the detection of O agglutinins the antigens must show a uniform turbidity after heating at 100° C for 2}% hr. Tests with the sera of more than 100 pseudotuberculosis patients with symptoms of acute appendicitis showed agglutinin titers between 1:80 and 1:12,800 (mostly in the range of 1:160-1:640) against living antigens. Heated antigens, on the contrary, were agglutinated only at very low titers or not at all.#® On account of the antigenic relationship and the presence of serological cross-reactions between Type IT and Factor 4 of the Salmonella B group, and between Type IV and Factor 9 of the Salmonella D group, a serological diagnosis of pseudotuberculosis can be made only with patients’ sera that have been absorbed with the corresponding strains of the Salmonella B or D groups. Slide agglutination: Slide agglutination tests using suspicious colonies or pure cultures made with type-specific O sera form an easy and rapid means of identifying strains of P. pseudotuberculosis and their type. The technic to be used is analogous with that used for the Salmonella groups. However, in order not to be misled by false positives, the isolates must be tested with P. pseudotuberculosis sera Types IT and IV absorbed with Salmonella strains of B or D group, and also subjected to biochemical tests as described below. B. Characteristics of P. pseudotuberculosis 1. Morphology and staining: Large rods and coccoid forms, 0.5 p by 1.5 to 5.0 p, occur in liquid media, mostly ovoid or coccoid forms on solid media. Neither spores nor definite capsules are formed. Most strains can be rendered motile by serial subcultivation on swarm agar®?814 at room temperature. For staining, all stains used on Enterobacteriaceae are suitable; like the latter microorganisms, the pseudotuberculosis bacilli are Gram-negative. 2. Cultural characteristics: Pseudotuberculosis bacilli grow aero- bically and anaerobically on all the usual media. For their isolation from heavily contaminated material, Leifson’s desoxycholate citrate agar (CM No. 57) has been recommended,® but, with one exception,” it has not been possible to isolate with the aid of this medium pseudo- tuberculosis bacilli from human specimens, such as lymph nodes, bile or feces.* The optimum temperature of incubation proves to range between 22° and 30° C. After 24 hr incubation, colonies of pinpoint size develop which a day later attain a diameter of about 1-2 mm. The colonies may correspond in appearance to the smooth, the intermediate or the rough type, but all of these can yield organisms that react PASTEURELLA INFECTIONS 361 specifically with O sera. In broth, S (smooth) strains produce uni- form turbidity and true R (rough) strains a granular type of growth, while intermediate strains show turbidity in addition to a granular sediment. 3. Biochemical reactions: As recorded in the literature (see the studies of 186 and 240 strains),”%18 pseudotuberculosis bacilli produce acid, but no gas, in media containing the following carbohydrates or alcohols: arabinose, galactose, glycerol, glucose, levulose, maltose, mannitol, rhamnose, trehalose, xylose, and usually but not invariably also in media containing melibiose, adonitol, salicin or dextrin. Acid formation does not take place in the case of erythritol, inositol or lactose, nor, with very rare exceptions, does it take place in dulcitol, inulin or sucrose; with more frequent exceptions, it does not take place in amygdalin, raffinose or sorbitol. The methyl red, potassium nitrate and urease reactions are positive. Hydrogen sulfide is not formed or is formed only in small amounts. The indole and Voges- Proskauer reactions are negative. Gelatin media are not liquefied. Table 1 shows the results of some tests distinguishing P. pseudo- tuberculosis from other microorganisms which it may more or less resemble. Details on differentiation between the microorganisms mentioned, on the preparation of the necessary media, and on the test Table 1—Differentiation of P. pseudotuberculosis and Other Gram- Negative Bacilli with Which It May Be Confused* P. pseudo- Test tuberculosis P. pestis P. multocida ~~ Salmonella | | Motility Melibiose Salicin Sorbitol Rhamnose Sucrose Urease Hydrogen sulfide Indole Methylene blue reduction Methyl red reduction Esculin19 Beta galactosidasel8 +++ * | + * | Wok 44 * LL * * * +11 1k+1 Ot * + AHH +++ + +++ + I+ © O =unknown. | *=rare exceptions from typical behavior. =positive reaction more frequent. pr F =negative reaction more frequent. 362 PASTEURELLA INFECTIONS methods to be used will be found in papers by Girard,?*2! Baltazard and associates? and Knapp,* and in the monograph on plague by Pollitzer.?2 As shown in these publications, up to the present time it has been impossible to make a fully reliable distinction of the micro- organisms mentioned by any of the enumerated carbohydrates or test methods alone, so that the combined results of several tests have to be evaluated. The evidence thus far in regard to the methylene blue reduction test?? and with melibiose is insufficient. 4. Phage test: All types, I to IV, of P. pseudotuberculosis are specifically lysed by the PST phage.242" The phage test is used to differentiate rapidly members of the Pasteurella group. 5. Animal inoculation: Guinea pigs are invariably killed by viru- lent strains of P. pseudotuberculosis. If inoculated intraperitoneally with 0.25-1.0 ml of a 48 hr broth culture, the animals die within 6 to 8 days, showing evidence of septicemia at autopsy. After injection of specimens obtained directly from infected individuals, death occurs within 10 to 30 days. Animals inoculated subcutaneously die within 2 or 3 weeks and the autopsy findings are chiefly characterized by the presence of nodules in the liver and spleen which are similar to lesions produced by Mycobacterium tuberculosis. Mice, the animals of choice for work with pseudotuberculosis toxin, often prove re- sistant to nontoxic strains. Rats, though resistant to pseudotubercu- losis infection, may succumb to the toxin of certain strains.”815 Avirulent strains can be identified only by biochemical and serological tests. For the routine diagnosis of pseudotuberculosis in human speci- mens the method of choice is intraperitoneal or intramuscular inocu- lation of guinea pigs. C. Evaluation of Findings A complete diagnosis should be based on isolation of the organism and serological and biochemical confirmation, as well as on tests for pathogenicity. According to recent observations, human pseudotuberculosis occurs not only in a rare septicemic or septic-typhoid form, but also in an appendicitis-like form, recorded particularly in boys. During the period 1954-1957 this new syndrome was diagnosed in 117 instances: in 13 through isolation of the microorganisms from the lymph nodes; in 2 through blood cultures; and in 94 through the correlation of the clinical signs, the findings at operation, and histological exam- ination or agglutination tests. In 7 patients with typical clinical and PASTEURELLA INFECTIONS 363 histological findings, agglutinins were not detected or were found only in titers from 1:20 to 1:40, and in one instance the histological re- action in the lymph nodes appeared to be absent.? Additional cases were reported by other authors, as noted by Knapp.#282° In most in- stances human infection was caused by strains of Type I. Antibody titers ranging from 1:80 to 1:12,800 will be found when the symptoms indicating pseudotuberculosis infection become mani- fest. Unless complications arise, depending upon the initial level of the titers, agglutination becomes negative after a period of from 1 to 4 months. Agglutination titers becoming manifest in the case of Type II or Type IV strains must be evaluated cautiously in view of the serological relationship between these types and Salmonella Groups B or D. In such cases close cooperation between the clinician and the laboratory workers is essential. Serological examination of 244 strains of P. pseudotuberculosis derived from different sources in Europe showed that 177 strains be- longed to Type I, 46 to Type II, 17 to Type III, 2 to Type IV and 2 to Type V.2! The possibility that strains belonging to new types will emerge in the future cannot be ruled out. The future incorporation of examinations for evidence of infection with P. pseudotuberculosis into the routine serological investigations of sera from patients with symptoms of typhoid and paratyphoid fever or of appendicitis, and in systematic surveys as well, will throw more light on the role played by this microorganism in human and animal disease. Il. PASTEURELLA MULTOCIDA The animal pathologist has reported, under the obsolete collective name “hemorrhagic septicemia” or the more specific “pasteurellosis,” infections of farm animals and domestic poultry associated with P. multocida or P. septica. In some cases this organism is evidently re- sponsible for the diseased state being investigated, but in others it seems to be merely a secondary invader. Some outbreaks display all the features of a serious epizootic, but single, isolated, sporadic cases do occur. Formerly it was customary to refer to the organisms of the pasteurellosis group according to the host from which they were isolated, 393! such as Pasteurella (Bacillus) avicida, P. boviseptica, P. suiseptica, or P. muricida. By biochemical reactions, but more decisively by serological reactions, members of the single species P. multocida can be subdivided into types dominated by a few commonly occurring members (four designated by Carter and Byrne? as A, B, Cand D). 364 PASTEURELLA INFECTIONS Pasteurellosis is a contagious, usually septicemic, disease charac- terized by hemorrhages of the mucous membranes and manifesting either high mortality or high morbidity rates. It is encountered in all classes of fowl as fowl cholera,®® in cattle in tropical countries, in reindeer, in water buffalo and bison.?*-3* P. multocida is not infre- quently found in the respiratory tract of calves or grown cattle with bronchopneumonia and in sheep and hogs with pneumonia. It also plays a role in infections among laboratory animals—rabbits,® mice®®37 and guinea pigs.?® Pasteurella is frequently found in the upper air passages of apparently healthy cattle, horses, swine, sheep, fowl, dogs, cats and rats.®® A pasteurella-like organism, P. pneumo- tropica, well adapted and latent in the lungs of mice, may be demon- strated through rapid serial intranasal inoculation of mice.*%4! Under the insult of virus infections or stress in the host, the saprophytic strains may assume pathogenic qualities. However, all pasteurellae are highly and more or less specifically pathogenic; the range of susceptible species is wide. Saprophytic strains that have acquired marked virulence through passage may rapidly resume their sapro- phytic qualities.*? More human infections are being reported. Diseases caused by P. multocida are widespread in the United States, Europe and prob- ably elsewhere.3943-47 An incomplete survey of published and un- published records brings the total up to 162 cases, with 5 deaths. Of this number, over 60 occurred in France between 1944 and 195446 and 30 in the United States. Approximately 50 per cent followed animal bites (principally cat bites,*8 rarely bites of both dogs and cats) ; 10 per cent were posttraumatic and in the remainder the means of infection is obscure. The recent finding of P. multocida in the sputum of patients suffering from bronchiectasis*®%® suggests that that organism should be more often sought in this disease. All of the cryptogenic cases were related to disease of the respiratory tract (e.g., pleurisy or empyema®®5!) implying latency of the organism in the mucosa of the rhinopharynx. The occurrence of Pasteurella meningitis*647.52.53 following trauma to the face or head suggests that at the time of trauma, Pasteurella may have been in the cranial sinuses.’*%¢ The microorganism has been found in the saliva of in- fected cats and other carnivores, and even on vegetables contaminated with animal excreta and on the meat from fowl which died of fowl cholera. These have been considered sources for human infection.*® A. Methods of Isolation P. multocida may be isolated from sputum, saliva, pleural exudate, pus, spinal fluid, blood, or tissues such as lung or lymph nodes. PASTEURELLA INFECTIONS 365 Specimens to be shipped some distance to a laboratory must be iced. Swabs of pus must be kept moist with a few drops of serum or blood broth. Tissues may be immersed in a 4 per cent solution of boric acid to prevent dehydration and to retard the growth of contaminants. Examination of films of pus, blood or exudate shows Gram-nega- tive rods about 1.5 to 2 pu long ; the staining may or may not be bipolar. All primary isolations must be made on blood agar or chocolate agar plates incubated at 35° C or by inoculation of animals, prefer- ably mice. 1. Culture—It is imperative that fairly large samples of the specimens be enriched in serum or blood broth and held for reference, because frequently primary cultivation on solid medium fails. These reference tubes may be held in the refrigerator or incubated at 35° C. Tissues should be triturated in a mortar with physiologic saline solu- tion or serum broth. Growth may or may not appear after 24 hr. At 22° C on plain agar, culture is rarely successful. It may also be overgrown by con- taminants. Reseeding from the serum broth reference tube as a rule will furnish good growth after 24 to 48 hr of incubation if P. multocida is present in the specimen. Some colonies may be about 0.5 mm in diameter, raised, opaque, bluish grey, with a sharply de- fined edge, and the growth may have a disagreeable earthy odor. Others may be discrete, small, dewdrop-like and colorless. Some isolates are large, raised, and show a marked tendency to coalesce; they become viscous and show a raised ring at the periphery after 72 hr of incubation. The dissociation pattern2:23:325758 with respect to colonies, anti- genic structure, capsule formation, reaction to acriflavine and hemag- glutination®® may be grouped as follows: 1) Mucoid (M), probably a phenotypic characteristic. Moist, white to reddish, opaque fluffy fluorescent colonies measuring up to 3 mm on serum agar in 24 hr at 35° C; cells well capsulated (+ +), slimy precipitate in acriflavine. Hemagglutination type A; inagglu- tinable in standard agglutination test. Highly pathogenic for mice and rabbits in small doses given intraperitoneally; some strains lack a toxic component and require 3 days to cause fatal infections. 2) Smooth (S) fluorescent—Diffuse growth. Hemagglutination type B. Greenish iridescent colonies measuring 1 to 1.5 mm after 24 hr. No flocculation in acriflavine. Capsule, plus. Cells remain in suspension. Pathogenic for mice and rabbits. 366 PASTEURELLA INFECTIONS 3) Smooth (S), nomiridescent—Intermediate antigenic type S. Capsulated. Discrete, small colonies. Blue colonies, noncapsulated. Partial flocculation in acriflavine. Hemagglutination type D. 4) Smooth (S), antigenic type SR—Granular, blue. No capsule. Coarse clumping in acriflavine. Hemagglutination type C. 5) Rough (R). Colonies measure 1 mm after 24 hr. Resemble smooth colonies but are drier and are difficult to emulsify. Agglu- tinate specifically, No capsule. Flocculation in acriflavine, Non- pathogenic for mice. 6) Dwarf. Rare. Pinpoint colonies near M colonies. On 5 per cent blood agar, hemolysis does not occur, but the colony is brownish. When the blood is reduced to 3 per cent, partial hemol- ysis may be observed. Certain isolates from the lungs of sheep and cattle with pneumonia (described as P. hemolytica) differ from P. multocida in that they produce beta hemolysis on rabbit or horse blood agar upon initial isolation, which property may disappear on sub- culture. This type ferments lactose and maltose and coagulates milk.%%-62 The above considerations pertain also to some isolates from the human respiratory tract. 2. Animal inoculation—The serum broth emulsion of tissues, pus or exudates is inoculated, in varying amounts not exceeding 0.5 ml, intraperitoneally into several white mice. When the specimen contains highly virulent P. multocida the animal dies within 24 to 48 hr and the microorganism may be readily demonstrated in and culti- vated from the heart blood or organs on blood or serum agar. If the microorganism is not capsulated and is of low virulence, the infection may proceed for several days. It is then advisable to sacrifice the mice and attempt cultivation from the peritoneal surfaces or from a few drops of peritoneal exudate, B. Identification of Isolates 1. Morphology—The microorganisms vary from small, short oval forms to coccobacilli with convex sides and rounded ends. They range in length from 0.3 to 2.0 p and in diameter from 0.15 to 0.25x and they appear singly or in pairs, small chains or clusters. Active, healthy organisms from cultures stain easily and diffusely with aniline dyes and are Gram-negative; but in films from animal tissues and fluids, the ends of the rod stain more intensely than the central portion. Films from diseased tissues fixed with alcohol and stained with methylene blue and fuchsin (so-called Wayson stain— PASTEURELLA INFECTIONS 367 see Section IV on plague) or any polychromatic eosin-methylene blue stain reveal the bipolarity. This staining reaction is of value in pre- sumptive diagnosis and is particularly important in the examination of inoculated test animals. Serologic Type B (Type 2)*° forms discrete rods two to four times as long as Type A or C. This charac- teristic alone differentiates this type from the others. Spores are not formed and P. multocida is not motile. In the M phase it has definite capsules that cannot be stained by the usual methods. Moist or dry India ink preparations show the capsule as a clear halo about the bacterium.%* Polysaccharide dyes such as Alcian blue, 8GS, phthalocyanin and Monastral blue tinge the capsular material of certain strains in the peritoneal exudate of infected mice distinctly but not intensely. 2. Growth and biochemical activities—P. multocida grows aero- bically on solid medium. In broth it produces turbidity or a slimy granular deposit. Neither gelatin nor coagulated serum is liquefied. Preliminary identification is made by inoculating urease test medium and triple sugar iron (TSI) agar with a loopful of the colonies to be identified. P. multocida is urease-negative and produces acidity of the slant, but an acid butt only after 48 to 72 hr in the TSI agar. From the TSI agar slant, inoculate 0.2 per cent tryptone (Difco) medium containing glucose, xylose, arabinose and dulcitol, peptone water and desoxycholate agar. This microorganism does not grow on desoxycholate agar but forms indole and H.S in varying small amounts. All members of the P. multocida group reduce nitrate and form ammonia. The catalase test is positive; the Voges-Proskauer reaction, negative. Little acid, but no gas, is produced from glucose, galactose, fructose, mannose, sucrose, sorbitol and mannitol. Some correlation between action on xylose, arabinose and dulcitol and the serologic types has been reported.5™#* As a general rule the Type A strains attack arabinose and dulcitol but not xylose, Types B and C ferment xylose but not arabinose or dulcitol. To identify the organism further and to guide the therapeutic pro- cedures to be instituted by the physician early in the course of the infection, test the great sensitivity of the isolate to penicillin and the tetracycline compounds by the paper disk or the test tube serial dilu- tion method.®* According to recent reports,**%¢ 0.3 to 0.4 units of penicillin or aureomyecin in a concentration of 0.1 to 1 mg per ml as a rule inhibits growth of isolates from human infections. 3. Serological identification—Studies reported recently®® have furnished considerable information about the serological and im- 368 PASTEURELLA INFECTIONS munological characteristics of P. multocida. The usual agglutination test proved unsatisfactory because it depends on an immunological reaction involving specific and nonspecific factors. - Strong cross- reactions made the unequivocal identification of types difficult. Since the S and F strains possess a type-specific capsular antigen, a routine typing technic using the precipitin and capsular swelling tests has been developed.%® In the hands of experienced workers the quellung reaction can be recognized readily. The precipitin test developed by Carter and Byrne®? also gives dependable information. The capsular material extracted with salt solution is adsorbed on human type O erythrocytes. These cells are specifically agglutinated when appro- priate dilutions of specific typing antisera are added (hemagglutina- tion test®7). This test might yield highly specific results, but many mucoid and uncapsulated strains are not typable. Furthermore, potent type-specific rabbit antiserum is available at only a few research laboratories (among them, the laboratory in Department of Bacteri- ology, Ontario Veterinary College, Guelph). Independent of animal or geographic source, four different serotypes, A to D, have been identified. The published records on the relation of serological types to animal species warrant the recommendation that in the future no effort be spared to type the isolates of P. multocida from human in- fections. Only Type C strains have been recovered from cats and dogs®7; Type A strains predominate as the cause of fowl cholera. 4. Pathogenicity tests—A complete study of an isolate from a human source includes pathogenicity tests on at least four laboratory animals: mice, guinea pigs, rabbits and pigeons. The injections are usually made subcutaneously with varying amounts of culture ma- terial. Strains of low virulence may be injected intravenously into rabbits. Pathogenicity varies according to the dissociation pattern of the culture, as outlined under Section IT A 2. As a general rule isolation by the inoculation of mice supplies adequate information about pathogenicity, so that special tests are not required. C. Serological Tests The conventional agglutination or complement-fixation test of the sera of animals or man infected with P. multocida as a rule has furnished little information of value. It is reported that in subacute or chronic human infection the homologous strain may be aggluti- nated in dilution as high as 1:3,200 one week after isolation of the microorganism from a pleural effusion of several weeks’ standing.®® When the isolate is autoagglutinable the complement-fixation test may establish some relationship between the infective microbe and the PASTEURELLA INFECTIONS 369 response of the host. For example, a titer of 1:384 was secured during the second month of an infection.® On the other hand, in other re- ports on subacute or chronic infection antibodies could not be demon- strated by methods that failed to take into account the immunological complexities of the capsulated and uncapsulated dissociates readily de- veloping in the test cultures. For practical purposes an infection with this organism can be identified serologically only if the newer typing reactions are taken into consideration. D. Allergic Skin Tests Atypical phlegmonous cutaneous inflammation without bite or scratch wound may be diagnosed with the skin test of Reilly™; it induces local and focal reactions and exerts therapeutic effects. E. Interpretation of Results A presumptive diagnosis is warranted when the specimen (a carcass from a poultry yard, material from pneumonic consolidations, pus or exudate, or sputum from mammals) reveals numerous small coccoid, oval bacilli, bipolar-staining and growing readily at 35° C on blood or serum agar, but not on desoxycholate agar, and forming slight acidity but no gas in glucose medium. A complete identification must include tests for urease activity, sensitivity to penicillin, pathogenicity for mice and rabbits and, when suitable antisera are available, deter- mination of the type of P. multocida in precipitin, capsular, quellung and hemagglutination tests. Ill. PASTEURELLA TULARENSIS The laboratory diagnosis of tularemia rests upon isolation of P. tularensis and the detection of antibodies. In general, results of sero- logical tests serve to confirm clinical impressions but are usually obtained too late in the illness to guide management of cases, On the other hand, isolation and identification of P. tularensis may be ac- complished relatively early and be of great value to the clinician in his treatment of the patient. This is especially true in instances where modified Ascoli tests are employed to identify the agent isolated. A. Collection and Handling of Specimens In order to collect specimens of real value to the laboratory, it is necessary to review briefly some of the clinical aspects of the disease in man, especially with relation to bacteremia and localization of the microorganisms.™-7® The onset of illness is sudden and is associated 370 PASTEURELLA INFECTIONS with a septicemia, which continues for 7 to 10 days. Thereafter the microorganisms are localized, and in only a few instances does secondary septicemia develop. Thus attempts to isolate P. tularensis from blood should be limited to the first 7 to 10 days after onset except in cases which manifest signs and symptoms indicating the presence of secondary septicemia, such as rapid enlargement of the liver and spleen, a widely remittent or continuous high fever, hyperpnea, cyanosis, meningitis or diar- rhea.”8% Tt is best to look for microorganisms in specimens from areas in which they are localized. Conjunctival scrapings in cases of oculoglandular tularemia readily yield the microorganism. The local lesions in the ulceroglandular type of illness contain large numbers of the microorganisms as do the sputum and pleural fluid in cases of typhoidal or pulmonary tularemia. Even in the absence of evidence indicating involvement of the respiratory tract, P. tularensis may be isolated from the sputum. Exudate from or scrapings from the wall of lymph nodes draining the site of initial infection may be employed for isolation; in untreated patients organisms may persist in these areas for months. Since the procedures for isolating and identifying P. tularensis are fraught with real danger to the technician, there has been a regret- table tendency to stress this so much as to discourage qualified indi- viduals from undertaking this work. When reasonable precau- tions are employed, however, the risk is minimized. Such precau- tions include attempts to decrease the probability of producing air contamination. ‘We have not found it necessary to employ masks, gowns, goggles or other protective clothing. Animals to be autopsied should be first immersed in a strong solu- tion of cresol or other disinfecting agent. Inoculating loops should be heated gradually rather than thrust into a flame to sterilize them, because the droplets of infected material dispersed in the air when loops are heated too quickly may be a source of infection. It has been our experience that when large volumes of virulent organisms are handled and when rabbits are used as experimental animals, infections are more likely to occur among laboratory personnel. Therefore, in diagnostic procedures rabbits should not be employed®? and in prepar- ing antigens smooth, avirulent strains should be selected.®? Specimens for serological examination should be collected with due regard to the fundamental principle of demonstrating rising antibody titers. An acute-phase specimen should be collected as soon as the patient is hospitalized or placed under treatment, and a second blood specimen 2 or 3 weeks after onset of illness. The development PASTEURELLA INFECTIONS 3N of antibodies is frequently delayed ; consequently a specimen collected after the third week of illness may be more informative than one col- lected earlier. It is best to collect serum samples in a sterile manner, thus eliminating the need for the addition of preservatives when they are to be stored or shipped to a central laboratory. No special pre- cautions need be taken in shipping the sera. B. Bacteriological Examination 1. Culture—P. tularensis may be isolated directly on culture media from infected materials, but often these materials may contain so many contaminating agents that the procedure is rendered extremely difficult or impossible. Pleural fluid, blood or pus from a local lesion which still has skin intact over it may be cultured on plates or slants of glucose cystine blood agar (CM No. 115). Even though the etiological agent has occasionally been isolated on other media, their use is not recommended, since glucose cystine blood agar has proved to be such an excellent medium for the isolation of P. tularensis. 2. Animal inoculation—In general, attempts to isolate the microorganisms by direct inoculation of artificial media are unsuc- cessful in comparison to those employing laboratory animals. The animals of choice are guinea pigs, mice or hamsters. These may be inoculated either subcutaneously or intraperitoneally with the speci- mens obtained from the patient. The subcutaneous route for guinea pigs has the advantage that the regional lymph nodes at the site of injection become enlarged and suggest the presence of P. tularensis in the material used to infect the animal. If the material is grossly con- taminated, it may be rubbed into the broken or unbroken skin of the laboratory animal, or penicillin may be added to the material before it is injected. The method of scarifying the skin is simple and may be employed with confidence. Animals usually survive 5 to 7 days but may succumb as early as 48 hr after inoculation. Guinea pigs in- jected subcutaneously show enlarged caseous lymph nodes at the site of injections, enlarged spleens, and necrotic foci on the liver and spleen. The lesions are typical in guinea pigs; they are less typical in mice and hamsters. For inoculation into animals, suspend specimens in physiological salt solution, using in the case of sputum about 10 to 20 volumes of the solution. When making the suspension, use a syringe fitted with a 20 gauge needle. Inoculate amounts varying from 0.1 to 0.5 ml subcutaneously into four to six white mice and one or two guinea pigs. Kill one or two mice after guinea pigs have developed enlarged lymph nodes. Enlargement of the regional lymph nodes in inoculated guinea 372 PASTEURELLA INFECTIONS pigs serves as a cue that at this time the number of microorganisms in the mice will be sufficiently large to make detection relatively easy. Remove liver and spleen. Culture heart blood and fragments of liver and spleen of the mouse or mice on plates or slants of glucose cystine blood agar (CM No. 115). Rub the cut surface of fragments of liver and spleen vigorously over the entire available surface of agar, using forceps or a heated bacteriological loop to grip the tissue. Pre- pare impression films of spleen and stain with Wayson’s stain; boil remainder of pooled liver and spleen with 2-3 volumes of salt solu- tion for 5 min for a modified Ascoli test. Filter extract through filter paper (Whatman No. 40). Perform precipitin tests with specific immune serum. The presence of precipitinogens in boiled extracts of tissue establishes a diagnosis, for the method is both sensitive and specific. These procedures, if successful, allow early laboratory diagnosis of tularemia. Although microorganisms are easily detected in tissues of infected mice, too many personal factors enter into the interpreta- tion of impression films to allow a reliable diagnosis.®* Suggestive findings merely indicate the possibility of the presence of P. tularensis. Precipitinogens are more easily detected in mice than in guinea pigs. Frequently P. tularensis may be isolated on cultures made from tissues of mice killed early in the course of experimental infection, thus obviating examination of animals which die later. The organisms isolated are identified by agglutination tests. For the test to be of diagnostic value, the microorganisms should be agglutinated to the same end point as an established strain of P. tularensis. 3. Culture of animal inoculation material—The medium most suitable for general laboratory procedures is glucose cystine blood agar. This may be made from infusion agar prepared in the labora- tory, but commercial preparations are also adequate. At the Rocky Mountain Laboratory, dehydrated cystine heart agar (Difco) is em- ployed. The medium is removed from the autoclave and brought to a temperature of 60° C in a water bath. Sterile defibrinated rabbit blood is added to a concentration of 8-10 per cent, and the whole is kept at 60° C for 12% hr before dispensing. Absence of growth on medium without cystine is a significant differentiating characteristic of P. tularensis. 4. Characteristics of P. tularensis—The microorganism re- sponsible for tularemia is classified as P. tularensis in Bergey's Manual of Determinative Bacteriology. It is a Gram-negative, non- PASTEURELLA INFECTIONS 373 motile organism occurring as rods measuring 0.2 by 0.7 p or as cocci measuring 0.2 p in diameter. It is noncapsulated and does not form spores. P. tularensis is aerobic and grows well on glucose cystine blood agar, less well on glucose cystine agar and coagulated hen’s egg yolk medium. Liquid media such as that devised by Snyder and associates®® support growth well but are not sufficiently sensitive to constitute tools for diagnostic work. On glucose cystine blood agar, viscous gray colonies appear in 24-48 hr. These may attain 4 mm in diameter. On media containing blood, a green discoloration is pro- duced in the immediate vicinity of the colony. Glucose, glycerol, maltose, mannose, fructose and dextrin may be fermented by P. tularensis, with production of acid but no gas; however, these reac- tions are not essential for identification of the microorganisms in routine work. On media containing cystine, HoS is produced. Smooth and rough variants occur. One of the interesting characteristics of these microorganisms is their solubility in sodium ricinoleate or such brand-name detergents as Dreft. Growth of P. tularensis occurs and may be maintained in serial passage in embryonated hen’s eggs. Since the use of eggs offers no advantages over animals for the isolation of this bacterium, the method will not be discussed further, C. Serological Examination The serological test of greatest value for the diagnosis of tularemia is the agglutination test. The precipitin test employing thermostable antigens will also be discussed in view of its value in establishing an early diagnosis of tularemia. Specific immune sera should be available for comparative purposes in performing serological tests. These sera may be obtained from human beings who have recovered from tularemia or from animals immunized with killed suspensions of P. tularensis. Larger diagnostic laboratories or research laboratories interested in work on the prob- lem of tularemia can usually supply limited quantities of serum. Rab- bits serve as a good source of serum when immunized with suspen- sions of killed organisms. The suspension of P. tularensis employed as antigen in the agglutination test is suitable for immunizing rabbits for the preparation of antiserum. The rabbits are given 1.0 ml of the antigen intravenously on 3 successive days each week for 2 weeks. After a rest period of a week the animals are bled and the serum is separated and tested for the presence of agglutinins. If these are present in sufficient titer, the animals may be exsanguinated and the serum harvested and stored for future use. 374 PASTEURELLA INFECTIONS 1. The agglutination test a. Selection and maintenance of strain for production of antigen— A smooth, avirulent strain of P. tularensis which grows readily on artificial media is preferable, These features are found in Strain No. 38, which was originally isolated at the National Institutes of Health and may be obtained from the American Type Culture Collection. To maintain the strain, a large inoculum of microorganisms is spread over the entire surface of a number of slants of glucose cystine blood agar, the tubes are incubated at 35° C for 48-72 hr, and if growth is profuse, tubes are subsequently stored at 2°-10° C. Subcultures are made once a month. Paraffined cotton stoppers prevent undue drying of the medium. The microorganism may also be maintained in a frozen and dried state. b. Production of antigen—Dispense 90 ml of glucose cystine blood agar (CM No. 115) into the desired number of Blake bottles. Incu- bate at 35° C for 48-72 hr. Subculture to new slants, preparing one slant for each Blake bottle. Incubate for 24-48 hr. Add 3 ml of sterile physiologic salt solution to each slant, suspend the microorgan- isms in the fluid, and transfer contents of each tube to a Blake bottle. Distribute inoculum over entire surface of medium and incubate at 35° C for 48-72 hr. At the end of the incubation period a thick, confluent gray sheet of microorganisms has formed. Add 15 ml of 0.5 per cent commercial formalin in salt solution to each bottle and suspend the microorganisms by rocking gently. Remove sus- pension and centrifuge to sediment microorganisms. Wash twice with salt solution containing 0.5 per cent formalin. Suspend final sediment in 15-20 volumes of salt solution with formalin and store in a tightly stoppered container in refrigerator as stock suspension. For use in agglutination tests, dilute stock antigen with physio- logic salt solution to correspond with a turbidity of 500 by the silica standard or tube No. 4 of the McFarland nephelometer. c. Technic of agglutination tesi—Place nine agglutination tubes in rack. Pipette 0.8 ml of salt solution into the first tube and 0.5 ml into each of the others. Pipette 0.2 ml of the serum under test into the first tube and mix thoroughly (serum dilution in Tube 1=1:5). Transfer 0.5 ml of diluted serum from Tube 1 to Tube 2, mix, and repeat through Tube 9. Add 0.5 ml of diluted antigen to Tubes 1 through 8. Shake tubes vigorously. Final dilutions of serum are 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640 and 1:1280. Tube 9 is retained without antigen and saved for further dilutions of serum if PASTEURELLA INFECTIONS 375 end point is not attained. An antigen control of 0.5 ml salt solution and 0.5 ml antigen is included in each day’s tests. Incubate in water bath at 35° C for 4 hr or at 52° C for 2 hr. Place in refrigerator overnight. Read the following morning. d. Reading the test—Results are graded 1+ to 4+ for each tube. A water-clear supernatant with a compact sediment constitutes a 4+ reaction, a slightly turbid supernatant with a compact sediment a 3 + reaction, a turbid supernatant with a relatively easily dispersed sedi- ment a 2+ reaction, and the presence of floccules only, a 1 + reaction. Only 4+ and 3+ reactions are considered positive. Prozone reac- tions occur only infrequently. 2. The precipitin test (modified Ascoli)®¢ Antigens are prepared from suspensions of liver and spleen of in- fected animals or from suspensions of microorganisms in physio- logical salt solution. They may be extracted from such sources by heating or by the addition of diethyl ether. To extract antigen from tissue by heat, grind tissues in a mortar with sterile sand and add 2-3 volumes of salt solution. Place in auto- clave with flowing steam for 15 min or boil gently for 5 min. Either centrifuge the heated suspension at 4,200 rpm for 30 min or filter through filter paper (Whatman No. 40). Retain clear fluid. To extract antigen with diethyl ether, add 2 volumes to the suspen- sion of tissues or microorganisms in salt solution. Use either a separatory funnel or a cork-stoppered tube, depending on the quantity of material to be handled. Shake vigorously. Keep at room tem- perature at least 1 hr. Harvest the aqueous phase and centrifuge at 4,200 rpm for 30 min. Retain clear fluid. The test is more easily performed employing the capillary tube method, the results of which are similar to those of the conventional precipitin test. Dilute antigen in test tubes, making twofold dilu- tions in salt solution. Draw serum into capillary tube to about one- third the length of the tube. Wipe with gauze pledget. Draw a similar amount of antigen into the tube. Invert and insert end of tube into rack containing plasticine or modeling clay. Clean outside of tube with water and wipe clean. Incubate at 35° C for 2 hr, transfer to refrigerator, store overnight, and read for the presence of precipi- tate the following morning. This procedure is used for the titration of antigen contained in tissues of animals or suspensions of microorganisms, By diluting serum rather than antigen, the titer of precipitins in sera may be de- 376 PASTEURELLA INFECTIONS termined. The precipitate may develop within a few moments after antigen and serum have been placed in contact, but usually not until the mixture has been refrigerated. The tests are read as positive or negative, depending upon whether or not a precipitate is formed. The relative amount of precipitate in positive tubes is not considered. Prozone phenomena do not occur. The isolation of P. tularensis from a patient either directly on culture media or by inoculation of laboratory animals confirms the diagnosis of tularemia. Demonstration of precipitative properties in tissues of laboratory animals inoculated with diagnostic materials from patients also constitutes a confirmation of the clinical diagnosis. In our experience, the test has yielded specific results; there have been no reactions against P. tularensis in sera containing antibodies specific for brucellosis, typhus fever, Rocky Mountain spotted fever, or plague; and antigens prepared from Brucella abortus, Br. suis, Br. melitensis, P. septica, P. pestis and Shigella dysenteriae produce no reactions when tested against the serum of tularemia patients. Furthermore, in our experience when precipitin tests of tissue ex- tracts are positive with P. tularensis serum, cultures of the tissues in- variably give corresponding results. The precipitin titer of extracts from animal tissues may be as high as 1:256, but ordinarily titers of 1:32 are encountered. However, even titers of 1:2 or 1:4 are of diagnostic significance, D. Evaluation and Reporting of Results The validity of results obtained from agglutination tests will be related to the time at which sera are collected. A single specimen may yield no information of value although the titer of antibodies is high. High agglutination titers persist for a considerable time after a patient has recovered from tularemia. Thus it cannot be said that, on the basis of results obtained from study of a single specimen of serum, the presence of agglutinative properties suggests a diagnosis of this disease. A rising titer of antibodies must be demonstrated. Agglutinative properties do not develop during the first week of ill- ness and may not be present even 14 days after onset. Before anti- biotic therapy it was unusual for agglutination titers to exceed 1:5,120. Currently, however, they have been known to exceed 1:40,000. The possibility of encountering antibodies incited by vaccination against tularemia must be considered, but the circumstances sur- rounding activities in which immunization is attempted should cause the laboratory to suspect this source of confusion. The subject of PASTEURELLA INFECTIONS 377 cross-reactions, especially with sera from cases of brucellosis, has been discussed frequently in the past, and the occurrence of cross- reactions has been greatly emphasized. In our experience this diffi- culty has not been significant. IV. PASTEURELLA PESTIS A. General Characteristics of Human Plague Man commonly contracts plague either as the result of the bite of an infective rodent flea or through inhalation of infectious airborne droplets. However, infection may also result from handling infected tissues or carcasses of rodents, performing autopsies on human plague victims or from working with virulent cultures of P. pestis. Although the incidence of plague has been low during the last few years, the disease is still perennially occurring in man, in India and adjacent countries, parts of the Middle East, Central and South Africa, and South America. In most of these areas the permanent reservoir of infection is wild rodents and their fleas. This type of infection exists also in the United States in 17 of the western states. As long as plague remains restricted to these “natural” foci of the infection, only sporadic manifestations of the disease in hunters or in other persons entering the affected localities need be expected. However, at the periphery of these localities a spread of the infection to peridomestic and domestic rodent species is apt to take place and may lead to epizootics among the latter, which, in their turn, may cause serious and widespread epidemics in man. The usual outcome of infection contracted by man directly from rodents, or—far more commonly—through the fleas of the latter, is the bubonic type of plague. Moreover in the later stages of this plague a secondary plague pneumonia may evolve which in its turn may lead to primary pneumonic plague among those in contact with the suf- ferers. The sputum of patients with pneumonic plague, secondary or primary, contains the causative microorganisms in large numbers and is highly infectious in the form of airborne droplets. The case of a boy with a plague bubo who, thought to be infected by staphylococci, was treated with penicillin instead of a drug effective in plague and who therefore succumbed to the infection illustrates the great importance of an early and accurate clinical diagnosis. Pre- sumption of the disease must be confirmed promptly by laboratory tests. Adequate (but not excessive) therapy must be administered immediately when plague is suspected. Proper public health meas- ures must be taken to control any possible spread of the disease. 378 PASTEURELLA INFECTIONS B. Principles of Identification of Plague 1. In arthropods and rodents—Complete procedures for the study of plague in arthropods and in rodents may be found in scientific literature and in manuals devoted to the subject®® and are beyond the scope of this discussion. However, the principles and methods given here may be applied directly to rodent tissues suspected of being infected. Technics of handling fleas will be briefly described. 2. In man—Laboratory confirmation of the clinical diagnosis of P. pestis infection is based either on isolation and identification of the bacilli from the tissues of the patients or on the demonstration of an increase of specific humoral antibodies within 1 to 5 weeks after onset of the disease. Sometimes a retrospective diagnosis can be based on a pattern of decreasing antibodies. In P. pestis infection in man a polymorphonuclear leukocytosis (20,000 to 25,000 per cu. mm) may develop but, except for the so- called septicemic type of the disease, an early-appearing and pro- gressive bacteremia is absent. Inflamed and enlarged lymph nodes, in which the bacteria are usually localized in large numbers, charac- terize the bubonic form. In the case of pneumonic plague the or- ganisms are usually abundant in the sputum. An intense septicemia usually occurs before fatal termination. When plague is suspected as the cause of death, every state in the United States authorizes the physician or health officer to demand an autopsy. A compilation of the laboratory methods for the diagnosis of plague used and recommended by investigators throughout the world has been recently published under the auspices of the World Health Organization.?? However, this text, intended to provide a base for the standardization of internationally acceptable procedures, contains a number of complex alternatives. Most of the restricted number of methods presently considered are identical with those recommended by the experts, but some have been modified and simplified. More- over, reference has been made to new diagnostic procedures recom- mended by Winter and Moody3"# and Moody and Winter. C. Collection and Handling of Specimens 1. Precautions—The clinician and his assisting staff must fol- low the precautions prescribed for highly contagious diseases. A face mask composed of six to eight layers of gauze is indispensable to guard against infectious airborne droplets. Personnel whose work constantly exposes them to P. pestis should be vaccinated. A commercial plague vaccine is available from Cutter PASTEURELLA INFECTIONS 379 Laboratories, at Berkeley, Calif.* Initial vaccination is given in two injections at 7 day intervals, and single booster injections are given biannually. The vaccination is effective in producing a partial im- munity?’ which in case of infection mitigates the severity of the disease and renders it more amenable to treatment. 2. Collection of specimens and preparation of films—Sputum, citrated or heparinized blood (5-10 ml), and fluid aspirated from the bubo should be obtained for etiological diagnosis. The yield of fluid from the bubo early in the disease is small. Puncture and aspiration are done (with the area under local anesthesia if possible) with a 20-22 gauge thin-walled, short-beveled needle well fixed on a 2 to 5 ml syringe. The needle and syringe are then thoroughly washed with a small volume of either sterile salt solution or broth, and the washings are combined with the fluid aspirated from the bubo for laboratory study. Tissues from necropsy should include pieces of the bubo, liver, spleen, lung and bone marrow. When autopsy is not performed, sufficient amounts of these tissues can be obtained by aspiration with a needle, and bone marrow may be obtained through puncture of the sternal bone or by digitotomy. Two impression films or smears of each tissue are made at the time specimens are collected. The slides are allowed to dry in air and are fixed with absolute methanol (2 to 5 min) or by heating; the alcohol fixation is preferable. After the films are fixed, they are stained with polychromatic stain for microscopic examination. The inexperienced should not make a diagnosis from microscopic examination of films alone, and workers not familiar with P. pestis should refer the slides and specimens to laboratories that employ experts. Plague is reportable to the nearest public health official by telephone, and he may be consulted about the proper handling and destination} of diagnostic specimens. If the specimens are to be sent to a distant laboratory, they must be placed in thick-walled glass or plastic vials. Fill the vials containing solid tissues with enough Broquet’s fluid so that little or no air space will be left above them. Stopper tightly with a well-fitted, sterile, paraffin-coated cork and—to avoid leaks during airmail travel—firmly tape the stopper (or screwtop cap) to the vial. * Other major sources of vaccine are the Haffkine Institute, Bombay, India, and the Pasteur Institutes in Paris and Tananarive, Madagascar. + Communicable Disease Center, Public Health Service, U. S. Department of Health, Education, and Welfare, San Francisco 18, Calif., is one of the groups that concerns itself mainly with plague. George Williams Hooper Foundation, University of California, and most of the state public health depart- ments are equipped for this work. 380 PASTEURELLA INFECTIONS Send the specimens in double mailing containers by the most rapid means, if possible by airmail. If the tissues are to be stored, keep them in a refrigerator but do not freeze them. Broquet’s Fluid GIFCErOl, CoP, wait orn miliinnsinsssismmsins oss ss § 55 aaemmnse bs inn 20 ml Disodium phosphate, anhydrous: .. seems sos risssewawsss sss ses 1.11 g CRricatid suis His Phen siivire WS SARE hliary mined Shlvmmeslel os roan 002 g DRSHCA WBE oo ufyvi ois win wmsnwrasn wig widiounminnn: ik cumin osm ia menial iuinthin aie Wien 100 ml Steam the finished fluid for 10 min at 100° C and keep in sterile bottles. Rodent specimens, whole or in parts, when properly wrapped, may be packed in dry ice and sent to the laboratory by air express. 3. Collection of fleas and inoculation of flea material —Fleas to be studied are obtained from rodents or from their burrows and nests, This task is best assigned to a field worker familiar with the procedures. In general, fleas are either killed or anesthetized. They are knocked off a dead rodent or are combed from an anesthetized animal into a white enamel pan. In the field fleas are killed by placing the host animal in a jar and then adding ether or DDT aerosol. The dead or anesthetized fleas are combed quickly from the animal over a 15X 10X35 in. pan, picked up from the pan and put into a 2 dram vial containing 5 ml sterile aqueous solution of 2 per cent sodium chloride. The addition of “Tween 80” to a concentration of 0.01 per cent facilitates the drowning of live, anesthetized fleas. A dead rodent may be placed in a fleaproof paper, cloth or plastic bag, which is then securely closed. On arrival in the laboratory the carcass in the bag is exposed to cyano-gas to kill the ectoparasites. The contents of the bag are then emptied into a large white enamel pan (at least 24X16X10 in.) and the dead insects are separated from the rodent by striking it with a heavy rod—say, a 10 in. triangular steel file, while holding the animal by the base of its tail over the pan. Fleas that have been in 2 per cent salt solution for a number of days must be washed before they are triturated for animal inocula- tion. The washing is best done with a 1:5 dilution of 3 per cent hydro- gen peroxide in sterile distilled water followed by rinsing with sterile physiologic salt solution. From 1 to 50 fleas may be triturated either in a mortar with a pestle or in a small tissue homogenizer with 1 to 2 ml of physiologic salt solution. The triturate is then inoculated sub- cutaneously into two to four mice or a guinea pig. Subsequently the animals are observed in the same manner as those inoculated with clinical specimens. Cultivation of pooled flea triturates is not recom- mended, since experience has shown that such cultures invariably be- PASTEURELLA INFECTIONS come overgrown by other microorganisms. For t scope of culturing fleas individually, see Quan et al.?! D. Identification of P. pestis and Its Antigens To identify P. pestis it must be isolated, cultiva chemical and biological properties determined. To ider these must be separated and their specific serol observed.?2-94 1. Procedures for the isolation of P. pestis a. Cultivation—Place 0.5 to 1.0 ml of blood into ez of infusion broth. Spread 0.25 ml of blood on thre extract agar slants or plates. Incubate these inoc 35° C and examine them for growth of P. pestis daily growth does not take place after 7 days, discard. Cultivate other tissues and tissue fluids similarly, sion broth to which either blood or sodium sulfite been added, and use blood agar in place of plain agar Triturate solid tissues with a small volume of sterile b handling. Growth characteristics: The plague bacillus is aerc tively anaerobic. The optimum temperature range f 25° to 30° C, but it multiplies from —2° to 45° C monly used for cultivation from infectious material) ; occurs at a pH range of 7.2 to 7.6, but the microorge pH of 5.0 to 9.6; pH 6.8 to 7.4 is generally use laboratory. Most kinds of broth—for example, brain heart inf 21), trypticase soy (CM No. 11) and heart or mea No. 5)—support growth of the plague bacilli well synthetic fluid media of simple amino acids with gluc are also suitable,?-97 but these are probably more use of vaccine production and research than for routine Pure broth cultures of P. pestis show a slight turb of incubation at 35° C. When these cultures are sh: vortex is formed, probably due to the chainlet grow to 4th day clumps of growth begin to appear and enlar precipitate is formed. P. pestis grows well but slov medium with blood, say, an ordinary agar base cont of defibrinated or citrated blood per 100 ml. The con able dehydrated blood agar bases are convenient and e Discarded human blood too old for transfusion is 381 he method and ted and its bio- tify its antigens ogical reactions ich of two tubes e or four plain ulated media at y for 7 days. If but use an infu- to 0.025%) has as solid medium. roth to facilitate bic and faculta- or its growth is (35° C is com- maximal growth nism tolerates a 1 in the clinical usion (CM No. t infusion (CM A number of ose and minerals ful for purposes work. idity after 24 hr aken, a feathery th. On the 3rd rge until a heavy vly on any agar aining 4 to 5 ml nmercially avail- conomical to use. satisfactory for 382 PASTEURELLA INFECTIONS growing plague bacilli, and blood from normal laboratory animals or from sheep or horses may be used if necessary. One of the characteristics that differentiate P. pestis from P. pseudotuberculosis and many other species of bacteria is the slow de- velopment of P. pestis on blood agar and still slower development on plain extract agar. Even after massive inoculation growth during in- cubation at 35° C for 18 to 24 hr is insignificant and can be detected at times only because of a darkening of the blood. Isolated colonies are hardly discernible without the aid of a lens. They are pinpoint (0.2 to 0.3 mm in diameter), thin, clear and transparent. Confluent growth after 2 days at 35° C becomes greyish; at room temperatures, greyish white, Isolated colonies become grossly visible in 2 days when they have grown to about 1 mm in diameter. They appear as shiny half- domes and have an entire edge. Magnified, they show a lobated, hammered-copper surface and a spreading, undulated edge. They are sticky and translucent. Well-isolated colonies continue to grow larger and their surface becomes rougher; in 7 to 10 days such a colony will have a diameter of 4 to 7 mm. Crowded colonies do not noticeably en- large beyond 2 to 3 mm, as seen about the 3rd day of cultivation. Some strains, especially when grown between 20° and 30° C, grow in colonies with a white dome-shaped summit which is surrounded at a lower elevation by a clear spread skirt, giving the colony on the 2nd or 3rd day the shape of a fried egg. It is important to note that many media become unsuitable for the cultivation of P. pestis after sterilization by autoclaving unless ade- quate reducing substances such as sodium sulfite are added (to a concentration of 0.025%). Although P. pestis grows poorly on crystal violet agar, the dye in a concentration of 1:40,000 inhibits the spread of Proteus; this is particularly useful when the dye is used in the media for the cultiva- tion of P. pestis from dead rodent tissues. Microscopical appearance: The plague bacillus is nonmotile and nonsporulating. Usually its envelope can be readily seen in India ink preparations made with cultures grown on blood agar at 35° C. P. pestis is a Gram-negative rod. Its bipolar character is seldom seen in films stained by Gram’s method. Any polychromatic stain, such as Wayson or Giemsa, will show it. The Wayson stain colors the bacteria more rapidly (in 5 to 10 sec) than the Giemsa and is pref- erable. When Wayson stain is not at hand, a satisfactory substi- tute can be improvised by first mixing 0.2 ml of liquefied phenol with 0.5 ml of Ziehl-Neelsen carbolfuchsin stain and then diluting the mixture with 4 ml of Loeffler methylene blue stain. ‘With the Wayson PASTEURELLA INFECTIONS 383 stain the polar bodies of P. pestis are dark blue, the remainder light blue to reddish. The forms and shapes of P. pestis, either in tissues or in cultures, are influenced by oxygen tension, drugs, chemicals, age and other factors. The shapes seen vary from a slightly flattened coccus to a boat-shaped rod ; the latter has been described as having the shape of a closed safety pin or as barrel-like. P. pestis varies in width from 0.5 to 0.8 1 and in length from 1.0 to 2.0 x Thus, in size and appear- ance the coccoid form looks like a staphylococcus, and the large bacil- lary form is similar to the smaller salmonellae. Films made from necrotic lymph nodes and spleens, especially films from tissues of a host who has received drug therapy, include many shapes and sizes of this pleomorphic microorganism so various as to give the impres- sion that the bacteria are of several different kinds. Under these conditions P. pestis can be identified (1) by its convex sides, formed about a straight axis to give a short, oval bacillus with rounded ends and (2) by the aforementioned bipolar staining. Here, as in most tissue films, this organism is predominantly single and is seldom seen in chains of more than two bacilli. In blood films the organism is more uniform. Fresh young agar and broth cultures also are com- posed of slightly varied populations. Organisms in stained films from a surface agar culture, however, differ from those obtained from a broth culutre. The former are smaller, being short rods scattered singly and in chains and clusters of two or more. From day-old broth the organisms are long and broad, similar in size to the salmonellae, and most of them appear in chains of 4 to 16 bacilli. Chain lengths of 6 to 10 units predominate—a single bacterium is rarely seen. Old cultures contain many poorly stained, involuted and bizarre forms. Biochemical reactions: These are no longer used extensively to identify P. pestis, but mainly to differentiate it from other pasteu- rellae, especially from the species P. pseudotuberculosis. The tests generally made are: growth in infusion broth, glucose, lactose, litmus milk, blood agar and urea medium; as well as the methylene blue, methyl red and hydrogen peroxide tests. The growth of the micro- organism produces either no change or a slightly alkaline reaction in litmus milk. It forms neither indole nor H,S. It does not hemolyze blood cells or liquefy gelatin. It produces acid but no gas from arabi- nose, glucose, glycogen, levulose and mannose. It does not ferment lactose, rhamnose, sucrose or sorbitol. The methyl red test is positive ; methylene blue is reduced slowly or not at all. The hydrogen peroxide test for catalase is positive. The microorganism grows in urea medium but does not hydrolyze urea.® 384 PASTEURELLA INFECTIONS Specific lysis of P. pestis with bacteriophage: A bacteriophage for P. pestis lyses all strains of this microorganism on which it has been used to date. Gunnison and her associates®® showed that high speci- ficity could be attained by application of the phage at 20° C. At higher temperatures the virus becomes active on other bacteria and thereby loses some of its specificity for P. pestis. This definite lysis by a specific bacteriophage makes it possible to recommend the use of completely in vitro procedures for rapid identi- fication. Positive phage action with serological and microscopical con- firmation can identify the plague bacillus. A procedure to facilitate the use of plague-specific bacteriophage is to apply the virus lyophil- ized on filter paper strips.?®* The activity of the phage on these strips is retained for at least 2-5 years, b. Animal inoculation—For rapid propagation of virulent plague bacilli, inject 0.5 to 2 ml of blood, sputum, tissue fluid or tissue tritu- rate intraperitoneally into two to six laboratory mice or one or two guinea pigs. Aspirate some peritoneal fluid with a capillary pipette or syringe and needle 24 hr after inoculation and examine the exudate for plague bacilli. If no microorganisms are found, aspirate again the next day. When P. pestis is present, the animals usually die within 2 to 7 days after inoculation and large numbers of P. pestis will be found in their blood, liver and spleen. To produce more typical pathological changes, inoculate a guinea pig subcutaneously in the inguinal region with 0.5 to 2.0 ml of a sus- pension of the sample. If the inoculum contains plague bacilli, an inguinal bubo develops, and fluid for microscopic examination and cultivation can be aspirated from the incipient bubo on the 2nd day. The enlarged lymph node is hard and can be distinctly palpated from 2 to 4 days after injection. Even if large numbers of virulent plague bacilli were present in the inoculum, the animals usually do not die before 4 or 5 days after infection. If the animals survive, they are sacrificed after an observa- tion period of 8 to 10 days. Guinea pigs which succumb to infection with virulent plague bacilli show the following postmortem changes : The inoculated area will be ulcerated, with or without abscess for- mation, generally surrounded with gelatinous edema. The regional lymph nodes are considerably swollen and inflamed, often are partly * Samples of these phage paper strips are available from San Francisco Field Station, Communicable Disease Center, Public Health Service, U. S. Department of Health, Education, and Welfare, San Francisco 18, Calif. The bacterio- phage is maintained also at the Hooper Foundation, San Francisco, the Haffkine Institute, Bombay, India, and the Institut Pasteur, Paris. PASTEURELLA INFECTIONS 385 necrotic. As a rule these buboes are surrounded with edematous and hemorrhagic tissue. The liver is generally pale and fatty and may be studded with yel- lowish pinpoint nodules. The spleen is enlarged, dark in color, and usually shows few to numerous white to yellowish nodules of about 0.5 to 3 mm in diameter. The margin of the organ is rounded, the pulp softened. The lungs may show congestion or more progressive lesions of secondary plague pneumonia, with nodules of pinpoint to 5 or 6 mm in diameter surrounded by a hemorrhagic zone or by extensive areas of consolidation involving entire lobes. Lung lesions are seldom seen in guinea pigs that die less than 5 days after infection. Smears and impression films made from heart blood, spleen, liver, bubo and/or lung usually show innumerable plague bacilli. Unless the subcutaneously inoculated dose causes death of labora- tory mice in less than 5 days, the lesions are somewhat similar to those seen in the guinea pig. When working with a suspension of unknown P. pestis content it is difficult to estimate a suitable dosage for the inoculation of mice. Little regard is given to adjusting the dosage, with 0.1-0.5 ml usually injected into each mouse. If the specimen is suspected of containing microorganisms other than P. pestis, as in the case of sputum, putrefied tissue or triturates of flea pools, mix the suspension to be inoculated with a solution of penicillin in a syringe. Inoculate mice intraperitoneally in such dosage that each mouse receives 1,000 units of penicillin.’®® Do not store unused specimen with penicillin, as this antibiotic would in time kill any plague bacilli present. 2. Storage and disposal of specimens and cultures Unused portions of specimens and cultures derived from clinical material should be kept in a refrigerator (4° to 10° C) until diagnosis is established. Agar slants of P. pestis cultures remain viable in the cold for years. For permanent storage, however, lyophilization and sealing either in vacuo or under dry nitrogen is best. The lactose salt solution developed by Hornibrook!! is an excellent menstruum for lyophilizing P. pestis. Disinfectants, such as dilute solutions of saponified cresol, sodium hypochlorite, or benzalkonium chloride, that are used in the laboratory are quite effective in destroying plague bacilli, but death times vary from a few minutes to several hours depending on density of the bacterial suspension, size of the clumps and other factors. Most of the antibiotics, including penicillin (which is not effective in the 386 PASTEURELLA INFECTIONS therapy of plague) in high concentrations, are bactericidal to the bacillus in vitro. Boiling for 5 min kills plague bacteria in aqueous suspension. Autoclaving is a very effective way to sterilize all ma- terials contaminated with P. pestis. 3. Serological examination a. Agglutination (1) Preparation of agglutination sera—If the anti- plague serum for agglutination tests is not obtainable from the public health laboratories, it may be prepared with the aid of a living aviru- lent strain according to the following procedure: Strain A1122 (Jawetz and Meyer, 1943)1%2 or another suitable strain is grown on infusion agar in Roux culturing flasks at 35° C for 2 or 3 days. The growth is suspended in 10 to 15 ml of sterile salt solution and shaken with about 15 to 20 sterilized round glass beads. The thick suspension is diluted with physiologic salt solution to 101° organisms per ml. One ml is injected slowly into the marginal ear vein of rabbits three times a week for 3 consecutive weeks, It is advantageous to give penicillin (about 5,000 units) in alternating in- jections beginning with the third dose. The serum is collected when a test bleeding indicates a high agglutinating titer, usually at 8 to 20 days after the final inoculation. A titer of about 1:160 is satisfactory. If the titer is too low, another series of injections is given for 2 weeks, with another test bleeding made 8 to 10 days after the last inoculation. If the result is satisfactory, the serum is collected. Such an antiserum remains active for years when stored refrigerated, and indefinitely when lyophilized. P. pestis contains somatic antigens in common with P. pseudotuber- culosis. These interfering antibodies can be removed by absorbing the antiserum with boiled antigens of either P. pestis or P. pseudo- tuberculosis. It is noteworthy, however, that, as stated in a recent paper by Winter and Moody,*** fully specific sera could be produced with the aid of a short immunization schedule by inoculation of rabbits with Fraction 1 or with whole-cell P. pestis antigens. As the two authors summarized, the conditions required for the production of a serum of the latter type involved culturing a fully virulent strain of P. pestis in casein hydrol- ysate glucose mineral broth for 72 hr at 37° C, killing the culture with 0.4 per cent formalin and immunizing rabbits with this antigen for a total of three in- jections given over a period of 1 week. 88b According to a second article by Winter and Moody," the globulin portion of this serum, obtained by precipitation with half-saturated ammonium sulfate at 0° to 4° C and conjugated with fluorescein PASTEURELLA INFECTIONS 387 isocyanate, could be used for the rapid identification of P. pestis. For this purpose heat-fixed films were covered with the conjugate and al- lowed to stand for 30 min in a petri dish fitted with a piece of moist filter paper. After rinsing 10 min in 0.85 per cent sodium chloride solution of pH 7.0, the films were blotted carefully and mounted under a cover slip with glycerol salt solution (9 parts glycerol, 1 part 0.85% sodium chloride buffered at pH 7.0 with 0.01 M phosphate buffer). Unstained films and those treated with normal rabbit globu- lin conjugate served as controls, The technic described by Moody, Goldman and Thomason'®® was used for fluorescence microscopy. Except for 3 strains, each of the 33 plague strains tested proved posi- tive with this method, whereas none of the pseudotuberculosis strains or other bacterial strains used as controls were stained with the reagent. As recorded by Winter and Moody®® in a preliminary statement, the use of the fluorescent antibody proved suitable for demonstrating P. pestis in the bubo exudate and blood of a plague patient 4 days after onset of illness. Likewise, according to a communication by Moody and Winter,%® the method proved adequate for the identifica- tion of plague bacilli in tissue impression films made from laboratory mice sacrificed within 2 days after intraperitoneal infection. 2) Agglutination tests with plague-suspect bacilli: To identify plague-suspect bacilli with the aid of the usual agglutination method, add to a suspension of the microorganisms to be examined, formalin to a concentration of 1 per cent.and allow to stand for 1 hr at room temperature. Centrifuge down the dead bacteria and thoroughly wash them twice with physiologic salt solution. Resuspend them in sodium chloride solution and test for agglutination, using a 1:150 dilution of antiplague serum. Agglutination indicates the presence of P. pestis or P. pseudotuberculosis unless the antiserum has been absorbed with plague somatic antigens. 3) Tests with the sera of patients: The antigen used for demon- strating the presence of agglutinins in the sera of patients is a known strain of P. pestis cultivated either in broth or on extract agar at 35° C for 1 or 2 days. An avirulent strain of low Fraction 1 content, such as No. 14 (Jawetz and Meyer)1%? or EV76 (Girard and Robic)* is recommended, but any P. pestis strain may be used. The bacteria in the suspension are killed by adding formalin to a con- centration of 1 per cent and allowing the disinfectant to act for 1 hr at room temperature. The dead microorganisms are then sedimented by centrifugation and washed twice with physiologic salt solution. 388 PASTEURELLA INFECTIONS The antigen is suspended in salt solution for use in the agglutination test either by the conventional macroscopic test tube procedure or by the more rapid slide technic. The following procedure combines the advantages of both the test tube and the slide technic, with only slightly more labor. Prepare 6 to 10 tubes of twofold dilutions of serum in 0.5 ml volumes of salt solution beginning with a 1:4 dilution of serum and ending at the penultimate tube. Add 0.5 ml of antigen suspension (turbidity, McFarland scale No. 4) to each tube. Shake well to mix. With a pipette or 74 ml syringe and needle, transfer a small volume (0.05-0.10 ml) from each tube to concavities of a glass slide (plate), beginning with the last tube of the series. Shake the slide gently on a rotating platform for 30 min. Read the results under low magnifi- cation. Place the test tube series in a water bath at 35° C for 2 hr and then at room temperature for about 18 hr, and this time read the results with the aid of a hand lens. b. Precipitation—The precipitin test for soluble P. pestis anti- gens is not very sensitive and necessitates the use of hyperimmune rabbit serum. By standardization of a high-titer serum with a soluble antigen, Fraction 1, this procedure is applied as a system of quanti- tative turbidimetry. The Fraction 1 content of many plague strains has been so determined. Any aqueous solution containing sufficient soluble antigens of P. pestis forms a precipitate in this test. Larson used this basic re- action in his modified Ascoli test to obtain evidence of plague infec- tion in dried or decomposed carcasses of rodents, For this purpose an ether-treated clear extract of the tissue is prepared for use as the antigenic solution. Hoyer and Courdurier'®® applied this procedure to tissues of plague-suspect dead bodies. Except for the antiserum, the procedure is similar to that for tularemia and is given in detail in Section IIT of this chapter. To make the tissue extract, cut the tissues (preferably liver, spleen, lymph nodes, lungs) into small pieces. Grind them in a mortar with sterile sand, moistening with salt solution if necessary, and make suspensions with about 3 volumes of salt solution. Transfer the sus- pensions to test tubes or Erlenmeyer flasks fitted with cork stoppers. Add approximately 2 volumes of diethyl ether, stopper, and agitate gently but thoroughly. Allow the mixtures to stand at room tempera- ture for 5 to 20 hr. Remove 0.5-2 ml of the supernatant fluids and centrifuge them at about 1,700XG (gravity) or 3,000 rpm for 30 min. In testing, use the clear fluid as an antigenic extract in capillary tubes in volumes of 0.03-0.1 ml with an equal volume of antiserum. PASTEURELLA INFECTIONS 389 Strongly positive extracts form precipitates within minutes; other- wise, incubate the tubes for 3 hr at 35° C and then overnight in the refrigerator before reading. c. Hemagglutination—The hemagglutination test with the envelope protein of P. pestis, Fraction 1, is highly specific and is the most sensitive of all the serological tests for antibodies against this micro- organism. Fraction 1 is not available commercially but may be pre- pared according to the method of Baker et al.* Antigen coating of the sheep red cells: To a volume of 5 per cent washed sheep erythrocytes in physiologic salt solution add an equal volume of 1:20,000 saline solution of tannic acid. Incubate the mixture in a 35° C water bath for 10 min and then centrifuge it gently (200X G, about 1,000 rpm at a 6 in. radius) for 3 min to settle the cells. Wash once with salt solution, then resuspend the tannic acid- treated cells to a concentration of 2.5 per cent in salt solution. Add a 1 mg:10 ml solution of Fraction 1 in salt solution in a volume equal to that of the suspension of the erythrocytes and allow the mixture to stand at room temperature for 15 min. Wash the cells twice (centri- fuge at 300XG) with salt solution containing a 1:250 dilution of in- activated, absorbed normal rabbit serum (for preparation, see the following). Finally, resuspend the cells to 2.5 per cent concentration in the 1:250 dilution of normal rabbit serum in physiologic salt solution. Inactivation and absorption of the normal rabbit serum and of the test serum: Inactivate sera by heating at 56° C for 30 min. Then mix each inactivated serum in the proportion of nine parts of serum to one part of washed and packed (800XG) normal sheep erythrocytes, al- lowing them to stand at room temperature for 30 min, This is done to absorb and thus remove antisheep antibodies. Serum may be diluted before inactivation and a lesser volume of packed cells may be used for absorption of antisheep antibodies from a diluted serum. Test procedure: Dilute the inactivated, absorbed test serum in a twofold series, using 0.5 ml volumes of a diluent containing a 1:100 dilution of inactivated, absorbed normal rabbit serum in physiologic salt solution. Then add to each tube containing serum dilution 0.05 ml of the antigen-coated sheep erythrocyte suspension, mix, and allow the mixtures to react and settle for 2 hr at room temperature. Read the sedimentation pattern for signs of hemagglutination. This test requires a control series for tannic acid-treated cells in addition to the usual controls for antigen and untreated cells. It does not have the disadvantage of the prozone phenomenon. 390 PASTEURELLA INFECTIONS When this procedure was applied at the microscale level, 300 to 400 sera were titrated a day with less than 10 ml suspension of antigen- coated red cells. d. Complement fixation—According to the best procedure, the anti- genic solution is prepared by making a 1:500 dilution of a stock solu- tion of 2.0 mg of Fraction 1 envelope protein per ml of salt solution. However, Fraction 1 is difficult to prepare and, as noted, is not avail- able commercially, By using the supernatant fluid from plague vac- cine, satisfactory results have been obtained.!®” Various samples of the supernatant fluid assayed contained from 0.02 to 0.48 mg of Fraction 1 to 1 ml of solution ; there were other soluble antigens in the samples. The technic otherwise follows the conventional procedure. E. Report and Its Evaluation After isolating and identifying P. pestis, the laboratory shall report its findings immediately to the physician. If blood has been cultured, the quantitative result should be given. The report from the labora- tory might be evaluated with the following description of plague in man: The disease is bubonic plague when it involves the swelling of peripheral lymph nodes. These nodes become intensely painful and inflamed. Most attacks of bubonic plague are initiated by bites of infective fleas, and the bubo results from necrotization of the lymph node by the bacteria which have drained from the site of infection. The microorganisms multiply in the lymph nodes and showers of them escape into the bloodstream whenever they cannot be confined. Early in the disease, fluid from the infected lymph node is usually full of plague bacilli, but as the bubo becomes necrotic, they diminish in num- bers and finally disappear. In the so-called septicemic type of plague no clinically manifest superficial buboes develop because the lymph nodes are overrun by a rapidly progressing infection. Heavy infections thus initiated in the blood overtax the filtering system and the bacteria multiply in the blood. More than 40 bacilli per milliliter of blood indicate a severe septicemia, fewer generally represent a mild and probably temporary bacteremia. The organisms are seldom seen in blood films; when present they are usually free, not phagocytized. The use of fluores- cent antibody staining will facilitate the identification of P. pestis in blood films. A positive film or culture from the blood does not always indicate a serious prognosis. P. pestis is usually present in the sputum in both primary and secondary pneumonic plague. The former is contracted by inhalation PASTEURELLA INFECTIONS 391 of infected airborne droplets, whereas the latter is acquired through invasion of the respiratory system in the course of bubonic plague. Regardless of the mode of initiation of the pneumonia, this form of the disease is the most contagious. The prognosis is good when the disease is diagnosed early and is forthwith treated intensively with effective drugs such as streptomycin and the tetracyclines. 108 As therapeutic measures and the defense mechanism of the body control the infection, the plague bacilli disappear and antibodies de- velop. As noted before, the hemagglutination test with Fraction 1 protein-coated cells is far more sensitive for the demonstration of these than the conventional agglutination test with a bacterial sus- pension. A hemagglutination titer of 1:10 can be obtained with the serum of pneumonic plague patients as early as the 2nd or 3rd day of the acute disease, and titers are 1:10,000 and higher in the late convalescent period. Agglutination titers of 1:10 dilution or less are not detectable until the 5th to 7th day of illness, and the titer seldom exceeds 1:200. Complement-fixing antibodies appear late in the disease and in low dilution, 1:2 to 1:64. A positive complement- fixation reaction is confirmatory; a negative reaction has little sig- nificance, Antibodies to the plague bacillus are greatly reduced or have completely disappeared in a few months after recovery from the disease. The difficulty of diagnosing plague lies not in the absence of promi- nent and precise road markers in the form of procedures for the identification of P. pestis, but in the frequent failure of those first seeing the patients to realize that plague is a diagnostic possibility. If the presence of the disease is not recognized until autopsy, plague may have an opportunity to become epidemic, particularly where secondary pneumonia has developed. The dismal history of plague has marked well the geographic areas where P. pestis has been and is now present. In these areas vigilance is especially necessary. Because of the epidemic potential and the mortality of the untreated disease, a per- ceptive eye must be kept on the perimeter of these regions and on other areas open to inroads of the infection. K. F. Meyer, D.V.M,, Pu.D., M.D,, Chapter Chairman F. A. HumpHREYS, D.V.Sc. W. Knapp, M.D. C. L. Larson, M.D., D.Sc. R. Porrrrzer, M.D. S. F. Quan, Pu.D. E. TrAL, V.M.D. 392 PASTEURELLA INFECTIONS REFERENCES 1. Meyer, K. F. In Bacterial and Mycotic Infections of Man (R. J. Dubos, Ed.). Philadelphia, Pa.: J. B. Lippincott, 1958, pp. 420-425. 2. Knapp, W. Pasteurella pseudotuberculosis als Erreger einer Mesenterialen Lymphadenitis beim Menschen. Zbl. Bakt. I Abt. Orig. 161:422-424, 1954. 3. ————————— Mesenteric Adenitis Due to Pasteurella pseudotuberculosis in Young People. New England J. Med. 259:776-778, 1958. 4. —————— Pasteurella pseudotuberculosis unter Besonderer Beruck- sichtigung ihrer Humanmedizinischen Bedeutung. Ergebn. Mikrobiol. Im- munitdts. und Exper. Therap. 32:196-269, 1959 (with more references of the world literature). 5. ——————— Die Diagnostische Bedeutung der Antigenen Beziehungen Zwischen Past. pseudotuberculosis und der Salmonella- Gruppe. Zbl. Bakt. I Abt. Orig. 164:57-59, 1955. 6. ————— Die Agglutinationsreaktion und Ihre Besonderheiten in der Serodiagnostik Menschlicher Infektionen mit Pasteurella pseudotubercu- losis. Z. Hyg. Infektkrh, 143:261-277, 1956. 7. TuAL, E. “Untersuchungen uber P. pseudotuberculosis.” Thesis. Univ. of Lund (Sweden), 1954. English summary in Nord. vet. med. 6:829, 1954. 8, ———— and CueN, T. H. Two Simple Tests for the Differentiation of Plague and Pseudotuberculosis Bacilli. J. Bact. 69:103-104, 1955. 9. Piecaaup, M. Un Nouveau Cas de Pseudo-Tuberculose Humaine. Ann. Inst. Pasteur 83:420-421, 1952. 10. ScruEerze, H. Bacterium pseudotuberculosis rodentium. Arch. Hyg. 100: 181-194, 1928. 11. ———— A System of Bacteriology, Vol. 4. London : British Medical Research Council, 1929, pp.446-482. 12. —————— Studies in B. Pestis Antigens II. The Antigenic Relationship of B. Pestis and B. pseudotuberculosis rodentium. Brit. J. Exper. Path. 13:289-293, 1932. 13. KaurrmaNN, F. Vergleichende Untersuchungen an Pseudotuberkulose, Paratyphosus- Pasteurella-, und Pestbacillin. Z. Hyg. Infektkrh. 114:97- 105, 1932. 14. —————— Enterobacteriaceae (2nd ed.). Copenhagen: Munksgaard, 1954. 15. ScHAR, M,, and THAL, E. Comparative Studies on Toxins of Pasteurella pestis and Pasteurella pseudotuberculosis. Proc. Soc. Exper. Biol. & Med. 88:39-42, 1955. 16. BHATNAGAR, S. S. Bacteriological Studies on Pasteurella pestis and Pasteurella pseudotuberculosis. Indian J. M. Res. 28:1-15, 1940. 17. Daniers, J. J. H. M. Enteral Infection with Pasteurella pseudotubercu- losis. Isolation of the Organism from Human Faeces. Brit. M. J. 2:997, 1961. 18. Morrarer, H. H., and LEMINoOR, L. Recherche de la Beta-Galactosidase chez les Différentes Pasteurellae et Consequentes quand Leur Taxonomie. Ann. Inst. Pasteur 102:649-652, 1962. 19. Parnas, J. L’Epreuve Esculinique dans le Diagnostique de la Peste et de la Pseudotuberculose. Ann. Inst. Pasteur 100:691-692, 1961. 20. Grarp, G. Projet d’Uniformisation de Méthodes permettant de dif- férencier P. Pestis de P. Pseudotuberculosis. WHO Bull. 9:650-653, 1953. 21. —————— and CHEVALIER, A. Classification Serologique de 56 souches de Pasteurella pseudotuberculosis dont 52 Isolees en France. Ann. Inst. Pasteur 88:227-229, 1955. PA 22. 23. 24. 25, 26. 27. 28. 29. 30. 3. 32 33. 34. 35. 36. 37. 38. 39. 41. 42. STEURELLA INFECTIONS 393 Barrazarp, M., et al. Recommended Laboratory Methods for the Diag- nosis of Plague. WHO Bull. 14 :457-509, 1956. PorLitzer, R. Plague. WHO Monograph Series No. 22. Geneva: World Health Organization, 1954. Larson, Carr L. Studies on Thermostable Antigens Extracted from Bacterium tularense and from Tissues of Animals Dead of Tularemia. J. Immunol. 66 (2) :249-259, 1961. GirarD, G. Sensibilité des Bacilles Pesteux et Pseudotuberculeux d’Une Part, des Germes du Groupe Coli-Dysenterique, d’Autre Part, aux Bactériophages Homologues. Ann. Inst. Pasteur 69:52-54. Kw~are, W. Untersuchungen mit Pasteurella pseudotuberculosis- und Pasteurella pestis Phagen. Ztschr. Hyg. 148:375-382, 1962. Apams, M. H. Bacteriophages. New York: Interscience Publishers, 1959. Dantes, J. J. H. M., and Daniers-Bosman, M.S.M. Lymphadenitis Mesenterialis, Veroorzaakt door Pasteurella pseudotuberculosis Verlopend onder het Beeld von Akute Appendicitis. Nederl. Tjidskr. geneesk. 104: 922-926, 1960. Morrarer, H. H. L’Adénite Mésenterique Aigue a Pasteurella pseudo- tuberculosis. Paris: Masson et Cie., 1962, p. 74. Breen, R. S.,, Murray, E. G. D. and Hrrcuens, A. P. In Bergey's Manual of Determinative Bacteriology (6th ed.). Baltimore, Md.: Wil- liams & Wilkins, 1948, pp. 546-549. Roserts, R. S. An Immunological Study of Pasteurella septica. J. Comp. Path. & Therap. 57 :261-278, 1947. CArTER, G. R., and ByrnNE, J. LL. A Serological Study of the Hemorrhagic Septicemia Pasteurella. Cornell Vet. 43:223-230, 1953. Prrrcaerr, I. W., Beauperte, R. R., and Hucues, T. P. The Epi- demiology of Fowl Cholera. IV. Field Observations of the “Spontaneous” Disease. J. Exper. Med. 51:249-258, 1930. Hutyra, F.,, Marek, J.,, and MANNINGER, R. In Special Pathology and Therapeutics and the Diseases of Domestic Animals (5th ed.). Chicago, T11.: Alexander Eger 1949, Vol. 1, pp. 95-152. WeBsTER, LL. T. The Epidemiology of a Rabbit Respiratory Infection. I. Introduction. J. Exper. Med. 39:836-841, 1924; II. Clinical, Pathological, and Bacteriological Study of Snuffles. Ibid. 39:843-856; IV. Susceptibility of Rabbits to Spontaneous Snuffles. Ibid. 40:109-116; V. Experimental Snuffles. Ibid. 40:117-127. Dincre, J. H. In Biology of the Laboratory Mouse (G. D. Snell, Ed.). Philadelphia, Pa. : Blakiston, 1941, Chapter 12, pp. 380-474. Greexwoon, M.; Newsorp, E. M.; Torrey, W. W. C.; and WiLsoN, J. On the Mechanisms by Which Protection against Infectious Disease is Acquired in “Natural” Epidemics. J. Hyg. 25:336-353, 1926. WricHT, J. Epidemic of Pasteurella Infection in Guinea Pig Stock. J. Path. & Bact. 42:209-212, 1936. Scurprper, G. J. Unusual Pathogenicity of Pasteurella multocida Isolated from the Throats of Common Wild Rats. Bull. Johns Hopkins Hosp. 81:333-356, 1947. . Jawerz, E. A Pneumotropic Pasteurella of Laboratory Animals. I. Bac- teriologic and Serological Characteristics of the Organism. J. Infect. Dis. 86:172-183, 1950. —— and Baker, W. H. A Pneumotropic Pasteurella of Labora- tory Animals. TI. Pathological and Immunological Studies with the Organ- ism. J. Infect. Dis., 86 :184-196, 1950. CARTER, G. R. Observations on the Pathology and Bacteriology of Shipping Fever in Canada. Canad. J. Comp. Med. 18:359-364, 1954. 394 43. 45. 46. 47. 48. 49. 50. 51. 52. 53. 55. 56. 57. 58. 59. 60. 61. 62. 63. PASTEURELLA INFECTIONS BearN, A. G., Jacoss, K, and McCarry, M. Pasteurella multocida Septicemia in Man. Am. J. Med. 18:167-168, 1955. Bezjak, V., and Mimica, M. Two Human Infections Caused by Pas- teurella multocida. Brit. M. J. 2:757-758, 1952. ReGAMEY, R. Les Infections Humaines a B. bipolaris septicus (Pasteurel- loses). Bern, Switzerland : Editions Hans Huber, 1939, p. 126. Remry, J, and Tournier, P. Les Pasteurelloses Humaines. Rev. prat. 4:1929-1937, 1954. TruMMERT, W., REMKyY, H., and A~pers, C. Uber Traumatische Pas- teurella-Phlegmonen beim Menschen. Munchen med. Wchnschr. 101 :34-37, 1959. BYRNE, J. J., Boyp, T. F,, and DaLry, A. K. Pasteurella from Cat Bites. Surg. Gynec. & Obst. 103:57-61, 1956. Morris, A. J.; Heckrer, G. B.; ScuAus, I. G.; and Scort, E. G. Pas- teurella multocida and Bronchiectasis. Report of Two Cases. Bull. Johns Hopkins Hosp. 91:174-177, 1952. OrsEN, A. M., and NeeprAM, G. M. Pasteurella multocida in Suppurative Diseases of the Respiratory Tract. Am. J. M. Sc. 224:77-81, 1952. Pestana, R. B., Arantes. M. and Rucai, E. Pasteurellose Humana. Rev. Inst. Adolfo Lutz 1:357-360, 1941. RecaMEy, R. Un nouveau Cas de Méningite Cérébrospinale 4 B. bipolaris septicus. Apercu des Cas Publies sous le Nom de Pasteurelloses Humaines. Schweiz. med. Wchnschr. 68 :666-668, 1938. ZeLLEr, W. W,, and Lepper, M. H. Meningitis Due to Pasteurella Other Than Pasteurella tularensis and Pasteurella pestis. Am. J. Med. 9:701- 706, 1950. Bartiey, E. O., and Hunter, K. Penicillin in Surgical Treatment of Pasteurella Sinusitis. Lancet 1:908-909, 1947. NEerer, E., DEKLEINE, E. H., and Ecan, P. W. Treatment with Aureo- mycin of Localized Pasteurella multocida Infection. J. Pediat. 38:242-243, 1951. NEeTER, E., GorzyNskI, G. A, and Cass, W, A. Aureomycin and Terramy- cin Treatment of Pasteurella multocida Infection and Neomycin’s in vitro Effects on P. multocida. Proc. Soc. Exper. Biol. & Med. 76:493-495, 1951. CarTER, G. R., and Bicranp, C. H. Dissociation and Virulence in Strains of Pasteurella multocida Isolated from a Variety of Lesions. Canad. J. Comp. Med. 17:473-479, 1953. ELserg, S. S., and CueNG-LEE, Ho. Studies on Dissociation in Pasteurella multocida. J. Comp. Path. & Therap. 60:41-50, 1950. CARTER, G. R. Studies on Pasteurella multocida. I1. Identification of Anti- genic Characteristic and Colonial Variants. Am. J. Vet. Res. 18:210-213, 1957. Beveringe, W. I. B. Note on an Infection of Sheep with a Pasteurella- like Organism. Austral. Vet. J. 13:155-157, 1937. Jones, F. S. A Study of Bacillus boviscpticus. J. Exper. Med. 34:561-577, 1921. MontcoMERIE, R. F., BoswortH, T. J., and Grover, R. E. Enzootic Pneu- monia in Sheep. J. Comp. Path. & Therap. 51 :87-107, 1938. HENRIKSEN, S. D., and Jyssum, K. A Study of Some Pasteurella Strains from the Human Respiratory Tract. Acta path. et microbiol. scandinav. 51:354-368, 1961. ScuaAus, I. G., and Forey, M. K. Diagnostic Bacteriology: A Textbook for the Isolation and Identification of Pathogenic Bacteria (4th ed.). St. Louis, Mo. : Mosby, 1952, pp. 330-331. PASTEURELLA INFECTIONS 395 65. 66. 67. 68. 69. 70. 7% 72. 73 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 85. 86. Lirtie, P. A., and Lyon, B. M. Demonstration of Serological Types within the Nonhemolytic Pasteurella. Am. J. Vet. Res. 4:110-112, 1943. CARTER, G. R. Type-Specific Capsular Antigen of Pasteurella multocida. Canad. J. M. Sc. 30:48-53, 1952. — Studies on Pasteurella multocida. I. A Hemagglutination Test for the Identification of Serological Types. Am. J. Vet. Res. 16:481- 484, 1955. THJ@TTA, T., and HENRIKSEN, S. D. Pneumonia and Empyema Caused by a Pasteurella of the Hemorrhagic Septicemia Group. Acta path. et micro- biol. scandinav. 23:412-414, 1946. SvENDSEN, M. Brain Abscess Caused by Pasteurella septica. Acta path. et microbiol. scandinav. 24 :150-154, 1947. RELY, J., TourNier, P., and Bastin, R. Le Réactions Allergiques au cours des Pasteurelloses Humaines, Leur Use en Evidence, Leurs Mani- festations, Leur Interét Diagnostique et Therapeutique. Ann. med. (Paris) 53:113-137, 1952. ArcHER, V. W., Brackrorp, S. D., and WissLer, J. E. Pulmonary Mani- festations in Human Tularemia. Roentgenologic Study Based on Thirty- Four Unselected Cases. J.A.M.A. 104 :895-898, 1935. FosHAY, LEE, Tularemia. Ann. Rev. Microbiol. 4:313-330, 1950. Francrs, EpwArp. Tularemia Francis 1921: A New Disease of Man. J.AAM.A. 78:1015-1018, 1922. Hunt, JouN S. Pleuropulmonary Tularemia: Observations on 12 Cases Treated with Streptomycin. Ann. Int. Med. 26 (2) :263-276, 1947. Linpekg, H. I, and Mammen, S. D. Oculoglandular Tularemia Treated with Aureomycin, J.A.M.A. 142(2) :99-100, 1950. PuLLEN, Roscoe L., and Stuart, Byron M. Tularemia: Analysis of 225 Cases. J.A.M.A. 129:495-500, 1945. Simpson, WarLter M. Tularemia. History, Pathology, Diagnosis, and Treatment. New York: Paul B. Hoeber, 1929, pp. 162. Stuart, Byron M., and PuLLeN, Roscoe L. Tularemic Pneumonia. Review of American Literature and Report of 15 Additional Cases. Am. J. M. Sc. 210:223-236, 1945. FosuAy, Lee. Cause of Death in Tularemia. Arch. Int. Med. 60:22-38, 1937. ——— Tularemia: A Summary of Certain Aspects of the Disease Including Methods for Early Diagnosis and the Results of Serum Treat- ment in 600 Patients. Medicine 19(1) :1-83, 1940. AsHBURN, L. L.,, and MLLER, S. E. Tularemia. A Report of a Laboratory Infection Fatal on the Fifth Day, with Early Pulmonary Involvement; Autopsy. Arch. Path. 39 :388-392, 1945. PARrRkER, R. R., and SpeNCER, R. R. Six Additional Cases of Laboratory Infection of Tularemia in Man. Pub. Health Rep. 41 (27) :1341-1355, 1926. EiceLssacH, H. T., Braun, WERNER, and Herring, R. D. Studies on the Variation of Bacterium tularense. J. Bact. 61:557-569, 1951. Larson, CArL L. Isolation of Pasteurella tularensis from Sputum. A Re- port of Successful Isolations from Three Cases without Respiratory Symptoms. Pub. Health Rep. 60(36) :1049-1053, 1945. SNYDER, T. L.; PenrieLD, R. A, ENcLEY, F. B.; and Creasy, J. C. Culti- vation of Bacterium tularense in Peptone Media. Proc. Sec. Exper. Biol. & Med. 63:26-30, 1946. Ber, J. Freperick; JeLLisoN, WiLriam, L.; Owen, Cora R.; and LAr- soN, Car. L. Applicability of the Ascoli Test to Epizootic Tularemia in Wild Rodents. J. Wildlife 23:238-240 (Apr.) 1959. 396 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. PASTEURELLA INFECTIONS Winter, C. C., and Moopy, M. D. Rapid Identification of Pasteurella pestis with Fluorescent Antibody. Bact. Proc. 1957, pp. 146-147. , and . A Rapid Identification of Pasteurella pestis with Fluorescent Antibody. (a): I. Production of Specific Anti- Serum with Whole Cell Pasteurella pestis Antigen. J. Infect. Dis. 104 :274— 280, 1959; (b): II. Specific Identification of Pasteurella pestis in Dried Smears. Ibid. 104 :281-287, 19509. Moony, M. D. and Winter, C. C. Rapid Identification of Pasteurella pestis with Fluorescent Antibody. ITI. Staining Pasteurella pestis in Tissue Impression Smears. J. Infect. Dis. 104 :288-294, 1959. Meyer, K. F. Recent Studies on the Immunity Response to Administra- tion of Different Plague Vaccines. WHO Bull. 9:619-636, 1953. Quan, S. F,, von FinteL, H., and McManus, A. G. Ecological Studies of Wild Rodent Plague in the San Francisco Bay Area of California. II. Efficiency of Bacterial Culture Compared to Animal Inoculation as Method for Detecting Pasteurella pestis in Wild Rodent Fleas. Am. J. Trop. Med. 7 :411-415, 1958. BAKER, E. E, et al. Studies on Immunization against Plague I. The Isola- tion and Characterization of the Soluble Antigen of Pasteurella pestis. J. Immunol. 68:131-145, 1952. Caen, T. H, and Meyer, K. F. Studies on Immunization Against Plague. X. Specific Precipitation of Pasteurella pestis Antigens and Anti- bodies in Gels. J. Immunol. 74 :501-507, 1955. Burrows, T. W. An Antigen Determining Virulence in Pasteurella pestis. Nature 177 :426-427, 1956. RockENMACHER, M., James, H. A, and ELBErG, S. S. Studies on the Nutrition and Physiology of Pasteurclla pestis. I. A Chemically Defined Culture Medium for Pasteurella pestis. J. Bact. 63:785-794, 1952. ENGLESBERG, E., and Levy, J. B. Studies on the Immunization against Plague. VI. Growth of Pasteurella pestis and the Production of the En- velope and Other Soluble Antigens in a Casein Hydrolysate Mineral Glucose Medium. J. Bact. 67 :438-449, 1954. HicucHl, K.,, and CaruiN, C. E. Studies on the Nutrition of Pasteurella pestis. 1. A Casein Hydrolysate Medium for the Growth of Pasteurella pestis. J. Bact. 73:122-129, 1957. Gunnison, J. B., Larson, A., and Lazarus, A. S. Rapid Differentiation between P. pestis and P. pseudotuberculosis by Action of Bacteriophage. J. Infect. Dis. 88:254-255, 1951. CavanaucH, D. C., and Quan, S. F. Rapid Identification of P. pestis Using Specific Bacteriophage Lyophilized on Strips of Filter Paper. Am. J. Clin. Path. 23:619-620, 1953. Brycoo, E. R. Le Diagnostique de la Peste par Inoculation a Souris de Produits Pathologiques Additiones de Penicilline. Bull. Soc. path. exot. 49: 409-414, 1956. HorNiBROOK, J. W. A Useful Menstruum for Drying Organisms and Viruses. J. Lab. & Clin. Med. 35:788-792, 1950. Jawerz, E., and Meyer, K. F. Avirulent Strains of Pasteurella pestis. J. Infect. Dis. 73:124-143, 1943. Moony, M. D., GoLomAN, M., and THoMAsoN, B. M. Staining Bacterial Smears with Fluorescent Antibody. I. General Methods for Malleomyces pseudomallei. J. Bact. 72:357-361, 1956. GirarDp, G., and Rosic, J. Vaccination Contre la Peste au Moyen d'une Souche de Bacilles de Yersin Vivants, de Virulence Atténuée. Bull. Acad. nat. med. 111:939-945, 1934. PASTEURELLA INFECTIONS 397 105. Larson, C. L.; Pap, C. B.; Wicar, W. C.; and HucHEks, L. E. Pre- cipitin Reactions with Soluble Antigens from Suspensions of Pasteurella pestis or from Tissues of Animals Dead of Plague. J. Immunol. 67 :280- 298, 1951. 106. Hover, B. H., and CoUuRDURIER, J. Detection of Plague Antigens in Tissues of Persons Dead from Plague. Bull. Soc. path. exot. 47:758-759, 1954. 107. Cuen, T. H., Quan, S. F,, and Meyer, K. F. Studies on Immunization against Plague. II. The Complement Fixation Test. J. Immunol. 68:147- 158, 1952. 108. McCrums, F. R., et al. Chloramphenicol and Terramycin in the Treatment of Pneumonic Plague. Am. J. Med. 14:284-293, 1953. CHAPTER 14 WHOOPING COUGH I. Isolation of the Etiological Agent A. Diagnostic Medium B. Directions for Obtaining Cultures 1. Cough plates 2. Nasopharyngeal swabs C. Incubation and Examination of Cultures D. Criteria for Identification of Colonies E. Reporting the Results of Cultural Examination 11. Pure Culture Studies A. Cultural and Staining Characteristics B. Agglutination Tests C. Reactions in Animals III. Serological Procedures in Diagnosis A. Agglutination B. Complement Fixation C. Opsonic Reaction References Bordetella pertussis* (Hemophilus pertussis)'-3 was first isolated from the sputum of an infant with whooping cough by Bordet and Gengou* in 1906. Koch’s postulates were not fulfilled until the work of Rich et al.’ with chimpanzees and of MacDonald and MacDonald® with children. The disease is a highly communicable infection of the respiratory tract characterized in its typical form by a series of spasmodic coughs, followed by a sudden forceful inspiration "(the * The bacterial species in this genus were formerly designated as Hemophilus pertussis, parapertussis bacillus or Hemophilus parapertussis, and Brucella bronchiseptica or Hemophilus bronchisepticus. For some years it has been con- sidered that the pertussis bacterium should not be taxonomically classified with the so-called influenzae group of bacteria that require one or both of the growth factors X and V. It does not require either factor. Further, Bordetella parapertussis and Bordetella bronchiseptica, which are closely related to pertussis and should be grouped with it, grow on nutrient agar without the addition of blood. For lack of a legitimate genus in which the three organisms could be grouped, Topley and Wilson! in 1946 tentatively placed parapertussis and bronchisepticus in the genus Hemophilus. In 1952, Moreno-Lopez2 pro- posed the genus Bordetella. The new genus is included in the seventh edition of Bergey's Manual and the species recognized are pertussis, parapertussis and bronchiseptica. Proom3 has furnished further justification for the new classifi- cation, in that bacteria of each species have an absolute requirement for nicotinic acid. 398 WHOOPING COUGH 399 whoop) and sometimes by vomiting. It is prevalent among infants and children. Sequelae manifested by retardation in physical and mental growth and by behavioral disorders are rare. Mortality rates have been declining: Average death rates per 100,000 population (U. S. Registration Area) were 3.84 for 1930-39 and 0.46 for 1949- 53. Additional information is given by Pittman.” Bordetella parapertussis is occasionally isolated from patients with symptoms of whooping cough, and there have been rare reports of B. bronchiseptica infections in human beings. In general the disease caused by B. parapertussis is mild and of short duration. Although the organism has been found in many areas of the world, information as to the true incidence is lacking. In Grand Rapids, Mich., a 16- year summary® showed that 2 per cent of whooping cough patients with positive laboratory findings had B. parapertussis infections. Lautrop?® reported a parapertussis epidemic in Copenhagen, and Brad- ford and Slavin! in New York found that B. parapertussis was the etiological agent in 5 per cent of the whooping cough cases studied during a relatively short period. The few B. bronchiseptica infections reported have been in persons who had contact with rabbits. These patients apparently developed the cough characteristic of whooping cough. The clinical course of whooping cough is variable but consists of three indistinct stages: the catarrhal, the spasmodic, and the con- valescent, each lasting for approximately 2 weeks. The local reaction caused by the microorganism and its toxic components in the epi- thelium of the trachea and bronchi results in the early catarrhal symptoms of sneezing and coughing, etc. When the organisms invade further into the deeper structures of the respiratory tract, peribron- chiolitis and interstitial pneumonia may result. Possible light on the essential lesion is obtained from the work of Gallavan and Good- pasture! on chick embryos infected with B. pertussis in which necro- sis of the basilar area of the epithelium with infiltration of polymor- phonuclear leucocytes occurred. In infants, secondary invading microorganisms such as pneumococcus, the hemolytic streptococcus Hemophilus influenzae, or staphylococcus may be the responsible agents when pneumonia develops. Edema, hemorrhage and obstruc- tion of the airways by mucous plugs induce atelectasis which, along with pneumonia, decreases oxygenation. The resulting anoxia ap- pears to be an important factor in the causation of brain injury where it occurs. The exact pathogenesis of the encephalitis which sometimes complicates the disease is not known; however, it differs from virus postencephalitis. 400 WHOOPING COUGH With the development of the paroxysmal cough, the white blood cell count is usually elevated, sometimes to an extreme degree. This is largely due to an absolute and relative increase in the number of lymphocytes. There is reason to believe that this lymphocytosis may be due to the heat-labile toxin, one of the antigenic components of the microorganisms. Extreme degrees of hyperleukocytosis (some- times over 100,000 cells per cu. mm of blood) may be present. A possible role of allergy in the pathogenesis of pertussis has been suggested by Toomey,'? who recognized that during the later stages of the disease the host’s cells become sensitized. Laboratory animals infected with B. pertussis antigens develop sensitivity to bacterial products (Eldering,'® and Parfentjev, Goodline and Virion), Mice injected intraperitoneally with pertussis vaccine (Parfentjev, Good- line and Virion?) or infected intranasally with B. pertussis (Pitt- man’) develop a 50- to 100-fold increase in susceptibility to hista- mine. On the other hand, guinea pigs and rabbits, normally very sensitive to histamine, become slightly less sensitive after injection of pertussis vaccine (Stronk and Pittman 17). It also appears (Bradford, Scherp and Tinker!®) that the characteristic changes in the blood are more pronounced in mice sensitized to the bacterial products. Because the disease is so variable in its manifestations, is easily “confused with a number of other conditions, and is so communicable at a time when diagnosis is most difficult, it is obvious that the etio- logical agent can be defined only by bacteriological methods. Further- more, the serious threat it poses to health and even to life during early infancy calls for immunization at an early age. I. ISOLATION OF THE ETIOLOGICAL AGENT B. pertussis is the usual etiological agent, with B. parapertussis found occasionally and B. bronchiseptica very rarely. The micro- organisms are recoverable from the trachea, bronchi, lungs, nasal passages and their secretions on Bordet-Gengou medium. Specimens for cultural examination are best obtained by the cough plate method of Mauritzen, described by Chievitz and Meyer! or by the plating of nasopharyngeal swabs as described by Bradford and Slavin?’ and used by various workers—for example, Saito, Miller and Leach. The pernasal swab is recommended, especially for use with young infants. For a detailed consideration of laboratory diagnostic methods in whooping cough, the reader is referred to a bulletin of the World Health Organization by Lautrop.?? WHOOPING COUGH 401 A. Diagnostic Medium General success in the isolation of B. pertussis has been attained only through use of either the original medium of Bordet and Gengou* or a modification of this formula. The medium recently recom- mended by Lacey?® has not had sufficient use to determine its practi- cality. The original formula of Bordet and Gengou called for a basal medium made up of glycerol potato extract combined with salt solu- tion (0.6%) and agar; for the final medium this base was mixed with an equal amount of rabbit or human blood. Since the medium did not contain peptone, it was considered less favorable to the develop- ment of putrefactive saprophytes. The large amount of blood re- quired makes this medium impractical in large-scale work. The Danish modification using 30 per cent blood has given excellent re- sults, especially for maintenance of stock cultures in an antigenic state. As a diagnostic medium, however, it has limitations because it is not possible to detect the hemolytic zone which is caused by the growth of B. pertussis. Recommended medium: For diagnostic work, reduction of the blood content to between 15 and 20 per cent with adjustment of the salt balance makes possible recognition of a hemolytic zone around the colony, a useful diagnostic criterion. Sheep blood is recommended. Such a modified formula has given very satisfactory results (CM No. 18). Penicillin in a concentration of 0.25 to 0.5 units per ml is an effective agent in reducing the growth of Gram-positive microorgan- isms on diagnostic plates. Occasional strains of B. pertussis are in- hibited by one unit of penicillin per ml, and perhaps rarely by even less. In order to increase the number of positive findings, it is suggested that two plates be used for each patient, one with and one without penicillin, Also, a nasopharyngeal swab may be streaked on plates of blood agar (CM No. 16) and Levinthal medium (CM No. 34 or CM No. 17) for examination for other microorganisms such as hemolytic streptococci, staphylococci and H. influenzae. B. Directions for Obtaining Cultures 1. Cough plates—Hold the uncovered plate of Bordet-Gengou medium 4 or 5 in. from the patient’s mouth during several explosive coughs. Cover the plate as soon as possible after obtaining the speci- men in order to avoid contamination. Send the plate to the laboratory as soon as possible to facilitate early incubation and early reporting. Exposed plates may be kept at room temperature for some time and 402 WHOOPING COUGH still be of use for diagnosis, but this procedure is not to be recom- mended because of the resulting delay. In transporting to and from the laboratory, the plates may be held together by heavy rubber bands and placed in manila envelopes with their accompanying history slips. Pyrex glass plates may be sent through the mail when wrapped in corrugated paper and placed in tightly fitted boxes. In Denmark and in the U. S. aluminum plates have been used by Sauer? in transporting medium and cultures through the mail. 2. Nasopharyngeal swabs—The swab consists of a small bit of cotton tightly wrapped about the end of a thin, flexible wire.* The prepared swab should be carefully inspected before sterilization to make certain that the cotton is secure on the wire and that the swab is very small and slender and entirely smooth. The sterile swab is passed gently through a nostril into the naso- pharynx of the child while his head is immobilized to prevent injury. Often a cough is induced, which increases the chances for successful results, especially if the swab is allowed to remain in place during a coughing paroxysm. If resistance is encountered because of a large turbinate, deviated septum or adenoids, force should not be used. It is occasionally not possible to pass the swab through either nostril. The inoculated swab is withdrawn from the nostril and streaked immediately over a portion of a plate of Bordet-Gengou medium. The inoculum is then spread over the entire plate with a platinum loop. If medium containing penicillin is used, the swab may be streaked directly over the entire plate. At this time other media may also be streaked, as suggested under Section IA herein. For these supplementary cultures, the use of a separate swab will obviate any possibility that penicillin picked up by a swab during the streaking of a pertussis diagnostic plate containing the antibiotic may inhibit growth. Transportation of nasopharyngeal swabs is limited by their rapid drying. Note on fluorescent antibody staining: Current reports suggest that direct slide preparations of nasopharyngeal specimens can be made from the pernasal swabs and stained with B. pertussis antiserum con- jugated with fluorescein isothiocyanate, Kendrick, Eldering and Eveland® believe this method may hold real promise for the rapid identification of B. pertussis in nasopharyngeal specimens but point * A wire that has been found satisfactory is braided bronze trolling line 2, No. 100B (300 ft winders) manufactured by Edwards Mfg. Co., 2215 South Michigan Avenue, Chicago 16, Ill. WHOOPING COUGH 403 out certain pitfalls. They recommend that diagnostic laboratories with the required equipment use the procedure—not alone, but in addition to standard cultural procedures. The slide preparations can be sent through the mail. C. Incubation and Examination of Cultures 1) Incubate the plates at 35° C and examine them several times during the first 48 hr to detect molds and other spreading colonies which might later overgrow the plate. With a sterile needle or scalpel remove the agar supporting and surrounding such spreaders. 2) Examine the plates after about 40 hr for colonies of B. pertussis. With a bright light and a hand lens, examine the plates by trans- mitted light to detect hemolysis and by reflected light for typical colony appearance. Examine the plates twice daily until B. pertussis is found. Most of the B. pertussis isolations will be obtained by the 4th day, and B. parapertussis by the 2nd or 3rd. Discard the plates after 5 or 6 days if no colonies have been identified. D. Criteria for Identification of Colonies 1. Colony appearance—B. pertussis colonies appear smooth, raised, glistening, pearly, almost transparent and are not over 1 mm in diameter. Colonies of Gram-positive cocci are generally duller, darker and more opaque. Colonies of B. pertussis are surrounded by a characteristic zone of hemolysis. The zone is not sharply delimited but merges somewhat diffusely into the surrounding medium. 2. Consistency of the growth—The consistency of the growth and the manner of its diffusion in water are typical of B. pertussis. The growth is homogeneous and when placed in a drop of water or salt solution, it spreads, showing first a momentary clumping effect which disappears with very slight agitation and leaves a smooth homogeneous suspension. 3. Morphology and staining reactions—Stained by Gram’s method B. pertussis decolorizes readily, even more readily than H. influenzae. Microscopical examination of a stained film will show small, faintly stained coccoid bacilli scattered evenly throughout the field which occur for the most part singly and in pairs, occasionally in short chains. The microorganisms are almost invariably less than 1p in length. Smooth, freshly isolated cultures show little pleomorphism. Strains maintained for long periods on laboratory media may show smooth-to-rough colonial changes with marked pleomorphism, thread- 404 WHOOPING COUGH like filaments, and thick bacillary forms, but these atypical micro- organisms are not encountered in diagnostic work. 4. Slide agglutination test—Suspend the suspected colonies in a drop of salt solution on one end of the slide. On the opposite end of the slide, mix several loopfuls of this suspension with several loop- fuls of antipertussis serum in appropriate dilution, usually to about a hundredth of the titer. For example, if the titer of the serum is 1:5,000, use a 1:50 dilution for the slide test. When the colonies are of B. pertussis, there will be almost immediate agglutination. The result is significant only if the control suspension without serum shows no clumping. Refer also to Section IIB for the technic of the quantitative agglutination test in tubes and for further comments. 5. Identification by subculture—If there is insufficient growth for a slide agglutination test, colonies of suspected B. pertussis may be transferred to another plate or to a Bordet-Gengou slant, After 24 hr incubation, there is usually sufficient growth for a slide agglu- tination test. 6. Differentiation of B. pertussis, B. parapertussis and B. bronchiseptica—As noted in the introduction, B. parapertussis is occasionally recovered from patients with pertussis-like symptoms. Also B. bronchiseptica has been reported from human beings in a few instances. Colonies of both these species on Bordet-Gengou medium resemble B. pertussis in all respects except that they develop more rapidly and grow to a larger size. These microorganisms are also morphologically indistinguishable from B. pertussis. Differentiation is based on agglutination tests and biochemical characteristics. Since cross-agglutination may occur to a relatively high titer, care must be taken in performing slide agglutination tests to use serum dilutions beyond the range of the cross-reactions; or, preferably, to use absorbed antisera for the three strains. Table 1 indicates the differ- ential cultural characteristics of the three species and those of bacteria of other genera in which one or more of these three microorganisms have at one time or another been classified. E. Reporting the Results of Cultural Examination 1. Positive report—As soon as plates show colonies that have cultural and morphological characteristics of B. pertussis and which agglutinate in specific antiserum, report as follows: “Bordetella per- tussis (Hemophilus pertussis) found.” Until the clinician becomes familiar with the newer terminology, the older name should also be WHOOPING COUGH 405 Table 1—Differential Characteristics of Bordetella Species and of Three Bacteria Formerly Thought Related B. B. per- B. para- bronchi- Alcal- H. influ- Characteristic tussis pertussis septica Brucella igenes enzae, etc. Growth on blood-free peptone agar _ + + XS + fet Browning of peptone agar on + 3 fg — _ Requires nicotinic acid + + + + — —~ Requires thiamine — — — + pr a= Requires growth fac- tors X and V — ee 2s one = oe Produces urease — + 4 hr + a ~~ Nitrate reduction — — + + of ee + Hemolysin + + ot een = ey ife Motility a et 0 fe 4 _ Citrate utilization — + + shown on the report. When B. parapertussis is found, report “Borde- tella parapertussis found” and include a negative report for B. pertussis. 2. Negative report—Plates showing no colonies of B. pertussis by the 4th day should be reported “Bordetella pertussis (Hemophilus pertussis) not found to date.” Such plates should be incubated for an additional day or two before being discarded. In the rare instance of a positive finding after the first report, an additional report may be forwarded: “Bordetella pertussis (H. pertussis) found after further incubation.” 3. Unsatisfactory result—If the diagnostic plate shows a very scanty inoculation or overgrowth with molds or other saprophytic microorganisms, making the report on B. pertussis unreliable, a nega- tive report should not be made. Report that the culture was un- satisfactory, give the reason, and request another specimen. Il. PURE CULTURE STUDIES A. Cultural and Staining Characteristics To preserve their antigenic stability, B. pertussis cultures should be maintained on Bordet-Gengou medium containing at least 15 per 406 WHOOPING COUGH cent blood. Some workers believe 30 per cent blood is preferable. For storage, it is recommended that cultures suspended in sterile skimmed milk be subjected to the freeze-dry process. It is possible to adapt cultures of B. pertussis to other media such as blood agar, chocolate agar, brain veal agar or infusion agar, but in the process of adaptation the microorganisms dissociate and lose the characteristics associated with smooth colony formation, assuming those of intermediate or rough colonies. The latter usually are non- pathogenic, show increased electrophoretic migration velocities, and are serologically and culturally altered as compared with the original parent strain. We are concerned herein only with smooth colony- producing cultures, the group designated Phase I by Leslie and Gardner.?® These cultures invariably are isolated from patients with pertussis, whereas dissociated forms result from laboratory manipu- lation. The colony appearance on Bordet-Gengou medium and the staining characteristics of B. pertussis have already been described. Capsules can be demonstrated by proper technic, perhaps the most satisfactory result being obtained using the stain devised by Lawson.?” Capsule swelling in antiserum has not been demonstrated. B. pertussis is strictly aerobic and grows best at a temperature of 34° to 37° C. A formula based upon that of Bordet-Gengou (CM No. 18) seems to be essential in providing for growth requirements and maintaining the characteristics of freshly isolated strains. How- ever, it has been demonstrated by several workers, including Horni- brook,?® Verwey and Sage,?® and Cohen and Wheeler,3® that smooth strains can be maintained for at least a few generations on defined liquid media which do not contain blood. With regard to the usual biochemical tests applied to bacteria, the pertussis microorganism is peculiarly inert. It does not ferment carbohydrates, form indole, reduce nitrates or liquefy gelatin. Litmus milk is slowly rendered alkaline. The production of alkali is a characteristic of all forms of B. pertussis, whether smooth or dissociated, and all media will have a final alkaline reaction of approximately pH 8.0. Catalase is pro- duced; tests for this enzyme may be made with H»O on washed, aerated suspensions of B. pertussis grown 48 to 72 hr on Bordet- Gengou medium, using a technic essentially as described elsewhere by Farrell.3! B. Agglutination Tests Agglutination procedures are used in identification of cultures, and in studies of relationships among strains, Recent observations in WHOOPING COUGH 407 different laboratories (Anderson,®? Lacey,?® Eldering et al.?*) indicate that freshly isolated cultures of B. pertussis do not comprise a homo- geneous serological group, as was previously believed; and it should be remembered that a culture may be isolated which is not agglutin- ated by the particular antiserum in use. Further work is needed be- fore there can be general agreement as to signficance of the serological differences observed by different workers. The following procedure for a rapid agglutination test is essentially the technic of Noble? applied to pertussis studies by Kendrick.?¢ I. Technic of Rapid Agglutination Test a. Antigen preparation—With a stiff bent needle, remove the growth of B. pertussis from Bordet-Gengou medium incubated for 36 to 72 hr and suspend in salt solution, If the suspension is not entirely smooth, filter through a thin layer of cotton. A technic for preparing simple filters for this procedure has been outlined by Kendrick. Adjust the turbidity of the suspension to approximately 20 billion microorganisms per ml, approximately 20 opacity units by photometric comparison with the pyrex glass opacity standard furnished by the National Institutes of Health. If a photometer is not available for adjustment of the turbidity of the suspension, it is suffi- ciently accurate for the agglutination test to consider McFarland density tube No. 3 as roughly equivalent to 10 billion B. pertussis per ml, b. Preparation of antiserum—Since it is difficult currently to find a source of pertussis diagnostic antiserum, the method of prepa- ration is outlined. Immunize a rabbit against a selected culture of B. pertussis. The antigen should be a suspension containing 10 bil- lion microorganisms per ml of culture grown on Bordet-Gengou medium (CM No. 18) for not more than 72 hr and killed by contact with a 1:10,000 solution of merthiolate or 0.5 per cent phenol for at least 48 hr at 4° C. Inject four doses of this vaccine—0.2, 0.4, 0.8 and 0.8 ml, respectively, per kg of weight—intravenously at 3 or 4 day intervals. One week after the fourth injection make a trial bleeding. If the agglutination titer against the homologous strain and several recently isolated strains is satisfactory, bleed the rabbit from the heart and collect the antiserum. The titer should be 1 :4,000 or more by the rapid test. Occasionally the titer will be found satis- factory after the third injection, and sometimes a rabbit will require more than the usual four injections. 408 WHOOPING COUGH c. Dilution of antiserum—DPrepare dilutions of the serum to cover the range of its activity, for example, 1:10 to 1:2,500. d. Agglutination test procedure—Mix 0.1 ml of each serum dilution with 0.1 ml of antigen in agglutination tubes, the measure- ments being made with graduated pipettes. For an antigen control, mix 0.1 ml of salt solution with 0.1 ml of antigen. Shake the serum-antigen mixtures by hand for 3 min, rocking the racks at a rate of approximately 60 back-and-forth motions per min (in such a way that the contents flow about three-quarters of the length up the tube) or in a shaking machine adjusted for the purpose. After the period of shaking, add 0.5 ml of physiological salt solu- tion to each tube to facilitate reading. The Hipple pipetting appara- tus, which is frequently used in the Kahn test, set to deliver 0.5 ml of salt solution, is convenient for large series of tests. e. Reading results—Read the reactions immediately after add- ing salt solution. Record each reaction as —, ==, 1, 2, 3 or 4 plus, according to the degree of agglutination. In determination of the titer, 2 plus is the lowest reading to be considered as an end point. It should be noted that the titers in this rapid test are not directly comparable to those obtained in other tests where the concentration of antigen, the total volume of reagents, and the time and temperature of incubation are dissimilar.3¢ C. Reactions in Animals Specific reactions in animals constitute helpful criteria for the study of B. pertussis. 1. Skin reactions—A skin reaction is produced in rabbits and guinea pigs by the intracutaneous injection of a living suspension of smooth B. pertussis. Within a few hours after injection there is an ischemic, indurated area at the site of injection. By 24 hr a purplish center appears, surrounded by an ischemic ring; beyond that, the outer part of the indurated zone appears inflamed and may show an arborization of capillaries. The central zone shows a dark purple necrotic area after 48 hr. A dose of a 0.1 ml suspension containing 2,000 million bacteria per ml induces a reaction about 1 cm in diameter. 2. Intraperitoneal injection—The intraperitoneal injection of mice and guinea pigs with a suitable dose of virulent B. pertussis is followed by death and characteristic gross and microscopic pathology WHOOPING COUGH 409 at autopsy. The fatal dose shows considerable variation with indi- vidual pigs. Usually 1 ml of a suspension containing 10,000 million bacteria per ml kills a 15 to 18 g mouse within 72 hr. A larger dose is usually required for guinea pigs. At autopsy there is observed a more or less extensive hemorrhagic area in the peritoneum at the site of injection and occasionally an area of necrosis in the skin. The peritoneal fluid is increased markedly in the guinea pig. Frequently all organs and membranes are covered with a sticky mucoid exudate. The heart, lungs, liver and spleen show no gross changes. B. pertussis may be recovered with ease from the peritoneal fluid, and cultures from the heart’s blood of the mice usually are positive. 3. Intranasal injection—The intranasal injection of susceptible strains of mice under anesthesia as described by Burnet and Tim- mins®? is followed by death of a large percentage of the animals. The dose must be determined for a particular set of conditions but is usually about 1,000 million microorganisms, or 0.04 ml of a 25,000 million per ml suspension. Nonfatal lung infection may be induced by fewer bacteria. At autopsy, pneumonia is indicated by gross and microscopical pathology. The pathological picture has been described in detail by Bradford.®® B. pertussis may be recovered in a large per- centage of the animals from the pleural fluid, lungs, trachea and heart’s blood. Reports on the use of the intranasal method of in- fecting mice have been made by Lawson,*® Miller and Silverberg*? and also by North, ef al.*! Intranasal infection of young rats with the production of experimental pertussis has been reported by Horni- brook and Ashburn.*2 4, Intracerebral injection—The course of disease in mice fol- lowing intracerebral injection of an infective dose of B. pertussis has been described by Kendrick, Eldering, Dixon and Misner.*3 There is an incubation period of several days after which the infected mouse shows a series of typical symptoms. Encephalitis is followed by death usually between 4 and 14 days after injection. The majority of freshly isolated strains in a dose of 107 microorganisms kill 16 to 20 g white mice. But more virulent strains are encountered: In some cases as few as 10% bacteria may be lethal. The intracerebral route for the infection of mice is useful as a method of determining the relative virulence of cultures; and, in protection tests, for studying the relative antigenicity of cultures. This route of infection has been used more successfully than any other in laboratory evaluations of the potency of pertussis vaccine, It 410 WHOOPING COUGH is used in the official test to determine the unit value of pertussis vac- cine in the United States. ill. SEROLOGICAL PROCEDURES IN DIAGNOSIS The demonstration of circulating antibodies by serological tests, that is, agglutination, complement-fixation, and opsonic tests, is of diagnostic help late in the disease or in atypical coughs of long dura- tion. Following vaccination, positive results with any of these tests are usually obtained in varying titers; their significance in terms of protection cannot be stated with certainty, As mentioned earlier, the potency of pertussis vaccine can be measured in terms of its active protection of mice, and evidence is reported by the Whooping-Cough Immunization Committee of the Medical Research Council** that mouse-protective activity is correlated with protection in children. Passive protection of animals with children’s sera following disease or vaccination has not been reported in large enough series for evaluation. In pertussis as in other diseases, it may be pointed out, occasional persons fail to produce demonstrable antibodies and a satisfactory explanation awaits further investigation, A few notes on the technic of several serological tests follow. A. Agglutination For testing a patient’s serum, the rapid agglutination technic already mentioned may be used except that lower serum dilutions should be included. A convenient series is 1:2, 1:4, to 1:128, etc. The series may be extended or modified to meet the requirements. A technic modified for testing children’s sera has been described by Miller and Silverberg.*s In two studies (Miller, Silverberg, Saito and Humber,*® and Sako*7), both using this technic, it was found that vaccinated chil- dren carrying high titers of agglutinins were clinically immune but, on the other hand, children with low titers were not necessarily susceptible, B. Complement Fixation A description of the technic of complement fixation is omitted, since this procedure has not been studied sufficiently with newer anti- gens made from smooth microorganisms and with the modern technic of complement fixation. Both technic and interpretation need re- investigation, Kristensen and Larsen*® reported on the use of com- plement fixation in 1926. Additional authors include Daughtry- Denmark,*® Paton,’ and Mishulow, Siegel, Leifer and Berkey.5! WHOOPING COUGH 411 C. Opsonic Reaction Opsonocytophagic tests have been used in pertussis studies by Kendrick, Gibbs and Sprick,’> by Singer-Brooks and Miller,’ by Bradford and Slavin,’ and by Rambar et al.?® The technic is given in the previous edition of this work.* There is a marked increase in opsonins following pertussis and also following injections of an active vaccine of B. pertussis. A high level is maintained for several months. The reactions may be slightly weaker after 6 months and relatively weaker as the interval after disease or immunization increases. The reaction in children with no record of a pertussis attack or immuniza- tion usually is negative or weakly positive. Pearn L. Kenbrick, Sc.D., Chapter Chairmant Harrie E. ALEXANDER, M.D. WiLLiam L. Braprorp, M.D. Grace ELDErING, Sc.D. Marcarer Prrrman, Pa.D. REFERENCES 1. Wison, G. S., and Mires, A. A. Topley and Wilson’s Principles of Bac- teriology and Immunity (4th ed.). Baltimore, Md.: Williams & Wilkins, 1957, Vol. I. Moreno-Lorez, M. El Genero Bordetella. Microbiol. Esp. 5:177-181, 1952. ProoM, H. The Minimal Nutritional Requirements of Organisms of the Genus Bordetella Moreno-Lopez. J. Gen. Microbiol. 12:63-75 (Feb.) 1955. 4. Borpert, J., and GeENcou, O. Le microbe de la Coqueluche. Ann. Inst. Pasteur 20:731-741, 1906. 5. RicH, A. R,, et al. The Experimental Production of Whooping Cough in Chimpanzees. Bull. Johns Hopkins Hosp. 58:286-306 (Apr.) 1936. 6. MacDonarp, H., and MacDonarp, E. J. Experimental Pertussis. J. Infect. Dis. 53:328-330 (Nov.-Dec.) 1933. 7. PrrrmaN, M. Pertussis and Pertussis Vaccine Control. J. Wash. Acad. Sci. 46 :234-243, 1956. 8. ELperiNG, G., and Kenbrick, P. L. Incidence of Parapertussis in the Grand Rapids Area as Indicated by 16 Years’ Experience with Diagnostic Cul- tures. A.J.P.H. 42:27-31 (Jan.) 1952. 9. Lautror, Hans, Parapertussis. Copenhagen: Munksgaard, 1954. 10. Braprorp, W. L., and Sravin, B. An Organism Resembling Hemophilus pertussis with Special Reference to Color Changes Produced by Its Growth upon Certain Media. A.J.P.H. 27:1277-1288 (Dec.) 1937. 11. Garravan, M, and GooprasTurg, E. W. Infection of Chick Embryo with H. pertussis Reproducing Pulmonary Lesions of Whooping Cough. Am. J. Path. 13:927-938, 1937. WN * Diagnostic Procedures and Reagents (3rd ed.). Prepared by the Sub- committee on Diagnostic Procedures and Reagents, American Public Health Assn. New York: The Association, 1950. + Note on authorship: The manuscript was prepared by P. L. Kendrick, Grace Eldering, and W. L. Bradford; reviewed and revised in consultation with other members of Whooping Cough-Influenza Bacilli Infections Committee (H. Alexander and M. Pittman). 412 WHOOPING COUGH 12. TooMEy, J. A. Mechanism of Whooping Cough. Am. J. Dis. Child. 56:469, 1938. 13. EvrperiNG, G. A Study of the Antigenic Properties of Hemophilus pertussis and Related Organisms. II. Protection Tests in Mice. Am. J. Hyg. 36, 3:294-302 (Nov.) 1942. 14. ParrFENTJEV, I. A., GoopLINE, M. A., and Virion, M. E. A Study of Sensi- tivity to Hemophilus pertussis in Laboratory Animals. II. Hemophilus pertussis Allergen and Its Assay on Laboratory Animals. J. Bact. 53:603- 611, 1947. 15. — A Study of Sensitivity to Hemophilus pertussis in Laboratory Animals. ITI. The Formation of Antibodies and the Development of Sensi- tivity in Laboratory Animals Injected with Flemophilus pertussis Antigens. J. Bact. 53:613-619, 1947. 16. PirrmaN, M. Sensitivity of Mice to Histamine During Respiratory Infec- tion by Hemophilus pertussis. Proc. Soc. Exper. Biol. & Med. 77:70-74, 1951. 17. StronNk, M. G., and Prrrman, M. The Influence of Pertussis Vaccine on Histamine Sensitivity of Rabbits and Guinea Pigs and on the Blood Sugar in Rabbits and Mice. J. Infect. Dis. 96:152-161 (Mar.-Apr.) 1955. 18. Braprorn, W. L., Scuere, H. W,, and Tinker, M. R. Effect of Extracts of Hemophilus pertussis on Leukocyte Counts in Normal and Sensitized Mice. Pediatrics 18:64-71, 1956. 19. CHievirz, J., and MEYER, A. H. Recherches sur la Coqueluche. Ann. Inst. Pasteur 30:503, 1916. 20. Braprorn, W. L., and Sravin, B. Nasopharyngeal Cultures in Pertussis. Proc. Soc. Exper. Biol. & Med. 43:590-593, 1940. 21. Sarto, T. M., MILLER, J. J., Jr.,, and LeacH, C. W. The Nasopharyngeal Swab in the Diagnosis of Pertussis. A.J.P.H. 32:471-474 (May) 1942. 22. Lautror, H. Laboratory Diagnosis of Whooping Cough or Bordetella Infections (with an annex by B. W. Lacey). WHO Bull. 23:15-35, 1960. 23. Lacey, B. W. A New Selective Medium for Haemophilus pertussis, Con- taining a Diamidine, Sodium Fluoride and Penicillin. J. Hyg. 52:273-303 (Sept.) 1954. 24. SAUER, Lours W. Whooping Cough: Resumé of a Seven Years’ Study. J. Pediat. 2(6) :740-749 (June) 1933. 25. Kenprick, P. L., ErperinG, G., and Everano, W. C. Application of Fluorescent Antibody Techniques: Methods for Identification of Bordetella pertussis. Am. J. Dis. Child. 101:149-154 (Feb.) 1961. 26. Lesuik, P. H., and GARDNER, A. D. The Phases of Haemophilus pertussis. J. Hyg. 31:423-434, 1931. 27. Lawson, G. McL. Modified Technique for Staining Capsule of Hemophilus pertussis. J. Lab. & Clin. Med. 25 :435-438, 1940. 28. Horni1BROOK, J. W. Cultivation of Phase I H. pertussis in Semisynthetic Liquid Medium. Pub. Health Rep. 54 :1847-1851, 1939. 29. Verwey, W. F., and Sack, D. An Improved Liquid Culture Medium for the Growth of Haemophilus pertussis. J. Bact. (Soc. Proc.) 49:520, 1945. 30. Coun, S. M.,, and WHEELER, M. W. Pertussis Vaccine Prepared with Phase I Cultures Grown in Fluid Medium. A.J.P.H. 36:371-376 (Apr.) 1946. 31. FarreLL, M. A. Studies on the Respiratory Mechanisms of the Streptococci. J. Bact. 29:411-435, 1935. 32. AnpersiN, E. K. Serological Studies on H. pertussis, H. parapertussis and H. bronchisepticus. Acta path. et microbiol. scandinav. 33:202-225, 1953. 33. Lacey, B. W. Antigenic Modulation of Haemophilus pertussis. J. Gen. Microbiol. 5:21, 1951. WHOOPING COUGH 413 34. 35: 36. 37. 38. 40. 41. 42. 43. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 53. ELperiNG, G., HornBECK, C., and BAKER, J. Serological Study of Bordetella pertussis and Related Species. J. Bact. 74:133-136, 1957. Nosre, A. A Rapid Method for the Macroscopic Agglutination Test. J. Bact. 14:287-300 (Nov.) 1927. Kenprick, P. L. Rapid Agglutination Technic Applied to B. pertussis. A.J.P.H. 23:1310-1312 (Dec.) 1933. Burner, F. M,, and Timmins, C. Experimental Infection with Haemophilus pertussis in the Mouse by Intranasal Inoculation. Brit. J. Exper. Path. 18:83-90, 1937. Braprorn, W. L. Experimental Infection in the Mouse Produced by Intra- tracheal Inoculation with Hemophilus pertussis. Am. J. Path. 14:377-383 (May), 1938. Lawson, G. McL. Immunity Studies in Pertussis. Am. J. Hyg. 28B:119- 131 (May) 1939. MILLER, J. J., JR, and SILVERBERG, R. J. H. pertussis Vaccines. The Effect of Washing and the Use of Mouse Protection Tests. J. Infect. Dis. 65: 16-23, 1939. NorrH, E. A.; KeocH, E. V.; ANDERSON, G.; and WILLIAMS, S. Passive Immunity in Experimental Pertussis. Am. J. Dis. Child. (Abstracts from current literature) 60,4:956-957 (Oct.) 1940. HornNiBroox, J. W., and AsuBurN, L. L. A Study of Experimental Pertussis in the Young Rat. Pub. Health Rep. 54 :439-444, 1939. Kenprick, P. L.; ErperiNg, G.; Dixon, M. K.; and MisNer, J. Mouse Protection Tests in the Study of Pertussis Vaccine: A Comparative Series Using the Intracerebral Route for Challenge. A.J.P.H. 37:803-810 (July) 1947. Medical Research Council, Great Britain. (a) Vaccination Against Whoop- ing Cough: Relation Between Protection in Children and Results of Laboratory Tests. Brit. M. J. 2:454 (Aug.) 1956; (b) Final Report. ibid. 1:994 (Apr.) 1959. MILLER, J. J., Jr, and SILverBERG, R. J. The Agglutinative Reaction in Rela- tion to Pertussis and to Prophylactic Vaccination Against Pertussis with Description of a New Technic. J. Immunol. 37:207-221, 1939. MILLER, J. J.; SILVERBERG, R. J.; Sarto, I. M.; and Humser, J. B. An Agglutinative Reaction for Hemophilus pertussis. I1. Its Relation to Clinical Immunity. J. Pediat. 22:644-651 (June) 1943. Sako, W. Studies on Pertussis Immunization. J. Pediat. 30:20-40 (Jan.) 1947. KristENSEN, M., and LARrseN, S. Production d’Anticorps aprés la vaccina- tion contre la Coqueluche. Compt. rend. Soc. de Biol. (danoise) 95:1110 (Oct.) 1926. DaveHTRY-DENMARK, L. Studies in Whooping Cough. Diagnosis and Immunization. Am. J. Dis. Child. 52:587-598, 1936. Paton, J. P. J. The Diagnosis of Whooping Cough. The Complement- Fixation and Intradermal Tests. Lancet 1:132-135 (Jan. 16) 1937. Misuurow, L.; Siecer, M.; LerFer, L.; and Berkey, S. A Study of Pertussis Antibodies. Am. J. Dis. Child. 63:875-890 (May) 1942. Kenprick, P. L., Gis, J., and Sprick, M. The Opsonocytophagic Test in the Study of Pertussis. J. Infect. Dis. 60:302-311, 1937. SiNGer-Brooks, C., and MILLER, J. J., JR. The Opsono-cytophagic Test in Children with Pertussis and in Children Vaccinated with H. pertussis Antigens. J. Clin. Invest. 16:749-761 (Sept.) 1937. Braprorn, W. L., and Sravin, B. The Opsono-Cytophagic Reaction of the Blood in Pertussis. J. Clin. Invest. 16:825-828, 1937. RaMBAR, A. C, et al. Studies in Immunity to Pertussis. J.A.M.A. 117:79-85 (July 12) 1941. CHAPTER 15 HEMOPHILUS INFECTIONS I. Introduction II. Methods for Isolation and Identification A. Culture Media 1. Blood Agar 2. Transparent Agar Media 3. Agents To Inhibit Gram-Positive Bacteria 4. Media for Determining Growth Factor Requirements B. Collection and Preparation of Specimens C. Identification Morphology Colonial Formation Determination of Type Specificity of H. influenzae Determination of Growth Factor Requirements 5. Other Bacteriological Procedures D. Maintenance of Cultures GR J00 IN pt References I. INTRODUCTION The bacteria in the so-called influenzal group of the genus Hemoph- ilus are divided into several species on the basis of their growth re- quirements for X and V factors (hemin and diphosphopyridine nu- cleotide) and their ability to hemolyze blood. The species that will be considered in this chapter are H. influenzae, H. aegyptius, H. parain- fluenzae, H. hemolyticus, and H. parahemolyticus. A—H. influenzae, which requires both X and V and does not hemo- lyze blood, is encountered in pathological conditions more frequently than any of the other species of this group. It occurs with or with- out a capsule, 1. Among the encapsulated H. influenzae, six distinct types have been observed. These are designated by the small letters a to f. Each type is characterized by a specific polysaccharide-like substance which may be demonstrated either by swelling of the capsule in the presence of type-specific antibody or by precipitation of the specific substance, dissolved in fluid medium or in exudates by type-specific antibody. Type b occurs in 90 to 95 per cent of the more severe H. influenzae pyogenic infections of childhood: meningitis, obstructive laryngitis, pneumonia, empyema, pyarthrosis, and osteomyelitis. None of these 414 HEMOPHILUS INFECTIONS 415 infections except the obstructive laryngitis has clinical features that differentiate it from corresponding infections caused by other species of bacteria. The laryngitis, however, appears to have a definite clinical entity and has come to be known as Type b H. influenzae epiglottitis. The onset is sudden and the course fulminating, Within a few hours dyspnea increases to a degree that makes tracheotomy imperative, Bacteremia is characteristic not only of all these infections but it frequently accompanies a mild upper respiratory infection. Types a and f are occasionally found associated with severe pyogenic infec- tions. The other types are infrequently encountered and then usually in milder infections or in normal throats. MN On onl AN {ola 2. The unencapsulated or nontype-specific strains of H. influenzae, hereafter designated as n.t.s., are found more frequently in apparently normal throats than are the type-specific strains. The incidence is re- ported to be 50 to 70 per cent.? Because of this distribution and the rare occurrence of these strains in the more severe H. influenzae pyogenic infections, there is a lack of agreement on their pathogenesis. It is certain that as a whole these strains are potentially less virulent than Type b strains. On the other hand, their recovery as the pre- dominant or sole microorganism in cultures taken from infected upper and lower respiratory lesions strongly suggests that if they are not primarily pathogenic, they play at times a significant role perhaps following tissue injury caused by a viral or another bacterial infection. Several cases of meningitis caused by these strains have been re- ported.3-% Otitis media, sinusitis and pneumonia in young children have also been reported to be caused by them.®? Mulder et al.®® con- sider that they play the role of a pathogen in chronic mucopurulent bronchitis ; Allibone et al.1® and Williamson and Zinnemann!! found them in sputa from the majority of children and adults with purulent bronchiectasis and concluded that they are responsible for keeping the chronic inflammatory process smoldering; Davis and Pittman? isolated them in pure culture from cases of acute conjunctivitis, There have been a few reports of the isolation of H. influenzae from cases of salpingitis. Hardy!® isolated H. influenzae from the vagina of children, and in most instances the same microorganism was isolated from the nose. Whether or not there are differences between the n.t.s. bacteria that are isolated from normal throats and those isolated from infected lesions is unknown. No systematic study has been reported. Some strains from pathologic sources have been found to have relatively high virulence for mice, 416 HEMOPHILUS INFECTIONS B—H. aegyptius (Koch-Weeks bacillus), the other nonhemolytic species that requires both X and V factors, is primarily associated with acute and subacute conjunctival infections.'* The endemic in- fections seem to be limited to the extreme southern areas in the United States and to tropical and subtropical climates throughout the world. C—H. parainfluenzae requires only factor V and is nonhemolytic. It is a normal inhabitant of the throat, although occasionally it is isolated from the cerebrospinal fluid of patients who have a brain abscess and from the blood of patients with endocarditis. Alexander! has divided the bacteria of this species into two groups, encapsulated and non- encapsulated, and states that there appears to be more than one type among the encapsulated forms. D—H. hemolyticus and H. parahemolyticus, the two hemolytic species, require factors X and V, and V, respectively. On blood agar the clear colorless zone around the colonies may cause them to be mistaken for hemolytic streptococci. Colonies of both species, how- ever, are usually more translucent than those of beta-hemolytic streptococci. Both microorganisms have been classified as H. hemo- Iyticus.®® Information about the species requiring both X and V factors, now designated as H. hemolyticus, is rather meager. FH. parahemolyticus may occur as the predominant microorganism in blood agar plate cultures obtained from patients who exhibit acute pharyngitis; their pathogenic role is not clear. So striking is the appearance of these colonies on the plate that the first impression is that the infection is due to Streptococcus pyogenes. E—Other small Gram-negative bacilli that are encountered in the laboratory may be confused with those in the hemophilic group. One of these is classified as Hemophilus aphrophilus. 817 Others have not been properly identified. Although they do not grow luxuriantly and may need special nutrients, these bacilli require neither X nor V factors for growth. They have been isolated from patients suffering from either endocarditis or meningitis, Some forms require CO. for isolation and cultivation. A hemophilic-like bacterium isolated from urine and from the lower genitourinary tract of men and women has been described by Leopold.’® Gardner and Dukes,'® Amies and Jones.2? Leopold found it associated with prostatitis and cervicitis ; Gardner and Dukes con- sider it to be the cause of a vaginitis (see Chapter 17). Isolation has been successful on Casman’s medium or on 10 per cent sheep blood agar plates incubated under increased carbon dioxide tension. The HEMOPHILUS INFECTIONS 417 organism is microaerophilic; colonies are tiny, pinpoint and colorless, surrounded by a zone of hemolysis. The exact growth requirements have not been reported. Certain requirements that are distinct from those bacteria in the so-called influenzal group, under consideration in this chapter, are indicated. Gardner and Dukes named this bacterium Hemophilus vaginalis. Differences between strains have been reported, and additional work is needed to determine whether more than one bacterium is involved. Hemophilic species have also been isolated from animals: Hemo- philus suis from swine influenza ;?* Hemophilus hemoglobinophilus from the preputial secretion of dogs; and others, from different species of animals. In Table 1 are given the differential characteristics of the influenzal species from human sources. Also given are the usual specimens from which the bacteria of the different species are isolated. Il. METHODS FOR ISOLATION AND IDENTIFICATION A. Culture Media When the growth factors are supplied, the bacteria of the influenzal group can utilize a variety of basal infusion media. Beef muscle, beef heart and brain-heart infusions are satisfactory. A pH of 7.5+0.1 is optimum. The most common source of X and V factors is blood. Hemin (X) and diphosphopyridine nucleotide (V') are available for purchase. Final concentration of 1:250,000 and 1:10,000,000, respectively, are adequate for promoting growth. The V factor may be prepared from brewer's yeast?> (CM No. 32); used for this purpose in a concentration of 5 per cent. For another source of X, media containing X and V factors (including blood) may be autoclaved for 5 min at 15 Ib pressure to destroy the V factor. 1. Blood agar—Although blood agar medium is not optimal for the growth of hemophilic bacteria, its use for diagnosis is indicated in order to differentiate and isolate other bacteria that may be present. Blood contains an enzyme that reduces or destroys the V factor, and the serum of some animals, and especially of man, is bactericidal. There may be no recovery of H. influenzae on media containing human or sheep blood. Rabbit and horse blood agars usually promote growth but the colonies are generally very small and dewdrop-like in appearance ; magnification is necessary to observe the relative number of colonies. Blood agar also facilitates isolation of the hemolytic species. Table 1—Differential Characteristics and Human Sources of Hemophilus Species sly Colonies, Required Transparent Agars Growth Mor- Semi- Hemol- Hemagglu- Species Factors phology solid Solid ysis tination ~~ Indole Serology Specimen H. influenzae: Capsule X,V cb,b,t If Semitrans- mn — Types a,b, 6 specific | Spinal fluid, blood, lucent, cf: plus; types sputum, throat and iridescent types d,e: conjunctival swabs, minus purulent exudates from joints, bones No capsule X,V cb,b g,sf Translucent, — — + Heterol- and genitourinary blue sheen ogous tract H. aegyptius X,V b,cb sf Translucent, — + — Homol- Conjunctival swabs blue sheen ogous H. para- Vv b g,sf Slightly — — — 2 types, Sputum, throat swab, influenzae opaque, dull others spinal fluid, blood or iridescent heterol- ogous H. hemolyticus X,V cb,b * Slightly + %* — * Sputum, throat swab, opaque blood ( ?) H. parahemo- Vv Pleo- g Dull, opaque + * — * Sputum, throat swab, Iyticus morphic blood Symbols: cb, cocobacillus; b, bacillus; t, threads; If, large fluffy; sf, small fluffy; g, granular. * Information inadequate. Note: Cultures of all species reduce nitrates to nitrites and in litmus milk containing growth factors there is no change or only slight acidity. Carbohydrate fermentation is weak; glucose is fermented by all species; xylose only by some strains of H. influenzae and H. para- hemolyticus, and polysaccharides are fermented only by H. parainfluenzae and H. parahemolyticus. SNTIHdOW3H SNOILD3IdNI HEMOPHILUS INFECTIONS 419 2. Transparent agar media a) Filtered heated blood medium—Although chocolate broth and agar are excellent for promoting growth, they are improved for diagnostic work if clarified (Levinthal medium, CM No. 34). b) Peptic digest of blood media—Fildes’ peptic digest of blood? (CM No. 31) is another excellent source of X and V. It is easily prepared and has the advantage that it may be kept for several years with little likelihood of contamination. In broth or solid agar medium it should be used for this purpose in a 2-3 per cent concentration; in semifluid agar (CM No. 15) add 0.05 ml to the top of the medium. c) Semifluid agar—Infusion broth containing 0.15 per cent agar to which 0.05 ml peptic digest of blood has been added to the top is valuable for primary isolation, for maintenance of cultures, and for differentiating colonial forms (see column 4, Table 1). To differen- tiate colonies stab the transferring loop into the medium after inocu- lating the top of the medium. Semifluid agar is also excellent in studies of pure cultures for making colony counts of broth cultures. The inoculum should contain not more than 20 bacteria. Twirl the tube after inoculation to distribute the bacteria and to aerate the medium. 3. Agents to inhibit Gram-positive bacteria—The use of 0.2 units of penicillin or 1.0 mg sodium oleate®* per ml of agar medium facilitates the isolation of pure cultures of hemophilic bacteria from sputum or throat cultures containing Gram-positive bacteria. Their use is particularly indicated in the case of transparent media, where some Gram-positive bacteria exert inhibitory action on hemophilic bacteria. 4. Media for determining growth factor requirements—Broth media are the best. Peptone broths are preferred for the basal medium. Extract and infusion media may contain traces of X factor. Use 2 ml of medium per tube in order to provide a relatively large surface area. To one tube add X and V, to a second V, and to a third X; a fourth tube without either factor serves to detect those bacteria which require neither factor. For sources and amounts, see first paragraph of this section (“Culture Media’). Agar media also may be used: autoclaved blood agar (X) and peptone agar (no X or V). The V factor is supplied by streaking a staphylococcus culture across the inoculated plate. Semifluid medium is less desirable because it has a lower oxidation-reduction 420 HEMOPHILUS INFECTIONS potential : Under reduced tension the hemophilic bacteria may require a lower concentration of X and additional transfers may be needed to eliminate the effect of X carried over in the inoculum. B. Collection and Preparation of Specimens Specimens in which the hemophilic bacteria may be present are listed in the last column of Table 1. The handling of cerebrospinal fluid is described in Chapter 16. Blood, sputum, throat swabs and purulent exudates are collected in the usual manner. Inoculate the blood specimen into a flask of broth medium which has a relatively large surface area. Streak sputum and throat cultures on one of the transparent agars and also on blood agar if indicated. An agent to inhibit the growth of Gram-positive bacteria is used when relatively pure cultures of hemophilic bacteria are desired. Incubate the plates for 20-24 hr. An atmosphere of 2-10 per cent carbon dioxide is often beneficial. Conjunctival cultures are made as follows: With the thumb pull the lower eyelid downward to expose the conjunctiva, while with the forefinger hold the upper lid to prevent it from blinking. Rub a small, sterile cotton swab moistened with broth across the exposed conjunctiva. Immediately place the swab in a tube containing about 5 ml of semifluid agar (plus 0.05 ml peptic digest of blood or some other source of X and V factors). Incubate the tube for about 4 hr. Then remove the swab and streak it across one side of a transparent agar plate—and also a blood plate if isolation of other bacteria is de- sired. With a loop, streak the inoculum over the remaining portion of the plate. Incubate the plates for 20 to 24 hr at 35° C. Return the primary tube to the incubator for possible recovery of bacteria that may be scarce in the culture, C. Identification The differential criteria of five species of Hemophilus are given in Table 1. 1. Morphology—The bacteria of the five species are relatively small Gram-negative rods. Only H. parahemolyticus can be differ- entiated morphologically. It is usually somewhat larger, stains more heavily, and is more pleomorphic; the bacteria frequently appear as long tangled threads with globoid areas.’ Dominant morphological types appear among the other species but there are overlappings among them. Nontype-specific (n.t.s.) H. influenzae are usually coccoid while the type-specific bacteria tend to appear slightly wider and HEMOPHILUS INFECTIONS 421 longer and to show more elongated or thread forms. FH. aegyptius tends to be a very slender, slightly elongated rod, but at times it may be coccoid and it stains faintly.'* H. parainfluenzae tends to be slightly longer than n.t.s. H. influenzae and to have slightly pointed ends ; and it stains more heavily than H. aegyptius. The limited descriptions of H. hemolyticus indicate that coccoid forms predominate. The mor- phology of the bacteria of all of the species is influenced by length of incubation period and the cultural medium. The descriptions given apply to the bacteria in the logarithmic growth phase from optimal media. 2. Colonial formation—On blood agar the colonies of nonhemo- lytic species of Hemophilus are dewdrop-like and usually pinpoint in size. Type-specific colonies can seldom be differentiated from colonies of other hemophilic strains. If the culture is contaminated, especially with staphylococci, the colonies are considerably larger in the neigh- borhood of the foreign colony (satellite phenomenon). On trans- parent agar, H. influenzae colonies may be 1.0 to 2.0 mm in diameter, and the colonies of type-specific strains are readily differentiated when they are observed in obliquely transmitted light (100 watt lamp). The type-specific colonies are iridescent, mucoid and slightly opaque, while the n.t.s. colonies have a bluish sheen, are nonmucoid and translucent. At times the bluish sheen of n.t.s. colonies may be mistaken for iridescence. When there is doubt, transfer to semifluid agar. Make the transfer to the top of the medium, then before with- drawing the loop make one stab. The type-specific strains produce a large fluffy colony. The n.t.s. strains usually produce a granular colony, although an occasional strain produces a small fluffy colony. On solid transparent agar or blood agar, H. aegyptius develops at a slightly slower rate than H. influenzae, but its colonies are indis- tinguishable from those of n.t.s. H. influenzae. In semifluid agar, however, they usually differ. The colonies start as granules and then each develops a small, fluffy comet-like tail. The colonies of H. parainfluenzae on transparent media tend to be smaller and more opaque than n.t.s. H. influenzae. They may or may not be iridescent in transmitted light; green predominates. They lack the mucoid transparency of type-specific colonies of H. influenzae. In semifluid agar most strains produce granular colonies. The colonies of H. parahemolyticus on blood or transparent agars are the largest of all the species. On blood agar they are slightly opaque and are surrounded by a clear zone of hemolysis. On trans- parent agar in transmitted light they are more opaque and they lack 422 HEMOPHILUS INFECTIONS the glistening character of H. influenzae colonies. They are also granular in semifluid agar. 3. Determination of type specificity of H. influenzae—The serological type of a strain of H. influenzae is determined by capsular swelling, precipitation of the soluble specific substance, or agglutina- tion of the microorganisms in the presence of type-specific antibody. Direct typing of the microorganisms in cerebrospinal fluid or some other exudates is performed by the first two tests. The capsular swelling test is the simplest and most rapid method for direct typing of microorganisms in cerebrospinal fluid.! Positive reactions may be observed when the bacteria are too scarce to be detected in a stained film. The method is described in Chapter 7. In the interpretation of a positive test obtained with exudates, it should be kept in mind that certain types of pneumococci may react with H. influenzae Types a, b or c antisera.’ When performing the capsular swelling test with cultures, whether from broth or agar, use only cultures that are in the logarithmic growth phase; after this phase the capsules degenerate. When the specimen inoculated contains only a few bacteria and the initiation of growth is retarded, a 24 hr culture may be satis- factory, while a transplant from a rapidly growing culture to broth may be ready for testing in a few hours. Occasionally type-specific bacteria in specimens from patients under antibiotic therapy fail to give a positive reaction, yet success may be obtained with the culture after several rapid transfers. For the precipitin test, use the clear supernatant from centrifugated cerebrospinal fluid, a broth culture or a salt solution suspension of bacteria from solid media. Layer the clear supernatant on the top of a 1:2.5 dilution of Type b antiserum. If a ring does not form at the juncture of the fluids within a few minutes, repeat test with other type-specific antisera. If no tube immediately shows a positive ring reaction, incubate the tubes for 1 hr at 35° C. Perform the agglutination test with young cultures that have been washed off solid medium with salt solution. Prepare several serial dilutions of the antiserum beginning with 1:5 or 1:10. Add the anti- gen to the tubes and shake vigorously, then incubate for 1 hr at 35° C. Read the reaction immediately or after overnight in a re- frigerator. When the culture is type specific, the agglutinated bacteria form a disk in the bottom of the tube. Do not use tempera- tures of incubation higher than 37° C, as they cause a dissolution of the capsule; the exposed somatic antigens may react with somatic antibodies in the antiserum to give a nontype-specific agglutination. HEMOPHILUS INFECTIONS 423 4, Determination of growth factor requirements—Inoculate the four media listed under Section ITA (4) of this chapter with a small inoculum of the culture under test and incubate for 24 hr at 35° C. If growth occurs only in the presence of X and V factors, it may be stated that the microorganism requires both factors. If growth appears in any of the other tubes, make a second or third transfer to eliminate the possibility of a carry-over of a growth factor in the original inoculum. With agar plates (Section ITA (4) of this chapter) inoculate with a washed culture, then streak halfway across the inoculum with a staphylococcus culture. If the culture grows only in the environ- ment of the staphylococcus and on both plates, it requires only the V factor; if only on the X plate, it requires both X and V. Determine the growth factor of all suspected hemophilic cultures from the blood of patients suffering from endocarditis and also of other hemophilic cultures that are not typical for the species. In a survey of respiratory tract bacteria, test all suspected hemophilic bacteria for their growth factor requirements. 5. Other bacteriological procedures—The bacteria of all five species under consideration are soluble in 1.0 or 2.0 per cent sodium desoxycholate. Add 0.1 and 0.2 ml of a 10 per cent solution of sodium desoxycholate to 0.9 and 0.8 ml, respectively, of a young broth culture and incubate for 2 hr at 35° C. Both tubes should be- come clear, except possibly for a slight opalescence. These five species of Hemophilus all reduce nitrates to nitrites. To demonstrate this property use peptone broth containing 0.1 potassium nitrate and the growth factors. Do not use infusion or extract broths; by the end of 24 or 48 hr the microorganisms may have re- duced any nitrite that was formed, in which case a negative nitrite test will be obtained. The fermentation reactions of the hemophilic bacteria are not strong—the lowest pH produced is around 6.4. Nevertheless the re- actions, when combined with other criteria, are of some differential value. Agar slants containing 1.0 per cent carbohydrate, Andrade indicator, and the growth factors may be used. The significant dif- ferential reactions are given in a footnote to Table 1. D. Maintenance of Cultures For laboratory maintenance, semifluid agar is better than slants or broth media. Transfer the cultures each week, or more often, and keep them in the incubator. They die rapidly when placed in the 424 HEMOPHILUS INFECTIONS refrigerator. H. influenzae usually remains viable a week at room temperature (22°-25° C), but H. aegyptius usually dies. Do not hold plate cultures in the refrigerator for subsequent fishings; hold them at room temperature. Type-specific H. influenzae tends to lose specificity on artificial media. Some strains may change to the nonencapsulated form within a week. Little is known about changes of the nontype-specific bacteria on artificial media. If the cultures are to be used for experimental work it is best to freeze-dry them immediately after isolation. Type- specific cultures may also be kept without apparent dissociation by transferring them biweekly in 1.0 ml of freshly drawn defibrinated rabbit blood while it is still warm. Incubate the blood culture over- night and store it in the refrigerator between transfers. Of the non- type-specific strains, only the mouse-virulent strains can be main- tained by this procedure. Marcarer PrrrmaN, PH.D. Chapter Chairman Harrie E. ALEXANDER, M.D. W. L. Braprorp, M.D. Grace Evpering, Sc.D. Peart L. Kenprick, Sc.D. REFERENCES 1. Arexanper, H. E. “The Hemophilus Group,” in Bacterial and Mycotic Infections of Man (2nd ed.). Philadelphia: J. B. Lippincott, 1952. 2. STRAKER, E., Hirt, A. B, and LoveLr, R. A Study of the Nasopharyngeal Bacterial Flora of Different Groups of Persons Observed in London and South-East England During the Years 1930 to 1937. Reports on Public Health and Medical Subjects No. 90. London: His Majesty’s Stationery Office, 1939. 3. Pittman, M. The Action of Type-Specific Hemophilus influenzae Anti- serum. J. Exper. Med. 58:683, 1933. 4. Goroon, J., Wooncock, H. E. pe C, and ZiNNEMANN, K. Meningitis Due to Pittman and non-Pittman Strains of H. influenzae. Brit. M. J. 1:779, 1944. 5. ZinNEMANN, K. A Survey of the Outcome of 20 Cases of H. influenzae Meningitis Related to Bacterial Type. Brit. M. J. 2:931, 1946. 6. TunevaLL, G. Oto-rhinological Infections in Childhood. Sero-bacteriologi- cal Studies of Paranasal Sinusitis and Suppurative Otitis with Special Reference to Haemophilus influenzae. Uppsala: Almqvist & Wiksell Boltryckeri AB, 1952. Also Suppl. 92, Acta paediat. 7. PrrrmaN, M. The Protection of Mice Against Hemophilus influenzae (Nontype-Specific) with Sulfapyridine. Pub. Health Rep. 54:1769, 1939. 8. Mutrper, J.; Gosrings, W. R. O.; van per Pras, M. C.; and Lops Carnozo, P. Studies on the Treatment with Antibacterial Drugs of Acute and Chronic Mucopurulent Bronchitis Caused by Hemophilus influenzae. Acta med. Scandinav. 143:32, 1952. 9. MuLbEr, J. Bacteriology of Bronchitis. Proc. Roy. Soc. 49:773, 1956. HEMOPHILUS INFECTIONS 425 10. 11. 12. 13. 14. 185, 16. 17. 18. 19. 20. 21. 22 23, 24. 23. Arvmeong, KE. C, Arvuson, P. R, and ZinNeEmaNN, K. Significance of H. influenzae in Bronchiectasis of Children. Brit. M. J. 1:1457, 1965. WirLLiamsoN, G. M., and ZiNnNEMANN, K. Noncapsulated Haemophilus wmfluenzae, a Major Factor in Chronic, Nontuberculous Respiratory Infec- tion. Abstr. VII Internat. Cong. Microbiol. Uppsala: Almqvist & Wiksell Boltryckeri AB, 18u, 1958. Davis, D. J., and Pittman, M. Acute Conjunctivitis Caused by Hemophilus. Am. J. Dis. Child. 79:211, 1950. Harpy, G. C. Vaginal Flora in Children. Am. J. Dis. Child. 62:939, 1941. Pittman, M,, and Davis, D. J. Identification of the Koch-Weeks Bacillus (Hemophilus aegyptius). J. Bact. 59:413, 1950. Prrrman, M. A Classification of the Hemolytic Bacteria of the Genus Haemophilus: Haemophilus haewmolyticus Bergey et al. and Haemophilus parahaemolyticus nov. spec. J. Bact. 65: 750, 1953. KaAamrAT, O. Endocarditis Due to a New Species of Haemophilus. J. Path. & Bact. 50:497, 1940. TosuAcH, S., and Bain, G. O. Acquired Aortic Sinus Aneurysm Caused by Hemophilus aphrophilus. Am. J. Clin. Path. 30:328, 1958. LeororLp, S. Heretofore Undescribed Organism Isolated from the Geni- tourinary System. U. S. Armed Forces M. J. 4:263, 1953. GarDoNER, H. L., and Dukes, C. D. Haemophilus vaginalis vaginitis. A Newly Defined Specific Infection Classified as “Nonspecific” Vaginitis. Am. J. Obst. & Gynec. 69:962, 1955. Awmies, C. R, and Jones, S. A. A Description of Haemophilus vaginalis and Its L Forms. Canad. J. Microbiol. 3:579, 1957. Lewis, P. A, and SHore, R. E. Swine Influenza. A Hemophilic Bacillus from the Respiratory Tract of Infected Swine. J. Exper. Med. 54:361, 1931. Tujoérra, T., and Avery, O. T. Studies on Bacterial Nutrition. II Growth- Accessory Substances in the Cultivation of Hemophilic Bacilli. J. Exper. Med. 34:97, 1921. Fipes, P. New Medium for Growth of B. influenzae. Brit. J. Exper. Path. 1:129, 1920. Avery, O. T. A Selective Medium for B. influenzae. Oleate Hemoglobin Agar. J.AM.A. 71:2050, 1918. EncBoEk, H. C. Pfeiffer’s Bacillus, Serological Studies with Special Refer- ence to Capsular Antigens. Acta. path. et microbiol. scandinav. 27:378, 1950. II. IIT. IV. VI VIL VIII CHAPTER 16 BACTERIAL MENINGITIS . Common Etiological Agents Handling and Transportation of Specimens Materials Needed and Their Preparation A. Culture Media B. Stains C. Chemical Reagents D. Sera Examination of Specimens A. Cerebrospinal Fluid Collection Microscopical Examination Cultural Examination Chemical Tests Serological Tests for Syphilis . Animal Inoculations B. Blood C. Petechiae D. Nasopharynx SRN aN . Identification of Pure Cultures A. Meningococcus and Other Neisseria 1. Bacteriological 2. Serological Reactions 3. Reporting 4. Other Neisseria B. Microorganisms Other Than Neisseria H. influenzae D. pneumoniae Strep. pyogenes Staph. aureus Proteus and Pseudomonas Species E. coli and Related Organisms Other Bacteria MOIR La) Oe Testing for Antibiotic Sensitivity Serological Examinations A. Cerebrospinal Fluid B. Patient’s Serum Other Methods Not in Common Use References 426 BACTERIAL MENINGITIS 427 The term “meningitis,” as used in this chapter, may be defined as an inflammation of the meninges, accompanied in varying degree by inflammation of the brain, spinal cord and/or their vascular supply, and frequently by manifestations involving other systems. In all instances there is inflammation of the meninges, with increased cell count in the spinal fluid and usually certain other features which are common to meningitis regardless of etiology: signs of spasticity of the erector muscles of neck and back, evidence of mental disturbance, and many less specific symptoms such as fever, headache, irritability, nausea and vomiting. In young infants the usual signs do not appear until late. A bulging fontanel, fever and vomiting may be the only signs. Early diagnosis is of the utmost importance—hence a high index of suspicion is necessary, and the spinal fluid should be studied without waiting for manifestations to become pronounced. In many instances infection of the meninges is only one part of a generalized systemic illness, whereas in others it is the predominant manifestation. In this chapter “inflammatory diseases of the meninges” will be considered synonymous with infection. It is immediately apparent that such infection can be caused by any of the six major microbial groups: viruses, rickettsia, bacteria, fungi, spirochetes and protozoa. This chapter will deal primarily with meningitis caused by bacteria. From the laboratory point of view the first important differentia- tion is made between purulent, or polymorphonuclear response, and nonpurulent, or mononuclear response in meningitis. In general only bacteria cause a type of meningitis in which there is a marked predom- inance of polymorphonuclear cells in the spinal fluid (often over 1000 per cu. mm), usually associated with a glucose content below normal. An exception to this general rule among bacteria is Myco- bacterium tuberculosis. Infections with this organism and those due to most viral, rickettsial, spirochetal, fungal and protozoal agents are characterized by a nonpurulent type of spinal fluid. Although the cell count is usually low, fluids from early tuberculous and viral meningitides may show a predominance of polymorphonuclear cells. The procedures to be followed in their identification will not be out- lined in this chapter. The meningeal involvement in these non- bacterial diseases is often part of a generalized illness, and the study of the spinal fluid is one facet of the over-all investigation. In very early stages of bacterial meningitis the spinal fluid may lack polymor- phonuclear characteristics. Therefore, all nonpurulent as well as purulent spinal fluids should have a bacteriological study, even though the likelihood of bacterial etiology is remote. 428 BACTERIAL MENINGITIS With the exception of petechial skin lesions frequently associated with meningococcus infections, there are no clinical signs which re- liably differentiate the meningitides. Laboratory examinations are therefore of crucial importance in establishing rapid and accurate etiological diagnosis. Speed and accuracy of identification enable clinicians to utilize optimal therapy for the organism involved. Il. COMMON ETIOLOGICAL AGENTS In principle, any bacteria that are capable of invading human tis- sues can cause infection of the meninges. Even where there is no obvious portal of entry, a large variety of microorganisms have found their way into the nervous system. Table 1 lists many kinds that have been found to do so. Obviously all these bacteria cannot be treated in detail in this chapter. Nine species of importance which are representative of the groups involved will be considered here. Discussion of these species will serve as a guide to the study of others more or less related to them. Those considered are: 1. The meningococcus N. meningitidis, which is responsible for epidemic cerebrospinal fever 2. H. influenzae, the next most common cause of meningitis 3. The pneumococcus D. pneumoniae 4. Strep. pyogenes 5. Staph. aureus 6. Proteus species 7. Pseudomonas species 8 9 . E.coli . M. tuberculosis It is important to keep in mind that many other species not listed may be involved (details concerning them may be found in other chapters of this volume). N. meningitidis—The normal habitat is the nasopharynx. Many persons harbor meningococci for periods varying from a few days to a number of years. Sometimes few such microorganisms are present, at other times they are predominant. Carrier surveys in which a single nasopharyngeal culture is made from each individual have little or no value. The significance of the recovery of the menin- gococcus from the nasopharynx, both in epidemic and endemic periods, depends on its serological group and its virulence. An ac- curate serological typing is essential. Some chronic carriers harbor strains of negligible virulence that do not seem to fall into any of the known groups. BACTERIAL MENINGITIS 429 Table 1—Bacteria Which Cause Meningitis Organism Comments A. Chief manifestation is a meningitis: Common Neisseria meningitidis (meningococcus) ....... a Hemophilus influenzae (Pfeiffer’s bacillus) Diplococcus pneumoniae (pneumococcus) M. tuberculosis Infrequent Staphylococcus aureus ....... oT Other Neisseria species Mimeae species ............... Escherichia coli «........... esezennre Listeria monocytogenes Streptococcus pyogenes Alcaligenes species Aerobacter-Klebsiella group Salmonella species Other Gram-negative rods of the colon intermediate group Bacteroides species Pseudomonas species Proteus species Nocardia asteroides Cause of epidemic cerebrospinal fever Most frequent cause of meningitis in infants and children when meningo- cocci are not epidemic A common cause of endemic meningi- tis in adults; less common than H. in- fluenzae in children Usually characterized by a nonpuru- lent spinal fluid—may be mixed with other bacterial infection Almost always a portal of entry such as otitis media is found Often confused at first with meningo- coccus Particularly in neonatal infants 430 BACTERIAL MENINGITIS Table 1—Bacteria Which Cause Meningitis (Continued) Organism Comments B. Chief manifestation is a meningitis with an artificial portal of entry: Common Staph. aureus Pseudomonas species Usually an artificial portal of entry E. coli, Aerobacter-Klebsiella directly into the meninges following group, Alcaligenes species, recent trauma, surgical procedure or and related microorganisms lumbar puncture, or caused by con- . genital malformation (such as spina Proteus species bifida) Enterococcus species Infrequently other bacteria.... Any bacteria capable of invading human tissue, including common pathogens such as Strep. pyogenes and pneumococcus, as well as saprophytic organisms and rare human pathogens, e.g., Pasteurella multocida C. Meninges are one of many tissues involved in a generalized infection process: Common N. meningitidis «.o.vovvnenennnns . Particularly fulminating infections H. influenzae ..... vss wn Most common of all when meningo- coccus is not epidemic D. pneumoniae M. tuberculosis .......oovueeninnn As part of miliary tuberculosis Infrequent Neisseria gonorrheae Salmonella, including S. typhosa Almost any generalized infection with Brucell i : EE ucella species bacteremia may develop a meningitis Staph. aureus Bacillus anthracis N. asteroides Cryptococcus neoformans ....... . A fungus, but mentioned here because of its importance as a cause of meningitis BACTERIAL MENINGITIS 431 Classification of the meningococcus into groups and types has undergone many changes since Weichselbaum first identified it in 1887 as the cause of meningitis. Recognition by Dopter in 1909 that all meningococci were not serologically identical was followed during the past half century by so many independent observations that some confusion ensued. The Subcommittee on Neisseriaceae of the Inter- national Committee on Nomenclature described a classification! which follows the International Code of Nomenclature.? This classification is used in the present discussion. Table 2 shows its relation to other previously used classifications. When cultures of meningococci are typed, no confusion need result if the relationships shown in this table are kept in mind. Table 2—Relationship Among the Most Common Classifications of Meningococci Recom- Gordon Nicolle, mended by and Griffith Debains In Common Inter- Dopter and Murray, and Scott, and Jouan, Use Since national Pauron, 1914 1915 1916 1918 1940 Committee Meningococcus I I A I A III Parameningo- IT 11 B coccus II B iv Iv D Cc Ila C D* * Relation of this Group D to other groups is unknown. The importance of serological classification of cultures from pa- tients and carriers is frequently underestimated. More than 90 per cent of cases of meningococcus infection during epidemic years have been due to Group A (I), whereas Groups B (II) and C (IIa) have + There has been no widespread epidemic since 1943 and few Group A strains have been encountered during these years, whereas infections with Groups B and C have occurred with equal frequency. 432 BACTERIAL MENINGITIS usually accounted for sporadic cases. By far the greater number of carrier strains recovered during nonepidemic periods are of Group B. Although rarely involved in epidemics, meningococci of Groups B and C are often very virulent and small outbreaks do occur. In consider- ing the question of quarantine and release of contact and convalescent carriers or patients, the facts presented here are fundamental. LH. influenzae—When meningococcus infections are not epi- demic, H. influenzae is the most common cause of meningitis in in- fants and children. H. influenzae occurs frequently in the upper air- ways. Most of these organisms are nontype-specific. Of particular importance in causing meningitis are members of Type b. This type is responsible for 90 to 95 per cent of H. influenzae meningitis, which is almost exclusively a disease of infants and very young children. Type a and nontypable strains occasionally cause meningitis. H. in- fluenzae meningitis is generally preceded by an infection of the respiratory tract and the microorganisms can be demonstrated in the nasopharyngeal mucus; from this focus bacteremia is the rule. In- fection in the lung, ear or joints will accompany meningitis in some patients. Since these infections are dangerous to infants and young children, patients with any of them should be separated from other young children in the wards. Meningitis of any origin is usually a reportable disease. Hemophilus infections in general are discussed in Chapter 15. Pneumococcus—The normal habitat for pneumococci is the upper respiratory tract of man. The carrier rate varies considerably for different types; the invasive characteristic, insofar as causing pneumonia is concerned, is greatest for Types 1, 2, 3, 5, 7, 8 and, in children, Type 14. But in the causation of meningitis any type may be involved. In infants the meningitis is secondary to a bacteremia, with or without other localization. In the older child and adult there is frequently an associated purulent focus continguous to the men- inges, such as otitis media, mastoiditis or sinusitis ; or other involve- ment, such as lobar pneumonia, empyema, pericarditis or endo- carditis. In general the meningitis accompanying severe systemic illnesses is more apt to be incited by a type generally associated with pneumonia, whereas those secondary to ear infection are more apt to be of Types 3 and 8, since these often cause otitis in children, Those secondary to injury into the upper airway may be any of the higher carrier types. For a discussion of pneumococcus infections, see Chapter 7. BACTERIAL MENINGITIS 433 Strep. pyogenes—Streptococci, especially those of Group A, often occur in the nose and throat of adults and children. Menin- gitis is usually the result of a direct spread of infection from the upper airway, particularly from the ear and/or mastoid cells, through the skull and into the meninges. Meningitis may complicate a septicemia following a respiratory or wound infection. General public health aspects of streptococcus infections, classifica- tion of these microorganisms, and the significance of serological groups are discussed in Chapter 5. Staph. aureus—Organisms of this group are found in the nose in about 50 per cent of the population. Despite this situation they are relatively rare as a cause of meningitis and when they are implicated, there was most likely an artificial portal of entry. Rarely a severe systemic infection with these microorganisms will develop an associated meningitis. Many serious micrococcal infections are traceable to hospital personnel and these may be due to antibiotic-re- sistant strains. This possibility is foremost in the requirements for aseptic precautions when the meninges are exposed or spinal punc- ture is made. A full discussion of staphylococcus infections may be found in Chapter 6. Pseudomonas and Proteus species—Species of Pseudomonas and Proteus usually invade the nervous system through artificial portals. These microorganisms are relatively resistant to most cur- rently available antibiotics, although Pseudomonas infections can at times be effectively treated with polymyxin. As with the staphylo- coccus, the occurrence of these infections emphasizes the need for careful asepsis. E. coli is an infrequent cause of meningitis, although it may enter the meninges under circumstances similar to those favoring the Pseudomonas. These microorganisms are of particular importance in infants in the neonatal period; they may cause a primary systemic infection of which meningitis may be the major manifestation. A full discussion of these last three named groups of bacteria may be found in Chapters 10 and 22. M. tuberculosis—Although meningitis incited by M. tubercu- losis is not uncommon, such infections are not among those considered in detail here, since they are generally characterized by a nonpurulent cerebrospinal fluid. However, mixed bacterial and tuberculous menin- gitis has been reported. It is therefore wise to look for mycobacteria 434 BACTERIAL MENINGITIS in the initial microscopical examination of all fluids when the patient has been shown to have a tuberculous infection elsewhere and to culture for tuberculosis all nonpurulent types of cerebrospinal fluid as well as the fluid of any patient with a purulent type not re- sponding as expected to treatment. Tuberculous meningitis probably occurs next in frequency to meningitis caused by H. influenzae in periods when meningococcus is not epidemic. Prompt diagnosis has assumed great importance, since modern drug therapy gives it a good prognosis if it is treated early. The reader is referred to the chapter on tuberculosis (Chapter 9) for detailed procedure. Il. HANDLING AND TRANSPORTATION OF SPECIMENS A. Collection of Specimens In cases of bacterial meningitis great care must be taken in the collection and handling of specimens if satisfactory findings are to be obtained. The time in the course of the infection at which the specimen is taken, the temperature at which it is held, and the amount of inoculum used for cultures are all of importance. The specimens usually submitted for laboratory examination are cerebrospinal fluid, blood, petechial scrapings (in meningococcus infections), nasopharyn- geal swabs, and other body fluids such as ventricular, cisternal or subdural fluid. Details for handling these specimens are presented in Table 3. B. Transportation Many of the microorganisms responsible for bacterial meningitis are sensitive to chilling so that it is important to keep all specimens near body temperature. In some instances, however, even the menin- gococcus and H. influenzae have been recovered after long journeys at variable temperatures. Use large inocula for cultures from shipped fluids more than 24 hr old. Nasopharyngeal swabs should be cultured immediately from the individual being examined. If this is impossible, the swab may be placed in a tube containing 1 ml of defibrinated horse blood or a small amount of semisolid agar until the laboratory can be reached. Sera for the agglutination test may be shipped with little if any loss in potency provided the cells are removed by centrifugation to prevent hemolysis. Details for the transportation of the various types of specimens are given in Table 3. Table 3—Collection and Transportation of Specimens, in Cases of Bacterial Meningitis Type of Specimen Time of Collection Method of Collection Amount Required Type of Test Transportation 1. Cerebro- spinal fluid 2. Blood: Culture Clotted 3. Petechial scrapings Concurrently with de- velopment of menin- geal symptoms As soon as infection is suspected Serial specimens—the first collected during the first 5 days; sub- sequent specimens taken at 4-5 day in- tervals Early as a diagnostic aid in doubtful cases and in post-mortem examination Lumbar punctures be- tween 3rd and 4th spaces to 3 sterile tubes. Size of needle depends on individual case, com- monly 18 gauge. Drawn from basilic vein to 1% citrate un- less culture can be made at bedside. Vein puncture to sterile tube. Separate serum from cells if the speci- men cannot be sent to the laboratory promptly. Fine Pasteur pipette or hypodermic needle into center of petechia. Avoid blood if possible. Culture on semisolid medium. 2-5 ml in each of 3 tubes 10-15 ml in 100 ml warmed broth in 250 ml flask 5-10 ml Tiny bit Cell count; glucose, protein, chloride con- centration. Culture Capsule swelling Gram-stained film Culture isolation Serological test: Agglutination or quellung Microscopic exam- ination and culture isolation Keep constantly warm and send to laboratory at once. Warm—send to lab- oratory immediately. Refrigerate at 5° C. If sent by mail re- move cells and ship serum only. Warm—transfer to culture medium im- mediately. SILI9NINIW 1VI¥d3ILOVHE SEP Table 3—Collection and Transportation of Specimens, in Cases of Bacterial Meningitis (Continued) Method of Collection Amount Required Type of Test Transportation Type of Specimen Time of Collection 4. Naso- As soon as carrier pharyn- is suspected geal swab 5. Other body fluids : Ventri- cular If fontanel is still open Cisternal If spinal canal is blocked Subdural If clinical suspicion of subdural collection of fluid Place swab along floor of nasal passage until it meets wall of naso- pharynx. Twirling mo- tion causes swab to wind itself on mucus and exudates. Or pass swab behind uvula from pharynx. Puncture Puncture Puncture in young chil- dren; trephine in older individuals if neces- sary. Place swab in Culture isolation tube with 1 ml defibrinated blood or semi- solid medium. Same as for spinal fluids 2-5 ml in each of 3 tubes If possible, cultures should be made on the spot; or keep warm and send to laboratory as soon as possible. Warm—send to lab- oratory immediately. 9Eb SILI9NINIWN TVI¥3ILOVE BACTERIAL MENINGITIS 437 Il. MATERIALS NEEDED AND THEIR PREPARATION A. Culture Media In patients whose cerebrospinal fluid shows organisms on Gram- stained films, a presumptive diagnosis can sometimes be made im- mediately on the basis of the stain and the capsule swelling. A suffi- cient variety of culture media should be used so that all the common pathogens have ample opportunity to grow. Even when a presump- tive diagnosis seems certain, the possibility of mixed infection indi- cates the use of more than one culture medium. In addition, specific technics should be used when there is reason to suspect an unusual microorganism such as in brucellosis; or, in the event of a secondary meningitis, where a portal of entry is available from contiguous mucous membranes; or in instances where surgical intervention has taken place. Basic media should comprise at least one medium containing blood, such as a blood agar plate or a blood broth, or a substitute nitrogen source, such as neopeptone or trypticase soy, and a semisolid agar medium in which many microorganisms grow especially well. In addition, it is wise to include a Levinthal broth or agar and Fildes peptic digest broth for H. influenzae. In order to isolate other Gram- negative rods it is advisable to use Endo or eosin-methylene blue agar or MacConkey’s agar. For the occasional anaerobe, a tube of Brewer's thioglycolate medium should be included. However, when anaerobes are definitely suspected, particularly if Veillonella is a probability, duplicate plates of meat base blood agar or chocolate agar or a tube of meat infusion broth used for aerobic cultures should be placed in an anaerobic jar. An alternate plan is to use a chopped meat medium or a liver broth with minced liver either in an anaerobic jar or one sealed with vaseline. When tuberculosis is suspected, Lowenstein’s, Petragnani’s, or Dubos’ broth medium should be included. Fungi are sometimes involved. Actinomyces will usually grow in suitable anaerobic cultures and Nocardia will usually appear on one of the aerobic plates if these are held at room temperature for several weeks. True fungi grow best on Sabouraud’s medium or on blood agar containing 0.1 unit of penicillin and 5 mg of streptomycin per ml of medium. For media used in the identification of individual species other than the meningococci the reader should refer to the appropriate chapter of this work. 438 BACTERIAL MENINGITIS For the fermentation reactions of the meningococcus a sugar-free semisolid meat infusion agar medium, with carbohydrate and indicator added, is satisfactory. Should a strain fail to ferment a sugar in this medium, a solid sugar-free meat infusion agar slant with carbohy- drate and indicator may be used; some workers prefer this. A buffered medium may mask feeble acid production. For maintenance of stock cultures lyophilization is the method of choice. Should this be impossible, stock cultures can be maintained on semisolid agar transplanted every 3 to 4 weeks, although virulence is quickly lost in this medium. Serum glucose agar slants, trans- planted every 3 to 4 days, and blood agar transplanted every 2 days, are both superior for the maintenance of virulence. For agglutination tests the microorganisms should be grown on serum agar with 0.5 per cent glucose or on buffered cornstarch agar with 0.5 per cent glucose. Formulas for all these media and a description of their preparation will be found in Chapter 4. B. Stains Reference to recommended stains is made in connection with the examination of specimens. See Chapter 4 for formulas and directions for use. C. Chemical Reagents For the preliminary six tube test for sugar [see Section IV A 2 (e)], Benedict's qualitative reagent is used. For confirmatory quantitative analysis use the solutions described in any of the texts on clinical laboratory diagnosis cited in the references.3-¢ These works also describe reagents for determination of protein and chloride con- centrations. D. Sera The details of preparing diagnostic sera vary according to the species of microorganism employed, but several general principles should be observed with all species. Rabbits are most commonly used for this purpose. No fixed rules can be formulated for the immu- nization of rabbits, since individual response in these animals varies; weight and condition of the animal are the safest criteria. The first requisite is a suitable antigen; the cultures should be smooth and specific. Young cultures 5 to 10 hr old should be used. Formalinized suspensions (i.e., 0.4% formalin in 0.85% NaCl) are generally employed for the first few injections, but living, freshly made cultures are substituted as soon as possible. BACTERIAL MENINGITIS 439 Second, it is important to begin with a very small dose. With bacteria requiring intravenous injections, a good start can often be made by giving the first dose intracutaneously. The size of the first dose depends to some degree on the species of microorganism used and the route of administration; with meningococci it should not be over 50,000,000 cells. Third, give the animals adequate rest periods. With the meningo- coccus the immunization schedule is usually as follows: 1. Three injections on successive days; rest 4 days. 2. Repeat this for 3 weeks; rest 1 week. 3. Repeat the 4 week program. 4 5 . Make trial bleeding from the ear vein. . Repeat the course if the titer is unsatisfactory. When a satisfactory antibody titer is obtained, the rabbits are bled from the heart. It is well to remember that the serological pattern will “spread” if immunization is long continued, and the sera become less specific as somatic agglutinative properties appear. Horses are sometimes used instead of rabbits and are especially good for making large amounts of multivalent sera. Chickens, also sometimes used,” are very helpful with Group B meningococci (which are often poor antigens) because they can withstand very large intra- venous doses of meningococci. With chicken sera it is best to use the rapid method of agglutination.’ The most satisfactory method of preserving immune sera is to place small quantities in ampuls, freeze, dry and seal in vacuo. No preservative is then required. Although the procedure given here is outlined as for the meningo- coccus, it can be adapted to any of the microorganisms that are dis- cussed, and details can be found in appropriate chapters of this work. IV. EXAMINATION OF SPECIMENS A. Cerebrospinal Fluid The laboratory should furnish the clinician at least three important kinds of data: (1) Report as soon as possible the type of cell which predominates—whether polymorphonuclear or mononuclear ; the con- centration of sugar in the spinal fluid; whether bacteria are seen, their morphology and their Gram-staining characteristics. (2) The initial microscopical study should be as complete as possible. If microorganisms are found, identification should be pursued, giving the performance of capsular swelling tests where indicated. The most precise report available should be telephoned immediately to the 440 BACTERIAL MENINGITIS clinician. Sugar concentration should be confirmed. (3) Cultural studies should be pursued to completion ; but if at any time a presump- tive diagnosis not heretofore reported can be made, it should be tele- phoned to the doctor in charge. All these steps are necessary, since proper treatment is dependent on the specific etiological diagnosis. Under optimal conditions a tentative diagnosis and assessment of the severity of infection can be made within 30 min after the cerebro- spinal fluid reaches the laboratory. In many other instances the in- fectious agent can be grown and identified within 24 hr. 1. Collection—Cerebrospinal fluid is collected in three sterile tubes as described in Table 3. The first tube may be used for micro- scopical examination, the second for cell counts and sugar determina- tions, the third for cultures. 2. Microscopical examination a) Cell counts on fresh unstained cerebrospinal fluid—Cell counts must be made immediately if they are to be of value. The sample of fluid should be free from blood and the cells should be evenly dis- tributed. Usually the fluid may be placed directly in the counting chamber without dilution. Nine large squares are counted and the sum is multiplied by ten-ninths to obtain the number of cells per cu. mm. If the specimen is contaminated by red blood cells, these may be removed by washing the pipette with glacial acetic acid and discharging the contents, leaving a thin film of acid on its surface. If the cells are then drawn into the pipette, the red cells will be lysed and the resultant fluid placed in the chamber will contain unlysed white blood cells. Where cells are too numerous to count by this method, the fluid may be diluted in a “white counting” pipette, as for a regular leukocyte count. b) Preparation, fixation and staining of film for differential count —A differential count for cerebrospinal fluid cells should be made at this time. With very cloudy fluid, films for this purpose can be made without centrifugation. If the fluid is only slightly cloudy it may be centrifugated first at 3,000 rpm for 20 min and the sediment used. Wright's stain may be used as for blood films, or May-Griinwald followed by Giemsa. c) Preparation, fixation and staining of films for examination of bacterie—A film of the centifuged specimen stained by Gram'’s method is an important factor in early diagnosis. The flattened Gram-negative diplococci of the Neisseria species may be abundant both within and without the leukocytes. On the other hand, a care- BACTERIAL MENINGITIS 441 ful search may be required to detect them and sometimes they cannot be found. A half-hour is none too long to look for them. Similarly, the lancet-shaped Gram-positive encapsulated organisms character- istic of pneumococci may be abundant or sparse. In general, they are somewhat easier to find than meningococci. H. influenzae is immediately suggested when Gram-negative pleo- morphic microorganisms are present, appearing as coccobacilli, diplo- cocci, or definite rods, short or long. Tt is much more common to mis- take H. influenzae for a pneumococcus or a meningococcus than to err in the reverse direction. With patients who have received anti- biotic agents, even in small doses, it is frequently impossible to find organisms in stained spinal fluids despite the fact that the disease itself is still severe. Because of the urgency of diagnosis the stained film should be examined as soon as possible following admission of the patient. A second film may be stained with methylene blue, as this dye is especially valuable in revealing morphology and in differ- entiating between pneumococcus, meningococcus and H. mfluenzae. The less frequently found Gram-negative rods of the coliform group or other cocci of the streptococcal and staphylococcal groups are occasionally recognizable on the stained film by characteristic mor- phology. A film stained by an acid-fast method (Ziehl-Neelsen) will demonstrate tubercle bacilli or other acid-fast organisms, including Nocardia. d) Preliminary identification in typing by capsular swelling is often possible in the case of the meningococcus, pneumococcus and fH. influenzae, as well as with Klebsiella, when these microorganisms hap- pen to be present. In every case where microorganisms are present in direct film, an attempt should be made to confirm the morphological diagnosis by specifically demonstrating capsular swelling with homol- ogous antisera, a technic now used routinely in the laboratory. In order to insure success there must be a sufficient number of bacteria in the sample of cerebrospinal fluid. Capsules can be demonstrated on meningococci in Groups A and C but none has yet been reported on Group B strains. One is almost always able to obtain capsular swelling when pneumococci are present in sufficient numbers, if antisera are available. Since almost all menin- gitis caused by H. influenzae is due to Type b, specific capsular swell- ing can usually be demonstrated. One should attempt to show specific capsular swelling with available Klebsiella antisera when Gram- negative rods other than H. influenzae are observed. This technic makes a quick diagnosis possible, thus saving valuable time. To perform the tests a drop from each serum and a drop of 442 BACTERIAL MENINGITIS cerebrospinal fluid sediment and of methylene blue are mixed on a cover slip which is sealed in a slide and incubated in a moist atmos- phere for 30 min at 30° to 37° C and then examined microscopically. e) Test for reducing sugar—Some laboratories are prepared to conduct the regular quantitative tests promptly and rapidly. Where this is not practicable, an indication of the severity of the infection may be furnished by making a rapid, preliminary semiquantitative estimate of sugar concentration using a six tube test which can be done directly on spinal fluid within a few minutes. This procedure as described by Alexander, Ellis and Leidy® is carried out as fol- lows :— (1) Pipette 1 ml of Benedict's qualitative reagent (the reagent used in routine urinalysis) into each of six test tubes. (2) Into the first five tubes place the amounts of cerebrospinal fluid indicated in Table 4. Leave Tube 6 for control. (3) Immerse all tubes in boiling water for 10 min. (4) Interpret the sugar concentration by the reduction of Benedict's solution as indicated in Table 4. Table 4—A Semiquantitative Test for Estimating Sugar in Spinal Fluid mg Sugar per 100 ml Fluid Cerebrospinal Tube Fluid Over Under No. (in ml) 50 40-50 30-40 20-30 10-20 10 1 0.05 4 0 0 0 0 0 2 0.1 + + 0 0 0 0 3 0.15 bo - =} 0 0 0 4 0.2 whe H= 3 + 0 0 5 0.25 + + + += + 0 + =Reduction of Benedict's solution. 0=No reduction of Benedict’s solution. f) Evaluation of findings and reporting of results—Mistakes are frequent when identification of microorganisms is made by micro- scopical examination of stained films only. HH. influenzae, because of its very pleomorphic nature, may be confused with other Gram-negative rods and Gram-negative cocci. When there is specific capsular swell- ing, a definite report can be given ; otherwise the approximate number, the morphology and the Gram-staining property of the organism, as well as the cell count and sugar concentration in the cerebrospinal fluid, should be reported at once, with a tentative identification empha- sizing that specific diagnosis must await further study. In a well equipped laboratory such a report can usually be made within an hour. BACTERIAL MENINGITIS 443 Further study consists of cultivation of the organisms on a suit- able medium and examining the morphology of the pure culture, the cultural characteristics, and the action upon specific sugars. Particu- larly in the case of the meningococcus, the action upon glucose and maltose is necessary to differentiate the microorganism from other species of Neisseria. Corroborating serological evidence should be obtained with pure cultures. In the case of the meningococcus, agglutination of the culture with a multivalent serum can be done; this is particularly valuable with strains in which the fermentative powers are not well developed. It should be remembered that some freshly isolated strains of meningococci are inagglutinable in some multivalent antisera. It is therefore not absolutely necessary that a strain agglutinate with multivalent antisera in order to be called a meningococcus when other characteristics are typical. More detailed discussion of serological identification is given in Section VII herein, 3. Cultural examination a) Preliminary treatment of cerebrospinal fluid—It is most de- sirable to collect cerebrospinal fluid before the patient receives specific antimicrobial therapy. Actually many samples are collected after such therapy has been established. In these cases, para-aminobenzoic acid and penicillinase should be added to the culture media. Enough para-aminobenzoic acid should be added to give a final concentration of 5 mg per 100 ml; enough penicillinase to give a final concentration of approximately 1 unit of penicillinase per ml. In most labora- tories it is customary to add penicillinase and para-aminobenzoic acid regularly to culture media used for clinical specimens. Despite the use of these antibiotic inhibitors, cultures of cerebrospinal fluids from patients who have received antibacterial agents may be very slow in growing or may appear sterile. It is best to incubate all cultures from cerebrospinal fluids obtained when antibiotics are being given for at least 5 to 7 days. b) Preparation of cultures—A generous inoculum should be used, that is, from 0.5 to 1.0 ml, depending on the nature of the cerebro- spinal fluid. Uncentrifugated fluid may be used when organisms are abundant in the stained film, but in most instances centrifugation will be necessary. This inoculum should be put (1) on a blood agar plate (CM No. 16), (2) into a tube of semisolid agar (CM No. 15) with or without enrichment, and (3) into a tube of Levinthal broth (CM No. 33). Other media should also be used when indicated. These cultures should be included without and within a candle jar; but if A444 BACTERIAL MENINGITIS a choice is necessary, then preferably within the jar. The cerebro- spinal fluid itself as a medium should not be ignored. Incubating it at 35° C overnight often results in the multiplication of microorgan- isms, particularly meningococci, so that it is easier to detect them on films. Cerebrospinal fluids must be kept warm while awaiting examination. ¢) Examination of cultures—Colonies of meningococci on blood agar plates are usually characteristic in appearance, being smooth and translucent, often much larger than textbook description would lead the reader to believe. Usually colonies are 2-3 mm in diameter, although they may be as large as 1 cm. Growth may be confluent. Colonies may be fished to semisolid media, to blood agar, to serum glucose agar slants, or to slants of Mueller’s medium (CM No. 42). On semisolid agar the meningococci grow as a pellicle at the surface of the medium. For the colony characteristics of pneumococci, H. in- fluenzae, E. coli, beta-hemolytic streptococci and Pseudomonas and Proteus species, as well as other causative microorganisms encoun- tered, the reader should refer to the respective chapters of this work concerning these organisms. d) Reporting—From the colonial characteristics of the microorgan- ism and the Gram-stained film from the colony, the microorganism may be tentatively reported to the person in charge. However, final specific identification should not be made until the pure culture is completely identified. 4. Chemical tests a) Sugar—Many laboratories make quantitative determinations of sugar in spinal fluids repeatedly and consider them important, not only as an aid in diagnosis, but in prognosis and in following the course of the infection. The amount of sugar is decreased in various forms of purulent meningitis, whereas it remains normal or increases in poliomyelitis, epidemic encephalitis, or lymphocytic choriomeningitis. The normal true sugar content is 40 to 60 mg per 100 ml. These figures are somewhat low when the total reducing substances are measured, as with the Benedict method. Persistent low sugar con- tent of the cerebrospinal fluid in meningitis is considered to be indica- tive of poor prognosis and an increase toward the normal is an en- couraging sign. There are several points to remember in the interpretation of sugar concentration in the cerebrospinal fluid. One is that when cerebro- spinal fluids are allowed to stand, almost any glucolytic system, in- BACTERIAL MENINGITIS 445 cluding many bacterial systems, will use up the sugar present. An- other is that there may be reducing substances other than sugar present. A third is that there is a relation between the sugar content of the cerebrospinal fluid and that of the blood. It is well to make a sugar determination on blood drawn at the same time the cerebro- spinal fluid is obtained. A preliminary six tube test has been described in Section IV A 2 (e) preceding in this chapter. The course of the infection should be fol- lowed by quantitative estimations as indicated.-® b) Protein and chloride—Estimates of protein and chloride con- tent are sometimes indicated. Descriptions of reagents and pro- cedures may be found in any standard laboratory manual.?-6 5. Serological tests for syphilis—Routine tests for syphilis should be performed on the first specimen of cerebrospinal fluid taken from the patient suspected of having meningitis. Such tests are de- scribed in the chapter on syphilis (No. 18). The results, when corre- lated with the blood test, history and physical findings, may make clear the diagnosis of an otherwise puzzling case. 6. Animal inoculations—Animals are occasionally inoculated with cerebrospinal fluid when a diagnosis of leptospiral, viral or tuberculous infection is being considered. Occasionally in order to recover pneumococci with fully developed capsules, the cerebrospinal fluid from a purulent meningitis should be injected into a mouse. B. Blood Blood cultures are a valuable aid in the diagnosis of all types of bacterial infections of the meninges except tuberculous, since these are frequently associated with a bacteremia. Blood cultures are particularly valuable in the very early stages of meningococcal infec- tion, especially in cases without meningitic symptoms and in those with a fulminating infection but with no extensive involvement of the central nervous system. Blood cultures should be made as soon as possible, preferably at the bedside. Into a sterile syringe 10 to 15 ml of blood are drawn from a vein and added to 100 ml of warm infusion broth in a 250 ml flask. This will give a relatively large surface, which is desirable. Numerical results may be obtained by adding 1 ml of the blood to a tube of infusion agar; melt and cool to about 45° C, mix and pour into a petri dish. Addition of 0.1 to 0.2 per cent glucose to these media may enhance growth. When initial cultures are obtained, identification of microorganisms proceeds as usual. Blood cultures should be held for at least 3 weeks. 446 BACTERIAL MENINGITIS C. Petechiae Petechial rashes are most characteristic of meningococcic menin- gitis; they are rarely found in other types of purulent meningitis. Although meningococci are present in the petechiae, isolation from them is seldom attempted. Material may be withdrawn with a fine Pasteur pipette or a small hypodermic needle from the center of the petechiae, avoiding contamination of the specimen with blood. Stained films are prepared from this and from cultures made in a semisolid agar, Occasionally after death, examination of the petechiae provides a diagnosis when other methods are unsuccessful. This is especially true in the case of many patients who have died without showing signs of meningitis and for whom no blood cultures were made. Microorganisms may be seen in properly stained sections (Gram- stained) and often they may be cultivated if a tiny bit of skin is placed in a semisolid medium. D. Nasopharynx Few hospital laboratories make nasopharyngeal cultures routinely from cases of meningitis. However, cultures from this area will frequently show the meningococci, H. influenzae, pneumococci, or beta-hemolytic streptococci. For the isolation of the three last-named microorganisms the reader should consult the appropriate chapters of this work. In the case of the meningococcus it is not known how con- stantly or for how long these microorganisms are harbored in the naso- pharynx of the patient. With the effective therapy now available it is likely that they will not persist for more than a few hours. How- ever, when the microorganisms are sought before the institution of therapy they are usually found. Nasopharyngeal cultures have more frequently been made for the purpose of detecting carriers. In either case, the technic is the same. Carrier studies should not be under- taken unless they can be made under the proper conditions and fol- lowed through to completion. a) Collecting the material —Equipment consists of (1) wooden tongue depressors; (2) cotton swabs applied to wooden applicators. Many prefer a wire swab with the cotton-covered end bent at an angle. Swabs should be of two sizes, small ones for the nasal passage and larger ones for the nasopharynx; (3) a head mirror or lamp; (4) a cellophane face mask; (5) plates of freshly poured blood agar. It is important that the medium be firm but moist. Meningococci re- quire abundant moisture for their growth. BACTERIAL MENINGITIS 447 Procedure—When a carrier survey is made, the demand that the work be expedited creates a constant temptation to take short cuts. Yielding to such temptation is fatal to the study. No procedure re- quires more strict attention to every detail. When the work cannot be done thoroughly, it should not be attempted. The results will not only be worthless, but misleading. Those who are to be examined come before the bacteriologist in single file in a quiet and orderly manner. They need not be seated but stand in front of the operator, who requests the subject to open his mouth wide. The dorsum of the tongue is held down, with the applicator put inside the mouth, and the subject is asked to phonate vigorously. This raises the uvula and permits the swab to be passed under and beyond it. By lifting the uvula more, using the lower teeth as a fulcrum, the swab is easily pushed up against the posterior nasopharyngeal wall—the region where the nidus of the meningococci is most likely to be. While the swab is pressing gently against the mucous membrane, it is given a slight twirling motion. This tends to wind up some of the local mucus on the swab. The bacteriologist withdraws the swab quickly, taking care to avoid bringing the tip into contact with anything. The entire procedure takes only two or three seconds. For specimens to be collected through the nostril the procedure is simple. The subject is told to look straight ahead and not to throw his head back. The tip of the nose is raised slightly with one hand and with the other the swab is passed along the floor of the nasal pass- age until it meets the posterior wall of the nasopharynx. A gentle twirling motion seems to cause the swab to wind up on itself the mucus and exudate required. If at any time during this procedure the subject begins to move his head around, merely drop or let go of the applicator ; the promise to take it out for him without hurting him generally induces him to cooperate. Which type—throat or nasal culture—is likely to get better results is not known. Sometimes one will be positive and the other negative. It is probable that two throat cultures or two nasal cultures would give about the same average. When such thoroughness is possible, both a throat and a nasal culture should be made. b) Plating out material —Blood agar (CM No. 16) is the medium of choice for isolation of the meningococcus from the naso- pharynx. Where blood is unobtainable, Mueller’s medium (CM No. 42) or trypticase soy agar (CM No. 12) can be used. Inoculation of the plate from the swab is done variously by different workers. The technic used by Rake in his carrier studies is recommended. The tip 448 BACTERIAL MENINGITIS of the swab is brought gently into contact and smeared on the blood agar over a very small area at one edge. Then a fresh clean sterile swab is taken and with it the inoculum is spread over one-half the plate, the excursions back and forth across the plate being made very close together or overlapping; a second fresh swab now spreads the material at a right angle for another one-fourth the area of the plate; a final swab is used at a right angle to finish covering the surface of the plate with the progressively decreasing inoculum. Platinum loops may be used in place of swabs after the plate has received the initial inoculum. These plates are incubated for 18-24 hr after being placed inside museum jars with wet cotton beneath them. Some workers introduce CO. into the jars. When jars of this type are not avail- able, some other container should be improvised which will insure a humid atmosphere. c) Fishing of colonies for further study—On the usual blood agar plate the translucent meningococcus colonies are easily dis- tinguished from those of the commoner inhabitants of the naso- pharynx, except for other members of the genus Neisseria which are often confused with those of the meningococcus. The yellow pigment of the chromogenic species is often slow in appearing. Occasionally meningococcus colonies may be very large. Differentiation of the meningococcus from other Neisseria often requires careful cultural biochemical and serological studies. This makes carrier detection a laborious procedure. Sometimes the proportion of meningococci to other bacteria in the nasopharynx will be very small; at other times they may be recovered in practically pure culture. The suspected colonies are ringed and transferred to blood agar, serum glucose agar or trypticase soy agar slants and incubated at 35° C. The following day the growth is stained by Gram’s method and if it shows typical Neisseria morphology, cultures are made to test its fermentation reaction. The culture is transferred to semisolid agar to keep it alive for future use and the remainder of the 18-24 hr growth is suspended in salt solution to use immediately for agglu- tination with both multivalent and type or group sera. If these tests cannot be performed immediately, the method of preparing antigen used for the agglutination test described in Section VII should be followed. The most specific results of agglutination are obtained with 5-6 hr cultures. If slide agglutination is used at this stage it should be confirmed later by the tube method. The oxidase reaction may be of practical value with nasopharyngeal cultures, as it facilitates recognition of the genus Neisseria. It is discussed in detail in Chapter 17. The addition BACTERIAL MENINGITIS 449 of tyrothricin, 1:1000 or 1:10,000, to blood agar inhibits growth of many Gram-positive species and facilitates isolation of both Neisseria and Hemophilus. When the virulence of the culture is to be studied, it should be kept on serum glucose agar slants and trans- ferred twice a week, or on blood agar slants transferred every other day. V. IDENTIFICATION OF PURE CULTURES A. Meningococcus and Other Neisseria 1. Bacteriological—The meningococcus is a Gram-negative coccus, usually occurring in pairs, having flattened adjacent sides, which ferments glucose and maltose with the production of acid. Five other characteristics may be included in the definition : character- istic colony formation, lack of pigment production, failure to grow well on plain agar, growth only at body temperatures, and agglutin- ability in multivalent antimeningococcus serum. Microorganisms having all these characteristics are certainly meningococci, but not all meningococci have each and every one of these characteristics. The cocci may occur singly or in groups as well as in pairs; the individual cells of a pair often vary greatly in size and in intensity of staining, although they are always Gram-negative when properly stained. Strains may be encountered that fail to ferment maltose or glucose or both of these sugars when they are first isolated. Such strains usually acquire this property sooner or later, although it may be lacking at the time when a diagnosis of meningococcus infection is being sought. When Gram-negative diplococci of typical morphology are obtained in pure culture, it is necessary to carry their identification through to completion, for all members of the genus Neisseria have a similar morphology, and species other than N. meningitidis are occasionally encountered in meningitis, The only other member of the genus Neisseria that produces colonies as translucent as the meningococcus is the gonococcus (N. gonor- rheae). These are usually smaller, growth is less luxuriant, and there is less tendency to confluence. The gonococcus causes meningitis more commonly than is generally thought. Colonies of N. catarrhalis are more opaque and whitish, whereas those of N. sicca are so dry and adherent that they are either taken up entirely by the inoculating needle or can be pushed about. Colonies of the pigmented members, N. flava, N. perflava and N. flavescens, may resemble the meningococcus on the first day but they gradually 450 BACTERIAL MENINGITIS become more opaque and develop a yellow color. This color is em- phasized when the culture is grown on Loeffler’s blood serum medium. Nutritional and temperature requirements are less exacting with these six species and these points may be of value in differentiation. Fermentation reactions are useful in identifying members of this group of bacteria. The meningococcus produces acid from glucose and maltose. Individual strains vary greatly in both actual and relative amounts of acid produced from these two sugars; at times, soon after isolation, the reaction will be so transient that the strain being tested may seem to produce no acid in one of these sugars, especially when a buffered medium is used. Fermentation nearly always becomes more typical after a period of laboratory maintenance. The gonococcus produces acid from glucose only; N. catarrhalis and N. flavescens ferment no carbohydrates. The four pigmented members of the genus are separated from each other chiefly by their fermentation reactions, although N. flavescens seems to be more homogenous serologically than the others. For these fermentation tests, growth from a pure culture is inocu- lated into special semisolid agar. Because an acid reaction may be transient, the cultures should be examined often during several days of incubation. The differential characteristics of the common species of Neisseria are given in Table 5. Table 5—Principal Diagnostic Characteristics of Neisseria Species Glucose Levulose Maltose Sucrose Pigment Comments N. flavescens oO oO oO Oo RS Serologically homo- genous N. subflava + Oo + Oo + Pigment often slight N. flava + + + O + N. perflava wpe a= = =} i Most common N. sicca oj + + + Oo Colonies often very dry and white N. catarrhalis Oo oO Oo oO oO Colonies colorless and more opaque than meningococci N. gonorrheae + oO oO oO oO Colonies delicate, translucent, dis- crete N. meningitidis + oO Ra oO oO Colonies translucent, coalescent + = Acid. O=No acid. BACTERIAL MENINGITIS 451 2. Serological reactions a) Identification by agglutination—The identity of the meningo- coccus should be confirmed by agglutination with multivalent serum. Growth from solid media is used, not more than 24 hr old and prefer- ably from 6 to 12 hr. Suitable media for this purpose are 0.5 per cent glucose agar, 5.0 per cent rabbit’s blood agar, buffered corn starch agar, or trypticase soy agar. Growth should be suspended in buffered 0.85 per cent sodium chloride solution and the suspension diluted to contain approximately 1 billion microorganisms per ml. Such a suspension corresponds to the No. 5 tube of the McFarland scale, or 500 turbidity units determined as described in Standard Methods for the Examination of Water and Wastewater.*® Any other method of estimating turbidity that gives similar results is satisfactory. It is important to know the pH of the salt solution used, since a culture that may be inagglutinable at pH 7.8 may agglutinate very well at pH 6.8. Serum dilutions should be 1:25 to 1:400 or higher, making the final dilutions after adding the suspension of bacteria 1:50 to 1:800. The total amount in an ordinary agglutination tube is usually 1 ml but may be 0.8 ml. A salt solution control should always be included, and often normal horse serum in 1:25 and 1:50 dilutions is also used. The whole set-up is incubated in a water bath at 37° C for 2 hr and read after storage in a refrigerator overnight. Multivalent sera that are made with “rough” stock strains often do not agglutinate “smooth” specific strains. Such smooth strains will seem to be inagglutinable. However, if a smooth strain should lose its specific capsular substances and become rough, it may be well agglutinated by the same multivalent serum that failed to agglutinate it in its smooth state. Agglutinating sera should be made with smooth strains. Another technic for agglutination which is popular in the field is the “rapid” or “short” method described by Noble.® The suspension of microorganisms contains five times as many bacteria, and the serum concentrations are five times as great as for the regular agglutination test. Only 0.1 ml serum dilution is placed in each small tube and 0.1 ml suspension added. The rack is inclined to an angle of about 90° and is shaken slowly for 2 min in such a way that the mixture of serum and suspension flows about 1 in. up the tube. Then 0.8 ml of 0.85 per cent sodium chloride solution is added to each tube and the agglutination is read immediately. The usual control tubes should be included. This technic has the advantage of giving results quickly. Some laboratories use microscopic-slide agglutination. The ad- vantage of this procedure is that individual colonies from a primary 452 BACTERIAL MENINGITIS culture may be used and a correct identification is arrived at promptly. The usual method for performing this test is a follows: Multivalent antimeningococcic horse serum and normal horse serum are diluted 1:10 with 0.85 per cent sodium chloride solution and a loopful of each is placed on a slide. Some of the suspected colony is rubbed up in each, or some of the colony is mixed with the normal serum, and a portion of this suspension is transferred to a drop of diluted immune serum. Agglutination may be observed macroscopically and microscopically. Microscopic agglutination should be confirmed by the macroscopic tube method. Microorganisms of characteristic morphology and staining which are agglutinated only by the immune serum may be tentatively considered meningococci. b) Grouping and typing—Determination of serological group is important from an epidemiological standpoint. Group determination is quickly made when good sera are available, and it is preferable that there be no delay. A much better idea of the antigenic pattern can be obtained immediately after isolation, since most strains tend to lose serological specificity on prolonged laboratory maintenance, making it more difficult later to determine accurately their group. In the event storage is necessary, as in extensive carrier surveys, such changes are reduced to a minimum when the strains are preserved by drying them from a frozen state. For “grouping®”, the sera should be prepared with smooth strains that have abundant capsular substance, for it is on this characteristic that group specificity seems to depend. The relative amount of this specific substance can be determined by the “halo” reaction described in Section VIII. Suspensions of meningococci to be studied are pre- pared as just described for agglutination with multivalent serum. Serum dilutions, however, are lower, usually from 1:10 to 1:320. The same procedure is followed as with the multivalent serum. Specific agglutination is favored by (1) typing the newly isolated strains promptly, (2) using very young cultures, preferably less than 12 hr old, and (3) incubating the test at 37° C for 2 hr. Although in- creased titer may be obtained by longer incubation at higher tem- peratures, such agglutination is much less specific and considerable “crossing” occurs. The rapid technic described by Noble? is often found useful, especially in the field, where water baths are not avail- able. * Although, correctly speaking, the term “type” applies to a subdivision of a “group,” the term “typing” has been used so long with meningococcus that strict insistence upon the term “grouping” does not seem indicated at this time. BACTERIAL MENINGITIS 453 It is essential that the sera be group-specific. The strains of menin- gococci with which the rabbits are immunized must be chosen with great care; they should correspond as nearly as possible to standard type strains and should be smooth. Preservation in a dried state is the simplest way of keeping the biological characteristics of such cul- tures unchanged. Information about the availability of standard type strains can be obtained at the present time from the Division of Bio- logics Standards, National Institutes of Health, at Bethesda, Md., or from the American Type Culture Collection in Washington, D. C. The preparation of these sera is discussed in Section III D of this chapter. In grouping or typing meningococci, the strains with which the sera were made should be included among the antigens in the test; a multivalent horse serum of good agglutinating titer should be used also for control purposes. c) Identification by capsular swelling—Determination of the sero- logical group of meningococci directly in the cerebrospinal fluid has been described in Section IV A 2 (d). This technic can of course be used with pure cultures also, and even with single colonies. A loopful of a 5 hr culture suspension (about 100,000,000 cells per ml) is substituted for the cerebrospinal fluid. A 10 per cent serum broth is an especially good medium for such cultures. This technic is rapid and economical and has become very popular. Capsular swelling has not been successfully demonstrated with Group B meningococci. It is essential that the serum used be known to contain capsular-swelling antibodies. 3. Reporting—When Gram-negative diplococci are seen in a stained film, they should be reported immediately as such but should not be labeled meningococci until further information is obtained. A strain may be reported as a meningococcus if it is not agglutinated by normal horse serum, does not form pigment, and ferments glucose and maltose only. However, if it fails to ferment one or both of these sugars—and some strains seem to fail when tested immediately after isolation—confirmation of serological identity must be obtained before reporting it certainly as a meningococcus. This report should be made as promptly as possible, without waiting for the grouping or typing to be finished. The group is reported as soon as it is known. 4. Other Neisseria—Other Neisseria species are sometimes the cause of meningitis. Before the advent of modern drug and anti- biotic therapy the gonococcus (N. gonorrheae) was responsible for 454 BACTERIAL MENINGITIS approximately 2 per cent of sporadic cases; now it is seen less often. Gonococcus grows more slowly and delicately on culture media, is more sensitive to adverse conditions than other Neisseria and fer- ments only glucose. There is considerable cross-agglutination be- tween the gonococcus and meningococcus, especially with menin- gococci of Group B. N. catarrhalis, N. sicca and the pigmented Neisseria are identified by cultural and biochemical reactions (see Table 5), except N. flavescens, which is serologically homogenous and may be identified by agglutination with specific sera. B. Microorganisms Other Than Neisseria 1. H. influenzae—Colonies of H. influenzae grow on blood agar as transparent, pinpoint dewdrops. On Levinthal’s agar they are somewhat larger and exhibit iridescence. If colonies of other organ- isms, especially staphylococci, occur on the plate, the Hemophilus colonies growing in the vicinity may be much larger, an occurrence known as the “satellite phenomenon.” Growth in broth is diffuse. Growth factors X and V are both essential for H. influenzae; these are discussed in the chapter on Hemophilus (Chapter 15). Hemophilus organisms are variable in morphology, sometimes ap- pearing as small roundish pairs, sometimes longer and more slender, or even in threads. They are always Gram-negative. Colonies should be picked to Levinthal or Fildes medium, or broth, semisolid or agar slants. Further identification, typing and maintenance of Hemophilus organisms are discussed in detail in Chapter 15. 2. D. pneumoniae—Colonies of pneumococci on blood agar are small and translucent, with well-defined edges. They are surrounded by a zone of greenish discoloration. In stained films made from these colonies the organisms appear as Gram-positive pairs or short chains. When in pairs, the outer side often seems pointed; when in chains the individual cells seem longitudinally oval. Pure colonies should be fished to blood agar slants or to broth, and growth from these examined for capsule swelling. If swelling does not occur in any group, serial mouse passage should be done and capsule swelling attempted on peritoneal fluid. Doubtful cultures are tested for bile solubility and for fermentation of inulin. Gram-positive diplococci of characteristic shape that produce green discoloration on blood agar and are bile soluble may be presumed to be pneumococci; capsule swelling specifically determines the type. BACTERIAL MENINGITIS 455 For details of these tests and for procedures to use in further studies, see Chapter 7. 3. Strep. pyogenes—The small grayish colonies of this species are usually beta-hemolytic on blood agar. Most likely to be mistaken for it in colonial appearance is H. hemolyticus. The streptococci of Lance- field’s Group A are usually round and occur in short or long chains, In broth the chains may be long, tangled filaments. Sometimes capsules are evident. Pure cultures should be obtained by fishing isolated colonies to blood agar slants or broth. Differentiation from other streptococci, as well as from other Gram-positive cocci, is discussed in detail in Chapter 5. 4. Staph. aureus—Colonies may not show the usual golden color on blood agar until the second or third day. They are often hemo- lytic but are usually distinguishable from streptococci at an early stage on account of their size and opacity. Arrangement of the cells in grape-like clusters in fluid media, instead of in chains, pairs or tetrads is characteristic. Pathogenic strains are almost invariably coagulase-positive. Meningitis from this microorganism is much dreaded because of its resistance to many antibiotics and sulfonamides, these properties vary- ing greatly from strain to strain. Complete identification of staphy- lococcus and determination of virulence are discussed in Chapter 6. 5. Proteus and Pseudomonas species—Both of these groups of Gram-negative rods, especially Proteus, tend to grow on culture plates as “spreaders.” Contaminating microorganisms sometimes become in- volved and special care is needed to insure pure cultures. Replating may be necessary. Spreading is less apt to occur when a high con- centration of agar is used. Both groups grow luxuriantly on the blood agar plates routinely used for culturing cerebrospinal fluid, but isolation is often made easier by including one of the special enteric media, such as Endo or EMB, made with at least 3 per cent agar. Most Pseudomonas produce a soluble green pigment when in pure culture. This is best accomplished by fishing colonies to slants of meat extract glycerol agar and incubating them at room temperature. Most Pseudomonas usually liquefy gelatin and all of them ferment glucose. Most but not all species of Proteus liquefy gelatin. A test for urease production probably offers the most reliable evidence that the organism under study belongs to this group. 456 BACTERIAL MENINGITIS Directions for a more detailed study of Pseudomonas are given in Chapter 22. 6. E. coli and related organisms—When these bacilli occur alone in the cerebrospinal fluid, isolation in pure culture is easily effected from the usual blood agar plate. Use of a special enteric medium gives some indication of the identity of the causative organism by permitting recognition of characteristic colony appearance. The Escherichia group of bacteria is discussed in detail in Chapter 10. 7. Other bacteria—Reference is made in Table 1 to microorgan- isms other than those discussed in this section. In the case of such groups as Salmonella, Alcaligenes, Aerobacter-Klebsiella, Entero- coccus and Brucella, refer to the appropriate chapter of this work, but for some of the other less often encountered species, a few com- ments may be helpful here: Mimeae species are frequently mistaken for meningococci, since in cerebrospinal fluids they may simulate diplococci soon after isolation. The tiny Gram-positive Listeria may be confusing; the strictly anaerobic Bacteroides may be overlooked ; the Nocardia may be mis- taken for other acid-fast organisms. Cryptococcus neoformans is by no means uncommon as a cause of meningitis, Methods for isolation and identification are described in the chapter dealing with these microorganisms. VI. TESTING FOR ANTIBIOTIC SENSITIVITY It is the opinion of some workers that any causative microorganism should be tested for its sensitivity to the various antibiotics considered for use in treatment of the disease. Other experienced workers believe that certain of the microorganisms commonly causing menin- gitis need not be tested routinely for sensitivity to the antibiotic agents to which they are usually sensitive except when a patient does not respond as expected. Whenever possible, the organism should be isolated before initiation of treatment. Antibiotic therapy is usually started before the results of sensitivity tests are available. In some cases it is valuable to test microorganisms isolated from later speci- mens of spinal fluid in order to follow the possible development of resistance. Four microorganisms may not require antibiotic testing routinely : 1. Meningococcus during epidemics, when most cases are caused by the same strain (some strains from sporadic cases are less uniform in response and frequently need to be tested) BACTERIAL MENINGITIS 457 2. H. influenzae 3. Pneumococcus 4. Hemolytic streptococcus Other microorganisms should be tested: N. meningitidis from sporadic cases, nonclassified and nonclassifiable enteric bacteria, Pseu- domonas, staphylococci, streptococci, M. tuberculosis, and others. In the case of some of these microorganisms which occur elsewhere more often than in meningitis, the laboratory should make sure that the one encountered is the true etiological agent. Technics for carrying out antibiotic assays and the interpretation of results are discussed in detail in Chapter 27. The disk method is more widely used than any of the others, since it is simpler to perform. However, it does not give complete information. It is therefore highly desirable to use the tube dilution method when sensitivity tests are undertaken on fastidious microorganisms such as N. meningitidis, H. influenzae, or M. tuberculosis. Selection of a suitable test medium, size of inoculum and time of incubation are also of the utmost im- portance. These details are presented in Table 6. Table 6—Suggestions for Testing Sensitivity of Fastidious Organisms to Antibiotics Inoculum: Time of Age of Culture Incubation Organism Medium Culture Dilution at 35°-37° C N. meningitidis Blood broth 4-5 hr 1:200 18 hr H. influenzae Levinthal broth 4-5 hr 1:200 18 hr M. tuberculosis Dubos broth 4-7 days 0.1 ml* 14 days * Of a density slightly less than that of a No. 1 barium sulfate nephelometer. Miller and Bohnhoff!! have reported strains of N. meningitidis which not only became rapidly resistant to streptomycin but even became streptomycin-dependent and failed to grow in its absence. This emphasizes the need for proper identification of the micro- organisms, since streptomycin, which may be contraindicated in meningococcus meningitis, is a very useful antibiotic (as in the case of H. influenzae meningitis). 458 BACTERIAL MENINGITIS Vil. SEROLOGICAL EXAMINATIONS A. Cerebrospinal Fluid 1. Direct precipitation upon the cerebrospinal fluid—Rake!'? has described such a test for meningococci in which he used univalent rabbit serum especially high in group precipitins. He emphasized the fact that negative results are apt to be obtained with cerebrospinal fluids in which there are few meningococci, thus causing very little specific substance to be present. This technic, which can be used with appropriate sera for pneumococcus and HH. influenzae, as well as with meningococci, is also suitable for use with multivalent sera. A posi- tive result with such a test can give a provisional diagnosis very early. A negative test may merely mean that the number of microorganisms or the amount of specific soluble substance of the meningococcus is too small to be demonstrable, or that the serum used is not sufficiently rich in precipitative properties, The conditions necessary for the successful performance of this technic limit its usefulness. IFalse- positive rings are obtained when sera are prepared with antigens grown on media containing human blood or ascitic fluid. 2. Capsular swelling—Typing of the infecting microorganisms directly in the cerebrospinal fluid by means of demonstrating capsular swelling with homologous antisera has already been described in Section V A 2 (¢) of this chapter. B. Patient's Serum 1. Agglutination—For over a decade several workers have made a study of the agglutinative properties in the serum of patients with meningococcic meningitis.!*-1* This procedure is particularly important in those instances where microorganisms cannot be found in the blood or cerebrospinal fluid; as a method of typing the infect- ing agent when univalent typing serum is unavailable; and finally in tracing the epidemiology of an outbreak. It may not be helpful early in the acute phase of the disease.’ When used as a diagnostic procedure, serial tests covering the various phases of the disease are found to be important; little or no reliance can be placed on the results obtained from a single specimen. In practically all instances where several specimens collected at appro- priate intervals are tested, significant titers are obtained. Sera col- lected early in the acute phase or late in convalescence are often negative. For a proper interpretation of the test as a diagnostic aid, the agglutinative properties must be correlated with the clinical picture, since in some cases the presence of significant titers may be BACTERIAL MENINGITIS 459 indicative of a latent meningococcic infection not necessarily the cause of the patient's current illness, or it may be due to anamnestic reactions. Agglutinative properties have also been observed in a few cases where the patient was infected with another member of the Neisseria genus such as the gonococcus. These tests are set up as usual. The following method has proved satisfactory for more than 12 years in the New York City Health Department: All sera are tested for Group A, B and C agglutinins. Standard type cultures are used to prepare the antigens ; these cultures should be maintained in the lyophilized state. Antigens are prepared by washing the 5 to 6 hr growth of meningococci from Mueller’s medium in pint Blake bottle slants, heating the suspensions at once in a water bath at 65° C for 1 hr and diluting them to a density of about 1,000,000,000 cocci per ml in 0.5 per cent phenolized, buffered salt solution. This density corresponds to tube No. 5 on the Mc- Farland scale. The diluted antigens are stored at 5° C. The specificity and agglutinability of each lot of cells are checked by testing them with homologous- and heterologous-strain rabbit antisera and normal human serum. Only those suspensions are used which will agglutinate in a homologous antiserum to titer and do not show spontaneous agglutination in buffered 0.85 per cent salt solution or agglutination in normal human sera in titers exceeding 1:50. Sufficient antigen can be prepared in each batch to last 2 or 3 months. Serum dilutions range from 1:50 to 1:6400. Each dilution is in- creased twofold. The tests are incubated at 35° C for 2 hr, centrifuged at high speed for 10 to 15 min, and kept in the refrigerator overnight. Readings are made immediately after centrifugation and also follow- ing overnight storage. The last tube showing definite ( + +) agglu- tination is considered indicative of the titer of the serum. Agglu- tination titers over 1:100 are regarded as significant for the presence of specific antibody, whereas those up to and including 1:100 are considered within the normal range. Other laboratories have secured satisfactory results by using living antigens and reading the test immediately after centrifugation, omit- ting the previous incubation at 35° C. In this case dilutions starting at 1:8 may be necessary, since the titers may not be high. Again it is essential that all reagents be accurately checked and controlled. 2. Other tests—Precipitation with the patient’s serum and known specific soluble substance may also be valuable. Similarly, capsular swelling tests using patients’ serum with known well-encapsu- lated type strains may offer a good diagnostic tool in the absence of bacteriological proof. 460 BACTERIAL MENINGITIS Vill. OTHER METHODS NOT IN COMMON USE A. Oxidase Test The method of applying the direct oxidase reaction to the differen- tiation of bacterial colonies is described in Chapter 17. All of the Neisseria produce this reaction, and some of the Hemo- philus, as well as other bacteria that produce an active oxidase. The practical value of the oxidase test in facilitating detection of such bacteria in mixtures is obvious. It has found its chief use in detect- ing the gonococcus on plates made from urethral and cervical dis- charges, but it is also a useful aid in the isolation of meningococci and other Neisseria from nasopharyngeal cultures. With these bacteria chocolate agar plates are generally used. B. The "Halo" Reaction The development of halos of precipitate around meningococcal growth on agar plates containing immune serum has several useful applications in the laboratory. The reaction is group-specific and has the virtue of identifying the noncapsulated Group B strains as well as those of Groups A and C. This method is applicable to a number of other kinds of bacteria, as well as to the meningococcus, provided suitable specific immune serum is available; and it can be extremely useful for preliminary grouping. By this means strains that are smooth, virulent and antigenic can be readily distinguished from those which have lost these properties. In general, the follow- ing technic is suggested :1® 1. Melt the agar in tubes, each tube containing approximately 15 to 16 ml. 2. Cool the agar to 50° C, add 0.8 ml of specific immune serum, and pour the mixture into sterile petri dishes. This gives approximately 5 per cent of the serum. 3. Collect a mass of growth, about 2 mm in diameter, from an 18 hr culture on glucose serum agar, or from a 5 hr culture on blood agar. 4. Place this mass upon the surface of the specific serum agar plate without spreading; 6 to 10 different cultures may be tested on one petri dish. 5. Incubate the plate at 35° C and examine it after 48 and 72 hr in a strong light against a dark background. The specific precipitating properties in the serum and the soluble specific antigens of the microorganisms will form a visible precipitate in the form of a halo around the bacterial growth. The intensity of the halo is recorded as 1 plus to 4 plus. This test is less often performed now than formerly because of the scarcity of specific immune serum. Sara E. BRanaaM, M.D, Pua.D., Chapter Chairman Harrie E. ALExANDER, M.D. CaroLyN R. Fark Mark H. Lepper, M.D. Lours WeInsTEIN, M.D., Pu.D. BACTERIAL MENINGITIS 461 REFERENCES 1 2 10. 11. 12 13. 14. 15. 16. Subcommittee on the Neisseriaceae. Internat. Bull. Bact. Nomenclature 4:95-101, 1954. International Bacteriological Code of Nomenclature. J. Bact. 55:287-306, 1948. KovLMER, J. A., SpauLpinG, E. H., and Rosinson, H. W. Approved Labora- tory Technic (5th ed.). New York, N. Y.: Appleton-Century-Crofts, 1951. GrabpwoHL, R. B. H. Clinical Laboratory Methods and Diagnosis (3rd ed.). St. Louis, Mo. : Mosby, 1943, Vol. I. Stitt, Epwarp R., CroucH, Paur W., and BranuaM, Sara E. Practical Bacteriology, Hematology and Parasitology (10th ed.). Philadelphia: Blakiston, 1948. Topp, James C., Sanrorp, ARTHUR H., and WELLS, Benjamin B. Clinical Diagnosis by Laboratory Methods (12th ed.). 1953. PHAR, J. J., SmrrH, DorotHY G., and Root, CuarrLorre M. Use of Chicken Serum in the Species and Type Identification of Neisseria. Proc. Soc. Exper. Biol. & Med. 52:72-73, 1943. NosLe, ArRLyLE. A Rapid Method for the Macroscopic Agglutination Test. J. Bact. 14:287-300, 1927. Arexanper, Hattie E., Ervis, CATHERINE, and LEmy, GRACE. Treatment of Type-Specific Hemophilus influenzae Infections in Infancy and Childhood. J. Pediat. 20:673-698, 1942. Standard Methods for the Examination of Water and Wastewater. (11th ed.). New York, N. Y.: American Public Health Assn., 1960. Miiier, C. P., and BounHorFr, MARJORIE. Development of Streptomycin- Resistant Variants of Meningococcus. Science 105 :620-621, 1947. Rake, Grorrrey. Studies on Meningococcus Infection. II. Monovalent Diagnostic Sera Prepared from “Fresh” and “Stock” Strains. J. Exper. Med. 57:561-569, 1933. Fark, CaroLyN R,, and AprpeLBauM, E. Type-Specific Agglutinins in Human Serums. I. Description of Method. Proc. Soc. Exper. Biol. & Med. 57:341- 343, 1944. Maver, R. L., and Dowring, H. F. The Determination of Meningococcic Antibodies by a Centrifuge Agglutination Test. J. Immunol. 51:349-354, 1945. Fark, CaroLyN R., and ApperBaUM, E. Type-Specific Meningococcic Agglutinins. TI. Relationship of Titers to the Course of the Disease. J. Clin. Invest. 24:742-748, 1945. PrrrMAN, MARGARET, BRANHAM, Sara E., and Sockriper, ELsie M. A Com- parison of the Precipitation Reaction in Immune Serum Agar Plates with the Protection of Mice by Antimeningococcus Serum. Pub. Health Rep. 53:1400-1408, 1938. IL III. IV. CHAPTER 17 THE VENEREAL DISEASES, EXCLUSIVE OF SYPHILIS . Gonococcal Infections A. Collection of Specimens 1. Outfits for Collection of Specimens History Slips General Directions Collection of Specimens from Men Collection of Specimens from Women Miscellaneous Specimens . Transportation of Specimens B. Examination of Specimens 1. General Directions 2. Films for Microscopical Examination 3. Cultures 4. Determination of Sensitivity to Antibiotics in vitro NoOUAE LN Chancroid A. Collection of Specimens 1. Films from Primary Lesions 2. Films from Buboes 3. Cultures B. Microbiology and Cultural Methods C. Intradermal Test 1. Ito-Reenstierna Reaction 2. Preparation of Skin Test Antigen D. Lesion Biopsy Granuloma Inguinale A. Films 1. Collection of Specimen 2. Staining 3. Microscopical Examination B. Tissue Sections 1. Obtaining Specimen 2. Staining 3. Microscopical Examination Nongonococcal Urethritis in the Male . Microorganisms Found in the Disease Examination for T. vaginalis Pleuropneumonia-like Organisms Hemophilus Species Staphylococci and Streptococci Diphtheroids . Coliform Organisms . Proteus and Pseudomonas Species Viruses TEoEmuOEy References 462 THE VENEREAL DISEASES 463 I. GONOCOCCAL INFECTIONS The diagnosis of gonorrhea is still a very important function of the public health laboratory. Penicillin and other antibiotics have greatly increased the cure rate but the incidence of new infections has not declined. Technics for culture of the gonococcus have made possible positive differentiation of gonococcal from nongonococcal urethritis, have facilitated diagnosis of the disease in the female, and are of great value in confirming apparent cures. The bacteriological procedures described here are those that have given the most uniform and reliable results in many comparative studies of methods for staining the gonococcus and of media, atmos- pheric requirements, and temperatures of incubation for growing the gonococcus. Diagnosis by stained film has in its favor the element of simplicity. It is relatively inexpensive because it requires little equipment and, where laboratory service is not available, it can be performed by the physician in his office. A diagnosis can be made within a few minutes, or, on the other hand, examination of films can be postponed in- definitely without affecting the results. The disadvantages, however, are serious enough to warrant the use of a more reliable method. Recent studies indicate that a markedly high percentage of positive findings can be made in cases where the results of microscopical ex- amination of stained films were negative. Of paramount importance for the control of gonococcal infection is the adoption of uniform diagnostic procedures. Films are often negative in chronic cases of gonococcal infection when the number of gonococci has been markedly reduced, and especially after chemo- therapy. Erroneous diagnosis may be made because of confusion with other Gram-negative cocci. Furthermore, the direct micro- scopical examination of slides does not distinguish viable from non- viable gonococci. Results vary with the degree of application, the skill of the technician, and the time devoted to the examination of films. The results cited in various reports comparing film and culture methods indicate that the culture method is not only a useful adjunct to the film method but is a necessary procedure for establishing accurate diagnosis. In chronic cases in both sexes, and in the detection of carriers, the cultural method is far more dependable. It is preeminent, however, in the diagnosis of the disease in females, where it has proved to be as much as 200 per cent superior to the film method.! 464 THE VENEREAL DISEASES False-positive results are avoided and, since the isolation of Neisseria gonorrheae in culture constitutes an unquestionable diagnosis, the test is acceptable as legal evidence. Neisseriae other than the gonococcus frequent the genitourinary tract and may produce symptoms similar to those observed in gonococcal infection. The culture method is essential in detecting Neisseria such as N. meningitidis or N. flava and in differentiating them from N. gonorrheae.®* In its present stage of development the culture method is limited to use in venereal disease clinics, hospital laboratories and communi- ties where specimens can be delivered promptly to a laboratory that is equipped to carry out the procedure. If media cannot be inoculated at the time the specimen is collected from the patient, cultures must be made shortly thereafter for best results. The special media and equipment needed for the test and the greater amount of time ex- pended in performing it increase the cost, although it is no higher than that for cultural tests used in the diagnosis of other infectious diseases. It is as yet primarily a procedure requiring a trained laboratory technician and for this reason cannot be utilized effectively in the office of the general practitioner. In view of the limitations of both the film and the culture methods, their simultaneous use is strongly recommended to secure a maximum of positive results. The film, however, is most valuable in the diagnosis of acute untreated urethritis in the male. The use of cul- tures is unnecessary in this type of case unless the information obtained is to be used for research or for medicolegal purposes. Examination of films prepared from prostatic secretion, on the other hand, does not yield dependable evidence. Because of the consistency of the secretion such films are more difficult to stain satisfactorily with Gram’s method. The complement-fixation test cannot be recommended as a means of diagnosis. Even the best technic sometimes gives negative results when the patient shows typical symptoms of gonococcal infection and films and cultures are positive. Furthermore, the majority of patients treated with penicillin are cured before antibodies develop. On the other hand, positive results are occasionally obtained in cases with no history of gonococcal infection and negative bacteriological tests. If used in conjunction with the film and cultural methods, however, it can be of some aid to the physician who is acquainted with its limita- tions. The most practical application at the present time is in the diagnosis of gonococcal arthritis, pelvic inflammatory disease, and chronic prostatitis. THE VENEREAL DISEASES 465 Further research on the biochemistry of the gonococcus and on re- lated aspects of the problem may produce a complement-fixation test as reliable as that for the diagnosis of syphilis. A dependable sero- logical test would be not only of practical value to the physician but an invaluable aid to the epidemiologist and microbiologist. Because the test is rarely employed in the diagnostic laboratory, the descrip- tion of the technic is omitted from this (4th) edition of the book. The reader is referred to the description of the technic appearing in the 1950 edition. A. Collection of Specimens 1. Outfits for collection of specimens—Cotton-tipped appli- cators, kept sterile in a stoppered test tube, and glass microscopic slides comprise the necessary materials for preparing films, When specimens are to be sent or delivered to the laboratory, a wooden or cardboard slide holder accommodating two slides and a mailing con- tainer or heavy manila envelope addressed to the laboratory should be provided. The swabs are made by tightly winding a small amount of absorbent cotton around the terminal 34 in. of a 6 in. wooden or aluminum applicator. The diameter of the swab should not exceed 34¢ in., and a diameter of 14 in. is preferable. The slides must be clean and free from grease—otherwise the film of exudate will not adhere to the glass. Slides with etched ends are satisfactory, espe- cially recommended where they are to be used more than once. If etched slides are not used, a diamond-point pencil should be available in the laboratory for marking the slides. Gummed labels or wax pencils are unsatisfactory. When cultures are to be taken, at least two sterile swabs are included in the outfit. Investigators report that the gonococcus is rapidly killed by contact with wooden applicator sticks for even short periods. This toxic action, however, may be eliminated as shown by Stuart, Toshach and Patsula.? Some prefer a cotton-tipped aluminum swab® for the collection of material for culture. A tightly stoppered tube or vial containing 1 ml of either an extract broth or the transportation medium of choice is also necessary. 2. History slips—A suitable history form calling for pertinent information is essential for accurate laboratory records. It should provide space for the patient’s name, address, age, sex, source of the specimen, date taken and type of examination desired. The duration of the disease and whether it is acute or chronic should also be re- corded. For cultural tests, the hour the specimen was taken should 466 THE VENEREAL DISEASES be noted. For statistical purposes it is well to include occupation, race and marital status. Space is of course provided for the name and address of the physician. A properly arranged history form may be used for both film and cultural examinations, although a different form is advisable for the complement-fixation test. The laboratory report of the bacteriological examination should provide for the date of examination, the name or initials of the examiner, and the signature of the person responsible for the results. The two items of information to be included in the report on films are (1) the presence or absence of Gram-negative diplococci and (2) the relative number of pus cells. The report of the cultural examination should state that the gonococcus was (or was not) isolated in culture. 3. General directions—In acute cases of gonococcal infection, specimens of exudate for examination by either the film or cultural method are generally taken from the urethra of the male or the cervix of the female. In chronic cases in the male, prostatic secretions and urine may also be submitted. In vulvovaginitis, specimens are obtained from the vagina. Other sources of infectious material are the conjunctiva, abscesses of Bartholin’s glands, the Fallopian tubes, pelvic lesions and rectal discharges. Cultures of blood and of joint and cerebrospinal fluids occasionally reveal the gonococcus. The cultural examination of cerebrospinal fluid from atypical cases of meningitis should not be overlooked. Not infrequently the gono- coccus can be recovered from the mucous membranes of the geni- tourinary tract, from prostatic secretions, or from urine sediment of patients who are symptom-free after treatment.” Specimens to be examined as a test for cure should not be obtained for at least 48 hr after the therapeutic agent could be expected to be completely elimi- nated from the body because minimal concentrations of sulfonamides or antibiotics in the inoculum inhibit growth of the organism. In taking specimens it is important to avoid accidental infection of the conjunctiva, which is especially vulnerable to invasion by the gonococcus. The use of rubber gloves is recommended, because the patient may be suffering from syphilis as well as from gonococcal infection. The usual cotton-tipped applicator is suitable for collecting most specimens. Separate swabs, however, should be used for obtaining exudates for the culture and the film. It is advisable to take first the specimen for culture because more exudate is required for this ex- amination. Aseptic technic is unnecessary except when specimens are THE VENEREAL DISEASES 467 obtained from the spinal canal, from joints, or from abscesses. Cerebrospinal fluid is taken as ordinarily collected for examination for evidence of meningococcal infection. The technic recommended for procuring joint fluid is as follows: 1) By means of a piece of sterile gauze or a sterile swab, apply tincture of iodine to the skin over the joint, 2) With a 2 ml sterile syringe and a hypodermic needle, inject 1 per cent procaine under the skin and into the subcutaneous tissues at the point of greatest fluctuation. 3) When satisfactory anesthesia has occurred withdraw the joint fluid by inserting into this area a 20 gauge needle of suitable length, using a 10 or 20 ml sterile syringe. To insure the greatest number of positive results the medium should be inoculated immediately after the specimen has been collected from the patient. If possible the specimen should be inoculated directly onto the surface of the agar plate to be employed for isolating the gonococcus. If the specimen cannot be cultured immediately, the swab with the exudate should be placed at once in a test tube con- taining 1 ml of meat infusion broth or any other suitable menstruum not injurious to the gonococcus. A 2 per cent solution of Proteose No. 3 broth* to which sodium chloride is added to yield a final con- centration of 0.5 per cent is satisfactory. Such a broth will maintain the viability of the gonococcus satisfactorily for only 4 or 5 hr, depend- ing upon the amount of inoculum, the number of gonococci in the specimen, the temperature, and the number and type of concomitant bacteria in the specimen. Although specimens of exudate may be inoculated directly onto the culture medium, there are several advantages in first suspending the material in a small amount of broth. It permits delayed inoculation when laboratory facilities are not immediately available; it dilutes the inoculum, thereby reducing overgrowth of the gonococcus with commensal organisms; it provides an increased amount of moisture to the surface of the solid medium. When the laboratory is distant from the patient and the specimen cannot be cultured for several hours after collection, a so-called “trans- portation medium” is essential.® Comparative tests for viability of the gonococcus on several media recommended for this use have demon- strated that, as the number of hours increases between collection of the specimen and inoculation of the medium at the laboratory, the number of strains isolated decreases. Nevertheless, under certain circumstances the shipment of specimens to a laboratory from con- siderable distances is warranted. * Prepared by Difco Laboratories, Inc., Detroit, Mich. 468 THE VENEREAL DISEASES The method of choice for such shipment is that of Stuart, Toshach and Patsula.? This is based on three principles: 1) Pretreatment of the swabs with charcoal to neutralize an inhibitory factor present in the agar used in the transport medium. 2) Prevention of oxidation and desiccation of material on swabs to maintain viability of the organism. 3) Absence of nutrient substances in the transport vehicle (CM No. 51) to prevent undesirable multiplication of other organisms. Three other semisolid media may also be used: gelatin egg albumin agar (CM No. 46),® gelatin blood agar (CM No. 45)? and bovine albumin agar (CM No. 50)? but they are less satisfactory. 4. Collection of specimens from men—Aseptic technic as a rule is unnecessary in obtaining specimens for culture from the male urethra. When such precautions seem necessary, the foreskin is re- tracted and the glans penis is cleansed with soap and water; a mild antiseptic, such as 70 per cent alcohol, may be applied. Care must be taken that even a minute amount of the antiseptic does not re- main in the meatus to be absorbed on the cotton swab when the urethral exudate is collected, thereby inhibiting growth of the gono- coccus. Pus at the meatus is removed with a sterile cotton swab. In the absence of visible exudate, the penile urethra is stripped with the thumb and forefinger and any resulting mucopurulent or mucoid exudate is cultured. Inasmuch as the gonococcus not infrequently can be isolated from urine sediment, the first 10 or 15 ml of voided urine serve as a useful specimen, especially when no urethral exudate is present.!! As a rapid, simple screening procedure for the detection of sus- pected cases of gonorrhea, an equal quantity of 10 per cent acetic acid is added to fresh urine specimens and the mixture is examined macroscopically for pus or shreds, the presence of which is taken as presumptive evidence of gonorrheal infection. Another aliquot of urine may be used for culture and the preparation of stained films from the sediments. In chronic cases prostatic fluid should also be cultured. To obtain this fluid the penile urethra is compressed with the thumb and finger to prevent loss while prostatic massage is carried out in the usual manner. Pressure is then released and the prostatic fluid in the urethra is permitted to flow directly onto a chocolate agar plate or into a test tube containing 1 ml of sterile broth for subsequent cultur- ing. If the quantity is scant, the exudate is collected from the meatus by means of a swab, which is then placed in a tube of broth, or preferably on a chocolate agar plate. As a test for cure, a single com- THE VENEREAL DISEASES 469 bined specimen of prostatic fluid and urine may be used, inasmuch as it permits detection of infection in the prostate as well as in the posterior and anterior urethra. From 10 to 15 ml of urine are col- lected in a sterile tube immediately after prostatic massage. Compara- tive tests have shown that the gonococcus is very seldom isolated from the prostatic secretion when it is not present in the anterior urethra. 5. Collection of specimens from women—Sterile preparation of the vulva and douching of the vagina are seldom necessary, If the urethral meatus appears normal and no exudate is present, films and cultures from this source are not indicated. The gonococcus is so rarely isolated from the urethra when it cannot be recovered from the cervix that duplicate films and cultures from both sources are un- necessary. This observation is borne out by a comparison of hundreds of cultures from the urethra and cervix. Therefore, in most venereal disease clinics only cervical films and cultures are examined, which is a saving in time and expense. If it is deemed necessary to culture the urethra, the following technic is employed: The urethral meatus is cleansed by a sterile cotton pledget. Digital pressure is applied with a gloved finger to the urethra and to Skene’s glands. Exudate is then collected on a sterile swab and immersed in a tube containing 1 ml of suitable broth or cultured directly on chocolate agar. To be satisfactory, it is most important that specimens collected for culture from the cervix be taken with great care by an experienced person. A bivalve vaginal speculum should always be used, without lubricant other than water or physiological salt solution (Fig 1). The cervix is cleansed with a cotton pledget on dressing forceps. The cervical plug, if present, is removed with a cotton-tipped applicator. The cervix is then gently compressed with the blades of the speculum to express secretions from the endocervical glands. Specimens are then collected by inserting a small sterile swab 0.5 to 1 cm into the cervix. A sufficient quantity of exudate should be obtained to insure successful results. During pregnancy the material should be collected from the cervical os. The swab is cultured as heretofore described. The collection of specimens from children and infants with vulvo- vaginitis may be carried out with a cotton-tipped applicator, but the use of a female glass catheter is preferable because it causes no trauma and permits collection of more material. The catheter, con- taining a small amount of physiological salt solution, is introduced into the vagina and moved about so as to permit approximately 0.3- 0.5 ml of vaginal secretion to flow into the catheter. A film is pre- pared from one drop and the remainder is suspended in 1 ml of broth for culture or is inoculated directly onto a suitable solid medium. 470 THE VENEREAL DISEASES ENDOCERVICAL GLANDS Figure 1—Sterile cotton-tipped applicator has been inserted through bivalve speculum into cervical canal (see text). 6. Miscellaneous specimens—In taking specimens from the anorectal region, aseptic technic is important. The area is cleansed with soap and water, an antiseptic applied, the anal sphincter dilated by means of an anoscope, and the specimen collected from the mucosa adjacent to the terminal portion of the anal canal. Pus from the conjunctiva and from abscesses may be inoculated directly onto the culture medium or suspended in extract broth. Joint and cerebrospinal fluids and urine are collected in sterile tubes with- out broth. Blood for cultures is obtained by venipuncture. One ml and 4 ml amounts are added to 100 ml of glucose ascitic fluid broth; 5 ml are mixed with 2-4 ml of 2.5 per cent sodium citrate to prevent coagulation for use in blood agar plates. 7. Transportation of specimens—No special precautions other than protection from dust are necessary in transmitting films to the laboratory, since they are unaffected by time, temperature or atmo- spheric conditions. Specimens of urine, blood, and exudate in broth, on the other hand, should be delivered promptly and cultures made as soon as possible. Not more than 6 hr should elapse between the time the specimen is collected and the time it is cultured, unless a THE VENEREAL DISEASES 471 special transport medium such as that of Stuart ef ol.’ (CM No. 51) is employed. When cultures cannot be made directly from the patient, the specimen should be kept in a refrigerator if possible—otherwise at room temperature, especially when Stuart’s medium is being used. Under no circumstances should the culture be incubated, because ex- posure to temperatures as high as body temperature favors the multi- plication of contaminating microorganisms, which soon overgrow the gonococcus and make isolation difficult. B. Examination of Specimens 1. General directions—As soon as the outfit is opened in the laboratory, the specimen should be given an accession number and the information checked with the accompanying history form. Care should be taken at all times to preserve the identity of the specimen and guard against errors in labeling, etc. 2. Films for microscopical examination a) Preparation—To prepare a suitable film, the exudate which has been obtained on a sterile swab is “rolled out” over the surface of two glass slides by rotating the cotton-tipped applicator between the thumb and index finger. The rolling procedure is superior to rub- bing the swab over the surface because the pus cells remain intact and more gonococci retain their intracellular position. The film should be placed near one end of the slide so that space remains available at the opposite (etched) end for labeling. The success of the examination depends to a great extent upon the care with which the film is made. Thick films are unsatisfactory and can be avoided by collecting only a small amount of exudate and by rolling the swab over the surface but once. A well-made preparation should be not more than one cell thick. When the film has been air- dried or fixed by gentle heating of the reverse side of the slide, it should be marked plainly with the patient’s name, source of the specimen and date, or with a code number. b) Staining—The procedure recommended is Hucker’s modifica- tion of Gram’s stain? The films should be stained individually for best results, although when large numbers are to be examined they may be stained in groups by the use of slide holders. If itis necessary to examine them collectively, slides with thick films should be removed and stained separately, as they require more decolorization than slides with thin films. To control the staining reaction, one loopful of a young broth culture of Staphylococcus aureus and one of Escherichia coli are 472 THE VENEREAL DISEASES placed on the slide adjacent to the film before staining. Organisms from solid media may be used if properly diluted with distilled water. While it may not be necessary to control the Gram stain in this man- ner in laboratories where numerous examinations are made daily, the provision is essential for laboratories where only an occasional film is examined. c) Microscopical examination—A compound microscope equipped with a mechanical stage and an oil-immersion lens is essential for making a satisfactory examination. When correctly stained, the nuclei of the pus cells should retain some of the violet dye, while the cytoplasm should be pink. The examiner notes first the staining re- action of the staphylococci and colon bacilli placed on the slide for control. (Staphylococci are Gram-positive, that is to say, they retain the initial violet dye; colon bacilli are Gram-negative, that is, they do not retain the initial dye but are decolorized and appear pink from the effects of the counterstain.) If the control films are not properly stained, the preparation is unsatisfactory for diagnosis. Gonococci are typically Gram-negative when correctly stained and appear as pink or orange-red cocci, usually arranged in pairs. The approximating surfaces are flattened, producing the well-known biscuit or coffee bean shape. The organisms may be inside or out- side the pus cells. The intracellular location is typical and has con- siderable diagnostic significance. As a rule, extracellular gonococci are found frequently in films prepared from cervical exudates. The entire film should be examined for gonococci if necessary. For an experienced observer the period of examination should be at least 3 min, preferably 5. Difficult slides may take more time. Special care is required in the examination of vaginal and cervical films from chronic cases in order to avoid missing the rare gono- coccus. All slides should be filed and kept for at least 6 months for reference and legal considerations. While making the micro- scopical examination the absence or presence of pus cells and their frequency should be reported. If spermatozoa are observed, this fact may also be recorded. d) Reporting results—Responsibility for interpreting the results of a microscopical examination is solely that of the physician in charge of the case. The laboratory examiner should report only what is observed in the film, that is: “Intracellular Gram-negative diplococci resembling gonococci were [were not] found.” If extracellular Gram- negative cocci resembling the gonococcus are observed, a continued search usually will reveal intracellular groups as well. If only extra- cellular forms are found, the report should read, “Extracellular Gram- THE VENEREAL DISEASES 473 negative diplococci resembling gonococci were found” and a request for a second film should be made. If the film is too thick for satis- factory staining or no exudate can be detected on the slide, this in- formation should be reported and a second film requested. The report should include a statement concerning the number of pus cells noted: “Many [Few] [No] pus cells present.” 3. Cultures a. Precautions in handling exudates before culturing: It is im- portant that precautions be taken to prevent drying of the exudate before suspension in transport media. Drying destroys the gono- coccus rapidly. Contrary to the general belief that specimens to be cultured for the gonococcus should be kept at body temperature prior to inocula- tion, better results are obtained when they are held at temperatures of from 4° to 10° C. If facilities for refrigeration are not available, cultures should be kept as cool as possible to avoid overgrowth of the gonococci with commensal organisms. It is emphasized that best re- sults are obtained when cultures are made immediately after the speci- men is collected; however, the findings are usually dependable if inoculation is not delayed for longer than 6 hr. b. Media: A comparative study under controlled conditions of media for the isolation of the gonococcus revealed that the greatest number of isolations were made on the three following media :13:14 1) Modified McLeod's agar with Nile Blue A and enriched with horse plasma and hemoglobin (CM No. 43).15 2) Proteose No. 3 agar with Nile Blue A and enriched with horse plasma and hemoglobin (CM No. 44).16 3) Bacto-GC medium base, with Bacto hemoglobin and supplement A or B (CM No. 41) .* Although these three media were equally as effective from the standpoint of growing the gonococcus, the chocolate agar prepared from the GC medium base and Bacto hemoglobin was more uniform in quality and more simply prepared than the other two media. The addition of either Bacto supplement A or B to the chocolate agar is essential. In the comparative study'* supplement B proved to be slightly superior to supplement A. These supplements are an especially prepared thermolabile yeast concentrate containing adequate amounts of growth-accessory factors, particularly glutamine, cocarboxylase and coenzyme, required by the more fastidious strains of the gono- * Prepared by Difco Laboratories, Inc., Detroit, Mich. 474 THE VENEREAL DISEASES coccus. Supplement A differs from supplement B only in that it con- tains sufficient crystal violet to yield a final concentration of 1:714,000 in the medium.* The supplements are essential because numerous strains of the gonococcus fail to grow unless the medium is augmented with glutamine.” Some strains of N. gonorrheae will proliferate in air in a fluid medium containing casamino acids and salts in the presence of small amounts of yeast extract. The latter can be replaced by a mixture of hypoxanthine, uracil and oxalacetate. The requirement for hypoxan- thine was found to be specific, and growth of the organism was proportional to the concentration of this substance. The dehydrated GC medium base with Bacto hemoglobin and either supplement A or B possesses several advantages over the other media described for the isolation of gonococcus. It is immediately available and can be readily prepared in either large or small quanti- ties, insuring a supply of fresh, moist medium essential for depend- able results. Furthermore the cultures may be examined after incu- bation for 24 hr instead of the 48 hr required by the other media. Certain laboratories still incubate cultures for 48 hr. The addition of tyrothricin to chocolate agar is another useful aid.!? A final concentration of 1:15,000 inhibits the growth of the Gram- positive organisms such as streptococci, lactobacilli and diphtheroids, thereby facilitating isolation of the gonococcus. Many strains of the gonococcus may be isolated on other media prepared from an infusion agar base enriched with blood, serum or ascitic fluid. Bovine albumin has been reported to enhance viability of the gonococcus. The purpose of the cultural method, however, is to isolate the maximum number of strains, so that the best special media must be employed if the procedure is to be effective. Stuart’s transport medium (CM No. 51) is prepared by dissolving 6 g of Bacto agar in 1,900 ml of sterilized distilled water. Add 2 ml of thioglycolic acid (3.3 g sodium thioglycolate)* and adjust to pH 72 (with 1 N NaOH). Add 100 ml of sodium glycerophosphate (20% w. v. in water) and 20 ml of a 1 per cent w. v. in water solution of CaCl... Readjust the pH to 7.4 with 1 N HCl. Add 4 ml of a 0.1 per cent aqueous solution of methylene blue. Dispense into small screw-capped vials, filling to capacity. Sterilize at 85° C for 1 hr and then secure the caps tightly. Prepare the swabs by boiling cotton- tipped wooden applicators in Sgrensen’s phosphate buffer solution at pH 7.4, then immediately dipping into a 1 per cent suspension of * Prepared by Difco Laboratories, Inc., Detroit, Mich. THE VENEREAL DISEASES 475 finely divided activated charcoal in water. If cotton-tipped aluminum applicators are used, the boiling process is omitted. Dry the swabs, place in tubes or glassine envelopes, and sterilize. After taking the specimen with aluminum applicator, push the cot- ton tip into the holding medium with sterile forceps. When wooden applicators are used, break off the cotton-tipped end into the medium and screw the cap tightly onto the vial. If a blue color can be detected in the holding medium, it indicates the presence of oxygen. This is toxic to gonococci and the medium is unsatisfactory. A recent extensive study of delayed culture results from N. gonorrheae showed a 67 per cent increase in the positive findings from cultures over stained films when effective transport media were employed.?2° c. Inoculation of media: As previously directed, specimens from the urethra and cervix should, if possible, be inoculated directly onto the surface of the agar plate. Inoculation of the plate is an important procedure and it requires skill and experience to obtain suitable cul- tures because the amount of inoculum collected on a swab varies. Care should be taken to effect good distribution of the inoculum in order to utilize the maximum surface of the agar and yet avoid over- growth with commensal bacteria. From one-fourth to one-third the surface of the agar plate should be streaked back and forth without rotation of the swab. A platinum or chrome wire loop or needle may then be used to spread the inoculum further, at right angles to the area originally inoculated, thereby covering the remainder of the plate surface. If the exudate has been suspended in a liquid medium, from 0.05 to 0.1 ml of inoculum, depending on the turbidity of the specimen, may be pipetted onto the agar and then spread over the surface of the plate with a sterile glass L-shaped rod. If the swab employed to collect the exudate is submitted in the tube of broth, the applicator should be rotated and compressed against the inner wall of the tube to suspend as much of the exudate as possible. From 0.05 to 0.1 ml of the suspension is then pipetted to the agar plate and inoculated onto the surface by means of a glass rod or wire loop. If exudate is scant, the suspension should be centrifuged and the sediment cultured. Joint and cerebrospinal fluids and urine are centrifuged, and the sediment is streaked on chocolate agar plates. In the case of blood cultures, citrated blood from the patient is made into blood agar plates by the following procedure: Infusion, hormone or Douglas’ agar is melted and then cooled to about 42°-45° C, at which temperature 10 per cent of ascitic fluid and 1 per cent of 476 THE VENEREAL DISEASES glucose are added; to 20 ml of this medium, 3 ml of citrated blood is added and a plate poured; to another 20 ml, 2 ml of citrated blood is added and a second plate poured. The inoculated chocolate agar plates and the blood plates are in- verted and stacked in jars for incubation in an atmosphere reinforced with carbon dioxide. Desiccators or museum jars fitted with covers may be used, and can be made airtight with vaseline or silicone lubri- cant.* Any container that has a tightly fitting cover and a mouth wide enough to permit the introduction of the petri dishes may be substituted. (Fig 2 shows a 1 gal wide-mouth glass jar fitted with a metal screwtop.) oo Figure 2—(a) Inexpensive wide-mouth jar, 1 gal size, showing method of reducing oxygen tension by use of lighted candle; (b) cover for jar fitted with stopcock for use with equipment as illustrated in Fig. 3. Reinforcement of the atmosphere with carbon dioxide is obtained in one of several ways: 1. A suitable stopcock, screwed into the cover of the jar, permits rubber hose connections to be made to a cylinder of compressed car- bon dioxide and to a vacuum pump. A mercury, open U-tube type of manometer is included to measure the pressure within the jar. Air * Dow-Corning Corp., Midland, Mich. THE VENEREAL DISEASES 477 is evacuated until the pressure within the jar has been reduced by 9 cm of mercury (removal of approximately 12% of the air), then slightly less carbon dioxide than removed air is released into the jar. This is accomplished by closing the stopcock when the pressure registers between 0.5 and 1 cm of mercury below atmospheric pres- sure (Fig 3). Figure 3—Diagram of equipment used for increasing the carbon dioxide content of the atmosphere surrounding the cultures made for the isolation of the gonococcus. A: faucet suction pump; B: museum jar containing petri dishes; C: mercury manometer; D: tank of compressed carbon dioxide. 2. A less costly and simpler method, but an equally satisfactory one, is to place a lighted smokeless candle approximately 1.5 to 2 in. long and 1 in. in diameter on the plates in the jar and then replace the lid. When the flame is extinguished by depletion of the oxygen, the con- centration of carbon dioxide is adequate for growth of the gonococcus. 3. Sodium bicarbonate and sulfuric acid may be used if this method is preferred. Note: It is important that the cultures be incubated in a moist atmosphere which may be insured by placing a thin layer of moist filter paper in the bottom of the jar. The jars containing the cultures are placed in an incubator at 35° or 36° C. The flasks containing blood in glucose ascitic fluid broth are 478 THE VENEREAL DISEASES likewise placed in an atmosphere with carbon dioxide and incubated at 35° C. A 36° C incubator is also satisfactory. Even 37° C may be used; however, it should be borne in mind that some strains of gonococci cannot be isolated at this temperature. The cultures may be inspected after 24 hr of incubation if necessary, at which time there is often sufficient growth for examination. Some strains of gonococci require 48 hr of incubation. d. Direct inspection of cultures: After 24 or 48 hr of incubation, plates are inspected for the presence of colonies of the gonococcus. On a suboptimal medium, only minute pinpoint colonies are observed. On chocolate agar the colonies are convex, transparent, and from 1 to 3 mm in diameter, with undulate margins (Fig 4). By their trans- parency and the character of their margins they can usually be differ- entiated from young colonies of streptococci or diphtheroids, which they simulate. From the colonies selected, films are prepared, stained and examined. e. The oxidase test: When no colonies typical of the gonococcus can be detected by direct inspection, the culture is subjected to the “oxidase” test, which is of especial value in detecting colonies of Neis- seria in mixed cultures (Fig 5). The test is based upon the presence of an enzyme oxidase produced by organisms belonging to the genus Neisseria. The enzyme is detected by the use of either the oxalate salt?* or monohydrochloride of the dye component, para-aminodi- methylaniline, which in the presence of oxidase produces a charac- teristic series of color changes: first pink, on further oxidation, maroon, and finally black. The oxalate salt of the compound possesses advantages over its monohydrochloride. It is less toxic for the gonococcus and during storage does not deteriorate as rapidly as the monohydrochloride. Likewise the oxalate salt does not form the marked black precipitate on chocolate agar sometimes observed from use of the monohydro- chloride, especially when the solution is not fresh. The oxalate salt is somewhat less soluble than the monohydrochloride and gentle heat- ing is required in the preparation of a solution. Direct inspection of an agar plate culture containing but a few gonococcus colonies obscured by a luxuriant growth of other microorganisms is almost valueless. The microscopical examination of films from many colonies is impractical. It is in such instances that the value of the oxidase test is greatest. A 1 per cent aqueous solution is prepared from the desired com- pound. Best results are obtained with a fresh preparation made daily, THE VENEREAL DISEASES 479 although a solution will continue to be suitable for several days when stored in the refrigerator. An experienced bacteriologist may desire to pick typical colonies of the gonococcus for pure culture studies before the reagent is applied. If there is uncertainty about the Figure 4—Surface colonies of N. gonorrheae and N. catarrhalis on chocolate agar, 48 hr. The gonococcus colonies are gray, with undulate margins. 480 THE VENEREAL DISEASES st Figure 5A—Culture from cervix on surface of chocolate agar plate: before oxidase test. colony type, a few drops of the reagent may be applied to the growth over a small area of the plate. If oxidase-positive colonies appear, subcultures can be made from similar uncontaminated colonies in an adjacent section of the plate. If no oxidase-positive colonies appear from the application of the reagent to a small area, 1 to 2 ml of the solution is dropped on the agar plate culture by means of a pipette and the plate tilted so that the entire surface is moistened. Where a large series is to be examined, a nasal atomizer provides a simple and economical way to apply the reagent. The plate is observed for a period of from 5 to 8 min for evidence of change in the color of the colonies. This usually occurs in less than 2 min, but a freshly prepared solution may delay the reaction slightly. The series of color reactions—pink, maroon and black—readily identi- fies the colonies of Neisseria. Films are made from oxidase-positive colonies, stained, and examined microscopically. If subcultures are THE VENEREAL DISEASES 481 i Figure 5B—The same culture as in Fig 5A, after oxidase test. to be made for further identification, the colonies should be picked as soon as they become pink because the dye component is toxic for the organisms. When oxidation has progressed until the colony is black, the cells are usually dead and subcultures fail to grow. The dye does not interfere with subsequent Gram stains, f. Interpretation of results: A Gram-negative diplococcus isolated from the genital canal, particularly from the male, and showing the typical morphological characteristics of N. gonorrheae may be pre- sumed to be a gonococcus when picked from an oxidase-positive colony. Further identification on carbohydrate media is necessary in all medicolegal cases and in doubtful cases when the history and clinical status of the patient are inconsistent with the bacteriological findings. Special care should be taken to differentiate the gonococcus from meningococcus and from Neisseria catarrhalis in cultures from the conjunctiva. Cultures from the lower birth canal and from prostatic 482 THE VENEREAL DISEASES secretions occasionally show oxidase-positive colonies other than those of the Neisseriae, but difficulty seldom arises in differentiating them because the majority are either Gram-positive or Gram-negative bacilli. Streptococci and diphtheroids, which are the most difficult or- ganisms to differentiate from N. gonorrheae by direct inspection of colonies, do not form oxidase. g. Identification by carbohydrate fermentation: For final identifica- tion of the gonococcus and differentiation from the other Neisseria, it is necessary to inoculate media containing the following carbohy- drates: glucose, lactose, sucrose, maltose, levulose and mannitol. The use of glucose and maltose only will differentiate the gonococcus from the meningococcus. Many basic media for incorporating carbohy- drates have been described, but ascitic fluid agar (CM No. 49) is the best. The ascitic fluid must be bile-free, with a pH range between 7.2 and 8.0. Ascitic fluid of high alkalinity inhibits the growth of the gonococcus ; therefore, pH should be determined before use and the fluid neutralized if necessary. Even though all these specifications are met, some lots of ascitic fluid are unsatisfactory. It is thus advisable to compare the growth-stimulating factor of each new supply with fluid known to be suitable. In laboratories where a supply of ascitic fluid is not available, serum may be substituted. Rabbit serum is the most suitable, although human serum may be used (the maltose con- tent of beef, sheep and horse sera make them unsuitable in carbo- hydrate medium as a growth-promoting substance). Typical colonies are selected from the original plate and sub- cultured on a suitable medium such as chocolate agar. When growth occurs, transfers are made to a series of slants of ascitic fluid agar containing Andrade’s indicator and 1 per cent of each carbohydrate. Rubber stoppers are inserted in the tubes to limit the oxygen supply; as growth develops, the carbon dioxide content within the tube is increased. The use of rubber stoppers insures moist, fresh media by preventing the evaporation of condensation water. Cultures are incubated for 48 hr at 35° C and then inspected. The fermentation of glucose only, which is indicated by the agar slant turning pink, differentiates N. gonorrheae from other members of the genus. Another satisfactory medium is Bacto phenol-red carbohydrate broth containing 0.5 per cent of the desired carbohydrate. A semi- solid medium is preferable, however. This is prepared from the carbo- hydrate broth base by adding 0.15 per cent agar.* A comparatively * Prepared by Difco Laboratories, Inc., Detroit, Mich. THE VENEREAL DISEASES 483 large amount of inoculum (several colonies) is required to establish growth. The inoculum should be placed in the top 0.5 cm of the semisolid agar column. In Table 1 are listed the carbohydrate fermentation reactions of the various species of Neisseria. Table 1—Fermentation of Carbohydrates by the Neisseriae Species Glucose Lactose Sucrose Maltose Levulose Mannitol N. gonorrheae + — = a it — N. meningitidis* “+ — - ule = — N. catarrhalist — — oo — = — N. sicca + — + + 4 eo N. flava Ro — — + = a N. perflava + —_— + + + & N. subflava + — = he — i N. flavescens — — — _ = - * N. subflava produces yellow pigment on agar which differentiates it from N. meningitidis. + N. catarrhalis is differentiated from N. flavescens by growth at 22° C; N. flavescens is reputed to fail to grow at that temperature. h. Distinguishing characteristics: The characteristics which aid most in distinguishing the gonococcus from other important members of the genus Neisseria should be mentioned. 1) The fermentation of glucose only, with the formation of acid, is the most dependable criterion for differentiating the gonococcus from other members of the genus Neisseria (Table 1). 2) A Gram-negative, biscuit- or coffee bean-shaped diplococcus, observed in films prepared from exudates from the genital canal or from oxidase-positive colonies from the same source, usually proves to be the gonococcus. 3) Colony formation often will distinguish N. gonorrheae from the other species of Neisseria when grown on ascitic fluid or chocolate agar. After incubation for 24 to 48 hr they are convex, transparent, 1 to 3 mm in diameter, and have undulate margins. They are grayish and slightly opaque when viewed by transmitted light. By their transparency and the character of their margins they can usually be differentiated from the young colonies of streptococci and diphtheroids with which they are most frequently confused. 4) Growth on slants of an enriched medium is usually delicate. 5) The failure of a Gram-negative coccus to grow on plain agar, especially a meat extract agar, at room temperature points to the gonococcus. Occasionally, a strain of N. gonorrheae grows well on unenriched media at room temperature. 484 THE VENEREAL DISEASES i. Evaluation of findings: Routinely it may be reported that a cul- ture of the gonococcus has been isolated from the specimen if three characteristics have been observed: —If the colony shows typical morphology (see { h(3) above). —If the colony is oxidase-positive. —If the organism is a Gram-negative, biscuit-shaped diplococcus. It should be emphasized that in medicolegal and doubtful cases, or when a Gram-negative gonococcus is isolated from sources other than the genital tract, final identification should be carried out on carbohy- drate media. j. Reporting results: Report that “the gonococcus (or N. gonor- rheae) was [was not] isolated in culture.” If the original specimen was unsatisfactory for culture, this fact should be reported and a second specimen requested. 4. Determination of sensitivity to antibiotics in vitro—The author has not observed a strain of the gonococcus that would grow in a concentration of greater than 0.08 units of penicillin per ml of broth culture.?>23 Several other investigators have reported cases of penicillin-resistant gonorrhea.?*-2" There is a possibility that anti- biotic-resistant strains of the gonococcus may be developing. Tests of antibiotic resistance should therefore be conducted, especially with organisms isolated from patients who are not cured by standard therapeutic dosages. In a recent WHO report®® the author has stressed the criteria upon which a diagnosis of infection with resistant organisms may be based. The two principal methods for determining the sensitivity of gono- cocci are given (see also Chapter 27) : a. Tablets and disks on solid media—Numerous manufacturers supply disks or tablets impregnated with varying concentrations of the common antibiotics and chemotherapeutic agents. These, placed on the surface of plate cultures of the organism under study, reveal by a zone of growth inhibition their efficacy against that organism. Because of the variety of test disks and tablets available commer- cially, details of technic will not be presented here. In the use of all these reagent disks we must emphasize that the width of the zone of inhibition is a measure of the diffusibility of the reagent—not neces- sarily the sensitivity of the organism to the antibiotic. b. Broth medium—3By the use of graded concentrations of anti- biotics in a suitable liquid culture, the sensitivity of a strain of the gonococcus can be determined with much greater accuracy. THE VENEREAL DISEASES 485 A satisfactory medium and technic for this determination is the following : Douglas’ broth (CM No. 47) with 0.05 per cent potassium nitrate, 0.04 per cent potassium dihydrogen phosphate, and 5 per cent rabbit blood is employed as the basic medium. A culture is tested in varying concentrations of penicillin as fol- lows: 0.2 ml of a 48 hr blood broth culture of each strain of gono- coccus is inoculated into a series of tubes of the blood broth con- taining, respectively, 0.005, 0.01, 0.02, 0.04 and 0.08 units of crystal- line penicillin G. A control tube without penicillin is likewise inocu- lated. The total volume of each tube is 3 ml. The culture is then incubated for 48 hr at 35° C. After incubation for 48 hr, subcultures are made on chocolate agar plates and incubated for 48 hr at 35° C under CO. tension. The plates are examined thereafter, and the first tube from which no sub- culture is obtained indicates the inhibiting concentration of penicillin. Il. CHANCROID Methodology in the accurate laboratory diagnosis of venereal diseases, particularly syphilis and gonorrhea, has progressed rapidly in the last decade. In the case of chancroid, however, this has not been true. Although adequate bacteriological methods have been in existence for some time, they are for the most part so precise in their application that the routine diagnostic laboratory experiences difficulty in their effective employment. Since chancroid is a disease en- countered primarily among individuals with poor hygienic habits, the similarities of the clinical lesions to those of granuloma inguinale and lymphogranuloma venereum can be confusing and must be borne in mind. Of no less perplexity are the taxonomy and botanical classi- fication of the etiological agent Hemophilus ducreyi. Although it requires some blood constituents for growth, this species does not morphologically resemble other members of the genus, nor is it hemophilic. The organism is commonly referred to as a “strepto- bacillus” despite the invalidity of this term as it relates to the genus Hemophilus in systematic bacteriology.*** Of significance are the re- cent studies of Boak on the etiology of penile ulcers among Armed Forces personnel in the Far East. By employing tissue culture methods she isolated herpes simplex virus from 56 per cent of male patients with penile ulcers typical of chancroid.2® The lesions of chancroid are usually nonindurated ulcers with echinate and undermined edges. In appearance they vary from a 486 THE VENEREAL DISEASES small herpes-like area to a large gangrenous crater. The base of the lesion is made up of purulent granulation tissue covered by a grayish exudate. Such lesions may be multiple and almost any area of the genitalia can be involved. However, these lesions often occur in areas where abrasions are present, such as the fourchette of the vulva or the edge of a phimotic prepuce. A. Collection of Specimens 1. Films from primary lesions—The Ducrey bacillus may sometimes be demonstrated in stained film preparations, and unfor- tunately it is the method by which most laboratory diagnoses are made, although the intrinsic difficulties have been emphasized by Teague and Diebert.?® The organisms are not easily seen in films from large older lesions which have most often undergone secondary bacterial infection. The ulcer must first be carefully cleansed with either salt solution or sterile water. This should be done in a manner to preclude bleeding if possible. The most satisfactory material for study should be obtained from the base or the echinate margins of the ulcer with a stiff wire loop. The material is then placed in a small drop of salt solution on a glass slide, spread, and allowed to air-dry. Films prepared from early, relatively clean lesions may be satis- factorily stained by Gram’s method. On the other hand, material obtained from old lesions usually contains a varied flora of bacteria and special staining by Unna-Pappenheim’s method is desirable. The Ducrey bacillus when stained by Gram’s method appears as a small Gram-negative rod, 0.5 by 1.5 to 2.0 pu, with rounded ends, occurring singly in short chains. Occasionally a “school of fish” formation of bacilli may be observed, but usually they occur in small clumps and clusters. The Unna-Pappenheim stain is of special value in examining films prepared from secondarily infected lesions. The Ducrey bacillus thus treated exhibits a bipolar “closed safety pin” appearance which distinguishes it from other concomitant rod forms. Because the characteristic exudative cell is polymorphonuclear, care- ful search for intracellular, bipolar-stained organisms should be made. While properly prepared and carefully examined films are useful in the detection of H. ducreyi, every effort should be made to culture the microorganism before a final report is rendered. It should be remembered that mixed infections incited by Treponema pallidum and H. ducreyi may appear in the same indi- vidual—indeed, in the same lesion. The ruling out of syphilis from a public health point of view is paramount, and dark-field preparations should be examined concurrently with the stained films for chancroid. THE VENEREAL DISEASES 487 2. Films from buboes—Inguinal lymph node enlargement occurs in approximately 50 per cent of patients with chancroid. The nodes become acutely inflamed, confluent, very painful, and eventually suppurate. If untreated, they become fluctuant and rupture spon- taneously, producing a large crateriform lesion infected with a variety of bacteria, Films prepared from these lesions are of little or no diagnostic value. Microscopical examination of pus from fluctuant buboes should be performed when possible. The pus must always be removed by aseptic aspiration. Spread on clean glass slides and apply Gram’s stain, Careful search for the morphologically characteristic Gram- negative rods is required because aspirated pus rarely yields more than a few organisms. However, when they are seen intra- and extracellu- larly, they may be considered to be Ducrey’s bacilli, particularly when similar microorganisms have been observed in the primary genital lesions. 3. Cultures—Inoculate culture media before preparing the films. This is especially important when seeding the culture media with aspirated bubo pus. B. Microbiology Following the isolation and description of H. ducreyi by Ducrey over fifty years ago, only sporadic attempts have been made to study the microbiology of this organism. By far the greater number of these studies have been directly associated and concurrent with attempts to improve cultural methods. Reymann?®! stated that Himmel in 1901 was among the first to employ heat-inactivated clotted rabbit blood as a culture medium. Teague and Diebert in 19203 used such a medium and recovered 140 morphologically “positive” mixed cultures from 274 cases of penile ulcer. Attempts to grow the organism on blood agar plates were reported in 1920 by Moore, who obtained positive results in 5 of 55 cases? In the succeeding decade the cultural method was seldom successful, and the view expressed in 1935 by Cole and Levin®? that “cultivation of the Ducrey bacillus is a most difficult procedure” was generally accepted. In 1938 Greenblatt and Sanderson®* found that the organism could be grown with moderate success in whole blood. Beeson and Hey- man in 1945% reported the use of defibrinated rabbit blood for pri- mary isolation and obtained positive cultures in 42 of 50 cases. Lind- berg et al. in 195138 reported excellent results employing 50 per cent tryptose phosphate rabbit serum broth. They found this medium 488 THE VENEREAL DISEASES especially useful in promoting formation of the characteristic parallel chains, or the so-called “railroad tracks.” The recent studies of Deacon®’ show promise in overcoming some of the inadequacies of previously described cultural methods. He reported impressive results using fresh clotted blood, which is in direct contrast to the long-accepted method of inactivating blood prior to use. In his hands a virulent bubo culture was found to grow in either fresh or inactivated blood, human or rabbit. On the other hand, avirulent stock cultures grew only in inactivated blood, and best in rabbit blood. In 18 cases of clinical chancroid, H. ducreyi was isolated in pure culture from fresh clots, whereas heavy contamina- tion was evident in every case when inactivated blood was used. Transfers from fresh clot cultures grew readily on solid substrates containing blood when incubated in candle jars. Conversely, subcul- tures from inactivated blood clots were never possible. Cultural methods 1. Fresh Clot Method of Deacon et al.®—Clot media are pre- pared as follows : Human or rabbit blood is drawn aseptically and dis- pensed in sterile, cotton-stoppered tubes (13 X 100 mm) in approxi- mately 3 ml quantities. Clots are considered to be fresh if used within 2 hr. When human blood is used, it is furnished by the patient, Material for culture is obtained from the lesion after it has been cleansed by rinsing with sterile salt solution and has been wiped with several changes of salt solution-moistened gauze. Inoculum is obtained from the base of the cleansed lesion using a small wire loop, and inoculation of the fresh clot consists of inserting the loop into the serum surrounding the formed clot, Cultures are incubated for 72 hr at 35° C. Observations are made at 24 hr intervals when sub- cultures and Gram-stained films are prepared. Subcultures may be made (1) on 1.5 per cent extract agar (CM No. 4) containing 15 per cent defibrinated rabbit blood in petri plates, or (2) in sterile de- fibrinated rabbit blood on slants of extract agar (CM No. 4). Trans- fers from positive clot cultures will grow readily on either of these media. The blood agar plates must be incubated in a candle jar. Characteristics and behavior of newly isolated cultures—The fol- lowing identifying features are among those observed in all cultures: a) Colonies grown on extract agar (1.5%) enriched with 15 per cent rabbit blood and incubated in a candle jar at 35° C for 48 hr appear slightly gray by reflected light and measure from 2 to 5 mm THE VENEREAL DISEASES 489 in diameter. Frequently colonies show some evidence of a rough surface and when touched with an inoculating loop tend to remain intact. b) Morphology of individual bacteria may vary considerably. Ex- tremes include coccoid forms and filamentous types. In general, stained preparations of colonies usually reveal a few short-chain “streptobacilli,” but often rods of various lengths and widths are observed. The familiar “streptobacillus” morphology, described as being typical of H. ducreyi, is best demonstrated in defibrinated rabbit blood extract agar (1.5%) slants. ¢) On blood agar plates none of the colonies display evidence of hemolytic activity. However, in fresh or inactivated clot culture and in defibrinated blood on agar slants, hemolysis is regularly noted after 24 hr. d) Conditions obtained by candle jar incubation appear to be opti- mal for growth of H. ducreyi in petri plates. The bacilli cultured in tubes fitted with screw caps grow as well as those in the plate cultures. Whether this is the result of decreased oxygen or increased carbon dioxide or a combination of both has not been determined. e) Peptone, agar and sodium chloride are necessary for satisfactory cultivation, 2. Method of Sanderson and Greenblatt®®—Agar slants in 15 mm-size culture tubes prepared with either extract (CM No. 3) or infusion broth (CM No. 5) with a pH between 7.4 and 7.6 are used as a base medium. Prior to inoculation, 1 ml of sterile defibrinated human or preferably rabbit blood is pipetted onto each slant. Loop scrapings or swabbings from a well-cleansed ulcer are inoculated directly into the blood. Incubation is at 35° C in a candle jar and cultures should not be discarded as negative until after 4 days. 3. Method of Beeson and Heyman®—This method requires only defibrinated rabbit blood dispensed in 1 ml quantities in 13 X 100 mm pyrex tubes, which may be stored in a refrigerator at 4° C until used. Material from primary genital lesions and aspirated buboes can be cultured in this medium, using a small amount of inoculum. Cul- tures are rarely pure, but the characteristic morphology of H. ducreyi serves to identify it when films are stained by Gram’s method. 4, Method of Lindberg®*—Tryptose phosphate broth (CM No. 35) with 50 per cent rabbit or human serum added is placed in 2 ml amounts in Kahn test tubes (12 X 75 mm). The tubes are inoculated 490 THE VENEREAL DISEASES with material from either primary genital lesions or aspirated buboes in the manner previously described. Cultures are incubated at 35° C in a candle jar until positive findings are obtained, or for 3 days. Re- sults obtained by this method closely parallel those of the Beeson- Heyman method, but it has the advantage that expensive rabbit serum may be replaced by pooled Seitz-filtered human serum recovered from serological laboratory discards. This results in a practical substrate which is available in any routine clinical laboratory. C. Intradermal Test 1. Ito-Reenstierna Reaction:***" For the diagnosis of chan- croid, this method employs an intradermal inoculation of H. ducreyi vaccine. In patients with chancroidal infection, a focal reaction of erythema and induration is considered evidence of a positive reaction. Although opinions have varied as to the sensitivity and specificity of the test, killed suspensions of Ducrey bacilli have been employed ex- tensively as diagnostic aids and have been generally recognized as useful. However, a positive reaction can be indicative of either present or postinfection. Hence, it should never be used alone in determining the diagnosis of chancroid, and it must always be per- formed concurrently with the Frei test for lymphogranuloma venereum, The application of the intradermal test is further complicated by the curtailment of commercially prepared antigens, Until recently, a prepared antigen (Lederle) was available. Until such time as com- mercial products are again available, prospective users must rely on homemade preparations.*! 2. Preparation of Skin Test Antigen*>—The method is as fol- lows: Pipette 1 ml of sterile defibrinated human blood onto the sur- face of a beef infusion agar (CM No. 6) slant and inoculate a stock culture of H. ducreyi into the blood. Heat the side of the tube gently and close with a rubber stopper to reduce the oxygen tension. Incu- bate at 35° C for 3 days, after which remove the blood and lake in 25 ml of sterile distilled water. Following centrifugation the sedi- ment is washed twice or until free of hemoglobin. Add to the sedi- ment of each tube 10 ml of physiological salt solution and kill the sus- pension of bacilli by heating at 60° C for 40 min. The suspension may be preserved with either 0.3 per cent phenol or 1:10,000 merthiolate. Interpretation will be guided by the following: A positive reaction to an intradermal injection of 0.1 ml of antigen is manifested by an THE VENEREAL DISEASES 491 area of induration surrounded by an erythematous halo at the site of injection. The induration should measure 7 mm in diameter in posi- tive cases. Greenblatt and Sanderson? have reported that occasion- ally the induration may be minimal but that in such cases the erythema must measure at least 14 mm in diameter to be considered positive. Any reaction which is smaller in size is considered doubtful and the test should be repeated after several days. Strong reactions some- times display a central pustular area. Generalized reactions have not been observed. Frank negative reactions show nothing but the trau- matic effect of injection. D. Lesion Biopsy The laboratory diagnosis of chancroid is sometimes difficult to ac- complish. The reliability of the film method is questionable, and cultural procedures are usually successful only when precise and exacting bacteriological methods are applied by experienced workers. The skin tests are dubious, and especially so when both the Frei and Ducrey tests are positive. The accurate differentiation of chancroid and lymphogranuloma venereum is fraught with difficulties. Conse- quently, it is usually necessary to perform the multiple methods de- scribed: films, cultures and skin tests. It has been stated** that if only one method is to be employed, the histopathological diagnosis of biopsied ulcer tissue is the most satis- factory. The histologic appearance of chancroid is sufficiently char- acteristic to permit reasonably accurate diagnosis. In patients with unusual or slowly healing lesions, biopsy is indicated in order to rule out the presence of neoplastic disease. Ill. GRANULOMA INGUINALE Granuloma inguinale is generally considered to be a venereal disease in view of its predilection for the genital area, frequent association with other venereal diseases, and occurrence in the age group in which venereal disease most commonly occurs, Question has been raised from time to time, however, whether this infection is truly a venereal disease in view of its rare occurrence among sexual contacts exposed to patients who have active lesions in which the causative agent Donovania granulomatis could be readily demonstrated. The etiological agent originally designated as the “Donovan body” was described first by Donovan, who mistakenly considered it to be a protozoan organism. It has been postulated by some workers that the disease results from an autogenous bacterial infection with fecal 492 THE VENEREAL DISEASES organisms belonging to the genera Aerobacter*® or Klebsiella.” Bergey's Manual of Determinative Bacteriology*® formerly described Klebsiella granulomatis, which was believed by Aragio and Vianna*? to be the etiologic agent of this disease. A more recent edition of this manual®® no longer mentions K. granulomatis. Serological studies®®51 have shown that members of the genus Klebsiella share an antigen in common with D. granulomatis now accepted as the specific infectious agent.’>5% The microorganism is now designated in Bergey's Manual®™ as a separate genus in the family Brucellaceae (formerly Parvobacteriaceae). Isolation of D. granulomatis is made by yolk sac culture from in- fected tissue. D. granulomatis may be cultivated in various yolk media as well as in chick embryos®:5% but will not grow on unenriched media. After cultivation in artificial media, however, D. granulomatis will not reproduce the lesion of granuloma inguinale.’® Inoculation of tissue®” and of pseudo bubo pus®®?® produced typical lesions in human volunteers, thus introducing some element of doubt that the true etio- logical agent has been cultivated artificially, The specific diagnostic significance of the “Donovan body” in films from infected tissue, how- ever, is unquestionable. Intradermal and complement-fixation tests have been the subject of study, and a high degree of sensitivity and specificity has been re- ported when suitable antigens are employed in complement-fixation tests.?0:60-62 Antigens for complement-fixation tests are equally effec- tive whether prepared from certain strains of Klebsiella pneumoniae or from D. granulomatis.?’ Serological tests and those for dermal sensitivity are of greater value for investigating the epidemiology of the disease than for routine laboratory diagnosis. For this reason subsequent discussion will be limited to a consideration of the use of films and biopsy sections in diagnosis. A. Films 1. Collection of specimen—The source of material is commonly the granulomatous lesion. Because secondary infection is almost uni- versal in the lesion and may obscure the agent, an area of the lesion should be cleaned with gauze to remove surface-contaminating micro- organisms. If the lesion is soft and friable, pressure of the flat surface of a clean slide upon fresh granulomatous areas usually re- sults in the adherence of sufficient tissue cells to permit identification of the microorganism. A more satisfactory procedure is to obtain a thin film of granulation tissue by scraping the surface of the lesion THE VENEREAL DISEASES 493 with the short edge of one glass slide, then spreading a thin film of the accumulated granulation tissue on another slide. The lesions are usually painless and such a procedure does not cause discomfort. A similar preparation, although somewhat less satisfactory, may be obtained by crushing a particle of the granulation tissue between two slides, or by rubbing the cut surface of biopsied tissue upon a slide after absorbing the blood with gauze. The film is stained by Wright's method. In rare cases where a subcutaneous abscess or a bone abscess is suspected, a thin film of pus should be spread on a slide and stained with Wright's stain in the usual manner. 2. Staining—After fixation the film is stained by Wright's" method. Better results are usually obtained with overstaining, that is, staining 3 min with the undiluted stain followed by a period of & min with the addition of water to the stain. Those familiar with the appearance of the microorganisms have no difficulty in identifying them in properly prepared films. 3. Microscopical examination—In scrutinizing the slide for D. granulomatis after staining, search should be made for either of the two morphological forms which the microorganisms manifest, What is believed to be the more immature phase of the microorganism is represented by the so-called “closed safety pin” forms, resulting from the characteristic blue bipolar staining of condensations of chromatin. The microorganism tends to bulge in the center and taper toward the ends, although frequently the cell wall is absent on one side between the bipolar masses of chromatin, making the microorganism appear as a curved rod with clubbing at both ends. This form of the micro- organism may lie free in the cytoplasm of large mononuclear cells or may be enclosed in cysts containing many pleomorphic forms. Frequently the microorganisms are surrounded by an unstained halo. A second form of the organism encountered in the cytoplasm of large mononuclear cells is observed in which the periphery of the halo stains from pink to purplish black, producing the appearance of a capsule around the microorganism, This form is more distinctive and should be sought whenever possible, inasmuch as the “safety pin” forms may resemble other bacteria (some believe that a positive diag- nosis should not be made if only the “safety pin” form is observed). Diligent search will usually reveal the encapsulated forms also.®® The encapsulated form is from 1 to 2 p in length and is oval in appear- ance. It bears some resemblance to the Leishman-Donovan bodies of leishmaniasis but lacks the eccentric nucleus and parabasal body of the 494 THE VENEREAL DISEASES latter. The encapsulated microorganism may be observed in clusters within the cytoplasm, frequently in cysts; or as separate micro- organisms distributed throughout the cytoplasm. Although such microorganisms may occasionally be encountered extracellularly, diagnosis should be based upon the presence of intracellular micro- organisms. In lesions of long standing, where epithelization of the surface has occurred and collagenous fibers have produced a firm, often keloid- like lesion, the organisms may be difficult to find, requiring removal of granulation tissue from beneath the surface in order to prepare a satisfactory film. B. Tissue Sections 1. Obtaining specimen—A biopsy of granulation tissue is obtained in the usual manner. Such specimens are less satisfactory than a film for identification of the microorganism, owing to its intra- cytoplasmic position, which is readily obscured when more than one layer of cells is visualized. 2. Staining—While those who are thoroughly familiar with the appearance of D. granulomatis stained with hematoxylin and eosin can identify it readily, the inexperienced will usually fail to observe it. For this reason, the examination of tissue sections stained with hematoxylin and eosin and examined in a routine manner will fre- quently miss the diagnosis. Silver impregnation employed by Dieterle®* for visualizing Treponema pallidum was adapted by Pund and Greenblatt®® for demonstrating D. granulomatis in tissue sections and is superior for tissue section diagnosis. 3. Microscopical examination—Employing the silver impreg- nation method?® the cell outline, nucleus, and cyst walls of the large mononuclear cells stain a pale yellow, while the Donovan bodies ap- pear as elongated ovoid forms with intense brown or black staining. Using this method, the microorganisms are more prominent and easier to identify than when stained with hematoxylin and eosin. The histo- pathology of the granuloma reveals, in addition to the numerous characteristic large mononuclear cells, many plasma cells and poly- morphonuclear leukocytes with a paucity of the small lymphocytes so characteristic of the primary lesion of syphilis. From the standpoint of identifying accurately the etiological agent with minimum effort, the biopsy section method is inferior to examination of films stained by Wright's method, as described previously. THE VENEREAL DISEASES 495 IV. NONGONOCOCCAL URETHRITIS IN THE MALE The great frequency with which urethritis occurs among males in the absence of the gonococcus®®-% necessitates further etiological studies. The disease is often designated as “nonspecific” urethritis, but in the absence of the gonococcus a more accurate terminology is “nongonococcal” urethritis, Because a specific etiology of non- gonococcal urethritis has not been verified, although it is thought to be microbial in origin, diagnostic studies must be made for several types of infectious agents. Microbiological studies of the urethral discharge from such cases of urethritis are justified and are necessary for a correct therapeutic regimen for the patient. Culturing a urethral exudate from a case of nongonococcal urethritis may yield practically a pure culture of a single organism or a predominating species in a mixed culture. Often, the clinical symptoms disappear when the observed organism is eliminated from the urethra by chemo- or anti- biotic therapy. The resolution of the etiology of nongonococcal urethritis, however, remains an important venereal disease study which is not without pitfalls. In view of the necessity to detect multiple infectious agents in non- gonococcal urethritis, a special method for the collection and exami- nation of specimens cannot be recommended. No specific serological or intradermal tests for the diagnosis of nongonococcal urethritis are available. A. Microorganisms Found in the Disease The following list tallies the microorganisms most frequently iso- lated from nongonococcal urethral exudates. Their significance in the etiology of the disease is as yet uncertain, . Chlamvdozoon oculogenitale88 . Coliform organisms . Diphtheroids . Hemophilus species89,70 Pleuropneumonia-like organisms?,72 Proteus species Pseudomonas species? . Staphylococci™ . Streptococci, alpha and gamma®6,73 . Trichomonas vaginalisT4-76 COON UTA LN — B. Examination for T. vaginalis 1. Collection of specimens—The external meatus is cleaned with 70 per cent alcohol, and the urethra is stripped to obtain dis- charge at the meatal orifice. 496 THE VENEREAL DISEASES 2. Preparation for examination—A clean microscope slide is touched to the drop of discharge and a loopful of physiological salt solution immediately mixed with it, or the discharge may be trans- ferred by a sterile platinum loop to a clean slide and mixed with a loopful of salt solution. A clean cover glass is placed over the material and the specimen is then examined under the microscope using the low-power objective to detect the presence of the large (up to 30 pn in length) actively motile flagellate. Films of the urethral discharge may be stained with Giemsa’s stain and examined microscopically for bacteria and for cellular content. 3. Cultures—Occasionally 7. vaginalis may be cultured when not observed microscopically in clinical material.” A drop of the discharge is transferred with a sterile cotton swab to a tube containing 10 ml of casein hydrolysate medium (CM No. 11), is thoroughly mixed, and is incubated at 35° C. A drop of the culture is examined microscopically after 24 and 48 hr. If the microorganisms are not observed, the entire culture is centrifugated for 15 min at 1,200 rpm and the sediment examined microscopically. Many media have been proposed and are suitable for cultivating T. vaginalis. In recent years the composition and preparation of media have been somewhat simplified. However, serum continues to be a necessary component of all media.™ C. Pleuropneumonia-like Organisms Directions for the detection of pleuropneumonia-like organisms are described in Chapter 22. D. Hemophilus Species In recent years Leopold” has described an organism closely related to Hemophilus which he isolated from cases of prostatitis with or without urethritis. Gardner and Dukes®® have isolated an organism designated Hemophilus vaginalis from cases of vaginitis as well as from the male urethra. The authors presented evidence to show that the infection was transmitted by sexual contact. Discharge from the urethral meatus or from the cervix is streaked directly onto Bacto proteose No. 3 agar containing 10 per cent de- fibrinated sheep blood. Freshly voided urine from the male may be centrifuged and the sediment similarly cultured. The cultures are incubated 48 hr in an atmosphere containing 10 per cent COs or in a candle jar. Subcultures should be made into thioglycolate broth if the organ- ism is to be maintained more than 2 days. THE VENEREAL DISEASES 497 E. Staphylococci and Streptococci Staphylococci, especially Staph. albus, are often present in the an- terior male urethra as part of the normal flora and are isolated fre- quently from urethral exudates. Alpha, beta and gamma types of streptococci may be isolated. The beta types occur less frequently than the alpha and gamma types. Most of the beta streptococci encountered belong to Lancefield’s Group D. Streptococcus fecalis is frequently observed. Detailed procedures for the identification of streptococci are given in the chapter on streptococci. The isolation of staphylococci and streptococci is made on blood agar (CM No. 16). F. Diphtheroids Like the staphylococci, diphtheroids are present occasionally as part of the normal flora of the anterior male urethra. They may be isolated on ordinary blood agar (CM No. 16) and recognized as species of Corynebacterium by their Gram-positive staining reaction and characteristic morphology. G. Coliform Organisms Coliform organisms may constitute a part of the normal bacterial flora of the anterior male urethra. They may also be isolated from nongonococcal urethral exudates. Special media are not required, as they grow readily on blood agar plates routinely employed for primary cultures of urethral exudates. Identification is made by sub- culturing the colonies to triple sugar agar (CM No. 53 or 53a) and by carrying out standard biochemical tests as may be required. H. Proteus and Pseudomonas Species Proteus organisms will be recognized on the plates inoculated with the original specimen by the characteristic swarming of the growth over the surface of the fresh, moist medium. More specific differ- entiation may be made by performing a urease test. Pseudomonas species are recognized by the “beaten copper” appear- ance of the colony surface, the soluble bluish green pigment, and the sweetish odor. The characteristic bluish green pigment is best de- tected by growing the organisms in extract broth or agar and mixing 1 ml of chloroform with the culture. The pigment is readily soluble in chloroform. 498 THE VENEREAL DISEASES J. Viruses Attempts to isolate a virus as the causative agent of nongonococcal urethritis have been unsuccessful. The demonstration by either Giemsa’s or Macchiavello’s stain of red-staining inclusion bodies within epithelial cells from the anterior urethra, however, has created interest in viral etiology. Specimens to be stained for inclusion bodies are best obtained by drawing the end of a sterile platinum spatula over the urethral mucosa and spreading the material on a glass slide, CuARLES M. CArPENTER, M.D., D.V.M.,, Pu.D., Chapter Chairman Harry E. Morton, Sc.D. Hexry Packer, M.D., Dr.P.H. Arvey C. Sanpers, Lt. Cor, U. S. ARMY REFERENCES 1. Leany, A. D., and CarPENTER, C. M. The Diagnosis of Gonococcal Infec- tions by the Cultural Method. Am. J. Syph. 20:347, 1936. 2. CarpeNTER, C. M., and CHArLES, R. Isolation of Meningococcus from the Genitourinary Tract of Seven Patients. A.J.P.H. 32:640, 1942. 3. CaArPENTER, C. M. The Isolation of Neisseria flava from the Genitourinary Tract of Three Patients. Proc. N. Y. State Assoc. Pub. Health Labs. 22(1) : 4, 1942. 4. WiLkinsoN, A. E. Occurrence of Neisseria Other Than the Gonococcus in the Genital Tract. Brit. J. Ven. Dis. 28:24, 1952. 5. Stuart, R. D,, TosuacH, S. R., and Parsura, T. M. The Problem of Transport of Specimens for Culture of Gonococci. Canad. J. Pub. Health 45:73, 1954. 6. BeakLEY, J. W. The Toxicity of Wooden Applicators for Neisseria gonorrhea. Pub. Health Lab. 29:11, 1957. 7. Arrison, S. D., Cuaarcres, R., and CARPENTER, C. M. The Survival Time of the Gonococcus in Urine from Male Patients with Urethritis. J. Ven. Dis. Inform. 23:283, 1942. 8. Buck, T. C, Jr. A Transport Medium for Neisseria gonorrhea. J. Ven. Dis. Inform. 28:6, 1947. 9. HirscuBerG, N. Use of Solid Medium for Transportation of Specimens for Gonococcus Culture. Am. J. Syph. 29:64, 1945. 10. Prizer, L. R., SteFrEN, G. I, and KrEIN, S. Simple and Efficient Transport Method for Gonorrheal Specimens. Pub. Health Rep. 64:299, 1949. 11. TacGArT, S. R. Gonorrhea Detection by Urine Examination. Pub. Health Rep. 70:245, 1955. 12. Manual of Microbiological Methods. Prepared by the Committee on Bac- terological Technic, Society of American Bacteriologists, New York: McGraw-Hill, 1957, p. 16. 13. CArPENTER, C. M,, et al. Evaluation of Twelve Media for the Isolation of the Gonococcus. Am. J. Syph. 33:164, 1949. 14. THAYER, J. D., Scuusert, J. H., and Buccs, M. A. The Evaluation of Culture Mediums for the Routine Isolation of the Gonococcus. J. Ven. Dis. Inform. 28:37, 1947. THE VENEREAL DISEASES 499 15. 16. 37. 18. 19. 20. 21. 22. 23. 24. 26. 27. 28 29, 30. 3, 32. 33. 34. 35. 36. 37. 38. Prrzer, L. R., and SterreEN, G. I. A Modification of the Horse Plasma Hemoglobin Agar for Primary Culture of the Gonococcus. Usefulness of Nile Blue A in This Medium. J. Ven. Dis. Inform. 23:224, 1942. CarpPENTER, C. M,, and Morton, H. E. An Improved Medium for the Isolation of the Gonococcus in 24 Hours. Proc. N. Y. State Assoc. Pub. Health Labs. 27:58, 1948. Lankrorp, C. E., and Sw~eLr, E. E. Glutamine as a Growth Factor for Certain Strains of Neisseria gonorrhoeae. J. Bact. 45:410, 1943. GrirrIN, P. J., and Racker, E. The Carbon Dioxide Requirement of Neisseria gonorrhoeae. J. Bact. 71:717, 1956. StokiNGeEr, H. E., AckerMAN, H., and CarpenTER, C. M. The Use of Tyrothricin in Culture Medium as an Aid in the Isolation of Neisseria gonorrhoeae. J. Bact. 45:31, 1943. De Boynton, E. Personal communication, Los Angeles County Health Department, 1950. CArPENTER, C. M,, SunrLAND, L. G., and Morrison, M. The Oxalate Salt of P-Aminodimethylaniline, an Improved Reagent for the Oxidase Test. Science 105 :649, 1947. CarpPENTER, C. M,, and HucHEes, R. P. Studies on Gonococcal and Non- gonococcal Urethritis among Troops in the Pacific Theater. Bull. U. S. Army Med. Dept. 7(8) :660, 1947. CARPENTER, C. M. Gonococcal Resistance to Penicillin in the Light of Recent Literature. WHO Bull. 4:321-326, 1961. Saunpers, R. L., and May, M. M. Penicillin-Resistant Gonorrhea. M. Clin. North America 29: 688, 1945. . Franks, A. G. Successful Combined Treatment of Penicillin-Resistant Gonorrhea. Am. J. M. Sc. 211:553, 1946. Hucues, R. P., and CarpenTER, C. M. Alleged Penicillin-Resistant Gonorrhea. Am. J. Syph. 32:265, 1948. Epstein, E. Failure of Penicillin in Treatment of Acute Gonorrhea in American Troops in Korea. J.A.M.A. 169 :1055, 1959. Bergey's Manual of Determinative Bacteriology (7th ed.). Baltimore, Md. : Williams & Wilkins, 1957: (a) p. 451; (b) p. 418. Boak, R. A. Studies on the Etiology of Penile Ulcers. Professional Report of the 406th Medical General Laboratory, 1955, pp. 198-198d. Teacug, O., and DieBert, O. The Value of the Cultural Method in the Diagnosis of Chancroid. J. Urol. 4:543-550, 1920. RevymaNN, F. A Study of the Growth Conditions of Haemophilus ducreyi. Acta path. et microbiol. scandinav. 24:208-212, 1947. Moore, J. E. The Diagnosis of Chancroid and the Effect of Prophylaxis upon Its Incidence in the American Expenditionary Forces. J. Urol. 4:169- 176, 1920. Core, H. N,, and Levin, E. A. The Intradermal Reaction for Chancroid with Chancroid Bubo Pus. J.A. M.A. 105:2040, 1935. GreenNBLATT, R. B., and Sanperson, E. S. The Intradermal Chancroid Bacillary Antigen as an Aid in the Differential Diagnosis of the Venereal Bubo. Am. J. Surg. 41:384-392, 1938. Beeson, P. B, and Heyman, A. Studies on Chancroid: Efficiency of the Cultural Method of Diagnosis. Am. J. Syph. 29:633-640, 1945. LinpBerG, R. B,, et al. Culture of Hemophilus ducreyi. Ann. Hist. Rep., 406th Medical General Laboratory (Tokyo), 1951. Deacon, W. E. Personal communication, 1955. SaxpersoN, E. S., and Greensratr, R. B. The Cultivation of H. ducreyi and Preparation of an Antigen for Intravenous Diagnosis. South. M. J. 30:147-149, 1937. 500 THE VENEREAL DISEASES 39. Ito, T. Klinische und Bakteriologische-Serologische Studien iiber Ulcus Molle and Ducreysche Streptobazillen. Arch. Dermat. u. Syph. Orig. 116: 341, 1913. 40. ReEnsTIERNA, J. Research on Bacillus ducreyi: Antiserum for Soft Chancre—Skin Reaction in Diagnosis. Arch. Dermat. u. Syph. Orig. 147: 362, 1924. 41. Karran, W.; Deacon, W. E.; OLANSKY, S.; and ArusritroN, D. C. VDRL: Chancroid Studies. III. Use of Ducrey Skin Test Vaccines on Rabbits. J. Invest. Dermat. 26 :415-419, 1956. 42. GrapwonL, R. B. H. Clinical Laboratory Methods and Diagnosis. St. Louis: C. V. Mosby, 1948, pp. 1494-1495. 43. GreenBLATT, R. B. and SanpersoN, E. S. Intracutaneous Test for Chancroid Infection: A Comparison of Antigens. J. Georgia M. A. 27 :218-219, 1938. i 44. Diagnostic Procedures and Reagents (3rd ed.). New York: American Public Health Assn, 1950, p. 304. 45. DonovaN, C. Ulcerating Granuloma of the Pudenda. Indian M. Gaz. (Calcutta) 40:414-418, 1905. 46. DEMonsreUN, W. A., and Gooprasturg, E. W. Further Studies on the Etiology of Granuloma inguinale. Am. J. Trop. Med. 13:447-470, 1933. 47. RaAkE, G. The Antigenic Relationships of Donovania granulomatis ( Ander- son) and the Significance of this Organism in Granuloma Inguinale. Am. J. Syph. 32:150-156, 1948. 48. Breep, R. S., Murray, E. G. D., and HircHENS, A. P. Bergey's Manual of Determinative Bacteriology (6th ed.). Baltimore, Md.: Williams & Wilkins, 1948, p. 459. 49. ARAGAO, DE BEAUREFAIRE H., and VIANNA, G. Pesquizas Sobre o Granuloma Venero. Mem. Inst. Oswaldo Cruz, Rio de Jan. 4:211-217, 1912. 50. DuraNEey, A. D., and Packer, HENry. Complement Fixation Studies with Pus Antigen in Granuloma Inguinale. Proc. Soc. Exper. Biol. & Med. 65:254, 1947. 51. GorpBerG, J. Studies on Granuloma inguinale. ITI. The Antigenic Hetero- geneity of Donovania granulomatis. Am. J. Syph. 38:330, 1954. 52. AnpersoN, K. The Cultivation from Granuloma Inguinale of a Micro- organism Having the Characteristics of Donovan Bodies in the Yolk Sac of Chick Embryos. Science 97 :560-561, 1943. 53. ——————, DEMonereuN, W. A., and GooprasTURE, E. W. An Etiologic Consideration of Donovania granulomatis Cultivated from Granuloma inguinale (3 Cases) in Embryonic Yolk. J. Exper. Med. 81:25-38, 1945. 54. Dienst, R. B,, GreensrATT, R. B,, and CHEN, C. H. Laboratory Diagnosis of Granuloma Inguinale and Studies on the Cultivation of the Donovan Body. Am. J. Syph. 32:301-306, 1948. . Duraxey, A. D., Guo, Kopa, and Packer, HENRY. Donovania granulomatis: Cultivation, Antigen Preparation, and Immunologic Tests. J. Immunol. 59:335-340, 1948. 56. Dienst, R. B.,, Caen, C. H., and GreensrATT, R. B. Experimental Studies of the Pathogenicity of Donovania granulomatis. Am. J. Syph. 33:152- 157, 1949. 57. McIntosH, J. A. The Etiology of Granuloma Inguinale. J.A. M.A. 87:996- } 1000, 1926. 58. Dienst, R. B.,, GREENBLATT, R. B.,, and SanpersoN, E. S. Cultural Studies on the Donovan Bodies of Granuloma inguinale. J. Infect. Dis. 62:112-114, 1938. 59. GreeNBLATT, R. B.; Dienst, R. B.; Punp, E. R.; and TorpiN, R. Ex- perimental and Clinical Granuloma inguinale. J. A.M.A. 113:1109-1115, 1939. [951 ut THE VENEREAL DISEASES 501 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72 73. 74. 73. 76. 77. 78. 79. AnpersoN, K., GooorasTUrg, E. W., and DEMonBrEUN, W. A. Immunolo- gic Relationship of Donovania granulomatis to Granuloma inguinale. J. Exper. Med. 81 :41-50, 1945. Packer, Henry, and GorLpBerG, Jurius. Complement Fixation Studies in Granuloma inguinale. Am. J. Trop. Med. 30:387-395, 1950. GOLDBERG, Jurius, WEAVER, R. H. and Packer, Henry. Studies on Granuloma Inguinale. II. The Complement Fixation Test in the Diagnosis of Granuloma Inguinale. Am. J. Syph. 37:71-76, 1953. CANNEFAX, GEeORGE. The Technic of the Tissue Spread Method for Demonstrating Donovan Bodies. J. Ven. Dis. Inform. 29:201-204, 1948. Dieterte, R. R. Method for Demonstration of Spirochacta pallida in Single Microscopic Sections. Arch. Neurol. & Psychiat. 18:73-80, 1927. Punp, E. R., and GReenBLATT, R. B. Granuloma venereum of Cervix uteri (Granuloma inguinale) Simulating Carcinoma. J.A.M.A. 108:1401-1402, 1937. CARPENTER, C. M., and Hugues, R. P. Non-Gonococcal Urethritis in the Male. Recent Advances in the Study of Venereal Diseases. A Symposium published by the Venereal Disease Education Institute in cooperation with the USPHS, 1948. Basiong, R. W,, and GramaM, R. S. Non-Gonococcal Urethritis in the Navy. Am. J. Syph. 36:480-482, 1952. Brisou, J. Non-Gonococcal Urethritis. A presentation at the International Union against the Venereal Diseases and Treponematoses, Monaco, 1954. GaroNEr, H. L., and Dukes, C. D. Haemophilus vaginalis Vaginitis. Am. J. Obst. & Gynec. 69:962-976, 1955. LeoroLp, S. Heretofore Undescribed Organism Isolated from the Geni- tourinary System. U. S. Armed Forces M. J. 4:263-266, 1953. Morton, H. E, Smita, P. F., and KEeLLER, R. Prevalence of Pleuro- pneumonia-Like Organisms and the Evaluation of Media and Methods for Their Isolation from Clinical Material. A.J.P.H. 42:913-925, 1952. SHEPARD, M. C. The Recovery of Pleuropneumonia-Like Organisms from Negro Men With and Without Non-Gonococcal Urethritis. Am. J. Syph. 38:113-124, 1954. WAGNER, B. M.,, Morse, W. H., and Kuans, D. M. Recent Studies on Nonspecific Urethritis. A.J.P.H. 43:853-859, 1953. Durer, P.; RorroN-RaTnNEr, V.; Sisourer, A.; and Sorer, C. Non- gonococcal Urethritis. Brit. J. Ven. Dis. 30:69-72, 1954. Durer, P., and SiBouLret, A. Non-gonococcal Urethritis in Males. A presen- tation at the Symposium on Non-gonococcal Urethritis organized by the International Union against the Veneral Diseases and Treponematoses, Monaco, 1954. Fro, L. G. The Incidence and Significance of Trichomonas vaginalis In- festation in the Male. Am. J. Trop. Med. 24:195-198, 1944. LasH, J. J, and Bert, E. A Cultural Method for the Diagnosis of Tri- chomonad Infestations. Am. J. Obst. & Gynec. 57:980-983, 1949. LasH, J. J. A Simplified Casein Hydrolysate Serum Medium for the Culti- vation of Trichomonas vaginalis. Am. J. Trop. Med. 30:641-642, 1950. Serince, H., and KupperBerG, A. B. The Nutrition of Protozoa. II. The Separation of Human Blood Serum into Two Fractions, Both Essential for the Sustained Growth of Trichomonas vaginalis. J. Bact. 53:441-447, 1947. CHAPTER 18 SYPHILIS I. Serological Tests for Syphilis . Selection of Tests Nontreponemal Tests Treponemal Tests . Biologic False-Positive Reactions Anticomplementary Reactions Procedures To Aid Interpretation of Serological Results . Cerebrospinal Fluid Examination . Rapid Reagin Tests VDRL Tests . One-Fifth Volume Kolmer Test . Quantitative Determination of Cerebrospinal Fluid Protein FRAC IQEHUNER 11. Microscopical Examination for 7. pallidum A. Dark-Field Examination 1. Collection and Submission of Specimens 2. Examination 3. Interpretation of Results References I. SEROLOGICAL TESTS FOR SYPHILIS The earlier hope! that the effectiveness of penicillin in curing syphilis would bring about control and the relegation of this disease to one of minor public health significance has not been realized. The downward trend in this disease observed subsequent to 1947 has leveled off and in recent years disturbing increases in syphilis includ- ing early disease have been noted. In view of these circumstances and of the continued importance of venereal syphilis and other treponematoses as public health problems in the foreseeable future, the maintenance of serological testing services is clearly indicated and warranted. Until 1949 the serology of syphilis was based on the use of lipid antigens. The isolation in 1941 of cardiolipin and the demonstration of reactivity when combined with lecithin and cholesterol? constitute the most significant achievement during this period. This contribu- tion was widely adapted to a variety of flocculation and complement- fixation serodiagnostic tests for syphilis. 502 SYPHILIS 503 In contrast to the progress that had been achieved with respect to lipoidal antigens, leading to their world-wide use, early experiences with treponemal antigens had been most disappointing. These ex- periences not only restricted their use but tended to discourage re- search in this area. The discovery of the immobilization of virulent Treponema pallidum by specific antibody in 19492 and the isolation of a protein antigen from the Reiter treponeme in 1953* again focused attention on the potentialities of treponemal antigens in the serodiag- nosis of syphilis. A. Selection of Tests With the availability of many tests using lipoidal, cardiolipin or treponemal antigens,’® a question naturally arises about the selec- tion of appropriate procedures for the clinical and public health laboratory. Rising costs for laboratory tests and demands for laboratory services not concerning the venereal diseases are factors which presently tend to influence this selection. A routine which would provide adequate serodiagnostic results for syphilis promptly, economically and with the minimum use of personnel would be most desirable. The authors have selected for inclusion in this chapter a group of tests, both reagin and treponemal, which appear to meet most of the current needs for syphilis serology. They are not unanimously agreed on details of methodology, including formulation of the cardio- lipin antigens. The proposed methods represent a decision of the majority rather than a consensus. Both experience and wisdom would dictate the continued employment of reagin* tests, even though we are more than a decade into the treponemal antigen test era. Some of the more important points leading to this decision might be enumerated here: 1. Reagin tests represent highly standardized, highly reproducible, widely used and relatively inexpensive tests. 2. They may be used both qualitatively and quantitatively, When used quantitatively, they are of value in following treatment, repre- senting a major criterion both for cure of early syphilis and for the determination of congenital syphilis in the newborn. 3. Present lack of knowledge precludes the use of treponemal tests as guides for treatment and cure. * The term “reagin” refers to antibodies that react with cardiolipin-lecithin- cholesterol antigens. 504 SYPHILIS 4. Reagin tests are better understood and interpreted by physicians than are the various treponemal procedures. 5. They are the earliest nonspecific indicators of certain chronic diseases and potentially may be developed as community diagnostic test procedures for chronic disease control programs. 6. For general use, none of the current treponemal tests offers significant advantage over the reagin tests for clinical purposes with two exceptions: (1) in differentiating biologic false-positive (BFP) reactions versus those related to treponemal infection, and (2) in indicating syphilitic origin of late sequelae of patients whose sera no longer react in reagin tests. B. Nontreponemal Tests For these reasons adequate laboratory serodiagnosis of syphilis should include reagin testing. To accomplish this most economically, consideration should be given to the use of the rapid reagin tests®89-12 as screen tests. Such tests, made possible by the use of unheated plasma, were originally designed to fulfill the need for rapid and economical screening of large numbers of persons in field studies of migrant groups, as they permitted immediate, on-the-spot, specific and prophylactic treatment of reactors. The principle of the original rapid plasma reagin (RPR) test? has been applied to serum. The rapid reagin test, using unheated serum,® is particularly adaptable as a screening procedure in a large laboratory. Those specimens re- active (R)* or weakly reactive (WR) with the screen test may then be tested with a more conventional reagin procedure, with quantita- tion of those that are reactive in the conventional tests, Although a variety of excellent tests for reagin are available,® the VDRLY slide test is included in this chapter because of its wide use and ease of performance. Furthermore, antigen suspensions for the rapid reagin tests are prepared from the same stock alcoholic solutions used for the VDRL test. C. Treponemal Tests Specimens giving reactive or weakly reactive results with the screen test should be routinely examined with a readily performed treponemal test, particularly when obtained from individuals not *To conform to nomenclature recommended by the National Advisory Serology Council and used in Serologic Tests for Syphilis, PHS Publication No. 411 (Revised 1959), the authors use the terms “Reactive” (R), “Weakly Reactive” (WR), and “Nonreactive” (N) in substitution for Positive (4), Weakly Positive or Doubtful (+), and Negative (—). 1 Venereal Disease Research Laboratory. SYPHILIS 505 previously diagnosed. Additional or supplemental treponemal testing should be undertaken only after proper steps have been taken to insure maximal usefulness of results. (See Heading F in this sec- tion.) The last decade has witnessed the development of numerous treponemal tests.>® These tests have demonstrated a multiplicity of antibodies occurring in syphilis infection and have presented new aids to serum diagnosis. Evaluation studies of the treponemal tests have indicated that they are neither absolutely specific nor infallibly indicative of past or present disease.!® Despite their various limita- tions, however, utilization of treponemal tests appears justified under proper circumstances. From all the treponemal tests available, the Reiter protein comple- ment-fixation (RPCF) or related Kolmer test with Reiter protein (KRP) has been selected because it represents a procedure most readily adaptable by the public health laboratory, since the technic is familiar and the cost is low. Antigen for this test is derived from the Reiter treponeme according to a procedure originally described by D’Alessandro.* The organisms are easily grown in culture, and rela- tively good standardization of the antigen may be effected. Approved commercial antigen is available from several sources. The sensitivity and specificity of this test as revealed by the SERA study® and other studies!*15 were satisfactory There may be situations where the clinician has need of other treponemal tests. This will seldom be the case when reagin and Reiter tests are in agreement. Practically all disagreement observed will be reagin-reactive, Reiter nonreactive, as specimens selected for testing by the Reiter procedure are those which react to a reagin test. Such discrepancies will most certainly occur. In some instances this type of discrepancy will be correlated with previous adequate treatment of the patient, as the Reiter antibody appears more sensitive to the effects of treatment than do other treponemal antibodies in general. In experimental syphilis of rabbits under certain conditions the results of reactive reagin and TPIT tests with a nonreactive RPCF test repre- sent our earliest serological manifestation of biological cure after treatment in these animals. On the other hand, some of the non- reactive Reiter results could represent inadequate standardization of the antigen (blocking agents in the serum) or improper test perform- ance. In any event, it is felt that current research will improve and clarify the situation considerably. It must be kept in mind that no test procedure is perfect or without certain deficiencies and disadvantages; however, the disadvantages in- + Treponema pallidum immobilization. 506 SYPHILIS dicated here which apply to Reiter procedures are not of sufficient magnitude to deny employment of this treponemal test for the pur- poses and under the conditions here set forth. Under circumstances needing further serological testing to clarify the status of the indi- vidual patient there are public health, university and commercial laboratories offering additional tests such as the fluorescent trepone- mal antibody (FTA)® test, Treponema pallidum immobilization (TPI)®1617 and T. pallidum complement-fixation (tpcf 50) tests.’ Recently the Reiter strain of treponeme has been used successfully in place of the Nichols strain in the fluorescent treponemal antibody test; thus a treponeme easily cultivated in vitro can be substituted for one requiring costly in vivo maintenance.!8 Knowledge of criteria for proper selection, use and interpretation of such procedures is primarily a responsibility of the physician rather than the laboratorian; however, both should be aware of the proper steps to be taken usually before requesting a treponemal test (see Heading F in this section). Such steps are essential to exploit the maximal usefulness of the various treponemal tests. Proper test selection, as well as interpretation of the results, may well depend upon the information so obtained. In all cases it will not be possible to obtain such information, but a sincere effort to do so is the mark of an informed and able clinician. D. Biologic False-Positive Reactions By a BFP, the authors mean the presence of serologically detectable reagin in persons who do not have or who have not had syphilis or a related treponematosis (yaws, pinta, bejel, etc.). It is helpful to consider BFP reactions as falling into one of two categories: 1. Acute—relatively transient (duration less than 6 months) and occurring in patients who are ill, taking certain drugs, recently vaccinated or possibly with certain other conditions not yet concisely evaluated. 2. Chronic—those occurring in patients or persons who are apparently well, with a duration beyond 6 months to life. The types of illnesses which may give rise to an acute BFP reaction are found among certain bacterial, viral, protozoal, rickettsial, spiro- chetal and other conditions as noted above. One must remember to inquire of those individuals with moderate to severe hypertension whether they are or have recently been taking hydralazine hydro- chloride. In a certain proportion of people taking this drug, BEP’s may be expected. Hypertensives most likely to be taking this drug are those with: moderate to severe essential hypertension at relatively fixed levels; early malignant hypertension; hypertension after sym- SYPHILIS 507 pathectomy; and hypertension associated with acute glomerulone- phritis and toxemia of pregnancy. In addition to being associated with illness, the most important other criterion of the acute BFP reaction is its transient nature. Also the titers usually tend to be low. In most instances the physician will recognize these acute BFP reactions when they occur during or shortly following one of the aforementioned conditions. In almost all cases, the reagin test will re- vert to nonreactivity within a period of six months without syphilo- therapy. Serological follow-up of the patient is recommended at monthly intervals with quantitatively titered tests until spontaneous reversal to nonreactivity. A falling titer during the follow-up period is to be expected. A rising titer is quite suspicious of syphilis and one must remember that the patient may have a BIFP reaction and also have acquired syphilis. A stationary titer during the serologic fol- low-up period would also suggest the possibility of concurrent syphilis or a chronic BFP reaction, which of course would have to be con- firmed. It should be kept in mind that a patient with a chronic BFP reaction may incidentally become ill with any of the above diseases. In most cases the chronic BFP reactor is an apparently well person with a reactive reagin test. A well person with a reactive reagin test is also what the physician sees when confronted by a case of latent syphilis. Chronic BFP reactions are characterized by the fact that they bear no relationship temporally to any known infectious disease except lepromatous leprosy; they do not disappear spontane- ously but persist for longer than six months—possibly years and even for the duration of the patient’s lifetime. The exact incidence and prevalence of this reaction is unknown, but guesses on scanty evidence suggest that such a reaction may be an- ticipated in approximately one out of every two to six thousand persons tested. Depending upon the prevalence of syphilis in the population tested, such a proportion of BFP reactions may or may not constitute a conspicuous problem. For example, if serological tests are performed on a particular population group having a reac- tivity rate of 15 per cent, as in a large, selective survey, the proportion of BFP reactors in this group would be minimal. On the other hand, when serologic tests are performed on patients from the upper socioeconomic and educational strata from the northern United States (in which the prevalence of syphilis is known to be low), the propor- tion of BEP reactors may be quite troublesome, Among such groups, it has been estimated, as many as 40 out of 100 reactors detected may indeed represent BFP reactors. 508 SYPHILIS Sufficient evidence has been developed to speculate that the chronic BFP phenomenon is one manifestation chiefly of collagen and vascu- lar tissue damage due to agents or antigens yet unknown. One of the first evidences of injury is dysgammaglobulinemia, eventually as- sociated in a rather large per cent of chronic BFP reactors with rheumatoid arthritis, systemic lupus erythematosus, or other collagen vascular disease and bizarre, usually serious, illnesses. A high in- cidence of BFP reactions among drug addicts is reported.l® E. Anticomplementary Reactions Anticomplementary reactions do not occur to any appreciable extent in a given laboratory using a standard procedure which experience has shown to be properly adjusted with respect to the various components of the test. An unusual number of anticomplementary findings can generally be traced to inferior reagents, unclean glassware, or failure to adhere to prescribed incubation temperatures. Anticomplementary reactions associated with a particular speci- men are caused by extrinsic or intrinsic factors. Among the ex- trinsic factors are bacterial contamination or the presence of chemicals such as soaps, acids, alkalies and anticoagulants. Little is known of the intrinsic factors involved beyond the demonstration that isolated human gamma globulin possesses anticomplementary properties.2® Related to this is the appreciable incidence of anticomplementary re- actions in conditions characterized in part by elevations in gamma globulin, such as cirrhosis of the liver, lymphogranuloma venereum, and lupus erythematosus. F. Procedures To Aid Interpretation of Serological Results The following steps and their order of presentation have been de- veloped particularly for the interpretation of a reactive reagin test for syphilis in a clinically negative patient. While it is not possible here to go into important details to be undertaken at each step, this information is available?! 1. Repeat of the test or tests—Mistakes do happen both in and out of the laboratory and it is essential to make sure that a first reactive test is confirmed, including quantitation if possible. 2. Repeat of the test, using cardiolipin antigen—This is usually applicable only where lipoidal antigens or highly sensitive screening procedures have been utilized for the initial observation. While only a few biologic false positives may be eliminated, this relatively simple task is worthy of recommendation as the second step in the diagnostic procedure. SYPHILIS 509 3. Adequate history—Here the clinician or professional V. D. in- terviewer must obtain detailed information as it might relate to syphilis, the treponematoses, the use of certain drugs, recent diseases or immunizations, etc., knowledge of which is so necessary to differ- entiate the persons with detectable reagin related to syphilis from those with serological activity unrelated to syphilis. 4. Physical examination—While initially we assumed that a patient is “clinically negative,” experience has shown that the patient may be “clinically negative” only because a careful examination, particu- larly pointed toward manifestations of syphilis, has not been made. 5. Other laboratory examinations—At this point the physician can readily determine what other tests available in the clinical laboratory may be appropriate for a given situation, depending upon the patient’s history and physical examination. For example, heterophil agglu- tination, skin tests, biopsies, albumin-globulin ratio and gamma globu- lin determination might be indicated. 6. Spinal fluid examination—If a diagnosis has not been reached at this point, the differential possibilities rest among asymptomatic neurosyphilis, latent syphilis and a chronic biologic false-positive re- action. To rule out the likelihood of asymptomatic neurosyphilis, a spinal fluid examination, properly carried out and including a cell count, total protein determination and a reagin test, is mandatory. 7. Epidemiological investigation—A professional V.D, investigator or physician should obtain for clinical examination and laboratory testing all available contacts (sexual and marital partners, siblings, parents and children). 8. Serological tests with treponemal antigens—If the diagnosis has not been resolved with the information obtained in the prior seven steps, treponemal tests should be run. Here again a reactive result should be repeated. The type of tests employed and the interpreta- tion of test results are much less likely to be in error, using the information obtained in this stepwise development. In many in- stances one will find often in retrospect that a valid diagnosis could have been reached without recourse to the Reiter or any other trep- onemal test. While the order of these eight steps may seem inconsistent with the suggestion that previously undiagnosed reagin reactors routinely be given a Reiter antigen test regardless of other considerations, there is no actual conflict because the results of the test should regularly be available for the clinician, on request, to assist him in studying the patient. 510 SYPHILIS Before undertaking the supplementary treponemal tests, which are complicated and costly, the following facts should be considered: 1. Reagin and Reiter nonreactive agreement practically excludes syphilis, except incubating and seronegative primary syphilis. 2. Reagin and Reiter reactive agreement, while indicating the presence of reagin and group-specific treponemal antibody, practically means syphilis in this country. 3. Reagin and Reiter disagreement should alert the physician immediately to the necessity of the type of intensive and somewhat specialized study of his patient proposed here. If he has not already undertaken such study, the disagreement should prompt him to do so without further delay, since in the absence of lesions he cannot possibly determine the significance of test results. Here an “informed guess” or intuition is no proper substitute for thorough, painstaking clinical work. From the preceding standpoint a nonreactive reagin (or trepone- mal) test does not necessarily exclude syphilis. Nor does a reactive reagin (or treponemal) test mean syphilis. Moreover, there is a long latent or “hidden” stage of syphilis during which the only manifesta- tion of the disease is a reactive serological test. Finally, reagin can be detected by serological tests in relation to certain diseases, drugs and immunizations ; following blood donations; or even in “normal” persons yielding biologic false-positive (BFP) reactions for syphilis in the absence of syphilis (discussed under Heading D in this section). G. Cerebrospinal Fluid Examination In the examination of spinal fluids three tests are considered obliga- tory by most syphilologists : cell count, total protein, and reagin tests. The cell count and total protein tests are nonspecific tests considered to reflect inflammation and degeneration within the central nervous system. The reagin test is, from a practical standpoint, pathognomic for syphilis. The cell count may be estimated using the Fuchs-Rosenthal cham- ber.2%23 Precautions to avoid nonessential admixture of blood cells should be taken. The cell count should be accomplished within an hour after the spinal fluid specimen has been obtained. The VDRL method for the quantitative determination of spinal fluid protein is included in this presentation because of its reproduci- bility and simplicity. Two technics for detecting reagin are given. The One-Fifth Volume Kolmer test may be used by those desiring complement fixation. For those preferring flocculation, the VDRL test with spinal fluid is described. Very limited studies on the use of treponemal tests with spinal fluids have been made.??> No recommendation regarding their use can therefore be made at this time. SYPHILIS 511 Colloidal tests of cerebrospinal fluid such as the colloidal gold test? have very limited value in the management of neurosyphilis. A first- zone curve definitely indicates parenchymatous involvement of the central nervous system. However, colloidal tests are of very little value in determining the activity of neurosyphilis, and in laboratories employing unstandardized technics, the results are valueless. Col- loidal tests, like spinal fluid reagin tests, may continue to show abnormal reactions for a long time after arrest or cure of neuro- syphilis. H. Rapid Reagin Tests®10:26-28 Equipment 1. Rotating machine, adjustable to 180 rpm, circumscribing a circle 34 in. in diameter on a horizontal plane. 2. Centrifuge, angle-head, Servall SS-1, type “XL” or equivalent.* 3. Tubes, stainless steel, 50 ml capacity, without flange. * Glassware 1. Micro test slide, Boerner.} 2. Disposable Pasteur capillary pipettes, 534 in. long.§ 3. Slides, 2 X 3 in., with paraffin rings approximately 14 mm in diameter. Reagents 1. Antigen (VDRL flocculation test antigen). 2. Saline solution, 1 per cent buffered (VDRL flocculation test buffered saline). 3. Phosphate (.02M), merthiolate** (2%) solution: Dissolve 1.42 g¢ Na,HPO,, 1.36 g KH,PO4, 1.00 g merthiolate in distilled water to a final volume of 500 ml. The pH of this solution should be 6.9. Store in the dark at room temperature. May be used for a period of 3 months. 4. Choline chloride solution (40%): (a) Dissolve 40 g choline chloride in distilled water to a final volume of 100 ml. (b) Filter and store at room temperature. May be used for 1 year. Refilter if visible particles form. * Jvan Sorvall Co., New York, N. Y. + Arthur H. Thomas, Philadelphia 5, Pa., Catalog No. 7049-M. § Scientific Products Co. Evanston, Ill, Catalog No. 67770; Harshaw Scientific Co., Cincinnati 13, Ohio, Catalog No. H55698. *#* Eli Lilly and Company, Indianapolis, Ind. 512 SYPHILIS 5. EDTA 0.1 M: Dissolve 3.72 g (ethylenedinitrilo) tetraacetic acid disodium salt to a volume of 100 ml in distilled water. May be used for 1 year. 6. Resuspending solution: To prepare 10 ml, combine the following ingredients— BOTA QF A. oviniin ris onmmisnes s Aas aamnis eso SuBuanens s spams 1.25 ml Choline chlorife TATE). oosvbdivns so slsnivame ve 3s 5 3 Spmamivas enamine 25 ml Phosphate (.02M) merthiolate (2%) ......covevinrnueennnnneennnn 5.0 ml DISHED WALEE . civwivs ws we immimimimintons « mins rmmnonin ssn ne mimrniis 4 5m aie Slaw 1.25 ml This solution is prepared freshly each time antigen suspensions are made. Preparation of Antigen Suspension 1) Prepare antigen emulsion as for the VDRL flocculation tests. 2) Centrifuge measured aliquots of the antigen emulsion in stainless steel tubes in an angle centrifuge at room temperature at a relative centrifugal force of approximately 2,000 G’s for 15 min. From 5 to 30 ml may be centrifuged in a single centrifuge tube. 3) Locate the sediment and decant supernatant by inverting the tube away from the side containing the sediment. While holding the tube in an inverted position, wipe the wall with cotton gauze, taking care not to disturb the sediment. 4) Resuspend with a volume of resuspending solution equal to that of the antigen suspension which was centrifuged. Blow solution directly onto the sediment. Agitate the centrifuge tube by hand to resuspend all sediment. 5) If more than one centrifuge tube is used, combine all re- suspended aliquots. This is the completed antigen suspension. 6) Check reactivity of antigen suspension by performing the rapid reagin test with control sera of graded reactivity. 7) Antigen suspension should be stored in a refrigerator and re- moved for daily use. It should be kept at room temperature for not less than 30 min before it is used. 8) Antigen suspension stored as described has been found to give satisfactory results for at least 6 months.?® However, if at any time the expected results with the graded control sera are not obtained, the antigen suspension should be discarded. Preparation of Control Sera—Although this test may be per- formed with plasma or serum, it is more convenient to prepare con- trols using sera. Serum dilutions are made in 0.85 per cent saline SYPHILIS 513 solution containing 0.01 per cent merthiolate. Dilutions of serum that produce reactive, weakly reactive, and nonreactive results in the RPR test should be selected by trial testing and maintained for daily use. These serum dilutions can be used over a period of 1 month if stored in the refrigerator. For daily use, they should be taken out and allowed to stand at room temperature for at least 30 min. Collection of Blood Specimens—To perform the RPR test on plasma, blood is collected in tubes containing an anticoagulant (heparin, potassium oxalate or potassium sequestrene). Vacuum tubes containing these substances are commercially available.* Potas- sium sequestrene is the preferred anticoagulant if blood is for sero- logical testing only.?? To perform the rapid reagin tests on unheated serum (USR), clotted blood is obtained. Preparation of Blood Specimens 1) Centrifuge blood specimens at room temperature at a force suffi- cient to separate the plasma or serum from the cellular elements. Generally, 1,500 to 2,000 rpm for 4 min is adequate. 2) Allow plasma or serum to remain in the original collecting tube. Note: The specimens are tested without heating. If specimens have been refrigerated, they should stand at room temperature for at least 30 min before testing. Preliminary Testing of Antigen Suspension—Antigen suspen- sion should first be examined with control sera of known reactivity by the appropriate method described under the heading which follows. Only those suspensions that have given the designated reactions in tests performed with control sera should be used. Performance of Rapid Reagin Qualitative Tests—For use in the field, for example to test a group of migrant laborers, the technic employing unheated plasma and disposable pipettes (RPR) is recom- mended. For use as a screen test in the public health laboratory, the technic employing unheated serum (USR) with conventional pipettes and paraffin-ringed slides is recommended. A. Performance of Rapid Plasma Reagin Test (RPR): 1) Withdraw plasma, using disposable pipette and being careful not to disturb cells. * Becton, Dickinson and Co., Rutherford, N. J.; Scientific Glass Instrument Co., Northfield, N. J. 514 SYPHILIS 2) Hold pipette in a vertical position and place 3 drops of the plasma in one concavity of a Boerner microtest slide. If drop size is too large, only 2 drops may be used to prevent spilling during rota- tion. If drop size is very small, more than 3 drops may be used. 3) Using disposable capillary pipette held in a vertical position, place 1 drop of antigen suspension on each plasma. Select a pipette that produces drops of moderate size. 4) Rotate slides for 4 min at 180 rpm. 5) Read tests immediately after rotation with a microscope at 100 X magnification. 6) Report as follows: Reactive (R) — Large clumps Weakly Reactive (WR) — Medium clumps Nonreactive (N) — Small clumps or less B. Performance of Unheated Serum Reagin Test (USR) : 1) Pipette 0.05 ml of unheated serum from the original collecting tube into one ring of a paraffin-ringed glass slide. 2) Add one drop (145 ml) of antigen suspension to each serum. Note: An 18 gauge needle without point which will deliver 45 drops per ml may be used. 3) Rotate slide for 4 niin at 180 rpm. 4) Read tests immediately after rotation with a microscope at 100 X magnification. 5) Report as described in Paragraph 4 (6) preceding. J. VDRL Tests VDRL Slide Flocculation Tests with Serum30:31 Equipment 1. Rotating machine, adjustable to 180 rpm, circumscribing a circle 34 in. in diameter on a horizontal plane. 2. Ringmaker, to make paraffin rings approximately 14 mm in diameter. 3. Slide holder* for 2 X 3 in. microscope slides. 4. Hypodermic needles of appropriate sizes, with or without points. * Catalog No. 66533, Scientific Products Company, Evanston, IIL SYPHILIS 515 Glassware 1. Slides, 2 X 3 in., with paraffin rings approximately 14 mm in diameter. Note: Glass slides with ceramic rings may also be used for the VDRL slide test, taking certain precautions. The rings must be high enough to prevent spillage when slides are rotated at prescribed speeds. Slides must be cleaned after each use so that serum will spread to the inner surface of the ceramic rings. This type of slide should be discarded if or when the ceramic ring begins to flake off, since these particles, in test sera, may be mistaken for antigen particle clumps, thereby leading to a false reactive report. 2. Bottles,} 30 ml round, screw-capped (Vinylite or tinfoil-lined) or glass-stoppered, with narrow mouth, Note: Some of the 30 ml glass-stoppered bottles now available are unsatisfactory for preparing a single volume of antigen emulsion for these tests due to an inward bulging of the bottom that causes the 0.4 ml of salt solution to be distributed only at the periphery. A satisfactory emulsion may be obtained if the 0.8 ml of salt solution covers the bottom surface of this type of bottle when double quantities of antigen emulsion are prepared. Round bottles of approximately 35 mm diameter with flat or concave inner bottom surfaces are satisfactory for prepar- ing single volumes of antigen emulsion. 3. Syringe, Luer type, 1 or 2 ml capacity. Caution: The low cost of plastic caps recommends against attempts to clean these for reuse. The use of an unclean stopper or cap can be the cause of unsatisfactory emulsions. Reagents Antigen: Antigen for this test is an alcoholic solution containing 0.03 per cent cardiolipin, 0.9 per cent cholesterol, and sufficient purified lecithin to produce standard reactivity. During recent years, this amount of lecithin has been 0.21 per cent == 0.01 per cent.* Each lot of antigen must be serologically standardized by proper comparison with an antigen of known reactivity. Antigen is dispensed in screw-capped (tinfoil or Vinylite liners) brown bottles or in hermetically sealed glass ampuls and stored at room temperature (73° to 85° I). The components of this antigen remain in solution at normal temperature so that any precipitate noted will indicate changes caused by such factors as evaporation or additive materials contributed by pipettes. Antigen containing precipitate should be discarded. + Catalog No. LG-1, MW-90525 (plain bottles) ; LG-1, MW-90530 (glass- stoppered bottles). Corning Glass Works, Corning, N.Y. * An otherwise identical antigen containing 0.27 per cent lecithin32.33 is preferred by some national and state agencies. 516 SYPHILIS Saline solutions: (1) A buffered salt solution containing 1 per cent sodium chloride should be used: Formalin, neutral, reagent Srade wu.ecesvss sessmsime sess same 0.5 ml Secondary sodium phosphate (Na,HPO,12H,0) ............... 0.093 g Primary potassium phosphate (KH,PO,) .......ccoovninnnann 0.170 g Sodium chlofide (A.C.8.) ..1ivemvivvecssmmmenn vd seaming os 100 g DUSTTICA SATE... vomiritimmmanvon hese smeeawsgs hehe [os i pared ost. fb medelleie 1,000.0 ml This solution yields potentiometer readings of pH 6.0 = 0.1 and is stored in screw-capped or glass-stoppered bottles. 2) For a 0.9 per cent saline solution, add 900 mg of dry sodium chloride to each 100 ml of distilled water. Preparation of Sera 1) Clear serum, obtained from centrifuged, clotted blood, is heated in a 56° C water bath for 30 min before being tested. 2) All sera are examined when removed from the water bath and those found to contain particulate debris are recentrifuged. 3) Sera to be tested more than 4 hr after the original heating period should be reheated at 56° C for 10 min. Preparation of Slides Clean 2 X 3 in. glass slides. Glass slides with concavities or glass rings are not recommended for this test. Paraffin rings are made by transferring heated paraffin to the slides by means of a hand-operated or an electrically heated ring-making machine, Care should be exercised to produce rings of the prescribed diameter, Preparation of Antigen Emulsion 1) Pipette 0.4 ml of buffered salt solution to the bottom of a 30 ml round glass-stoppered or screw-capped bottle. 2) Add 0.5 ml of antigen (from the lower half of a 1.0 ml pipette graduated to the tip) directly onto the salt solution while continuously but gently rotating the bottle on a flat surface. Temperature of buf- fered salt solution and antigen should be in the range of 23° to 29° C at the time antigen emulsion is prepared. Note: Antigen is added drop by drop but rapidly, so that approxi- mately 6 sec are allowed for each 0.5 ml of antigen. Pipette tip should remain in upper third of bottle and rotation should not be so vigorous as to splash salt solution onto pipette. Proper speed of rotation is SYPHILIS 517 obtained when outer edge of bottle circumscribes a circle 2 in. in diameter approximately 3 times per sec. 3) Blow last drop of antigen from pipette without touching pipette to salt solution, 4) Continue rotation of bottle for 10 more sec. 5) Add 4.1 ml of buffered salt solution from 5 ml pipette. 6) Place top on bottle and shake from bottom to top and back approximately 30 times in 10 sec. 7) Antigen emulsion is then ready for use and may be used during 1 day. Double this amount of antigen emulsion may be prepared at one time by using doubled quantities of antigen and salt solution. In this case, a 10 ml pipette should be used for delivering the 8.2 ml volume of salt solution. If larger quantities of antigen emulsion are required, more than one mixture should be prepared. These aliquots may then be tested and pooled. Testing antigen-emulsion delivery needles—It is of primary importance that the proper amount of antigen emulsion be used. For this reason the needle used each day should be checked. Practice will allow rapid delivery of antigen emulsion but care should be exercised to obtain drops of constant size. For use in the slide qualitative test and slide quantitative test A, antigen emulsion is dispensed from a syringe fitted with an 18 gauge needle without a point which will deliver 60 drops of antigen emulsion per ml when syringe and needle are held vertically. For use in the slide quantitative test B, antigen emulsion is dis- pensed from a syringe fitted with a 19 gauge needle without a point which will deliver 75 drops of antigen emulsion per ml when syringe and needle are held vertically. } When allowed to stand, antigen emulsion should be gently mixed before use by rotating the bottle. Preliminary testing of antigen emulsion—Each preparation of antigen emulsion should first be examined by testing sera of known reactivity in the Reactive, Weakly Reactive, and Nonreactive zones. This is accomplished by the method described under “VDRL Slide Qualitative Test with Serum,” the heading which follows. These tests should present typical results and the size and number of antigen particles in the Nonreactive serum should be optimum. Only those antigen emulsions that have produced the designated reactions in tests performed with control sera (Reactive, Weakly 518 SYPHILIS Reactive, and Nonreactive) should be used. If antigen particles in the Nonreactive serum tests are too large, the fault may be in the manner of preparing antigen emulsion, although other factors may be responsible. An unsatisfactory antigen emulsion should not be used. VDRL Slide Qualitative Test with Serum 1) Pipette 0.05 ml of heated serum into one ring of a paraffin-ringed glass slide. 2) Add one drop (¥%o ml) of antigen emulsion onto each serum. 3) Rotate slides for 4 min. (Mechanical rotators that circumscribe a circle 34 in. in diameter should be set at 180 rpm. Rotation by hand should circumscribe a circle 2 in, in diameter 120 times per min.) Note: Serum controls of graded reactivity (Reactive, Weakly Re- active, and Nonreactive) are always included during a testing period to insure proper reactivity of antigen emulsion at the time tests are run, Reading and reporting results—Read tests microscopically with low-power objective at 100X magnification. The antigen particles appear as short-rod forms at this magnification. Aggrega- tion of these particles into large or small clumps is interpreted as degrees of reactivity. Reading Report No clumping or very slight roughness = Nonreactive (N) Small clumps = Weakly Reactive (WR) Medium and large clumps = Reactive (R) Zonal reactions, caused by an excess of reactive serum component, are recognized by irregular clumping and the loosely bound character- istics of the clumps. The usual Reactive finding is characterized by large or small clumps of fairly uniform size. Experience will allow differentiation to be made between this type of reaction and the zonal picture wherein large and/or small clumps may be intermingled with free antigen particles. A zonal reaction is reported as “Reactive.” In some instances, this zoning effect may be so pronounced that a weakly reactive result is produced by a very strongly reactive serum. It is therefore recommended that all sera producing weakly reactive results in the qualitative test be retested using the quantitative pro- cedure before a report of the VDRL slide test is submitted. When a Reactive result is obtained in some dilution of a serum that pro- duced only a Weakly Reactive result as undiluted serum, the report is “Reactive.” SYPHILIS 519 VDRL Slide Quantitative Tests with Serum All sera that produce Reactive or Weakly Reactive results in the qualitative VDRL slide test should be quantitatively retested by one of the two methods referred to as quantitative tests A and B. Since both of these procedures in most instances provide for direct measure- ments of serum and salt solution, either method is efficient in terms of technician time and amount of glassware required. Since quanti- tative test A uses serum dillutions of 1:2.5, 1:5, 1:10, etc., the al- ternate quantitative test B has been added for those laboratories desiring the doubling serum dilution scheme of 1:2, 1:4, 1:8, 1:16, etc. VDRL Slide Quantitative Test A 1) Place four 2 X 3 in. glass slides containing twelve 14 mm para- fin rings in a five-place slide holder (see Fig 1). 2) Place a glass slide with two parallel strips of masking or ad- hesive tape in the center space of the slide holder. 3) Prepare a 1:10 dilution of each serum to be tested quantitatively by adding 0.1 ml of heated serum to 0.9 ml of 0.9 per cent salt solu- tion, using a 0.2 ml pipette graduated in 0.01 ml divisions. 4) Mix serum and salt solution thoroughly, then allow pipette to stand in test tube. 5) Using this 0.2 ml pipette, transfer 0.05 ml, 0.02 ml and 0.01 ml quantities of the 1:10 dilution of the first serum into the 4th, 5th and 6th rings, respectively. 6) With the same pipette, transfer 0.05 ml, 0.02 ml and 0.01 ml quantities of the first serum, undiluted, into the 1st, 2nd and 3rd ringed areas, as illustrated in Fig 1. ‘ 7) Repeat this procedure with each serum and the accompanying 1:10 serum dilution until each of the eight sera is pipetted onto the slides. 8) Add 1 drop (0.03 ml) of 0.9 per cent salt solution to the 2nd and 5th rings of each serum, by vertical delivery, from a 15 gauge hypodermic needle fitted to a glass syringe. Needles should be checked for proper drop size. 9) Add 1 drop (0.04 ml) of 0.9 per cent salt solution to the 3rd and 6th rings of all eight sera by vertical delivery from the syringe fitted with the 13 gauge needle. The six mixtures of each serum are then equivalent to dilutions of 1:1 (undiluted), 1:2.5, 1:5, 1:10, 1:25 and 1:50. 520 SYPHILIS Figure 1—VDRL Quantitative Tests: Illustration—Slides in Slide Holders Tabulation—Serum Dilutions Quantitative Test A Serum Serum (Un- Serum Dilutions diluted) Solution (ml) (ml) (ml) 0.05 0 1:1 (a) 0.02 0.03 1:25 (b) 0.01 0.04 1:3 (©) (Diluted 1:10) 0.05 0 1:10 (d) 0.02 0.03 1:25 (e) 0.01 0.04 1:50 (f) Quantitative Test B (Un- diluted) 0.04 0 1:1* (a) 0.02 0.02 1:2 “(b) 0.01 0.03 1:4 (c) (Diluted 1:8) 0.04 0 1:8 (d) 0.03 0.02 1:16 (e) 0.01 0.03 1:32 (f) 1 (a) 2 (a) 3 (a) 4 (a) 1 (b) 2 (b) 3 (b) 4 (b) 1 (c) 2 (0) 3 (0) 4 (c) 1 (d) 2 (d) 3 (d) 4 (d) 1- (2) 2 (e) 3 (e) 4 (e) I.) 2 (f) 3 (f) 4 (f) Numbers: ii slide holder show order of speci- mens. Squares in chart represent the 12 paraffin rings on each of the four slides. Numbers denote sera. Letters denote di- lutions. 5 a) 6 (a) 7 (a) 8 (a) 5 (b) 6 (b) 7 (b) 8 (by 5 (co) 6 (c) 7 (c) 8 (c) 5 (d) 6 (d) 7 (d) 8 (d) 5..(e) 6 (e) 7 (e) 8 (e) 5 (f) 6 (f) 7 {DH 8 (f) * That is, undiluted. SYPHILIS 521 10) Rotate slides gently by hand for about 15 sec to mix the serum and salt solution. 11) Add 1 drop (¥o ml) of antigen emulsion to each ring, using a syringe and needle as described in the technic for the slide qualitative serum test. This manner of preparing serum dilutions by adding serum and salt solution directly to the slides is outlined in Fig 1. 12) Complete tests by rotation of the slides in the manner pre- scribed under the heading “VDRL Slide Qualitative Test with Serum” preceding. 13) Read results microscopically. The highest serum dilution giv- ing a Reactive result (not Weakly Reactive) is reported as the re- activity end point of the serum, for example: Reactive, 1:25 dilution; or Reactive, 25 dilutions. 14) If all serum dilutions tested give Reactive results, prepare a 1:100 dilution of that serum by diluting 0.1 ml of the 1:10 serum dilution with 0.9 ml of 0.9 per cent salt solution. 15) Pipette 0.05 ml, 0.02 ml and 0.01 ml amounts of this 1:100 serum dilution onto each ring and add enough salt solution to bring volumes to 0.05 ml. Serum dilutions of 1:100, 1:250 and 1:500 are thus prepared. Test these dilutions of serum exactly as the lower dilutions are tested. VDRL Slide Quantitative Test B 1) Place four 2X3 in. glass slides with 12 paraffin rings in five-place slide holder (see Fig 1), with a numbered slide in the center space exactly as described previously. 2) Prepare a 1:8 dilution of each serum by adding 0.1 ml of the heated serum to 0.7 ml of the 0.9 per cent salt solution using a 0.2 ml pipette graduated in 0.01 ml divisions, 3) Mix the serum and salt solution thoroughly and then allow the pipette to stand in the test tube. 4) Using this pipette, transfer 0.04 ml, 0.02 ml and 0.01 ml quanti- ties of the 1:8 serum dilution into the 4th, 5th and 6th paraffin rings, respectively. 5) With the same pipette, transfer 0.04 ml, 0.02 ml and 0.01 ml of the undiluted serum into the 1st, 2nd and 3rd paraffin rings, respectively. 522 6) 7) 8) 9) 10) 11) 12) SYPHILIS Repeat this procedure with each serum and the accompanying 1:8 serum dilution until each of the eight sera are pipetted into their respectively numbered places on the slides. Add 2 drops (0.01 ml in each drop) of 0.9 per cent salt solution to the 2nd and 5th rings of each serum by vertical delivery from a 23 gauge hypodermic needle fitted to a glass syringe. Needles should be checked for proper drop size. Salt solutions may be delivered from a 19 gauge needle (0.02 ml per drop) and a 15 gauge needle (0.03 ml per drop). Add 3 drops of 0.9 per cent salt solution (delivered in the same manner) of the same size to the 3rd and 6th rings of each serum. Rotate slides gently by hand for about 15 sec to mix the serum and salt solution. Add 1 drop (Ys ml) of antigen emulsion to each ring, using a syringe and needle of appropriate size. (Caution: Note that the amount of antigen emulsion used in this method has been reduced to ¥5 ml to correspond with the reduced serum volume of 0.04 ml.) Complete tests in the manner described for the VDRL slide qualitative test with serum and read results microscopically im- mediately after rotation. By this method, the dilutions of each serum are 1:1 (undiluted), 1:2, 1:4, 1:8, 1:16 and 1:32. If all serum dilutions tested produce Reactive results, prepare a 1:64 dilution of that serum in salt solution. Add seven parts of saline solution to one part of the 1:8 serum dilution, and test in three amounts, as was done with the 1:8 serum dilutions. Dilu- tions prepared from the 1:64 dilution will be equivalent to 1:64, 1:128 and 1:256. Reading and reporting results—Read tests microscopically at 100X magnification as described for the qualitative procedure. Re- port results in terms of greatest serum dilution that produces a Re- active (not Weakly Reactive) result in accordance with the examples shown in Table 1. VDRL Tests with Spinal Fluid®* Equipment—XKahn shaking machine (must be operated at 275 to 285 oscillations per min). Reagents Antigen: VDRL slide flocculation test antigen, as herein described. SYPHILIS 523 Table 1—Laboratory Reporting Based on Test Results Method A Un- diluted Sey Serum Dilutions 1:1) 1:25 1:5 1:10 1:25 Report as— R WR N N N Reactive, undiluted only, or 1 dil* R R WR N N Reactive, 1:2.5 dilution, or 2.5 dils R R R WR N Reactive, 1:5 dilution, or 5 dils WR N N N N Weakly Reactive, undiluted only, or 0 dils WR R R WR N Reactive, 1:5 dilution, or 5 dils Method B 12 1:4 1:8 1:16 R WR N N N Reactive, undiluted only, or 1 dil R R WR N N Reactive, 1:2 dilution, or 2 dils RB R R WR N Reactive, 1:4 dilution, or 4 dils R=Reactive, WR =Weakly Reactive, N=Nonreactive. Note: Under conditions of high temperature and low humidity which sometimes occur during the summer months in certain areas, antigen emulsion may be stored in the refrigerator but should be brought to room temperature before use. To avoid surface drying under these conditions, tests should be completed and read as rapidly as possible. Slide covers containing a moistened blotter may be employed. * See section on reading and reporting quantitative test results for explanation of abbreviation. Saline solutions: (a) 1 per cent buffered salt solution. Prepare as for the VDRL slide flocculation tests. (b) 10 per cent sodium chloride solution. Dissolve 10 g of dry sodium chloride (A.C.S.) in 100 ml of distilled water. Preparation of spinal fluid 1) Centrifuge and decant each spinal fluid. Spinal fluids which are visibly contaminated or contain gross blood are unsatisfactory for testing. 2) Heat spinal fluid at 56° C for 15 min. Cool to room temperature before testing. Preparation of the sensitized antigen emulsion 1) Prepare antigen emulsion as described for the VDRL slide flocculation tests under “Preparation of Antigen Emulsion.” 2) Add one part of 10 per cent sodium chloride solution to one part of VDRL slide test emulsion. 524 SYPHILIS 3) Mix well and allow to stand at least 5 min but not more than 2 hr before use. VDRL Qualitative Test with Spinal Fluid 1) Pipette 1.0 ml of heated spinal fluid into a 13 X 100 mm test tube. Include Reactive and Nonreactive spinal fluid controls in each test run. 2) Add 0.2 ml of sensitized antigen emulsion to each spinal fluid. Resuspend the sensitized antigen emulsion immediately before use by inverting container several times. 3) Shake racks of tubes on Kahn shaking machine for 15 min. 4) Centrifuge all tubes for 5 min at a force equivalent to 1,800 rpm in No. 1, or 1,600 rpm in No. 2, I.LE.C.* centrifuge with hori- zontal heads. 5) Return tubes to Kahn shaking machine and shake exactly 2 min. Reading and reporting qualitative test results 1) Read test results as soon as possible after the secondary shaking period by holding tubes close to the shade of a desk lamp having a black background. Note: Each tube may be held motionless or shaken gently during the reading. Excessive agitation should be avoided. 2) Report results as follows: Reactive: Definitely visible aggregates suspended in a water-clear or turbid medium. All borderline reactions where the observer has doubt regarding visible clumping should be reported as Nonreactive. Nonreactive: No aggregation, complete dispersion of particles, ap- pearance turbid or slightly granular. Definite silken swirl on gentle shaking. VDRL Quantitative Test with Spinal Fluid Quantitative tests are performed on all spinal fluids found to be Reactive in the qualitative test. Prepare spinal fluid dilutions as follows: 1) Pipette 1.0 ml of 0.9 per cent sodium chloride solution into each of five or more tubes. 2) Add 1.0 ml of heated spinal fluid to tube 1, mix well, and transfer 1.0 ml to tube 2. * International Equipment Co., Boston, Mass. SYPHILIS 525 3) Continue mixing and transferring from one tube to the next until the last tube contains 2 ml. Discard 1.0 ml from the last tube. The respective dilution ratios are 1:2, 1:4, 1:8, 1:16, 1:32, etc. 4) Test each spinal fluid dilution as described for the VDRL quali- tative test with spinal fluid. Reading and reporting quantitative test results 1) Read each tube as described under the heading “VDRL Quali- tative Test with Spinal Fluid.” 2) Report test results in terms of the highest dilution of spinal fluid producing a Reactive result. The abbreviation “dils,” which ex- presses the same dilution reactivity end point, may be applied. As an example: Spinal Fluid Dilutions Report 1:2 1:4 1:8 1:3 N N N N N Reactive,* undiluted only, or 1 dil R R R N N Reactive, 1:8 dilution, or 8 dils R R R R N Reactive, 1:16 dilution, or 16 dils 6 1:32 R=Reactive, N=Nonreactive. * Reactive finding as with undiluted spinal fluid in the qualitative test. K. One-Fifth Volume Kolmer Test This small-volume test'415:23:35 conserves reagents and may be used with cardiolipin or Reiter protein antigens. Equipment—Test tube racks of galvanized wire, for 72 tubes. Glassware 1. Test tubes, pyrex, 12 X 75 mm outside dimensions. 2. Tubes, centrifuge, graduated, 15 ml capacity, pyrex. 3. Tubes, centrifuge, round-bottom, 50 ml capacity. 4. Pipettes, 0.25 ml or 0.5 ml capacity. Reagents Antigens: a) Cardiolipin antigen®® for the Kolmer tests is an alcoholic solution containing 0.03 per cent cardiolipin, 0.05 per cent lecithin, and 0.3 per cent cholesterol. Each new lot of this antigen should be tested in + An antigen containing 0.0175 per cent cardiolipin, 0.0875 per cent lecithin, and 0.3 per cent cholesterol32,33,37,38 is preferred by some national and state agencies. 526 SYPHILIS parallel with a standard antigen in both qualitative and quantitative tests on Reactive, Weakly Reactive, and Nonreactive sera before being placed in routine use. b) Reiter protein antigen is obtained by cryolysis and ammonium sulfate fractionation of a culture of Reiter treponemes.*38 Saline solution: 1) Weigh 8.5 g of dried sodium chloride (A.C.S.) and 0.1 g of magnesium sulfate or chloride crystals for each liter of salt solution. 2) Dissolve salts in distilled water. Freshly prepared salt solution should be used for each test run. 3) Place portion of salt solution sufficient for diluting complement, to be used for completing the tests, into refrigerator, allowing re- mainder to stand at room temperature (73° to 85° I). Sheep red cells (See Manual of serologic tests for syphilis.®) : Freshly collected sheep blood should be refrigerated for 48 hr before being used. Hemolysin (See Manual of serologic tests for syphilis.®) Complement serum (See Manual of serologic tests for syphilis.®) Preparation of Sera 1) Centrifuge blood specimens and separate serum from the clot by pipetting or decanting. 2) Heat serum at 56° C for 30 min. Previously heated sera should be reheated for 10 min at 56° C on day of testing. 3) Recentrifuge any serum in which visible particles have formed during heating. Preparation of Spinal Fluid 1) Centrifuge and decant all spinal fluids to remove cellular and particulate debris. Spinal fluids which are visibly contaminated or contain gross blood should not be tested. 2) Heat all spinal fluids at 56° C for 15 min to destroy thermolabile anticomplementary substances. Preparation of Sheep Red Cell Suspension 1) Filter an adequate quantity of preserved sheep blood through gauze into a 50 ml round-bottom centrifuge tube, SYPHILIS 527 2) Add 2 or 3 volumes of salt solution to each tube. 3) Centrifuge tubes at a force sufficient to throw down corpuscles in 5 min (I.E.C.* Centrifuge No. 1 at 2,000 rpm; I.E.C. Centrifuge No. 2 at 1,700 rpm). 4) Remove supernatant fluid by suction through a capillary pipette, taking off upper white cell layer. 5) Fill tube with salt solution and resuspend cells by inverting and gently shaking tube. 6) Recentrifuge tube and repeat the process for a total of three washings. If supernatant fluid is not colorless on third washing, cells are too fragile and should not be used. 7) After supernatant fluid is removed from third washing, cells are poured or washed into a 15 ml graduated centrifuge tube and centri- fuged at previously used speed for 10 min in order to pack cells firmly and evenly. 8) Read the volume of packed cells in the centrifuge tube and care- fully remove supernatant fluid. 9) Prepare a 2 per cent suspension of sheep cells by washing the corpuscles into a flask with 49 volumes of salt solution. Shake flask to insure even suspension of cells. Example: 2.1 ml (packed cells) x49=102.9 ml (salt solution required) 10) Pipette 15 ml of the 2 per cent cell suspension into a graduated centrifuge tube and centrifuge at previously used speed for 10 min. A 15 ml aliquot of a properly prepared cell suspension will produce 0.30.01 ml of packed cells. Caution: Use only centrifuge tubes that have been tested for proper calibration in 15 ml and cell-pack volume zones. Note: When the packed cell volume is beyond the tolerable limits given above, the cell suspension concentration should be adjusted. The quantity of salt solution which must be removed or added to the cell suspension to accomplish adjustment is determined according to the following formula: Actual reading of centrifuge tube . SHS .Of seaming X Volume of cell suspension Correct reading of centrifuge tube equals Correct volume of cell suspension * International Equipment Co., Boston, Mass. 528 SYPHILIS Example 1: Volume of cell suspension .......oovvvuvininnnnnn. 100 ml Centrifuge tube (15: ml) reading ..oivvuvsxainsinss 0.27 ml 0.27 ml X 100 ml = 90 ml 0.3 ml Therefore, 10 ml of saline solution should be removed from each 100 ml of cell suspension. Salt solution may be removed by centri- fuging an aliquot of the cell suspension and pipetting off the desired volume of salt solution for discard. Example 2: Volume of cell SUSPENSION ... vv. seus viniein aie sin sins 100 ml Cenirifuge tube (15 ml) reading ....cvvrsnseivasss 0.33 ml .33 ml 033 ml 100 ml = 110 ml 0.3 ml Therefore, 10 ml of salt solution should be added to each 100 ml of cell suspension. An adjusted cell suspension should be rechecked by centrifuging a 15 ml portion. Note: Place flask of cell suspension in refrigerator when not in use. Always shake before using to secure an even suspension, as the corpuscles settle to the bottom of the flask when allowed to stand. Preparation of Cardiolipin Antigen Dilution Place the required amount of salt solution in a flask and add anti- gen drop by drop while continuously shaking the flask. Rinse pipette. The amount needed may be calculated from the number of tubes con- taining antigen in the test and titrations. The test dose constitutes 0.1 ml of the antigen dilution indicated on the label of the bottle, which is usually 1:150.* Antigen dilution is kept at room temperature in a stoppered flask. The diluted antigen should stand at room temperature for at least 1 hr before it is used. Preparation of Reiter Protein Antigen Dilution3® 1) Place the required amount of salt solution in a flask or tube. * The corresponding dilution for an alternative antigen32.33,39 is 1:130. It is prepared by measuring salt solution and antigen into separate beakers, pouring salt solution onto antigen and mixing thoroughly by pouring from one beaker to the other ten times. SYPHILIS 529 2) Draw up 0.05 ml or more of Reiter protein antigen in the bottom half of a 0.1 ml pipette graduated to the tip, and add to the salt solution. (The test dose is 0.1 ml of antigen dilution, indicated on the bottle label, as determined by titration. The amount needed may be calculated from the number of tubes containing antigen in the test.) 3) Mix well by filling and emptying the pipette a few times in the diluted antigen solution. Preparation of Stock Hemolysin Dilution Prepare 1:100 stock hemolysin dilution as follows: SEI BOMIIBH. ov cwmcen mio ® © wives ocd SPS SE STR 8 ATT 0 oF ETAT 0% 94.0 ml Phenol solution (5% in saline solution) .........coouovennn. o- 4.0 ml Glycerinized hemolysin (B0%) vos vsiinsnnvsnis savin wvnss vs sssaines 2.0 ml Phenol solution should be mixed well with the salt solution before glycerinized hemolysin is added. This solution keeps well at refrig- erator temperature but should be discarded when found to contain precipitate, Each new lot of stock hemolysin dilution (1:100) should be checked by parallel titration with the previous stock hemolysin dilution before it is placed in routine use. Dilutions of hemolysin of 1:1,000 or greater are prepared by further diluting aliquots of the 1:100 dilution. After these reagents are prepared, the complement and hemolysin titrations may be assembled. Hemolysin and Complement Titrations 1) Place 10 tubes (numbered 1 to 10) in a rack. 2) Prepare a 1:1,000 dilution of hemolysin in tube No. 1 by adding 0.5 ml of the 1:100 stock hemolysin to 4.5 ml of salt solution, meas- uring from point to point in a 1 ml pipette. Discard pipette and, with a clean one, mix thoroughly, being sure to wash down all hemolysin solution adhering to the wall of the tube. 3) Pipette 0.5 ml of 1:1,000 hemolysin solution into tubes 2 through 5. 4) Add the following amounts of salt solution to tubes 2 through 10. Salt Solution Tube No. 2 3 4 5 6 7 8 9 10 Amount per tube (ml) 0.5 1.0 1.5 20 0.5 0.5 0.5 0.5 0.5 530 SYPHILIS 5) Proceed as follows: Final Hemolysin Tube No. Process Dilution 1 1: 1,000 2 Mix 1: 2,000 3 Mix and transfer 0.5 ml to tube 6 1: 3.000 4 Mix and transfer 0.5 ml to tube 7 1: 4,000 5 Mix and transfer 0.5 ml to tube 8 1: 5,000 6 Mix and transfer 0.5 ml to tube 9 1: 6,000 7 Mix and transfer 0.5 ml to tube 10 1: 8,000 8 Mix 1:10,000 9 Mix 1:12,000 10 Mix 1:16,000 6) Perform the hemolysin and complement titrations simultaneously in the same rack. 7) Use 12 X 75 mm tubes. Place 10 tubes (numbered 1 to 10) in one side of the rack for the hemolysin titration and 6 tubes (numbered 1 to 6) in the other side for the complement titration. 8) Pipette 0.1 ml of each of the hemolysin dilutions (1:1,000 to 1:16,000) into each of the corresponding 10 tubes of the hemolysin titration. 9) Prepare a 1:50 dilution of complement by adding 0.1 ml of guinea pig serum to 4.9 ml of salt solution measuring from point to point in a 0.2 ml pipette. Discard pipette. With a clean 1 ml pipette, mix thoroughly, being sure to wash down all serum adhering to the wall of the tube. 10) Pipette 0.1 ml of 1:50 complement into each of the 10 tubes of the hemolysin titration. 11) Add the following amounts of 1:50 complement and salt solution to the complement titration tubes. 1 2 3 4 5 6 Complement 1:50 (ml)* 0.25 0.20 0.15 0.12 0.10 0.0 Salt solution (ml) 0.25 0.30 0.35 0.38 0.40 0.50 * The complement dilution should be delivered to the bottom of the tubes. The quantity of 1:50 complement in the first two tubes may be measured with a 0.5 ml or 0.25 ml pipette, and in the last three tubes with a 0.2 ml pipette, SYPHILIS 531 12) Complete the hemolysin titration by adding 2 per cent sheep cell suspension and salt solution in the amounts and in the order indicated : Sheep Red Cell Hemolysin, Complement, Suspension Salt Tube No. 0.1 ml 1:50 (2%) Solution (ml) (ml) (ml) 1 1: 1,000 0.1 0.1 04 2 1: 2,000 0.1 0.1 0.4 3 1: 3,000 0.1 0.1 04 4 1: 4,000 0.1 0.1 04 5 1: 5,000 0.1 0.1 0.4 6 1: 6,000 0.1 0.1 0.4 7 1: 8,000 0.1 0.1 04 8 1:10,000 0.1 0.1 04 9 1:12,000 0.1 0.1 04 10 1:16,000 0.1 0.1 04 13) Shake each tube of hemolysin titration to insure even distribution of cells and place rack containing the two titrations in the 37° C water bath for 1 hr. 14) Remove rack from water bath and read hemolysin titration. The unit of hemolysin is the highest dilution that gives complete hemolysis. 15) Prepare a quantity of diluted hemolysin containing 2 units per 0.1 ml sufficient for the complement titration as shown in the following: Highest Dilution Giving Complete Dilution That Stock Hemolysin Saline Hemolysis Will Contain Solution, 1:100, Solution (1 u./0.1 ml) 2 u./0.1 ml Required Required (ml) (ml) 1: 4,000 1:2,000 0.1 1.9 1: 5,000 1:2,500 0.1 24 1: 6,000 1:3,000 0.1 2.9 1: 8,000 1:4,000 0.1 3.9 1:10,000 1:5,000 0.1 4.9 1:12,000 1:6,000 0.1 5.9 1:16,000 1:8,000 0.1 7.9 16) Add 0.1 ml of diluted hemolysin (containing 2 units of hemoly- sin) to each of the first five tubes of the complement titration. 17) Add 0.1 ml of 2 per cent sheep red cell suspension to each tube of the complement titration. 532 SYPHILIS 18) Shake each tube of the complement titration to insure even dis- tribution of cells and return rack to the 37° C water bath for 15 hr. The complete complement titration is shown in the tabulation below : Sheep Red Complement Salt Cell Sus- 1:50% Solution Hemolysin ~~ pension Tube No. (ml) (ml) Incubation 2 1) (2%) Incubation (ml) (ml) 1 0.25 0.25 0.1 0.1 1 hr 2 0.20 0.30 1 hr in 0.1 0.1 secondary 3 0.15 0.35 37° C 0.1 0.1 incubation 4 0.12 0.38 water 0.1 0.1 n37 C 5 0.10 0.40 bath 0.1 0.1 water 6 0.0 0.50 None 0.1 bath * All measurements should be made to the bottom of the tube. The quantity of diluted complement in the first two tubes may be measured with a 0.5 ml or 0.25 ml pipette and in the last three tubes with a 0.2 ml pipette. The smallest amount of 1:50 complement giving complete hemolysis is the exact unit. For use in the test, complement is diluted so that 2 exact units are contained in 0.2 ml quantities, Example: FN0EL 0 rin te thr wrt mndes hans wok Sipe Jame 0 3p I Rr 8 18 AE 0.15 ml Two exact wnls (AOS), «+ 1 oimmumien om wun save cibioe o's be 0.30 ml Complement dilution used in titration ................. 1:50 ml The dilution of complement to be employed in the test proper may be calculated by dividing 50 by the dose and multiplying by the volume in which the dilution is to be contained, that is, 50 —X 0.2 = 33, 0.3 or a 1:33 dilution of guinea pig serum. Other examples may be given as follows: Exact Unit Two Exact Units Test Dose (ml) (ml) 0.25 0.5 0.2 ml of 1:20 dilution 0.2 04 0.2 ml of 1:25 dilution 0.15 0.3 0.2 ml of 1:33 dilution 0.12 0.24 0.2 ml of 1:42 dilution 0.10 0.2 0.2 ml of 1:50 dilution SYPHILIS 533 Qualitative Tests with Serum and Spinal Fluid 1) Arrange 12 X 75 mm test tubes in wire racks so that there are two tubes for each serum or spinal fluid to be tested. Control sera of predetermined reactivity must be included. Number the first row of tubes to correspond to the serum or spinal fluid being tested. Three additional test tubes are included for reagent controls (antigen, hemo- lytic system, and corpuscle). 2) Prepare a 1:5 dilution of each serum by adding 0.2 ml of serum to 0.8 ml of salt solution. Mix well. Spinal fluid is tested undiluted. 3) Complete the One-Fifth Volume Kolmer test. Comple- Sheep Test Subst Salt Aus (2 Hemol- Cell 5 SHDsiance Solu- fii Shaking Hen Incubation ysin (2 Suspen- and Tube No. ton gen exact units) sion units) (2%) erum (1:5) I ( 3 ( #1 (test) :0.2 None 0.1 0.2 0.1 0.1 #2 (control) :0.2 0.1 None 0.2 0.1 0.1 g . Shake rack Incubate 15- pinal fluid well. Allow 18 hr at 6°- #1 (test) 0.1 0.1 01 to stand at) 45 (10° C fol-| gy 0.1 #2 (coniroD) 0.1 02 None [room tem-] 3 flowed by 107 go 0.1 perature for min in a 37° eagent controls 10-15 min. water bath. Antigen 0.2 0.1 0.2 0.1 0.1 Hemolytic system 0.3 None 0.2 0.1 0.1 Corpuscle 0.6 None J L None | L None 0.1 Note.—All quantities are in ml. 4) Mix contents of tubes thoroughly and return to 37° C water bath. The period of secondary incubation will be determined by the length of time necessary to reproduce the predetermined reactivity pattern of the control serum. In all instances the reading time should be at least 10 min more than the time necessary to hémolyze the anti- gen and hemolytic system controls and should not exceed a total in- cubation time of 60 min. 5) Remove each rack of tubes from the water bath at the end of the secondary incubation period and immediately place in an ice bath. Using reading standards, read and record observed hemolysis. Report 534 SYPHILIS the results of the qualitative test as indicated below, except in those in- stances where inhibition of hemolysis is noted in the control tube: Test Control Tube Tube Reading Reading Report as— 4 - Reactive 3 — Reactive 2 — Reactive 1 — Reactive = — Weakly reactive — — Nonreactive 4 4 Anticomplementary 4 3 Anticomplementary 4 2 Weakly reactive 4 1 Reactive 3 3 Anticomplementary 3 2 Anticomplementary 3 1 Weakly reactive 3 = Reactive 2 2 Nonreactive 2 1 Nonreactive 2 = Weakly reactive 1 Nonreactive gh ve = Nonreactive All sera and spinal fluids showing inhibition of hemolysis in the control tube should be returned to the 37° C water bath. Read con- trol tubes at 5 min intervals and record test results when complete hemolysis is observed in the control tube (not longer than 1 hr of secondary incubation). Record test results in accordance with pre- ceding tabulation. Quantitative Tests with Serum and Spinal Fluid 1) Place 12 X 75 mm test tubes in racks, allowing eight tubes for each serum and six tubes for each spinal fluid to be tested. Reagent control tubes are the same as for the qualitative tests. SYPHILIS 535 2) For each serum, pipette 0.2 ml of salt solution into tubes No. 2 through No. 7 and 0.1 ml into tube No. 8. 3) Pipette 0.2 ml of serum (diluted 1:5) into tubes 1, 2 and 8. 4) Mix the contents of tube 2, transfer 0.2 ml to tube 3, and so on to tube 7. Mix contents of tube 7 and discard 0.2 ml. 5) For each spinal fluid, pipette 0.3 ml of salt solution into tube 1, 0.2 ml into tubes 2 through 5, and 0.1 ml into tube 6. 6) Pipette 0.3 ml of spinal fluid into tube 1, mix well, and transfer 0.2 ml to tubes 2 and 6. Mix tube 2, transfer 0.2 ml to tube 3, and so on to tube 5. Mix tube 5 and discard 0.2 ml. 7) Add 0.1 ml of diluted antigen to the first seven tubes of each serum test and to the first five tubes of each spinal fluid test. 8) Add 0.2 ml of diluted complement to all tubes, 9) Shake the racks to mix thoroughly and place in the refrigerator at 6° to 10° C for 15-18 hr. 10) Complete tests the following morning, as indicated for the qualitative tests. 11) The end-point titer is the highest dilution giving a Reactive re- sult. With both sera and spinal fluids, the first tube is considered to be undiluted, or 1 dilution. Additional dilutions may be pre- pared and tested if no end point is obtained. Retesting of Anticomplementary Sera Anticomplementary sera may be retested by preparing serial two- fold dilutions in salt solution as described for the quantitative test, beginning with the 1:5 dilution and ending with 1:80. Each dilution of serum in 0.2 ml amounts is tested in two tubes, one for test and one for control, as described for the quantitative test. Results of these tests may be interpreted as Reactive, without reference to titer, if the first serum dilution showing complete hemolysis in the control tube has a 3 plus or 4 plus reaction in the tube containing antigen. All other reactions would be reported as Anticomplementary. Reading Standards 1) Heat tubes of hemoglobin solution (saved from the titration) in the 56° C water bath for 5 min. 2) Prepare a 1:7 dilution of 2 per cent sheep cell suspension by add- ing 0.5 ml of 2 per cent suspension to 3.0 ml of salt solution. 536 SYPHILIS 3) Prepare reading standards by mixing hemoglobin solution and cell suspension in proportions given below : 1:7 Corpuscle Hemoglobin Equivalent Complement Fixation Suspension Solution Per cent Record (ml) (ml) 0.7 None 100 4+ 0.35 0.35 50 3+ 0.175 0.525 25 24 0.07 0.63 10 1+ 0.035 0.665 5 + None 0.7 0 — L. Quantitative Determination of Cerebrospinal Fluid Protein#0-4! Equipment—Photoelectric colorimeter and photoelectric colorim- eter cuvettes to accommodate 5 ml volumes or less. Glassware—Test tubes, 13X100 mm outside dimensions. Reagents Trichloracetic acid solution (10%) : Dissolve 10 g of trichloracetic acid (C.P.) in 100 ml of distilled water. Filter into a glass-stoppered flask and store at room temperature. Standard serum: Select serum free of gross bacterial contamination or hemolysis and determine the total protein concentration by Kjel- dahl analysis. : Preparation of Spinal Fluid Centrifuge spinal fluid and decant. Spinal fluids containing visible contamination or gross blood are unsatisfactory for testing. Performance of the Test 1) Pipette 2.0 ml of spinal fluid* into a 13 X 100 mm test tube. A protein solution of known concentration should be included each time tests are performed. 2) Add 2.0 ml of 10 per cent trichloracetic acid solution. 3) Invert the tube twice to mix contents. Avoid foaming. 4) Allow tube to stand for 10 min in 37° C water bath. * Lesser amounts of spinal fluid may be tested in the same proportion, pro- vided that the photoelectric colorimeter employed is adaptable to the use of smaller cuvettes. SYPHILIS 537 5) Again invert the tube and pour fluid into photoelectric colorim- eter cuvette. 6) Determine percentage of light transmission at a wave length of 420 p with the unknown, using a water blank at 100 per cent transmission. 7) Convert per cent transmission of unknown to milligrams per cent total protein by reference to a calibration chart. Note: If spinal fluids contain concentrations of protein greater than 60 mg per cent, they should be appropriately diluted with 0.9 per cent sodium chloride solution and retested. Values obtained from the calibration chart are then multiplied by the dilution factor. Preparation of the Calibration Chart 1) Prepare 10, 20, 30, 40, 50 and 60 mg per cent protein solutions from the standard serum. Example: 1f protein content of serum (Kjeldahl) is 7,325 mg per cent, to find dilution factor for 60 mg per cent standard, divide 60 into 7,325. 7,325 ———=122, or dilution factor. Therefore, 1 part of serum is diluted with 121 parts of 0.9 per cent sodium chloride solution to make 60 mg per cent standard solution. 2) Test each standard solution in the same manner as described for spinal fluid. 3) Plot the light transmission values thus obtained on semiloga- rithmic paper. 4) Draw a line through the points on the graph. 5) A conversion chart also may be prepared showing the total protein values for each possible light transmission reading. Il. MICROSCOPICAL EXAMINATION FOR T. PALLIDUM A diagnosis of syphilis can be made by initiated observers with the demonstration of T. pallidum in suspected and accessible lesions or regional lymph nodes using dark-field microscopy. In primary syphilis it may be possible to identify the etiologic agent and to diag- nose the disease before the appearance of measurable antibodies. Hence the earliest and empirically most specific means of diagnosing 538 SYPHILIS syphilis is by dark-field microscopy. Every genital lesion should be considered syphilitic until proved otherwise. Such lesions should be examined by dark-field microscopy, and any observable lesion of the skin or mucous membranes should be so examined if there is the slightest suspicion of syphilis. Only primary and secondary (or early lesion) stages of syphilis will be found to harbor T. pallidum. The prerequisites for identification of T. pallidum are proper equipment, adequately trained personnel, and perseverance. Ideally, the dark-field examination should be made on the spot. Where proper facilities are lacking for this purpose a capillary tube speci- men should be carefully collected and promptly submitted to the nearest laboratory prepared to perform such examinations. Pro- longation of the interval between obtaining the specimen and exam- ining it reduces the likelihood of finding T. pallidum. A. Dark-Field Examination 1. Collection and submission of specimens—Rubber gloves and other necessary precautions should be used to obtain the specimen in such a manner as to avoid accidental infection. If the lesion is covered with a scab or crust, this should be removed. Cleanse the lesion with a gauze sponge which has been wetted with plain tap water or physiological salt solution. The use of antiseptics or soap should be avoided. After drying the area, the lesion is abraded with a dry sponge to provoke slight bleeding and serous exudation. As oozing occurs, wipe away the first few drops, particularly if they contain appreciable amounts of blood, and await the appearance of relatively clear serum. It is sometimes necessary to squeeze the base of the lesion or to apply a suction cup in order to promote the appear- ance of serum. It is more desirable to obtain the serum from the depths of the lesion rather than from its surface, Where a history of local therapy is elicited from the patient, it may be difficult or impossible to demonstrate treponemes in serum from the lesion. Compresses of boric acid or warm physiological salt solution for 24 hr often will facilitate finding the organisms. For direct examination, several clean cover slips or slides are applied to the oozing lesion, or a capillary pipette may be used to transfer serum from the lesion to glass slides. Cover slips and slides are pressed firmly together with the exudate between. The cover slip should be evenly depressed or flattened with the blunt end of a cotton swab stick or pencil point in such a manner as to remove air bubbles. The edges of the cover glass may be ringed with vaseline to minimize evaporation. SYPHILIS 539 When it proves impossible to demonstrate the organism from the lesion after examining several specimens, a sample may be obtained from a regional lymph node, particularly if it is palpable. To do this, either wash out or introduce about 0.5 ml of physiological salt solution into a small sterile syringe to which a small hypodermic needle is attached. Sterilize the skin overlaying the node by painting with iodine and alcohol or some other suitable agent. Hold the gland firmly and insert the needle well into the node. The ability to manipu- late the needle freely is a good indication that the capsule of the node has been pierced. Inject the salt solution and gently manipulate the needle in various directions to macerate the tissue, aspirating as much fluid as possible. Discharge the aspirated material on slides for direct examination or into capillary tubes for transmission. To send a speci- men to another laboratory, a capillary tube containing the exudate or aspirate is sealed on both ends with beeswax or a mixture of bees- wax and vaseline, The tube is then encased in a glass tube of suit- able size. Such outfits are generally supplied by state health depart- ments. Measures should be taken to facilitate the prompt delivery of specimens to the examining laboratory, as the feasibility of such de- layed dark-field examinations is questionable when no motility of the treponeme is observed. 2. Examination—Using properly adjusted dark-field equip- ment, examine the specimens for organisms of characteristic mor- phology and motion. A careful and exhaustive search should be made before rendering a negative report. While it is not possible here to review in detail all the technical aspects of dark-field microscopy, a summary of the more frequent sources of error in the use of this technic may be helpful as a check list for the beginner. a. Errors in preparation —Inclusion of too many refractile elements (red blood cells, air bubbles, tissue fragments, etc.). —Dirty or defective glassware (fine scratches on slides and cover slips). —Slides and cover slips too thick (1.3-1.5 mm slide thick- ness with No. 1 cover slip is usually satisfactory; however, the proper slide thickness is generally marked on the condenser). —Excessive fluid between glass slide and cover slip, with too rapid flow of liquid across the field of vision and too great a depth to scan. —Too little fluid between glass slide and cover slip, with ac- centuation of evaporation effects, 540 SYPHILIS —TForgetting to place oil between condenser and slide and/or between objective and cover slip. b. Errors in use of condenser —Not properly centered. —Not properly focused. c. Errors in use of objective —Too high a numerical aperture used. —PFailure to compensate high numerical aperture with funnel stop or iris diaphragm. d. Inadequate light source As indicated previously and reemphasized here, definitive differ- entiation of 7. pallidum from other treponemes and spirochetes de- pends not only upon size and other morphological characteristics, but —of equal importance—upon characteristic motility. 7. pallidum appears to be a small, tough spirilliform (corkscrew shaped) organism with regularly spaced, fairly tightly wound coils. The length is usually 5-15p, averaging 7p. This is slightly larger than the average diameter of a red blood cell. An occasional red blood cell in the preparation can be used as the practical criterion of length. The thickness seldom varies from 0.25 p, and the pitch of the spirals ap- pears to be 1 up. The coiled appearance is maintained despite active motility. Characteristic movements are forward and backward (translation), rotation about the long axis like a corkscrew, and slow bending, twisting or undulation from side to side. There is no waving or flat- tening, as can be noted in larger saprophytic spirochetes. The most common bending is to be noted in the middle of the organism, stiffly executed like the bending of a coil spring, which comes back in place when released. Many other spirochetes have a whip-like lashing movement and rapidly move across the microscopic field, unlike T. pallidum. A practical criterion for differentiating 7". pallidum from non- pathogenic treponemes and spirochetes found commonly in the mouth and not uncommonly upon the genitalia is that when multiple organ- isms are observed in microscopic fields of the slide and all are quite uniform in size, shape and motility (as described), these will most usually be T. pallidum. On the other hand, saprophytic organisms are quite usually mixed, so that any one preparation will contain spiral forms of various sizes, shapes and motility. It must be re- membered, of course, that some lesions will contain mixed flora and SYPHILIS 541 T. pallidum. When in doubt under such circumtances, the aspirate of the enlarged regional lymph node draining the site of the lesion will contain only T. pallidum (if present) and no saprophytic forms. 3. Interpretation of results—For practical purposes in this country the demonstration of treponemes of characteristic mor- phology and motility constitutes a positive diagnosis of syphilis. Sub- sequent follow-up of dark-field positive seronegative cases should show a development of reactivity within several days to several weeks in untreated patients who have primary syphilis. Other stages of early, dark-field positive syphilis should be routinely seroreactive. Failure to find the organism does not mean that the patient does not have syphilis. Such negative dark-field examination results may have several meanings: that organisms were not found in sufficient num- bers in the test specimen; that the patient received antitreponemal drugs locally or systemically; that the lesion is “fading” or examina- tion was made just before natural resolution or disappearance; that the lesion is one of late syphilis; or, finally, that the lesion is not syphilitic. Serologic follow-up should be continued for about four months—at weekly intervals for the first month and at biweekly in- tervals thereafter. Since treponemes found either normally in the mouth or in nonsyphilitic oral lesions bear such a close resemblance to T. pallidum, the results of an examination of oral lesions in sus- pected cases are of questionable significance where the site of the lesion is at or near the edge of the gums or where special precautions have not been taken in collecting the specimen. Caution should be exercised in interpreting results on slides con- taining numerous artifacts or refractile objects. The uninitiated or the unwary may be deceived by miscellaneous pieces of cellular debris, cotton strands, flagellae, cilia or fine scratches on glass slides. These scratches and certain forms, similar to treponemes, made of spiral fibrin filaments can, with Brownian movement, be quite deceptive. Other elements which can be readily recognized may, when present in great numbers, make difficult the demonstration of 7. pallidum. These are air bubbles, powdery hemokoniae, platelets, epithelial cells, leukocytes and erythrocytes with or without rouleaux formation. WaRFIELD GARsoN, M.D., M.P.H., Chapter Chairman Ap HARrTS Jou~n F. Kent, Pu.D. Josep Portnoy, PH.D. Danie. WipELock, Pu.D. 542 SYPHILIS REFERENCES 1. MaHsoNEY, J. F., and ZwaLry, M. R. “Serodiagnosis of Syphilis,” in 10. 11 12. 13. 14. 15, 16. 17 18. 19. 20. 21. Diagnostic Procedures and Reagents (3rd ed.). New York: American Public Health Assn., 1950, p. 291. PancBorN, M. C. A New, Serologically Active Phospholipid from Beef Heart. Proc. Soc. Exper. Biol. & Med. 48:484-486, 1941. NeLson, R. A. and Maver, M. M. Immobilization of Treponema pallidum in vitro by Antibody Produced in Syphilitic Infection. J. Exper. Med. 89:369-393, 1949. D’ALEssANDRO, G., and DArpANoni, L. Isolation and Purification of the Protein Antigen of the Reiter Treponeme. Am. J. Syph. 37:137-150, 1953. Serology Evaluation Research Assembly. U. S. Department of Health, Education, and Welfare, Public Health Service, 1956-1957. PHS Pub. No. 650, Washington 25, D. C.: Gov. Ptg. Ofc. Serologic Tests for Syphilis. 1959 Manual. U. S. Department of Health, Education, and Welfare, Public Health Service. PHS Pub. No. 411 (1959 Rev.), Washington 25, D. C.: Gov. Ptg. Ofc., 1959. GARrsoN, W. The Treponemal Tests for Syphilis. South. M. J. 50:911-918, 1957. ———————— Recent Developments in the Laboratory Diagnosis of Syphilis. Ann. Int. Med. 51:748-758, 1959. Portnoy, J., Garson, W., and Smith, C. A. Rapid Plasma Reagin Test for Syphilis. Pub. Health Rep. 72:761-766, 1957. Portnoy, J.; Bossak, H. N.; Farcong, V. H.; and HArris, Ap. Evalua- tion of Rapid Reagin Test with Unheated Serum and New Improved Antigen Suspension. Presented at the Twelfth Annual Symposium on Recent Advances in the Study of Venereal Diseases, April 13-14, 1961, New York, N.Y. ANDUJAR, J. J., and Mazurex, E. E. The Plasmacrit (PCT) Test on Capillary Blood. Am. J. Clin. Path. 31:197-204 (Mar.) 1959. ScammTt, H., and BeEntzoN, M. W. Investigations on the Rapid Plasma Reagin Test. WHO Bull. 19:563-568, 1958. National Advisory Serology Council. Statement. Pub. Health Lab, 17:122- 123, 1959. CANNEFAX, G. R., and Garson, W. Reiter Protein Complement-Fixation Test for Syphilis. Pub. Health Rep. 72:335-340, 1957. Bossaxk, H.; Farcong, V. H.; Duncan, W. P.; and Harris, Ap. Kolmer Test with Reiter Protein Antigen. Pub. Health Lab. 16,2:39-48, 1958. RosenAv, B. J., and Kent, J. F. Procedure for Increasing the Sensitivity of Tests for Specific Treponemal Antibody. Lab. & Clin. Med. 51:664— 666, 1958. Kent, J. F., and DEWEEerpT, J. B. Diagnostic Utility of the 40-Hour T.P.I. Test. Am. J. Clin. Path. 35:526, 1961. Kent, J. F,, et al. Evaluation of Fluorescent Treponemal Antibody (RFTA and FTA II) and Other Tests (RPCF and TPI) for Syphilis. Proc. Soc. Exper. Biol. & Med. 109 :584-589, 1962. Boak, R. A., CARPENTER, C. M., and MILLER, J. N. Biologic False-Positive Reactions for Syphilis among Narcotic Addicts. J.A.M.A. 175:326, 1961. Davis, B. D.; Kasar, E. A.; Harris, A.; and Moore, D. H. The Anti- complementary Activity of Serum Gamma Globulin. J. Immunol. 49:223- 233, 1944. MacnusoN, H. J. Interpretation of Positive Serologic Tests for Syphilis in Clinically Negative Patients. J. Michigan Med. Soc. 53:744-748, 1954. SYPHILIS 543 22. 23. 24. 25 26. 27. 28. 29. 30. 31. 32. 33. 34. 3. 36. 37. 38. 39. 40. 41. Standard Methods of the Division of Laboratories and Research of the New York State Department of Health. Baltimore: Williams & Wilkins, 1947. KoLMmEr, J. A. Sepaurning, E. H., and Rosinson, H. W. Approved Laboratory Technic (5th ed.). New York: Appleton-Century-Crofts, 1951. KosTtaNT, G. M.; Lanpy, S. E.; MILLER, J. N,, and KeLcec, L. C. Trepo- nemal Tests on Cerebrospinal Fluid. A.M.A. Arch. Dermat. 80:439-441, 1959. Harris, A.; Bossakx, H. M.; Deacon, W. E.; and Buwncu, W. L, Jr Treponemal Tests for Syphilis in Spinal Fluids. Brit. J. Ven. Dis. 36:178- 180 (Oct.) 1960. Laboratory Procedures for Modern Syphilis Serology. Department of Health, Education, and Welfare, USPHS, VD Branch, CDC, Alanta, Ga., July 1961, 35 pp. Portnoy, J., BREWER, J. H., and Harris, A. H. Rapid Plasma Reagin Card Test for Syphilis and Other Treponematoses. Pub. Health Rep. 77:645-652, 1962. PorrNoOY, J., and Garson, W. New and Improved Antigen Suspension for Rapid Reagin Tests for Syphilis. Pub. Health Rep. 75:985-988, 1960. PorTNOY, J. Personal communications. Harris, A. RosenBerG, A. A. and Rieper, L. M. A Microflocculation Test for Syphilis Using Cardiolipin Antigen. Preliminary Report. J. Ven. Dis. Inform. 27:169-174 (July) 1946. HAaRrris, A., RosexBerG, A. A, and DEL Vecchio, E. R. The VDRL Slide Flocculation Test for Syphilis. II. A Supplementary Report. J. Ven. Dis. Inform. 29:72-75 (Mar.) 1948. Kent, J. F., et al. Differentiation of the Antilipids Occurring in Nontrepo- nemal Diseases and Syphilis. J. Chronic Dis. 7:36-42, 1958. Kent, J. F., Otero, A. G., and Harrigan, R. E. Relative Specificity of Serologic Tests for Syphilis in Mycobacterium leprae Infection. Am. J. Clin. Path. 27:539-545, 1957. RoseNBERG, A. A., Harris, A., and Haroing, V. L. A Macroflocculation Spinal Fluid Test Employing Cardiolipin-Lecithin Antigen. J. Ven. Dis. Inform. 29:359-361 (Dec.) 1948. KoLMER, J. A. The Technic of the Kolmer Complement-Fixation Tests for Syphilis Employing One-Fifth Amounts of Reagents. Am. J. Clin. Path. 12:109-115, 1942. —————— and Lyn~cH, E. R. Cardiolipin Antigens in the Kolmer Com- plement-Fixation Test for Syphilis. J. Ven. Dis. Inform. 29:166-172 (June) 1948. MALTANER, ErizaBerH, and MALTANER, FRANK. The Standardization of the Cardiolipin-Lecithin Cholesterol Antigen in the Complement-Fixation Test for Syphilis. J. Immunol. 51 :195-214, 1945. WALLACE, A. L., and HARrris, A. Preparation of Reiter Protein Antigen. Pub. Health Lab. 16,2:27-38 (Mar.) 1958. Kent, J. F, Bovyp, H. M,, and Sanpers, R. W. Cardiolipin Antigen in the Kolmer Complement-Fixation Test for Syphilis. Bull. U. S. Army Med. Dept. 8:284-293, 1948. Bossak, H. M., RosENBERG, A. A., and HArrrs, A. A Quantitative Turbidi- metric Method for the Determination of Spinal Fluid Protein. J. Ven. Dis. Inform. 30:100-103 (Apr.) 1949. Harping, V. L., and Harris, A. The Effect of Temperature Variants on Quantitative Turbidimetric Determinations of Spinal Fluid Protein Using Trichloracetic Acid. J. Ven. Dis. Inform. 30:325-327 (Nov.) 1949. CHAPTER 19 LEPTOSPIROSIS I. Collection and Handling of Specimens II. Bacteriological Examinations A. Demonstration in Body Substances by Microscopical Technics 1. Morphology 2. Dark-Field Microscopy 3. Stained Preparations B. Isolation by Direct Culture 1. Nutrition of Leptospiras 2. Selection of Medium and Glassware 3. Technics C. Isolation by Animal Inoculation Methods 1. Choice of Animal 2. Preparation of Specimens for Injection 3. Technics D. Maintenance of Stock Strains III. Serological Technics - Preparation of Antigens (General) Preparation of Standard Control Antisera Leptospira Serotypes Employed Microscopical Equipment Specific Reactions The Microscopical Agglutination Test With Live Antigens With Formalin-Fixed Antigen IV. Evaluation and Reporting of Results References Leptospiral infections caused by various serotypes of leptospiras in- clude a number of similar acute, febrile, septicemic, protean diseases of man and certain mammals. The natural reservoirs of these organ- isms apparently are various wild and domesticated mammals, par- ticularly rodents. Weil! described the first cases of leptospirosis in man in 1886. In 1916, Inada and his associates? isolated the causative agent from human cases in Japan. In 1917 Noguchi® found in American wild rats a spirochete morphologically and immunologically identical with the Japanese strains. In 1918 he further described the morphology of this organism and named it Leptospira icterohemorrhagiae.* 544 LEPTOSPIROSIS 545 In 1922 Wadsworth® described the first case of leptospirosis in man in the United States. Meyer® in 1938 reported the occurrence of Canicola fever in man in California and established the dog as the reservoir of the causative agent Leptospira canicola. Jungherr” in 1944 found microscopical evidence of bovine leptospirosis in cattle in this country. Since that time an increasing number of cases of lepto- spirosis in man and animals due to a variety of serotypes of these organisms has been reported in the United States. That leptospirosis is an occupational hazard and hence a public health problem has been acknowledged in numerous countries including the United States.®?® Laborers have been legally compensated as a result of disabling or fatal leptospirosis incurred while at work in occupations involving risk of exposure.’ The pathogenic leptospiras are classified on the basis of their serological properties according to the schema prepared by Wolff and Broom in 1954.11 At that time 20 serogroups comprising 32 sero- types were recognized. Since then, new strains have been observed, and more than 80 serotypes are presently known.'%1% The micro- scopical agglutination (agglutination lysis) test of Schiiffner and Mochtar!* is the oldest and most reliable of the available serological methods and is employed throughout the world for classification of leptospiras, as well as for detection and preliminary identification of leptospiral antibodies in human and animal sera. Diagnostic labora- tories should be capable of performing procedures designed to isolate leptospiras in addition to providing confirmation or denial of clinical impressions. Technics will be outlined for isolation and cultivation of leptospiras. Serological technics of proved specificity and reliability will be spelled out. The reader should refer to the current scientific literature for details of the newer, recently reported serological methods cur- rently undergoing evaluation. These technics when adequately evalu- ated will probably provide vastly improved and simplified diagnostic procedures requiring revision of this chapter. These more promising, newer methods include the macroscopic agglutination procedure of Stoenner,'® Galton et al.,'® and Muraschi'”; the complement-fixation tests of Terzin'® and of Rothstein and Wolman'?; and the hemolytic procedures of Chang et al?***' and Cox* employing erythrocyte- sensitizing antigens.??* Some of these newer tests!®21:22 employing alcohol-extracted antigens exhibit generic specificity. The protean nature of the clinical manifestations of leptospirosis has been described®-27 and is now recognized by the medical and veterinary professions. Leptospirosis in the United States can be 546 LEPTOSPIROSIS caused by any one of at least ten?®* antigenically distinct serotypes of leptospiras. Because of this protean nature of the clinical symptoms and the multiplicity of distinct serotypes, a successful diagnosis re- quires the close cooperation of clinician and laboratorian. Diagnostic facilities need to be greatly expanded at federal, state and local levels. The extent and importance of the public health and economic prob- lems incident to these infections will become apparent only when adequate laboratory diagnostic facilities are provided to the medical and veterinary clinicians. I. COLLECTION AND HANDLING OF SPECIMENS For most effective and efficient examination, samples must be properly collected and shipped to the laboratory. A serological diag- nosis of leptospirosis is virtually impossible unless at least two blood samples are furnished. The first sample is collected as early as possible in the course of the disease, the second 7 to 17 and preferably about 14 days later. A third serial serum sample is always desirable, if feasible, and should be collected about the 21st day of the disease. Equally important is a clinical history of the patient, provided by the physician or veterinarian, which should contain name, sex, age; possible contacts with animals or fomites contaminated with lepto- spiras; specific treatment already undertaken; relationship between the dates samples were collected and clinical course; and, finally, the manner in which the specimens have been treated prior to their arrival at the laboratory, including the addition of any preservatives, approxi- mate temperature in transit, inactivation of sera, date and time of shipping. A complete discussion of the clinical manifestations of the lepto- spiroses in man and animals is beyond the scope of this report. However, a brief discussion of pertinent biological properties of leptospiras will provide a basis for the precautions to be taken and the methods to be followed in the collection and handling of speci- mens. The choice of methods will depend on the stage of the disease during which the samples are collected. In a majority of instances the organisms can be isolated most readily from the blood and occasion- ally the spinal fluid during the initial febrile stage of the disease in man and animals. This leptospiremic phase persists for approximately 1 week and is generally terminated when antibodies appear in the blood. * Two additional serotypes have been recognized since this chapter was written. LEPTOSPIROSIS 547 The disappearance of the organisms from the blood often is associ- ated with remission of fever. At this time the organisms may be detected in the urine. Antibodies may appear at various times in different individuals ranging from about 5 days after onset to about 21 days, with an average of about 9 days. Antibodies may also be demonstrable in the spinal fluids and in the urine. In addition to blood and urine, organisms may be cultured from the anterior chamber fluid in cases of iridocyclitis in human beings®:3% or periodic oph- thalmia in horses!; from the spinal fluid in cases of meningitis®*33; from the fetus or fetal membranes in cases of aborting animals3*3%; from the milk during the acute systemic phase of the disease in lactat- ing animals®®; and from other tissues. Resistance to Various Agents As a rule leptospiras are very fragile in vitro.8"® In a variety of artificial media they usually are killed within 1 to 12 hr by freezing, drying, lyophilization (freeze-drying), heating, high salt concentra- tions, soaps, detergents, bile salts, acids, disinfectants, anaerobic con- ditions, and microbial contamination. In general, their resistance to physical and chemical agents approximates that of the vegetative forms of bacteria. Methods of Preservation Leptospiras remain viable and virulent for many days to weeks in uncontaminated tissues stored at 5° C.2® In defibrinated uncontamin- ated blood kept at room temperature (20°-25° C), they may survive for a week.*® They will survive in fluid raw milk for a number of hours and in diluted milk for a period of days.3¢ Upon rapid freezing and maintenance at —70° C they have survived up to 5 years in cultures and infected blood and liver tissue.*'*2 Considerably fewer organisms have survived freezing at — 15° C and storage at —70° C.#! Certain antibiotics, including penicillin, streptomycin and aureomycin, may be leptospirostatic or leptospirocidal in vitro and in vivo. General Precautions All materials must be handled so as to minimize destruction of the organisms. Preferably all materials, whether for culture, animal inoculation or serology, should be collected aseptically and examined promptly. It is essential that materials for culture be treated in this manner. Since serology may involve the addition of living lepto- spiras, heavy contamination of the serum might cause immobilization 548 LEPTOSPIROSIS or destruction of the spirochetes by a rapidly growing contaminating microorganism, Even a trace of soap or detergent may be fatal to leptospiras, so that glassware and syringes should be scrupulously clean, Stuart*? recom- mends that culture tubes be washed and rinsed in the usual way and then steeped in phosphate buffer at pH 7.6 for 24 hr before being rinsed and sterilized. However, this procedure is not essential if equipment is properly cleaned. Stuart*® considers sulfaguanidine superior to either sulfanilamide or sulfadiazine for inhibiting growth of contaminating microorgan- isms. A mixture of 4 parts of a saturated solution of sulfadiazine in basic medium with 6 parts of basic medium is satisfactory. Arm- strong#* employed crystalline cyclohexamide (0.1 g per liter of medium) to suppress growth of contaminating fungi and bacteria. Other antibiotics, such as chloramphenicol, that exert little effect on leptospiras may be advantageously used. If contaminants are suspected in clinical materials, they may often be separated efficiently by passage through bacteriological filters or by animal inoculation.®® The material is passed through any one of several types of bacteriological filters (such as fritted glass, Seitz or Selas filters) which permit passage of the spirochetes but retain the contaminating microorganisms. Most of these filters are available in 2 to 5 ml capacity, which are the most suitable sizes for working with small quantities of fluid. Separation may also be accomplished by injecting either the contaminated blood or culture intraperitoneally into a young guinea pig or mouse and 5 or 10 min later withdrawing blood from the heart for culture. The heart’s blood should contain the spirochetes, which rapidly invade the bloodstream.*® Recently “colonial” growth of leptospiras on a solid medium has been described.*® This and similar media have been effectively em- ployed to separate leptospiras from contaminating microorganisms.*’ Collection and Handling of Body Substances Blood—Blood is employed for culture, subinoculation into animals, and serology. A number of centrifugation procedures have been described for concentration of the organisms for culture. However, there is considerable doubt at present whether the concen- tration achieved by these procedures is worthwhile in view of the de- structive effects of centrifugation on leptospiras. Therefore, until definitive data are available on these points conservative procedure is advocated. It is known that with aseptic collection of blood and prompt culture of sufficient amount of untreated or defibrinated blood LEPTOSPIROSIS 549 in a suitable medium, isolations may be achieved consistently, How- ever, for best results blood should be drawn very early in the disease before specific antibodies appear in the circulation. After the 4th day of disease it may be more advantageous to employ minimal inocula (0.05 ml of blood to 5-10 ml of medium) to minimize the inhibitory effects of accumulating specific antibodies in the blood. This question of a relationship between the presence of antibodies in the blood and success of culture is still an open one. For details on actual cultural procedures see Section IT (Bacteriological Examinations). It is preferable to culture blood immediately at the bedside or upon collection from an animal. For this purpose, it is more convenient to provide sterile diaphragm-capped bottles (see Section IIB2) so that the blood may be inoculated directly into the medium. If the blood must be transmitted to the laboratory it may be defibrinated or mixed with anticoagulants. Defibrination is achieved by gentle manual swirling of beads in the bottle. Anticoagulants (sodium oxalate, 0.5 ml of a 1% solution to 5 ml of blood; or “liquoid” Roche, 1 ml of a 1% solution in sterile salt solution to 5 ml of blood) can be used. Citrate solutions are inhibitory.3? When culture or animal inoculation cannot be performed immedi- ately, every effort should be made to minimize delay in processing specimens. Unfortunately definitive information is unavailable on the viability of leptospiras under various conditions. Leptospiras are reported to have remained viable for at least 6 days in coagulated blood ; however, it is generally believed that delay in cultivation les- sens the chance for recovery of microorganisms. If blood has clotted, the clot may be triturated and the homogenate used for animal inoculation. The greatest danger inherent in this pro- cedure is the introduction of contaminating microorganisms, which as a rule rapidly destroy leptospiras. Serum—Blood should be handled as aseptically as possible for serology. Ideally, no preservative is added to serum when it is to be employed for serology, as there is danger of interference with the reaction. If it is absolutely necessary to employ a preservative—for instance, when it is impossible to collect sera aseptically—add 1 part of glycerol to 1 part of serum or 1 part of 1:1,000 aqueous merthio- late solution to 9 parts of serum. To avoid lipemic serum, the blood should not be collected immediately after mealtime. Serum may be sent by ordinary mail. If collected aseptically or preservative is added, it is not necessary that the serum be refrigerated. Note that serum can also be absorbed onto filter paper and sub- mitted in the dried state. The sample can be extracted from the paper 550 LEPTOSPIROSIS and tested.*®*® This provides a convenient method when only small amounts of serum are available and when samples are submitted over long distances. The filter paper strips require no refrigeration, can be stored in an ordinary desiccator in a relatively small space. Blood can be similarly collected, but such samples are unsuitable for com- plement-fixation tests. Others—Specific instructions for the handling of body fluids and tissues, including blood and urine, are given in the section of this chapter on bacteriological examinations, which follows. Il. BACTERIOLOGICAL EXAMINATIONS* In the laboratory diagnosis of leptospirosis, isolation of the organ- isms is very important, chiefly because identification of the serotype involved in a particular infection cannot be made with certainty by serological examination of the patient’s serum. Isolation of the or- ganisms is also important in epidemiological and ecological investiga- tions, particularly those involving the question of legal compensation for fatal or disabling leptospirosis contracted in occupations that entail unusual risk of exposure to these spirochetes. The general methods for the demonstration of leptospiras include (1) isolation by direct culture or animal inoculation, and (2) micro- scopical examinations, using dark-field technics or suitably stained films and tissue sections. Of these the microscopical examinations may often yield falsely positive results. However, since they are employed in connection with both cultural and animal inoculation procedures, the microscopical technics will be discussed first. A. Demonstration in Body Substances by Microscopical Technics 1. Morphology—Leptospiras are among the smallest of the spirochetes, usually measuring from 6 to 12 p in length and 0.1 p in diameter. There is little variation in diameter, although individuals may vary from 4 to 40 p in length. The body of the spirochete is tightly coiled on its long axis and the terminal portions are hooked so as to give the form of the letter C or S. Occasionally there is only one terminal hook or, more rarely, none.’° The amplitude of the single coils is small, and the light reflected from the top of each coil gives the organism the appearance of a brightly shining string of minute beads. * The methods outlined here have been successfully tried in at least two or more laboratories. Others perhaps as good or, better but not as widely used will be mentioned briefly. Details may be obtained by consulting the reference material. LEPTOSPIROSIS 551 In young cultures particularly, leptospiras are extremely active; the rapid rotation of the organism on its long axis often results in a “buttonhole” appearance at either end. Directional motion across the field and flexion of the body are also characteristic of Keptospira. More recently examinations by electron microscopy have demon- strated the presence of an axial filament entwined uniformly along the body of these spirochetes.5-5* Distinction among various strains of Leptospira cannot be made by morphological examinations, even those made with the electron microscope. 2. Dark-field microscopy—Direct dark-field examination, par- ticularly of blood, results so frequently in misdiagnosis that it is not to be recommended as a single diagnostic procedure. Even the most experienced workers often find it impossible to distinguish from leptospiras the “pseudospirochetes” (protoplasmic strands and fibrils of several origins) observed in blood specimens, and many false-posi- tive diagnoses have been made as a result. On the other hand, since the concentration of leptospiras in many specimens is small, one may fail to see the organisms in the samples examined and thus make a false-negative diagnosis. In the case of urine specimens from certain species and also with rodent kidney emulsions, dark-field examinations may yield unques- tionably positive results, since profuse numbers of leptospiras are so often present. If such specimens are to be examined, they should be fresh and of neutral or slightly alkaline reaction to maintain the typical morphology and motility by which leptospiras are identified. All dark-field diagnoses should be confirmed by cultural or animal inoculation methods. 3. Stained preparations—Leptospiras may be demonstrated on occasion in films of various body fluids and in tissue sections, fixed and stained by appropriate technics. Since the same pitfalls are to be expected in this procedure as in dark-field microscopy, special care must be taken in the fixation process to avoid distortions in the mor- phology of the organisms. Stavitsky,% after employing a number of different technics, has recommended a dilute Giemsa stain for films. A number of other procedures have been used successfully. Details may be found in the references.56-59 For the demonstration of spirochetes in tissue sections, various modifications of the Levaditi silver-impregnation procedure (e.g., Fontana’s) and several other methods have been used. For details of some of these technics the reader is referred to the work of Kerr, Steiner and Steiner, Campbell and Rosahn,®? and Meyer et al.® 552 LEPTOSPIROSIS In summary, dark-field microscopy and examination of stained preparations may yield very useful information in certain instances but should not be used as single diagnostic procedure. The results of such examinations should always be confirmed by cultural and/or serological methods. B. Isolation by Direct Culture 1. Nutrition of leptospiras—There is a dearth of basic informa- tion regarding the metabolism and growth requirements of these organisms. All the media generally used for their isolation and main- tenance have at least two essential ingredients in common—a buffer system and a final concentration of 5 to 10 per cent of mammalian serum. Most workers are agreed that slightly hemolyzed rabbit serum is superior to that of other species ; however, equine and bovine serum have been used by some investigators. In our experience, unpre- dictable failures and spotty growth frequently occur when Leptospira organisms are grown in media prepared with sera of a species other than the rabbit. If serum from horses, cattle or swine is to be used, it should always be pretested for leptospiral antibody and for ability to support growth. Attempts to substitute various amino acids, vitamins and other “growth factors” for whole serum or some fraction thereof have been consistently unsuccessful. There has been some agreement that certain concentrations of thiamin, nicotinic acid or its amide, ribo- flavin, asparagine, yeast extracts and vitamin B-12 may stimulate somewhat the growth of the particular Leptospira strains studied, while many other amino acids, carbohydrates, vitamins and a variety of other materials inhibit growth or affect it not at all.#3:%4-72 Tn many instances the concentration of the stimulatory substance was found to be quite critical. Marshall” demonstrated that the oxygen uptake of concentrated L. icterohemorrhagiae cultures was stimulated by the addition of rab- bit serum although not by a variety of other substances tested, includ- ing other constituents of the culture medium. The extensive work of Greene and Schneiderman and their co- workers®-70 has provided some evidence that the albumin fraction of rabbit serum contains those components essential for growth of their test strains. These investigators report that among the serotypes, L. canicola, L. pomona, L. ballum, L. sejroe and L. icterohemorrhagiae could be subcultured on a simple medium containing only salts, aspara- gine, thiamin and rabbit serum albumin, LEPTOSPIROSIS 553 Subsequently, Helprin and Hiatt™ found that serum protein in medium supplied and also detoxified fatty acids, which stimulated the respiration of L. icterohemorrhagiae. Trace amounts of various fatty acids stimulated respiration in the presence of albumin. In the absence of albumin some of these acids are toxic. Woratz™ successfully grew L. canicola in a serum-free medium containing casein hydrolysate and minute amounts of fatty acids in gelatin; however, these results were not reproducible in another laboratory.” 2. Selection of medium and glassware—Formulas for several satisfactory media are given in Chapter 4 of this book (CM Nos. 88 through 92). There appears to be little choice among them, although the semisolid form offers certain advantages over a fluid medium for direct culture methods. In media containing a small amount of agar, leptospiras usually concentrate in a ring a few millimeters below the surface, thus facilitating the detection of growth by gross inspection. All glassware used in work with leptospiras should be thoroughly cleaned and rinsed with distilled water until free from any trace of detergent. For general use approximately 3 to 5 ml of medium is dispensed in 16X125 mm screw-cap tubes. If a fluid medium is selected, the smaller volume is recommended to give a more desirable ratio between depth of medium and area of exposed surface (leptospiras grow well only under aerobic conditions). If medium for blood culturing is to be shipped to the field, dia- phragm-stoppered vaccine vials are convenient for its packaging. Ap- proximately 12-15 ml of medium is dispensed in 30 ml capacity vials. After the stopper has been cleaned with disinfectant, the medium may be inoculated directly by puncturing with the needle of the syringe used for collecting the blood. Caution: Some rubber stoppers are coated with substances lethal to Leptospira organisms. Stoppers should be thoroughly cleaned and boiled before use. Leptospiras have been isolated by direct culture from cerebrospinal fluid, bone marrow, the anterior chamber of the eye, bladder urine, and various tissues, as well as peripheral blood (see prior Section I, “Collection and Handling of Specimens,” for timing and procedures most likely to yield successful isolations with various types of speci- mens). Since blood is usually most readily available, the technics outlined below are for blood culture, although they may be adapted for use with any material which is free of bacterial contamination. In smaller mammals and experimental animals, sterile samples of urine may be obtained by direct bladder tap.” 554 LEPTOSPIROSIS 3. Technics—Inoculate each of four to six tubes of culture medium with a drop or two of the specimen.* Incubate the tubes at 25°-30° C and examine for growth at weekly intervals. All tubes should be incubated for at least 4 or 5 weeks before they are discarded as negative. Examinations for growth are made in the following manner: In- spect all tubes for evidence of turbidity at the top of the medium. From each suspicious tube, place a large loopful of fluid (or a drop from a capillary pipette) on a slide and cover with a cover slip. Both slide and cover should be scrupulously clean. Examine by dark-field technic, using approximately 430>X magnification. The leptospiras are recognized by their characteristic morphology and motility. Transfer positive cultures to fresh medium and determine serotype by appropriate serological methods described elsewhere. C. Isolation by Animal Inoculation Methods As indicated elsewhere in this chapter, attempts to culture lepto- spiras from urine, milk, tissue emulsions or other materials which are not free from bacterial contamination almost invariably result in failure. Consequently animal inoculation procedures must be em- ployed for examination of such specimens. 1. Choice of animal—Several genera and species of rodents (both wild and laboratory strains) have been used for isolation pro- cedures. Young guinea pigs, which were among the animals in earliest laboratory use, are still perhaps the most generally used. However, in recent years the weanling hamster3343¢ has appeared to be a better choice for most of the serotypes of Leptospira en- countered in North America. Van der Hoeden has reported the unusually great susceptibility of a species of Meriones®? to all strains of leptospiras tested, but these animals may not be generally available. Any laboratory animal selected should be known to be free of inter- current infections, This is especially important in the case of mice, which frequently are found to harbor L. ballum.3®3%% Young indi- viduals, regardless of species, appear to be definitely more susceptible to infection than older individuals, so that selection should be made with this in mind. * The optimum amount of inoculum has not been clearly defined. The presence of “inhibitors” (probably antibody) in blood, urine or kidney emulsion has been suggested and the practice of using relatively small inocula in multiple tubes of medium developed as a result.78-80 Tt is quite probable that larger amounts of blood may be used without deleterious effect during the first few days of disease. However, the small inoculum suggested has proved quite satisfactory.81 In some instances, e.g., urine and kidney emulsion, cultivation of higher dilutions may increase chances for recovery of leptospiras. LEPTOSPIROSIS 555 The 1 to 2 day old chick has been successfully used to isolate leptospiras.8® Following intraperitoneal inoculation of infective material, leptospiremia extending up to a period of 3 weeks has been observed in chicks, which are not, however, susceptible to infection with all serotypes. The 17 day old chick embryo may also be useful for isolation procedures and for other laboratory investigations.8¢ Generally, a larger number of organisms is required to establish in- fection in chick embryos and wet chicks than in hamsters.8788 2. Preparation of specimens for injection—Freshly voided urine specimens, if injected immediately, need no preliminary treatment. When urine must be transported to the laboratory, the pH of the specimen should be neutral or slightly alkaline. The proper pH may be arrived at by alkalinizing the patient before collecting the specimen or by careful and immediate adjustment of the pH following collec- tion. It should be emphasized that animal inoculation at the bedside is the preferable procedure and any delay in injecting the specimen greatly diminishes the chance of successful isolation. Emulsions of tissue are prepared by grinding in a small Waring blendor, tissue grinder or sterile mortar, using sufficient culture medium or physiological salt solution to make a suspension fluid that will pass through a 22 to 25 gauge hypodermic needle. (The addition of a little sterile sand to the mortar aids in grinding the tissue into smaller particles.) Recently, Stuart™ found that the presence of antibodies in urine may decrease the chances of recovering leptospiras. Rudge? made analogous observations on the recovery of leptospiras from kidney emulsions. Consequently, the chances for recovery of microorgan- isms may be increased by employing minimal inocula or by attempting isolation from multiple dilutions, such as 1:10, 1:100, 1:1000.8° Broom and his co-workers, using animal inoculation methods, have had considerable success in isolating leptospiras from clotted blood specimens which had been in transit for a period up to 3 days.®® These workers prefer to use the clot rather than serum and prepare it by grinding in a sterile mortar with a small amount of physiological salt solution. 3. Technics—Divide the specimen into portions of 0.3 to 1.0 ml (depending on age, size and species of animal chosen). Inject one portion intraperitoneally into each of four to six animals. Some workers advise daily determinations of weight and temperature of inoculated animals; in at least some cases signs of infection may be detected readily by these procedures. However, fever, loss of weight, 556 LEPTOSPIROSIS appearance of jaundice or other “typical” clinical signs cannot be relied upon as criteria for infection, and all animals should be ex- amined as follows : Anesthetize lightly and obtain blood from the heart for culture and microscopical examination on the 4th and 6th day and then at 3 to 4 day intervals up to the 20th day postinoculation, if death has not occurred previously. Inoculate each of four to six tubes of culture medium with a drop or two of blood delivered directly from the syringe immediately upon withdrawal. Incubate and examine tubes as outlined previously. Some investigators recommend daily dark-field examination (be- ginning on the 3rd day after inoculation) of peritoneal fluid and blood collected from the ear of the animal before bleeding for culture.33:85,89-92 Tp the hands of experienced workers this procedure gives good results, although it is rather difficult and impractical when hamsters or animals other than the guinea pig are used. In infections with strains particularly virulent for the test animal, death frequently occurs within a week to 10 days. On the other hand, infection may occur with little or no sign of illness or may result in complete recovery after a relatively mild course. All animals remain- ing alive at the time of last bleeding should be sacrificed and examined both grossly and by isolation technics for evidence of infection. Blood, bladder urine, emulsions of liver and kidney tissue should be ex- amined by the cultural and animal inoculation technics outlined above. Some workers have recommended three successive animal passages at intervals of about 2 weeks before making a negative report. This procedure has yielded some positive cultures which otherwise would have been missed, especially with serotypes and strains of relatively low virulence for the test animal employed. On occasion, spirochetes have been demonstrated in sections of liver and kidney of animals from which no spirochetes could be isolated. Histological examina- tions may be particularly helpful in instances where specific lesions are observed and cultural methods have failed. Autopsy findings may vary greatly from the classical picture most frequently seen in L. icterohemorrhagiae infections in guinea pigs. Obvious jaundice, for example, is more frequently absent than present, especially when dealing with serotypes other than L. ictero- hemorrhagiae. The lesions most usually found and which should be sought are: numerous petechial hemorrhages in subcutaneous and muscle tissue, mucous membranes and viscera ; swollen adrenals ; and some enlargement of the spleen, liver and kidneys. The adrenals, liver and kidney are often abnormal in color, tending to appear more orange or brownish than bright red. Hemorrhagic spots in the lungs LEPTOSPIROSIS 557 are perhaps the most striking single lesion; usually they are sharply defined foci which suggest the appearance of a butterfly. Many in- vestigators consider “butterfly lungs” as practically pathognomonic of leptospiral infection. D. Maintenance of Stock Strains* A number of attempts to preserve leptospiras in a viable condition for long periods of time by such means as freezing, drying and storage on various kinds of media have been reported.39:42:47.93-97 None of these methods has proved entirely successful and some pro- cedures apparently result in almost immediate death of the spirochetes. The technic of freezing with glycerol as described by Hollander and Nell®® for preservation of Treponema pallidum and certain bacteria should be investigated for applicability to Leptospira cultures. Until this method is proved successful, however, or until other more effec- tive means of preserving leptospiras are developed, stock strains must be maintained by serial transfer in the artificial culture media. Semisolid media, either Fletcher's (CM No. 92) or Chang’s (CM No. 91), are preferred for the maintenance of stock cultures. Transfers are made at 6 to 8 week intervals into medium that has been previously examined for sterility and ability to support growth of leptospiras. One ml inoculum is aspirated from the concentrated linear area of growth and is distributed into each of two tubes of medium (4 to 5 ml per tube). Inoculated cultures are incubated at 30° C for 2 to 3 weeks, at which time they are examined for typical ring formation. Cultures may then be stored at room temperature in the dark, until the next transfer. Some strains may be viable in semisolid media for a considerable period of time, up to 1 and 2 years. Cultures maintained in fluid media must be transferred at 3 to 6 week intervals. The inoculum should comprise 5 to 10 per cent of the final volume of subculture. Extreme care must be exercised in the transfer of large numbers of strains to prevent bacterial contamination and to assure accuracy in labeling tubes. In at least some instances labeling errors have given rise to confusion and discrepancies in results of work from one labora- tory to another. The composition of the basal medium and species of animal serum used in preparing the medium should be carefully controlled and kept constant in transferring stock strains. Until more data are available regarding the effect of the constituents of the medium (especially the * The Department of Agriculture and the U. S. Public Health Service require a permit for importation or interstate shipment of cultures of leptospiras. 558 LEPTOSPIROSIS species of animal serum used) on the antigenic composition of lepto- spiras, the possibility remains that such factors may be important in determining serological characteristics. As a point of practical interest, Leptospira cultures contaminated with other bacteria may usually be recovered by passing them through any one of several types of bacteriological filters* (fritted glass, Seitz, Selas or equivalent) as well as by animal passage. The growth of leptospiras on solid media also provides a means of eliminating con- taminating bacteria.*6:47 Technics 1. Filtration: It is frequently helpful to centrifuge the culture for 10 to 15 min at about 3,000 rpm before filtration. The usual procedure, especially when Seitz filters are to be used, also involves prewetting the filter with a small amount of sterile salt solution or medium and a final rinsing with about two volumes of culture medium. The total filtrate is distributed among fresh culture tubes, incubated, and examined as outlined elsewhere. 2. Animal passage: 0.5 to 1.0 ml of contaminated culture is in- oculated into a guinea pig or hamster by the intraperitoneal route; 10 to 15 min later the animal is bled from the heart and the blood used as inoculum for a number of tubes of culture medium. lll. SEROLOGICAL TECHNICS Various types of serological phenomena—agglutination, complement fixation, hemagglutination, hemolysis—have been employed in diag- nostic tests for leptospirosis. Of the tests heretofore described, the microscopical agglutination test, using either live or formalin-treated antigens, has been the most widely employed.2833 The microscopical agglutination test using live antigens (agglutination-lysis test) is con- sidered to be the best single test for the demonstration of leptospiral antibodies.®® However, many laboratories have substituted formalin- treated antigens in this procedure to facilitate use of antigens and avoid infection hazards. The preparative technics and readings of these tests vary from laboratory to laboratory, but the basic phe- nomenon—agglutination—is a universally accepted criterion of sero- logical activity and possesses recognized diagnostic significance. Various complement-fixation tests have been devised.18:19,100-103 However, none has been used widely over a period of years by a * Most of these filters are available in sizes of 2 to 5 ml capacity, which are most suitable for working with small quantities of culture. LEPTOSPIROSIS 559 number of laboratories. Some of the antigens are difficult to prepare and/or may have limitations regarding specificity, sensitivity and stability. The recently developed macroscopical agglutination antigens!®17.104 have not been used widely enough to warrant recommendation as a standard technic. The hemolytic antigens?*2* have been advantage- ously used with human sera but have limitations in testing animal sera. Furthermore, the value of these antigens in epidemiological surveys is equivocal. For these reasons, only the microscopical technics are described here. Preparation of Antigens (General) Antigens for the microscopical tests consist of young, actively growing cultures of leptospiras propagated in fluid medium. Media containing suspended agar are not considered satisfactory for the preparation of antigens, since the agar particles interfere with the serological reaction as well as the reading. The cultures used as anti- gens should be 4 to 7 days old. They can be cultivated conveniently in screw-cap culture bottles or resistance-glass prescription bottles. Where tests are not performed frequently enough to warrant serial transplants of antigen cultures in large quantities, then small-volume seed cultures should be transplanted regularly at the same interval of time selected for propagation of the large-scale antigen cultures, to keep the leptospiral strains in a uniform, active state of growth. The formulas for these media are given in Chapter 4 (CM Nos. 88 through 92). Most workers in this field have found it advisable to use amounts of inoculum equal to 5 to 10 per cent of fresh medium for transplant of antigen cultures to insure sufficient density of growth by the end of the incubation period. Most laboratories will find it advantageous to transfer antigen cultures regularly each week. Some strains of leptospiras, especially in older cultures, tend to form microcolonies or “breed nests,” which consist of tangles or packed masses of active leptospiras. Generally, the formation of microcolonies is prevented by the frequent, regular transplant of anti- gen cultures. Some Leptospira strains form many microcolonies, even after frequent transplants. In such cases it is advisable to obtain new strains, of the same serotype, which do not tend to form ag- glomerations. When only a few microcolonies are present in an antigen culture, they may be removed by differential centrifugation at 1,500 to 2,000X G for 15 to 30 min, since the microcolonies tend to be thrown out of suspension more rapidly than individual leptospiras. The whole live culture may be used as antigen for the microscopical agglutination. 560 LEPTOSPIROSIS Source: Gsell (Reference 27) Figure 1—Leptospirosis: (a) Control suspension without serum; (b) agglutination, many cells unaffected; (c) agglutination and partial “lysis”; (d) “lysis”; (e) discard, do not use; and (f) agglutination, occasionally observed with live antigen and usually with formalin-treated cells. LEPTOSPIROSIS 561 Two types of reaction for positive serum are observed micro- scopically : agglutination and so-called “lysis.” Agglutinated cells are generally circular in outline, although occasionally some antigens may agglutinate along their longtitudinal axis, giving a “frayed rope” appearance. When “lysis” is manifest, few freely moving leptospiral cells are seen and small refractile granules in which individual cells may not be discernible are present. Formerly believed to be cellular residues of lysed cells, these lysis granules are now known to be tightly packed conglomerations of intact cells'® (see Fig 1). Positive re- actions with formalin-treated cultures (final concentration of formalin =0.3%) are manifest by agglutination only. Before each series of tests is to be conducted, the culture should be examined microscopically to determine its condition. Cultures containing a large proportion of leptospiras which are disintegrated or agglutinated because of nonspecific reactions should be discarded. Contaminated cultures should not be used. Generally, live cultures retain uniform sensitivity for several days when kept at the tempera- ture of incubation after attainment of peak density. With care in preparation, formalinized cultures can almost always be used reliably for more than a week when kept refrigerated. However, both types of antigens should be observed microscopically and tested serologi- cally at 2 to 4 day intervals to insure that they are being maintained in a uniform state of morphology, dispersion and serological activity. Cultures containing too few leptospiras may yield false-positive results or an exaggerated end-point titer. Very dense cultures tend not to be sensitive enough and may depress the end-point titer of the antisera tested. The recommended density is roughly 50 to 100 million organisms per ml. Methods of determining and adjusting the density are given by Borg-Petersen.10 Although microscopical observation of antigen appearance and standardization of antigen density are valuable adjunctive means of controlling quality, definitive determination of sensitivity can best be accomplished by titration against standard homologous antisera. In laboratories conducting frequent or daily runs of tests, the positive and negative serum control series will serve to evaluate antigen quality. In laboratories conducting tests 3 days a week, the antigens should be titrated against standard homologous antisera before the test run. Control antisera are prepared according to procedures given later herein. Antigen sensitivity tests are conducted according to the same schema given in the description of the diagnostic test. Antigens yielding a titer greater or less by fourfold than the standard titer of the control homologous antiserum should not be used. With live 562 LEPTOSPIROSIS antigens, adjusting the density may correct hyposensitivity if the culture is not senescent. However, hypersensitivity of either type of antigen or hyposensitivity of the killed antigen indicates that physico- chemical instability has rendered the antigens unreliable for sero- logical use. Preparation of Standard Control Antisera Because of the marked agglutinin production which is characteristic of its immune response, the rabbit is the most suitable laboratory animal for the production of standard antisera. The regimen of inoculation recommended by Wolff?® consists of the injection of live cultures of leptospiras, at 4 or 5 day intervals, into the marginal ear veins. The initial dose is 0.5 to 1 ml, and succes- sive doses are 1 to 2 ml, 2 to 4 ml, and 4 to 6 ml. The last dose can be repeated once at option of the worker. Precautions should be taken in the handling of animals inoculated with live cultures. Such animals may become renal shedders of lepto- spiras and may be a source of leptospiral infection to handlers. In laboratories where established procedures militate against the use of live inoculum, whole Leptospira cultures killed by repeated slow freezing and thawing may be used as the immunizing antigen. The cultures are frozen in the freezing compartment of a mechanical refrigerator. Slow thawing is accomplished at room temperature. After two or three cycles of freezing and thawing, the culture is divided into convenient doses in separate containers. The doses are then frozen and kept in that state until the time of administration. After thawing, the antigen dose is inoculated into the marginal ear vein of the test animal. The suggested regimen consists of four doses: 0.5 ml, 1 ml, 1.5 ml and 2 ml given at 4 day intervals. Formalin- treated cultures or washed, fixed-cell suspensions from these can also be used as antigens. In any regimen the animals are bled by cardiopuncture or are ex- sanguinated 7 to 10 days after administration of the last antigen dose. The serum is separated from the clot, centrifuged free of cells, and preserved by refrigeration, preferably in the frozen state. Some laboratories sterilize the serum by filtration, for example, through Seitz filters, prior to storage. The serum is best preserved by refrigera- tion and aseptic handling but it can also be preserved chemically by the addition of glycerol or merthiolate as specified elsewhere in this work. Serum may also be preserved by freeze-drying. The serum should be titrated against fresh antigen, using a twofold dilution in- terval. The titration should be repeated to assure validity. LEPTOSPIROSIS 563 Leptospira Serotypes Employed At least 80 serotypes of Leptospira have been differentiated. Sero- logical and bacteriological studies of human and animal infections indicate that at least 12 serotypes or serogroup representatives have been isolated in the United States: L. icterohemorrhagiae L. grippotyphosa L. canicola L. hardjo L. powmona L. mini (Georgia) L. autumnalis L. australis (australis A) L. ballum L. hyos L. paidjen L. atlantae Numerous serotypes have global distribution and it is probable that many additional serotypes occur in the United States. To insure detection of leptospiral antibodies that may be elicited by any of the numerous diverse serotypes, it is necessary to employ a battery of screening antigens that encompass cross-reactions with known sero- types as well as with serotypes not heretofore recognized in this country. Preferably the selection of antigens should include types that have already been isolated. The following 10 serotypes are recommended for use as test antigens : L. icterohemorrhagiac L. hyos (bakeri) L. canicola L. australis (australis A) L. pomona L. alexi (pyrogenes serogroup) L. bataviae L. wolffi L. grippotyvphosa L. autumnalis To render more comprehensive diagnostic service, additional sero- types may be used: L. celledont L. borincana L. djasimin L. ballum L. javanica L. butembo Microscopical Equipment The basic instrument of critical importance in the reading of agglu- tination and agglutination-lysis tests is the dark-field microscope. The syphilology type possessed by most U. S. diagnostic laboratories, which has a 1.2 numerical aperture oil-immersion condenser, is not entirely practical. The necessity for using oil between slide and condenser and the critical focus make reading of microscopical tests unnecessarily tedious. Magnifications of 100 to 200X are ample for reading the microscopical tests ; therefore dry-system dark-field condensers of 0.6 to 0.8 numerical aperture are adequate. Furthermore, elimination of the oil-immersion feature saves much time in the reading of tests. 564 LEPTOSPIROSIS The ordinary bright-field Abbé condenser will render illumination adequate for reading the test when equipped with the dark-field con- version diaphragm supplied with most standard microscopes. The laboratory planning extensive work in leptospiral serology will find it advantageous to procure a microscope specifically for test read- ing purposes. The “reversed stand” microscopes with integral illum- ination systems are best suited for this work because of the accessi- bility of the stage and the fixed focus and centering of the illumina- tion. Proper selection of microscope equipment increases the effi- ciency of operation and accelerates work flow. Special equipment may pay for itself in savings of man-hours, Specific Reactions The reactions in the microscopical agglutination test can be graded conveniently in the following manner (see Fig 1): 4+ =all except occasional single cells agglutinated. 3+ =the majority of organisms are agglutinated—many clumps present in each field. 2+ =the number of leptospiras agglutinated is approximately equal to the number unaffected—at least one definite specific clump to each field. 14 =occasional small clumps or small stellate aggregations. The microscopical agglutination test is not a sharp-titered reaction and the degree of agglutination may fade through a range of serum concentrations. The presence of microcolonies may be confusing. However, this type of clumping is quite different from that due to positive serological reactions and can be differentiated from the latter by a comparison with positive and negative controls. There is con- siderable diversity among laboratories in the interpretation of end titer reaction and in the selection of dilution schema. Generally, the criterion for significant positive reaction varies from 1 plus to 4 plus agglutination at a 1:100 level of serum dilution (final dilution after addition of antigen). Many laboratories prefer to use an unequivocal reaction of 3 plus agglutination or greater as a basis for a positive reading. Typical grades of reactions are shown in Table 1 in tests using a fourfold dilution scheme starting with a final serum dilution of 1:100. The end titer is the highest dilution of serum that shows a 3 plus reaction. Note that prozone phenomena do occur, the prozone occurring in the greater serum concentration. In the case of serum A, and possibly of sera B and J, further dilutions would be required to de- termine accurately the antibody titer. In the case of sera H and I, dilutions of 1:25 might be made for stronger reactions. However, many laboratories consider reactions at final serum dilution levels of LEPTOSPIROSIS 565 Table 1—Examples of Agglutination Reactions with Leptospiras Reaction in Final Dilution Serum 1:100 1:400 1:1,600 1:6,400 Titer A 4+ 4+ 4+ 4+ >1:6,400 B 4+ 4+ 4+ 3+ 1:6,400 C dep A, 3+ 2+ 1:1,600 D 4+ 3+ 2+ 1+ 1:400 E 44 2+ 1+ 0 1:100 F 3+ 2+ 1+ 0 1:100 G 3+ 1+ 0 0 1:100 H 2+ 1+ 0 0 <1:100 (suspicious reactor) 1 1+ 0 0 0 Negative J 2+ 3+ 44 3+ 1:6,400 (prozone) K 1+ 4p 3+ 14+ 1:1,600 (prozone) less than 1:100 to be equivocal. This is true for sera from both human beings and livestock. The antibody in canine sera may not be detect- able at dilutions of 1:100, so that in these samples it may be desirable to initiate tests at 1:25 dilution levels. Although titers higher than 1:6,400 are not uncommon in active leptospirosis, the test described should be adequate to detect rising titers—the only reliable serological criterion of current infection. It is emphasized that no one titer can be considered diagnostic of current leptospirosis. Serial serum samples should be taken from the patient during the acute and later stages of the disease and should be tested simultaneously to confirm the diagnosis of leptospirosis by exhibiting a rising titer, This does not mean that sera taken during the acute stage should not be tested immediately to attempt to gain diagnostic indications. The microscopical agglutination test is highly serogroup- and sero- type-specific, often affording differentiation of the etiological agent by virtue of the comparative titers. For instance, the serum of a person infected by L. icterohemorrhagiae may show a 1:6,400 titer for this 566 LEPTOSPIROSIS species, may show a 1:1,600 titer for L. canicola, and may be nega- tive against the other serotypes used. Occasionally in the acute stage of a disease, however, more heterologous serum reactions may be found, and the patient may have even higher serum antibody titers against heterologous serotypes. Later in the course of the disease, antibodies for the specific serotype generally predominate. With certain serogroups, antigenic relationship is so close as to prevent etiological definition by differential titer. In this case, cross-agglu- tinin absorption tests may be helpful. For this technic the reader is referred to the monograph by Wolft.33 In general, conduct of the microscopical tests is enhanced by at- tention to detail, using aseptic procedures. Excessive bacterial con- tamination is to be avoided in the live antigen test because bacterial clumps make the reading of tests uncertain and the presence of some bacteria may cause nonspecific lysis of the leptospiras. Oven-baking of pipettes and tubes before use reduces contamination. Hemolysis or contamination in sera may not prevent agglutination ; nevertheless one cannot rely fully on the titers obtained with such specimens. Proper collection and preservation of specimens on the part of the clinician or field investigator paves the way for valid laboratory results. Although the initial establishment of facilities for the microscopical agglutination test with live cultures may seem tedious, the work be- comes simple once the routines are established. Reading and grading of tests require time and consideration on the part of the neophyte, but eventually, experience makes possible the grading of reactions at a glance. The Microscopical Agglutination Test with Live Antigens (Agglutination-Lysis Test) Preparation of Antigen 1) Use Leptospira cultures of maximum growth density (5 to 7 day old cultures preferred). Examine microscopically for bacterial contamination and nonspecific agglutination. If either of these changes is noted, do not use the antigen. Occasional clumps due to nonspecific agglutination can be removed by centrifugation, which renders the antigen suitable for use. 2) Examine for growth density. A good rule of thumb is 100 to 200 leptospiras for each microscopic field (using a 10X ocular and 45X objective). Do not use sparsely grown antigens. LEPTOSPIROSIS 567 3) Titrate each antigen against standard homologous antiserum to determine sensitivity, using the technic of the diagnostic test. Use only antigen of standard sensitivity. Very dense antigens may be hyposensitive but this can be corrected by dilution with medium. 4) After initial titration, the antigen can be used for several days or until morphological changes, autoagglutination, or changes in sensitivity occur, as indicated by microscopical examination or by the saline and serum control systems of the diagnostic tests. The Diagnostic Test—Since the microscopical tests are not sharp-titered, fourfold dilution intervals are employed to reduce the tedium of reading and the amount of materials used. These intervals are adequate to determine the titers of sera as well as to check for diagnostic rise of titer. 1) Place 9.8 ml of 0.85 to 0.9 per cent salt solution in a clean test tube and add 0.2 ml of test serum. Mix thoroughly with a pipette. The serum dilution thus prepared is 1:50. Known negative (normal) serum and standard Leptospira antiserum against the serotypes em- ployed may be similarly diluted for use as control sera. (Some labora- tories prefer to use as diluent a salt solution buffered with phosphate salts to pH 7.4.) 2) Prepare serial fourfold dilutions from a portion of the initial 1:50 serum dilutions to provide test dilutions of serum of 1:50, 1:200, 1:800 and 1 :3,200. 3) Prepare a series of four agglutination tubes for each of the antigens to be tested with the serum. Add 0.2 ml of the 1:50, 1:200, 1:800 and 1:3,200 serum dilutions respectively in the first, second, third and fourth tube of each series of the agglutination tubes for the diagnostic test. 4) Place 0.2 ml of antigen in each tube of the serum dilution series. Shake for a few seconds to mix. The final test dilutions, with addi- tions of the antigens, are 1:100, 1:400, 1:1,600 and 1 :6,400. 5) Incubate tests 2 to 3 hr at 32° C (some laboratories prefer to incubate tests overnight at room temperature). 6) Shake each tube briefly, place a drop on a slide with a dropper or pipette, spread to flatten and examine microscopically (10X objec- tive and 15X ocular) by dark-ground illumination without the use of cover slips. 568 LEPTOSPIROSIS The Microscopical Agglutination Test with Formalin-Fixed Antigen Preparation of Antigen 1) Vigorously growing 5 to 7 day old cultures of leptospiras are ex- amined microscopically to determine that the culture is of suitable density, of normal morphology, and free of excessive amounts of autoagglutination. 2) Add reagent-grade, neutral formalin to a concentration of 0.3 per cent by volume and allow antigen to stand 2 to 3 hr for complete fixation. Centrifuge for 5 to 10 min at approximately 500X G to remove larger aggregates, 3) Titrate against standard sera, using all the twofold dilutions given in the diagnostic test procedure. Hyposensitive, very dense anti- gen should be adjusted to proper density by the addition of medium base or salt solution containing 0.3 per cent formalin. Antigen should yield the standard titer of the controlled anti- serum within a twofold dilution. Variations of fourfold or greater cast doubt upon suitability of the antigen. 4) If formalinized antigen is not used for daily tests, and thereby not subjected to surveillance in the diagnostic test serum control systems, it should be retitrated every 3 or 4 days to assure stability. 5) Before each use, examine microscopically to determine whether morphology and dispersion of organisms are normal. Remove spontaneously agglutinated leptospiras by centrifugation. The Diagnostic Test—The technical procedure for diluting sera and adding antigen is the same as that outlined for the microscopical agglutination test with live antigens. The antigen-serum dilution mixtures are incubated at 35° to 45° C for 3 to 4 hr and examined microscopically as described. The end point is the highest dilution of serum (final dilution) showing agglutination. Nonspecific agglu- tination in the salt solution or serum controls invalidates the test for the antigens showing such aberrant reaction. Generally the reactivity of formalin-treated antigens is less sensitive than that of live antigens, and titers will be slightly lower. Although the over-all specificity of the live and fixed antigens is similar, the fixed antigens cross-react more broadly with diverse serotypes. IV. EVALUATION AND REPORTING OF RESULTS The best evaluation of any laboratory test can be made only in the light of all the pertinent clinical, epidemiological and technical infor- LEPTOSPIROSIS 569 mation available in the particular situation. In the case of leptospiro- sis, critical evaluation of laboratory results is of especial importance. here is a dearth of basic knowledge in many important areas: the biology of the microorganisms themselves; and the mechanisms in- volved in the manifold interrelationships of the host, the parasite, and the environment, In addition, it must be realized that while certain of the laboratory procedures described here (when performed by competent personnel) are considered to be highly specific, the tools employed in these tests are not well standardized. Few data are available regarding possible nonspecific or anamnestic reactions, There have been no extensive studies with leptospiral antigens on the sera of individuals having any of a number of other infections which may simulate leptospirosis clinically. These and other limitations of the available body of knowl- edge must be considered in attempts to define the meaning and/or significance of the results of all laboratory examinations. Bacteriological Examinations Isolation attempts—So far as is now known, the isolation of leptospiras from the bloodstream or cerebrospinal fluid is reliable evidence for laboratory confirmation of a clinical diagnosis of current illness. Recovery of leptospiras from human urine is usually indicative of an infection within the preceding 10 days to several months, although the duration extremes of leptospiruria in man are unknown. On the other hand, many animals shed organisms in their urine for extra- ordinarily long periods of time after an inapparent infection, as well as following clinical illness. The isolation of leptospiras from animal urine, therefore, gives no definitive information regarding probable date of infection or whether these organisms were actually responsible for a particular clinical syndrome, past or present. The significance of negative results following isolation attempts is even more difficult to define. If collection of the specimens has been properly timed and optimum conditions have been maintained throughout collection and handling, failure to recover the organisms on repeated attempts probably indicates no infection in a large majority of instances. Direct dark-field examination and staining technics—The possibility of failure or misdiagnosis by these methods is great. Posi- tive results should always be confirmed by cultural methods ; negative results may never be interpreted to mean absence of infection. 570 LEPTOSPIROSIS Serological Examinations Single specimens: A positive titer* in a single test constitutes presumptive evidence of infection with the organism dating back from the preceding week to several years. The height of the titer and the degree of cross-reaction may give some indication as to the recency of infection but cannot be relied upon to do so because agglutinating antibodies may persist at detectable levels for many years. Generally a titer of 1:1,600 or greater, determined according to outlined criteria, provides strong presumptive evidence of recent or current infection. A negative result does not rule out the possibility of infection. If the complement-fixation test is the technic employed, a positive result may limit more definitely the probable time of infection. In human infections, complement-fixing antibody detectable by present- day antigens and technics usually but not necessarily disappears from the bloodstream in from 3 weeks to 2 months after initial infection. Serial specimens with significant rises in titer: Demonstration of a significant rise in titer in the examination of at least two properly timed serum specimens is good evidence of recent infection with Leptospira. The definition of a “significant” titer rise must be made for each technic employed; in general, it may be defined as a rise ex- ceeding the limits of experimental error in the particular test. A four- fold or greater rise in the microscopical agglutination or agglutination- lysis test is usually considered to be significant. Serial specimens positive, but no rise in titer: Such results may be interpreted in one of at least three ways: (1) The illness observed was not leptospiral in etiology and the positive reaction represents some past infection with leptospiras; (2) the infection was with a serotype of Leptospira not used in the antigen battery and the titers observed were incited by heterologous strains; (3) the initial speci- men was obtained after the maximum rise in titer had occurred. There is suggestive evidence that some types of antibiotic therapy may sup- press antibody formation in leptospiral infections. This possibility may be of considerable importance and should be investigated more thoroughly. All serial specimens negative: Such results are good evidence for absence of infection with the antigen strains tested. However, certain strains of Leptospira are antigenically distinct and give little * The significance and specificity of titers of less than 1:100 are still in question. The term “positive titer” as used here means definite agglutination in serum dilutions of 1:100 or greater. LEPTOSPIROSIS 571 cross-reaction with other types. It is possible, therefore, that nega- tive results may not indicate absence of infection. The patient may have been infected with a strain of Leptospira antigenically unrelated to the serotypes employed in the tests. Furthermore, some persons apparently respond poorly to various antigenic stimuli. This individual peculiarity may explain the very occasional anomaly of the patient from whose bloodstream leptospiras have been isolated, even though all serological studies made on his serum, including those with the isolate, were negative. In summary, the following points should be emphasized: 1. Awareness of the limitations of the available basic information of all kinds including laboratory technics is essential to intelligent evalua- tion of laboratory findings. 2. Differential clinical diagnosis and epidemiological information must be combined with the results of bacteriological and serological examinations in arriving at an accurate diagnosis of leptospiral infec- tions, 3. A number of factors may affect the outcome of the laboratory tests used as diagnostic aids in the identification of leptospirosis: —The selection, timing of collection, and handling of the specimens to be examined, —The number of specimens examined, —The sensitivity and degree of standardization of the technics employed, —The experience and training of the laboratory technician, and —In some cases, apparently, the species of animal from which the specimens are taken. 4. The results of any serological test on a single specimen have limited significance. 5. The serotype of the infective strain of Leptospira cannot be de- termined with certainty by serological examination of the patient’s serum but must rest upon antigenic analysis of the organism itself. 6. Whenever feasible, laboratory confirmation of leptospiral infec- tion should include both bacteriological and serological findings. These, in turn, should be critically evaluated in the light of all other available data. Reporting of Results In many instances the type and amount of information given in the report of laboratory examinations for leptospirosis will be determined by the technics used and the purpose for which the tests are performed. 572 LEPTOSPIROSIS Any such report should contain (1) all data related to the identi- fication of the specimen, name of person submitting the specimen, patient’s name, date of onset of illness, specimen number, nature of the material to be examined, date of collection, date of arrival, and date reported; (2) designation of the laboratory technics employed; and (3) results of the examination. In the case of a positive bacteriological examination, a preliminary report should be made as soon as possible and should include a nota- tion that designation of the serotype of the strain will follow after typing procedures have been completed. The reporting of results of serological tests may vary somewhat, depending on whether the serum specimens from the tested individual were single or paired. For example, in the case of surveys for anti- body prevalence in a particular human or animal population, the presence or absence of detectable antibody at a certain level may be the only point of concern. In such cases, perhaps only one or two dilutions of serum will have been tested, and the report could con- ceivably consist only of a listing of “positive” and “negative” results, with a statement of the dilutions that have been tested. The primary concern in the results of serological examinations is whether or not a significant rise in titer was demonstrated; conse- quently all data should be tabulated so that this question can be answered at a glance. In addition to identification of the specimens there should be noted the duration of the patient’s disease at the time of collection of each specimen and the specific titers with each antigen strain used. If several different tests are performed on a particular specimen, the results of all tests should be tabulated in such a manner that they can be quickly compared and summarized. The particular design of the report form is largely a matter of individual - preference so long as all pertinent data are given in a clear and concise manner. Inclusion on the report form of a statement defining the significance of various laboratory findings is highly recommended, since it may be of considerable assistance to the person responsible for final evalua- tion of the particular case. RomerT H. Yacer, Lt. CoL.,, U. S. ArMY, Chapter Chairman AAroN D. ALEXANDER, PH.D. WirLiaM S. GOCHENOUR, Jr., Cor. U. S. ARMY Karr R. ReinuARrRD, D.V.M.,, PH.D. ABrAM B. Sravirsky, M.V.D., Pu.D. MartaA K. Warp, Sc.D. LEPTOSPIROSIS 573 REFERENCES 1. ~ 10. 11 12 13. 14. 15, 16. 17. 18. 19. 20. WEIL, A. Uber eine Eigenthumliche, mit Milztumor, Icterus und Nephritis Einhergehende, Akut Infectionskrankheit. Deutsches Arch. Klin. Med. 39:209-232, 1886. InapA, R., ¢t al. The Etiology, Mode of Infection, and Specific Therapy of Weil's Disease (Spirochactosia icterohaemorrhagica). J. Exper. Med. 23:377-402, 1916. Nocucur, H. Spirochaeta icterohaemorrhagiae in American Wild Rats and Its Relation to the Japanese and European Strains. J. Exper. Med. 25:755-763, 1917. ————— Morphological Characteristics and Nomenclature of Lepto- spira (Spirochaeta) icterohacmorrhagiae (Inada and Ido). J. Exper. Med. 27 :575-592, 1918. WabnswortH, A., et al. Infectious Jaundice Occurring in New York State; Preliminary Report of an Investigation, with Report of a Case of Accidental Infection of the Human Subject with Leptospira ictero- haemorrhagiae from the Rat. J. A.M. A. 78:1120-1121, 1922. Meyer, K. F., SteEwArT-ANDERSON, B., and Eppie, B. “Canicola Fever,” a Professional Hazard. J. Am. Vet. M. A. 93:332, 1938. JuNGHERR, E. Bovine Leptospirosis. J. Am. Vet. M. A. 105:276-281, 1944. Stites, W. W., and Sawyer, W. A. Leptospiral Infection (Weil's Disease) as an Occupational Hazard. J.A. M.A. 118:34-38, 1942. StEIGNER, K. F. Leptospirose als Berufskrankheit. Zbl. Bakt. I Ref. 162: 431-463, 1957. KortHOF, G. Infectie met Leptospira icterohaemorrhagiae bij den Hond. Trop. Dis. Bull. 28:309-310, 1931 (abstract). Worrr, J. W., and BrooMm, J. C. The Genus Leptospira Noguchi, 1917. Problems of Classification and a Suggested System Based on Antigenic Analysis. Documenta Med. Geograph. et Trop. 6:78-95, 1954. Breep, R. S., Murray, E. G. D., and SmrtH, N. R. Bergey’s Manual of Determinative Bacteriology (7th ed.). Baltimore: Williams & Wilkins, 1957. ALEXANDER, A. D., et al. Leptospirosis in Malaya. II. Antigenic Analysis of 110 Leptospiral Strains and Other Serologic Studies. Am. J. Trop. Med. 6:871-889, 1957. ScuUrFFNER, W., and MocHTAR, A. Versuche zur Aufteilung von Lepto- spirenstimmen mit Einleitenden Bemerkungen iiber den Verlauf von Agglutination und Lysis. Zbl. Bakt. Abt. I Orig. 101 :405-413, 1927. StoENNER, H. G. Application of the Capillary Tube Test and a Newly Developed Plate Test to the Serodiagnosis of Bovine Leptospirosis. Am. J. Vet. Res. 15:434-439, 1954. Garton, M. M.; Powers, D. K.; Harr, A. D.; and CornELL, R. G. A Rapid Macroscopic-Slide Screening Test for the Serodiagnosis of Lepto- spirosis. Am. J. Vet. Res. 19:505-512, 1958. MurascHr, T. F. Latex-Leptospiral Agglutination Test. Proc. Soc. Exper. Biol. & Med. 99:235-238, 1958. TerziN, A. L. Leptospiral Antigens for Use in Complement Fixation, Boiling of the Cultures and Acetone Treatment. J. Immunol. 76:366-372, 1956. RorusteIN, N., and WorLman, F. Studies of the Immunochemistry of Leptospires. III. Serologic Characterization of the Complement-Fixation Antigen. J. Infect. Dis. 105:280-287, 1959. Cuang, R. S., and McCowms, D. E. Erythrocyte-Sensitizing Substances from Five Strains of Leptospirae. Am. J. Trop. Med. 3:481-489, 1954. 574 21. 22 23. 24. 25. 26. 27. 28. 29. 30. 3. 32 3. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. LEPTOSPIROSIS CHANG, R. S,, et al. The Use of Erythrocyte-Sensitizing Substance in the Diagnosis of Leptospirosis. II. The Sensitized Erythrocyte Lysis Test. Am. J. Trop. Med. 6:101-107, 1957. Cox, D. D. Standardization and Stabilization of an Extract from Lepto- spira biflexa and Its Use in the Hemolytic Test for Leptospirosis. J. Infect. Dis. 101:203-209, 1957. , ALEXANDER, A. D., and Murpay, L. C. Evaluation of the Hemolytic Test in the Serodiagnosis of Human Leptospirosis. J. Infect. Dis. 101:210-218, 1957. Suarpe, C. F. Laboratory Diagnosis of Leptospirosis with the Sensitized- Erythrocyte Lysis Test. J. Path. & Bact. 76:349-356, 1958. WoobpwArp, T. The Protean Manifestations of Leptospirosis. Symposium on the Leptospiroses. Medical Science Pub. No. I 57-68. Washington, D. C.: Gov. Ptg. Ofc, 1953. AvrstoN, J. M. and Broom, J. C. Leptospirosis in Man and Animals. Edinburgh: E. & S. Livingstone, 1958. GskeLL, O. Leptospirosen. Bern: Verlag Hans Huber, 1952. Garton, M. M. Identification of Two Leptospiral Serotypes New to the United States. Pub. Health Rep. 74:141-148, 1959. Beeson, P. B,, Hankey, D. D., and Cooper, C. F. Leptospiral Iridocyclitis. Evidence of Human Infection with Leptospira pomona in the U. S. J.AM.A. 145:229-230, 1951. ALEXANDER, A., et al. Leptospiral Uveitis. Report of a Bacteriologically Verified Case. Arch. Ophth. 48:292-297, 1952. Yacer, R. H. Discussion in Symposium on the Leptospiroses. Medical Science Pub. No. I 81-82. Washington, D. C.: Gov. Ptg. Ofc, 1953. Beeson, P. B., and Hankey, D. D. Leptospiral Meningitis. Arch. Int. Med. 89:575-583, 1952. Worrr, J. W. The Laboratory Diagnosis of Leptospirosis. Springfield, 111. : Charles C Thomas, 1954. Bryan, H. S., Ruoapes, H. E., and WiLLican, D. A. Studies on Lepto- spirosis in Domestic Animals. II. Isolation of Leptospira pomona from Aborted Swine Fetuses. Vet. Med. 48:438, 1953. Pooewarre, G. D., et al. Isolation of Leptospira pomona from Three Aborted Bovine Fetuses. Vet. Med. 50:164-165, 1955. BAKER, J. A. and Lirtie, R. B. A Virus Causing Abnormal Milk in Cattle. Proc. Soc. Exper. Biol. & Med. 63:406-407, 1946. WiLson, G. S., and Mites, A. A. Principles of Bacteriology and Immunity. Baltimore : Williams & Wilkins, 1946. UnLENHUTH, P., and Fromme, W. “Weilsche Krankheit.” In Handbuch der Pathogenen Mikroorganismen (3rd ed.). Edited by W. Kolle, R. Kraus, and P. Uhlenhuth. Jena: Gustav Fisher, Vol. 7, pp. 487-660. StaviTsky, A. B. Preservation of Leptospira icterohemorrhagiae in Vitro. J. Bact. 50:118-119, 1945. UnrenuaUuTH, P., and Fromme, P. Zur Aetiologie der Sog. Weilschen Krankheit (Ansteckende Gelbsucht). Berl. klin. Wchnschr. 54:269-273, 1916. WEINMAN, D., and McALLISTER, J. Prolonged Storage of Human Patho- genic Protozoa with Conservation of Virulence: Observations on the Storage of Helminths and Leptospiras. Am. J. Hyg. 45:102-121, 1947. WoLrr, J. W. Personal communication. Stuart, R. D. The Preparation and Use of a Simple Culture Medium for Leptospirae. J. Path. & Bact. 58:343-349, 1946. ARMSTRONG, J. C. Studies on the Colonial Growth of the Leptospirae. Master's thesis, University of Missouri, 1959. LEPTOSPIROSIS 575 45. 46. 47. 48. 49. 50. 51. 52 53. 54. 53, 56. 5. 58. 59. 60. 61. 62. 63. 65. 66. 67. ScHUFFNER, W. Meerschweinchen als Lebende Schnellfilter fiir Ver- unreinigte Leptospiren-Kulturen. Zbl. Bakt. Abt. T Orig. 145:341, 1940. Cox, C. D, and Larson, A. D. Colonial Growth of Leptospirae. J. Bact. 73:587-589, 1957. KirRsCHNER, L., and GraHAM, L. Growth, Purification and Maintenance of Leptospira on Solid Media. Brit. J. Exper. Path. 40:57-60, 1959. Brepe, H. D. Qualitativer Nachweis von Leptospiren—Antikorpern in Trockenblutproben. Zbl. Bakt. Abt. I Orig. 167 :21-37, 1956. Worrr, H. L. The Filter Paper Method for Shipping Blood Samples for Serological Examination. Trop. & Geog. Med. 10:306-308, 1958. Basupierr, B., and Casterrr, N. La Morfologia di Leptospira celledons. Rendi. d’ist. Sup. di Sanita 21 :698-700, 1958. Mousert, E. Elektronmikronskopishe Untersuchungen zur Morphologie von Leptospiren. Z. Hyg. Infektkrh. 141:82-90, 1955. Braprierp, J. R. G., and Cater, D. B. Electron-Microscopic Evidence on the Structure of Spirochaetes. Nature 169 :944-949, 1952. Basupieri, B. The Morphology of the Genus Leptospira as Shown by the Electron Microscope. J. Hyg. 47 :390-392, 1949. Breesg, S., GocHENour, W. S., Jr. and YAcGer, R. H. Electron Micros- copy of Leptospiral Strains. Proc. Soc. Exper. Biol. & Med. 80:185-188, 1952. Stavitsky, A. B. Studies on the Pathogenesis of Leptospirosis. J. Infect. Dis. 76:179-192, 1945. Frovisuer, M., Jr. “Dorner’s Nigrosin Stain,” in Fundamentals of Microbiology (6th ed.). Philadelphia: Saunders, 1957, p. 109. DeLAMATER, E. D., Haaves, M., and Wicearr, R. H. Studies on the Life Cycles of Spirochetes. II. The Development of a New Stain. Am. J. Syph. 34:505-518, 1950. Vaco, S. C. Intensified Form of the Mercurochrome-Methyl-Violet Stain for Spirochetes. Stain Tech. 28,2:87-88, 1953. Levine, B. S. Staining Treponema pallidum and Other Treponemata. Pub. Health Rep. 67:253-257, 1952. Kerr, D. A. Improved Warthin-Starry Method of Staining Spirochetes in Tissue Sections. Am. J. Clin. Path. (Tech. Suppl.) 2:63, 1938. STEINER, C., and STEINER, G. New Simple Silver Stain for Demonstration of Bacteria, Spirochetes, and Fungi in Sections from Paraffin-Embedded Tissue Blocks. J. Lab. & Clin. Med. 29 :868-871, 1944. Cameeerr, R. E.,, and RosauN, P. D. The Morphology and Staining Characteristics of the Treponema pallidum. Review of the Literature and Description of a New Technique for Staining the Organisms in Tissues. Yale J. Biol. & Med. 22:527-543, 1950. Meyer, K. F., Stewarrt-ANDERSON, B., and Ebppie, B. Canine Lepto- spirosis in the United States. J. Am. Vet. M. A. 95:710-729, 1939. RosenreLp, W. D., and Greene, M. R. Studies on the Metabolism of Leptospira. J. Bact. 42:165-172, 1941. CuANG, S. L. Studies on Leptospira icterohaemorrhagiae. I. Two New Mediums for Growing L. icterohaemorrhagiae, L. canicola, and L. biflexor, and a Method for Maintaining the Virulence of L. icterohaemorrhagiae in Culture, J. Infect. Dis. 81:28-34, 1947. . Studies on Leptospira icterohaemorrhagiae. 111. The Growth Rate of, and Some Biochemical Observations on, Leptospira ictero- haemorrhagiae in Culture. J. Infect. Dis. 81:35-47, 1947. Warp, T. G., and StarBuck, E. B. Enhancing Effect of Nicotinic Acid and Cysteine Hydrochloride on Growth of Leptospira icterohaemorrhagiae. Proc. Soc. Exper. Biol. & Med. 48:19-21, 1941. 576 68. 69. 70. 71. 72 73. 74. 75. 76. 77. 78. 79. 80. 81. 86. 87. 88. 89. LEPTOSPIROSIS Greene, N. R,, CamieN, M. N, and Dunn, M. S. Studies on the Nutri- tion of Leptospira canicola. Proc. Soc. Exper. Biol. & Med. 75:208-211, 1950. ScHNEIDERMAN, A. et al. Nutrition of Leptospira canicola. 11. A Chemically Defined Basal Medium Containing Purified Rabbit Serum Albumin. Proc. Soc. Exper. Biol. & Med. 78:777-780, 1951. ——— Nutrition of Leptospira canicola. 111. Utilization of Vitamins and Amino Acids. Proc. Soc. Exper Biol. & Med. 82:53-56, 1953. Furron, J. D., and SprooNER, D. F. The Metabolism of Leptospira ictero- hemorrhagiae in Vitro. Exper. Parasitol. 5:154-177, 1956. Basubpieri, B., and Zarpr, O. Ricerche su Alcuni Fattori di Crescita per Leptospire Patogene e Saprofite. Atti IX Congr. Naz. de Microb. Palermo, April 5-7, 1956. MarsHALL, P. B. Measurement of Aerobic Respiration in Leptospira icterohaemorrhagiae. J. Infect. Dis. 84:150-152, 1949. Heverin, J. J, and Hriarr, C. W. The Effect of Fatty Acids on the Respiration of Leptospira icterohemorrhagiae. J. Infect. Dis. 100:136-140, 1957. WoraTz, H. Wachstumversuche mit Fettsduren an Leptospira canicola. Zbl. Bakt. Abt. I Orig. 169 :269-274, 1957. ALEXANDER, A. D. Personal communication. Menges, R. W., Garton, M. M,, and Harr, A. D. Diagnosis of Lep- tospirosis from Urine Specimens by Direct Culture Following Bladder Tapping. J. Am. Vet. M. A. 132:58-60, 1958. Stuart, R. D. The Importance of Urinary Antibodies in the Diagnosis of Leptospirosis. Canad. J. Microbiol. 2:288-297, 1956. Runge, J. M. Observations on the Efficiency of Animal Inoculation for Isolating Leptospirae from Kidney Tissue. New Zealand Vet. J. 6:15-16, 1958. Kensy, S. C, et al. Detection of Viable L. pomona in Bovine Kidneys after Leptospiruria Had Apparently Ceased. Vet. Med. 53:647-648, 1958. GocHENOUR, W. S., Jr.; Yacer, R. H.; Wermorg, P. W.; and HicH- TOWER, J. A. Laboratory Diagnosis of Leptospirosis. A.J.P.H. 43:405-410, 1953. . VAN per HorpEN, J. The Pathogenicity of Leptospiras to Field Rodents in Israel (A New Test Animal for Use in Leptospira Research). J. Infect. Dis. 95:213-219, 1954. . YAGer, R. H.; GocHENOUR, W. S., JR.; ALEXANDER, A. D.; and WETMORE, P. W. Natural Occurrence of Leptospira ballum in Rural House Mice and in an Opposum. Proc. Soc. Exper. Biol. & Med. 84 :580-590, 1953. StoENNER, H. G., and MacLean, D. Leptospirosis (L. ballum) Contracted from Swiss Albino Mice. Arch. Int. Med. 101 :606-610, 1958. . Hoag, W. G., GocHENour, W. S., Jr, and Yacer, R. H. Use of Baby Chicks for Isolation of Leptospires. Proc. Soc. Exper. Biol. & Med. 83:712-715, 1953. Grerser, C. A. Jamnes, W. and Byrne, R. J. Avian Leptospirosis: Studies on Chick Embryo Culture. Cornell Vet. 45:296-304, 1955. Division of Veterinary Medicine, Walter Reed Army Institute of Re- search, Unpublished data. Fisuer, G. W.,, Powers, D. K,, and Garton, M. M. Studies of Technics for the Isolation of Leptospires. I. The Suitability of One to Two Day Old Baby Chicks as Compared to Hamsters for the Isolation of Various Leptospiral Serotypes. J. Infect. Dis. 103:150-156, 1958. Broom, J. C. Leptospirosis in Tropical Countries. A Review. Tr. Roy. Soc. Trop. Med. & Hyg. 47:273-291, 1953. LEPTOSPIROSIS 577 90. a1. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. GocHENOUR, W. S., Jr, Greiser, C. A., and Warp, M. K. Laboratory Diagnosis of Leptospirosis. Ann. New York Acad. Sc. 70:421-426, 1958. YAGER, R. H., and GLEISER, C. A. Unpublished data. Van TuiIeL, P. H. The Leptospiroses. Leiden: Univ. of Leiden Press, 1948. Turner, T. B., and Freming, W. L. Prolonged Maintenance of Spiro- chetes and Filtrable Viruses in the Frozen State. J. Exper. Med. 70:629, 1939. Monrux, W. S. The Incidence of Leptospirosis in the Rat Population of Ithaca, New York, and Vicinity. Cornell Vet: 38:57-69, 1948. Woratrz, H. Ein Fester Nidhrboden fiir Dauerkulturen Pathogener Lep- tospiren. Z. Hyg. Infektkrh. 134:78-80, 1952. SHIGEKAWA, J. M., and Stockton, J. J. Studies on the Preservation of Leptospira icterohaemorrhagiae by Freezing. Am. J. Vet. Res. 16:619- 622, 1955. ANNEAR, D. I. Observations on the Preservation by Drying of Leptospirae and Some Other Bacteria. Austral. J. Exp. Biol. & M. Sc. 36:14, 1958. Horranper, D. H., and NEL, E. E. Improved Preservation of Treponema pallidum and Other Bacteria by Freezing with Glycerol. Applied Microbiol. 2:164-170, 1954. Diagnosis and Typing of Leptospirosis. Report of Study Group. WHO Tech. Rep. Ser. No. 113, 1956. Yacer, R. H., et al. Complement Fixation in Diagnosis of Human Lep- tospirosis. Fed. Proc. 10(1) :424-425, 1951. Muraschr, T., CLemons, O., and Tompkins, V. Ethylene Glycol Extracts of Leptospirae in Complement-Fixation Tests. Proc. Soc. Exper. Biol, & Med. 92:274-277, 1956. Pike, R. M,, Owens, H. B.,, and Humes, W. The Plate Complement- Fixation Test with Leptospiral Antigens. J. Lab. & Clin. Med. 44 :609-613, 1954. Scuusert, J. H.; CarriNGTON, L. B.; CoNNER, E.; and HoLpEMAN, L. V. Whole Leptospira Suspensions as Antigens in the Complement-Fixation Test for Leptospirosis. Am. J. Hyg. 63 :254-260, 1956. StoENNER, H. G. A Capillary-Tube Test for Leptospirosis. Am. J. Hyg. 57(3) :316-327, 1953. LAWRENCE, J. J. The Lysis of Leptospires by Antiserum. Austral. J. Exp. Biol. & M. Sc. 33:91-101, 1955. Borg-PeTERSEN, C., and Facrarus, A. The Influence of the Antigen Density and Other Factors on the Serum Titer in the Agglutination- Lysis Test for Leptospirosis. Acta path. et microbiol. scandinav. 26:555- 567, 1949. CHAPTER 20 ANTHRAX I. Introduction II. Collection and Handling of Specimens ITI. Bacteriological Examination Identification of B. anthracis A. Microscopical Examination B. Cultural Methods C. Animal Inoculation IV. Serological Examination V. Evaluation and Reporting of Results References I. INTRODUCTION Anthrax, one of the oldest and most destructive infectious diseases of animals, is of great historic interest to the bacteriologist because of its close association with early discoveries that led to the develop- ment of the modern science of bacteriology and immunology. Bacillus anthracis, the specific cause of anthrax, is a Gram-positive nonmotile, aerobic, spore-forming rectangular-shaped bacterium of relatively large size (4-8uX1-1.5u). The bacilli possess a high degree of virulence, under certain conditions forming highly resistant spores which may retain their viability and virulence in contaminated animal products and other material for many years. This fact is of great hygienic importance and is the chief reason for the widespread geo- graphic distribution of the disease and for the difficulty encountered in its control and eradication. Virtually all warm-blooded animals are susceptible to anthrax in some degree. Natural infection occurs chiefly in herbivorous animals such as cattle, sheep, goats, horses and mules, which are highly susceptible and which usually develop the disease in an acute or sub- acute generalized, septicemic form. Omnivorous types—man and swine—possess a greater natural resistance, swine usually acquiring the disease in a chronic localized form. Although highly resistant, 578 ANTHRAX 579 dogs, cats, mink and other carnivora, as well as birds, may become in- fected under certain conditions. Laboratory animals such as mice, guinea pigs and rabbits are very susceptible, while rats are less so. In herbivorous animals, infection is usually the result of grazing on infected pasture land. The infection may also be caused by con- taminated feeds or by the bites of contaminated flies. Although primarily a disease of animals, anthrax is transmissible to man by contact with infected animals or their carcasses or by manipulation of infected animal products, such as hides, hair, wool, ivory and bone or other contaminated material. The disease may occur in man as a cutaneous, pulmonary or in- testinal infection. A meningitic form has also been reported but is rarely observed. The external carbuncle is the most common form, occurring as a primary localized infection of the skin, chiefly on ex- posed areas like hands, arms, neck or face. The lesion first appears as a small pimple, which rapidly develops into a large vesicle with a black necrotic center, often referred to as a malignant pustule. If un- treated, the localized infection may become generalized, resulting in a fatal septicemia. The pulmonary form (referred to as woolsorter’s disease) chiefly affects the lungs and is caused by inhalation of spores during the processing of hair and wool. An intestinal form of an- thrax, which is rare in the United States, sometimes follows the con- sumption of contaminated meat. The pulmonary and intestinal forms of the disease frequently terminate fatally, Importance of Laboratory Examination A presumptive diagnosis of anthrax in man or animals based on clinical symptoms should always be confirmed by laboratory diagnosis. The laboratory examination on which final and conclusive diagnosis is based therefore entails a grave responsibility and should be promptly and carefully carried out. For the laboratory diagnosis of anthrax the methods commonly used in identifying the organism include, first, a microscopic examina- tion of blood or tissue films properly stained with polychrome dyes for the detection of encapsulated bacilli having the morphologic charac- teristics of B. anthracis; second, a cultural examination on plain agar and blood agar plates for characteristic anthrax colonies showing no or only slight hemolysis, followed by tests for motility, Gram-stain reaction, and reactions on different types of media; and, third, regardless of whether the microscopical and cultural tests are negative or positive, pathogenicity tests carried out on guinea pigs or mice, preferably the former. 580 ANTHRAX In old and contaminated animal material, where it is difficult to isolate the organism—for example, hides suspected of being contamin- ated—the thermoprecipitin test of Ascoli! is sometimes employed to identify the antigens. Il. COLLECTION AND HANDLING OF SPECIMENS Animals—Tissue specimens selected for laboratory examina- tion should be collected a short time following death of the animals, since specimens from carcasses showing evidence of considerable de- composition are unsuitable for laboratory examination. Specimens for laboratory examination should if possible include (1) blood or spleen films on clean glass slides; (2) sterile cotton swabs, gauze or suture tape saturated with spleen pulp; (3) thick dried blood film from a peripheral blood vessel; or (4) a few drops of blood drawn with a sterile syringe and transferred to a sterile vial and sealed. Specimens should be placed in clean sterile containers labeled “Sus- pected Anthrax” and sent to the laboratory in sealed metal mailing tubes. All specimens should be properly identified and the history of the case or outbreak furnished when available. Ears and spleen tissue collected a short time after death and brought to the laboratory within a few hours constitute very satisfactory material for examination. In cases of localized anthrax in animals, specimens obtained from the edematous swellings should be submitted for examination. When suspected cases of chronic anthrax in swine are encountered, speci- mens of the cervical lymph nodes packed in borax should be sub- mitted for examination, as anthrax organisms can rarely be demon- strated in the bloodstream in this species. Man—In suspected cases in man, specimens may be obtained from cutaneous lesions by making films or taking swab specimens from the vesicles or necrotic center of the anthrax carbuncle or by taking stab specimens from edematous lesions. In septicemic cases, blood samples should likewise be collected for examination. The specimens should whenever possible be collected before treatment of the patient with antibiotics, Industrial products—Specimens of industrial material sub- mitted for laboratory examination, such as hair, fur, wool, pelts, bone meal, meat scraps, blood meal, dust, soil or mixed feeds, should repre- sent a composite sample of the suspected material. Laboratory personnel engaged in handling specimens, films, cul- tures, laboratory animals and the like should take every precaution to ANTHRAX 581 avoid contracting the infection or contaminating the laboratory. Where possible all laboratory work should be carried out in an iso- lated unit (see Chapter 3). Ill. BACTERIOLOGICAL EXAMINATION Identification of B. anthracis A. Microscopical examination—From fresh tissue specimens that show little or no contamination with extraneous organisms, a tentative diagnosis of anthrax can usually be made by a preliminary examination of properly stained films of blood or other tissue. In cases where the affected animal dies of generalized anthrax the bacilli usually are numerous in the blood and in all the organs con- taining blood, especially the spleen. However, fatal cases may occur in some animals such as swine, horses and dogs in which few or no microorganisms appear in the general bloodstream. In these in- stances the localized edematous areas and the lymph glands, particu- larly in the region of the neck in swine and dogs, should be examined. Putrefactive anaerobic bacilli and others having a morphological resemblance to B. anthracis are sometimes mistaken for the latter. A tentative diagnosis based on morphological findings, therefore, should always be confirmed by cultural and animal inoculation tests. In films of blood or other tissue made on glass slides and stained with any of the common dyes, B. anthracis appears as large, straight- sided, square-ended rods occurring singly or in chains of two or more bacilli. The bacilli found in tissue specimens are usually en- capsulated. In films stained with ordinary dyes, the capsule is not well defined but often appears as a halo or as poorly stained granular material surrounding the microorganism. Giemsa’s or Wright's stain is excellent for revealing the pinkish-colored capsule which surrounds the dark bluish-colored bacillus. The M’Fadyean® methylene blue reaction, which is often used in the field for making a rapid tentative diagnosis, consists of fixing fairly thick films of blood or spleen pulp, after drying, by passing through a flame several times and staining with polychrome methyl- ene blue for a short time. This staining technic, when properly carried out, usually works well with fresh specimens, and if the slide is held up to the light and examined it will be noted that the film is stained a characteristic purplish color resulting from disintegration of the capsule by heat. When examined microscopically, the microorganisms appear as straight, square-ended dark blue rods surrounded by an amorphous 582 ANTHRAX purplish, stained material which represents the disintegrated capsules —the M’Fadyean reaction (see Fig 1). When stained films are prepared from dry material like blood or spleen pulp on swabs, microscopic examination may reveal disinte- grated microorganisms, shadow forms, sporulating bacilli or the small oval-shaped refractile spores either in chain formation or as free spores, singly, in pairs or in groups. Films made from the broken- down center of an anthrax carbuncle may likewise reveal sporulating bacilli (Fig 2). The belief that anthrax bacilli in tissues never show spores unless the tissue has been exposed to the air for some time is one widely accepted in the past, but a number of investigators (Howie and Cruickshank? Soltys,* and Nordberg®) as well as the authors have demonstrated sporulating anthrax bacilli in the blood and tissues of animals following inoculation with spore suspensions. Figure 1—Stained films showing anthrax bacilli: A, encapsulated bacilli in sheep blood (methylene blue), magnification 1,700 Xx ; B, bacilli in guinea pig blood (Giemsa stain), magnification 1,600 X. Figure 2—Photomicrographs of stained anthrax spores: A, sporulating organisms in chain formation from spleen swab (bovine field case), magnifica- tion 1,400 X ; B, free anthrax spores from culture, magnification 1,900 Xx. ANTHRAX 583 Of considerable importance to remember in handling anthrax slides is that spores in films on slides may remain viable after fixing and staining. B. Cultural methods—Since B. anthracis will grow well on all ordinary media, it can be cultivated readily from infected tissue speci- mens providing such specimens are not heavily contaminated with extraneous microorganisms that crowd out, overgrow or obscure the growth of anthrax bacilli. A suspension is made from the blood or tissue specimen with sterile salt solution and a few drops are streaked or inoculated on the surface of a series of plain agar plates, pH 7.2, with a platinum spreader. The plates are incubated 18-24 hr. If B. anthracis is present in specimens showing little or no contamination with extraneous microorganisms, characteristic colonies representing a pure culture are usually obtained. The colonies of virulent strains of B. anthracis isolated directly from infected animal blood or tissues, when growing on the surface of plain agar plates, are characteristic but not diagnostic. They are the typical rough type, relatively flat and granular, with a dull, grayish white surface and an irregular margin. They are small or medium-sized and irregular in shape. When examined with trans- mitted light the colonies are opaque and have a cut-glass appearance. Colonies have a tough stringy consistency and when transferred with a platinum loop they tend to come away in threads or strips (Fig 3). Figure 3—Anthrax colonies on surface of plain agar plate, 18 hr growth, seeded direct from salt solution suspension of bovine anthrax spleen: A, typical rough, flat, granular, grayish white irregular-shaped colonies half natural size; B, small colony; C, large comet-shaped colony. Note rough ground-glass appearance of both colonies, magnification 10 Xx. 584 ANTHRAX Examination under low magnification shows the colony to be com- posed of a mass of closely twisted filaments resembling bundles of wavy hair or sheep wool, with a dense center and a somewhat lighter, wide wavy margin. Under high magnification the typical hair lock (so-called Medusa head) appearance may be observed at the margin of the colony. The filaments are revealed as parallel-arranged loops consisting of closely packed long chains of square-ended bacilli arranged end to end. The wavy segmented chains of bacilli have a tendency to turn back into the mass of the colony but with few, if any, loose ends at the margin (Fig 4). a a od om * # wm Tey Figure 4—Photomicrographs of a stained anthrax colony: A, colony from anthrax field strain, magnification 45 X ; B, extreme margin of a portion of the border shown in A (note the wavy, hairlike parallel arrangement of bac- terial filaments, magnification 400 Xx ; C, loop shown in B highly magnified (1,600 x) to show arrangements of individual bacteria composing the long chains. (All illustrations reduced about 3.) This description applies only to virulent field strains of B. anthracis which occur in the rough form. Dissociated, avirulent or vaccine strains often appear in the smooth or intermediate form with very different colony characteristics. Except in cases of vaccine anthrax in animals and chronic anthrax in swine, however, these differences have no significance in diagnosis. A number of the nonpathogenic aerobic spore-bearing bacilli, com- monly found in the soil, in dust, and as laboratory contaminants, form colonies which resemble those of B. anthracis. Some of these anthrax- like colonies can be differentiated from anthrax colonies by placing a flamed cover glass on the suspected colony and examining it with the 4 mm objective. If motility is detected at the periphery of the colony and numerous short crisscrossing filaments are observed instead of ANTHRAX 585 the long wavy filaments characteristic of anthrax colonies, the possi- bility of its being anthrax is eliminated. For the principal points of difference between R-type anthrax colonies and anthrax-like colonies, see Table 1. Descriptions of colony forms of anthrax and anthrax- like organisms are given by Hagan® and Stein.” B. anthracis in gelatin stab cultures shows a characteristic “inverted fir tree growth” with slow liquefaction of the medium. Tests for hemo- lysis, reduction of methylene blue, and fermentation of salicin are of some value in helping to distinguish anthrax organisms from some of the anthrax-like organisms, especially when considered with motility and colony characteristics. For example, a microorganism that pro- duces rapid, marked hemolysis in 24 hr and that reduces methylene blue broth or produces marked acidity in salicin within 48 hr is not likely to be B. anthracis. The differentiation of B. anthracis from the anthracoid bacilli is discussed by Eurich and Hewlett,® Nordberg,? Burdon,? 1° and Stein.!! C. Animal inoculation—Inoculating laboratory animals is the most reliable method of isolating and identifying anthrax bacilli in infected tissue and industrial material. However, the test may mis- carry in instances where the material containing anthrax bacilli is con- taminated with extraneous microorganisms antagonistic to anthrax or with anaerobes that cause death of the experimental animals before anthrax infection has had time to develop. White mice and guinea pigs are generally used as test animals, the latter preferred on two counts—their greater resistance to anthrax- like bacilli such as B. cereus and B. mycoides and their high suscepti- bility to anthrax. Specimen material should be inoculated subcutane- ously in the thigh, dorsal region or abdominal region in doses of 0.1 ml for mice and 0.25 ml for guinea pigs. Special caution should be taken to avoid injecting the material into the peritoneal cavity. When the inoculum is contaminated with microorganisms producing putre- faction, the so-called scratch method of inoculation should be used. This consists of rubbing the inoculum into scarified areas of the skin. This method of superficial inoculation favors the development of anthrax bacilli when these are present but discourages the growth of anaerobes. Inoculated animals usually show little evidence of the disease for 24 to 30 hr except the appearance of doughy swellings at the site of inoculation. Acute illness usually appears 2 or 3 hr before death. Inoculated animals generally die in less than 48 hr but deaths may occur after 3 to 5 days or longer. When animals die in less than 24 hr, death is usually due to infection with anaerobes. Table 1—Chief Points of Difference Between Bacillus anthracis (R Type) and Anthrax-Like Organisms! Characteristic B. anthracis B. cereus B. stamensis B. tropicus B. mycoides B. subtilis B. mesentericus Pathogenicity for guinea pigs +2 = po — wo -— - Motility (hanging drop method) — + or ‘+ +4 + —3 +++ +t Reduction of methylene blue broth, 48 hr —- ++ of seeds — V4 —_ Marked acid in salicin, 48 hr — + or —5 — + AA V4 + Complete hemolysis of sheep blood cells® (tube test) —7 +++ +++ wj=dls —7 —T7 st Liquefaction of gelatin Slow Rapid Rapid Rapid Slow Slow Slow Action on litmus milk (peptonization) Slow Rapid Rapid Rapid Slow Slow Slow Slightly General broth character- Clear or turbid, then istics slightly turbid Turbid Turbid Turbid Clear clear; pellicle. Turbid Growth on agar Texture tena- Texture soft—easily removed from medium. Rhizoid Dry, Texture soft cious—comes growh ad- wrinkled —easily re- off medium in heres to and growth moved from threads. penetrates adheres to medium. medium. medium. 1 Characteristics given are those common to most strains examined. 2 Usually encapsulated in blood and tissues of infected animals. 3 Some strains are reported to be motile, ¢ V=variable (+ or =). 5 Most strains are acid-positive. ¢ In 5% suspension. 7 Some strains exhibit very slight hemolysis, ur bd oo XVIHLNV ANTHRAX 587 Lesions of Anthrax in Experimental Animals For performing a post-mortem examination of laboratory animals that die of suspected anthrax, the carcass is first tied out on a leak- proof tray and the abdomen wetted down thoroughly with a disin- fectant solution such as 1:1,000 bichloride of mercury or 5 per cent phenol to lay the hair or fur. The skin is then incised down the median line and reflected from the entire abdominal floor. At the point of inoculation a yellow gelatinous exudate will usually be found in posi- tive cases. In some instances the exudate is quite extensive, spreading through the connective tissue over most of the abdomen. The blood vessels in this area are engorged and there may be hemorrhages at the point of inoculation. The tissues, however, are not reddened, as in many of the phlegmons produced by anaerobic bacilli, and gas is never present. The blood is darker in color than normal, and usually unco- agulated. Generally, the mesenteric vessels and internal organs are con- gested. The most characteristic change is observed in the spleen, which is greatly enlarged, soft, and much darker than normal. A congested, dark mahogany-colored liver is also a constant finding. Anthrax bacilli can be demonstrated in stained films made from any of the tissues and especially from the engorged spleen. The microorganism can be readily recovered from the blood and other tissues of the dead animal for study of its cultural features to confirm its identity. Examination of Industrial Material for the Presence of B. anthracis Certain industrial material such as wool, hair, bristles, fur, skin, bone meal, blood meal, meat scraps, mixed feeds containing animal proteins, soil and dust suspected of being contaminated with anthrax spores are often submitted for laboratory examination. Since samples of such materials are usually heavily contaminated with extraneous microorganisms of different types, specimens are subjected to special treatment prior to bacteriological examination. Specimens of hair, wool, bristles or skin (hides or pelts) are first cut into small fragments and placed in a sterile flask with several volumes of sterile physiological salt solution, then shaken thoroughly in a mechanical agitator. If the material appears to contain consider- able fat or grease, it should be treated with an emulsifying agent such as a 3 per cent solution of potassium hydroxide (caustic potash) to facilitate the recovery of anthrax spores. Lloyd!? recommends a hydrocarbon sulfonate detergent in a 1 per cent sterile aqueous solu- tion, known as Duponol MP-189, for this purpose because of its ex- 588 ANTHRAX cellent properties of emulsifying fats, low alkalinity and nonirritating effect when injected into mice, In handling other types of industrial material like bone meal, soil or meat scraps, suspensions are first prepared from samples by adding 10 to 20 g of the material to 250 ml of physiological salt solution and thoroughly agitating for several minutes. The suspension prepared from different types of industrial material as described is allowed to stand for a short time to permit the heavy particles to settle out. The supernatant fluid is decanted and centri- fuged. The sediment is then resuspended in a small amount of normal salt solution and the suspension heated at 80° C for 10 min or at 65° C for 30 min to destroy nonsporulating microorganisms. After cooling the suspensions, proceed as follows: 1) Inoculate laboratory animals subcutaneously and by scratch method with the suspension. 2) Inoculate a series of plain agar and 5 per cent horse blood agar plates with the suspension and incubate 18 to 24 hr. 3) Pick nonhemolytic anthrax-like colonies and transfer to agar slants, extract broth, and gelatin. 4) Check broth culture for motility, morphology, Gram stain and purity. If cul- ture is pure and appears to be B. anthracis, inoculate into differential media for further studies. 5) If B. anthracis has not been recovered from animals inoculated with the heated suspension, inoculate others with broth cultures or with a suspen- sion of the agar slant culture. 6) If laboratory animals die, examine blood and spleen films for B. anthracis. Owing to the great difficulty encountered in isolating B. anthracis from industrial material, special cultural technics to facilitate its re- covery have been advocated and used with varying degree of success. Hagan and Bruner? have pointed out that the observation of deep colonies in poured agar plates inoculated with material suspected of containing anthrax spores is sometimes helpful in recovering the organism. The deep colonies of B. anthracis appear brownish, ragged and stringy, resembling small wisps of colored cotton or moss, while the deep colonies of most anthrax-like microorganisms are compact and some fail to grow. In a similar method advocated by Jones'* known as the sandwich agar plate method, the plates are composed of three layers of hardened agar, the middle layer being inoculated with the treated suspension. Examined by this method, the anthrax colonies appear like short pieces of cotton thread. Stein” has demonstrated that treatment with phenol of suspensions from animal tissue containing anthrax spores but heavily contamin- ated with extraneous microorganisms is an excellent method of de- stroying spreaders and other vegetative forms. In this method 0.5 ANTHRAX 589 ml of the suspension is thoroughly mixed with 5 ml of a 2 per cent phenol solution and allowed to stand at room temperature for 30 min. The resuspension is then streaked on plain agar plates and these plates are examined for colonies of anthrax bacilli after 18 to 24 hr incubation. Good descriptions of various methods employed for the isolation of B. anthracis from industrial material and soil samples are given by Llovd.'? Jones,'* DeKock et al.'® and McGaughey and St. George.!® IV. SEROLOGICAL EXAMINATION The thermoprecipitation test for the diagnosis of anthrax was de- veloped and first used by Ascoli! for the recognition of anthrax in- fection in animal tissue. While the test has been widely used in some European countries, principally for the detection of anthrax infection in hides and putrefactive material, it is not employed as routine diag- nostic procedure in laboratories in the United States. Careful technic is required for successful use of the test and only a highly potent, specially prepared precipitating serum can be used. The commercial antianthrax sera are generally unsatisfactory for this purpose. In applying the test, suspected tissue specimens are cut into small sections; 5 g of tissue or other suspected material to be tested are placed in about 10 ml of physiological salt solution and boiled 5 to 10 min, during which time the heat-stable specific antigens are ex- tracted if the material contains anthrax bacilli. The extract is cooled and filtered. The filtrate is then layered in narrow tubes over a highly potent anthrax-precipitating serum which has been proved by test. If anthrax antigens are present in the extract, a positive reaction is obtained within a few minutes, as in- dicated by the formation of a whitish ring of turbidity at the junction of the two fluids. In recent years evidence that the test is not absolutely specific has been reported by Palmeiro,'™ Emsbro and Plum,'® Eurich and Hewlett,® Hailler and Heicken,' Hausam,?® and Lloyd.'? V. EVALUATION AND REPORTING OF RESULTS Positive or negative laboratory findings in suspected outbreaks in animals or cases in man should always be confirmed by laboratory animal inoculation tests. Regardless of how typical the microorgan- ism may appear to be morphologically and culturally, animal virulence tests should be carried out before concluding that it is B. anthracis. 590 ANTHRAX When a positive report based on morphological findings or cultural tests or both is made, it should be qualified by definite statements that these findings are preliminary, are not conclusive, and are being checked by virulence tests on laboratory animals. In evaluating and reporting on the bacteriological examination of industrial material for the presence of B. anthracis, the following facts should be taken into consideration: First, unless the contaminated material contains numerous anthrax spores, the isolation of B. anthracis is usually very difficult. Second, since the small sample of such material subjected to examination con- stitutes only a minor fraction of the quantity under suspicion, nega- tive findings cannot be taken as conclusive evidence that the entire lot was free from contamination with B. anthracis. CrarReNCE D. SteIN, V.M.D., Chapter Chairman WiLLiaM Hagan, D.Sc, D.V.M. Raymond Ranparr, D.V.M. REFERENCES 1. Ascori, A. Die Prizipitindiagnose bei Milzbrand. Zbl. Bakt. I Abt. Orig. 58:63-70, 1911. 2. M’FADYEAN, J. A Peculiar Staining Reaction of the Blood of Animals Dead of Anthrax. J. Comp. Path. & Therap. 16 :35-40, 1903. 3. Howig, J. W,, and CruicksHANK, J. Effect of Shock-Producing Substances on Experimental Anthrax Infection in Mice. J. Path. & Bact. 59:127-135, 1947. 4. Sortys, M. A. Anthrax in a Laboratory Worker, with Observations on the Possible Source of Infection. J. Path. & Bact. 60:253-257, 1948. 5. NoroBerG, B. K. Studies of Bacillus anthracis in Regard to Its Properties of Diagnostic and Pathogenic Importance. Thesis, Royal Vet. College, Stockholm, 1951 (116 pp. In English). 6. HacaN, W. A. The Diagnosis of Anthrax from Putrefying Animal Tissue. J. Bact. 5:343-352, 1920. 7. Stein, C. D. Studies and Observations on the Laboratory Diagnosis of Anthrax. Vet. Med. 38:130-139, 1943. 8. Eurich, F. W,, and Hewrerr, R. T. Bacillus anthracis—A System of Bacteriology. London: Medical Research Council of Great Britain, May 1930, p. 448. 9. Burpnon, K. L. Rapid Isolation and Identification of Anthrax Bacilli. Proceedings, Symposium on Anthrax in Man, Philadelphia, Pa., Oct. 1954, pp. 45-54. 10. ——— Useful Criteria for the Identification of Bacillus anthracis and Related Species. J. Bact. 71:25-42, 1956. 11. Stein, C. D. Differentiation of Bacillus anthracis from Nonpathogenic Aerobic Spore-Forming Bacilli. Am. J. Vet. Res. 5:38-54, 1955. 12. Lroyp, R. S. Occurrence of Anthrax Bacilli in the Carpet Wool Industry in the United States. Arch. Indust. Hyg. & Occup. Med. 6:421-434, 1952. 13. HacaN, W. A, and Bruner, D. W. “Anthrax,” in The Infectious Diseases of Domestic Animals (3rd ed.). London: Bailliére, Tindall & Cox, 1957, pp. 176-190 ANTHRAX 591 14. 15. 16. 17. 18. 19. 20. JonEs, E. R. Diagnosis of Bacillus anthracis. J. Path. & Bact. 54:307-314, 1942. DeKock, G. v.o.W., SterNE, MAX, and Rosinson, E. M. Sterilization of Bone Meal. J. South African Vet. Med. A. 11:138-141, 1940. McGaucHEYy, C. A., and St. GrorGE, C. Isolation of Bacillus anthracis from Soils: The Use of Pearce and Powell Selective Medium. Vet. Record, 67: 132-133, 1955. PALMER, J. M. A reagdo de Ascoli, considerada como reagdo de grupo é insuficiente para o diagnostico do carbinculo. Repos. Trab. Lab. Central Patol. Vet. (Lisbon) 5:99-103, 1942. Emssoro, P., and Prum, N. Laboratory Diagnosis of Anthrax. Maanedsskr T. Dyrl 45:113-137, 1933. Hamrer, E., and Hricken, K. Untersuchungen zur Bekdmpfung des Gewerblichen Milzbrandes. VI. Mitteilung, Zur Frage der Milzbrandgefahr- lichkeit Auslandischer Schaf—Und Ziegenfelle. Z. Hyg. Infektkrh. 131 :443- 459, 1950. HausaMm, W. Die Milzbrandfrage in der Lederindustrie. Zbl. Bakt. I Abt. Orig. 155:352-361, 1950; abstracted in Bull. Hyg. 26:917-918, 1951. CHAPTER 21 GLANDERS AND MELIOIDOSIS 1. Introduction II. Collection of Specimens ITI. Bacteriological Examination A. Microscopical B. Cultural C. Animal Inoculation IV. Skin Test V. Serological Tests VI. Melioidosis (Pseudoglanders) References I. INTRODUCTION Although glanders is now considered practically extinct in North America, we can afford neither to forget this disease entity nor to abandon the technical knowledge necessary for the laboratory diag- nosis. Several sporadic cases of naturally occurring human glanders!-? and laboratory infections® have been reported during the past two decades. The most recent case was reported* in a resident of Rochester, N. Y., in 1948; however, this case is suspected of having been contracted outside the United States. Glanders is still endemic in many parts of the world, particularly eastern Europe, southeastern Asia and isolated areas of South America and Africa. Natural infection with Malleomyces mallei (Actinobacillus mallei),” the causative agent of glanders, occurs in solipeds (horses, asses, mules and donkeys), and in man as a result of contacts with these animals. Carnivora may be infected by feeding on carcasses of dis- eased solipeds. Herbivora other than solipeds are seldom if ever infected naturally,® although many may be infected artificially by in- oculation. The disease is manifested in equines in two forms, known respec- tively as glanders and farcy. In glanders, the lesions are found in 592 GLANDERS 593 the upper respiratory tract and lungs, with frequent metastases to other organs. In farcy, the lesions involve primarily the superficial lymphatic vessels, nodes of the subcutaneous tissues, and, secondarily, the skin, by breakdown of the nodes with ulceration to the surface. The formation of nodules or infectious granulomas, histologically somewhat resembling those of tuberculosis, is characteristic of glanders in whatever form it occurs. Breakdown of these nodules results in the discharge of a glutinous pus which is highly infectious and by means of which the disease is often transmitted directly or indirectly. The nasal membranes in the horse are usually involved and the result- ing profuse catarrhal discharge is considered extremely infectious. Most cases of human glanders have occurred in persons who have had considerable contact with horses. Numerous infections have been reported among laboratory workers.!® It is generally conceded that M. mallei is a dangerous agent in the laboratory, and special pre- cautions must be taken when working with virulent strains. Human glanders is a protean disease that may be manifest in many different ways.10-12 Lung lesions and lesions in the respiratory mucosa similar to those of horses are most common in the acute cases. In the more chronic forms there are nodules which break down, forming abscesses and fistulas in the subcutaneous tissues, muscles, joints and internal organs. The untreated acute disease frequently runs a short, fulmi- nating, fatal course; the chronic disease may run a course of several months or persist with remissions and exacerbations for many years. In the days when glanders was prevalent, most of the diagnosed cases terminated fatally, although a very few cases are on record as having recovered spontaneously. Today, however, a diagnosis of glanders is no longer a death sentence. Sulfadiazine has been demon- strated to be effective in the treatment of human glanders,® while the newer wide-spectrum antibiotics inhibit M. mallei in vitro but have not as yet been evaluated clinically. ll. COLLECTION OF SPECIMENS The following materials should be collected for the purpose of making a complete laboratory study for evidence of a Malleomyces infection, Blood for culture, during the acute phase Blood for serology, one sample in the acute phase and another at 4 weeks Pus or exudate from lesion Excised superficial nodule Nasal swab or catarrhal discharge oan oe 594 GLANDERS Ill. BACTERIOLOGICAL EXAMINATION A. Microscopical Films should be made of pus or exudate from the superficial lesion(s). When present, the etiological agent appears as a Gram- negative slender pleomorphic rod which stains irregularly and is often beaded. Carbolfuchsin should be used as the counterstain rather than safranin in Gram’s technic, since stains containing alkali or phenol give best results. The characteristic beading is best seen in films stained with Loeffler’s alkaline methylene blue, Pus from old lesions may result in negative films even though the same material may yield positive cultures. B. Cultural M. mallei will grow poorly, if at all, on media that do not contain glycerol, especially on primary isolation. Beef extract or infusion broth containing 4 per cent glycerol and adjusted to a final pH of about 6.8 will yield good growth in 48 to 72 hr when incubated at 35° C. On agar the colonies are smooth, raised, convex and translu- cent, With prolonged incubation the colonies tend to become opaque and the centers take on a brownish discoloration. The growth in broth is not particularly characteristic. Cultures are made from lesion exudates or, better still, from the pus of an incised superficial node. When a node is excised for biopsy a portion of it may be ground aseptically and used for both culture and animal inoculation. It is rather rare to isolate the organism from the bloodstream, but in the acute stage blood cultures should be attempted. C. Animal Inoculation Of the common laboratory animals, the guinea pig and hamster are the most susceptible to J. mallei infection. Nevertheless, because of individual variation in susceptibility it is necessary to inoculate these animals in groups of three. Male animals are inoculated intraperi- toneally with the same material used for the cultural examination. The animals are sacrificed on the 4th day after inoculation. A positive result is frequently indicated on the 2nd and 3rd day after inoculation by the development of an orchitis or Straus reaction observed as swelling and inflammation of the scrotal sacs. In addition to the orchitis, the mesenteric and iliac lymph nodes become enlarged and small abscesses develop in the spleen and liver. The Straus re- action, although characteristic of Malleomyces infection, is not diag- nostic, since other agents (Brucella) may produce a similar reaction. GLANDERS 595 It is necessary therefore to culture material from the animal lesions to demonstrate conclusively the presence of an organism having the cultural characteristics of M. mallei. IV. SKIN TEST Mallein, the concentrated filtrate of old glycerol broth cultures of M. mallei, produces a tuberculin-like reaction in both glanders and melioidosis (see Section VI). Skin testing of human beings is per- formed intracutaneously with 0.1 ml of a 1:10,000 dilution of “intra- dermic mallein” and the test is read at 48 hr. The mallein is produced and distributed by the Veterinary Division, Army Medical Service Graduate Scheol, Walter Reed Army Medical Center, Washington 12, D.C V. SEROLOGICAL TESTS The agglutination and complement-fixation tests are excellent diag- nostic aids for Malleomyces infections. But, like the mallein skin test, the serological tests are specific only for the genus Malleomyces and do not permit a differentiation between M. mallet and M. pseudo- mallet infections, Sera from normal persons may agglutinate Malleomyces organisms in titers as high as 1:320. However, a significant rise in agglutinins can be demonstrated 2 to 3 weeks after infection, Agglutination titers of 1:1,280 and greater are obtained with antigens prepared from the homologous strain. The complement-fixation test is considered more reliable than the agglutination test, since there has been no demonstration of comple- ment-fixing antibody in normal sera. The complement-fixation test becomes positive at about the 4th week of the disease. Intradermic mallein diluted 1:100 may be used as antigen in the complement- fixation test, so that this test can be performed in any laboratory equipped for routine serological testing. VI. MELIOIDOSIS (PSEUDOGLANDERS) This is a glanders-like disease of man which was thought for many years to occur only in southeast Asia. Several hundred cases have been described since the first case reported by Whitmore and Krishnaswami'® in 1912 from Rangoon. The first cases recognized in other parts of the world in persons who had not visited the endemic area in Asia were among military personnel on the Island of Guam 596 GLANDERS in 1946.1 In the following year the first of several cases was recognized in the United States among persons who had not been out- side the Western Hemisphere.'7-18 The causative agent of melioidosis is M. pseudomallei (Pseudo- monas pseudomallei)” which morphologically resembles J. mallei. An important differential characteristic is that M. mallei is atrichous and nonmotile, while M. pseudomallei is motile, with 1 to 4 polar flagellae. Further, M. pseudomallei liquefies gelatin, whereas M. mallet usually does not. M. pseudomallei does not require glycerol for optimum growth and will grow well on standard laboratory media. In addition, it is catalase-positive and reduces methylene blue. The young colonies of M. pseudomallei resemble those of M. mallei, but after 72 hr incu- bation, colonies of the former become rough, dry and crenated. Con- fluent growth takes on a corrugated or honeycomb appearance, Im- munologically M. mallei and M. pseudomallei are very closely related.® Several papers20-2® have attempted to show a relationship of M. pseudomallei to members of the genus Pseudomonas. Reports of pigment production by certain strains of M. pseudomallei have not been substantiated. However, the cultural and morphological similari- ties of M. pseudomallei to some Pseudomonas species should be noted, since the latter occur frequently as contaminating microorganisms in clinical laboratories. Melioidosis in man may be acute or chronic. The acute disease is rarely diagnosed during life. The symptoms are characterized by sudden onset, high fever, chills, prostration, general aching pains, diarrhea, lymphadenopathy, signs of bronchopneumonia, collapse and death in from 3 to 7 days. The mortality is about 95 per cent. Autopsy shows multiple miliary abscesses throughout the soft tissues and especially in the lungs, liver and other viscera. The chronic form of the disease often is an extension of the acute process. This is characterized by multiple sinuses in the soft tissues, with extensive abscess formation. Treatment with sulfa drugs and antibiotics has not been uniformly successful; further evaluation is required. The source and mode of infection are not known. Rodent infec- tions occur naturally, and it has been surmised that human cases may be due to contamination of their food by rats. It has been shown that the rat flea, Xenopsylla cheopis, may be a transmitting agent, but this is not thought to be the usual mode of transmission. Cases have been recognized in horses in Malaya?* and in sheep in Australia.?526 Person to person transfer of the disease among human beings is not known to occur. GLANDERS 597 The laboratory diagnostic methods for melioidosis are the same as those discussed for glanders. As with glanders, it should be noted that normal human sera may show agglutinins of low titer to MM. pseudomallei. WitLiaMm Hacan, D.Sc, D.V.M., Chapter Chairman Leo Cravirz, Dr.P.H. Raymond Ranparr, D.V.M. REFERENCES BuracEss, J. F. Chronic Glanders. Canad. M.A.]J. 34:258-262, 1936. MEenDELSON, R. W. Glanders, Ann. Int. Med. 10:43-48, 1936. 3. Herorp, A. A., and Erikson, C. B. Human Glanders: Case Report. South. M. J. 31:1022, 1938. 4. Bourop, M. G., Cravrrz, L., and Harrerr, J. J. Human Glanders: Report of a Case. Proc. N. Y. State Assoc. of Pub. Health Lab. 29(2) :49, 1949. 5. Womack, C. R, and WeLLs, E. B. Co-existent Chronic Glanders and Multiple Cystic Osseous Tuberculosis Treated with Streptomycin. Am. J. Med. 6:267-271, 1949. 6. Howe, C., and MirLer, W. R. Human Glanders: Report of Six Cases. Ann. Int. Med. 26:93-115, 1947. 7. Bergey's Manual of Determinative Bacteriology (7th ed.). Baltimore, Md.: Williams & Wilkins, 1957, (a) p. 417 and (b) p. 100. 8. Huryra, F., and MARek, J. Special Pathology and Therapeutics of the Disease of Domestic Animals. Chicago and London: Alex Eger, 1926. 9. McGiLBray, C. D. The Transmission of Glanders from Horse to Man. Canad. Pub. Health. J. 35:268-275, 1944. 10. BernstEIN, J. M., and CaAruinG, E. R. Observations on Glanders with a Study of Six Cases and a Discussion of the Methods of Diagnosis. Brit. M. J. 1:319-325, 1909. 11. Dupceon, L. S., Symonps, S. L., and WiLkiN, A. A Case of Glanders in the Human Subject (Experimental Inoculation of the Horse and Mule, and a Comparison of the Blood Immunity Reactions). J. Comp. Path. & Therap. 31:43-51, 1918. 12. GaiGer, S. H. Glanders in Man. J. Comp. Path. & Therap. 26:223-236, 1913. 13. Cravrrz, L. Unpublished data, 1949. 14, ——————— and Miter, W. R. Immunologic Studies with Malleomyces mallet and Malleomyces pseudomallei. 11. Agglutination and Complement Fixation Tests in Man and Laboratory Animals. J. Infect. Dis. 86:52-62, 1950. 15. WHrrMorg, A., and Krisunaswamri, C. S. An Account of the Discovery of a Hitherto Undescribed Infective Disease Occurring Among the Popula- tion of Rangoon. Indian M. Gaz. (Calcutta) 47:262-267, 1912. 16. Mirick, G. S., et al. Melioidosis on Guam. J.A. M.A. 130:1063-1067, 1946. 17. McDoweLL, F., and VaArNEY, P. L. Clinical Notes, Suggestions and New Instruments-—Melioidosis. Report of First Case from the Western Hemis- phere. J.A.M.A. 134:361-362, 1947. 18. BrAMER, P. R., et al. Melioidosis. Report of Second Case from the Western Hemisphere, with Bacteriologic Studies on Both Cases. Am. J. Path. 24:717-718, 1948. PY foi 598 GLANDERS 19. Cravirz, L., and Mier, W. R. Immunologic Studies with Malleomyces mallei and Malleomyces pseudomallei. I. Serological Relationships between M. mallet and M. pseudomallei. J. Infect. Dis. 86:46-51, 1950. 20. LeGroux, R., and GeNEvrGy, J. Etude Comparative entre le Bacilli de Whitmore et le Bacilli Pyocyanique. Ann, Inst. Pasteur 51:249-264, 1933. 21. Branc, G., Derack, B., and MarTIN, L. A. Etude Comparative de Charac- teristiques Biochemiques et Serologiques du Bacilli de Whitmore et du Bacilli Pyocyanique. Ann. Inst. Pasteur 69 :65-74, 1943. 22. Brycoo, E. R., and RicuArp, C. Pouvoir Chromogene de Malleomyces pseudomallei Ann. Inst. Pasteur 83:822-825, 1952. 23. Ziskinp, J. Pizzoraro, P., and Burr, E. E. Characteristics of a Strain of Malleomyces pseudomalletr from Chronic Melioidosis. Am. J. Clin. Path. 24:1241-1245, 1954. 24. Davig, J, and WeLLs, C. W. Equine Melioidosis in Malaya. Brit. Vet. J. 108:161-167, 1952. 25. Correw, G. S., SuthHerLAND, A. K., and MeenaN, J. F. Meliodosis in Sheep in Queensland—Description of an Outbreak. Austral. Vet. J. 28:113- 123, 1952. 26. Lewis, F. A, and Oups, R. J. Melioidosis in Sheep and a Goat in North Queensland. Austral. Vet. J. 28:145-150, 1952. RECOMMENDED (but not keyed to text) Topley and Wilson's Principles of Bacteriology and Immunity (3rd ed.). Balti- more Md.: Williams & Wilkins, 1946, Vol. 1, pp. 486-496; or Ibid (4th ed.). 1955, Vol. 1, pp. 576-585. StantoN, A. T. and FrercuHErR, W. Melioidosis. Stud. Inst. M. Research Federated Malay States Bull. No. 21, 1932. CHAPTER 22 MISCELLANEOUS INFECTIONS I. Erysipelothrix Infections II. Klebsiella Infections III. Listeria Infections IV. Mimeae Infections V. Infections Due to Pleuropneumonia-like Organisms (PPLO) VI. Pseudomonas aeruginosa Infections VII. Sodoku VIII. Streptobacillus moniliformis Infections IX. Toxoplasmosis X. Vibrio fetus Infections Section |—Erysipelothrix Infections . Introduction . Collecting and Handling Specimens . Bacteriological Examination . Serological Examination . Evaluation and Reporting of Results HOOW» References A. INTRODUCTION Erysipelothriz rhusiopathiae® is a very slender rod-shaped bac- terium. The rods are sometimes single or paired, and straight, but most often are unevenly curved or sigmoid. The microorganism has a tendency to produce long, slender, unevenly flexuous, often tangled filaments which may thicken and show granules with special staining technics. They are nonmotile, Gram-variable,f and microaerophilic but grow well under aerobic conditions. They are catalase-negative and negative to all common biochemical tests except those for nitrate reduction and hydrogen sulfide production, in which the reactions are * Cf. Bergey's Manual of Determinative Bacteriology (6th ed.). Baltimore: Williams & Wilkins, 1948. + When working with most keys, E. rhusiopathiae should be considered Gram- positive. 599 600 MISCELLANEOUS INFECTIONS variable. Their saccharolytic powers are weak and irregular, slight to moderate acid usually being produced without gas from glucose, and more irregularly in descending order from lactose, galactose, fructose, maltose and mannose. Other “sugars” are not attacked. The organism is somewhat fastidious. Growth is favored by glucose. It grows well between 37° and 20° C, with no growth at 4° C (refrigerator tem- perature). The characteristic bottle-brush growth is produced in gelatin stab cultures. Many strains are pathogenic for white mice and pigeons, while rabbits are susceptible to some strains and in in- fections not acutely fatal develop a circulating monocytosis. Guinea pigs are not susceptible. E. rhusiopathiae is widely distributed in the animal kingdom,'? associated with disease in wild birds and mammals, in fish (including shellfish and crustaceans), and fish products, in meat and meat products, in hides and skins, in sewage waste from abattoirs, in poultry feeds, houseflies, putrid carcasses and parts thereof. There is no conclusive evidence that Erysipelothrix is capable of self-sustained existence in soil or vegetable matter. The microorganism gives rise to important veterinary problems in swine, turkeys and sheep, the disease produced in swine ranking among the most important infections of that species. Swine are an important reservoir of the infection in nature, since a high percentage of apparently healthy pigs harbor the microorganism in their tonsils. Wayson* described an epizootic of Erysipelothrix infection in meadow mice in California and there are many scattered reports in the literature of natural infections in mammals and birds. In the last few years E. rhusiopathiae has become a widespread disease in turkeys. The literature on E. rhusiopathiae infection in man has been re- viewed by Van Es. In man, as a rule, no more than benign skin lesions are produced—usually on a hand—but occasionally more generalized and serious disease results, with joint and cardiac in- volvement, extensive skin involvement and, on very rare occasions, other pathology. While the source of infection in a few human cases cannot be traced, the disease as a whole has a tendency to be occupa- tional, being found in fishermen, especially salt water fishermen, veterinarians, abattoir workers, fish cannery workers, butchers and agricultural workers. B. COLLECTING AND HANDLING SPECIMENS In collecting specimens for culture, precautions should be taken to guard against gross contamination with other infective material and MISCELLANEOUS INFECTIONS 601 disinfectants. From the time of collection until laboratory examina- tion, specimen material is best preserved by freezing. Freezing is not essential, however, since Erysipelothrix survives well in the presence of putrefaction, The thermal death point of the microorganism is fairly low (52° to 70° C) and overheating of specimens should be avoided. Since it is sensitive to penicillin and other antibiotics, specimens are best collected prior to antibiotic therapy; otherwise thought should be given to adding penicillinase to the initial culture medium. Specimen material for culture might include blood, swabs of exu- date from skin lesions and paronychia, biopsy material from an ad- vancing edge of an area of erysipeloidal erythema ; vegetative lesions from heart valves; fluid from arthritic joints; or material from any other suggestive lesion. In field surveys of pigs, rats or other animals it is well to keep in mind that apparently healthy animals may harbor the microorganism in such locations as tonsils and intestinal glands. Collection of material from other potential reservoirs must be left to the good judgment and ingenuity of the collector. Collect and handle specimens for serological study in the usual manner. C. BACTERIOLOGICAL EXAMINATION First, make and Gram-stain films of the specimen material, then examine for microorganisms suggestive of E. rhusiopathiae. More often than not the preliminary examination of films will be unre- warding. If present, however, the microorganisms are invariably numerous in endocardial vegetations; often numerous in the fluid of arthritic joints; and often demonstratable in the blood films of septi- cemic cases. Second, give consideration to inoculating animals (white mice or pigeons). Triturate specimen material with saline solution, using a mortar and pestle or blender, in the proportions of approximately 1 g of material to 10.0 ml of salt solution. Inoculate four or more mice each with 0.25 ml of the suspension intraperitoneally, or two or more pigeons each with 0.5 ml intraperitoneally. Most strains of Erysi- pelothrix kill white mice in 2 to 4 days. Pigeons usually die in 3 to 5 days. After death, culture heart blood, liver, spleen and kidney for Erysipelothrix. Some strains will fail to kill the inoculated animals, so that the method is not completely reliable. Failure to kill experimental animals does not mean that the microorganism is a potentially non- 602 MISCELLANEOUS INFECTIONS pathogenic strain, because E. rhusiopathiae is a microorganism that will change readily from a nonpathogenic to a pathogenic state. Often the change from nonpathogenicity to pathogenicity will take place spontaneously in the test tube. Hence if an animal-inoculation method is used, it should be used in conjunction with direct culture of the specimen material. In culturing, the media and procedure of choice will depend to some extent on the specimen material. Erysipelothrix will grow on infu- sion and extract media. While growth is enhanced by the addition of serum or blood, and glucose, it is not luxuriant on any medium. When contamination is light, blood agar will suffice for primary plating. Cystine-glucose blood agar (CM No. 115) as used for Pasteurella tularensis works well. Owing to the nature of much specimen material, however, contamination poses a problem, in which event a less direct procedure is indicated. The method of Connell and Lang- ford? gives good results and is recommended where extensive cultur- ing for E. rhusiopathiae is being undertaken. A glucose extract broth (CM No. 93) is the basic medium used. Agar media (modified Packer’s) for plating can be prepared from this base broth with certain additions and changes (CM No. 94). Modified Packer’s medium is strongly inhibitory and is the medium of choice when plating in the presence of contaminating microorganisms. Modified Edwards’ medium (CM No. 97) permits better growth, is preferable for making subcultures and carrying cultures, and is the medium of choice when culturing specimen material of low con- tamination. Erysipelothrix produces a characteristic growth in gelatin stab cultures. A special gelatin medium is not required. However, more rapid growth is obtained in a gelatin medium containing glucose (CM No. 95). Procedure Specimen material should be plated directly on modified Edwards’ medium or any other suitable medium of low inhibiting power. If contamination appears to be extensive, add 1 g amounts of trit- urated specimen material to tubes of broth (CM No. 93), shake tubes well, and place in refrigerator at 4° C for 7 to 14 days or longer. Ery- sipelothrix survives this procedure well, while the populations of the more serious contaminating microorganisms die out or are greatly reduced. After the holding period, remove tubes from refrigerator, shake well, and spread five loopfuls (loop 4 mm in diameter, wire 22 gauge) MISCELLANEOUS INFECTIONS 603 of fluid from each tube on separate plates of modified Packer’s agar. Incubate plates aerobically at 35° C for 24 hr and another 24 hr at room temperature. Examine the plates for small colonies, which should be grayish by reflected light but highly transparent by trans- mitted light, using a dissecting microscope at magnifications of 10 to 90X if necessary. Colony morphology may vary from tiny, dis- crete, and dewdrop-like to tiny, flat, spreading medusoid formations. The colonies may or may not be surrounded by narrow zones of alpha hemolysis. Gram-stain films of likely looking colonies and examine microscopically. The staining and morphology of Erysipelothrix are quite characteristic (see Introduction). With a small platinum loop pick suspected colonies and spread them on modified Edwards’ agar. Several colonies may be spread on a single plate, but be careful to avoid overlapping. Examine after in- cubating aerobically for 24 hr and after standing another 24 hr at room temperature. Colonies that are quite opaque, that cause black- ening of the medium (hydrolysis of esculin), or that produce marked greening are likely to be cocci and may be ignored. Colonies that are more transparent but blacken the medium (the blackening often being merely a pattern of tiny black granules directly beneath the colonies) are Gram-positive rods (usually Corynebacteria) and can be ignored. Small colonies grayish by reflected light and of low opacity by trans- mitted light that do not blacken the medium are likely to be Ery- sipelothrix, especially if surrounded by narrow zones of alpha hemolysis. Some species of Corynebacteria that do not hydrolyze esculin produce colonies of low opacity that resemble Erysipelothrix. Make and examine Gram-stained films. Inoculate likely colonies onto slopes of modified Edwards’ medium and incubate. After growth appears, make gelatin stab cultures from the slopes. Incubate the gelatin tubes at 20° C, avoiding higher temperatures, which will melt the gelatin. The appearance of a bottle-brush type of growth within 10 days or less is almost certain indication of Erysipelothrix. To confirm further the identification of Erysipelothrix, inoculate “sugars” (CM No. 98) and carry out catalase, hydrogen sulfide (CM No. 96) and nitrate reduction (CM No. 64) tests. Compare results with the description in the introduction to this section. D. SEROLOGICAL EXAMINATION Contrary to much that is reported, E. rhusiopathiae lends itself to the making of satisfactory antigens for serological testing. In human infections the agglutination test would doubtless constitute a useful 604 MISCELLANEOUS INFECTIONS aid in diagnosis. Antibodies, as a rule, appear in the blood about 5 days after the onset of infection and a well-marked titer with a tested antigen is strongly indicative of infection. American workers have described the preparation of antigen for tube and rapid agglu- tination tests,%® and Rice et al.? in Canada have described a method for preparing tube antigen and technics for getting around the tend- ency of Erysipelothrix antigen to clump spontaneously. Strains of E. rhusiopathiae appear to differ in quantity and spatial arrangement of component antigens but not in quality of antigen that would permit classification of strains into true serological types.l® Selection of strains for antigen production may be made partly on this basis and partly on the basis of smoothness, low tendency to produce filamentous growth, and spontaneous clumping. Production of antigen—Grow individual strains in flasks of heart infusion broth (CM No. 21) containing 2.0 per cent serum. Centrifuge 24 hr cultures, decant, suspend microorganisms in salt solution, centrifuge again and decant. Resuspend the packed micro- organisms to one-tenth of the original volume in distilled water con- taining 0.05 per cent merthiolate (Lilly). Pool the different lots of antigen strains. For use in the agglutination test, thoroughly shake the antigen suspension with glass beads to break up clumps and fila- ments, then dilute with distilled water to match Tube 2 of McFarland’s nephelometer. Technic of agglutination test—To 0.5 ml of serial twofold dilutions of serum beginning at a 1:50 dilution, add 0.5 ml of E. rhusiopathiae antigen. Shake tubes and incubate in water bath at 42° C for 4 hr followed by 18 hr at 35° C in an incubator. In reading the tests agitate the tubes gently, as the agglutinated clumps are easily broken up and vigorous shaking is likely to resuspend the micro- organisms, resulting in a missed reaction. Positive control serum may be prepared by inoculating rabbits with suspensions of the micro- organism in salt solution killed by the addition of 0.05 per cent merthiolate and adjusted to the density of Tube 2 of McFarland’s nephelometer. Four intravenous doses (1, 2, 3 and 3 ml) at 4 day intervals, bleeding from the heart 10 days after the last injection, yield a good positive serum. E. EVALUATION AND REPORTING OF RESULTS Recovery of E. rhusiopathiae from lesions is suggestive of infec- tion, and while the microorganism may be present owing to con- tamination or as a secondary invader, it should be regarded with MISCELLANEOUS INFECTIONS 605 suspicion. Recovery from apparently healthy reservoir hosts should likewise be regarded with suspicion, since the organism is capable of suddenly changing from a nonvirulent to a virulent state. Recoveries from other animals and animal matter in nature generally warrant con- sideration from the epidemiological standpoint. Negative and low serological titers should be interpreted with caution. High titers (1:100 and higher) indicate infection. The titer at which to read the agglutination tests will vary with the antigen. If a commercial antigen is used, the recommendations of the manu- facturer should be followed. RoBerT ConNELL, D.V.M. REFERENCES 1. Van Es, L, and McGratH, C. B. Swine Erysipelas. Univ. of Nebraska Coll, Agr. Research Bull. No. 128, 1942. 2. Levine, N. D. “Erysipelothrix Septicemia (Gefluegelrotlauf).” In Diseases of Poultry (2nd ed.). Ames, Iowa: Iowa State College Press, 1948, pp. 379-386. 3. ConnEeLL, R., and LAancrorp, E. V. Studies of Swine Erysipelas. V. Presence of Erysipelothriz rhusiopathiae in Apparently Healthy Pigs. Canad. J. Comp. Med. 17,11:448-453, 1953. 4, Wavson, N. E. An Epizootic Among Meadow Mice in California Caused by the Bacillus of Mouse Septicemia or of Swine Erysipelas. Pub. Health Rep. 42:1489-1493, 1927. 5. Vax Es, L. Swine Erysipelas Infection in Man. Univ. of Nebraska Coll Agr. Research Bull. No. 130, 1942. 6. Breen, F. Precipitation Test in Swine Erysipelas Diagnosis. North Am, Vet. 13,8:29-30, 1932. 7. ScuoeNiNG, H. W,, CreecH, G. T., and Grey, C. S. A Laboratory Tube Test and a Whole-Blood Rapid-Agglutination Test for the Diagnesis of Swine Erysipelas, North Am. Vet. 13,12:19-25, 1932. 8. Grey, C. G.,, Osteen, O. L., and ScuoenNing, H. W. Swine Erysipelas, the Agglutination Test for Its Diagnosis, and a Report on the Study of Arthritis in Swine. Am. J. Vet. Res. 2:74-76, 1941. 9. Rice, C. E.; ConneLL, R.; ByrnE, J. L.; and BouLANGER, P. Studies of Swine Erysipelas. IV. Serological Diagnosis in Swine. Canad. J. Comp. Med. 16,6:209-215, 1952. 10. Rick, C. E., et al. Studies of Swine Erysipelas. ITI. Antigenic Characteristics of Strains of Erysipelothrix rhusiopathiae Isolated in Different Areas in Canada. Canad. J. Comp. Med. 16,5:195-204, 1952. 606 MISCELLANEOUS INFECTIONS Section Il—Klebsiella Infections . Introduction Collection and Handling of Specimens Bacteriological Examination . Serological Examination . Evaluation and Reporting of Results HOO®Wp> References A. INTRODUCTION To many clinicians the designation “Klebsiella infection” is synony- mous with infection due to Friedldnder’s bacillus and is associated primarily with pathologic states of the meninges and the respiratory tract. “Aerobacter infection” is commonly thought of as disease in- duced by A. aerogenes and is associated primarily with infections in- volving the genitourinary and gastrointestinal tracts. In an effort to find the historical reasons for this taxonomic differentiation based on anatomic location of infected site, Obrinsky and associates® suggested that when the Friedldnder’s bacillus was isolated in 1882 from patients with pneumonia, it was studied primarily from the morphologic stand- point. On the other hand, Escherich, who first described A. aerogenes in 1886, emphasized its close similarity in biologic activity to Escherichia coli. Thus from the very first, 4. aerogenes was identified more closely with E. coli than with Friedldnder’s bacillus, a relation- ship upon which subsequent studies have cast considerable doubt. Extensive investigations during past years point to the fact that while A. aerogenes is related in some respects to certain strains of E. coli, it is more closely related, biochemically and serologically, to Friedlinder’s bacillus. While no useful purpose will be served by detailing all the reports supporting this view, reference should be made to the more comprehensive and definitive studies. As presently classified in Bergey's Manual? differentiation of Klebsiella pneumoniae from many strains of A. aerogenes is im- possible. The only differentiating characteristic given for the two species is motility—the former being described as nonmotile, the latter motile or nonmotile. As early as 1905, MacConkey?® reviewed the literature on Bacterium lactis aerogenes of Escherich and concluded that the organism had always been considered as nonmotile. He re- affirmed this viewpoint in 1909.* The excellent statistical study by Levine in 1918° also showed A. aerogenes to be nonmotile. It would thus appear that on historical grounds 4. aerogenes should be limited to include only nonmotile forms. MISCELLANEOUS INFECTIONS 607 In 1929, Edwards® reported on the morphological and biochemical similarity of Friedlinder’s bacillus and cultures which, on the basis of criteria existing at the time, were classified as A. aerogenes. This similarity was so close that in his opinion there was no basis for generic differentiation of the two organisms. Other investigators?™3 subsequently confirmed his findings. Julianelle’s'* view that K. pneumoniae and A. aerogenes could be distinguished on the basis of their somatic antigens is now untenable in the light of studies by Kauffmann,'® Oerskov,'® and Edwards and Fife.!® Consequently in- clusion of Friedldnder’s bacillus and A. aerogenes into a single genus has been advocated ,®1%17 with the recommendation that the combina- tion be assigned the designation Klebsiella on the ground of pri- ority.1®-1%:18 This recommendation has been adopted by the writer, so that the term “Klebsiella infection” as used herein is to be interpreted as referring to infection by Klebsiella organisms as defined by Kauft- mann!! regardless of the anatomic site from which these micro- organisms may be isolated. Thus the Klebsiella group comprises . a large group of serologically related Gram-negative, nonsporing and nonmotile rods which usually possess capsules and form mucus. Usually, they do not form indole. They split adonitol and inositol. Often they decompose urea, give a positive Voges-Proskauer reaction but a negative methyl red reaction. They usually grow on ammonium citrate agar and ferment lactose.10 The foregoing criteria permit ready identification of organisms as Klebsiella; however, much confusion will be avoided if it is borne in mind that while classification of bacteria into sharply delineated divisions is dictated by practical necessity, such divisions are in reality wholly artificial. Consequently it is to be expected that not all cultures which are encountered will fall neatly into one or another of these divisions. While biochemical pattern serves to differentiate Klebsiella from other groups in the family Enterobacteriaceae classification of mem- bers within the group is accomplished serologically, specifically by recognition of somatic and capsular antigens. In view of the facts that Klebsiella capsular antigens are heat-stable and that capsulated forms are inagglutinable in O sera, the determination of O antigens requires the isolation of acapsular forms, a procedure which is time- consuming and may lead to the production of variants with altered antigenic composition.!® As a consequence, it has been recom- mended!®® that in practice identification of individual Klebsiella types be made on the basis of differences in capsular antigens, each of which is designated by an arabic numeral. In such a diagnostic 608 MISCELLANEOUS INFECTIONS schema “specific names of these types would be superfluous.”?® For details of antigenic classification consult Edwards and Fife,'® Kauff- mann,'® Brooke’? Worfel and Ferguson,'® Goslings and Snijders, Edwards and Ewing,'” Edmunds,?! and Oerskov.!® Objections to the adoption of a serological typing schema for Klebsiella have been raised by Henriksen.?? B. COLLECTION AND HANDLING OF SPECIMENS In addition to its significance as an incitant of infection in the upper and lower respiratory tracts, Klebsiella has also been isolated from suppurative conditions in various parts of the body. These in- clude appendicitis, cystitis, pyelonephritis, ulcerative endocarditis, brain abscess and meningitis. Bacteremia and generalized septicemia may also be caused by Klebsiella. As a consequence the laboratory may be required to search for these organisms in such body fluids as blood, sputum, bronchial aspiration, cerebrospinal fluid, urine, and peritoneal, pleural, cervical and vaginal pus. In most cases, the examination of these specimens requires no special methods; procedures which are commonly applied to these specimens in the search for pathogenic bacteria in general are satis- factory. Autopsy materials such as lung, liver, lymph node and kidney should be ground aseptically before inoculation into suitable media. C. BACTERIOLOGICAL EXAMINATION Since klebsiellas are not fastidious in nutritional requirements, they are readily cultivated on a variety of simple infusion base media. Agar plates, with or without blood, such as are available in most laboratories, are excellent. Beef extract agar containing lactose with phenol red or bromthymol blue as indicator and desoxycholate agar may also be used additionally for their differential and/or selective value. These are streaked with the clinical specimen and incubated for 24 hr at 35° C. On blood agar plates, colonies are large, gray, mucoid, heaped-up and nonhemolytic. In consistency the colonies are soft; strains producing large amounts of slime substance “string up” in long threads when touched with an inoculating needle, Nonmucoid strains, both smooth and rough, also may be encountered (for details of colony variation see References 17 and 18). On bromthymol blue lactose agar, colonies are large, moist, shiny and confluent, and yellow in color. On standing at room temperature for several days, such colonies increase in size, may acquire a greenish blue color, become MISCELLANEOUS INFECTIONS 609 very mucoid, and take on a heaped-up appearance. On desoxy- cholate medium colonies are large, moist, shiny, mucoid and confluent, may have a red center and colorless periphery or be solidly pink in color. Suspicious colonies are picked and inoculated into appropriate media to determine whether they conform to the typical biochemical pattern of Klebsiella strains shown in the following tabulation: Indole 0 H,S oO Methyl red 0 Adonitol + Voges-Proskauer + Inositol + Citrate he Motility O Urea 0 + =positive. O =negative. D. SEROLOGICAL EXAMINATION From an academic standpoint, the serological examination of Klebsiella depends upon four antigenic components: 1. Capsular or K antigen 2. Somatic smooth or O antigen 3. Somatic rough or R antigen 4. Mucoid or “slime” envelope or M antigen On the basis of these components Kauffmann!® represents the antigenic and cultural forms known to occur as follows: a. Smooth Forms: MKO = Mucoid, capsulated, with O antigen KO =Nonmucoid, capsulated, with O antigen MO = Mucoid, noncapsulated, with O antigen O=Nonmucoid, noncapsulated b. Rough Forms: MKR=Mucoid, capsulated, without O antigen KR =Nonmucoid, capsulated, without O antigen MR = Mucoid, noncapsulated, without O antigen R =Nonmucoid, noncapsulated, without O antigen That the K and M antigens are serologically related and probably identical in a given culture was first shown by Edwards and Fifel® and later confirmed by Wilkinson and associates? The R antigen is weakly antigenic and its presence is not manifested in agglutination tests with smooth forms having fully developed O antigens, but in- vestigation still remains to be done before its significance is fully understood.’® For these reasons and because the determination of O antigens requires the isolation of acapsular forms—a difficult and time- consuming process that may lead to the production of rough variants with altered antigenic composition—the practical serological identi- fication of Klebsiella is based upon determination of the capsular (K) antigens. 610 MISCELLANEOUS INFECTIONS Demonstration of Capsules'® 1) Place a drop of a 4-6 hr broth culture or a drop of a dilute salt solution suspension from a colony on a slide. 2) Adjacent to it, place a small droplet of Pelican India ink.* 3) Join the two drops with a loop but do not mix. Cover with a cover slip. 4) Varying degrees of density of ink will result; some areas should permit ready recognition of capsules microscopically. Preparation of Capsular Antisera'® The culture used in the preparation of suitable immunizing anti- gens must comprise a majority of organisms possessing well-de- veloped capsules. The plating medium of Worfel and Ferguson? modified by the addition of 2 per cent agar has been found very use- ful for the selection of capsulated forms.” It is claimed that cultures which are regularly capsulated but which produce capsules of moder- ate size are more satisfactory than cultures producing very large capsules. Capsular nonmucoid forms are preferable to highly mucoid, very slimy cultures. a) Select a suitable colony and inoculate it into 50 ml of infusion broth con- taining glucose in a 0.2 per cent concentration. b) Incubate 4-6 hr at 35° C and kill the organisms by the addition of formalin to a concentration of 0.5 per cent. c¢) Examine the culture by means of India ink preparations to determine the predominance of capsular forms and the size of capsules. d) Use the killed culture as an immunizing antigen to inject the desired number of rabbits with amounts of 0.5, 1.0, 2.0, 3.0, 3.0 and 3.0 ml at intervals of 4 days. e) Test-bleed the rabbits 4 days following the last injection and test the serum for the quellung reaction. If the serum possesses a titer of 1:16 or higher, it is suitable for use. Sera revealing titers of lower magnitude indicate the need for additional immunizing injections. If this is the case, give two additional 3.0 ml injections at 4 day intervals and retest the serum. Discard animals which fail to attain a suitable titer following the second series of injections and repeat the procedure, utilizing a newly prepared antigen. f) Bleed rabbits with sera of adequate titer 5-7 days after the last injection, preserving the sera with merthiolate or glycercl if desired. Since many cross- relationships exist among the different capsule types,10,12,16 test each serum with all known capsular types. Many cross-reactions observed may be weak and low in titer and may not be apparent in routine typing procedures. These may be ignored. However, when cross-reactions are so strong as to cause confusion, it is necessary to absorb the sera. * Manufactured by Gunther Wagner, Hanover, Germany. MISCELLANEOUS INFECTIONS 611 Preparation of Specific Absorbed Capsular Serum 1) Grow the desired absorbing organism on a slant of Worfel- Ferguson agar.'® 2) Suspend the growth in 1.0 ml salt solution. 3) Add this suspension dropwise to 1.0 ml of serum to be absorbed, shaking continuously during the addition. As the first drops of antigen suspension are added, agglutination takes place almost instantly ; as additional antigen is added, the reaction occurs pro- gressively more slowly. 4) When the supernatant fluid shows a slight turbidity, discontinue the addition of absorbing antigen. 5) Centrifuge the serum, remove the clear supernatant by aspiration, and retest the serum by agglutination and quellung tests with the absorbing and homologous antigens. Note: Occasionally one encounters sera which require absorption with more than one antigen. In such cases absorptions are done successively, first with one and then with the other heterologous antigens. Determination of Capsular Antigens®:'7 The determination of capsular type may be made by a variety of methods, among them slide agglutination, tube agglutination, and quellung. In diagnostic work slide agglutination tests have proved convenient and reliable. 1. Slide agglutination a) Prepare a dense suspension in phenolized salt solution of well- capsulated organisms from a colony on a blood agar or infusion agar plate. b) Place a loopful on a slide and add a loopful of capsular serum. Mix thoroughly. ¢) Make as many preparations described above as are necessary, using a different capsular serum in each instance. d) Agglutination, when it occurs, is usually immediate. To insure that the observed agglutination is a capsular and not a somatic reaction, confirmation by the quellung test is required. 612 MISCELLANEOUS INFECTIONS 2. Quellung tests 1) Place two drops of a freshly prepared dilute suspension of organisms (4-8 cells per oil-immersion field) on a slide. 2) To one drop add India ink for a moist mount; to the other, add a loopful of antiserum and mix well. 3) Cover each drop with a separate cover slip. 4) Compare the size of the capsule on the organisms that have been mixed with antiserum to those on the organisms in the India ink preparation in order to determine whether the quel- lung reaction occurs. 3. Tube agglutinations a) As antigen use a 4-6 hr culture of organisms in infusion broth containing 0.2 per cent glucose which is preserved by the addi- tion of formalin to a 0.5 per cent concentration. b) Dilute antiserum serially, beginning with a dilution of 1:4. c) Into each of a series of serological test tubes pipette 0.5 ml of serum dilution. d) To each tube add 0.5 ml of antigen suspension. Mix well. Include antigen and antiserum controls. e) Incubate overnight at 48° C and read. Alternatively incubate at 35° C for 2 hr, refrigerate overnight, and read. Agglutination is evident by the appearance of a firm disk precipi- tate which disperses with difficulty. E. EVALUATION AND REPORTING OF RESULTS Report as “Klebsiella species” those organisms which conform to the biochemical pattern, morphology and staining characteristics as defined above. Report individual serotypes the identification of which is based on the use of specific capsular antisera as “Klebsiella capsular type (1,2, 3, etc.)” as the case may be. Report as “Klebsiella, not encapsulated and therefore not typed” those organisms which are typical Klebsiella on the basis of mor- phology, staining characteristics and biochemical pattern but which fail to produce capsulated forms despite repeated efforts to isolate them. GeorGE M. EISENBERG, Sc.D. MISCELLANEOUS INFECTIONS 613 REFERENCES 1 Hw ow 0 NI 10. 11, 12 13. 14. 15. 16. 17. 18. 19. 20. 21. 2. 23. OBrINSKY, W.; CorncoNT, R. E.; FowLer, R. E. L.; and RUHSTALLER, F. Friedlander-Aerogenes Infections in Infancy. Am. J. Dis. Child. 80:621, 1950. Bergey’s Manual of Determinative Bacteriology (7th ed.). Baltimore, Md.: Williams & Wilkins, 1957. . MacConKEY, A. Lactose-fermenting Bacteria in Feces. J. Hyg. 5:333, 1905. —————— Further Observations on Differentiation of Lactose-ferment- ing Bacilli with Special Reference to Those of Intestinal Origin. J. Hyg. 9:86, 1909. Levine, M. Classification of Colon-Cloacae Group. J. Bact. 3:253, 1918. Epwarps, P. R. Relationships of Encapsulated Bacilli with Special Refer- ence to Bact. aerogenes. J. Bact. 17:339, 1929. . Parr, L. W. Coliform Bacteria. Bact. Rev. 3:1, 1939. Bowman, E. K, Stuart, C. A, and WHEELER, K. M. Taxonomy of Family Enterobacteriaceae. J. Bact. 48:351, 1944. PerkINS, R. G. Bacillus mucosus capsulatus: A Study of the Groups and an Attempt at Classification of the Varieties Described. J. Infect. Dis. 1:241, 1904. KAUFFMANN, F. On Serology of Klebsiella Group. Acta path. et microbiol. scandinav. 26:381, 1949. —————— Enterobacteriaceae (2nd ed.). Copenhagen: Munksgaard, 1954. Brooke, M. S. Further Capsular Antigens of Klebsiella Strains. Acta path. et microbiol. scandinav. 28:313, 1951. WEiss et al. Klebsiella in Respiratory Disease. Ann. Int. Med. 45:1010, 1956. JuniaNeLLE, L. A. Immunological Specificity of Bacterium aerogenes and Its Antigenic Relation to Pneumococcus, Type II, and Friedlander’s Bacillus, Type B. J. Immunol. 32:21, 1937. Orrskov, I. O Antigens in the Klebsiella Group. Acta path. et microbiol. scandinav. 34:145, 1954. Epwarps, P. R,, and Fire, M. A. Capsule Types of Klebsiella. J. Infect. Dis. 91:92, 1952. Epwarps, P. R,, and Ewing, W. H. Identification of Enterobacteriaceae. Minneapolis, Minn. : Burgess Publishing Co., 1955, p. 165. KaurrMANN, F. The Differentiation of Escherichia and Klebsiella Types. Springfield, Ill. : Charles C Thomas, 1951, p. 1. WorreL, M. T., and Fercuson, W. W. A New Klebsiella Type (Capsular Type 15) Isolated from Feces and Urine. Am. J. Clin. Path. 21:1097, 1951. GosLings, W. R. O., and Swijpers, E. P. Untersuchungen iiber das Scleroma Respiratorium (Sklerom) ; IV Mitteilung. Die Antigene Struktur der Skleromstamme im Vergleich mit den Anderen Kapselbakterien. Zbl. Bakt. 1 Abt. 136:1, 1936. Epmunps, P. N. Further Klebsiella Capsule Types. J. Infect. Dis. 94:65, 1954. HeNrIkSEN, S. D. Studies on the Klebsiella Group (Kauffmann). VI. Conclusions. Acta. path. et microbiol. scandinav. 34 :281, 1954. WiLkinsoN, J. F., Ducuip, J. P., and Epmunps, P. N. Distribution of Polysaccharide Production in Aerobacter and Escherichia Strains and Its Relation to Antigenic Character; with a Note on the Influence of Potassium Deficiency upon Production of Polysaccharide by Aerobacter aerogenes. J. Gen. Microbiol. 11:59, 1954. 614 MISCELLANEOUS INFECTIONS Section Ill—Listeria Infections A. Collecticn and Handling of Specimens 1. Types of Specimens 2. Procedures B. Bacteriological Examination C. Serological Examination D. Evaluation and Reporting of Results References Listeriosis is being reported with increasing frequency in the United States! and throughout the world.? This probably reflects an added awareness of the disease rather than an actual increase of human infections. Purulent meningitis or meningoencephalitis and septic granulosis of the newborn account for 71 per cent of the clinical syndromes re- ported by Seeliger.®? Septicemia is often associated with meningitis but may occur separately, frequently in debilitated adults or pregnant women. Listeriosis cannot be differentiated clinically from other forms of bacterial infection, so that an accurate laboratory diagnosis is of paramount importance. A. COLLECTION AND HANDLING OF SPECIMENS I. Types of Specimens a. Cerebrospinal fluid—Collect two tubes of cerebrospinal fluid aseptically, For direct examination, count the number of cells per ml of fluid, using standard technic. The cell count in cases of proved listeriosis has been known to vary from 100 to 150,000 cells per ml, showing a preponderance of neutrophils. For biochemical tests, determine the glucose content (see Chapter 1) and perform a Pandy test for globulin as follows: PANDY TEST In a small test tube add 1 drop of spinal fluid to 1 ml of saturated aqueous solution of phenol (6.25 ml melted phenol crystals made up to 100 ml with distilled water, stored at 35° C for several days, and shaken occasionally to facilitate solution). A bluish white ring is MISCELLANEOUS INFECTIONS 615 immediately formed if an excess of globulin is present. The glucose content is markedly reduced and the Pandy test is positive in listeriosis. For bacteriological studies, take the following steps: 1) Centrifuge the second tube of fluid at high speed for 15 min. 2) Make a Gram-stained film of the sediment. The microorganisms are frequently seen in such preparations, 3) Inoculate any acceptable medium for cerebrospinal fluid cultures with a large loopful of sediment, 4) Refrigerate the remainder of the sediment in a tightly stoppered tube. b. Blood—Obtain about 15 ml of venous blood aseptically and proceed as follows: 1) Inoculate each of two flasks, which contain about 50 ml of any acceptable blood culture medium, with approximately 5 ml of the patient’s blood. 2) Incubate one flask at 35° C. 3) Refrigerate the second flask. 4) Place the remaining blood in a sterile tube, After the clot has formed, remove serum and save for serological tests if later desired. c. Specimens from the uterus, cervix and vagina—Cultures should be made from the reproductive tract of the mother in all cases where an infectious abortion, a stillbirth, or a postnatal in- fection of the infant has occurred. 1) Obtain twos swabs from each area indicated. 2) Use one swab to prepare a thin film and Gram-stain. Listeria may be present in abundance in such material. 3) Place the second swab in a tube of infusion broth medium. d. Tissues—Tissues from all fetuses, premature babies, and in- fants coming to autopsy as a result of undiagnosed infections should be examined for listeria. These microorganisms have been most fre- quently found in the brain, liver and spleen. 1) Request the pathologist to remove aseptically pieces of these tissues about 20 g in size; place them in a sterile container and send to the laboratory. 616 MISCELLANEOUS INFECTIONS 2) Grind about 5 g of each tissue in a sterile mortar with a small amount of sterile broth or salt solution, or macerate in an electric blender. 3) Inoculate two blood culture flasks with this emulsion. 4) Incubate one flask at 35° C. 5) Store the second in the refrigerator. 6) Place the remaining emulsion in the refrigerator. 2. Procedures a. For culture— Listeria monocytogenes is not difficult to grow. Any accepted enriched medium—brain heart infusion (CM No. 21 or 22), tryptose phosphate medium (CM No. 35), trypticase soy broth (CM No. 11 or 12)—is satisfactory for the original cultures. All material that is placed in the refrigerator should be subcultured weekly for at least 1 month. Gray* has shown that initial refrigera- tion is often essential for the isolation of these microorganisms from tissue or even from cultures. b. For bacteriological identification 1) Incubate all inoculated media at 35° C for 24 hr. 2) After subcultures are made, reincubate for an additional 24 hr. Repeat subcultures if the originals show no growth. 3) Examine Gram-stained films of all cultures. 4) Streak a blood agar plate with a large loopful of each culture. Plates made with sheep blood show the beta hemolysis well but other bloods are satisfactory. Note: The addition of 0.03-0.05 per cent potassium tellurite (CM No. 79) is helpful when werking with contaminated ma- terial. Incubate tellurite plates for 72 hr, as listeria grows slowly on this medium.3 5) Incubate all subcultures at 35° C for 24-48 hr. c. For biochemical identification > Carbohydrate fermentation: Inoculate a meat extract-peptone base containing bromcresol purple as an indicator and 0.5 per cent of the desired carbohydrate (CM No. 1). Incubate at 35° C for 1 week before making the final reading. Additional tests: Indole, HS, citrate, urea, nitrates to nitrites, methyl red, Voges-Proskauer, oxidase, litmus milk, and gelatin tests are carried out following accepted procedures (see Chapter 1), d. For motility—The motility of listeria differentiates it from the other diphtheroids, most of which are nonmotile. 1) Stab-inoculate two tubes of semisolid agar (CM No. 69) with 0.5 per cent glucose. MISCELLANEOUS INFECTIONS 617 2) Incubate one tube at room temperature and the other at 35° C. Motility at 24° C is visible as the microorganisms spread from the stab. Later the bacteria form a characteristic disk about 4 mm below the surface of the medium. Motility is minimal at 35° C. B. BACTERIOLOGICAL EXAMINATION 1. Morphology—L. monocytogenes, which is seen in Gram-stained films of the various cultures, appears as small (1-2 1xX0.3-0.5 wu), somewhat pleomorphic, nonacid-fast, nonspore-forming, unencapsul- ated Gram-positive rods. The microorganisms from rough colonies tend to form chains. 2. Colony appearance—Colonies on blood agar are small (1 mm-2 mm in diameter), circular, smooth, entire, raised and translu- cent. All strains readily produce a narrow zone of beta hemolysis on sheep blood agar. This characteristic may be less marked when other blood is used. Colonies on infusion agar are similar to those on blood agar but are somewhat smaller. 3. Biochemical reactions—Carbohydrate fermentation occurs as follows: Acid without gas is produced promptly in glucose and salicin, is usually prompt in maltose and rhamnose is produced slowly if at all in lactose and sucrose, and rarely in mannitol and sorbitol. Other characteristics should be noted. Among the most useful: Indole is not produced, HoS is usually not produced, there is no growth on Simmons citrate agar, and urea is not hydrolyzed. It is helpful at times to know that nitrates are not reduced to nitrites; the methyl red test is positive; Voges-Proskauer is negative (O’Meara), positive (Coblantz)?; oxidase is negative; litmus milk is reduced; gelatin is not liquefied. 4. Animal pathogenicity—Listeriosis is a common disease of animals so that animal pathogenicity studies are in general unsatis- factory for the diagnostic laboratory. A modification of the Anton ophthalmic reaction described by Julianelle® may be used. This is a dependable diagnostic test if freshly isolated microorganisms are em- ployed. Ophthalmic reaction a) Select a young healthy rabbit. b) Turn the lower lid of the eye outward over an applicator stick. 618 MISCELLANEOUS INFECTIONS ¢) Inoculate the membrane with a cotton swab which has been dipped in a 24 hr broth culture of the suspected organism. d) Maintain the animal in isolation and examine daily for 10 days. Smooth cultures of listeria produce a purulent conjunctivitis usually within 5 days. Infection: Local keratitis often occurs and is detected by the de- velopment of corneal opacity following the initial conjunctivitis. The opposite eye is usually not involved. Unless the animal develops a generalized infection, the inoculated eye heals sponstaneously, al- though the sight may be lost. Some rabbits develop a febrile illness following ocular infection and the organism may then be isolated from the blood. Tf the animal succumbs to the infection, culture the liver, spleen and brain. C. SEROLOGICAL EXAMINATION Serological studies are not required for the identification of L. monocytogenes and when they are done, the results are difficult to in- terpret.>®7 Description of procedures can be found in the monograph Human Listeriosis® if desired. Several factors make serological studies impractical for the diag- nostic laboratory: 1. The antibody titer is usually low even in acute infections.2 2. The antigen is complex and unabsorbed antilisteria serum made from the whole microorganism is unsatisfactory.8 3. Many persons who have had no evidence of clinical listeriosis have significant antilisteria titers.? 4. Cross-reactions are demonstrated with several associated microorganisms, in- cluding other diphtheroids, enterococcus, and Staphylococcus aureus.8:10 Serological division into types gives a useful epidemiological tool but such determinations are not recommended for the diagnostic laboratory.!* The 100 American cultures of listeria which have been submitted to the Communicable Disease Center at Atlanta, Ga., have been typed by Dr. Seeliger of Bonn, Germany. Of these cul- tures, 24 and 71 per cent are classified as Type 1 and Type 4b, re- spectively, This differs markedly from the European picture, where Type 1 accounted for 147 of the 156 cultures studied by Potel.'! Typing service is now available in the United States in the laboratory of Dr. M. L. Gray .* *Dr. Gray may be reached at the Veterinary Research Laboratory, Agri- cultural Experiment Station, Montana State College, Bozeman, Mont. MISCELLANEOUS INFECTIONS 619 D. EVALUATION AND REPORTING OF RESULTS Human listeriosis still carries a high mortality despite the fact that most of the organisms are sensitive to the action of the sulfa drugs and to chloramphenicol, erythromycin, streptomycin and the tetracyclines, and irregularly so to penicillin. The infection, which most frequently affects debilitated adults or infants, builds up rapidly so that therapy must be instituted promptly to be effective. The laboratory must be alert to the possibility of listeriosis and should give the clinician a tentative report when diphtheroids are found in body fluids. Therapy can be given, con- comitantly with the laboratory procedures, for definitive identifica- tion. The final report of L. monocytogenes can then be given in writing. When facilities are not locally available for identification of L. monocytogenes, the culture should be sent to the Communicable Disease Center reference laboratory through the local state board of health. Marron Hoop, Pu.D., D.Sc. REFERENCES 1. King, E. O,, and SeeLiGer, H. P. R. Serological Types of Listeria monocy- togenes Occurring in the United States. J. Bact. 77:122-123, 1959. 2. SEELIGER, H. P. R. Listeriosis. New York: Hafner Publishing Co., 1961. 3. —————— and CHErry, W. B. Human Listeriosis. U. S. Dept. Health, Education and Welfare. Communicable Disease Center, Atlanta, Georgia, 1957. 4. Gray, M. L,, et al. A New Technique for Isolating Listerellae from the Bovine Brain. J. Bact. 55:471-476, 1948. 5. Jurianerie, L. A. Biological and Immunological Studies of Listerella. J. Bact. 42:367-383, 1941. 6. Gray, M. L. Listeriosis in Animals—Listeriosen Symposiom. Berlin & Hamburg : Paul Parey, 1958. 7. Porter, J. Die Listeriose beim Menschen. Listeriosen Symposiom. Berlin & Hamburg: Paul Parey, 1958. 8. Hoop, M. Listeriosis. Report of Ten Cases. Am. J. Clin. Path. 28:18-26, 1957. 9. Oserorp, J. W, and Sawyer, M. T. Agglutinating Antibodies for Listeria monocytogenes in Human Serum. J. Bact. 70:350-351, 1955. 10. SeeLicer, H. P. R. Die Serodiagnostik der Listeriose. Listeriosen Sym- posiom. Berlin & Hamburg : Paul Parey, 1958. 11. Porter, J. Zum Gegenwirtigen Stand der Listeriose Forschung. Halle Wess: Math. Nat. VI/2, 1957, pp. 311-334. 620 MISCELLANEOUS INFECTIONS Section IlV—Mimeae Infections . Introduction Sources Morphology . Cultural and Biochemical Characteristics . Antibiotic Sensitivity Serology Pathogenicity OmmuNwE References A. INTRODUCTION Many Gram-negative microorganisms now frequently encountered were seldom noted before the advent of the antibiotic era. Among these are the two species included in the so-called tribe Mimeae. Few microorganisms have been as adequately described under as many different names. Few described species already occupying their ap- pointed place in accepted bacterial classification have as many strains available for study. Few microorganisms are so ubiquitous, so com- monly isolated, and so completely lacking in correct nomenclature. The oldest recognized description of these microorganisms was published in 1906 by von Lingelsheim,! who described them under the name Diplococcus mucosus. This designation has also been used by Cowan,? Gubler,® and Wolff.* In 1940 Audureau® described a “new” species under the name Moraxella lwoffi which she separated into two varieties, var. brevis and var. bacteroides. In the same year Stuart et al® described an anaerogenic group which they designated as BSW. They considered these microorganisms to be Shigella-like. DeBord? in 1942 described three “new” microorganisms for which he created the tribe Mimeae in the family Bacteriaceae. The three genera in the tribe Mimeae each contained one species, Mima polymorpha, Herellea vaginicola, and Colloides anoxydana. Schaub and Hauber® described a group of “unidentifiable” Gram-negative bacilli to which they gave the name Bacterium anitratum in 1948. Lemoigne?® described a group of soil organisms in 1952 under the name of Neisseria winogradskyi. In 1949 Ewing? called attention to the similarity between mem- bers of the tribe Mimeae and B. anitratum. Piéchaud et al** in 1951 studied Stuart’s and Audureau’s cultures and proposed the name MM. lwoffi var. glucidolytica. Henriksen? has linked M. lwoffi with both B5W and B. anitratum. Seeliger'® in 1953 recognized D. mucosus to be the same as B. anitratum. Villecourt and Jacobellil* recognized N. winogradskyi as B. anitratum. These and others, notably Brisou et al.15-1" Murray and Truant,'® Aiken et al,'® Lutz et al ***' and Klinge,?* contributed discussions toward solving the classifica- MISCELLANEOUS INFECTIONS 621 tion problem and establishing acceptable nomenclature for the two species described here. Brisou has further suggested the names Achromobacter lwoffi or anitratum® and Acinetobacter anitratum' to add to the growing list. In this discussion the terminology of DeBord? is used, since this appeared in the sixth edition of Bergey's Manual of Determinative Bacteriology.?® All mention of these microorganisms has been dropped from the seventh edition.?* These microorganisms are more like two species than two genera, but to distinguish between them the generic terms Mima and Herellea will be used. Mima refers to the micro- organism also designated as M. lwoffi var. brevis, while Herellea is otherwise designated as D. mucosus, B. anitratum, BSW, M. lwoffi var. glucidolytica, N. winogradskyi, Achromobacter amitratum or lwoffi, and Acinetobacter anitratum. DeBord’s third genus, Colloides, is now generally regarded as belonging to the family Enterobacteria- ceae and will not be discussed further here.1%1? B. SOURCES The sources from which these microorganisms are isolated are quite varied. A compilation of sources, as listed in studies of large numbers of Herellea strains,'®25-28 shows that approximately 47 per cent were isolated from the urinary tract and 16 per cent from the respiratory tract. Other sources (in per cent) are: blood, 6; cere- brospinal fluid, 5; abscesses, 5; feces, 5; wounds, 3; ears, 3; eyes, 1; genital tract, 1; and miscellaneous sources, 8 per cent. Mima sources were equally diverse, but a large percentage of the strains from case reports were isolated from blood and cerebrospinal fluid. That these organisms are widely distributed in nature is indicated by Lemoigne et al. who isolated Herellea frequently from soil samples in France, and by Kenner and Kabler,?® who isolated Mima from river water. C. MORPHOLOGY?:12,18,19,25.27 The microorganisms are Gram-negative, with a slight tendency to retain the Gram-positive stain. They are surrounded by a small capsule, but an occasional strain may be mucoid and show heavy encapsulation. They are nonmotile and nonspore-forming. The typical morphological form of freshly isolated strains of both Mima and Herellea is coccoid or diplococcoid. They resemble Neisseria, although rod forms may occur either frequently or so seldom as to require a diligent search. An occasional strain may be extremely pleomorphic, showing, besides the coccoid forms, long, 622 MISCELLANEOUS INFECTIONS wavy filaments with swollen areas which frequently retain the crystal violet, D. CULTURAL AND BIOCHEMICAL CHARACTERISTICS The microorganisms grow well on simple media and are therefore easily isolated. On blood agar (CM No. 16) the colonies of Herellea may attain a diameter of 2 to 3 mm in 24 hr at 35° C, while Mima colonies are usually somewhat smaller. Colonies of both species are low, convex, smooth, entire, butyrous, gray-white, opaque and usually nonhemolytic, although a few strains show zones of beta hemolysis. Rough or mucoid variants may occur. Mucoid variants of Herellea occasionally show large capsules and may be mistaken for Klebsiella. A characteristic unpleasant odor is frequently noted. No pigment is produced.*? A heavy, uniform turbidity with a slight ropy sediment and delicate pellicle occurs in liquid media. Rough strains produce a flocculent sediment and a heavy pellicle. The cultural and biochemical characteristics have been adequately described for Herellea®!3:1925.27 and for Mima.>11® Both species usually grow well on MacConkey (CM No. 60), EMB (CM No. 54), and desoxycholate citrate agar (CM No. 57). Growth is delayed on bismuth sulfite agar (CM No. 58) and usually does not appear on SS agar (CM No. 59) except as noted later. On TSI (CM No. 53) slants they produce a neutral butt and an alkaline slant. The majority of Herellea strains grow well on Simmons citrate agar (CM No. 66). The majority of Mima strains grow with small yellow colonies along the line of inoculation but fail to change the indi- cator.®®1% A few Mima strains may be either frankly citrate-positive or -negative, Both species are strongly catalase-positive. Herellea strains are uniformly oxidase-negative, but Mima strains are variable, a few being strongly oxidase-positive; a few positive when first tested and negative thereafter; and the majority negative. Urease may or may not be produced by either species and may be demonstrated in a weakly buffered medium such as Christensen’s urea agar (CM No. 68). Nitrate (CM No. 64) is never reduced, and the presence of un- reduced nitrate should be demonstrated by the use of zinc dust. Hydrogen sulfide (CM No. 53), indole (CM No. 2), and acetyl methyl carbinol (CM No. 112) are not produced. The reported results of the methyl red test for Herellea are not consistent owing to variations in the performance of the test.!® When tested in Difco MISCELLANEOUS INFECTIONS 623 MR-VP broth (CM No. 112) after incubation for 4 or 5 days, they are MR-negative or doubtful (orange color), never frankly positive. Most strains of both species fail to liquefy gelatin (CM No. 13) or do so very slowly. Some strains liquefy in 2 to 7 days. It is in- teresting to note that frequently gelatin-positive strains are beta- hemolytic and grow well on SS agar. These three characteristics are frequently but not always associated.’® In litmus milk (CM No. 14) the reaction is variable, Herellea strains frequently acidify and occasionally clot the milk. Mima strains usually produce an alkaline reaction. A few strains of both species may peptonize milk, but often there is no change. Mima and Herellea strains differ most markedly from each other in their action on carbohydrates. Herellea strains produce acid from certain carbohydrates by an oxidative process, while Mima strains fail to do so. This manner of carbohydrate utilization by the Herellea strains differentiates them sharply from the microorganisms of the enteric group, which utilize carbohydrates independently of the presence of oxygen by a true process of fermentation. This basic difference in carbohydrate utilization may easily be demonstrated by using the method of Hugh and Leifson.3? Two tubes of OF medium (CM No. 73) are inoculated, and one is sealed with sterile petrolatum. All oxidizing microorganisms will produce acid only in the open tube, while fermenters produce acid in the sealed tube also. It may also be noted that true fermenters produce strong acid reactions in the butt of TSI slants, while oxidizers produce an alkaline slant and a neutral butt, with an occasional strain showing a late weak acid re- action on the slant only if lactose or sucrose is utilized. Herellea strains will oxidize fairly promptly certain carbohydrates in extract broth (CM No. 1): glucose, xylose, arabinose, galactose and mannose. Oxidation of lactose and rhamnose is delayed, while the reaction is very late and variable in maltose. Carbohydrates and alcohols not oxidized are: sucrose, levulose, trehalose, mannitol, adonitol and dulcitol. Because Herellea strains produce strong alka- line reactions from peptones, and since the oxidative utilization of carbohydrates does not yield strongly acid reactions, it is best to use a low peptone medium such as the OF (CM No. 73) to demonstrate these oxidative reactions. Mima strains will produce no acid from carbohydrates in extract broth, in OF medium or on 10 per cent glucose (CM No. 70) or lactose slants?! Previously it had been shown by the use of a synthetic broth containing no nitrogen source that Mima and Herellea strains were able to utilize the same carbohy- drates.® Unfortunately this work cannot now be duplicated and 624 MISCELLANEOUS INFECTIONS utilization of carbohydrates by Mima can no longer be demonstrated.?? The important differentiating characteristics are found in Table 1. Many confusing organisms may be eliminated by means of the mo- tility and nitrate tests alone, Table 1—Important Characteristics of the Mimeae Common characteristics : 1. Morphology—Gram-negative cocci with occasional rod (freshly isolated). 2. Nonmotile. 3. No action on nitrates. 4. If carbohydrates are attacked, it is by an oxidative process. Herellea Mima Test: Catalase + + Oxidase — — or + Glucose Acid — Xylose Acid — Mannitol — — Lactose — (or + late) : Sucrose — — Maltose — — 10% glucose slant Acid Alkaline 10% lactose slant Acid Alkaline MR-VP — rn Indole — — H,S — — Urease Variable Variable Medium: MacConkey “- + (occ. —) SS — (occ. +) — (occ. +) Simmons citrate + Variable TSI Alkaline/alkaline Alkaline/alkaline E. ANTIBIOTIC SENSITIVITY Several studies have reported on the sensitivity to antibiotics of large numbers of Herellea strains.?%:26:33.3¢ Results are difficult to correlate owing to the different technics and amounts of antibiotics used in the tests, Most workers agree, however, that all strains are predominantly resistant to penicillin, chloromycetin, bacitracin, eryth- romycin; vary in their resistance to streptomycin and tetracycline; and are predominantly sensitive to terramycin, aureomycin, neomycin and polymyxin B. MISCELLANEOUS INFECTIONS 625 Antibiotic sensitivity studies of only five Mima strains have been reported in the literature.3*-37 A recent study3? with 35 Mima and 6 Herellea strains using the disk method (Difco disks) showed that Mima strains fell into two groups: The first group contained 21 strains which were very sensitive to most of the eight antibiotics used. A few strains showed resistance to penicillin, streptomycin or aureomycin. In the second group all 14 strains were resistant or only partially sensitive to many of the antibiotics used, with the exception of polymyxin B. Results of these tests are shown in Table 2. Table 2—Antibiotic Sensitivity* of the Mimeae Mima Herellea Sensitive, Resistant, Antibiotic and 21 strains 14 strains 6 strains Concentration S PS R S PS R S PS R Aureomycin 5-10-30 ug 19 2 0 3 3 8 0 0 6 Chloromyecetin 5-10-30 ug 21 0 0 8 5 1 1 1 4 Streptomycin 2-10-100 ug 16 1 4 7 2 3 0 5 1 Erythromycin 2-5-15 ug 21 0 0 9 4 1 1 4 1 Penicillin 2-5-10 u 12 0 9 2 0 12 0 0 6 Polymyxin B 50-100-300 u 21 0 0 14 0 0 6 0 0 Terramycin 5-10-30 ug 21 0 0 5 9 0 1 2 3 Tetracycline 5-10-30 ug 21 0 0 3 4 5 0 0 6 S=Sensitive PS =Partially sensitive R=Resistant * Bacto sensitivity disks for antibiotics, Difco Laboratories, Detroit 1, Mich. F. SEROLOGY The antigenic formula of these microorganisms has not been worked out in detail, but Ferguson and Roberts?® were able to show by agglutination and absorption tests at least 10 capsular types in the 626 MISCELLANEOUS INFECTIONS Herellea group. Herellea capsular-diagnostic sera are easily pre- pared and are remarkably tribe-specific.!® Two multivalent pools con- taining 12 different Herellea antisera have been shown to agglutinate about 95 per cent of approximately 400 strains tested.!® Herellea strains will agglutinate in antisera prepared from certain types of Klebsiella, notably Types 5, 6, 17, 45, 47 and 67. A limited number of Klebsiella strains of these types have failed to agglutinate in Herellea antisera.’® Only rarely is a microorganism other than Herellea or Mima encountered which will agglutinate in Herellea serum. Serological confirmation of the Mimeae is more difficult. A few strains will agglutinate in Herellea-diagnostic antisera,'*'? but the same methods useful for Herellea do not give good results with Mima. Carey, Lindberg and Faber®®3® have recommended a method for slide agglutination of both species which requires sodium hydroxide treat- ment of the antigen. G. PATHOGENICITY The published results of animal pathogenicity tests are conflicting because of the wide variation in the pathogenicity of various strains. Some strains have been found to be pathogenic for mice and guinea pigs on intraperitoneal inoculation, while others are nonpatho- genic.8:20:21,26,37.,40-42 Typical hepatic lesions have been described in rabbits.2%2! In the past there has been considerable doubt as to their pathogenicity for man, but more recently there are increasing num- bers of case reports in which both Herelleat!-45 and Mima35-37:40,46,47 are being incriminated as the primary cause of various infections, notably meningitis*® and septicemia. That they are pathogenic, at least for certain individuals, seems evident. Evrzasera O. King REFERENCES 1. LingersHEIM, W. von. Die Bakteriologischen Arbeiten der Kgl. Hygienis- chen Station zur Beuthen O. Schl. Wihrend der Genickstarreepidemie in Oberschlesien im Winter 1904-05. Klin. Jahrgb. 15:373-488, 1906. 2. Cowan, S. T. Unusual Infections Following Cerebral Operations, with a Description of D. mucosus (von Lingelsheim). Lancet, Nov. 5, 1938, p. 1052. 3. Guster, H. U,, and HassiG, A. Epiphytare Neisserien als Meningitiserreger Schweiz. Zentralbl. allg. Path. 13:610-615, 1950. 4. Worrr, L., and TeuscH, W. Meningitis durch Diplococcus mucosus. Med. Monatsschr. 5:839-841, 1951. 5. Aupureau, A. Etude du Genére Moraxella. Ann. Inst. Pasteur 64:126-166, 1940. 6. Stuart, C. A. FormaL, S. and McGaNN, V. Further Studies on B5W, an Anaerogenic Group in the Enterobacteriaceae. J. Infect. Dis. 84:235-239, 1940. MISCELLANEOUS INFECTIONS 627 7 10. 1 12. 13. 14. 16. 17 18. 19. 20. 21. 22 23. 24. 25. 26. 27. 28. 29. DEeBorp, G. Descriptions of Mimeae trib. nov. with Three Genera and Three Species and Two New Species of Neisseria from Conjunctivitis and Vaginitis. Iowa State Coll. J. Sci. 16 :471-480, 1942. ScHAUB, I. G., and Hauser, F. D. A Biochemical and Serological Study of a Group of Identical Unidentifiable Gram-Negative Bacilli from Human Sources. J. Bact. 56:379-385, 1948. LemoioNE, M., Girarn, H. and JacoBeLL, G. Bacterie du Sol Utilisant Facilement le 2-3 Butanediol. Ann. Inst. Pasteur 82:389-398, 1952. Ewing, W. H. The Relationship of Bacterium anitratum and Members of the Tribe Mimeae (DeBord). J. Bact. 57:659, 1949. Precuaun, D., Precaaun, M., and Seconp, L. Etude de 26 Souches de Moraxclla lofi. Ann. Inst. Pasteur 80:97-99, 1951. HenrikseEN, S. D. Moraxella: Classification and Taxonomy. J. Gen. Microbiol. 6:318-328, 1952. SEELIGER, H. Zur Systematik des Bacterium anitratum (Schaub and Hauber). Zbl. Bakt. T Orig. 159:173-176, 1953. ViLLecourt, P., and JacoseLrr, G. Bacteries Pathogénes ou Saprophytes Transformant le Glucose en Acide Gluconique (Bacterium anitratum, BSW, Moraxella lwoffi var. glucidolytica, Neisseria winogradskyi). Ann. Inst. Pasteur 86:493-502, 1954. Brisou, J., and MoricHAU-BEAUCHANT, J. Identité Biochemique entre Certaines Souches de B. anitratum et M. lwoffi. Ann. Inst. Pasteur 82:640- 643, 1952. — ——— and GiMENEZ, J. Mimeae et Formes Courtes des Bacteries. Ann. Inst. Pasteur 84:814-816, 1953. Brisou, J. Contribution a L’Etude des Pseudomonadaceae. Précisions Taxonomiques sur le Genere Acinetobacter. Ann. Inst. Pasteur 92:134-137, 1957. Murray, R. G. E., and TruanT, J. P. The Morphology, Cell Structure, and Taxonomic Affinities of the Moraxella. J. Bact. 67:13-22, 1954. Aiken, M. A., Warp, M. K,, and King, E. O. A Study of a Group of Gram-Negative Bacteria Resembling the Tribe Mimeae (DeBord). Pub. Health Lab. 14 :126-136, 1956. Lurz, A.; GrootEN, O.; VEUL, M.; and VELU, H. Récherches sur des Bac- teries du Type B. anitratum (BSW) et Leur Sensibilité aux Sulfamides et aux Antibiotiques. Rev. Immunol. 20:215-230, 1956. ——————— Role Pathogéne et Fréquence des Bacteries du Type B. anitratum. Ann. Inst. Pasteur 91:413-417, 1956. Kuringe, K. Zur Systematik, Gramnegativer, Pleomorpher, Kahlehydrate nicht Spittender und Ozydase-negativer Diplobakterien. Arch. Hyg. u Bakt. 142:171-179, 1958. Bergey's Manual of Determinative Bacteriology (6th ed.). Baltimore, Md. : Williams & Wilkins, 1948, page 595. Ibid. (7th ed.). Baltimore, Md.: Williams & Wilkins, 1957. Fercuson, W. W,, and Roserrs, L. F. A Bacteriological and Serological Study of Organisms BSW (B. anitratum). J. Bact. 59:171-183, 1950. Brooke, M. S. The Occurrence of BSW (B. anitratum) Strains in Denmark. Acta path. et microbiol. scandinav. 28 :338-342, 1951. Simpson, M. A. and Crossiey, V. M. B. anitratum (B5W) Strains Iso- lated in Ontario. Canad. J. Pub. Health 45:259-263, 1954. Scorr, E. G., and MAHONEY, B. A. The Occurrence of Members of the Tribe Mimeae in Human Infections. Delaware State M. J. 25:22-24, 1953. Kenner, B. A, and KaerLer, P. W. Members of the Tribe Mimeae Isolated from River Water. J. Bact. 72:870, 1956. 628 MISCELLANEOUS INFECTIONS 30. HucH, R., and LerrsoN, E. The Taxonomic Significance of Fermentative versus Oxidative Metabolism of Carbohydrates by Various Gram-Negative Bacteria. J. Bact. 66:24-26, 1953. 31. Cuicron, M. L., and Furton, M. Presumptive Media for Differentiating Paracolon and Salmonella Cultures. J. Lab. & Clin. Med. 31:824-827, 1946. 32. King, E. O.: Unpublished data. 33. Lunn, E. Sensitivity of B. anitratum to 8 Antibiotics as Determined by Means of the Tablet Method. Acta path. et microbiol. scandinav. 34:329- 335, 1954. 34. Brooks, B. E, and Sanpers, A. C. “Unidentified” Gram-Negative Rods and the Tribe Mimeae, U. S. Armed Forces M. J. 5:667-672, 1954. 35. Frep, H. L.; ArLen, T. D.; Hessel, H. L.; and Horrzman, C. F. Meningi- tis due to Mima polymorpha. Arch. Int. Med. 102 :204-206, 1958. 36. OrarssoN, M., Leg, Y. C., and AserNerHY, T. J. Mima polymorpha Meningitis. Report of a Case and Review of the Literature. New England J. Med. 258:465-470, 1958. 37. ScHULDBERG, I. I. Clinical and Pathological Simulation of Meningococcic Meningitis. Am. J. Clin. Path. 23:1024-1027, 1953. 38. Cary, S. G., LinDpBeRrG, R. B., and FABER, J. E. Typing of Mima polymorpha by a Precipitin Technique. J. Bact. 72:728-729, 1956. 39. — Slide Agglutination Technique for the Rapid Differentiation of Mima polymorpha and Herellea from the Neisseriae, J. Bact. 75:43-45, 1958. 40. DeBorp, G. G. Mima polymorpha in Meningitis. J. Bact. 55:764-765, 1948. 41. Waace, R. B. anitratum (BSW) Isolated from Cerebral Abscesses. Acta path. et microbiol. scandinav. 33 :268-270, 1953. 42. Deacon, W. E. A Note on the Tribe Mimeae (DeBord). J. Bact. 49:511- 512, 1945. 43. Ino, J., and NEUGERAUER, D. L. Isolation of a Species of Genus Herellea from a Patient with Chronic Synovitis. Am. J. Clin. Path. 26:1486-1489, 1956. 44. MinzTer, A. Human Infections Caused by the Mimeae Organisms. Report of a Case of a Presumably Healed Bacterial Endocarditis Due to Herellea vaginocola. Arch. Int. Med. 98:352-355, 1956. 45. RocHA, H., and Guzg, L. B. Infections Due to B. anitratum. Arch. Int. Med. 100:272-275, 1957. 46. Faust, J., and Hoon, M. Fulminating Septicemia Caused by Mima poly- morpha. Report of a Case. Am. J. Clin. Path. 19:1143-1145, 1949. 47. Townsenp, F. M., Hersey, D. F., and WiLson, F. W. Mima polymorpha as a Causative Agent in Waterhouse-Friderichsen Syndrome. U. S. Armed Forces M. J. 5:673-679, 1954. 48. Warrg, C. L,, and Kung, A. H. Mima polymorpha Meningitis, Report of a Case and Review of the Literature. Am. J. Dis. Child. 98 :379-384, 1959. MISCELLANEOUS INFECTIONS 629 Section V—Infections Due to Pleuropneumonia-like Organisms (PPLO) A. Collection of Specimens B. Bacteriological Examination 1. Culture Medium 2. Incubation 3. Colony Characteristics 4. Staining Reactions References The role of pleuropneumonia-like organisms (PPLO) as primary etiological agents of infectious processes in man remains to be demon- strated conclusively. However, the cultivation of these microorgan- isms in pure culture from infectious processes in man and animals makes it necessary to take cognizance of their possible presence and importance when attempting laboratory diagnoses of infectious diseases. Their presence in conditions diagnosed as vulvitis, vaginitis, cervicitis, endometritis, salpingitis, tubo-ovarian abscess, Bartholin gland abscess, urethrocystitis, prostatitis, nongonococcal urethritis, fusospirochetal gangrene of the penis, phagedenic balanitis, and epi- didymitis has been summarized.! While the majority of the reports of isolating PPLO from man (and frequently from animals) have involved specimens taken from the genitourinary tract, other regions of the body have been involved. These microorganisms have been isolated from brain abscesses, cere- brospinal fluid, blood, joint fluids, and the conjunctivae. PPLO which resemble in appearance those isolated from man are frequently isolated from animals with which man has contacts. These animal sources include mice, rats, cattle, sheep, goats, swine, dogs, cats, chickens, turkeys, pigeons, parakeets, ducks, guinea pigs and horses. In some species of animals, infections with PPLO are of great economic importance. Saprophytic strains also have been isolated. Another important aspect of PPLO is the presence of these micro- organisms in supposedly pure cultures of tissue cells, It is not always an easy task to eliminate or prevent contamination of tissue cell cultures by these microorganisms.? A. COLLECTION OF SPECIMENS Little definitive information can be gained from the microscopical examination of clinical specimens, as the size of PPLO is at the limit of resolution of the light microscope and smaller, The organisms stain poorly by the usual bacteriological stains, which is a further 630 MISCELLANEOUS INFECTIONS deterrent to microscopical examination. Clinical specimens to be ex- amined for PPLO practically always are taken for the purpose of cultivation in the laboratory. On artificial medium the organisms have characteristic growth requirements, produce characteristic colonies, and stain differentially. They have a very thin, fragile struc- ture which functions as a cell wall, in contrast to the thicker and more rigid cell walls of bacteria and fungi. These characteristics make it imperative that the microorganisms be handled gently and with great care. Material to be cultured for PPLO is best transferred to appropri- ate solid medium by means of an inoculating loop or cotton swab. If direct cultivation is not possible, the clinical material should be placed in broth, such as that used for their cultivation with or without the proteinaceous supplement. A matter of hours at room or refrigerator temperatures does not appear to be harmful. No studies have been made on the survival of these organisms in transportation media. B. BACTERIOLOGICAL EXAMINATION The detection of PPLO in clinical specimens depends upon the demonstration of characteristic colonies of the organisms which de- velop on appropriately enriched solid medium. I. Culture Media An infusion of beef heart muscle appears to be a necessary in- gredient in media for supporting the best growth of PPLO from human sources. Infusions prepared from 500 g of fresh beef heart muscle per liter of water or from 50 g of dehydrated beef heart for infusion (such as Bacto-beef for infusions) appear to be equally suitable.? The brand of peptone is important. It has been found that either Bacto-peptone or Bacto-tryptose in a final concentration of 1 per cent is the most satisfactory. Each liter of medium should contain 5 g of sodium chloride. Since the colonies of PPLO grow into the medium, in contrast to bacterial colonies, the medium should be slightly softer than when culturing bacteria. A concentration of 14 g of agar per liter of medium has proved satisfactory, whether the medium is subsequently enriched with 25 per cent ascitic fluid, 10 per cent blood serum, or 1 per cent serum fraction. Some lots of agar intended for bacterio- logical use may be inhibitory to the growth of PPLO. It is therefore desirable to determine the suitability of each lot of agar for support- ing the growth of PPLO.* MISCELLANEOUS INFECTIONS 631 The pH should be not less than 7.8 after sterilization. The medium should be filtered not more than once, as repeated filtrations may render it unsatisfactory for growth of PPLO. Sterilize in the autoclave at 121° C for 15 min. Earlier workers recommended adding 2 per cent packed red blood cells to the agar base, melted and cooled to 60° C for the purpose of neutralizing toxic substances which might be present. The mixture is heated in a boiling water bath and clarified by centrifugation, the supernatant decanted. This precaution is unnecessary with materials currently available and the few red blood cells remaining in the medium when so treated may be disconcerting to the technician search- ing for small colonies of PPLO (see CM No. 76 and No. 77). A dehydrated agar base containing the above ingredients, requiring merely solution in water and sterilization, is Bacto PPLO agar. The incorporation of fresh yeast extract into the medium has been found useful in growing some of the more fastidious strains.’ For growing the parasitic strains, the basal medium must be en- riched with some proteinaceous material. Ascitic fluid in a concen- tration of 25 to 30 per cent of total volume of the final medium may be used. It has the disadvantages that it is variable in its chemical composition, it entails working with large quantities, and it is some- times difficult to obtain in adequate and sterile supply. For growing the PPLO strains from man, ascitic fluid has the advantage that the protein is of human origin. Blood serum from various species of animals in final volume of 20 per cent may be used for enrichment, but sera from the various species may be toxic and fail to support growth of PPLO. Sera from different individuals in the same species may likewise be toxic. It is possible to fractionate serum chemically to obtain the growth-promoting fraction free of the toxic fraction.® A serum fraction is available commercially (Bacto-PPLO serum fraction) and is added to the basal medium in a concentration of 1 per cent.” There is no single substance which is an adequate enrichment for cultivating all strains of PPLO. Occasionally a strain may be en- countered which requires the presence of blood in the medium for primary isolation.® The addition of blood to the medium is not a practical step, as its presence makes the medium opaque. Examina- tion of colonies of PPLO on an opaque medium by transmitted light is extremely difficult. The metabolism of PPLO is quite different from that of most bacteria, so that it is possible to inhibit the growth of bacteria while 632 MISCELLANEOUS INFECTIONS permitting growth of PPLO to proceed. This often is desirable when working with clinical specimens or with contaminated cultures. The addition of thallium acetate to the medium in a final concentration of 1:1,000 or 1:2,000 is often very helpful. Since PPLO are the most penicillin-resistant microorganisms known, penicillin in the range of 100 to 1,000 units per ml of medium may be used to suppress bac- terial growth while at the same time permitting growth of PPLO. A combination of thallium acetate and penicillin has been recommended.? 2. Incubation Incubation at 36° C is desirable. An aerobic atmosphere during in- cubation is usually satisfactory. However, an anaerobic atmosphere may be more beneficial for an occasional strain when conditions for growth are not optimal. The presence of 10 per cent carbon dioxide in the atmosphere is usually not beneficial and in some cases may be inhibitory.? It is important that the medium and cultures of PPLO be protected from drying. It may be necessary to incubate cultures of PPLO for 3 to 5 days in order to obtain maximum growth. 3. Colony Characteristics The typical colonies of PPLO are best searched for by means of the low-power magnification of the microscope (about 100X) and transmitted light. When the organisms are growing satisfactorily on the medium and the colonies are well separated, the colonies usually but not always have a “fried egg” appearance. That is, the centers of the colonies are darker than the peripheries. This is caused by the fact that the central portion of the colony is growing into the medium while the peripheral portion of the colony grows on the surface of the medium. The diameter of colonies varies from 100 to 25 uw. In other cases—for example, when colonies are crowded on the medium or owing to the peculiarity of the strain—the colonies may be small, round and somewhat refractile, without obvious cellular structure. Sometimes the colonies are vacuolated. This gives them a lacy effect and the appearance is well brought out by either reflected or trans- mitted light.10 A second characteristic of PPLO colonies is that they are not com- pletely wiped off the medium with an inoculating needle or loop as are bacterial colonies. To make subcultures, a block of the agar contain- ing typical colonies of PPLO is cut out of a plate, placed inverted on the surface of fresh medium, and rubbed back and forth over the medium to be inoculated. MISCELLANEOUS INFECTIONS 633 A third characteristic of these colonies is that they stain by the method of Dienes (see Chapter 1). Cover glasses containing a dry film of the stain may be stored for a long time. The staining pro- cedure is carried out in one of two ways: (1) One method is to place a block of agar cut out of a petri dish culture containing colonies sus- pected of being PPLO onto a microscope slide, with the colonies uppermost. A cover glass treated with the stain is placed on top of the agar block so that the dry film of stain is in contact with the colonies. Colonies of bacteria and of PPLO stain blue within a very short time. After about 15 min, bacterial colonies lose the deep blue color because of the reduction of the methylene blue to its colorless state by the metabolizing bacteria; the colonies of PPLO retain the deep blue color for days if the agar block is protected from evapora- tion. (2) A second method is to allow a 1:100 dilution of the Dienes stain to flow over the surface of petri dish cultures. A fourth characteristic of PPLO colonies is that when a stained preparation is examined by means of the oil-immersion lens, the usual definite bacterial forms, such as rods and cocci, are not observ- able. There are unstained vacuoles among a matrix of fine particles which range in size from slightly less than 1 u to below the limits of resolution of the light microscope. Under the electron microscope PPLO from man appear spheroidal to ellipsoidal in shape.!! 4. Staining Reactions Individual cells of PPLO stain poorly using the common bacterio- logical stains. The most frequently employed stain for PPLO is the Dienes stain. It is more helpful to stain the colonies of PPLO in situ, as described in the detailed procedures for Dienes stain in Chapter 1 and in the preceding section, which deals with PPLO colonies. Giemsa’s stain as used for bacteria will also stain PPLO but not differentially as does the Dienes stain. Wayson’s stain, as described in Chapter 1, Section F, has been used by some investigators for staining PPLO. Harry E. Morton, Sc.D. REFERENCES 1. MortoN, H. E. “The Pleuropneumonia and Pleuropneumonialike Organ- isms,” Chapter 29 in Bacterial and Mycotic Infections of Man (Rene J. Dubos, Editor, 3rd ed.). Philadelphia: J. B. Lippincott, 1959. 2. Hearn, H. J, Jr.; OFFICER, J. E.; ELsNER, U.; and Brown, A. Detection, Elimination, and Prevention of Contamination of Cell Cultures with Pleuropneumonialike Organisms. J. Bact. 78:575-582, 1959. 634 MISCELLANEOUS INFECTIONS 3. Morton, H. E.,, SMmrirH, P. F,, and LeBerMAN, P. R. Investigation of the Cultivation of Pleuropneumonia-like Organisms from Man. Am. J. Syph. 35:361-369,1951. 4. LynN, R. J, and Morton, H. E. The Inhibitory Action of Agar on Certain Strains of Pleuropneumonia-like Organisms. Applied Microbiol. 4:339-341, 1956. 5. Epwarp, D. G. ff. A Selective Medium for Pleuropneumonia-like Organ- isms. J. Gen. Microbiol. 1:238-243, 1947. 6. Smita, P. F., and Morton, H. E. The Separation and Characterization of the Growth Factor in Serum and Ascitic Fluid Which Is Required by Certain Pleuropneumonialike Organisms. J. Bact. 61:395-405, 1961. 7. Morton, H. E., Smita, P. F., and KeLLer, R. Prevalence of Pleuro- pneumonia-like Organisms and the Evaluation of Media and Methods for Their Isolation from Clinical Material. A.J.P.H. 42:913-925, 1952. 8. Prorres, D. M., Morton, H. E, and Fro, L. G. Unusual Pleuropneumonia- like Organisms Isolated in a Study of Trichomonas vaginalis from Cases of Chronic Urethritis. J. Bact. 73:398-401, 1957. 9. Morton, H. E., and Lecce, J. G. Selective Action of Thallium Acetate and Crystal Violet for Pleuropneumonialike Organisms of Human Origin. J. Bact. 66 :646-649, 1953. 10. NEeLsown, J. B. Association of a Special Strain of Pleuropneumonia-like Organisms with Conjunctivitis in a Mouse Colony. J. Exper. Med. 91:309- 320, 1950. 11. Morton, H. E.; Leccg, J. G.; Oskay, J. J.; and Coy, N. H. Electron Microscope Studies of Pleuropneumonialike Organisms Isolated from Man and Chickens. J. Bact. 68:697-717, 1954. MISCELLANEOUS INFECTIONS 635 Section Yl—Pseudomonas aeruginosa Infections The genus Pseudomonas is composed of Gram-negative, polar- flagellated, straight or slightly curved rods capable of utilizing glucose under aerobic conditions, often with the formation of moder- ate amounts of acid. Although ethanol may be utilized, acetic acid is not produced in appreciable amounts. Most species produce water- soluble pigments, usually green, yellowish green or blue in color. Occasionally brown or reddish pigments occur and some strains lack pigmentation. Most of the species of this genus are ubiquitous saprophytes widely distributed in soil and water, from which sources they commonly gain access to foods and other materials. A few species are frequently or occasionally parasitic and some are actual or poten- tial pathogens.! The species most commonly encountered in clinical materials is Pseudomonas aeruginosa. This organism is frequently found in the intestinal tract? and can also be isolated from the surface of the body, mouth and throat in the absence of clinical symptoms. It is constantly present in sewage and can sometimes be isolated from soil and water, particularly when they have been fertilized or contaminated. The organism is also found in lower animals and is capable of producing a specific disease in the tobacco plant.>* As a human pathogen P. aeruginosa has been commonly isolated from cystitis,® intestinal in- fections,® infected wounds and burns,” generalized infections® and, apparently with increasing frequency, from the respiratory tract.® Since this organism has unusual resistance to many antibiotics and to the quaternary ammonium sanitizing compounds, it may become the predominant organism when these agents curtail the growth of competing bacteria either in association with the body or in the ex- ternal environment. A detailed account of clinical infections has recently been published by Forkner.1? Microscopically the organism is rod-shaped and measures approxi- mately 0.3 to 0.5 by 1 to 3 nu. It occurs singly, in pairs, and in short chains. Cultures on blood agar or other enriched media may show bi- polar staining to the same degree as do plague bacilli,** and occasion- ally intracellular granules are present. The organism is Gram-negative and no special staining procedures are necessary. Capsules are usually absent, although mucoid encapsulated strains have been described.'? The organisms are as a rule actively motile. Most strains appear to be monotrichous. P. aeruginosa can be cultivated on a wide variety of culture media and pure cultures are readily obtained, since the organism is inhibi- tory to many other bacteria through the production of antibiotic sub- 636 MISCELLANEOUS INFECTIONS stances. Colonies are low, greyish, rounded and soft, with a marked tendency to confluescence. Although colonies are not pigmented, they are often surrounded by a colored area from diffusion of pig- ment into the medium. On fresh moist media they generally grow as a thin spreading film, like that of Proteus, rather than as discrete colonies. Indeed, there is some danger that the thin film may be overlooked if it covers the entire plate. Colonies of many strains show a metallic, iridescent surface with a rather ragged moth-eaten pattern. While the exact nature of this phenomenon has not been clearly proved, it appears to be associated with the formation of a lipid or other material which lyses some of the cells. Despite this appearance, the lysis is not caused by the presence of bacteriophage and its presence or absence shows no correlation with source, bacterio- phage type, or cultural reactions. A chloroform-soluble blue pigment, pyocyanin, is produced in vary- ing amounts by cultures of P. aeruginosa but not by other species of the genus. Other pigments which may be produced by this or other species of Pseudomonas are soluble in water but not in chloroform. The addition of a few milliliters of chloroform to cultures on either liquid or solid media will result in extraction of the pigment, showing the same shade of blue as that seen in dilute copper sulfate solutions. In the presence of acid the pigment turns red and becomes insoluble in chloroform. This red color is sometimes confused with a positive indole test when the acid-type reagents are used. This organism does not form indole. The presence of pyocyanin has also caused confusion with hydrogen sulfide production in Kligler’s medium, since the red of the indicator in this medium and the deep blue produced by strains forming large amounts of pyocyanin give a black color. However, extraction with chloroform will cause disappearance of the black color by dissolving the pyocyanin. Sulfide, of course, is not soluble in this reagent. The amount of pigmentation varies with the strain and with the culture medium (see Table 1). The pigment is masked on blood agar and may be formed in only small amounts on some common media. Sabouraud’s maltose agar'® (CM No. 105) and the “Tech” medium of King et al.'* stimulate production of pyocyanin. Other pigments, particularly fluorescein, are better developed on the “Flo” medium of King.'* In some strains the production of other pigments may mask or even supplant the formation of pyocyanin. This is particularly true when the red chloroform-insoluble pyorubrin is formed. This should not be confused with the acid form of pyocyanin which it closely re- sembles in color. MISCELLANEOUS INFECTIONS Table 1—Media for Pseudemonas 637 a. Asparagine enrichment medium (for feces, sewage, etc.) : ASPETFABHIE ... . sositsmmnmmio n » wivi-siiei sd o's 30ETamias) 1 5 o FEABHEFE 2h % 8 2 g EK HPO, (2rliydrous) ic. be smuimns b+ 2 + semaines s £6 suamins arid 1 g KaS0, (anliydrons). woe: ssemvmmm nso es wrndmnes vos ouster: » 10 g MESO THAD Li inns os wi osnions §5 5s sutaimmings k + lism i934 05 ¢g GIBEIOL CB... commons vs 35 sprees 2 2 8% base + 3 5 SAREE 3 8 ml WBE woes iiss Gsvemesis i SUROEETE 2 t 53 SSEERS 1s & § Rniiemies 2 ¢ 5. 1,000 ml Adjust the pH to 6.9-7.2 and autoclave at 121° C for 15 min. b. Acetamide confirmatory medium, Buhlman et al.,'» modified : NACL vom: ve 3 225 mens v5 15 3 08 WHEE £55 5 SEE 2 § 8 ROUEN £528 5 g KLHPO, (anhydrons) «.::::eammine ss vosnmbinnss 5s o memidosies s 2s 1.39 ¢g BH,PO, (SOBRGATOURY . .. communes ss bnmmsne so ns wieanbonts so 3 on 0.73 g Phenol 18 ..;vvwwmmmen vos pumas ve noe Sen HSER b 3b bs HEEEERS BEE 28 0.012 g Acetamide oo... i ea 10 g Distilled. WISE ..ovebiommein x » wonmiaisim « » » » aiotwmnsos Sa smamared 1 3 40 1,000 ml Adjust to pH 6.9-7.2 and autoclave at 121° C for 15 min. c. “Flo” medium (to enhance fluorescein production) :14 Proteose peplone £3 co oteoimmiss vasa se awiinmdoe sss 20 g BED AHL. «covccmrmstitrecn sre bhnswn msasvoms ih mitt AIRE st gt is EVE 008 15 g Clyeeral C. Po. o.vvvimmmun vanmmenwimss boss b unlit totes bi sb 10 ml KPO, (anliyQrons) cvesossvmmisssismmmsmmimes smelmanss ise 153 g DIGS OUTTA ....cmsecncsmmmmimmmmmsmucrmn sis mms Simos wrist od 15 zg Adjust pH to 7.2, slant with generous butt. The correct peptone is very important. d. “Tech” medium (to enhance pyocyanin production) :14 Bacto PEDLONE sons avs sas waiass so shes mein asian ees wet ana 20 BEGID BBE ...omwiwio wrasse sims meses ssi nit 1s fp ao Thi ss BTA Sos 8 15 Glycerol CT. Bl vuvve sr sume vey 55s 1 sommsin ps gs Bn S64 4:8 SETH 10 MgCl, (anhydrous) ssovvmwsensss somes sv ssesniwsms st eninse 14 BnS0, (auhiydrons) :auscwsonssrsesmesisnst sommimsinisine.s 3 osisione 10 g g ml g g Adjust to pH 7.2, slant with generous butt. The correct peptone is very important. e. Potassium Gluconate Medium (for identification of P. aeruginosa) :16 TEUPIONE « coininis « i» 5S EEHIRAD £ 3 La SmSval08 & § LESS § 4.4 Pied 1.5 YEE BRITAE crv v5 2 mosis 5.8 + mimi #4 5 » Smiling » ¥ 5% 3mm 1.0 BotIPO, neon worn s pumommnne + 5 5 soins £5 8 Empat ts $55 1.0 Potassiom FIUCONRLE us smmmens sds poesia fs 2 5a smiows + « samens 40.0 DIStIIEA WALEE oii v5 5 5 ms ive un 2 + + 3 5 mimminiw 5.5 + 4 5% BRR £4 5 § 3 3 Ga 1,000 8 0 0] 0Q 09 1 Adjust pH to 7.0, place 100 ml in a 300 ml flask, incubate on a rotary shaker at 28°-30° C, and test for the presence of reducing substances after 48 hr incubation by means of Benedict's reagent. Incubate for an additional 48 hr at 35° C and observe for slime production. 638 MISCELLANEOUS INFECTIONS Most strains of P. aeruginosa have a very noticeable odor which is characteristic for the species—a rather cloyingly sweet, fruity aroma. This, together with the characteristic production of pyocyanin, leads to rapid identification of most isolates. Several specific characteristics are useful in the identification of P. aeruginosa, particularly valuable for nonpyocyanogenic strains: 1) Ability to grow at 42° C, which eliminates almost all other species in the genus. 2) Rapid reduction of nitrates to nitrites and gaseous nitrogen. 3) Production of moderate and often transitory acidity from glucose but not from sucrose or lactose. 4) Conversion of potassium gluconate to ketogluconate,16 as shown by the formation of reducing compounds when tested by means of Benedict's and similar tests. 5) Formation of abundant slime following further incubation in the same medium. 6) Lack of fermentation and a flat, shiny appearance on triple sugar iron agar. Urea is not hydrolyzed but the organisms are actively proteolytic. A species-specific extracellular toxic substance demonstrable by agar gel diffusion has been reported by Liu,'7 although it may not be practical for routine use. Studies on bacteriophage typing of P. aeruginosa have shown the existence of five distinct groups as well as a number of bacteriophage- sensitive cultures which do not fit any group.'® About 20 per cent of this species are not sensitive to bacteriophages currently available from the American Type Culture Collection. Epidemiological studies have implicated hospital environment as being the source of some P. aeruginosa infections, with organisms of one or more types that appear to be implanted in a given institution.® CuARrRLEs H. DrakE, Pu.D. Jorn C. Horr, PH.D. REFERENCES 1. Bergey's Manual of Determinative Bacteriology (7th ed.). Baltimore, Md.: Williams & Wilkins, 1957. 2. RingGeN, L. M,, and DrAKE, C. H. A Study of the Incidence of Pseudomonas aeruginosa from Various Natural Sources. J. Bact. 64:841, 1952. 3. Errop, R. P,, and Braun, A. C. Pseudomonas aeruginosa; Its Role as a Plant Pathogen. J. Bact. 44:633, 1942. 4. Horr, J. C, and Drake, C. H. Susceptibility of Pseudomonas polycolor to Pseudomonas aeruginosa Bacteriophages. J. Bact. 80:420, 1960. StanLey, M. M. Bacillus pyocyaneus Infections. Am. J. Med. 2:253, 1947. Hun~TER, C. A., and EnsioN, P. H. An Epidemic of Diarrhea in a Newborn Nursery Caused by Pseudomonas aeruginosa. A.J.P.H. 37:1166, 1947. 7. GraBer, C. D. Burn Center U. S. Army Surgical Research Unit, Fort Sam Houston, 1960. Personal communication. oven MISCELLANEOUS INFECTIONS 639 8. 9. 10. 11. 12, 13. 14. 15. 16. 17. 18. 19. DAuER, C. C. Epidemiological Notes—Septicemia. Pub. Health Rep. 74:354, 1959. GouLp, J. C. Department of Bacteriology, University of Edinburgh, Scot- land, 1960. Personal communication. ForkNER, CLAUDE E., Jr. Pseudomonas aeruginosa Infections. Modern Medi- cal Monographs No. 22. New York and London: Grune & Stratton, 1960. Drake, C. H,, and Harr, E. R. Department of Bacteriology and Public Health, Washington State University, 1960. Personal communication. Scuwartz, L. H., and Lazarus, J. A. An Unusual Strain of Pseudomonas aeruginosa. J. Bact. 54:30, 1947. MAarTINEAU, B., and ForGer, A. Routine Use of Sabouraud Maltose Agar for the Rapid Detection of the Bluish-Green Pigment of Pseudomonas aeruginosa. J. Bact. 76:118, 1958. King, E. O., Warp, M. K,, and Raney, D. F. Two Simple Media for the Demonstration of Pyocyanin and Fluorescein. J. Lab. & Clin. Med. 44:301, 1954. BunLMmAaN, X.,, ViscHErR, W. A., and BruHIN, J. Identification of Apyo- cyanogenic Strains of Pseudomonas acruginosa. J. Bact. 82:787-788, 1961. Haynes, W. C. Pseudomonas aeruginosa—Its Characteristics and Identifi- cation. J. Gen. Microbiol. 5:939, 1951. Liu, P. Y. Identification of Pathogenic Pseudomonads by Extracellular Antigens. J. Bact. 81:28, 1961. Horr, J., and Drake, C. H. Paper read before the Laboratory Section of the American Public Health Assn. at the Annual Meeting, San Francisco, Calif., November, 1960; also Ph.D. thesis, in press. Gourp, J. C, and McLeop, J. W. A Study of the Use of Agglutinating Sera and Phage Lysis on the Classification of Strains of Pseudomonas aeruginosa. J. Path. & Bact. 79:295, 1960. 640 MISCELLANEOUS INFECTIONS Section ViIl—Sodoku* Infection in man with Spirillum minus usually results from the bite of a rat, mouse or other rodent. Less frequently, the bite of a weasel, cat or other carnivore that presumably has been feeding on rodents causes infection. The history of an animal bite, a recrudescent wound after apparent healing, and the systemic symptoms suggest a diagnosis of sodoku. Although there are several claims in the literature that the micro- organism has been cultured on tissue-free media, these are not gen- erally accepted. Growth in tissue culture has not been reported. Diagnosis of the disease in man depends upon demonstration of S. minus in exudate from the initial lesion, in adjacent lymph nodes, or in the blood. When microorganisms cannot be seen in materials from the patient, laboratory animals should be inoculated either sub- cutaneously or intraperitoneally with freshly collected specimens. Mice, white rats, guinea pigs and rabbits are susceptible but may not become ill. The blood of experimental animals should be examined before inoculation, since S. minus is sometimes present in laboratory animals. Occasionally single microorganisms or objects indistinguishable from S. minus are found in films of blood or tissue from experimental animals where infection is not suspected. Attempts to establish the infection in such instances by serial passage have given consistently negative results. Such bodies may be intestinal spirochetes or tissue fibrils. The writer prefers to detect numerous typical organisms before making a diagnosis. In white mice, .S. minus is most abundant in the blood about 14 to 17 days after inoculation. S. minus may be readily demonstrated by dark-field examination but the writer prefers thin or thick blood films stained with Giemsa or re- lated stains. In 1949, Hughes! of the Rocky Mountain Laboratory demonstrated that there is a high concentration of microorganisms, 5 to 50 per microscope field, in impression films of the heart muscle of infected mice, Mus musculus. Comparable numbers of microorganisms were seen in impression films stained with Giemsa and in sections stained by silver impregnation. Such concentrations have also been found consistently in laboratory white mice 30 days to 1 year postinocula- tion when microorganisms were scarce or were not demonstrable in the blood. The Hughes technic! has proved extremely useful and re- * Ratbite fever induced by Spirillum minus Carter. MISCELLANEOUS INFECTIONS 641 liable in our laboratory work, and it has been used successfully, to a limited extent, in surveys for infection in wild mice and rats. It has also facilitated the development of a mouse protection test. A useful modification of the Hughes technic is the staining of fresh preparations of crushed heart tissue with Thedane blue solution T-5, a product of Allied Chemical and Dye Corporation, New York. This stain contains saponin, which quickly destroys the erythrocytes and some other cellular elements but stains .S. minus within a few minutes, usually by the time the slide is placed on the microscope. Serological tests for the diagnosis of S. minus infections include one in which immune serum immobilizes live microorganisms in a fresh preparation. It is not dependable according to some workers and is not routinely used in the United States. Complement-fixation tests for syphilis have been reported positive in many cases and nega- tive in others. This discrepancy may be due to confusion of two types of ratbite fever, namely, sodoku and Haverhill fever.2 S. minus infec- tion in rabbits gives rise to a positive Weil-Felix reaction with Proteus OXK strains, according to Savoor and Lewthwaite.3 We have con- firmed this with a Montana strain, An active lytic principle has been observed in immune sera and this suggested the possibility of a mouse protection test. We were able to show that convalescent sera from monkeys and rabbits would neutralize an infectious suspension of mouse heart inoculum when in- cubated 1, 2 or 3 hr at 35° C before inoculation into mice (unpub- lished data). Mice were then kept for 30 days and examined by heart impression films. Convalescent sera protected in dilutions of diag- nostic significance and end points were sharp. Sera from normal animals gave no protection. It was not possible to obtain enough human sera to evaluate the test. Sodoku is probably much more common than published reports of cases in the United States would indicate. WiLLiaMm L. Jervison, Pu.D. REFERENCES 1. Jeruson, W. L.; ExeBor, PaurL L.; Parker, R. R.; and HucGHEs, LynpaHL E. Rat-Bite Fever in Montana. Pub. Health Rep. 64,52:1661-1665 (Dec.) 1949. 2. Dorman, C. E.; Kerr, D. E.; CHANG, H.; and SHEARER, A. R. Two Cases of Rat-Bite Fever due to Streptobacillus moniliformis. Canad. J. Pub. Health, 42:228-241 (June) 1951. 3. Savoor, S. R,, and LEwrHWAITE, R. The Weil-Felix Reaction in Experi- mental Rat-Bite Fever. Brit. J. Exper. Path. 22:274-292 (Oct.) 1941. 642 MISCELLANEOUS INFECTIONS Section VIIl—Streptobacillus moniliformis Infections . Introduction Specimens . Identification . Special Methods . Serological Tests Animal Inoculation . Interpretation and Evaluation of Results QEEUO®Ep References A. INTRODUCTION Streptobacillus moniliformis is frequently found as a normal in- habitant of the nasopharynx of laboratory and wild rats.! Rats are generally refractory to infection but they may show involvement of the lungs and middle ear. Normal mice do not carry the micro- organism, but epizootic disease in laboratory and wild mice due to the streptobacillus occurs.?® Therefore transmission from mice to man must be considered. Streptobacillus moniliformis is transmitted to man most frequently by the bite of rats, occasionally by handling of rats, bites of other rodents, and probably also by ingestion of con- taminated food, particularly milk.* The human disease caused by the streptobacillus is referred to in different ways: (a) as Haverhill fever (erythema arthriticum epi- demicum, (b) as ratbite fever, and (c) by a clinical description of disease, for example, “subacute bacterial endocarditis” caused by Streptobacillus moniliformis. 1) Haverhill fever—The name originates from the description of an epidemic outbreak of human streptobacillus infection in Haver- hill, Mass., probably related to contaminated milk.> The term is still frequently used, particularly when history of a ratbite cannot be elicited. 2) Ratbite fever—There are two etiological agents of ratbite fever. The most commonly found in the U. S. is Streptobacillus moniliformis. Ratbite fever caused by Spirillum minus occurs more frequently in the Orient but is also encountered in the United States (see Section VII of this chapter preceding). Occasionally infection with Streptobacillus moniliformis clinically not typical of Haverhill fever or ratbite fever has been reported in human patients. A few cases of subacute bacterial endocarditis in- cited by Streptobacillus monilif ormis have been reported. MISCELLANEOUS INFECTIONS 643 In each of these illnesses, whether it is classical ratbite fever or another form of infection, the streptobacillus may be cultivated from the blood. If arthritis develops, as is frequently the case in Haverhill fever and ratbite fever, the organism may be recovered from the joint fluid. Abscess formation also occurs and the organism may be demonstrated in the pus. The laboratory examinations that aid in the diagnosis of Strepto- bacillus moniliformis infections are (1) microscopical examination of joint fluid or pus; (2) cultivation of the microorganism from blood, from fluid of involved joints, or from abscesses; and (3) agglutina- tion of streptobacillus antigen in the patient's serum. B. SPECIMENS I. Collection In the case of blood specimens, 15 ml of blood should be added to a small flask containing 4 to 5 ml of 2 per cent sodium citrate. Joint fluid should also be citrated, as it often clots. Pus from abscesses may be collected on a swab, or aspirated pus may be placed in a sterile tube. 2. Handling All specimens should be inoculated on culture media as soon as possible after collection. a. Blood—In addition to the routine procedure for blood cul- tures of inoculating a flask of broth, a tube of thioglycolate medium (CM No. 19) and two tubes of melted agar for pour plates, it is sug- gested where streptobacillus infection is suspected that several tubes of broth (5 ml) containing ascitic fluid (20%) or serum also be in- oculated, with 1 ml of blood as inoculum for each tube. Any good broth such as tryptose phosphate (CM No. 35) or glucose starch (Difco) or trypticase soy (BBL) (CM No. 11) with a final pH of 7.3 is satisfactory. Agar (1.5%) may be made with any of these broths as a base. The tubes of broth should be incubated aerobically and in a candle jar. b. Joint fluid—Films should be stained by Gram’s and Way- son’s methods and examined microscopically. Depending on the quantity of material available, enriched thioglycolate medium and tubes of enriched broth should be inoculated with 1 to 2 ml of fluid. Freshly prepared agar plates containing 20 per cent ascitic fluid or horse serum should be inoculated on the surface with 0.1 ml of joint 644 MISCELLANEOUS INFECTIONS Figure 1 A and B—Streptobacillus moniliformis: A (above), film of culture grown in liquid medium. Wayson stain (see B, photomicrograph on facing page 645, right) impression preparation from colonies grown on 20 per cent ascitic fluid beef infusion agar. fluid and this distributed by tilting of the plate. The agar should be extremely clear to facilitate microscopical examination of the plates. If serum or ascitic fluid is not available, coagulated blood agar (CM No. 17) is a good substitute. However the L; colonies are not as readily demonstrable on this as on transparent media (see “Special Methods”). The plates and broths should be incubated aerobically and in a candle jar (again refer to “Special Methods”). The plates to be incubated aerobically should be sealed with Parafilm or cellulose tape to preserve moisture. c. Pus from abscesses should be examined and handled as di- rected for joint fluid. If there is very little material to work with, it is best to inoculate at least one agar plate, one tube of broth, and one tube of thioglycolate medium, all enriched with serum or ascitic fluid. The plate and broth should be incubated in a candle jar, MISCELLANEOUS INFECTIONS 645 Photomicrographs courtesy Brown and Nunemaker C. IDENTIFICATION I. Morphology and Staining Reactions Streptobacillus moniliformis is a pleomorphic, Gram-negative rod, nonacid-fast, nonmotile, 2 to 3 u in length, with branching filaments up to 30-40 pn long; these filaments, which may be segmented, vary in width and often have large spindle-shaped swellings and “knobs.” The name assigned to this microorganism is quite descriptive, since the rods form chains that resemble a string of beads or a necklace. Giemsa stain and Wayson’s plague stain demonstrate very well the extremely pleomorphic nature of the streptobacillus (see Fig 1, both prints)—one of its most distinguishing features. The Wayson stain is preferred by the author because of the speed and ease with which the procedure can be carried out. The microorganisms appear blue when they are viable and stain pink when nonviable. Inter- mediate forms are often encountered. Microscopic preparations from 646 MISCELLANEOUS INFECTIONS pus or joint fluid may fail to show the pleomorphic spindle-shaped and filamentous elements seen in cultures but may show only very small, slender bacillary forms. 2. Growth Requirements The streptobacillus has an extremely high enrichment require- ment. Its inability to grow on unenriched culture media is one of the important criteria for identification. It will not grow even for one transfer unless the enrichment requirement is met. Addition of 20 per cent of ascitic fluid or serum or of 10 per cent whole blood to the culture media will fulfill this requirement. Reduced oxygen tension, obtained in a candle jar, will facilitate growth on primary isolation, while subsequent transfers will grow aerobically and anaerobically as well. Better growth is obtained when cultures on solid media are kept in a moist atmosphere. This is important, as initial growth may be slow (1-2 weeks). 3. Appearance of Growth In broth—On primary isolation the streptobacillus grows in broth cultures as discrete colonies which resemble “fluff balls.” In blood culture broth tubes, the colonies are seen resting on the surface of the sedimented red blood cells. It is well to remember that these tubes should be inspected very carefully under a good light for evi- dence of growth and should not be disturbed by shaking or tilting. The supernatant of the broth may be perfectly clear and may reveal no microorganisms on staining. If a “colony” is withdrawn with a capillary pipette and stained, its true structure is seen as a mass of tangled filaments and rods described above. It is much easier to observe the colonies in tubes of broth than in flasks. As the culture is transferred, it loses its “fluff ball” appearance and grows in a more dispersed fashion. Growth then begins at the bottom of the tube and extends up along the sides. Solid media—On blood agar and coagulated blood agar the colonies are small, round and grayish, appearing on the 2nd or 3rd day. In the depths of blood agar the colonies appear in 5 to 7 days, are 1 mm in diameter, and are surrounded by a zone of poorly defined partial hemolysis. On serum or ascitic fluid agar the colonies usually appear after 48 hr of incubation. They are small, round, colorless and granular (see Fig 2) and when observed under the low power of a microscope, the filamentous bacterial forms are demonstrable at the periphery. MISCELLANEOUS INFECTIONS 647 Photomicrographs courtesy Brown and Nunemaker Figure 2—Streptobacillus moniliformis colonies. 4. L; Component Two to 4 days after development of the streptobacillus colonies on plates of serum or ascitic fluid agar, a new type of colony appears. These colonies are extremely small. They are just barely visible to the unaided eye or with a hand lens but should be searched for and studied by direct examination of the inverted plates with a micro- scope, using a magnification of 100>X. These microscopic colonies are the L; or pleuropneumonia-like colonies (see Section V of this chapter) found in cultures of Streptobacillus moniliformis. They may appear between the bacterial colonies or in close proximity to them but are found most frequently in the center of these colonies. Young L; colonies appear tiny and somewhat darker than the bacterial colonies. Fully developed L; colonies have a characteristic appear- ance, showing a dark center, with a lighter, lacy, vacuolated periphery (see Fig 3). They grow into the agar. Films stained with Gram’s or Wayson’s stain are unrevealing. Im- pression films of IL; colonies stained with Giemsa’s or Wayson’s stains show only an amorphous mass of particles of varying size with no definite bacillary elements. 5. Other Properties The cultural features described are more helpful in identifying the streptobacillus than are the orthodox methods for classifying other bacteria. Carbohydrate fermentation is not diagnostic, although some 648 MISCELLANEOUS INFECTIONS Photomicrographs courtesy Brown and Nunemaker Figure 3—L, colonies separated from Streptobacillus moniliformis strain, strains are capable of breaking down glucose, levulose, maltose, salicin and starch. If desired, these determinations should be made in an enriched semisolid medium to which the carbohydrate has been added. A suitable indicator may be added to demonstrate the fermentation. The streptobacillus causes a marked drop in the pH of broth within 24 to 48 hr. The acidity which is produced kills the bacilli and after this period a successful transfer is impossible. In order to maintain viable cultures it is necessary to subculture the organism every 24 hr. The bacillus may also be preserved by freezing broth cultures at a temperature of —20° to —30° C. Viable organisms may be recovered from frozen cultures after 2 to 3 months’ preservation at this temperature, D. SPECIAL METHODS 1. Blind transfers—If an infection with Streptobacillus monili- formis is suspected, but after 2 or 3 days neither broth nor plate cul- tures show the typical growth described above, it is suggested that “blind transfers” be made from the original broth to broth as well as solid media for 3 to 5 successive days. Using this method, one can occasionally obtain growth that did not become apparent on primary culture. MISCELLANEOUS INFECTIONS 649 This method of blind transfer appears particularly important in the case of specimens from patients who have had one or more doses of penicillin before specimens were obtained. Most strains of Strep- tobacillus moniliformis are extremely sensitive to penicillin. How- ever, strains of pleuropneumonia-like organisms, including the IL, are quite resistant to penicillin. Penicillin therapy may result in disappearance of the bacterial forms, but with persistence of the L; component of the streptobacillus. In view of this and of the fact that the L; component is recognizable on solid media only, blind transfers from broth to solid media should be carried out several times before a culture is discarded as negative. 2. Agar cutout—This method may be used for transfer from solid to solid or liquid media. It also provides an excellent means for examination of bacterial and 1; colonies under an oil-immersion lens. This is important, especially when cultures are not on transparent media. A small platinum spade serves well for cutting the agar and lifting the block. A colony is first located under the microscope; or if the medium is not transparent, an area of apparent growth is selected. A block of agar approximately 1 cm square is cut out. For transfer, this block is either placed in a tube of appropriate broth or it is placed, colony side down, on an agar plate. Then it is gently pushed across the plate by the spade or by a sterile swab, Growth will occur along the lines of the streak. For microscopical examination the cutout is placed, colony side up, on a slide. A small loopful of Wayson’s stain is applied to the block and a cover slip is placed gently on the preparation. The preparation is then sealed at the border by melted paraffin. This preparation can be examined under the low-power or oil-immersion lens. For per- manent stained preparations the method of Salaman is recommended.® 3. Large inoculum—It is advisable to use a large inoculum (0.5 to 1 ml) when making blind transfers and when transferring broth cultures. Whole colonies should be cut out and used as in- oculum, rather than transfer by the usual method of touching a wire to a portion of a colony. 4. Impression films—An impression film may be made by plac- ing a flamed, sterile cover slip on a colony and leaving it there for a few seconds. The cover slip should be lifted gently with forceps, avoiding undue sliding, and flamed to fix the preparation. The cover slip is then placed, film side up, on a slide and sealed to it with melted paraffin. It may then be stained with Giemsa or Wayson stain (see Fig 1 B on page 645). 650 MISCELLANEOUS INFECTIONS E. SEROLOGICAL TESTS Agglutinins are formed during the course of a streptobacillus in- fection. These are demonstrated by the usual technic for tube agglu- tination. A titer of 1:80 is considered diagnostic. Titers as high as 1:5,120 have been reported. The examination of two or more speci- mens at intervals of 5 to 10 days is recommended to demonstrate change in titer, especially if agglutination occurs in a dilution no greater than 1:80 of the first specimen. The only technical difficulty in preparing a satisfactory strepto- bacillus antigen is the fact that the organism tends to grow in aggre- gates. This tendency can be overcome by adding glycerol to the culture medium. Preparation of antigen—To 80 ml of broth add sufficient glycerol to give a final concentration of 3 per cent. After steriliza- tion add 20 per cent of serum or ascitic fluid and inoculate with 2 ml of an 18 to 24 hr culture of the streptobacillus. At the end of 3 or 4 days’ incubation, add 1.25 ml of formalin and mix well. Centrifuge the culture and wash the sediment three times with 0.85 per cent salt solution. Resuspend in salt solution. The turbidity of the antigen should be adjusted to an optical density of 200 (Coleman) or to a McFarland Standard 1. Agglutination test—Follow the standard procedure for tube agglutinations (see Chapter 1). Incubate the tubes overnight in a 56° C water bath. Refrigerate them for 2 to 3 hr. Shake the tubes vigorously and read the results immediately. As in all agglutination tests, include for purposes of control, tests with normal and known agglutinating sera. The rabbit is a satisfactory animal for preparing known serum with a high titer. F. ANIMAL INOCULATION Animal inoculation is usually not necessary to establish a laboratory diagnosis of infection with the streptobacillus, although the micro- organism is pathogenic for mice. In acute infections in mice an over- whelming septicemia occurs, with early death. In the chronic type of infection, arthritis, anemia and conjunctivitis are the main features. Mice are also susceptible to intraperitoneal infection with Spirillum minus. For completeness of diagnosis it is therefore advisable to inoculate mice in cases with a history of ratbite in addition to the cultural and serological procedures outlined above. The mice are in- MISCELLANEOUS INFECTIONS 651 oculated intraperitoneally with 0.5 ml of citrated blood. It is neces- sary to ascertain that they are free of spontaneous .S. minus infection prior to inoculation. G. INTERPRETATION AND EVALUATION OF RESULTS The isolation of Streptobacillus moniliformis from blood, joint fluid or pus establishes the diagnosis of an infection incited by Strep- tobacillus moniliformis. An agglutination titer of 1:80 gives presumptive evidence of an infection with Streptobacillus moniliformis. Demonstration of a change in titer is essential for definite diagnosis, as agglutinins have been shown to persist for several years. Lucie B. RoBIiNson REFERENCES 1. StrancEwAayvs, W. I. Rats as Carriers of Streptobacillus moniliformis. J. Path. & Bact. 37:45, 1933. 2. Freunor, E. A. Streptobacillus moniliformis Infection in Mice. Acta path. et microbiol. scandinav. 38:231, 1956. 3. WiLLiams, S. An Outbreak of Infection due to Streptobacillus moniliformis among Wild Rats. M. J. Australia 1:357, 1941. 4. Brown, THOMAS McP., and NUNEMAKER, JouN C. Rat-Bite Fever. Bull. Johns Hopkins Hosp. 70,3:201, 1942. 5. Pracg, E. H,, Surren, L. E,, and WiLLNER, O. Erythema Arthriticum Epi- demicum. Preliminary Report. Boston Med. & Surg. J. 194:285, 1926. 6. SaLaMAN, M. H., et al. The Isolation of Organisms of the Pleuropneumonia Group from the Genital Tract of Men and Women. J. Path. & Bact. 58:31, 1946. 652 MISCELLANEOUS INFECTIONS Section IX—Toxoplasmosis A. Introduction 1. Clinical Features 2. The Agent 3. Pathological Lesions B. Isolation of the Parasite 1. Cerebrospinal Fluid 2. Tissue C. Immunological Diagnosis 1. Dye Test 2. Hemagglutination Test 3. Complement-Fixation Test 4. Skin Test A. INTRODUCTION Toxoplasma gondii is probably a protozoon which possesses the unique characteristic of being able to invade all cells of mammals and birds with the possible exception of non-nucleated erythrocytes. Evi- dence has been obtained which suggests that infection with this para- site occurs throughout the world and is widely distributed among both domesticated and wild animals. Its role in human disease was not proved until 1939, but great strides have been made during the past decade in obtaining information about the parasite and the ill- nesses attributable to it. In human beings, toxoplasma causes either acquired or congenital disease; the former may be inapparent or clinically active. The results of a number of serological epidemio- logical surveys suggest that inapparent infections are the most fre- quent. Not enough is known about the manifestations of the clinically apparent acquired disease to discuss with full confidence its entire clinical spectrum. I. Clinical Features a) Congenital toxoplasmosis—This disease probably results from the infection of a fetus as a complication of parasitemia in an inapparently infected pregnant woman. Infection of the fetus may result in a stillbirth or in a viable infant born either prematurely or at term. The infant may be ill at birth, with such findings as fever, maculopapular rash, splenomegaly, hepatomegaly, hydrocephalus, microcephaly, microphthalmia, chorioretinitis, cerebral calcification or convulsive disorder. None of these signs may be noted at birth— MISCELLANEOUS INFECTIONS 653 they may appear first within the neonatal period, or clinical suspicion may not be aroused until some weeks or months following delivery, when the infant may be found to have psychomotor retardation, abnormal variation in head size, chorioretinitis, convulsions or cere- bral calcification. b) Acquired toxoplasmosis—This may be manifested by a short or prolonged febrile course, with malaise, maculopapular rash, generalized lymphadenopathy, encephalitis, myalgia and myocarditis. These symptoms may occur in any combination but usually not all will be present in a single patient. Toxoplasma has been isolated from the adult eye and structures strongly resembling toxoplasma have been seen in sections from eye cases. The frequency with which acquired toxoplasmosis produces uveitis or chorioretinitis is unknown and is one of the most important questions about this disease that have yet to be answered. c) Inapparent toxoplasmosis—This is probably the commonest form of infection and presumably explains the frequency with which antibodies are detected in various normal populations. 2. The Agent T. gondii is considered to be a protozoon and is elongate, oval, crescentic or pyriform. The organisms are 6-7 p in length, 2-4 p in width, and are readily sedimented in the centrifuge. The nuclear struc- ture is distinct and readily identified from the cytoplasm. The para- site is quite delicate and is usually killed by freezing and thawing as well as by chemical bactericidal agents. However, it may be pre- served by freezing under special conditions. Multiplication is by binary fission. T. gondii is stained best with Wright or Giemsa stains. 3. Pathological Lesions The central nervous system is most severely injured in the con- genital form of toxoplasmosis but histological changes may be found in almost all tissues. Areas of necrosis are especially common in the heart, lungs, skeletal muscle, spleen, central nervous system, retina and choroid in the congenital disease. Organisms occur in tissues in two forms, either as isolated parasites or as pseudocysts. The pseudocysts represent aggregations of toxoplasma contained within a membrane. The surrounding tissue frequently shows no reaction. Parasites re- main viable for many years in pseudocysts. 654 MISCELLANEOUS INFECTIONS B. ISOLATION OF THE PARASITE Isolation is best accomplished by injecting suspect material into laboratory-reared mice. We isolated one strain of 7. gondii from pigeons which did not infect eggs until it had become well adapted to the mouse. This and similar experiences of others make the egg less desirable for initial isolation attempts than mice. I. Cerebrospinal Fluid When isolation is attempted from cerebrospinal or ventricular fluid the entire sample should be rapidly transferred to mice by both the intracerebral and the intraperitoneal routes. If the sample is large, concentrate by centrifugation for 30 min at 3,000 rpm. Resuspend sediment in small amounts of supernate and divide among three, preferably six, mice. At 14 day intervals, assuming that the mice remain well, sacrifice two animals, remove brains and spleens asepti- cally, pool and effect passage to a new group of six mice. At least three blind passages should be performed before considering the isolation trial a failure. Mice which become sick or develop ascites should be sacrificed and passaged. Organisms may be visualized in mice or in the original spinal fluid by staining films of ascitic or spinal fluid sediment with Wright or Giemsa stains. Examine each mouse passage for toxoplasma in stained impression films of the spleen, lungs and brain. 2. Tissue Mince human or animal tissues with sharp scissors and suspend in 10-20 per cent concentration in sterile skim milk, 10 per cent rabbit serum salt solution (rabbit serum should be inactivated for 30 min at 56° C) or salt solution with 0.5 per cent gelatin. Use fresh tissues and handle aseptically. Allow mixture to settle for a few minutes, take the bulk up into a syringe, and divide equally among six mice. Inoculate intraperitoneally and intracerebrally into the same animals. The remainder of this routine is as given in the preceding para- graph. Alternatively, tissues may be digested with pepsin solution (0.26 g of pepsin, 0.5 g of NaCl, 0.7 ml of concentrated HCI, and HO to 100 ml) for 2 hr at 35° C. Use approximately 125 g of tissue for 100 ml of pepsin solution. After incubation, filter through cloth, centrifuge, and wash sediment with salt solution twice. Resuspend in a small volume of salt solution and inoculate aliquots into mice. MISCELLANEOUS INFECTIONS 655 C. IMMUNOLOGICAL DIAGNOSIS I. Dye Test A sensitive indicator of antibody is the dye test, based upon the principle that living toxoplasmas killed by antibody plus heat-labile serum factor (probably the properdin system) are not stained by alkaline methylene blue. Suitable activator has been found only in human serum, for animal, bird and fish sera contain nonspecific killing factors which preclude their use in the dye test. Mouse serum does not possess activator, which may explain the unusual susceptibility of this species to infec- tion with the parasite. The following reagents are required for the dye fest: a. Activator (fresh human serum) b. Alkaline methylene blue (pH 10.5-11.0) c. Heparin (1:100 in salt solution) d. 0.9% NaCl in distilled water Activator—Prepare by obtaining 500 ml of blood from a human donor previously shown not to have dye test antibody. Blood may be allowed to clot overnight at 4° C, at which time the serum is removed, dispensed in 2 or 5 ml amounts (depending upon the anticipated daily test load), and rapidly transferred to a dry-ice box. Blood may be processed more rapidly by defibrinating it as it is obtained, spin- ning off the cells and rapidly freezing the serum. Whichever method is employed, keep blood and serum cool during the various manipula- tions. Once frozen in COs ice, activator will retain its activity for at least several years. Alkaline methylene blue—Stock is saturated solution of methylene blue in 95 per cent ethyl alcohol. To make alkaline methy- lene blue, 3.0 ml of stock solution are mixed with 10.0 ml of soda- borax buffer solution with a pH of 11.0. Commercially prepared buffer tablets may be substituted for the soda-borax buffer. Alkaline dye is ready for use after mixing. Use fresh solutions, since they become unsuitable after aging for more than 2 or 3 days. Heparin—Prepare stock heparin solution by suspending powder in sterile 0.9 per cent salt solution in a concentration of 1:100. Sterilize this solution by filtration through a bacteriological filter ; add no preservative. Store in cold and dispense as needed. Physiological salt solution—This is standard 0.9 per cent sodium chloride in distilled water. Use as a diluent throughout the procedure. 656 MISCELLANEOUS INFECTIONS Procedure Make fourfold dilutions of serum by mixing 0.1 ml of inactivated (30 min at 56° C) serum with 0.3 ml of salt solution and then serial dilutions by removing 0.2 ml from each dilution tube. Use 0.1 ml for the next dilution and place the remaining 0.1 ml in the tube in which the test will be performed. Observe standard serological rules for the mixing and carrying over of concentrated serum to tubes contain- ing dilutions. When all serum dilutions have been prepared, obtain parasites by removing ascitic fluid from intraperitoneally infected mice, preferably on the 3rd or 4th day after passage. Avoid including mouse blood. Mice may be killed first with ether or tapped while alive. If they struggle too much, bleeding may ensue. The fluid should be liquid, not thick, with many free parasites and relatively few intracellular organisms. Exudate must be used within 1 hr after being drawn from mice. If fluids from several mice are pooled, examine a drop of each before mixing to select those with the largest number of para- sites for the mixture. If the exudate is satisfactory, transfer one part to four parts of freshly thawed activator serum which has had enough 1 per cent heparin added to make a 1:50 final dilution of the anticoagulant. Im- mediately thereafter add 0.1 ml of this mixture to tubes containing similar amounts of the test serum dilutions. Include in each run negative (0.1 ml of salt solution or heated activator serum) and positive (0.1 ml of known positive serum) controls, Shake all tubes, transfer to 37° ‘C water bath, and incubate for 1 hr. Then add 0.02 ml of alkaline methylene blue to each tube to stain parasites and stop reaction. Remove a drop of stained suspension from each dilution tube and examine with high, dry magnification (fluorite or apochromatic lenses are advantageous), using a cover slip. To determine the proportion of stained and unstained parasites, 100 extracellular toxoplasmas are counted. The negative control tube should have at least 90 per cent stained parasites and the positive at least 90 per cent unstained parasites. Proportions lower than these lead to difficulties in interpreting the remainder of the test and it is best to discard such a run. The titer of the serum is the initial serum dilution in which 50 per cent of the parasites are unstained. Pro- zones of 1:64 or more are unusual. Sera may be screened at 1:4, 1:16 and 1:64, or 1:16-1:256. With practice, it is possible to select the dilutions near the end point by inspection, making it unnecessary to count the parasites in each tube. Intracellular organisms will remain stained in the presence of MISCELLANEOUS INFECTIONS 657 antibody and “unstained” organisms may have a small nuclear dot of blue but the cytoplasm should be unstained. Other technical modi- fications have been reported. We usually select a titer of 1:16 as being the minimal positive dilu- tion. When active infection is suspected, serial samples should be obtained at frequent intervals, preferably weekly, Titers of 1:1,024 and higher are common for several years after either acquired or con- genital infections. Passively transferred antibody (maternal) will diminish markedly by about 3 months of age and will disappear almost completely by 6 months of age. If the dye test is to be employed, it is best to select laboratory per- sonnel who already have antibodies, for laboratory-acquired infections can be serious. 2. Hemagglutination Test Antibody levels similar to those determined with the dye test have been reported by hemagglutination. Tannic acid-treated sheep or human type O, Rh-negative erythrocytes can be used. Antigen is prepared from water-lysed parasites, previously washed to remove other components of the toxoplasma-containing exudate. Antigen is unstable in the liquid state but keeps well frozen or lyophilized. Ex- perience with this test is limited. The authors recommend the follow- ing procedure: Inactivate all test and rabbit sera for 30 min at 56° C. Absorb test sera, and the rabbit sera used for dilution, overnight with equal volumes of washed, pooled sheep erythrocytes. Use 2 per cent normal rabbit serum salt solution for serum dilutions. Determine optimal antigen dilution by titration against a standard serum in a grid experiment. Select that dilution (frequently 1:200) which yields the highest antibody level, using a 2 plus hemagglutina- tion pattern as the end point. Suspend 1 ml of washed sheep erythrocytes (stored in modified Alsever’s solution) in 50 ml of buffered salt solution, pH 7.2. Mix with 50 ml of tannic acid 1:80,000 and incubate in 37° C water bath for 15 min. Wash red cells in salt solution, pH 7.2; sensitize by add- ing 1 ml of tannic acid-treated cells to 1 ml of antigen diluted with 4 ml of salt solution buffered at pH 6.4. After 15 min at room tem- perature, centrifuge and wash cells with 2 ml of 1 per cent rabbit serum in salt solution. Resuspend in 1 ml of the same diluent. Use 12X75 mm selected hemagglutination tubes for tests. To 0.5 ml of each serum dilution add 0.05 ml of sensitized red cells, shake well, and incubate for 2 hr at 37° C. After recording hemagglutina- 658 MISCELLANEOUS INFECTIONS tion patterns (4+ complete to 0, negative), resuspend and allow to resettle overnight at room temperature. Record final reading; the titer of the serum is the last tube in which 2+ hemagglutination is noted. 3. Complement-Fixation Test Antigens for complement fixation may be prepared either from frozen and thawed and centrifuge-clarified chorioallantoic membranes of infected embryonated eggs or mouse peritoneal exudate treated similarly. Use any standard complement-fixation system with the usual controls. Complement-fixing antibodies develop more slowly and disappear more rapidly than those measured in the dye test, Thus one may en- counter a high dye test titer and negative complement-fixation test, either early in an acute infection or several years after the subsidence of active disease. 4. Skin Test The skin test may be performed using the antigens prepared for the complement-fixation test. With egg material, normal membrane extracts may be used for control, while with mouse antigen an extract of spleen is used as the control. There is no suitable method for standardizing antigen for the skin test. We try to incorporate a standard amount of complement-fixing antigen in the 0.1 ml injected intradermally. Reactions are of the delayed, tuberculin type. A positive reaction requires a negative control and an area of erythema of at least 10X10 mm at 36-48 hr. Value of the skin test is limited in the individual patient because there is no positive correlation between the degree of skin reactivity and the serum antibody titer. There may be a negative skin test in persons with high dye test titers. We have noted neither increases in serum antibody titers nor the stimulation of antibodies (dye test) following the injection of egg antigen in indi- viduals who did not have antibodies before this test. Harry A. FeLoman, M.D. MISCELLANEOUS INFECTIONS 659 Section X—Vibrio fetus Infections Vibrio fetus is the etiological agent of a type of infectious abortion and impaired fertility among cattle and sheep. Vibriosis is prev- alent among domesticated ruminants and is world-wide in distribu- tion. VV. fetus has been cultured from several human sources: from a cheek pustule,® from the blood of three women with placentitis,? and from the blood of seven patients? who experienced febrile in- fections. Four intestinal tract infections with vibrios “related” to I”. fetus have been reported.® V7. fetus resembles Vibrio comma in cellular morphology. The cells are short, curved rods measuring 0.2 to 0.5 by 1.5 to 5.0 x. S-shaped forms are common; some cells may be coccoid or filamentous. A single polar flagellum is present, although bipolar flagellation may occur. The vibrio is actively motile and Gram-negative. Blood, exudates and body fluids should be examined promptly after collection. Prepare films of such materials and stain either by Gram’s method or by the following: 1) Fix the film by gentle heat. 2) Flood with aqueous 0.5 per cent Victoria blue 4R (Chicago Apparatus Co.) for 3 min. 3) Wash in tap water. 4) Decolorize, using 0.05 per cent sulfuric acid for 15 sec. 5) Wash in tap water, dry and examine. Direct or phase microscopy as well as dark-field examination of infected material may also prove helpful. V7. fetus is micro-aerophilic in nature. A suitable environment may be provided by incubating cultures at 35° C in an atmosphere contain- ing 10 to 15 per cent carbon dioxide or by incubation in a candle jar. The following semisolid media are recommended for isolation: liver infusion broth (CM No. 87) containing 0.3 per cent agar, Thiol containing 0.1 per cent agar (CM No. 86) (Difco),’ thioglycolate medium (CM No. 19) with 0.07 per cent agar (Baltimore Biological Laboratories) ,® or Brucella broth (Albimi) with 0.15 per cent agar added” (CM No. 40). The tubes should be filled so that the medium is approximately 3 in. in depth. Inoculate 0.05 to 0.1 ml of blood or exudate along a stab from the base of the tube to the surface of the medium. At least five tubes should be inoculated. Growth appears as a gray, fluffy haze at or just below the surface of the column of medium. The sur- face of blood agar plates should be liberally inoculated with suspected material. Growth appears as small gray, convex, translucent, non- hemolytic colonies or as a fine, almost transparent gray film; growth 660 MISCELLANEOUS INFECTIONS is seldom evident before 48 hr. MacConkey’s agar (CM No. 60) has been useful? Inoculated media should be incubated at least 14 days before being discarded as negative. Most strains produce catalase® and some hydrogenase.® Nitrates are reduced.’® Carbohydrates are not attacked!!; indole and urease are not produced; litmus milk and gelatin remain unchanged.® Litmus milk containing 0.1 per cent agar after 4 days shows an al- kaline surface zone and reduction of litmus.!® Hydrogen sulfide is generally not produced.® Growth can be observed in media containing 5 to 10 per cent ox bile.!? I, fetus does not grow in 0.1 per cent agar broth containing 3.5 per cent sodium chloride.? The microorganism is pathogenic for pregnant guinea pigs and hamsters following intraperitoneal inoculation, and abortions fre- quently result.!® Pathogenicity for embryonating hen’s eggs has been reported.!* Considerable antigenic heterogeneity among strains is evident. Sera from naturally infected animals may agglutinate only the homologous or infecting strain. The interpretation of routine agglutination tests for vibriosis is difficult and routine testing of animal sera is not advisable. A demonstrable serological relationship exists between some VV. fetus strains and the Brucellae™® and Salmonellae.? Erskine V. Morse, D.V.M., Pu.D. REFERENCES 1. Warp, B. Q. The Apparent Involvement of Vibro fetus in an Infection of Man. J. Bact. 55:113, 1948. 2. VinzenT, R. Une Affection Méconnue de la Grossesse I'Infection Placentaire a Vibrio foetus. La Presse Medicale 57 :1230, 1949. 3. King, E. O. Human Infections with Vibrio fetus and a Closely Related Vibrio. J. Infect. Dis. 101:119, 1957. 4. Prastrince, W. N. Cultural and Serological Observations on Vibrio fetus. J. Bact. 42:816, 1941. 5. Hupnreson, I. F. A Satisfactory Medium for the Isolation, Cultivation, and Maintenance of Viability of Vibrio fetus. J. Bact. 56:508, 1948. 6. Hansen, P. A, Price, K. E,, and CLEMENTS, M. F. Suitable Thioglycolate Media for the Cultivation of Vibrio fetus. J. Bact. 64:772, 1952. 7. Morse, E. V.; Ristic, M.; Rosirrstap, G. W.; and ScuHNEIER, D. W. Cross-Agglutination Reactions among Brucella, Vibrio and Other Micro- organisms. Am. J. Vet. Res. 14:324, 1953. 8. BRYNER, J. H, and Frank, A. H. A Preliminary Report on the Identifica- tion of Vibrio fetus. Am. J. Vet. Res. 16:76, 1955. 9. ReicH, C. V.,, Morsg, E. V., and WiLson, J. B. Gaseous Requirements for Growth of Vibrio fetus. Am. J. Vet. Res. 17 :140, 1956. 10. Kuzpas, C. D., and Morse, E. V. Physiological Characteristics Differen- tiating Vibrio fetus and Other Vibrios. Am. J. Vet. Res. 17:331, 1956. 11. Prastrince, W. N., et al. Vibriosis in Cattle, Storrs (Conn.) Agr. Exp. Sta. Bull. No. 281, 1951. MISCELLANEOUS INFECTIONS 661 12 13. 14. 15 ScuNEmER, D. W., and Morse, E. V. The Growth and Viability of Vibrio fetus and Related Vibrios in Media Containing Ox Bile. Cornell Vet. 45:84, 1955. Risric, M.; Morsk, E. V.; Wier, L.; and McNutt, S. H. Transmission of Experimental Vibrio fetus Infection among Guinea Pigs and Hamsters. Am. J. Vet. Res. 15:309, 1954. Wesster, H. D., and Traore, F., Jr. A Study of the Pathology of Em- bryonating Chicken Eggs Inoculated with Vibrio fetus. Am. J. Vet. Res. 14:118, 1953. Kiceins, E. M.; Prastrince, W. N.; Wirriams, L. F.; and EASTERBROOKS, H. L. Cross Agglutination between Vibrio fetus and Brucella abortus. Am. J. Vet. Res. 16:291, 1955. CHAPTER 23 ANAEROBIC INFECTIONS I. Collection of Specimens II. Anaerobic Technic III. Special Media and Selective Inhibitors IV. Culture Media V. General Technic VI. Special Remarks and Keys Appendix A: Testing for Pathogenicity Appendix B: Testing for Clostridial Toxins References To many bacteriologists the term “anaerobic infections” will sug- gest gas gangrene, tetanus and possibly botulism, but certainly nothing more. It must be realized, however, that quite apart from these syndromes (which are of rare occurrence in civilian practice), there exist a large number of other diseases which are also caused by anaerobic bacteria. Clinically these infections tend to be subacute or chronic, to be complicated by septicemia or pyemia, and they are limited usually to the skin and mucosae. Inevitably many species of aerobic bacteria occur in these lesions, but so far as the anaerobes are concerned, non- sporulating species—in particular streptococci and Bacteroides— predominate. Unless special methods of culture are employed in testing, anaerobic organisms will be overlooked. This chapter will indicate the character of the pathogenic anaerobes, setting forth simple and effective technics by which they may be isolated and identified. For a more detailed account of these bacteria, the reader is referred to McClung,! Smith,2 and Weinberg et al.3* I. COLLECTION OF SPECIMENS The organisms of primary concern in anaerobic cultures from wounds or infections are the clostridia, but the anaerobic cocci and * The Histotoxic Clostridial Infections of Man (MacLennan, Reference 13 of this chapter) has been published since this chapter was written. The reader is referred to it for a comprehensive description of these infections. 662 ANAEROBIC INFECTIONS 663 various nonspore-forming bacilli must also be considered. The speci- men of choice in wound cultures is a portion of tissue collected during debridement or at the time of dressing change. Less desirably, swabs from appropriate tissue surfaces may be cultured if tissue samples are unavailable. Fluids aspirated from tissue spaces of wounds, and blood cultures, should be examined for the presence of anaerobes. The optimal condition for recovery of anaerobes is to inoculate cul- ture media in the operating room or the ward; where this is not pos- sible, it is essential that specimens be transported to the laboratory with the minimum of delay. It is recommended that all initial cultures be seeded into chopped meat broth (CM No. 114). In most cases it is also advisable to carry out direct plating on blood agar plates (CM No. 16). For details see Section V of this chapter. . Wound cultures (biopsy specimens) : The use of sterile technic, even in debridement of grossly contaminated wounds, is essential for significant results. Cleansing and sterilization of skin and tissue surfaces prior to debridement should be thorough. Wash with tincture of green soap and rinse with sterile physiological salt solu- tion. Use fresh sterile instruments to obtain samples of muscle, since contamination by the skin flora is a constant hazard. Place blocks of tissue in individual petri dishes or other sterile containers for prompt transport to the laboratory. If sterile dishes are not avail- able, samples can be protected by placing individually between several layers of sterile 4X4 in. gauze flats. It is particularly important that swab samples be cultured soon after collection. In the event that several wounds are to be examined, samples should be taken from as many sites as possible. Ideally, every injury large enough to merit debridement should be cultured. The site of each culture should be carefully designated. In each case, selection of samples is controlled by the extent of the injury, but should, when possible, include subcutaneous tissue and superficial and deeper muscle layers. When large, avulsed wounds are present, with damage to or loss of relatively large amounts of tissue, single samples usually give an inadequate picture of the bacterial flora present. Several samples should be collected in such cases. In the case of missile wounds, samples at various levels of the track of penetration should be obtained, including a sample from the wound of exit. When foreign bodies or sequestra are encountered, they are cultured, if desired, by transfer of all or part of the specimen to enrichment medium. 664 ANAEROBIC INFECTIONS Cultures of body fluids: Purulent exudates and other fluid ele- ments associated with injuries should be aspirated, the sample placed in a sterile container and transported promptly to the laboratory. Blood cultures may infrequently yield clostridia or other anaerobes. The rubber-sealed blood culture bottle is inoculated with 5 ml of blood ; subcultures to anaerobic enrichment medium are made at 24 and 48 hr, then at weekly intervals for 3 to 6 weeks. Collection of other types of specimens: Vomitus and feces may at times be subject to study for anaerobes. Specimens are preferably collected in sterile wide-mouth glass containers. When such containers are not available, clean cardboard specimen boxes may be used. When foodstuffs are to be cultured, the specimen to be taken depends on whether or not the food is in the original container. Canned or bottled foods are opened aseptically in the laboratory ; processed foods are collected in sterile glass bottles and transported promptly to the laboratory for culture. Initial culture in enrichment broth: Divide samples of tissue aseptically, using sterile forceps and scissors or scalpel, for initial inoculation of enrichment medium. Pieces of tissue up to 10 mm in diameter from each sample are placed in two tubes (preferably screw- capped) of chopped meat medium (CM No. 114). Swabs are in- oculated into chopped meat medium by snapping off the stick of the swab so that the entire specimen is cultured. Tissue fluids are inocu- lated with a pipette where volume permits or several loopfuls of material may comprise the inoculum. Other samples are similarly planted in amounts of 1 g or more of sample per tube. Incubate for 48 hr at 35° C, then heat one tube at 80° C for 30 min in the water bath. These two tubes are to be used as master cultures, and subse- quent isolation attempts for anaerobes are made from these cultures. The master cultures are kept tightly sealed at 4° C when not in use. Direct plating of anaerobic culture specimens: The enrichment broth technic outlined here produces a greater yield of anaerobes than does direct plating. However, initial platings of samples should also be made on blood agar plates. For details of cultural methods, see Section V of this chapter. Il. ANAEROBIC TECHNIC 1. Agar plates—In the cultivation of the anaerobic bacteria, provision must be made for the exclusion of atmospheric oxygen. Although certain species are more tolerant than others with regard ANAEROBIC INFECTIONS 665 to oxygen potential, the assumption must be made that enrichments or samples contain species which require strict anaerobic conditions for proper cultivation. In initial cultivation of sample material, it is suggested, as noted in Section I, “Collection of Specimens,” that chopped meat broth be used as a primary enrichment in addition to direct plating. Although formerly much use was made of the technic isolating colonies from deep columns of agar in the purification of anaerobic species, modern workers now use surface or deep colony cultivation with plates incubated in one or another of a variety of efficient anaerobic systems. Unglazed porcelain lids for petri dishes may be used as an aid in keeping the plating surface dry; this will allow incubation of the plates agar side down. Following inoculation, place plates in an anaerobic system as quickly as possible (within 20 min), especially when vegetative inocula are used. The recommended anaerobic system is the Brewer anaerobic jar. Place cultures in the jar and seal the lid to the jar with plasticine or Cello-Seal. Attach the jar through a mercury manometer to a water vacuum pump or other source of vacuum and evacuate until the difference in mercury columns is 20 cm. Close connection to vacuum source and admit hydrogen gas. Activate catalyst by connecting the jar lid to 110-volt AC or DC current. Allow the jar to remain connected to gas and electricity sources for 30 min before closing the clamp to the gas line. Disconnect and incubate the jar at 35° C for 24 hr. It saves time to have electrical outlets in the incubator which may be connected with the jar through a time switch. If a Brewer jar is not available, or in an emergency, other anaerobic systems may be used, although some experience may be necessary to obtain satisfactory results (see Chapter 1). It is advisable to include in an anaerobic jar some indicator of anaerobiosis, that is, reduction potential. Of these the simplest is a mixture of the following three solutions: 1) N/10 NaOH, 6 ml; water to 100 ml. 2) 0.5% methylene blue (in water), 3 ml; water to 100 ml. 3) Glucose, 6 g; water to 100 ml. Although these solutions will keep indefinitely, it is recommended that a crystal of thymol be added to the glucose solution to inhibit microbial growth. For Use: Add 1 ml of each solution to a test tube, boil until colorless, then place in the jar immediately before sealing. Some color may return to the upper layers of the indicator during evacua- 666 ANAEROBIC INFECTIONS tion, but any tinge of blue after incubation indicates the presence of free oxygen. 2. Fluid media—Most fluid media intended for cultivation of anaerobic bacteria should be freshly exhausted of oxygen before inoculation. This may be accomplished by steaming at atmospheric pressure or by placing in boiling water for 10 min and rapidly cooling. For most satisfactory results inoculate from previous liquid cultures by transferring with a 2 ml rubber bulb approximately 0.1 ml to the bottom of the new tube by means of a long-tipped pipette drawn from 9.0 mm soft glass tubing. All liquid media should be tubed in deep columns and a sample tube pretested for growth-sup- porting ability by inoculation of a known species. Unless the medium contains reducing substances or is in such physical state that oxygen absorption is hindered, it is customary to layer the medium with an anaerobic seal (a mixture of vaseline and paraffin) following inoculation, Fluid thioglycolate medium, con- taining 0.03 to 0.05 per cent thioglycolic acid or sodium salt and 0.075 per cent agar (CM No. 20) may be used without heat exhaustion and may be incubated without anaerobic seals. Other reducing agents may be used instead of thioglycolate. An acceptable substi- tute consists of a strip of iron wire or ‘“‘stovepipe” iron freshly sterilized by flaming; powdered iron (iron by hydrogen, Merck) is also used. Care is needed in the observation of media containing iron, as turbidity may occur even in the absence of growth. Ill. SPECIAL MEDIA AND SELECTIVE INHIBITORS Purpose of Inhibitory Media The recovery of anaerobes, especially clostridia, from specimens harboring aerobic bacteria is often difficult, since many aerobes grow well under anaerobic conditions. Aerobic organisms are usually present on tissue samples from contaminated wounds and interfere with anaerobe isolation both by obscuring clostridia and by necessi- tating many additional colony pickings and screening procedures. By using selective inhibitors it is possible largely to suppress such interfering aerobic growth, thus facilitating the recovery of anaerobes. However, it must be borne in mind that, as with virtually all selective inhibitors, certain strains of clostridia may fail to tolerate the mini- mum inhibition dose used. To avoid missing such strains, standard media must always be used in parallel with inhibitory media. The aerobic groups most often interfering with the recovery of clostridia are Bacillus, Corynebacterium, Enterobacteriaceae (especially Pro- ANAEROBIC INFECTIONS 667 teus), Pseudomonas, staphylococcus and streptococcus. Most of these interfering organisms can be avoided by the use of appropriate inhibitors. 1. Chloral hydrate and sodium azide—These two compounds have often been used to suppress the growth of Enterobacteriaceae. Used singly, however, they are sometimes ineffective. Combination of the two in blood agar is more effective in inhibiting growth of Gram- negative bacteria as well as many strains of staphylococci and bacilli. Plates of chloral hydrate-sodium azide blood agar are streaked from initial enrichment tubes or from thioglycolate broth subcultures and incubated anaerobically for 48 hr. Plates are handled in the manner described for the isolation of clostridia on noninhibitory media. Strains isolated on any inhibitory medium should be carefully checked for purity before definitive identification, since a carry-over of viable but suppressed contaminants may occur, Colony form and hemolytic activity of clostridia on inhibitory media may not be identical with those shown on noninhibitory media. 2. Sorbic acid and polymyxin—The use of sorbic acid to inhibit unwanted catalase-positive facultative anaerobes in wound cultures was found to be very helpful by Lindberg et al.,* who investigated large numbers of wounds during the Korean war. Blood agar plates containing sorbic acid are inoculated from enrichment broth cultures and are run in parallel with unmodified isolation plates and chloral hydrate-sodium azide plates. Pseudomonas strains are not fully suppressed by any of the in- hibitors thus far described. These organisms, especially common in older wounds, may seriously interfere with the isolation of anaerobic organisms. Almost all strains of Pseudomonas, however, are in- hibited by 15 ug of polymyxin per ml, although most clostridia will tolerate twice this concentration. The antibiotic may be added to blood agar to give this concentration, and these plates streaked from en- richment cultures. A combination of sorbic acid and polymyxin in thioglycolate broth offers a convenient means of suppressing mixed contamination, Inoculate this medium with 0.2 to 0.5 ml of liquid from the chopped meat enrichment culture, incubate 48 hr at 35° C, and subculture to blood agar and to chloral hydrate-sodium azide blood agar plates. The four inhibiting substances mentioned in the foregoing may be combined by inoculating unmodified and chloral hydrate-sodium azide blood agar plates and sorbic acid-polymyxin thioglycolate broth from the chopped meat enrichment broth. After 24 to 48 hr of incuba- 668 ANAEROBIC INFECTIONS tion, this inhibitory broth is subcultured to unmodified and chloral hydrate sodium azide blood agar plates. The progressive inhibition provided by this sequence will often aid in the prompt recovery of clostridia from grossly contaminated specimens. 3. Concentrated agar and deep plates are useful in selective isolation of clostridia. Suppression of swarming by both aerobes and anaerobes is a primary objective in the isolation of these organisms. Concentrated agar, from 4 to 6 per cent, may be used to plate out cultures which contain swarming strains. The morphology of the colonies may be atypical on such media, and it is difficult to obtain a smooth dispersion of blood in the agar. All strains do not grow on concentrated agar. Deep agar plates are prepared either by suspending a portion of enrichment broth in melted agar and then pouring at once, or by streaking the plate and then overlaying it with additional melted agar. Deep colonies will be held in place and spreading thus avoided. Re- peated platings are necessary to ensure purity of cultures. IV. CULTURE MEDIA Blood agar plates (CM No. 16)—The most satisfactory base is a meat infusion, such as beef heart-peptone agar, adjusted to pH 7.0-7.2. Whole rabbit, horse, or preferably sheep blood should be added to the agar, which has been cooled to about 45° C, to make 4 to 5 per cent whole blood. Egg yolk or lecithovitellin agar plates—Scrub the shell of a fresh hen’s egg and sterilize with dilute mercuric chloride. Aspirate white of egg to discard tube and then aspirate yolk to sterile tube or flask, add an equal volume of salt solution, and mix by gentle shaking. Test sterility by plating 1 ml amounts. If sterile, the preparation may be stored in the cold up to 2 weeks. Add 10 ml of this egg yolk suspension to 90 ml of sterile agar medium and pour plates. The agar medium may be the same medium used as a base for blood agar, although somewhat better results are obtained with the following, as proposed by McClung and Toabe :? g NHPO, .oooanmonnn bs onsmns ves SURRIERGEE Eb oh Bis csmbi ve mo sen 5 ..g BHPO, ooo icvnmm vr cos ssmmumern £6 sa smpmons ts 3 STSmma es 05 3 5hamn 1 g NAS sicivi is snmmonss ins emi se 5 3 3 eiiees s ¢ £ oyswrernsy § § 35 THR 2 g MEEBO, «outs vmmnrsnss ses pammevsmmin s « 28 wmmnen oo mms « ¢ + 2g 01g GIGOBE ov v5 & sumiinie ais § 1 a abinhbions sd oh § Sitiusmaons on nv winpmmmevw s § ov eon 2 g UIA wimmons s 24 5 2 POMBE TF 75 SEHAIAAE E55 SERAIARDE E278 Bistmotii #5 TF 5 0 owe 25 g Distilled. WHEE . voniiuns «ts mmission § 54 8 Swim we 45 3 HERS 50% 25 2 50 1,000 ml Adjust to pH 7.6 and autoclave. ANAEROBIC INFECTIONS 669 Chopped meat medium (CM No. 114)—Grind finely 1 1b of beef heart or muscle, free of fat. Add meat to 500 ml of boiling N/20 sodium hydroxide (N/15 for muscle) and boil for 20 min. Allow to cool, remove the fat, and strain through muslin. Allow the meat to dry partially. Adjust the pH of the fluid to 7.5 and add 1 per cent peptone. Tube the meat, about 2 in. deep in each tube, and add broth until the level is about 1 in. above the level of the meat. Heat in a boiling water bath for 30 min and sterilize by autoclaving. Chloral hydrate-sodium azide blood agar—Melt 100 ml of blood agar base, add blood when suitably cooled, then add 1.0 ml of a 1 per cent solution of chloral hydrate and 2 ml of a 1 per cent solution of sodium azide. Final concentrations of inhibitors are 0.01 per cent chloral hydrate and 0.02 per cent sodium azide. The inhibitor solutions are sterilized by Seitz filtration. Best results are obtained with 2 per cent agar. The concentration of chloral hydrate may be varied with the degree of contamination present, from a final con- centration of 0.05 to 0.005 per cent. Sorbic acid medium—Dissolve 0.12 g of sorbic acid (2-4- hexadienoic acid) * in 100 ml of blood agar base. Sterilize at 15 1b pressure for 15 min, cool, add blood, and pour. The pH, which will be between 5.6 and 5.9, should not be adjusted. Sorbic acid (0.12%) may also be added to thioglycolate broth prior to autoclaving. Sorbic acid-polymyxin broth-—Add 0.12 g of sorbic acid to 100 ml of thioglycolate broth, After autoclaving, add 1.5 mg of poly- myxin to give a final concentration of 15 ug per ml. Tube aseptically in screw-cap tubes. Differential media—As a base for fermentation tests, use thioglycolate medium (CM No. 20) without added glucose. Add the sugars aseptically as 10 per cent solutions (5% for salicin), sterilized by filtration, to a final concentration of 1 per cent. Inoculum for fermentation media, as for the other differential media, should be 0.1 ml of a young culture in chopped meat medium. Acid production as an index of carbohydrate fermentation is detected by removing por- tions of the culture from time to time with a sterile pipette and adding to small quantities of an aqueous solution of bromthymol blue (0.05%) in a spot plate. Thioglycolate medium without added glucose may also be used for indole tests. Incubate the cultures 3 days and test with the usual * Sorbic acid can be obtained from the Eastman Kodak Co., Rochester, N. Y. 670 . ANAEROBIC INFECTIONS indole reagents. The same medium with 0.1 per cent sodium nitrate and 0.1 per cent added glucose may be used to test for reduction of nitrate to nitrite. For the determination of gelatin liquefaction, add 10 per cent gelatin to infusion broth, adjust to pH 7.4, tube and auto- clave. A strip of stovepipe metal or a few pieces of iron wire may be added for “iron gelatin.” For a milk medium, litmus milk may be used. Loeffler’s or Dorset’'s medium is satisfactory when coagulated serum or albumin medium is desired. V. GENERAL TECHNIC I. Preliminary Procedure In infections suspected of harboring anaerobic bacteria, the first problem is to isolate the organism or organisms present in pure culture. Three preliminary operations are desirable: Master cultures—As described in the preceding section, an essential first procedure is to prepare master cultures. Chopped meat medium in deep tubes should be boiled for 5 to 10 min and rapidly cooled. These tubes are then heavily inoculated with wound exudate or fragments of tissue from the wound. These master cultures should be incubated not less than 24 hr at 35° C. When it is important to obtain pure cultures in the shortest possible time, plates, as described in the following, should be prepared from the master cultures after they have been incubated a minimum of 24 hr. If this is done, it is desirable to prepare a second set of plates after the master cultures have been incubated 48 to 72 hr. When the presence of clostridia is suspected, it is often advan- tageous to heat one of the 48 hr master cultures (or a sample from it) at 80° C for 15 min in order to destroy the nonspore-forming species. Plates are then made from this heated sample in the usual way (see below). Preliminary plates—It is frequently desirable to make blood agar plates from the same wound material used to inoculate master cultures. These plates should be incubated and examined as described below for plates prepared from master cultures. Results from these plates will never be conclusive but they may give an early indication of the organisms predominating in the wound. If haste in arriving at a diagnosis is not a pressing consideration, these preliminary plates may be omitted. Final analysis of the bacteriology of the wound will depend upon plates prepared from the master cultures. ANAEROBIC INFECTIONS 671 Direct microscopic films—It is also desirable to make direct films stained by Gram’s method from the wound exudate or from tissue fragments. The presence of large Gram-positive rods in such preparations strongly suggests Clostridium. If the rods are short, plump and encapsulated, it is probable that the primary infecting organism is Clostridium perfringens. As spores are seldom formed in clostridia in wounds, this is approximately all that can be deter- mined by direct microscopic examination. However, failure to demonstrate characteristic organisms in direct microscope preparation does not exclude the possibility that anaerobic bacteria are present in the wound. 2. Plating from Master Cultures on Blood Agar Inoculation of blood agar plates—Before inoculation, the surface of the blood agar plates should be dried. This is done satis- factorily by using unglazed porcelain petri dish covers and incubating the plates for 1 to 2 hr at 35° C. In the absence of absorbing covers, the drying may be effected by tilting the inverted plate on its cover and incubating. Drying the surface of the agar is an essential precaution to prevent spreading on the plates. The worst offenders are members of the genus Proteus, which are frequently present in infected wounds. But some species of Clostridium are also troublesome in this respect, particularly Cl. tetani, Cl. sporogenes, Cl. septicum, and Cl. bifer- mentans. As moisture tends to accumulate in anaerobic jars, place a small amount of some desiccant in the bottom of the anaerobic jars. A petri dish cover partly filled with well dried Drierite or calcium chloride and placed in the bottom of the jar serves the purpose. If the surface of the plates is moist and an active spreader is present in the inoculum, it is extremely difficult to isolate pure cul- tures. In some instances a plate may appear on superficial examina- tion to have developed discrete colonies, although detailed examination will show that the spreader has extended over the entire surface of the plate and has overrun the apparently discrete colonies. This seldom occurs when the surface of the agar is kept properly dry. Antispreading agents—In grossly mixed cultures or in obsti- nate cases of spreading, it may be necessary to add various anti- spreading agents or selective inhibitors to the plating medium, as described in this chapter. Inoculating blood agar plates from master cultures—The in- oculum should be taken from near the bottom of the master culture 672 ANAEROBIC INFECTIONS tube. The best tool for the purpose is a relatively small-bore Pasteur pipette. Well-isolated colonies are usually obtained when three or four plates are inoculated from one master culture. Place a drop of inoculum on the first plate and spread with a large loop or, better, with a slender, bent glass rod; then streak the remaining plates with the same rod. This provides a graded series, from heavily to lightly inoculated plates. Deep agar plates from master cultures—Submerged colonies of clostridia are generally more discrete than surface colonies and in skilled hands isolations may be made earlier than from surface colonies. A clear 1.5 per cent agar in an infusion broth with 2 per cent proteose peptone is a suitable medium. In plating from master cultures a drop of fluid from the bottom of the master culture tube should be added to a tube of broth and one drop of the diluted culture added to 20 ml of agar which is held in solution at about 45° C. The latter is mixed well and poured into a petri plate, Incubation—The inoculated plates should be placed in anaer- obic jars with a minimum of delay. As soon as a jar is loaded it should be evacuated, filled with hydrogen, and the heating element turned on. Some clostridia will form characteristic surface colonies in 24 hr but unless time is pressing it is desirable to incubate plates made from the master cultures for 48 hr at 35° C. Deep agar plate cultures should not be incubated more than 24 hr. 3. Examination of Blood Agar or Deep Plate Cultures The only adequate examination of anaerobic plates is carried out with the aid of a binocular microscope of about 25 diameters magni- fication. With opaque plating medium such as blood agar this must be done with reflected light. Examination by the naked eye is entirely inadequate. A good hand lens is of considerable aid but the magni- fication is not sufficient for detailed work and is not suitable when colonies are being fished from the plates. Anaerobic plates made from wound exudates or from master cultures thereof are likely to develop a variety of species of bacteria, including facultative anaerobes such as streptococcus, staphylococcus, Proteus, Aerobacter, and Escherichia, together with obligate anae- robes. However, only the obligate anaerobes are considered in this section. The members of the genus Clostridium grow on blood agar or in deep, clear agar in fairly distinct colony forms which have been ANAEROBIC INFECTIONS 673 described by several authors. Accurate differentiation by this technic requires considerable experience. Those interested may consult the original papers, particularly the report by Reed and Orr. Hemolytic reactions—The species of Clostridium which pro- duce soluble toxins produce zones of hemolysis about the colonies when grown on blood agar. The clear zone is generally somewhat wider than the colony. The most distinctive reaction is produced by Cl. perfringens. This organism ordinarily produces a double zone of hemolysis, an inner zone of complete clearing of the medium 5 to 10 mm wide, and a wider outer zone of partial hemolysis. The nontoxin-forming species of Clostridium for the most part do not hemolyze blood in the underlying or surrounding medium. The most conspicuous exception is Cl. sporogenes, which, though not forming toxin, produces a narrow zone of partial hemolysis around colonies on blood agar. 4. Plating from Master Cultures on Egg Yolk or Lecithovitellin Medium At the same time that the master cultures are plated on blood agar, it is desirable also to plate on egg yolk media as a further aid in the identification of species. Or such media may be used in secondary plating. These media were originally designed as tests for lecithinase action, but subsequent work, particularly by McClung and Toabe,® showed that plates inoculated with members of the genus Clostridium in several important instances give distinctive species-specific reactions. Egg yolk or LV agar: Plates of this medium should be in- oculated from master cultures in the same manner as blood agar plates. In order to determine the characteristic reactions, it is neces- sary to have well-isolated colonies. LV medium reactions: On this medium colonies of clostridia present the same range of form as on blood agar, although slightly less distinctive, but in addition three types of reaction occur in the medium under or surrounding the colonies, as described by McClung and Toabe.b Reaction 1—The colonies are surrounded by a wide zone of opaque white precipitate with a sharply defined margin about 8 mm in diameter. There is no luster. After the plates have been held in the air for several hours, concentric zones of precipitate appear around the initial zone. These secondary zones tend to be more intense than the primary zone although the outer margin is less sharply defined. 674 ANAEROBIC INFECTIONS This type of LV reaction is characteristic of Cl perfringens, CL bifermentans, CI. sordellii, Cl. hemolyticum, and Cl. novyi Type B. The individual colonies in the precipitation zones may be distin- guished as on blood agar. Reaction 2—Colonies are surrounded by a wide zone of intense precipitation. The characteristic feature is an area of iridescent luster marked by radiating striations extending over the colony and part way over the precipitation zone. After the plates have been held in air for several hours, further concentric zones of precipitation appear around the initial zone and additional luster zones appear. This type of reaction is characteristic of CI. novyi Type A and of Cl. botulinum. The colony forms of CI. novyi and Cl. botulinum on blood agar are quite different. Moreover, the L'V reaction of Cl. botulinum differs slightly from that of CL nmovyi. Cl. botulinum Types C, D and E produce wide zones of precipitation but the luster zone does not extend beyond the colony margin. CI. parabotulinum Types A and B produce relatively narrow zones of precipitation and the luster zone extends nearly to the outer margin of precipitation. Reaction 3—A precipitate is deposited in the medium under the colony but rarely extends beyond the colony margin. The colony shows an iridescent luster which, however, does not extend beyond the margin of the colony. Rhizoids frequently extend beyond the precipitation and luster zone. This reaction is characteristic of CI. sporogenes. The following species of clostridia which may occur in wounds produce characteristic colonies on LV medium but produce no pre- cipitation, luster or any other visually detectable change in the medium: Cl. tetani, Cl. septicum, Cl. tertium, Cl. histolyticum, CI. capitovale, Cl. chauvei, and Cl. cochlearium. It should be noted that several aerobic species give zones of pre- cipitation on the LV medium. This is particularly true of several species of the genus Bacillus. 5. Isolation of Pure Cultures Colonies from blood agar, deep agar plates, or LV medium should be fished and replated. Picking of surface colonies is best done under the binocular dissection microscope in order to make certain that the inoculum is taken from a single colony. The greatest care should be taken to exclude spreading film or thread-like growths, which not infrequently overrun what may appear to be discrete colonies. It is occasionally necessary to make several successive ANAEROBIC INFECTIONS 675 fishings to establish a pure culture. In obstinate cases, as noted earlier, it may be necessary to fish to blood agar containing sodium azide before discrete colonies can be obtained. In any case it is necessary to have several successive plates showing well-isolated colonies of exactly similar type before a pure culture may be assured. Submerged colonies in clear agar also may be fished with a loop and replated, as in the case of surface colonies, but generally more satisfactory results are obtained with an ordinary Pasteur pipette redrawn to make a fine capillary tip turned at right angles. With the selected colony centered under the binocular microscope, the colony may be stabbed with the capillary. The tip of the capillary is then broken against the bottom of a tube of chopped meat medium. Once pure cultures have been obtained, a discrete colony should be heavily seeded into a tube of chopped meat medium. From this culture transfers into the various differential media may be made. 6. Cellular Morphology Cells of members of the genus Clostridium are in general large cylindrical rods with truncated or rounded ends. They vary greatly in length from 2 or 3 to 7 or 8 pu. In breadth they vary from 0.4 to slightly over 1 pu. The vegetative bacilli are ordinarily straight rods, although the longer rods are occasionally curved. They are generally arranged singly, but end-to-end pairs or chains are not infrequent and occasionally they occur in parallel bundles, Irregular forms are common in most species, especially in old cultures. These include boat-shaped forms, swollen lemon-shaped forms, and long straight or snake-like filaments. Capsules: Two species of Clostridium form capsules, Cl. perfrin- gens and Cl. fallax, but only the former is commonly found in in- fections. In microscopic preparations from infected tissues, CI. perfringens ordinarily appears as a plump rod with rounded ends 3 to 5 plong by 1 pin breadth surrounded by a capsule two or three times the diameter of the rod. Such forms, in direct films from wounds, are sufficiently distinctive to be strongly suggestive of CI. perfringens. In freshly isolated cultures on media containing blood or serum, the capsules are generally equally evident, but in old cultures or in cultures on media without serum, capsules are frequently absent. Motility: All clostridia which occur in infected wounds, with the exception of Cl. perfringens, are motile by means of peritrichal flagella. The movement, however, is slow compared with the darting 676 ANAEROBIC INFECTIONS motion of many small aerobic species. The demonstration of motility is made by observing a drop from a 6 to 12 hr culture in a chopped meat or serum broth medium under a cover slip. It is frequently impossible to demonstrate motility in cells from old laboratory cultures. Sporulation: Although all species of Clostridium produce spores, there is great variation in the readiness with which different species sporulate and in the form of the spore and its position in the cell. Even within a species some strains sporulate much more readily than others. Spores are either spherical or oval in shape and terminal or subterminal in position. To obtain spores, it is sometimes necessary to grow strains for a number of days in chopped meat medium in the anaerobic jar. Spores are not formed by most strains if the pH of the medium drops below 6.4; hence there should be no fermentable carbohydrate in any medium in which spore formation is desired. Staining: The vegetative cells of all species from young cultures stain readily with the ordinary dyes and usual staining procedures. The Gram stain is retained to give a deeply staining Gram-positive reaction. Acetone or acetone and alcohol should not be used as de- colorizing agents, for they generally produce irregular results. Micro- scopic preparations from old cultures of most species stain irregularly. Moreover, some species of clostridia lose their ability to retain Gram stain after 12 to 24 hr growth, particularly in broth. VI. SPECIAL REMARKS AND KEYS |. Clostridia The organisms to be presumptively identified in the key which follows represent those anaerobic spore-forming, usually Gram- positive bacilli which are most commonly found in pathological speci- mens; or types with which such bacilli might be confused. These species will not grow on nutrient agar exposed to atmospheric oxygen, yet they grow with relative ease in or on the common anaerobic media incubated under conditions which exclude atmospheric oxygen. Those readers needing a more complete key or additional characteristics for confirmation of species identification are referred to the seventh edition of Bergey's Manual of Determinative Bacteriology, from which the key given here has been adapted; for pathogenic species, consult Smith,2 who provides additional identifying characteristics. Characteristics generally needed for identification include: spore shape and relation to sporangium ; motility; colony characteristics on ANAEROBIC INFECTIONS 677 blood and egg yolk agar; action on gelatin (or glucose gelatin) co- agulated albumin and litmus milk; fermentation of glucose, sucrose, lactose, salicin, mannitol and glycerol; and toxicity on feeding or injection of filtrate of chopped meat medium culture to guinea pigs and mice. Those who do not work routinely with anaerobic species should secure authentic identified cultures to use as test organisms. Persons who have difficulty in identifying cultures are invited to make inquiries of a member of the chapter committee and to send cultures for confirmation of identification. Strictly Anaerobic (will not grow on rich media exposed to atmos- pheric oxygen) A. Motile rods with central to subterminal ovoid spores a. Rods distinctly swollen at sporulation b. Gelatin or glucose gelatin not liquefied. Glucose, lactose and sucrose fermented. c. Milk stormily or at least actively coagulated. d. Glycerol not fermented. Blood agar not hemolyzed. Poor, if any, growth on egg yolk agar in the absence of additional carbohydrate; with growth, no reaction. CL butyricum. dd. Glycerol fermented. Blood agar hemolyzed. Action on egg yolk agar not recorded. CI. multifermentans. cc. Milk slowly coagulated (not stormily). Glycerol not fermented. Blood agar not recorded. No action on egg yolk agar. Cl. fallax. ccc. Milk not coagulated. Blood agar not hemolyzed. No action on egg yolk agar, CL difficile. bb. Gelatin or glucose gelatin liquefied. c. Coagulated albumin not liquefied. Glucose fermented. d. Milk coagulated, clot not digested. e. Glycerol, mannitol and sucrose not fermented. Lactose and salicin fermented. Blood agar hemolyzed. Characteris- tic irregularly round colony with filamentous edge often spreading to form film but without reaction on egg yolk agar. Cl. septicum. ee. Glycerol, mannitol and salicin not fermented. Sucrose and lactose fermented. Blood agar hemolyzed. Circular to slightly irregular colonies without reaction on egg yolk agar. Cl. chauvei. eee. Glycerol fermented. Lactose, sucrose and salicin not fermented. Blood agar extensively hemolyzed. On egg yolk agar, large zone of intense precipitation without luster. Cl. hemolyticum. dd. Milk acidified but not coagulated, not digested. e. Blood agar hemolyzed. Smooth colonies with irregular edges, having a precipitation zone under the colony and surrounding it in a regular circle, with an iridescent luster area covering the colony but not the entire pre- cipitation zone. Exotoxin produced which is toxic on injection but not on feeding. CI. novyi. ee. Blood agar hemolyzed. On egg yolk agar, flat irregular- edged colonies with wide zone of precipitation and narrow 678 ANAEROBIC INFECTIONS luster zone following colony edge. Exotoxin produced which is toxic on injection and feeding (for toxin type determination, conduct specific antitoxin neutralization tests). CL. botulinum. cc. Coagulated albumin slowly to rapidly liquefied. Glucose fer- mented. d. Sucrose, glycerol and mannitol not fermented. e. Lactose fermented. Blood agar not hemolyzed. No action on egg yolk agar. Cl. aerofetidum. ee. Lactose not fermented. Blood agar hemolyzed. Irregular, rough, dry, rhizoid colonies with precipitate zone under the colony and slight luster covering the colony. No ex- otoxin active on feeding filtrates. Cl sporogenes. dd. Glycerol fermented. Sucrose, lactose and mannitol not fer- mented. e. Blood agar hemolyzed. On egg yolk agar, raised irregular- edged colonies with an area of precipitation under the colony and slightly beyond, and a luster zone extending to edge of precipitate. Exotoxin produced which is toxic on injection and on feeding (for toxin type determination conduct specific antitoxin neutralization tests). CL para- botulinum. ccc. Coagulated albumin not digested. Glucose, lactose, sucrose, glycerol and mannitol not fermented. Milk clot digested. No hemolysis on blood agar. Action on egg yolk agar not recorded. Cl. hastiforme. aa. Rods not swollen at sporulation. b. Motile. Glucose fermented; sucrose, lactose and mannitol not fer- mented; glycerol and salicin variable. Coagulated albumin and gelatin liquefied. Milk slowly coagulated and digested. Blood agar hemolyzed. On egg yolk agar, slightly raised, often shiny, irregular edged, chalky white colonies with wide zone of precipitation but without luster. CL bifermentans. AA. Motile rods with terminal ovoid spores a. Gelatin or glucose gelatin and coagulated albumin not liquefied. Milk aa. aaa. AAA. slowly coagulated, clot not digested. Glucose, lactose and sucrose fermented; glycerol and mannitol not fermented. Blood agar not hemolyzed. Cl. paraputrificum. Gelatin or glucose gelatin and coagulated albumin not liquefied. Milk unchanged. Carbohydrates not fermented. Blood agar hemo- lyzed. Cl. cochlearium. Gelatin or glucose gelatin and coagulated albumin liquefied. Milk usually coagulated, clot not digested. Glucose fermented; sucrose, lactose, mannitol and glycerol not fermented. Blood agar not hemo- lyzed. Cl. capitovale. Motile rods with terminal spherical spores a. Gelatin and glucose gelatin liquefied; coagulated albumin slowly liquefied. Carbohydrates not fermented. b. Milk may show soft coagulation; clot not digested. Blood agar hemolyzed. No action on egg yolk agar. Filtrates toxic on in- jection. Cl. tetani. bb. Milk shows soft coagulation; clot slowly digested. Blood agar hemolyzed. No action on egg yolk agar. Filtrates not toxic on injection or feeding. Cl. lentoputrescens. ANAEROBIC INFECTIONS 679 aa. Gelatin may or may not be liquefied (records vary); coagulated albumin not liquefied. Glucose fermented. Milk unchanged. Filtrates not toxic on injection or feeding. CI. tetanomorphum. AAAA. Motile rods with subterminal, becoming terminal, spherical spores a. Gelatin or glucose gelatin and coagulated albumin not liquefied. Glucose, lactose and salicin fermented; glycerol not fermented; sucrose variable. Milk slowly coagulated, clot not digested. Blood agar hemolyzed. No reaction on egg yolk agar. Cl. sphenoides. AAAAA. Nonmotile rods not swelling at sporulation; sporulation rarely observed—Glucose, sucrose and lactose fermented. Glycerol and salicin variable; mannitol not fermented. Coagulated albumin not liquefied; gelatin liquefied. Milk usually stormily fermented. On egg yolk agar, colonies round and usually smooth with wide zone of precipitation without luster. Toxin types identified by specific toxin neutralization and other tests. Cl. perfringens. Aerotolerant (will grow sparsely on rich media exposed to atmos- pheric oxygen, sporulation principally under anaerobic conditions) A. Motile rods with central to subterminal ovoid spores—Rods swollen at sporulation. a. Gelatin or glucose gelatin and coagulated albumin not liquefied. Glucose, sucrose, lactose fermented; glycerol and mannitol not fer- mented. Blood agar hemolyzed. No reaction on egg yolk agar. Cl. carnis. aa. Gelatin or glucose gelatin liquefied; usually producing orange-red coloration in iron gelatin. Coagulated albumin slowly liquefied. Carbo- hydrates not fermented. Blood agar hemolyzed. No reaction on egg yolk agar. Cl. histolyticum. AA. Motile rods with terminal ovoid spores—Rods swollen at sporula- tion. a. Gelatin or glucose gelatin and coagulated albumin not liquefied. Glucose, sucrose, lactose and mannitol fermented; glycerol not fermented. Blood agar hemolyzed. No reaction on egg yolk agar. Cl. tertium. 2. The Anaerobic Cocci Anaerobic cocci are encountered with surprising frequency in clinical specimens. They may be recovered from putrid abscesses, blood cultures, infected wounds and comparatively minor lesions, and from the mouth, intestinal tract and genital tract of entirely normal individuals. Although it is seldom possible to demonstrate pathogenicity with the anaerobic cocci in laboratory animals, it seems quite likely that some of them may be pathogenic for man, at least under special conditions. These organisms are most commonly found in infections of the gastrointestinal tract and are somewhat less frequently encountered in pleuropulmonary infections and infections of the genital tract. 680 ANAEROBIC INFECTIONS Morphologically, the anaerobic streptococci (peptostreptococci) are much like their aerobic counterparts, retaining the Gram stain well and appearing in the form of chains of short to medium length. The anaerobic staphylococci (peptococci) are usually grouped in masses, as are the aerobic staphylococci, but some lose the ability to retain the Gram stain quite early in culture. Although cultures of 6 to 12 hr incubation are clearly Gram-positive, they may be as clearly Gram- negative when examined after 24 hr incubation and are almost uni- formly Gram-negative when examined from blood agar plates. Isolation of the anaerobic streptococci is most easily made by cul- ture on blood agar plates incubated in anaerobic jars, Colonies of the anaerobic streptococci after 2 days of incubation are 1 mm or less in diameter, opaque, convex, and grayish to whitish, with entire margins. A few strains form transparent gray colonies, but this development does not seem to be correlated with species. There is usually no hemol- ysis of the red blood cells in the agar. If blood agar plates are left exposed to the air after removal from the anaerobic jar, a small amount of greening often appears around the colonies. These are strains which are producing hydrogen peroxide. Microscopic exami- nation of colonies of anaerobic streptococci usually shows organisms in pairs and short chains; long chains are infrequently found. Transfer should be made to chopped meat broth which is covered with sterile vaseline or else incubated in an anaerobic jar. After 2 days of incubation, growth is usually evident around the particles of meat in the bottom of the tube. Microscopic examination will show chains longer than those found in films made from colonies. All cultures should be streaked on blood agar plates which are incubated aerobically, as well as on plates incubated anaerobically, to demon- strate the anaerobic character of the strains. Inoculation from 2-day-old cultures should be made into glucose broth, gelatin, milk, peptone water (1% peptone, 0.3% yeast extract) and blood broth for tentative identification. It is not uncommon for strains to be encountered which are unlike described species in one or more characteristics. Often, little can be done with such strains so far as identification is concerned, other than noting their re- semblance to the known species. Many of the strains isolated from pathologic specimens and from blood cultures can be tentatively identified from the following key. The characteristics listed in Bergey's Manual should also be deter- mined, however, if there is doubt. ANAEROBIC INFECTIONS 681 Key to the genus Peptostreptococcus I. Milk clotted A. Gelatin liquefied ; anaerobic on first isolation, becoming facultative dur- ing culture. Peptostreptococcus evolutus. B. Gelatin not liquefied, remains strictly anaerobic 1. No growth at room temperature, slight or no acid from glucose or galactose. Peptostreptococcus parvulus. 2. Slow growth at room temperature, active fermentation of glucose and galactose. Peptostreptococcus intermedius. II. Milk not clotted A. Gas produced from peptone water 1. Large ovoid cells with pointed ends. Peptostreptococcus lanceolatus. 2. Spherical cells a. Acid from maltose. Peptostreptococcus anaerobius. b. No acid from maltose. Peptostreptococcus fetidus. B. No gas from peptone water 1. Small cells (0.2-0.4 ux); no gas or putrid odor in blood broth. Pep- tostreptococcus micros. 2. Medium-size cells (0.8 u); gas and putrid odor from blood broth. Peptostreptococcus putridus. Anaerobic staphylococci are not so commonly encountered as are anaerobic streptococci. Like the latter, however, they seem to be part of the normal bacterial flora of the body, including the skin, and are occasionally involved in pathologic processes. The only species in this group which are likely to be pathogenic for man are Pepto- coccus anaerobius, Peptococcus activus, and Peptococcus aerogenes. Isolation and identification of the anaerobic staphylococci follow much the same steps as do the isolation and identification of the anaerobic streptococci. On blood agar plates, after 2 days’ anaerobic incubation, the colonies of the anaerobic staphylococci are circular, 0.5 to 2 mm in diameter, smooth, entire and convex, usually opaque, although sometimes translucent, white to grayish white in color, and buttery in consistency. There is no hemolysis of the blood agar. Coagulase is not formed, but catalase is produced. From the colonies on blood agar, tubes of chopped meat medium should be inoculated, as well as aerobic and anaerobic blood agar plates, to determine anaerobic requirements. Irom the chopped meat medium, cultures should be inoculated into glucose broth, lactose broth, lactate (1% sodium lactate) broth, peptone water, gelatin, and shake tubes of nutrient agar. Fermentation of lactate is evi- denced by an increased amount of growth and of gas production. The pH of the medium does not drop but rises slightly. The following key may aid in tentative identification of the more commonly en- countered species. See also Foubert and Douglas.” 682 ANAEROBIC INFECTIONS Key to the genus Peptococcus I. Produces gas from peptone A. Ferments lactate. Peptococcus lactilyticus (Veillonella alcalescens?). B. Does not ferment lactate 1. Colonies in shake cultures blackened by 6th day of incubation. Pep- tococcus niger. 2. Colonies not blackened a. Gelatin liquefied. Peptococcus activus. b. Gelatin not liquefied 1) Indole produced, nitrate reduced. Peptococcus aerogenes. 2) Indole not produced, nitrate not reduced. Peptococcus prevotii. II. Gas not produced from peptone A. Glucose fermented 1. Lactose fermented. Peptococcus grigoroffii. 2. Lactose not fermented. Peptococcus saccharolyticus. B. Glucose not fermented 1. Gelatin not liquefied, cell size uniform. Peptococcus anaerobius. 2. Gelatin liquefied, cell size variable. Peptococcus variabilis. 3. The Nonsporulating Anaerobic Bacilli Like the anaerobic cocci, the nonspore-forming anaerobic bacilli are far more common and of greater importance clinically than is gener- ally realized. Many of these organisms, including at least some species of pathological importance, are normal inhabitants of the mouth and alimentary tract, of the female genitourinary tract and, very probably, of the respiratory passages. Although it is in these sites, where they are often present in very large numbers, that they most frequently cause disease, they have been found associated with infective processes in almost every area of the body and in almost every type of tissue. Such infections, which tend to be of a subacute or chronic nature, are typically foul, gangrenous and purulent and are not uncommonly complicated by septicemia or pyemia. The infecting organisms may be present in pure culture, but much more usually they are found in association with other bacteria, of which cocci (both aerobic and anaerobic), other anaerobic bacilli, spirochetes and various species of Enterobacteriaceae are the most important, It is presumably this fact, together with the extreme difficulty of cultivating and isolating them, that has tended in the past to obscure the relative importance and frequency of these organisms in disease. Most of the species likely to be found in clinical material may be grouped in four genera: Dialister, Fusobacterium, Bacteroides and Actinomyces (Bergey). This classification, which is primarily mor- phological, can at best be regarded as tentative. It is of considerable ANAEROBIC INFECTIONS 683 value in the routine identification of strains but is probably without much taxonomic significance or permanence. Moreover, from time to time obligately anaerobic bacilli will be found which, except for their oxygen requirements, appear to belong to other well-defined bacterial genera—for example, Corynebacterium, Vibrio or Spirillum. There is little doubt that some of these strains are pathogenic, but their exact status is uncertain and too little is as yet known about them to justify their detailed consideration here. Finally, mention must be made of various spirochetal forms—usually classified as Treponemata—which are constantly present in the mouth and, more irregularly, in other parts of the body and which, particularly when acting in symbiosis with certain fusobacteria, may assume pathogenic powers. Although some species have been grown in the laboratory (usually in mixed culture), their differentiation is still essentially microscopical. Because all these organisms are extremely difficult to cultivate and their primary classification and differentiation are morphologic, direct microscopical examination becomes of great importance. Fresh samples of pus, exudate or infected tissue should be insisted upon and several films (both thick and thin) prepared from these and stained by Gram’s method. It is important to use dilute carbol- fuchsin as the counterstain, since these anaerobic bacteria do not readily take up the other common red dyes. For detailed cytological study, a second film, stained with carbolthionin, is of considerable value. The films, which often exhibit a vast and varied array of microorganisms, must be examined at length and with care, with particular attention to the following points: the length and thickness of the various organisms present, whether filamentous, straight, curved or tapered; reaction to Gram’s stain; presence of true branch- ing; occurrence of knobs, swellings or “involutional forms”; occur- rence of intracellular granules; relative numbers of the different morphological types. In the case of spirochetal infections, a dark- field examination may also be of importance and occasionally is essential. At the same time the films are made, samples of the pathological material should be heavily inoculated into the following media: A. For Aerobic Culture: One or two heart infusion blood agar plates (CM No. 16). B. For Anaerobic Culture: a. Two heart infusion blood agar plates in series, i.e, without intermediate flaming of the loop. 684 ANAEROBIC INFECTIONS b. Where on morphologic or other grounds (see below) the presence of Actinomyces is suspected, two brain-heart infusion plates (CM No. 22), in series. c. Where on morphological or other grounds the presence of fusobacteria is suspected, one ascitic fluid-crystal violet agar plate (Rosebury et al.8). d. Except in oral bacteriological work, it is rarely necessary to attempt the cultivation of spirochetes and even then the procedure is so tedious and specialized as to be almost a research project. A most satisfactory medium is the ascitic fluid-cysteine-kidney agar described by Rosebury et al8 For details of technical methods, this paper should be consulted. e. One or more of the following fluid media: chopped meat broth with 1 per cent glucose, beef infusion broth with sheep blood, semisolid brain- heart infusion agar. The aerobic plates may be examined after 24 hr, but anaerobic media should be incubated at least 2 to 3 days. It will frequently be observed that more profuse and varied growth occurs in the fluid cultures, and these may be used as rough controls of the findings obtained from a study of the agar plates. Indeed, it is probably advantageous to make routine transfers of the mixed broth cultures every 5 to 7 days, the older tubes being then sealed with paraffin and kept in the refrigerator. In this way a series of reference cultures is obtained which is often of great assistance in the detailed analysis of these heterogeneous infections. There is no short cut to the isolation of the nonsporing anaerobes. The various solid media must be carefully and repeatedly examined (preferably under the dissection microscope), the individual colonies identified, picked off and replated, and the whole process repeated as often as necessary until pure cultures are obtained. Each stage should be controlled by microscopical examination, by aerobic cul- tures, and by reference to the original findings. In this respect, three points require special emphasis here. 1) Many of these bacilli tend to lose their distinctive morphology in artificial culture, with the result that after a few transfers they all appear as short Gram-negative rods, quite unexceptional and quite indistinguishable one from another. 2) The sensitivity of many of these organisms to oxygen is con- siderable and examination of anaerobic plates and colony selection must be carried out with a minimum of delay. 3) It will be found that with increasing purity of culture, many of these organisms become more difficult to grow. Once pure cultures have been secured they should be transferred to tubes of semisolid brain-heart infusion agar, preferably containing serum or ascitic fluid. Thereafter various special media are heavily inoculated for the determination of biochemical activities. With ANAEROBIC INFECTIONS 685 such slow-growing and fastidious organisms these tests are often difficult to carry out and to interpret. Especial care must be taken that the basal medium used is adequate; cysteine-heart-infusion broth (sugar-free) is probably the most generally useful, but with individual strains this may have to be supplemented in various ways. To this medium are then added the test substrates in suitable amount. Final readings of biochemical reactions should be taken only after a minimum of 5 days of satisfactory growth. Unfortunately, in a fair number of cases the reactions observed will prove to be atypical—in other words, they will not agree with those commonly listed as characteristic of any known organism. In some instances, such aberrant findings are due to impure cultures or to variations in the technics and criteria employed, but usually there is no such simple explanation. It must, however, be clearly realized that the nonspore-forming anaerobes as a whole have been so inadequately studied that neither the specific nor the generic status of various superficially similar forms, to say nothing of the degree of variability possible or permissible within a single species, has as yet been satisfactorily established. The result of this neglect renders it impossible for the average microbiologist to identify accurately all the strains he is likely to come across. The best he can do is to indicate their general relationships—and this should always be possible. In this connection, serological methods have proved of relatively little assistance owing to the high immunological specificity of the individual strains. Pathogenicity for experimental animals is also of slight diagnostic importance: It is usually difficult to demonstrate and, in any case, seems to be readily lost in pure culture. Indeed, there is now considerable evidence that many of these organisms are not pathogenic in themselves but only when acting in symbiosis with other species, aerobic or anaerobic, or when playing the role of secondary invader. It should be emphasized, however, that these facts in no way lessen the importance of these organisms in disease. In the light of the foregoing remarks, it will be clear that the data set out below will have to be interpreted with care and discrimina- tion. Those wishing more detailed information on individual species or on aberrant forms are referred to the review of Dack® and to the monographs of Weinberg, Nativelle and Prévot,®> MacDonald, ® Pré- vot,1112 and Smith.? 4. Individual Organisms Dialister—Within this genus only one species, D. pneumosintes, has received general recognition. However, it is probable that under 686 ANAEROBIC INFECTIONS this name are included a number of related forms, some of which at least merit specific rank. All are minute Gram-negative rods, non- motile and nonsporulating. They are so small as to be capable of passing through most bacterial filters—a fact that can be of consider- able importance in their isolation and identification. D. pneumosintes is not a very difficult organism to grow, although initially most strains require blood, serum or ascitic fluid in the medium. On blood agar the organism forms tiny “dewdrop” colonies which are nonhemolytic. Of the commoner sugars, only glucose, maltose, lactose and, more irregularly, galactose are fermented, without the formation of gas. The organism is not proteolytic and fails to produce indole or to liquefy gelatin. Although originally isolated from cases of influenza, D. pneu- mosintes is not currently thought to play any part in the pathology of this condition. In fact, it appears to be a normal inhabitant of the upper respiratory passages. In human disease it has been most com- monly found in various chronic purulent infections, particularly of the brain and meninges, but also involving other parts of the body. In such cases it may occasionally be recovered from the bloodstream, although primary septicemia has not been recorded. Apart from its strictly anaerobic requirements, D. pneumosintes closely resembles Hemophilus influenzae. Its very small size prevents it from being mistaken for any other obligate anaerobe. Fusobacterium—This genus is characterized by the spindle form of the adult cells. Although commonly described as Gram- negative, it is probable that some strains at least are Gram-positive in very young cultures. The organism is nonmotile and nonspore- forming but more or less distinct granules may be discerned within the cells of most strains. In human pathological lesions fusiform bacilli are usually, though not invariably, found in association with treponemata. It should be stressed that the typical tapering form of these organisms can be more or less readily lost in artificial culture. The fusobacteria are not very difficult to grow—{far easier, for example, than the Bacteroides—but their isolation in pure culture is often a long and tedious process. This proceeding can be greatly facilitated by the use of ascitic fluid agar containing crystal violet, which effectively suppresses most of the contaminating strains. On solid media the fusobacteria produce small, irregularly round, felt-like colonies, often with a domed and shiny center. On blood agar most strains show a narrow zone of alpha hemolysis which on exposure to the air may slowly change to true beta hemolysis. ANAEROBIC INFECTIONS 687 Biochemically these organisms have little obvious activity. Most strains have a negligible effect on the commoner protein media and even fail to liquefy gelatin. However, some produce a very foul odor in artificial culture, usually due to the evolution of H,S or indole. Saccharolytic powers are equally meager and are often very difficult to make out owing to the small amount of acid formed; gas is never produced. All strains appear able to ferment glucose, but reactions with other sugars are more variable (see Table 1). Largely on the basis of cultural reactions, a number of species of fusobacteria have been differentiated, but the validity of many of these is questionable. Bergey recognizes six species and while the status of even these is open to doubt, Table 1 may prove of some service. Table 1—Cultural Characteristics of the Four Species of Fusobacteria Species Foul Odor Glucose Maltose Lactose Indole FE. fusiforme — + + of ont FE. polymorphum + }- m= = + F. nucleatum + + — + E EF. biacutum — + + + — Of the species listed, the most important is probably F. fusiforme. This and the other fusobacteria of the mouth, usually but not always in association with certain spirochetes, and Bacteroides melanino- genicus can set up chronic gangrenous infections involving the oral, pulmonary or intestinal mucosa. They may also be involved in the infections resulting from human bites and, much more rarely, have been recovered from other chronic purulent conditions.. Bacteroides—Although the Bacteroides are the most important pathogens among the nonsporing anaerobic bacilli, they are the least well known and by far the most difficult to study. The genus as a whole is ill-defined and there can be little doubt that included within it today are representatives of other genera which will eventually be grouped elsewhere. Prévot!! has made a notable attempt to bring some sort of order out of the chaos, and while his classification (which depends in a number of cases on quite inadequate descriptions) is probably overelaborate and cannot be considered as final, his mono- graph is nevertheless a most useful compendium which may be 688 ANAEROBIC INFECTIONS consulted with profit by any worker in this field. Here we shall largely follow the listing of Bergey's Manual, but this is done for convenience and simplicity without consideration of the taxonomic soundness. In the animal body most strains of Bacteroides are Gram-negative, but a few Gram-positive forms of some importance (e.g., Bacteroides ramosus) are rather illogically included in the genus in Bergey's Manual. Although of variable length, these organisms are nearly all fairly slender bacilli, nonspore-forming and, with a few doubtful exceptions, nonmotile. In some species, long, tangled filamentous forms and round, swollen “fruiting bodies” (whose exact nature is uncertain) may be observed and are of some importance in differentia- tion. Such pleomorphism usually becomes much more pronounced in artificial culture. Intracellular granules, similar to those seen in the fusobacteria, are not uncommon. However, the absence in the Bacteroides of any tapering of the cells, their much more variable size and shape, and the frequent presence of various round, swollen bodies should always make presumptive identification under the microscope a fairly straightforward matter. Culturally these bacteria are extremely difficult to handle. Growth is always slow and scanty, even under conditions of the strictest anaerobiosis, and with purification becomes, as a rule, still more irregular and tenuous. Most strains require blood, serum or (prefer- ably) ascitic fluid. It is a good rule to inoculate several tubes of media at each transfer and to maintain at least some of these cultures under continuous anaerobic incubation for a period of 7 to 10 days. For isolation, most success has been achieved by the use of deep agar cultures, preferably in pour plates incubated anaerobically. Tt is again to be emphasized that most strains are very delicate and may be readily lost in culture. On solid media colonial appearance differs considerably from species to species, but most colonies are very small (0.5-2 mm), irregularly round, and slightly raised. Nearly all are translucent or transparent, but in at least one species, B. melaninogenicus, well- marked black pigmentation slowly develops on blood agar. Hemolysis is not common and is never pronounced. In most strains of B. funduliformis, however, there is a narrow zone of greening around the colonies which may change into true beta hemolysis on exposure to the air. Again, in certain other species which are nonhemolytic on first examination a slight zone of alpha hemolysis may slowly de- velop in the presence of oxygen. ANAEROBIC INFECTIONS 689 In the determination of individual cultural and “biochemical” reactions, the following points should be noted : A number of trials may be necessary in order to establish the most suitable basal medium in which to carry out the tests. Suitability varies too much to permit of any generalizations here, although cysteine-heart infusion broth (sugar-free) is probably the most generally useful. Such a medium may, how- ever, have to be supplemented in various ways and this can be done only as a result of direct experiment. Very heavy inocula are often essential, and even then a certain number of failures to grow out are to be anticipated. In many cases growth itself is slow, with a lag period of several days. In fermentation studies it is frequently advisable to estimate quantitatively the amount of carbohydrate consumed rather than to depend on the production of acid and/or gas. Table 2 lists the more important pathogenic species. However, the comments already made concerning the variability of cultural reactions among the nonsporing anaerobic bacilli apply with especial force in the case of the Bacteroides and any worker engaged in the study of these organisms is strongly advised to carry out all his tests in dupli- cate or triplicate and to consult closely the monographs cited. Table 2—Cultural Characteristics of Some Species of Bacteroides Gas Pro- Species duction Glucose Lactose Gelatin Indole B. fragilis RA 4 + or — —_ -— B. serpens + wl LE += g= B. funduliformis + + — — + B. vulgatus + + + he — B. tumidus — + + + — B. melaninogenicus — “he + x + B. gulosus — : “+ + + It may be convenient to remark here that in a number of laboratories in this country and elsewhere, the practice has developed of dividing the Gram-negative Bacteroides into two main groups, largely on the basis of the morphological appearances. In one—the B. funduliformis group—the occurrence of filamentous forms and large, round “fruit- ing bodies” is common; in the other—the B. fragilis group—no such pleomorphism is seen. From a strictly taxonomic point of view this approach has little to recommend it and has undoubtedly contributed to our current and continuing ignorance of the genus. On the other hand, it must be conceded that this differentiation appears to have 690 ANAEROBIC INFECTIONS some validity from the clinical standpoint, for the nature and course of the infection differ considerably as between the two types of microorganisms, Furthermore, in small laboratories poorly equipped for anaerobic work a differentiation of this sort may be all that is technically feasible. Yet we feel that this practice is one which is to be deplored. Essentially it is an admission of failure. Actinomyces—The pathogenic anaerobic actinomycetes are considered by some bacteriologists as being divided into three species : A. bovis, A. israelii, and A. baudetii, which are differentiated on the basis of colony consistency and hyphal staining. Others consider that the situation is adequately met by a single species named 4. bovis. Because it makes little difference to the clinical bacteriologist, and for the sake of simplicity, the term A. bovis will be used in this section as ‘referring to anaerobic actinomycetes, regardless of colony consistency or staining of hyphae. A. bovis usually grows in the tissues in the form of definite colonies or “sulfur granules,” and isolation is most easily made from them. They may vary from 0.3 to 3 mm in diameter and have a characteristic lobulated structure. Usually the small granules are whitish gray in color, although the large ones are yellowish or yellowish brown. If granules are not readily found in a specimen, the pus should be diluted with saline solution and slowly filtered through gauze, to which the granules will adhere. Examination with a 10X hand lens will aid in disclosing small granules, Several granules should be transferred to a clean slide, a drop of salt solution added, the granules crushed and covered with a cover slip. On microscopical examination radiating filaments may be seen, the outer ends of which appear to have a high index of refraction. The cover slip is then removed, the material spread more thinly, dried and Gram-stained. Gram-positive material in beads or bands along the rods may be seen, or Gram-positive hyphae or mycelial frag- ments. The club-shaped structures at the outer ends of the filaments, which are quite prominent in the wet preparations, do not take the Gram stain. : Material from a lesion suspected of being actinomycotic in origin should always be Gram-stained, for granules with club-shaped bodies may be found in infections caused by other organisms. Likewise, actinomycotic infections may be encountered in which no granules can be found, but in which branching, Gram-positive organisms are present. Some granules may be encountered that do not have clubs at the ends of the radiating filaments. ANAEROBIC INFECTIONS 691 Isolation is most easily made by crushing several granules in a small amount of saline solution and inoculating this mixture to several plates of brain-heart infusion agar to which 1 per cent glucose has been added. It is better to use comparatively small granules, as the larger ones may be highly calcified. Incubation should be carried out in anaerobic jars with 10 per cent carbon dioxide in the atmosphere. Deep agar shakes may also be used, but if an appreciable number of other organisms are present, difficulty will be encountered in isolating A. bovis. The plates should be incubated for at least 5 days before examination. Colonies with a “heaped up” appearance, resembling bread crumbs, should be examined with care. As a rule these do not emulsify easily and are difficult to pick from the agar. Colonies re- sembling those of staphylococci should also be examined. Colonies of A. bovis are not pigmented. The rods composing the rough colonies usually show appreciable twig-like branching. The cells resulting from the fragmentation of the branched mycelium are often joined to form L-, V-, and Y-shaped structures. The organisms composing smooth colonies may be almost bacterial in appearance, but so long as they are thin Gram-positive rods, the possibility of their being Actinomyces must be considered. Identification should never be made solely on morphological grounds, since some strains of propionibacteria exhibit quite similar branching, particularly when grown in the presence of oxygen. Tubes of semisolid medium or chopped meat medium should be inoculated from isolated colonies composed of branching organisms. After growth is apparent in these subcultures, differential media should be inoculated. Strains of 4. bovis do not clot or peptonize milk, do not liquefy gelatin, do not form indole, do not reduce nitrate to nitrite, do not produce hydrogen sulfide or pigment. Glucose is promptly fermented and lactose, maltose and sucrose are usually fermented somewhat more slowly. Fermentation of salicin, mannitol and glycerol may vary from one strain to another. Cultural study is always required before an organism is identified as A. bovis, for strains of other organisms are not infrequently con- fused with it. Probably the organism most commonly mistaken for A. bovis is Propiomibacterium (Corynebacterium) acne, which is the most common organism on the human skin and which is often found elsewhere in nature. It can readily be distinguished from A. bovis by its ability to liquefy gelatin, to peptonize milk, and to produce hemo- lysis on blood agar plates. 692 ANAEROBIC INFECTIONS APPENDIX A Testing for Pathogenicity Most of the anaerobic bacteria that are involved in human infec- tions are pathogenic for laboratory animals, and knowledge of this property is quite helpful in evaluating the role an organism may be playing in certain pathological conditions. For the clostridia, guinea pigs are commonly used, an injection of 0.5 ml of a young broth culture being made preferably into the muscle of the thigh. It is well to inject through the inner side of the leg, where the skin is less tough. The culture to be used should be grown in a suitable broth medium and should be injected as soon as definite turbidity appears. For most strains, this will be a culture of 5 to 8 hr incubation from a heavy inoculum. With weakly pathogenic strains, it may be necessary to add an equal volume of sterile 5 per cent calcium chloride solution to the broth culture immediately before inoculation. The calcium chloride causes sufficient tissue damage to provide excellent conditions for the growth of clostridia in the body. The injection of a virulent strain of CI. bifermentans (CI. sordellii) into a guinea pig is usually followed by death within 24 hr. On post- mortem examination, the most pronounced symptom is a gelatinous subcutaneous edema, clear or rose-colored, lying between the ab- dominal muscles and the skin. At the site of injection in the thigh, the muscle is markedly hemorrhagic and contains an appreciable amount of gas. The hemorrhagic area extends from the muscle of the leg into the abdominal muscles. There is a marked putrefactive odor, quite characteristic of Cl. bifermentans. Some strains do not produce lesions when they are inoculated in the absence of tissue debilitants, but when these strains are inoculated together with calcium chloride, marked digestion of the muscle takes place. The animal does not usually die, for the digestion of muscle is usually restricted to the inoculated limb. Most strains of Cl. bifermentans, however, are not pathogenic for guinea pigs. Guinea pigs injected with a culture or a culture filtrate of CI. botulinum or Cl. parabotulinum may show symptoms as early as 2 hr or as late as 4 days after injection. There is usually a progressively developing paralysis, with labored respiration and excessive salivation. The abdomen becomes noticeably pendulent, due to relaxation of the abdominal muscles. The pupils are dilated. The animal usually dies shortly after symptoms become evident. On post-mortem examination there are few, if any, characteristic changes. ANAEROBIC INFECTIONS 693 The injection of a culture of Cl. carnis usually causes death within 20 hr. There are no symptoms to be observed before death other than a slight swelling of the injected limb. On post-mortem examination the muscles of the leg into which the culture was injected will appear almost normal, although slightly swollen. A thin, glossy edema will be present between the abdominal muscles and the skin. This may be slightly blood-stained on the posterior part of the abdominal wall, The adrenal glands will be slightly enlarged and slightly hemorrhagic. Guinea pigs injected with cultures of Cl. chauvei may survive 2 or 3 days, or may perish in less than 24 hr depending upon virulence of the strain and age of the culture. No symptom is usually noticeable before death other than a marked swelling and tenderness at the site of inoculation. On post-mortem examination, there is usually a spreading, hemorrhagic edema, usually watery in character, in the subcutaneous tissues. There may be a small amount of gas at the site of inoculation and the muscle at this site may show an area blue-black in color, of normal consistency, and with a dry and slightly spongy surface on being cut. This is most apt to be evident when the animal has died the second or third day after inoculation. There is no putre- factive stench, but instead a slight butyric, sweetish odor that is characteristic of infection with Cl. chauvei. On the surface of the liver and elsewhere in the abdominal cavity will be found Gram- positive rods, isolated or in short chains of two to four organisms of the same length. Cl. hemolyticum causes the death of guinea pigs in 1 or 2 days after intramuscular injection. There are no characteristic symptoms to be seen before death. On post-mortem examination there will be found a moderate amount of hemorrhagic edema fluid between the skin and the abdominal muscles, which are redder than usual. There may be some bubbles of gas in the inoculated limb, but not elsewhere. The lungs are often slightly hemorrhagic and congested. There is usually some enteritis. If more than 1 day elapses between the injection of the culture and the death of the animal, hemoglobinuria is usually found. Examination of the heart blood will usually demonstrate that the great majority, if not all, of the erythrocytes have undergone lysis. Droplets of fat are commonly found on the surface of the heart blood and on the surface of the edema fluid near the site of injection, There is no characteristic odor. With CI. histolyticum, pathogenicity is usually restricted to smooth strains, although even these will vary, some of them producing no more than small localized lesions which heal spontaneously. With most smooth strains, however, it is noted on the day following injec- 694 ANAEROBIC INFECTIONS tion that the guinea pig does not use the leg which has been injected. There is no other sign of illness, the appetite being unimpaired and the animal apparently not suffering any pain. The second day after injection the bone of the thigh may be felt through the skin, an in- dication of the progressive digestion the muscle is undergoing. Usually the skin is digested the second or third day; it is then found that the muscle of the leg has been completely liquefied and that only a reddish fluid occupies the space between the skin and the bone. If the infection does not pass the hip joint, the animal will not die, and in such a case the microbiological amputation of the leg is the terminal result. If the infection does pass the hip joint, it will progress to the abdominal muscles and death will result. This ex- tensive tissue digestion is due to the action of the beta toxin of CI. histolyticum, an extremely active proteolytic enzyme capable of digesting collagen. Some strains of Cl. histolyticum produce ap- preciable amounts of alpha toxin, which is lethal only, and but small amounts of the collagen-digesting beta toxin. The injection of such strains into guinea pigs is usually followed by death within 24 hr of inoculation. There is only a small amount of digestion of muscle to be found post-mortem. A putrefactive odor is usually noted. After the injection of a culture of Cl. mnovyi, the guinea pig usually dies in 1 to 3 days, depending upon the virulence of the strain and the amount of toxin injected. There are no ante-mortem symptoms of significance. After death, the subcutaneous tissue between the abdominal wall and the skin is found to be infiltrated by a gelatinous edema, transparent and colorless or rose-colored, often a centimeter or more in thickness and extending anteriorly to the neck. Usually this edema fluid is hemorrhagic around the site of injection but not elsewhere. Small gas bubbles may be found in this site. The viscera may be congested, but no characteristic changes are to be seen except, perhaps, in the liver, where sharply outlined necrotic areas are sometimes found. The extensive edema is due to the action of the lethal alpha toxin of CI. novyi. There is no putrefactive odor, but instead a rather sharp, unpleasant smell. Cl. perfringens is pathogenic for all the commonly used laboratory animals, including pigeons. The intramuscular injection of a virulent strain into a guinea pig usually results in death within 1 day, although death may be delayed for several days with strains of slight virulence. On post-mortem examination, there is usually a large pocket of gas in the injected limb, and extensive blood-stained edema in the sub- cutaneous tissue, with globules of fat floating on the surface. Similar ANAEROBIC INFECTIONS 695 fat droplets are found on the surface of the heart blood and in the muscle tissue. Small bubbles of gas occur within the muscle tissue itself, which is soft and pulpy due to the action of the collagen- digesting kappa toxin on the collagenous membranes. Strains of Cl. perfringens that do not produce kappa toxin do not induce this soften- ing and pulping of the muscles, but instead produce a thin, glossy edema. There is a marked butyric odor. Some strains of Cl. per- fringens do not produce enough alpha toxin to cause death. The in- jection of such strains may produce no more than slight to marked swellings which spontaneously subside within a few days. With CI. septicum, death usually occurs within 20 hr after injec- tion of the culture. On post-mortem examination there is a consider- able amount of blood-stained edema, with a pocket of gas at the site of inoculation. Small bubbles of gas are found in the affected muscle, which is deep red in color. There is usually a noticeable enteritis. Characteristically, chains of organisms will be found on the surface of the liver. If the animal is examined only shortly after death, these chains will be very largely restricted to the anterodorsal portion of the diaphragmatic surface of the liver. The organisms within the chains will usually be of appreciable length and organisms of varying length will be found within a single chain. There is no putrid odor associated with Cl. septicum infections. Injection of a culture of CI. tetani usually produces tonic muscular spasms in 24 hr or less, depending upon the amount of toxin in the culture fluid. With this organism it is often well to use cultures several days old, since one is looking primarily for the effect of the preformed toxin and, with some strains, toxin production is not ap- preciable in young cultures. The muscular spasms usually start near the site of inoculation and soon progress to the opposite side of the body, and anteriorly. The progression is most rapid on the side of the body on which the injection was made, so that a noticeable curva- ture of the body toward the inoculated side may be noted. Death occurs from a few hours to several days after the first appearance of symptoms, depending upon the amount of toxin injected. Usually on post-mortem examination there are no symptoms to be seen other than a slight reddening of the muscle at the site of inoculation. Of the nonspore-forming anaerobic bacteria, only B. funduliformis is likely to be found pathogenic for laboratory animals, and this organ- ism seldom is able to infect guinea pigs. Rabbits and mice are sus- ceptible, however, and death often results from subcutaneous or in- traperitoneal inoculation. White mice may survive 2 to 10 days after subcutaneous inoculation and 1 to 5 days after intraperitoneal inocula= 696 ANAEROBIC INFECTIONS tion. After subcutaneous inoculation into white mice, the infection tends not to be sharply localized but is represented by a slowly spread- ing necrosis. Post-mortem, little is to be found except that multiple abscesses in the liver are sometimes seen. Following intraperitoneal inoculation, abscesses may be present on the surface of the intestine as well as in the liver. The organisms may be demonstrated in these abscesses microscopically, and they may also be cultivated from the heart blood. A. bovis or A. israelit species seldom causes a progressive infec- tion in either guinea pigs or rabbits following a single injection. Young male albino mice seem to be more susceptible, as do hamsters. How- ever, determination of the pathogenicity of a strain of Actinomyces recovered from a clinical condition is seldom made for there is gen- erally little to be gained by determining the pathogenic propensities in laboratory animals of a single strain. The anaerobic cocci are so seldom pathogenic for laboratory animals that there is little point in testing for this property unless a hemolytic strain of an anaerobic streptococcus is encountered. Such hemolytic strains seem to be of more than usual pathogenicity. APPENDIX B Testing for Clostridial Toxins In testing the toxicity of cultures of the clostridia, attention must be paid to the age of the culture. For Cl. histolyticum, Cl. perfringens, Cl. septicum and Cl. hemolyticum, toxin concentration is at a maxi- mum at the end of the logarithmic phase of growth and may drop rapidly thereafter. For Cl. bifermentans, Cl. botulinum, Cl. novyi and Cl. tetani, toxin concentration does not reach its maximum for several days, for with these organisms the toxin is not released into the medium until after lysis of the bacterial cells. Although special media have been devised for high toxin produc- tion by each of these species, toxin may generally be demonstrated in filtrates of cultures grown in chopped meat medium with 1 per cent added glucose. The culture should be centrifuged and filtered through Seitz sterilizing pads or sintered-glass filters and examined for toxin without delay. If this cannot be done immediately, the filtrates should be neutralized and stored in the cold. Dilutions should be made in diluent containing 0.5 per cent gelatin or peptone. Salt solution or phosphate buffer without added protein is often unsatisfactory. ANAEROBIC INFECTIONS 697 Toxicity is best tested by the intravenous inoculation of 0.2 ml of culture filtrate or a dilution thereof into mice, Intraperitoneal inocula- tion is also satisfactory, although larger doses will be required. Still larger doses will be required for inoculations made subcutaneously or intramuscularly. Intradermal inoculations into guinea pigs or rabbits are satisfactory only for the necrotizing toxins; they are unsatis- factory for the neurotropic toxins. All of the clostridial toxins, except those of Cl. botulinum and Cl. tetani, kill mice within a short time. A single lethal dose of a neurotropic toxin, however, may take a number of days to cause death. Guinea pigs, hamsters and rabbits may also be used for the demonstration of toxin, although they are usually less convenient than mice. Also, intravenous inoculation is quite difficult to perform in hamsters and guinea pigs. Neutralization of toxic filtrates may easily be accomplished by mixing 1 ml of culture filtrate with 0.5 ml of specific antitoxin, letting the mixture stand 30 min at room temperature, and injecting 0.3 ml intravenously. For Cl. novyi, Cl. tetani, Cl. bifermentans, Cl. septi- cum, Cl. histolyticum and Cl. hemolyticum, species-specific antitoxins are satisfactory. For Cl. botulinum, it is necessary to use type-specific antitoxins. Since these are not readily available, it is necessary to send the culture to some laboratory that is equipped to perform toxin- type identification tests. (See also Chapter 11, “Bacterial Food Poisoning.”) The very great majority of strains of CL perfringens encountered in clinical medicine will belong to Type A, for which antitoxin is com- mercially available. Most of the strains of Type A encountered will produce very little toxin when grown in ordinary media. This does not necessarily indicate that they are of low virulence. However, strains of Types D and F have been isolated from man. If one suspects that a strain of a type other than Type A has been isolated, it should be inoculated heavily into freshly prepared chopped meat medium containing 1 per cent glucose. Growth should be complete after 5 to 7 hr of incubation. The cultures should be centrifuged and the supernatant fluid adjusted to neutrality and filtered through a bacterial filter. To one portion of the filtrate 0.5 per cent trypsin (Difco) should be added and this mixture incubated at 35° C for 1 hr. Type A antiserum should be added to both the trypsinized and untrypsinized filtrate, allowed to stand at room temperature for 30 min, and inoculated into mice. Death of the animals inoculated with mixtures of either the trypsinized or untrypsinized filtrates and Type A antitoxin indicates 698 ANAEROBIC INFECTIONS that the strain under study belongs to a type other than Type A. It should then be sent immediately to a laboratory that is equipped for Cl. perfringens toxin typing. Jorn D. MACLENNAN, M.D., Chapter Chairman L. S. McCrLung, Pu.D. Louis D. S. Smita, PH.D. REFERENCES 1. McCrung, L. S. The Anaerobic Bacteria, with Special Reference to the Genus Clostridium. Ann. Rev. Microbiol. 10:173-192, 1956. 2. Smtr, L. D. S. Introduction to the Pathogenic Anaerobes. Chicago: Univ. of Chicago Press, 1955. 3. WEINBERG, M., NATIVELLE, R., and Prevor, A. R. Les Microbes Anaérobies. Paris: Masson et cie, 1937. 4. LinpBerG, R. B., Mason, R. P.,, and CurcHins, E. Selective Inhibitors in the Rapid Isolation of Clostridia from Wounds. Bact. Proc. 1954, pp. 53-54. 5. McCrung, L. S.,, and Toase, R. The Egg Yolk Plate Reaction for the Presumptive Diagnosis of Clostridium sporogenes and Certain Species of Gangrene and Botulinum Groups. J. Bact. 53:139-147, 1947. 6. Reep, J. B.,, and Orr, J. H. Rapid Identification of Gas Gangrene Anaerobes, War Med. 1:493-510, 1941. 7. Fousert, E. L., Jr, and DoucrLas, H. C. Studies on the Anaerobic Micrococci, I. Taxonomic Considerations. J. Bact. 56:25-34, 1948. 8. RoseBury, T.; Crark, A. R.; Enger, S. G.; and Tercis, F. Studies of Fusospirochetal Infection. 1. Pathogenicity for Guinea Pigs of Individual and Combined Cultures of Spirochetes and Other Anaerobic Bacteria Derived from the Human Mouth. J. Infect. Dis. 87:217-235, 1950. 9. Dack, G. M. Nonspore-forming Anaerobic Bacteria of Medical Importance. Bact. Rev. 4:227-259, 1940. 10. MacDonaLp, J. B. The Motile Nonsporulating Anaerobic Rods of the Oral Cavity, Univ. of Toronto Press, 1957. 11. Prevor, A. R. Manual de Classification et de Détermination des Bactéries Anaérobies. Paris: Masson et cie, 1948. 12. ——————. Biologie des Maladies Dues aux Anaérobies. Paris: Flam- marion, 1955. 13. MAcLENNAN, J. D. The Histotoxic Clostridial Infections of Man. Bact. Rev. 26:177-276, 1962. CHAPTER 24 FUNGUS INFECTIONS* I. Introduction 11. Identification of Fungi 111. Fungi Causing Infection of Hair Collection of Specimens Examination of Specimens Key to Fungi by Direct Examination of Specimens Culture of Specimens Examination of Cultures Key to Identification of Fungi by Examination of Cultures (The organization of each of Sections IV, V, VI, VII and VIII parallels that of Section III.) ocunbhLN~ IV. Fungi Causing Superficial Infection of the Skin and Nails V. Fungi Causing Infection of Mucous Membranes, Skin and Subcutaneous Tissues VI. Fungi Causing Pulmonary Infections VII. Fungi Causing Meningitis VIII. Fungi Recovered from Blood or Bone Marrow References I. INTRODUCTION The identification of a pathogenic fungus is based on its morphology in tissue or clinical specimens and in culture. Most of the fungi which cause disease in man are dimorphic, that is, the morphology of the tissue or parasitic form of the fungus is quite different from its culture or saprophytic form. Both forms must be examined for con- clusive identification. Biochemical methods of identification, such as fermentation and carbon and nitrogen assimilation patterns, are of value in the separa- tion of species of Candida, the yeasts, and actinomycetes. These methods are rarely used for other fungi. Growth factor dependence characterizes certain species of dermatophytes. Differential staining * Delays in publication, together with new information and the development of new methods, have necessitated a drastic revision of this chapter, for which the Chairman of the chapter committee assumes entire responsibility. 699 700 FUNGUS INFECTIONS by Gram’s method is not generally useful because most fungi are variably Gram-positive, depending upon the age of a cell. Some fungi, however, may be separated from closely related forms by partial acid-fastness—for example, species of Nocardia (aerobic actino- mycetes). The terminology and nomenclature used follow that of Emmons, Binford and Utz.! With few exceptions this nomenclature has been generally accepted, although there is still disagreement among medical mycologists about the correct names of a few fungi. These disagreements are, in effect, differences in interpretation and evalua- tion of variations in the morphology of fungi. The size of fungi, the extent of morphological specialization, temporary variation in form and color (depending upon culture media), and permanent mutational changes in form and color are more apparent in fungi than in bacteria. Depending upon one’s viewpoint, these factors simplify or compli- cate the systematic classification and the identification of fungi. For the medical mycologist and the physician, a thorough background knowledge of mycology is desirable, but for practical purposes the ability to recognize the 50 or more fungi which cause human disease is a workable substitute. Text and reference books!” should be consulted for more detailed information than can be included in this chapter. Pronunciation—There is a deplorable lack of uniformity among mycologists in the pronunciation of names of fungi. The rules gov- erning pronunciation of Latin (with few exceptions) require that the accent fall on the antepenult, that is, on the third syllable from the end of the word, unless the penultimate syllable has a long vowel. Webster’s unabridged New International Dictionary (Second Edi- tion) follows this rule. Familiar examples are Tri-choph’y-ton men- ta-groph’y-tes, Mi-cro-spo’rum aud-o-uin’ii, E-pi-der-moph’y-ton floc- cos’um and Ac-ti-no-my’ces is-rael’i-i. Il. IDENTIFICATION OF FUNGI Few special instruments are needed in a mycological laboratory for the microscopical examination and identification of fungi. In ad- dition to the usual equipment found in most bacteriological labora- tories, two stiff, sharp, heat-resistant needles are required for the transfer of cultures and for the preparation of specimens from a culture for microscopical examination. These can be made by filing to a sharp point a piece of heavy-gauge nichrome wire clamped in a FUNGUS INFECTIONS 701 suitable holder or sealed into the end of a tapered heavy-wall glass tube such as a broken 5 ml pipette. They are used to spread out the mycelium in a drop of mounting fluid. One of the needles is suitable for transfer of cultures. For occasional use a bacteriological wire loop will be needed. A safety hood is necessary for the handling of such pathogens as Coccidioides. A 10 per cent sodium or potassium hydroxide solution is the best mounting medium for microscopical examination of fungi. The hydroxide is used also to clear clinical materials such as skin, hair, nails and sputum. If a stain is desired, mix equal parts of Parker’s Superchrome blue-black ink and 20 per cent aqueous solution of sodium hydroxide. Lactophenol-cotton blue also stains fungi deeply and is commonly used in the preparation of mounts from culture material. A dropping bottle of 95 per cent alcohol should be at hand to moisten the mycelium and prevent entrapment of air bubbles. Prepare a clean slide by rubbing its surface with a silicone-coated paper and place adjacent drops of 10 per cent sodium hydroxide or lactophenol-cotton blue and 95 per cent alcohol on the slide. Holding a sharp sterile needle at a narrow angle to the culture, re- move in a single pass over the surface some of the aerial mycelium from an area containing young sporophores and spores. Touch this mycelium to the drop of alcohol and remove immediately to the hydroxide solution. With two needles, carefully spread the mycelium in the hydroxide. Place a cover slip on the drop, select a suitable field with the low power of the microscope and examine under high power for type and manner of sporulation. Mix pathologic materials with India ink, which may need to be diluted 1:1 with water, for the demonstration of budding cells with capsules in yeast-like cultures, cerebrospinal fluid, ventricular fluid, sputum or pus. The usual bacteriological stains and blood stains are occasionally used. Gram’s, acid-fast, Wright’s, Wilson's or Giemsa’s stain should be available for staining pus, sputum, spinal fluid or blood films. The Ziehl-Neelsen stain must be modified by destaining cautiously with 0.5 per cent aqueous sulfuric acid rather than acid alcohol in order to preserve the partial acid-fastness of Nocardia asteroides. The periodic acid-Schiff and Gridley fungus stains can also be adapted to examination of these preparations and are especially useful in the staining of tissue sections. Gomori’s methenamine-silver nitrate stain will demonstrate Histoplasma in sections of old lesions where it is not visible with other stains. Most of the fungi of medical importance grow on a wide variety of media, including modified Sabouraud’s (CM No. 105a), mycophil 702 FUNGUS INFECTIONS (CM No. 108), potato glucose (CM No. 107), cornmeal (CM No. 106), and Littman’s oxgall agar (CM No. 109). Throughout this chapter Sabouraud’s or modified Sabouraud’s agar refers to the 1 per cent neopeptone, 2 per cent glucose agar modification proposed by Emmons (CM No. 1052). Its reaction should be pH 6.5 to 7.0. Hypersensitivity and Serology Intradermal tests are widely used in diagnosis and in determining the prevalence of past and present mycoses. They have been used more widely and more profitably in coccidioidomycosis, histoplasmosis and blastomycosis than in other systemic mycoses and superficial fungal infections. Antigens may be prepared from either the mycelial or parasitic growth form of dimorphic fungi. Usually the mycelial form has been used. Inoculate flasks of glucose asparagine broth (CM No. 111) by floating spores or mycelial fragments on the surface and incubate 1 to 3 months at 30° C, or room temperature. Before harvesting shake the culture to wet the surface of the floating mycelium and spores. After 24 hr pour off the broth, spin down any hyphae present, pass the supernatant through a sintered-glass filter, and test for sterility. Add merthiolate to a final concentration of 1:20,000. Store this antigen in vaccine bottles at 4° C. Dilute 1:1,000 for preliminary testing. Titrate the antigen in experimentally infected guinea pigs and then in persons whose reaction has been previously determined. Coccidioidin is available commercially from Cutter Laboratories; histoplasmin from Parke, Davis & Co., Eli Lilly & Co., and the Divi- sion of Laboratories, State of Michigan Department of Health; blastomycin from Parke, Davis & Co.” Stock antigens are usually adjusted by dilution so that a final dilution of 1:100 is made for the intradermal test. Inject 0.1 ml of the diluted antigen intradermally and read tests at 24 and 48 hr. Disregard purely erythematous reactions. A delayed tuberculin type of reaction with an area of edema exceeding 5 mm is read as positive. It is generally interpreted as indicating that the patient has or has had an infection (either clinical or subclinical) caused by the fungus used in preparing the antigen. In a diagnostic test, coccidioidin, blastomycin and histoplasmin should be used simultaneously, employing needles and syringes that have not been used with other antigens. There are cross-reactions among these antigens and a diagnostic interpretation should be made with caution. Serological examinations are useful auxiliary tests in determining the diagnosis and the prognosis of a mycosis. In mild cases of pul- FUNGUS INFECTIONS 703 monary mycosis in which no sputum is raised, or in any cases where it is not possible to obtain pathological exudates or tissues, the diag- nosis may have to depend upon a serological test. The prognostic interpretation is based upon the observation that a patient who reacts to an intradermal test but does not have a significant titer of circulat- ing antibodies has a better prognosis than the patient who does not react to an intradermal test but has a high titer of complement-fixing antibodies. Precipitins and agglutinins usually appear early in a mycosis. Collodion-particle agglutination is a useful test in the hands of an ex- perienced investigator or technician. Antigens for complement fixa- tion may be prepared from either the yeast form or the mycelial form of a dimorphic fungus. Actually, both types of antigens should be used because they apparently measure different types of antibodies. Complement-fixation tests are carried out according to standard methods and either the 100 or 50 per cent end point may be read. Optimum dilution of antigen must be determined by test. Titers of 1:4 or 1:8 are on the borderline of significance. A dependable diagnostic interpretation cannot be made from a single serological test. Ideally, a serum sample should be taken early in the illness, one at the height of the illness, one during convalescence, and one several weeks after recovery. A sharp rise and subsequent fall in titer is usually acceptable diagnostic evidence, although cross- reactions with a heterologous antigen may appear early in the illness and may be confusing. Usually the titers of antibodies detected by the heterologous antigens decrease as the disease progresses. Ill. FUNGI CAUSING INFECTION OF HAIR I. Collection of Specimens Tinea capitis: Use of a Wood's light facilitates selection of infected hairs for examination and culture. Hairs infected by Microsporum audouinii and M. canis exhibit a greenish fluorescence. Hairs in- fected by Trichophyton fluoresce poorly or not at all. Remove fluorescent hairs or hair stubs with forceps and place in a petri dish, or fold securely in a clean paper for transport to the laboratory. Write the patient’s name, history number, location from which hair was selected, and date of collection on the package. A specimen from dermatophytosis does not deteriorate rapidly on drying and is suitable for examination and culture hours or days after its collection. It should be allowed to dry in order to decrease growth of contaminants. 704 FUNGUS INFECTIONS Trichomycosis axillaris: Under ordinary light, inspect hairs of the scalp and beard in suspected piedra, and hairs of the axillary and pubic regions in trichomycosis. Cut off with scissors any hairs with nodules adherent to the shaft, place them in a petri dish or test tube, or wrap securely in paper, and carry to the laboratory. Tricho- mycosis axillaris is usually bacterial in nature, although it was errone- ously believed at one time to be mycotic. 2. Examination of Specimens The Wood’s light may be used in the laboratory for selecting from previously collected specimens infected hairs for examination. Place basal 2-5 mm of hair in a drop of 10 per cent sodium hydroxide on a microscope slide. Place cover glass over the preparation and gently heat slide over the low flame of a Bunsen burner. Repeated gentle heating will drive out air bubbles and clear the specimen for im- mediate microscopical examination. Examine all such preparations with the low and high dry objectives. 3. Key to Fungi by Direct Examination of Specimens A. No nodules on hair; fungus grows in follicle and in and/or on hair shaft. Fungus hyphae grow downward in hair but do not reach bulb. Parasitized hairs break at variable distances above skin surface. 1) Hyphae and chains of arthrospores inside hair shaft and also forming a sheath of spores on the surface of the hair. a. Sheath surrounding shaft of hair formed of spores 2-3 u in diameter, crowded together into a mosaic pattern—Fig 1 (a) : Microsporum (several species). b. Sheath surrounding shaft of hair formed of parallel rows of spores 3-4 pu in diameter—Fig 1(b): Trichophyton (small-spored ectothrix species). ¢. Sheath surrounding shaft of hair formed of parallel rows of spores 4-6 pu in diameter—Fig 1(c): Trichophyton (large- spored ectothrix species). 2) Hyphae and spore chains inside hair shaft and not forming a conspicuous external sheath. a. Fungus inside of hair shaft seen as parallel, branching hyphae which break up into spores—Iig 1(d): Tricho- phyton (endothrix species). b. Fungus inside of hair shaft replaced by tunnels and spaces which fill with air after disintegration of fungus hyphae— Fig 1(e) : Trichophyton schoenleinii (favus). FUNGUS INFECTIONS 705 B. Nodules formed on hair shaft well above skin surface. 1) Nodules along shaft of scalp hair above skin surface (tropical distribution). a. Black, discrete, hard, adherent mycelial masses on hair shaft composed of wide, short-celled hyphae—LFig 1(f) : Piedraia hortai (black piedra). b. White, soft mycelial masses on hair shaft composed of wide, short-celled hyphae—Fig 1(g): Trichosporum beigelii (white piedra). 2) Nodules along shaft of axillary or pubic hair above skin surface (wide geographic distribution). a. Yellow, red or black nodules on hair shaft composed of masses of delicate hyphae 1 p in diameter—Ifig 1(h): Species of Corynebacterium, Micrococcus and Nocardia ( ?) (trichomycosis). 4. Culture of Specimens Hairs from piedra are sufficiently distinctive to permit immediate identification by microscopical examination without resorting to cul- tural technics. Since only one fungus, Piedraia hortai, causes black piedra and only one fungus, Trichosporum beigelii, causes white piedra, identification of these two fungi may be made on the appear- ance of the nodules on the infected hair. The microorganisms which cause trichomycosis vary with the color and texture of the granules and must be isolated in culture for identification. Although the appearance of the infected hairs in tinea capitis often allows generic identification of the invading fungus (see key), the species involved can be identified only by a critical examination of the fungus after it has been isolated in culture. Infected hairs should be planted on modified Sabouraud’s agar with chloramphenicol (CM No. 105b) and on cycloheximide agar (CM No. 105¢) in slants or petri dishes. It is desirable to use only the basal 2-5 mm of infected hairs and to space hair fragments so that well-separated colonies will develop. Incubate cultures at room temperature or at 30° C for at least 3 weeks before they are discarded as negative. If cultures are contaminated, subculture on Sabouraud-chloramphenicol medium from the edges of young colonies or from spores in order to obtain pure cultures for examination and identification. Enrich the medium with thiamine for easier isolation and identification of Trichophyton VErrucoSum. 706 FUNGUS INFECTIONS Figure 1—(a) Microsporum hair (b) Trichophyton hair, small-spored ectothrix (c) Trichophyton hair, large-spored ectothrix (d) Trichophyton hair, endothrix (e) Trichophyton hair, favus (f) Piedraia hortai, black piedra (g) Trichosporum beigelii, white piedra (h) Corynebacterium tenuis, trichomycosis hair 707 INFECTIONS FUNGUS SCHOOL OF MEDICINE PHOTO COURTESY DUKE UNIVERSITY 708 FUNGUS INFECTIONS 5. Examination of Cultures Take aerial mycelium or, if necessary, fragments of the substratum mycelium of a fungus colony with a short, stiff, straight, sharp needle fastened firmly in an appropriate holder. Holding the transfer needle at an acute angle to the surface of the culture, remove aerial mycelium in one pass across the colony surface. Bacteriological loops are worth- less for obtaining material from filamentous cultures and damage the colony for further examination. Place the mycelium from the culture momentarily in a small drop of alcohol on the microscope slide, transfer immediately to a drop of 10 per cent sodium hydroxide, hydroxide ink or lactophenol-cotton blue and tease apart carefully with dissecting needles to make a thin preparation. Place a cover glass over the preparation and examine it with the low- and high-power objectives of the microscope. 6. Key to Identification of Fungi by Examination of Cultures®? A. Culture filamentous, cottony to powdery, yellow or brown in reverse of colony. Macroconidia spindle-shaped, microconidia clavate. This group contains the species of Microsporum. 1) Colony slow-growing with dense, matted mycelium close to agar surface and dull, reddish brown pigmentation in the agar. Old cultures sometimes have radial grooves. Macroconidia when present (yeast extract added to medium increases spore production) are large, spindle-shaped, with no or few septa, imperfectly formed; microconidia (rare) 2-4X3-6 pu; “rac- quette hyphae” numerous—Fig 2(a) : Microsporum audouinii. 2) Colony fast-growing with wooly aerial mycelium and yellow- brown pigmentation in the agar. Numerous spindle-shaped, thick-walled, multiseptate (6-14 cells) macroconidia (8-15X 40-150 p) with roughened ends and clavate microconidia (2-4X 3-6 1) are produced; “racquette hyphae” numerous—Fig 2(b) : Microsporum canis (M. lanosum*). 3) Colony fast-growing with close, brown, powdery surface and dull orange to light brown pigmentation in the agar. Numerous spindle-shaped, rough, thick-walled, multiseptate (4-5 cells) macroconidia (8-12X30-50 pn) are produced; racquette hyphae numerous—Fig 2(c) : Microsporum gypseum. B. Culture filamentous, cottony to powdery or granular, with pig- ment in reverse of colony varying from yellow or red-brown to * Synonyms, when given, are not optional names—they are names used form- erly but incorrectly. FUNGUS INFECTIONS 709 purplish, or lacking. Macroconidia, when present, thin-walled and clavate with 0 to 5 septa. Microconidia clavate to subspherical. This group contains the mentagrophytes, rubrum, and crateriform species of Trichophyton. 1) Colony fast-growing, cottony or granular to powdery, surface mycelium white to cream color, reverse pale yellow, light brown or buff. Numerous subspherical microconidia develop on short conidiophores, both lateral and terminal, simple or branched, or along the sides of the hyphae, either sessile or on short, simple conidiophores; a few to several clavate, smooth, thin-walled, multiseptate macroconidia (4-6X10-50 up) are also found. Coiled hyphae, antler-like hyphae, nodular bodies (ascogonia) and racquette cells are numerous in some strains. From glabrous skin or from small-spored ectothrix hairs—Fig 2(d): T7i- chophyton mentagrophytes (T. gypseum.). 2) Colony may be fast-growing with cottony white aerial mycelium (some strains with glabrous margins) and deep red to purplish pigmentation in the substrate mycelium; or slower growing, somewhat velvety with deep red to purplish pigmentation in the aerial as well as the substrate hyphae. Numerous clavate mi- croconidia are produced on short conidiophores arising from the sides of the hyphae; on blood agar (CM No. 16) or modi- fied Sabouraud’s agar fortified with vitamins a few to several narrow clavate, thin-walled, multiseptate macroconidia (4-6X 10-30 pn) are also found. From glabrous skin and nails—Fig 2(e) : Trichophyton rubrum (T. purpureum). 3) Colony slow-growing, flat or heaped and folded; at times cerebriform, crateriform or acuminate, the surface covered with a yellow to reddish brown, short, velvety or powdery aerial mycelium and a pale yellow or brown pigmentation in the agar on reverse. Clavate microconidia are produced on short lateral conidiophores from the hyphae. Irom glabrous skin and endo- thrix hairs—Fig 2(f) : Trichophyton tonsurans. C. Culture slow-growing, glabrous, smooth and waxy, heaped or folded with a tendency to grow deep into the medium and split the agar. The surface may become velvety on prolonged growth or transfer. Sporulation may be lacking, but some strains produce mi- croconidia and macroconidia on enriched media. This group con- tains the “faviform” species of Trichophyton. 1) Colony slow-growing, heaped, folded, glabrous and waxy with cream colored to light brown pigmentation. The surface may 710 Figure 2—(a) (b) FUNGUS INFECTIONS Microsporum audouinii colony on Sabouraud’s agar, 21 days. Microsporum canis colony on Sabouraud’s agar, 10 days. (c) Microsporum gypseum colony on Sabouraud’s agar, 7 days. (d) (e) (£) (2) (h) (i) Trichophyton mentagrophytes colony on Sabouraud’s agar, 10 days. See Manual of Clinical Mycology. Philadelphia: Saunders, 1944. Trichophyton rubrum colony on Sabouraud’s agar, 12 days. See Zinsser’s Textbook of Bacteriology. New York. Appleton- Century-Crofts, 1948, p. 894, Fig 239. Trichophyton tonsurans colony on Sabouraud’s agar, 35 days. See Zinsser’s Textbook of Bacteriology. New York: Appleton- Century-Crofts, 1948, p. 894, Fig 240. Trichophyton schoenleinis colony on Sabouraud’s agar, 27 days. See Manual of Clinical Mycology. Philadelphia: Saunders, 1944, p. 254, Fig 119a. Trichophyton ferrugineum colony on Sabouraud’s agar, 14 days. Trichophyton wiolacewm colony on Sabouraud’s agar, 19 days. See Bacterial and Mycotic Infections of Man. Philadelphia: Lippincott, 1948, p. 591, Fig 41 (bottom left). m FUNGUS INFECTIONS PHOTO COURTESY DUKE UNIVERSITY SCHOOL OF MEDICINE 712 FUNGUS INFECTIONS become velvety on repeated transfer. Typical “favic chande- liers” and chlamydospores are numerous. On grain media or enriched media, a few clavate microconidia are produced. From favus—VFig 2(g) : Trichophyton schoenleinii. 2) Colony slow-growing, wrinkled, with a glabrous but sometimes velvety surface, white to light ocherous in color. Irom bovine dermatophytosis and from human beings exposed to infected cattle. Primary isolation difficult, few or no spores produced on Sabouraud’s agar. On vitamin-enriched media the colonies grow more rapidly and produce an aerial mycelium in which microconidia and macroconidia are developed. From large- spored ectothrix hair: Trichophyton verrucosum (T. faviforme, T. album, T. discoides, T. ochraceum,). 3) Colony slow-growing, heaped, folded, glabrous and waxy with deep reddish yellow to orange pigmentation—Iig 2(h): T7i- chophyton ferrugineum. 4) Colony slow-growing, folded, glabrous and waxy with deep violet pigmentation. From endothrix hair—Fig 2(i): T7i- chophyton violaceum. IV. FUNGI CAUSING SUPERFICIAL INFECTION OF THE SKIN AND NAILS I. Collection of Specimens Clean lesions on the glabrous skin with an alcohol (70%) sponge to remove surface bacterial and fungus contaminants and allow the area to dry before collecting specimens. Place materials obtained for ex- amination and culture in a petri dish or fold securely into a clean paper for transport to the laboratory. Tinea pedis—Remove the macerated skin between the toes with paper toweling or other materials and discard. Collect scales for examination from the advancing border of the lesion by peeling off with forceps, clipping with scissors, or scraping with a scalpel or the end of a microscope slide. Scrape brownish, discolored plaques occurring over the ball of the foot and heel and remove the tops of vesicular or vesicopustular lesions with scissors. Tinea cruris—Take material from the erythematous advanc- ing border of the lesion by scraping across the edge of the lesion FUNGUS INFECTIONS 713 toward the healthy skin. Include the tops of vesicles in material for ex- amination and culture. Tinea corporis—Typical circular lesions, with scaly, thickened or healing centers and erythematous, vesicular borders (ringworm) on the glabrous skin. Scrape specimens from the periphery of the lesions. Tinea nigra (tropical distribution)—Scrape epithelial scales from the black areas occurring on the palm of the hand. Tinea unguium—Take material from the discolored thickened areas of the infected nails by deeply scraping the surface, by digging out friable material from pits or grooves, and by removing accumu- lated keratinaceous material from beneath the nail. Also, where there is paronychial involvement, obtain exudate if present. Tinea versicolor—Take material from the superficial fawn- colored, furfuraceous patches by scraping with a scalpel or the end of a microscope slide. Hold another slide perpendicular to and against the skin directly below the lesion being scraped to insure catching the thin, greasy scales. Erythrasma—Take material from the reddish or reddish brown lesions by scraping the area with a scalpel or the end of a microscope slide. Hold another slide against the skin as for tinea versicolor. 2. Examination of Specimens Place thin fragments of skin or nail scrapings in a drop of 10 per cent sodium hydroxide on a slide. Place a cover glass over the preparation and heat the slide gently over the low flame of a Bunsen burner. Examine at once under low and high dry lens of the micro- scope. Scales from erythrasma and tinea versicolor may be treated with ether to remove fat before examination in sodium hydroxide. Such scales are so thin that they may be stained directly with methylene blue, Giemsa or lactophenol-cotton blue for microscopical examination. 3. Key to Fungi by Direct Examination of Specimens A. Branching, wide, septate mycelium in the material. 1) Colorless mycelium in the skin—Fig 3(a) : Trichophyton, Mi- crosporum, E pidermophyton floccosum. 2) Colorless mycelium in the nail—Fig 3 (a): Trichophyton, E pi- dermophyton floccosum. 4 FUNGUS INFECTIONS Figure 3—(a) Potassium hydroxide preparation of skin, showing fungus hyphae. i (b) Potassium hydroxide preparation of skin, showing “mosaic fungus,” an artifact. (¢) Lactophenol-cotton blue stained scales, showing Malassezia furfur (tinea versicolor). FUNGUS INFECTIONS 715 PHOTO COURTESY DUKE UNIVERSITY C SCHOOL OF MEDICINE 716 FUNGUS INFECTIONS 3) Dark-colored mycelium in the skin (tinea nigra) : Cladosporium werneckit. B. Branching, delicate (1 np) mycelium in the skin (erythrasma): Nocardia minutissima. C. Short, angular, septate hyphae intermixed with thick-walled spherical cells (3-8 pn) in the skin (tinea versicolor)—TFig 3(c): Malassezia furfur. D. Hyphae and oval, budding, yeast-like cells (4-8 pn) in skin or nails : species of Candida. E. Amorphous or crystalline material in mosaic arrangement out- lining epidermal cells (“mosaic fungus,” an artifact)—Fig 3(b). 4. Culture of Specimens Since the etiological fungi of tinea versicolor and erythrasma do not grow in vitro, do not attempt culture. The appearance of Malas- sezia furfur and Nocardia minutissima in sodium hydroxide or stained preparations is sufficiently distinctive to allow direct microscopic diagnosis. The three genera of the dermatophytes (Microsporum, Trichophyton and Epidermophyton) in skin and nails, however, are indistinguishable. All appear as septate, branching hyphae, and the fungi must be isolated in culture for specific identification. In addi- tion to the dermatophytes, there are several miscellaneous fungi which may infect the skin and nails. These are included in the key which follows. After washing the lesion with a 70 per cent alcohol sponge and collecting specimens, cut large pieces of skin into fragments with a scalpel and plant these on slants of Sabouraud’s agar with or without antibiotics (CM No. 105a). Shave or subdivide thick nail parings with a scalpel and plant on the same medium. Hold all cultures for at least 3 weeks at 30° C or room temperature and if necessary make transfers to slants of Sabouraud’s agar with chloramphenicol from the edge of the developing colonies for pure cultures and identification. 5. Examination of Cultures Note rate of growth, color on surface and reverse of colony, texture of surface, and type of surface configuration of colony. Prepare a clean microscope slide by wiping with silicone-treated paper, place a drop of 10 per cent sodium hydroxide on the slide, and place a drop of 95 per cent alcohol nearby. Take aerial mycelium only, when pos- sible, for microscopical examination. Touch the bit of mycelium to the alcohol and immediately transfer to and carefully spread it in the FUNGUS INFECTIONS 717 drop of hydroxide. Place a cover slip on the drop and examine first with low-power objective to find a sporulating area, then under higher magnification. 6. Key to Identification of Fungi by Examination of Cultures®® Since many of the dermatophytes which infect the hair also invade the skin and nails, a description of such species of Microsporum and Trichophyton may be found in the key to the identification of fungi which infect the hair. Some of the dermatophytes, however, do not infect the hair (Trichophyton concentricum and Epidermophyton floccosum) and species of Microsporum usually do not infect the nails. A. Culture filamentous, fast-growing, powdery, greenish yellow, and quickly overgrown with a white, cottony, sterile, aerial mycelium. No microconidia. Numerous clavate, smooth, thick-walled, septate (1-4 cells) macroconidia (7-12X20-40 pn) are produced—Fig 4(a): Epidermophyton floccosum. B. Culture glabrous, slow-growing, smooth and folded. The surface may become velvety on prolonged growth, at first white, but later brownish in the center. No typical spores are produced—LFig 4(b) (from tinea imbricata, tropical distribution): Trichophyton concen- tricum. C. Culture dark-colored, fungus extremely pleomorphic, at first black, glistening, and yeast-like ; later, aerial mycelium produced which is olivaceous to black. Black, oval, yeast-like cells in young cultures; dark-colored, thin hyphae in old cultures produce Cladosporium-like spores (from tinea nigra) : Cladosporium werneckii. D. Culture cream-colored, soft, with distinct yeast-like odor, fast- growing (48-72 hr) with oval, budding cells on the surface of the medium and pseudomycelium penetrating the agar. Chlamydospores produced on cornmeal agar (CM No. 106)—Fig 4(c): Candida albicans. V. FUNGI CAUSING INFECTION OF MUCOUS MEMBRANES, SKIN AND SUBCUTANEOUS TISSUES I. Collection of Specimens Nature of specimen and technic of collection vary according to type of lesion and of mycosis. See directions under specified disease head- ings. Examine a portion of all materials collected microscopically in fresh or stained preparations and culture the remainder on suitable media. ns FUNGUS INFECTIONS Figure 4— (a) Epidermophyton floccosum colony on Sabouraud’s agar, 12 days. See Zinsser’s Textbook of Bacteriology. New York: Appleton- Century-Crofts, 1948, p. 887, Fig 225. (b) Trichophyton concentricum colony cn Sabouraud’s agar, 18 days. See Am. J. Trop. Med. 20:293 (1940), Fig 6. (¢) Candida albicans colony on Sabouraud’s agar, 21 days. FUNGUS INFECTIONS 719 Cc PHOTO COURTESY DUKE UNIVERSITY SCHOOL OF MEDICINE 720 FUNGUS INFECTIONS Actinomycosis—Allow pus from draining sinuses to run into a sterile test tube held at the border of the lesion. Obtain small quanti- ties of pus with a sterile loop. Scrape or biopsy the walls of sinus tracts. Look for “sulfur granules.” Maduromycosis—Collect as for actinomycosis. Look for mycetoma granules. Rhinosporidiosis—Swab, lightly scrape, or gently squeeze with forceps the granulomatous lesions on the conjunctivae or the polypoid masses on the mucous membranes of the nose to release the sporangia and spores from the surface of the lesion. Coccidioidomycosis (cutaneous)—Aspirate pus from subcutaneous abscesses with sterile needle and syringe. Open microabscesses at the border of verrucous lesions and remove pus or obtain tissue by biopsy. Chromoblastomycosis—Remove superficial crusts from verrucous lesions with scalpel or forceps and examine for hyphae and sclerotic cells. Obtain pus from ulcerated lesions with sterile loop. Biopsy of ulcerated or verrucous lesions is most informative. Histoplasmosis (cutaneous and mucosal lesions)—Scrape or swab ulcerated lesions of the oral and nasopharyngeal mucous membranes, inoculate agar slants, and spread on slides for Giemsa staining. Take tissue by biopsy from granulomatous skin and oral lesions. Candidiasis (moniliasis, thrush, mycotic vaginitis )—Obtain macer- ated skin in intertriginous skin areas, pus from paroynchial lesions, and material from grayish patches on the mucous membranes of the mouth or vagina by scraping with loop, scalpel or swab. Sporotrichosis—Transfer pus and exudate with loop or swab from the primary, ulcerated, chancre-like lesion on the skin to agar slants and slides, to be stained with periodic acid-Schiff reagent. Obtain pus with sterile needle and syringe from unopened subcutaneous nodules along the lymphatics and obtain tissue by biopsy from one of these nodules. Cryptococcosis (cutaneous)—Take material from ulcerated skin lesions with a sterile bacteriological loop or swab and from granuloma- tous skin lesions by biopsy. Blastomycosis—Aspirate pus from subcutaneous abscesses with sterile needle and syringe. Open microabscesses at the border of verrucous lesions with scalpel and remove pus. Obtain tissue by biopsy from the border of the lesion. FUNGUS INFECTIONS 721 2. Examination of Specimens Examine exudates and pus from ulcerated lesions and from sub- cutaneous abscesses or nodules in stained films (actinomycosis and histoplasmosis) or in so-called fresh preparations. To prepare the latter, mix the specimen without drying in a small drop of 10 per cent sodium hydroxide on a microscope slide and place a cover glass over it. Clear crusts by warming in a drop of 10 per cent sodium hydroxide on a slide. Hydroxide ink mixture may be used to make the fungi more apparent by selective staining. Examine all preparations with the low and high dry objectives. Carefully regulate the amount of light used during microscopical examination of clinical materials. Too bright a light often obscures fungus cells in fresh preparations of pus or other exudates. When actinomycosis is suspected, stain films by Gram’s method. When histoplasmosis is suspected, stain film with Wright's, Wilson's or Giemsa’s stain. If cryptococcosis is supected, mix pus or exudates in a drop of India ink diluted 1:1 with water and examine as a wet preparation under a cover slip to demonstrate the diagnostic capsule of Cryptococcus neoformans. Prepare some of the material obtained by biopsy for sectioning, using the remainder for direct examination and culture. For direct examination, grind a small fragment of tissue in salt solution and ex- amine as for pus or exudates. 3. Key to Fungi by Direct Examination of Specimens A. Granules present. 1) Granules 0.5-3 mm in diameter, white or yellow, usually with peripheral “clubs,” granule composed of delicate Gram-positive hyphae 1 p or less in diameter, which when granule is crushed break up into diphtheroid and short branching elements—Fig 5(a) (b) : Actinomyces israelis and A. bovis. 2) Granules 1-3 mm in diameter, light colored (white or yellowish), clubs lacking or poorly developed, composed of Gram-positive hyphae 1 p or less in diameter: Nocardia brasiliensis. a. Granules yellow to pink: Streptomyces madurae. b. Granules coral red: S. pelletierii. c¢. Granules yellow to black: S. somaliensis. 3) Granules light colored, without true clubs but usually sur- rounded by eosinophilic material; composed of wide, septate hyphae 2-3 p in diameter and containing numerous enlarged 722 FUNGUS INFECTIONS Figure 5—(a) Actinomyces israeli granule in pus. Refer to Manual of Clinical Mycology. Philadelphia: Saunders, 1944, p. 11, Fig 7a. (b) Actinomyces israelit granule crushed and stained by Gram’s method. (c) Coccidioides immitis: endospore-filled spherule in pus. See Manual of Clinical Mycology. Philadelphia: Saunders, 1944, p. 64, Fig 32. (d) Phialophora pedrosoi: dark brown fungus bodies in pus. (e) Cryptococcus neoformans: budding encapsulated cells in pus, fresh preparation and India ink preparation. Refer to Am. J. Med. 2:600 (1947), Fig. 5. (f) Blastomyces dermatitidis: thick-walled budding cells in pus. FUNGUS INFECTIONS 723 PHOTO COURTESY DUKE UNIVERSITY SCHOOL OF MEDICINE 724 FUNGUS INFECTIONS cells or chlamydospores; the hyphae do not break up into bacillary elements: Allescheria boydii (Monosporium apiosper- mum). 4) Granules dark, composed of coarse dark, septate hyphae: Ma- durella spp. B. Granules not present, hyphae 1 u or less in diameter, partially acid-fast: Nocardia asteroides. C. Granules not present, fungi not bacterial in dimensions. 1) Fungus bodies do not reproduce by budding. a. Large, round, hyaline, thick-walled sporangia 40-300 un in diameter, containing small, spherical, hyaline spores 4-9 pn in diameter (cannot be cultured) : Rhinosporidium seeberii. Large, round, hyaline, thick-walled spherules 15-80 un in diameter, containing numerous endospores 2-4 p in diameter produced by progressive cleavage. Hyphae and cells in- termediate between hyphae and spherules may be found in pulmonary cavities—Fig 5(c) : Coccidioides immitis. Large, barrel-shaped to rounded hyaline cells 8-15 pu in diameter, and rectangular cells (arthrospores) 4-6X8-12 p in size: Geotrichum sp. Small, round, brown, thick-walled bodies 6-12 p in diameter, which reproduce by elongation and septation—Fig 5(d): Phialophora pedrosoi, Ph. compacta, or Ph. verrucosa. 2) Fungus bodies reproduce by budding. a. Small, oval, thin-walled, budding, uninucleate cells, 2-4 p in size, usually intracellular in large macrophages and best demonstrated by blood stains : Histoplasma capsulatum. Oval to spherical, budding, uninucleate cells 4-12 p in size, intracellular in giant cell and macrophages: Histoplasma duboisii. Small, oval, thin-walled, budding cells, 2-3X6-8 pu in size, and fragile hyphae (pseudohyphae) : Candida (Monilia) spp. Small, thin-walled, spherical, oval and fusiform bodies 1.5X 4-6 p in size, both intracellular and extracellular, best demon- strated by periodic acid-Schiff stain (infrequently seen in human materials) : Sporotrichum schenckii. Large, thin-walled, round, uninuclear budding cells 5-15 pu in diameter, surrounded by a wide mucopolysaccharide cap- FUNGUS INFECTIONS 725 sule best demonstrated in India ink preparations, Connec- tion between older cell and bud, narrow—TFig 5(e) : Crypto- coccus neoformans. f. Large, thick-walled, round, multinuclear budding cells 5-20 pu in diameter, lacking capsule. Connection between older cell and bud, wide—Fig 5(f) : Blastomyces dermatitidis. g. Large, thick-walled, round, multiple budding cells 10-60 pn in diameter: Paracoccidioides brasiliensis (Blastomyces brasiliensis). 4. Culture of Specimens Rhinosporidium seebert, the cause of rhinosporidiosis, does not grow in vitro. The characteristic appearance of this fungus in microscopical preparations, and especially in section, is diagnostic. In suspected actinomycosis, plant granules from draining sinus tracts, abscesses, or biopsies anaerobically in thioglycolate broth (CM No. 19), ground meat medium (CM No. 114), and deep shake cultures of beef infusion agar (CM No. 6) with 1 per cent glucose and incubate at 30° and 35° C. Also spread crushed and diluted granules on brain-heart infusion agar plates (CM No. 22) and incu- bate at 30° and 35° C under anaerobic conditions and with the addition of 5 per cent CO». Such cultures should produce growth of the mi- croaerophilic Actinomyces israelit and A. bovis. Granules should be cultured also on beef infusion glucose agar slants, blood agar plates, and Sabouraud’s agar slants without antibiotics at 30° and 35° C for isolation of the aerobic species of Nocardia. Pus, exudates and ground tissue from lesions suspected of contain- ing Candida species, Cryptococcus neoformans, Blastomyces dermatit- idis, Paracoccidioides brasiliensis, Histoplasma capsulatum, Coccid- i0ides immitis or Sporotrichum schenckii should be cultured on Sabouraud’s agar at room temperature. If the parasitic growth forms of Blastomyces, Paracoccidioides and Histoplasma are desired, ma- terial must be planted on glucose cysteine blood agar or Francis’ cystine blood agar (CM No. 115) and incubated at 35° C, When the material to be cultured is contaminated, chloramphenicol (0.05 mg per ml) should be added to Sabouraud’s agar (CM No. 105b). Nocardia is inhibited by these antibiotics, however. Cycloheximide agar (CM No. 105¢) is useful for the isolation of some pathogenic fungi from materials heavily contaminated with bacteria and sapro- phytic molds. On this medium most pathogenic fungi develop with their typical morphology and pigments and usually can be identified 726 FUNGUS INFECTIONS without subculturing to other media. It should be remembered, how- ever, that cycloheximide agar cannot be used to isolate Cryptococcus neoformans, Allescheria boydii and Aspergillus fumigatus, since these pathogens are inhibited by its fungistatic action. Materials from chromoblastomycosis should be cultured on Sab- ouraud’s agar at 30° C or room temperature. With the exception of Candida species, Sporotrichum schencki, Cryptococcus neoformans, Coccidioides immitis, and Absidia, Rhizo- pus and other Phycomycetes, the pathogenic fungi develop slowly in culture. All cultures should be held for at least 3 weeks before dis- carding. 5. Examination of Cultures Make microscopical preparations of molds and yeast-like cultures as previously given—in 10 per cent sodium hydroxide with or without Parker’s ink, in water or in lactophenol-cotton blue. Most fungi may be handled safely using ordinary sterile technics and taking the usual precautions. Coccidioides immitis, however, which becomes very powdery with the development of arthrospores in old cultures, should be handled only under a hood and with extreme caution. Whenever possible only young cultures should be examined; old petri dish cul- tures should not be opened. Arthrospores do not begin to develop in most strains until the cul- ture is 8 or 10 days old, at which time cultures can be opened with comparative safety. When old cultures must be opened for transfer, this should be done under a hood, preferably one which is closed. Transfers may be made by picking up on the sterile needle a tiny block of agar from the sterile tube and touching the latter gently to the edge of the old culture. The mycelium in an old culture of Coccidioides can be moistened by injecting slowly through the flamed cotton stopper sterile salt solution to which has been added a loopful of Tween &0. Do not pour or pipette salt solution into the open mouth of the tube (especially if the face of the slant is upward) because this procedure releases a cloud of infectious spores. 6. Key to Identification of Fungi by Examination of Cultures A. Culture bacteria-like, some closely resembling pigmented, sapro- phytic, acid-fast bacteria: The Actinomycetes. 1) Culture anaerobic, colonies raised, smooth or rough and granu- lar, grayish to yellowish on streaked plates of brain-heart in- fusion agar at 35° C under 5 per cent COs; bacteria-like, 1-3 mm in diameter in 4-6 days, with delicate hyphae 1 1 or less in FUNGUS INFECTIONS 727 2) 2) 3) 4) diameter ; these form radially oriented, branching, Gram-positive hyphae which fragment into diphtheroid Gram-positive ele- ments!*—Fig 6(a) : Actinomyces israelii, A. bovis. Culture aerobic, colonies covered with short, white aerial hyphae or wrinkled, granular, glabrous, and yellow to orange in color on Sabouraud’s agar; colony slow-growing, with delicate hyphae 1 p or less in diameter, which may fragment into bacillary and short, branching Gram-positive or partially acid-fast forms— Fig 6(b) : Necardia asteroides.* Culture aerobic, yeast-like at both 35° C and room temperature. Colony cream-colored, pasty, with distinct yeast-like odor; colony growth rapid, with small, oval, thin-walled, budding cells 2-3X4-8 pn on the surface of the medium and pseudomy- celium on the surface and penetrating the agar, especially corn- meal agar—Fig 6(c) (d) : Candida spp.T Colony white to yellowish, pasty, with yeasty odor, budding cells, no hyphae on cornmeal agar: Torulopsis spp. Colony white to cream-colored, with dry, mealy surface; colony growth rapid, with septate hyphae which by septation become numerous rectangular, barrel-shaped and rounded cells 4-12 pu in diameter—Fig 6(g) (h) : Geotrichum sp. Colony cream-colored to light tan, mucoid, glistening and with- out odor; growth rapid, with large, round, thick-walled budding cells 5-15 p in diameter, attachment of bud to parent cell narrow, with extracellular polysaccharide capsules. Growth at 35° C and virulent for mice—Fig 6(e) (f) : Cryptococcus neoformans. Culture yeast-like at 35° C, but filamentous at room temperature. Colony yeast-like, smooth, white to cream-colored, and moist at 35° C on glucose blood agar slants or Francis’ cystine blood agar slants; growth rapid, with small, oval, thin-walled, uni- nuclear budding cells 2X4 p in size. On Sabouraud’s agar at room temperature or at 30° C, culture filamentous, cottony and white at first, then buff to brown; growth slow, with septate hyphae which bear, on lateral conidiophores, rough-walled microconidia 2-3 p in diameter, and numerous spherical (oc- casionally pyriform), thick-walled macroconidia 8-20 p in diameter and covered with finger-like appendages. Fungus con- * For identification of Nocardia species, see Table 1. + For identification of Candida species, see Table 2. 728 FUNGUS INFECTIONS Figure 6-—(a) Actinomyces israelii colony on beef infusion glucose agar, pH 7.8, 5 days. (b) Nocardia asteroides colony on Sabouraud’s agar, 14 days. (c) Candida albicans colony on Sabouraud’s agar, 20 days. See Bac- terial and Mycotic Infections of Man. Philadelphia: Lippincott, 1948, p. 601, Fig 51. (d) Candida albicans from Sabouraud’s agar. See Am. J. Med. 2:601 (1948), Fig 6. (e) Cryptococcus neoformans colony on Sabouraud’s agar, 12 days. (f) Cryptococcus neoformans from Sabouraud’s agar. (g) Geotrichum candidum colony on Sabouraud’s agar, 10 days. See Zinsser’s Textbook of Bacteriology. New York: Appleton- Century-Crofts, 1948, p. 866, Fig 204. (h) Geotrichum candidum from Sabouraud’s agar. Refer to Am. J. Med. 2:604 (1947), Fig 11. (i) Hastoplasma capsulatum colony on Sabouraud’s agar, 12 days. See Bacterial and Mycotic Infections of Man. Philadelphia: Lippincott, 1948, p. 612, Fig 62. (j) Histoplasma capsulatum from potato glucose agar. Refer to Pub. Health Rep. 63:176 (1948), Fig 3. (k) Blastomyces dermatitidis colony on Sabouraud’s agar, 12 days. (1) Blastomyces dermatitidis from Sabouraud’s agar. Refer to Manual of Clinical Mycology. Philadelphia: Saunders, 1944, p. 39, Fig 22b. (m) Sporotrichum schenckii colony on Sabouraud’s agar, 12 days. (n) Sporotrichum schenckii from Sabouraud’s agar. Refer to Am. J. Med. 2:602 (1947), Fig 7a. (0) Coccidioides immitis colony on Sabouraud’s agar, 7 days. See Zinsser’s Textbook of Bacteriology. New York: Appleton- Century-Crofts, 1948, p. 859, Fig 197. (p) Coccidioides immitis from Sabouraud’s agar, Refer to Zinsser’s Textbook of Bacteriology. New York: Appleton-Century- Crofts, 1948, p. 859, Fig 198. (q) Allescheria boydii (Monosporium apiospermum) colony on Sa- bouraud’s agar, 15 days. See Zinsser’s Textbook of Bac- teriology. New York: Appleton-Century-Crofts, 1948, p. 880, Fig 218. (r) Allescheria boydit (Monosporium apiospermum) from Sa- bouraud’s agar. Refer to Manual of Clinical Mycology. Philadelphia: Saunders, 1944, p. 187, Fig 89b. (s) Phialophora pedrosoi colony on Sabouraud’s agar, 20 days. (t) Phialophora pedrosoi from Sabouraud’s agar. Refer to Manual of Clinical Mycology. Philadelphia: Saunders, 1944, p. 100, Figs 52b and 52d. (u) Phialophora compacta colony on Sabouraud’s agar, 20 days. (v) Phialophora compacta from Sabouraud’s agar. Refer to Manual of Clinical Mycology. Philadelphia: Saunders, 1944, p. 104, Fig 54b. (w) Phialophora verrucosa colony on Sabouraud’s agar, 20 days. See Zinsser’s Textbook of Bacteriology. New York: Appleton- Century-Crofts, p. 877, Fig 215. (x) Phialophora verrucosa from Sabouraud’s agar. 729 INFECTIONS FUNGUS PHOTO COURTESY DUKE UNIVERSITY MEDICINE OF SCHOOL Table 1—Characteristics of Five Aerobic Actinomycetes Nocardia Nocardia Streptomyces Streptomyces Streptomyces Characteristics asteroides brasiliensis madurae pelletieri somaliensis Tissue: Granules —* + (yellow) + (yellowish + (red) + (yellow to white to red) black) “Clubs” — rare + — — Partially acid-fast + + x — — Culture: Color Glabrous and Yellow to yellow- White to red Rose to red White, becoming Utilization of : Paraffin} Urea Mannitol Xylose Decomposition of casein Hydrolysis of starch Experimental patho- genicity orange, or with short white aerial hyphae orange +1 +++ 4 or + or — +++ I+ 11+] brown or black * Soft, poorly organized mycelial aggregates have been reported in rare cases. Reports of N. asteroides from mycetoma may he due to failure to differ- entiate N. asteroides from a similar species. + Coat glass rods with sterile paraffin and place aseptically in tubes of broth which lacks all other carbon sources. + There are discrepancies between the observations of Mariat!! and those of Gordon and Mihm.12 Mariat reported that N. asteroides utilizes mannitol and decomposes casein but not starch. 0€L SNONNH SNOILO3IANI FUNGUS INFECTIONS Table 2—Characteristics of Species of Candida* 731 C. pseudo- Medium C. albicans C. tropicalis tropicalis C. kruset Sabouraud’s agar Creamy, bubble Not characteris- Not character- Flat, dry craters, my- tic istic celial fringe Sabouraud’s broth No surface Surface film No surface Surface film, growth with bubbles, growth wide band on narrow band tube wall on tube wall Blood agar Medium-sized, Large gray Colonies small, Colonies small, dull gray colonies sur- not character- irregularly colonies rounded by istic shaped, flat mycelial or heaped fringe Cornmeal agar Branched my- Mycelium well Mycelium “Crossed sticks” celium with developed, poorly de- mycelium dense clusters bearing veloped of blasto- numerous spores, chla- scattered mydospores blastospores Glucose AG AG AG AG Maltose AG AG — — Sucrose A AG AG — Lactose — RA AG oe Medium C. parapsilosis C. stellatoidea C. guilliermondii Sabouraud’s agar Creamy Creamy Creamy Sabouraud’s broth Blood agar Cornmeal agar Glucose Maltose Sucrose Lactose No surface growth Colonies small, brilliant white Mycelium well de- veloped, abundant delicate branches AGT No surface growth Colonies star-shaped Mycelium with large ball-like clusters of blastospores, rare chlamydo- spores AG AG No surface growth Medium-sized dull gray colonies Mycelium well developed * Adapted from Martin, Jones, Yao, and Lee.13 + Occasionally acid only. i Langeron and Guerral* report acid and gas produced in glucose and sucrose when cultured at 25° C and held 20 days. 732 2) 3) 4) 1) FUNGUS INFECTIONS verted from mycelial to yeast form by transfer to blood agar and incubation at 35° C or by animal passage—Fig 6(i)(j): Histoplasma capsulatum. Colony yeast-like, wrinkled, waxy and gray to yellowish brown on blood agar slants at 35° C; growth rapid, with large, thick- walled, round, multinuclear budding cells 5-20 p in diameter; attachment of bud to parent cell, wide. Colony filamentous, cottony and white at first, then buff to brown on Sabouraud’s glucose agar at room temperature; growth slow with septate hyphae, numerous small, smooth, spherical to pyriform lateral conidia 2-8 pu in size. Fungus converted from mycelial to yeast form by transfer to blood agar and incubation at 35° C or by animal passage—Fig 6(k) (1) : Blastomyces dermatitidis. Colony yeast-like, wrinkled, waxy, and white to gray on blood agar slants at 35° C; growth rapid, with large, thick-walled, round, multinuclear, multiple budding cells 6-30 p in diameter; attachment of bud to parent cell, narrow. Colony filamentous, small and heaped, with short nap of aerial mycelium on Sabouraud’s agar at room temperature; growth slow, with septate hyphae, many chlamydospores and few if any conidia. Fungus converted from mycelial to yeast form by transfer to blood or other agar and incubation at 35° C or by animal pas- sage: Paracoccidioides brasiliensis (Blastomyces brasiliensis). Colony yeast-like, soft, moist and cream-colored on Francis’ cystine blood agar at 35° C; growth rapid, with small, fusiform or oval budding cells 1-4X3-6 p. On Sabouraud’s agar at room temperature, colony filamentous, smooth, leathery, lacking cottony aerial mycelium, dark brown or black but losing pig- ment by sectorial mutation after repeated subculture. Growth rapid, with delicate, septate hyphae 1-1.5 p in diameter and small, pyriform conidia 2-3X3-4 p borne in clusters on the ends of lateral conidiophores and, in older cultures, on the sides of conidiophores and hyphae. Fungus converted from mycelial to yeast form by transfer to blood agar and incubation at 35° C— Fig 6(m) (n) ; Sporotrichum schenckii. . Culture filamentous at both 35° C and room temperature. Colony filamentous, cottony, white at first, then tan to brown on Sabouraud’s agar; growth rapid, sporulation in 7-14 days, with conidiophores and hyphae segmenting into numerous cylindrical or cask-shaped spores (arthrospores) 2.5-3X3-4 pn in size—Fig 6(0) (p) : Coccidioides immitis. FUNGUS INFECTIONS 733 2) Colony filamentous, cottony, white at first, then grayish on Sabouraud’s agar, septate hyphae and brownish ovoid to clavate conidia 5-8X8-10 u borne singly on the ends of conidiophores which may be aggregated into coremia. Most strains produce also ascospores in cleistothecia (closed ascocarps)—Fig 6(q) (vr): Allescheria boydii (Monosporium apiospermum,). 3) Colony filamentous, flat to dome-shaped, covered with short, felt-like aerial mycelium, dark olivaceous to brown in color on Sabouraud’s agar. Growth slow, with black to olivaceous septate hyphae and three spore types: ovoid and shield-shaped spores in loose, branching chains from the terminal cells of conidiophores (Hormodendrum type) ; spores from the sides of conidiophores (Acrotheca type) ; and, rarely, spores from the bottom of the cup-shaped tip of tubular or flask-shaped phialides (Phialophora type)—Fig 6(s) (t) : Phialophora (Hormodendrum) pedrosoi.t® 4) Colony filamentous, heaped, brittle, with very short coarse aerial mycelium, dark olivaceous to brown in color on Sabour- aud’s agar. Growth slow, with black to olivaceous septate hyphae and three spore types: subspherical spores in compact, branching chains from the ends of conidiophores (Hormoden- drum type); rarely, lateral conidia (Acrotheca type); and spores from the cup-shaped tip of phialides (Phialophora type) —Fig 6(u) (v) : Phialophora compacta. 5) Colony filamentous, flat to dome-shaped, covered with short, felt-like aerial mycelium, dark olivaceous to brown in color on Sabouraud’s agar ; growth slow, with black to olivaceous septate hyphae, thin-walled spores 1.5-4 pu, predominantly from phialides but, rarely, in terminal branching chains (Hormodendrum type) or laterally (Acrotheca type)—Fig 6(w) (x): Phialophora verrucosa. VI. FUNGI CAUSING PULMONARY INFECTIONS I. Collection of Specimens Obtain a morning specimen of sputum after the patient has washed or rinsed the mouth thoroughly with an antiseptic. Collect the sputum in a sterile petri dish or sterile 1-oz, wide-mouth, screw-cap jar for immediate transportation to the laboratory. The specimen should be examined and cultures made promptly. A 24 hr specimen is worthless for mycological study because of the rapid overgrowth of Candida and bacteria in a specimen held at room temperature. 734 FUNGUS INFECTIONS Send bronchoscopic material to the laboratory immediately, after wrapping the trap with sterile gauze. Divide tissue obtained by biopsy during bronchoscopy for microscopical examination and culture and for sections. Obtain pleural fluid with sterile needle and syringe, transfer to a sterile test tube. Collect pus from thoracic sinus tracts after the skin around the opening has been cleansed with iodine and washed with alcohol (70%) to remove surface contaminants. Allow the pus to run into a sterile test tube held against the skin at the sinus opening. Collect stomach contents by gastric lavage in the morning before the patient has eaten. Neutralize or send immediately in a suitable sterile container to the laboratory. 2. Examination of Specimens Gastric washing should be neutralized as promptly as possible after collection and before making cultures and inoculating animals. Ex- amine sputum, bronchoscopic ‘specimens and pus from draining thoracic sinuses by placing a cover glass over a small quantity of the material mixed with hydroxide on a microscope slide. Microscopical examination with low and high dry objectives and reduced light from the microscope condenser should reveal fungus cells if present. Spread sputum or pus from sinuses on slides and stain with Giemsa for Histoplasma or by Gram’s method for easier identification of the delicate, branching hyphae or diphtheroid elements of Actinomyces israelii, A. bovis and hyphae of Nocardia species. Stain all such ma- terials showing Gram-positive branching filaments of bacterial dimen- sion with a modified Ziehl-Neelsen stain in which the film is cautiously destained with 0.5 per cent aqueous sulfuric acid in order to demon- strate the partially acid-fast, branching filaments of Nocardia aster- oides. If cryptococcosis is suspected, mix a loopful of material with a drop of India ink diluted 1:1 with water for demonstration of the extracel- lular capsule of Cryptococcus neoformans. Centrifuge gastric washings and pleural fluids at high speed for 30 min and examine the sediment in fresh preparations for fungus cells and in stained films for Gram-positive or acid-fast branching filaments. Grind tissues obtained during bronchoscopy in sterile salt solution and examine for fungus cells. 3. Key to Fungi by Direct Examination of Specimens With the exception of Rhinosporidium seeberi, Phialophora ped- rosoi, Phialophora compacta and Phialophora verrucosa, all the fungi FUNGUS INFECTIONS 735 listed in Section V 4 preceding (fungi causing infection of mucous membranes, skin and subcutaneous tissues) may produce pulmonary infections. Therefore, the key to identification of fungi found in that section will serve to identify also the fungi found in pulmonary infections. 4. Culture of Specimens With the exception of the fungi mentioned above, the cultural tech- nics described for the isolation of fungi from mucous membranes, skin, and subcutaneous tissues (Section V 4 preceding) are applicable also to fungi causing pulmonary infections. However, it is important to culture sediment from centrifuged gastric lavage immediately. It also should be borne in mind that all of these materials except pleural fluid may be heavily contaminated with bacteria. Therefore, they should be cultured on media containing chloramphenicol or penicillin and streptomycin, as well as upon media without antibiotics. The anti- biotics inhibit growth of the actinomycetes. In culturing sputum, ulcers, feces or contaminated material, it is often useful, in addition to making direct cultures, to inoculate mice intraperitoneally with a suspension of the specimen. This is especially useful in the diagnosis of histoplasmosis. Penicillin and streptomycin at the level of about 200 units of each per ml of the specimen can be added when pneumococci or other bacteria pathogenic for mice are present. Kill the mice after 4 weeks and make cultures from liver, spleen and abscesses observed. Incubate cultures at room temperature or at 30° C. Incubation at 35° C decreases the probability of obtain- ing a primary isolation on even enriched media. However, some of the material from histoplasmosis, for example, can be incubated at both 30° C and 35° C in order to propagate the fungus in its tissue phase as well as in its mycelial phase. Blastomyces dermatitidis, how- ever, grows well at 35° C. An exception may be made also in the case of aspergillosis and mucormycosis. Aspergillus fumigatus, Rhizopus species, and Absidia corymbifera grow well at temperatures as high as 42° C, and incubation at 35° to 45° C may facilitate isolation of these pathogens from sputum contaminated by saprophytic molds. 5. Examination of Cultures Microscopical preparations are made as described for fungi from mucous membranes, skin, and subcutaneous tissues (Section V 2 preceding). 736 FUNGUS INFECTIONS 6. Key to Identification of Fungi by Examination of Cultures The key found in Section V 6 preceding is applicable to fungi iso- lated from pulmonary infections. VII. FUNGI CAUSING MENINGITIS I. Collection of Specimens Transfer cerebrospinal fluid obtained by lumbar puncture to a suit- able sterile container for transportation to the laboratory, or transfer 0.5 ml directly to each of several slants. In some cases it is necessary to culture 5-10 ml of cerebrospinal fluid in order to isolate the fungus. 2. Examination of Cerebrospinal Fluid Centrifuge spinal fluid at high speed for 30 min and examine the sediment as a fresh preparation. Such preparations are suitable for demonstrating Blastomyces dermatitidis, Coccidioides immitis, Can- dida albicans, and Cryptococcus neoformans, Occasionally, however, cells of Cryptococcus neoformans are mistaken for and are counted as lymphocytes in differential cell counts of spinal fluid. This may be avoided by mixing the centrifuged sediment with India ink to demon- strate the polysaccharide capsule of Cryptococcus neoformans. Stained films of the centrifuged sediment should reveal the Gram- positive filaments of Actinomyces israelii and the partially acid-fast filaments of Nocardia asteroides, if present. Organized granules of A. israelii are rarely if ever found in the spinal fluid. 3. Key to Fungi by Direct Examination The following fungi may be encountered in spinal fluid: Actin- omyces israeli, Nocardia asteroides, Coccidioides immitis, Histo- plasma capsulatum, Blastomyces dermatitidis, Paracoccidioides brasi- liensis, Cryptococcus neoformans and Candida albicans. C. neofor- mans (Torula histolytica—Cryptococcus hominis, etc.) is the most fre- quent cause of mycotic meningitis. Meningitis caused by this fungus resembles that caused by Mycobacterium tuberculosis and can be differentiated only by laboratory studies during which one or the other of these microorganisms can be demonstrated either directly or by culture. The fungi listed here may be identified by the key found in Sec- tion V3 preceding. 4. Culture of Specimens The fungi mentioned above may be grown in culture from spinal fluid using the technics described in Section V 4 preceding. FUNGUS INFECTIONS 737 5. Examination of Cultures Microscopical preparations are made as described for fungi from mucous membranes, skin and subcutaneous tissues in Section V 5 preceding. 6. Key to Identification of Fungi by Examination of Cultures The key in Section V 6 preceding is applicable to cultures obtained from spinal fluids. VIII. FUNGI RECOVERED FROM BLOOD OR BONE MARROW Although many fungi cause systemic diseases and undoubtedly metastasize by hematogenous spread, blood cultures are not usually highly productive. Mycotic endocarditis, caused by Candida, Histo- plasma or Actinomyces is an exception. I. Collection of Specimens Collect venous blood and sternal bone marrow in the usual way. 2. Examination of Specimens Spread blood or bone marrow on slides and stain with Giemsa’s stain or any preferred stain selective for fungi. 3. Key to Fungi by Direct Examination of Specimens See key in Section V 3 preceding. Histoplasma, if present, may be found in macrophages (rarely in polymorphonuclear cells). Histo- plasma cells are egg-shaped, with a cup-shaped protoplast (stained by Giemsa) near the larger end of the cell. Attached buds may be found. Leishmania may be differentiated from it by the presence of demon- strable nucleus and kinetoplast. The nucleus of Histoplasma may be found only under special conditions of preparation in which good fixation and careful processing minimize shrinkage and distortion. 4. Culture of Specimens Make cultures of blood or bone marrow by transferring directly from the syringe to the surface of several blood or Sabouraud’s agar slants (0.5 ml of blood or marrow per slant), and incubate at 30° C or at room temperature. Small, discrete, granular to fluffy colonies develop slowly and the cultures should be held for at least 3 weeks before discarding as negative. Transfer the fungus to Sabouraud’s agar slants to obtain better development of the typical and characteris- tic tuberculate macroconidia. 738 FUNGUS INFECTIONS 5, 6. Examination of Cultures See Section V 5 preceding. Key to Identification of Fungi by Examination of Cultures See Section V 6 preceding. Cursteir W. Emmons, Pu.D., Chapter Chairman Lisero AjeLrLo, PH.D. Ruopa W. BexuaMm, Pu.D. Norman F. Conant, Pu.D. HerBerT G. JouNstoNE, M.D., PH.D. Do~arp S. Martin, M.D., Dr.P.H. CuArLes E. Smita, M.D., D.P.H. REFERENCES 1 2 3 10. 1. 12, 13. 14. 15. Emmons, C. W., Binrorp, C. H., and Utz, J. P. Medical Mycology. Phila- delphia, Pa.: Lea & Febiger, 1963. CoNANT, N. F,, ¢t al. Manual of Clinical Mycology (2nd ed.). Philadelphia: Saunders, 1954. Lewis, G. M.; Hopper, M. E.; WiLson, J. W.; and PrLunkert, O. A. An Introduction to Medical Mycology (4th ed.). Chicago: Yearbook Publishers, 1958. Lirrman, M. L,, and ZimmerMAN, L. E. Cryptococcosis. New York: Grune & Stratton, 1956. Fiese, M. J. Coccidioidomycosis. Springfield, Tll.: Charles C Thomas, 1958. WiLson, J. W. Clinical and Immunologic Aspects of Fungous Diseases. Springfield, II. : Charles C Thomas, 1957. Division of Biologics Standards, N.I.H. Biological Products. Pub. Health Svce. Pub. No. 50. Fed. Register Aug. 7, 1959. Georg, L. K. Dermatophytes: New Methods in Classification. Atlanta, Ga.: U. S. Dept. Health, Education, and Welfare, 1958. Emmons, C. W. Dermatophytes: Natural Grouping Based on the Form of Spores and Accessory Organs. Arch. Dermat. u. Syph. Orig. 30:337-362, 1934. Rosepury, T., Epps, L. J, and CrLark, A. R. A Study of the Isolation, Cultivation and Pathogenicity of Actinomyces israelii Recovered from the Human Mouth and from Actinomycosis of Man. J. Infect. Dis. 74:131-149, 1944. MariaT, F. Physiologie des Actinomycetes Aerobies Pathogenes. Myco- pathologia et Mycol. Appl. 9:111-149, 1958. GorooN, R. E., and M1uwm, J. M. A Comparison of Nocardia asteroides and Nocardia brasiliensis. J. Gen. Microbiol. 20:129-135, 1959. Martin, D. S.; Jones, C. P.; Yao, K. F.; and Leg, L. E. A Practical Classification of the Monilias. J. Bact. 34:99-129, 1937. LanceroN, M. and Guerra, P. Nouvelles Recherches de Zymologie Medicale. Ann. de parasit. hum. comp. 16:36-179, 4290-525, 1938. Binrorn, C. H, Hess, G., and Emmons, C. W. Chromoblastomycosis. Report of a Case from Continental United States and Discussion of the Classification of the Causative Fungus. Arch. Dermat. u. Syph. 49 :398-402, 1944. CHAPTER 25 MALARIA I. Life Cycle A. Sexual Phase—Sporogony (in the Mosquito) B. Asexual Cycles 1. Exoerythrocytic Schizogony (Tissue Phases) 2. Erythrocytic Schizogony (Peripheral Blood Phase) II. Collection and Handling of Specimens A. Slides B. Blood Films 1. Types of Blood Films The Thin Film The Thick Film Making Blood Films Shipping Blood Films Malaria Survey Films Staining Blood Films Making Permanent Preparations Using Diaphane Sui iN III. Microscopical Examination of the Blood Film A. Equipment B. Identification of Species 1. In the Thin Film 2. In the Thick Film 3. Sources of Confusion or Error in Stained Films C. Enumeration of Parasites 1V. Reporting Results of Examination V. Comments References Malaria, once a very important economic and health problem in the United States, particularly in the southeastern section, has disappeared from this country as an endemic disease. During recent years the return of servicemen and the arrival of travelers from malarious areas of the world have been responsible for most of the positive diagnoses. The use of new drugs, which prevent relapses? of treated cases, makes the reappearance of the disease most unlikely. Notwithstanding the scarcity in numbers of cases, the technician should be prepared to take specimens properly, to stain and examine them with assurance and dispatch. 739 740 MALARIA The parasites which cause malarial fevers in man all belong to the Phylum Protozoa, Class Sporozoa, Genus Plasmodium. There are four recognized species of the parasites (colloquial names follow in parenthesis) : (1) Plasmodium vivax (benign tertian, simple tertian malaria) ; (2) Plasmodium malariae (quartan malaria) ; (3) Plasmo- dium falciparum (estivo-autumnal, malignant tertian, subtertian, tropical, pernicious malaria) ; and (4) Plasmodium ovale (ovale ma- laria). I. LIFE CYCLE A. Sexual Phase—Sporogony (in the Mosquito) In each of the species there is a sexual phase passed in the female anopheline mosquito, the process of development being termed “sporogony.” This phase begins after the mosquito ingests infected human blood containing, among the other parasites, some mature male and female forms of the parasite (gametocytes). Gametocytes give rise to gametes which, after sexual union within the mosquito’s stomach, produce a series of developmental stages, terminating (after 10 to 21 days) in sporozoites. These forms of the malaria parasite are injected into another human being by the mosquito when it takes another blood meal. B. Asexual Cycles 1. Exoerythrocytic schizogony (tissue phases)—The sporo- zoites disappear within half an hour from the human blood® and parasites do not appear again in the blood for several days. In P. vivax this prepatent period is about 10 to 12 days; in P. falciparum, about 8 to 12 days; in P. malariae, about 21 days. During this period the following occurs: A few days after the sporozoites are introduced into the body there can be found in the parenchymal cells of the liver 4% a series of stages of parasite growth called pre-erythrocytic forms (primary tissue phase). From the mature pre-erythrocytic stage, young forms erupt which give rise to two cycles in man—one in the parenchymal cells of the liver called exoerythrocytic schizogony (secondary tissue phase), the other in the red blood cells, erythrocytic schizogony. It is only under experimental conditions that any of the tissue stages in man have been found, so that they are only of general interest to most technicians. In some species of malaria the exoerythrocytic stages in the liver may act as a source of parasites in relapses of the disease.” The erythrocytic cycle in the circulating blood is responsi- MALARIA 741 ble for the familiar symptoms of the disease and produces the stages with which the technician is concerned. 2. Erythrocytic schizogony (peripheral blood phase)—Each malaria parasite consists of a nuclear mass called chromatin and an area of cytoplasm; with the recommended stains, the former appears red or purplish red, the latter blue. The asexual forms in the blood are divided into trophozoites, schizonts, and mature schizonts. The term ‘“‘trophozoite” covers all the earlier, growing erythrocytic forms, from the youngest stage (called a “ring” because of its appear- ance) up through the large stage, where vegetative growth is complete (but the chromatin is as yet undivided). The growth of the tropho- zoite is gradual and consumes the greater portion of the life cycle. The parasite during this stage assumes many shapes, particularly in P. vivax, and ingests the hemoglobin of the host cell, which it metabo- lizes into granules of hematin, yellow-brown to black in color, depend- ing on species and stage. These granules, called “malarial pigment,” are dispersed throughout the cytoplasm of the trophozoite. The amount of pigment and size of the granules increase with the growth of the trophozoite, hence pigment can be used as an indication of age of the parasite; pigment is important, too, in the differentiation of species. Following the trophozoites are the schizonts, large forms in which the chromatin shows evidence of schizogonic division. There may be such forms with two, four, or more chromatin masses, but division is not complete. As the division progresses, the cytoplasm shows progressive separation, and in P. vivax and P. ovale the pigment ex- hibits a tendency to accumulate into fewer and fewer masses. In P. malariae the pigment clumps late in the schizont stage. The mature schizont is that stage in which division of both chroma- tin and cytoplasm is complete, forming within the red blood cell small individual new rings, or merozoites, the next generation of parasites. At this stage the pigment is usually clumped into one or two masses. The pigment of P. falciparum differs in that complete clumping is accomplished as early as the late-growing trophozoite stage and con- tinues throughout the rest of the life of the parasite. This is at times a valuable diagnostic clue. Shortly after division is completed, the mature schizont bursts the red blood corpuscle, the merozoites are liberated into the bloodstream along with the pigment, a possible residue of cytoplasm, and perhaps toxic materials produced by the parasite. It is just after this simulta- neous multiple red blood cell destruction that the subject is likely to experience the paroxysm, or the chill-followed-by-fever characteristic 742 MALARIA of malaria. The pigment and the residual cytoplasm are phagocytized by the monocytes, sometimes by the neutrophils. Not all the mero- zoites survive after the bursting of the cell, but those which do are found shortly thereafter in newly parasitized red blood cells, where they start again the erythrocytic asexual cycle. As the disease con- tinues, the number of parasites is increased by geometrical progres- sion, until, frequently, enormous numbers develop. More than one parasite may attack a single cell. Circulating in the blood with the asexual forms at times may be found the sexual forms: macrogametocyte (female) and micro- gametocyte (male). Whether these forms develop from certain mero- zoites of the erythrocytic schizonts or from some of the merozoites of the exoerythrocytic phase is not settled. Growth of the gametocytes of all the species takes place almost entirely in the internal organs, only an occasional immature one being seen in the peripheral blood. The gametocyte remains within the membrane of the red cell for the period of its life in the blood of man, which is thought to be only a few days; it degenerates and dies unless taken up by the mosquito. P. vivax and P. malariae gametocytes occur, in relatively fewer num- bers, along with the asexual forms. Those of P. vivax usually appear about 4 or 5 days after the first erythrocytic forms, and those of P. malariae occur later in the attack. The parasites of P. falciparum occur in waves, the first gametocyte wave about 7 to 10 days after the first trophozoite wave. The first trophozoite wave recedes as the gametocyte wave rises. Il. COLLECTION AND HANDLING OF SPECIMENS The microscopical detection of malaria parasites in stained thin and thick blood films is at this time the most reliable and accurate method of diagnosis of malaria. or easier and more dependable work certain rules which govern slides, blood films and staining must be observed. A. Slides Meticulously clean, unscratched slides are essential. Any acid or alkali on slides will interfere with the staining. Thin films will not spread evenly on an oily surface and thick films will not adhere to it in staining. Scratches can take the stain and can be misleading in diagnosis, so that new slides are preferable, although these must be washed to remove traces of alkali or oil left from the polishing process of manufacture. MALARIA 743 Previously used or very dirty slides: Drop slides one at a time into dichromate cleaning solution and let stand 8 hr or longer. Remove and let tap water run over them for several hours until the acid is thoroughly washed away, shifting their position occasionally. Continue the procedure with directions given below for new slides. New slides: Wash individual surfaces of the slides with a soft brush or cloth in warm water containing Alconox* (1 tablespoonful to 1 gal warm tap water). Rinse thoroughly in warm running tap water or in distilled water if tap water contains excessive chemicals. Let drain briefly on a clean towel. Place in 95 per cent ethyl alcohol. Dry and shine with a soft lintless cloth. (During the entire process avoid chipping edges of slides.) Protecting clean slides: Make slides into dustproof packages by rolling slide over slide in roll toilet tissue or by making packages of a few slides rolled in squares of plain white paper. B. Blood Films I. Types of blood films Two types of blood films are used—the thin film and the thick film. Although it is convenient to have them both on the same slide, better details can be obtained from films made on separate slides and stained separately. The thin film, generally employed for blood cell study, is familiar to most laboratory workers. It is sufficient for diagnosis of malaria when parasite density is high but usually fails entirely to reveal parasites when density is low. It is particularly suitable for learning morphological details of stages and species of malaria para- sites before attempting to study them in the thick film; for making differential leukocyte counts; and for otherwise studying the ac- companying blood picture (end of Section ITI B 1 following). The thick film, once not so well known, is now in general use. Through the period covering and following World Wars I and TI its superiority in the diagnosis of malaria was so definitely established that any well-informed technician should be unwilling to remain ignorant of its possibilities and its use. The thick film is a method by which a relatively large quantity of blood is placed in a small area and stained so that the hemoglobin is dissolved from the red cells and the blood film rendered sufficiently * Standard Scientific Supply Corp., 33 West Fourth Street, New York, N. Y. 744 MALARIA transparent for examination by transmitted light. The number of blood cells in a single microscopic field is enormously increased by the thick film and the increase in efficiency in showing parasites has been variously reported to be 3 to 22 times as great as in the thin film, depending upon thickness of the film, density of the parasites in the blood, and accuracy of the technician. It has been reported that symptoms of malaria are produced by as few as one parasite in 100,000 red corpuscles, a number which can hardly be scanned in a thin film in less than 30 min. If a technician contrasts the number of parasites found in the thin film with the number found in the thick film, in a given time, usually nothing further is necessary to convert him to the use of the thick film for diagnosis. On a positive thick film 100 microscopic fields usually but not always reveal parasites when they are scanty. Tf anything suggestive of parasitism is seen, the number of fields should be doubled or tripled. In nearly all patients who show active clinical symptoms of malaria, parasites will be found in thick films unless they have been reduced to a microscopically undetectable level by antimalarial drugs. How- ever, in persons of extreme susceptibility or in some cases of rapidly developing P. falciparum infections, symptoms may occur before para- sites can be found, so that subsequent films on successive days should be examined. The thick film has a number of important uses : —For diagnosis of malaria infections where parasites are sparse or scanty in number, as in new or chronic cases —For greater confidence in negative diagnoses, where the small amount of blood in the thin film gives no information —For parasite enumeration, when used with the white blood count taken at the same time (Section III C of this chapter following) —For large survey examinations of population groups (only one slide per person is made and many slides can be processed together) —For more rapid detection of white cells containing malarial pigment —TFor percentage estimate of eosinophils —TFor rapid detection of other organisms (filaria, trypanosomes, and spirochetes of relapsing fever) 2. Making blood films The first film on a malaria suspect is, for obvious reasons, fre- quently made during or immediately following the paroxysm. If positive diagnosis cannot be established at this time, subsequent films should be made at 8 to 12 hr intervals rather than wait for the next peak in the fever cycle, since such delay may be dangerous for the MALARIA 745 patient. Quite frequently species differentiation is impossible on films made at the time of the paroxysm because the parasites are nearly always in the young “ring” stage, with no differentiating characteris- tics. The films taken at 8 to 12 hr intervals will often reveal stages of development by which species can be determined. If it is feasible to hold off treatment for this period, there will be no danger of driving the parasites from the peripheral blood before species diagnosis can be made. Preparations for making the film: Clean well the skin of the finger (or other member) to be pricked, with gauze or cotton moistened in 70 per cent ethyl alcohol. Dry the skin to prevent the blood from spreading over a wide area. Puncture the finger with a quick, controlled motion, using an individual, heat-sterilized instru- ment (see Chapter 1, “General Procedures”). The puncture should allow the blood to well up into rounded droplets under gentle pressure. Making the thin film: Handle the clean slide by the edges to avoid getting oil from the fingers on the slide. Near one end of the slide obtain a small drop of blood, which can be spread thinly over about half the length of the slide, leaving room for the thick film on the opposite end. Have the angle between spreader slide and blood drop about 30° so that cells are spread separately, not overlapped or massed. With a spreader slide slightly narrower than that on which the blood is placed, draw the film out to a feather edge. Do not stop short to save space for the thick film, thus piling up the cells. A very thin film is most important. Malaria parasites are most frequently found in the thin film at the thin edge. Making the thick film: Wipe the finger dry again. Gently press out a large, rotund drop of blood. If both films are spread on one slide, grasp the sides of the thin-film end of the slide (thin film down) and touch the opposite end of the slide to the crest of the drop of blood (never touching the patient’s finger). Rotate the slide with a circular motion in the crest of the blood drop until a circular film of blood about the size of a dime is obtained; or take several droplets and puddle these with the corner of a clean slide into a film of the same size. Stop the spreading motion with the contact in the center of the circle of blood. This gives the film a thin edge and thicker middle. If the thick film is made on a separate slide, place it about a quarter in. from the end of the slide so that it is easily stained with a small amount of stain but is not so near the end of the slide that the mechanical stage prevents examination of the entire film. 746 MALARIA Caution: Do not make the film so thick that it will crackle and peel off when dry. If newsprint can just be deciphered through the wet thick film when the slide is placed on a printed page, then the blood is the right thickness. Marking the slide: Identify the slide by writing a number or accepted character with a lead pencil or waterproof ink in the thicker end of the thin film, or with a red wax pencil or glass-marking pencil on the slide, away from the blood film. Drying the thick film: Lay the slide flat until dry and protect it from dust and insects (in a petri dish in the laboratory; in a closed slide box, supported in a level perpendicular position, outside the laboratory). Tilting the blood while wet will cause such an uneven distribution that portions may be too thick and so peel off when dry, or the film may not be sufficiently penetrated by stain in the later dehemoglobinization and staining process. Dust in the film will introduce confusing artifacts. To facilitate quick drying, the slide may be placed in an incubator at 35° C until no longer visibly moist, or it may be exposed to the warm air from an electric hand hair dryer held not too close to the wet films.® Do not apply excessive direct heat to any thick film, as this “fixes” the red blood cells and prevents dehemoglobinization. 3. Shipping blood films In instances where films must be shipped to the laboratory, wrap them as soon as they are thoroughly dry to protect them from dust, pack them in such a manner that they cannot be broken, and ship immediately. Thick films may be “fixed” by summer temperatures. Single slides or a few slides, when well packed, may be mailed in slide containers or mailing tubes such as those supplied by many state health departments. Quantities of slides may be mailed in slide boxes, the boxes well wrapped in corrugated paper. After putting slides in the box, strips of Scotch tape or adhesive tape are drawn taut across the edges of the slides and on over the ends of the box, before closing the top to keep the slides from moving and lessen the possibility of breakage. The slides may be wrapped when dry, slide over slide in roll toilet tissue; packages made in this way are secured with a rubber band or tape and placed in a corru- gated box well padded with packing material. Broken slides will mean no examination at all or an inadequate examination and, at best, will entail extra work in staining. MALARIA 747 Old films do not stain so easily or so well as fresh films and if they are too old, the hemoglobin will not be dissolved from the thick films. 4. Malaria survey films For preparing large quantities of films, such as survey slides, for mass staining see Wilcox? or the previous edition of this work.1? 5. Staining blood films a) Transfer of parasites between slides in staining: There is always a possibility that malaria parasites or flakes of malarial blood may be transferred from one slide to another in staining. The slide may be greasy ; the blood may be so thick that it flakes off or so fresh that it has not adhered well to the slide; the hematocrit of the blood may be low, which prevents close adhesion to the slide; or the slide may be handled too roughly in staining. To obviate the possibility of transfer, where malaria is suspected or known, stain the slides on individual patients separately from other blood slides. If quantities of blood films are to be stained, as in malaria surveys or research projects, add to the buffered water, before adding the stain, a 0.5 per cent concentration of Triton X-30,'* or 0.1 per cent of X-100 to reduce to a minimum the possibility of transferring parasites between slides. This additicn of Triton will also accentuate Schiiffner’s stippling and other detail in the thin and thick film. b) A short method of staining: The technician in a hospital, doctor’s office or clinic must stain and examine very fresh films within a short time after they are taken. Many quick methods of staining thick blood films have been devised and publicized. Some of them give excellent results in certain hands or under certain conditions but are not consistently good on all slides, at all times, and in all hands. The difference between a good and a poor stain is frequently tantamount to revealing or not revealing parasites, particularly when parasites are scanty. For this reason too much stress cannot be put upon details and a little extra time in staining is well spent since it assures greater accuracy in diagnosis of species and stage of parasite. A method of staining is given here which, though not by any means the quickest available method, gives unusually good, consistent results in a reasonably short time. It is particularly effective on freshly dried thick films—not as brilliant on older ones. Thin films, being fixed, do not stain as well by a quick method as by a longer method. * Rohm & Haas, Philadelphia, Pa. 748 MALARIA 1) If both films are on one slide, fix the thin film by dipping only that end of the slide into methyl alcohol or by letting the alcohol from a medicine dropper run over the thin film without touching the thick film (since fixing the red cells in the thick film must be avoided). 2) Stand the slides on the thin film end until the alcohol dries. 3) Dip the whole slide (thick and thin films) quickly in and out of methylene blue-phosphate mixture.* This preserves the contours of both white cells and parasites, which are otherwise rather ragged in a freshly made thick film. The mixture causes the fresh thick film to adhere more readily to the slide and gives greater brilliance to the colors of the parasites and cells in both thick and thin films than one finds when this step is omitted. t 4) Wash the slide by waving it gently a few seconds in a vessel containing distilled water buffered to pH 7.0-7.2 (see tabulation which follows). 5) Raising the slide from the water, touch the lower end briefly to a paper towel or piece of gauze to absorb excess moisture. 6) Place slide in a Coplin jar and cover the film with Giemsa stain diluted in proportion of 1:50 with distilled water of pH 7.0-7.2. Stain for 20 min. 7) Remove slide and place it thick-film end down in a Coplin jar containing enough distilled water (pH 7.0-7.2) to cover the entire slide. Immediately pour off all the water except enough to cover the thick film. Allow the thick film to stand in the water at the bottom of the jar for 4 or 5 min. 8) Remove. To hasten drying, stand slide on end on absorbent paper—in winter near a warm microscope lamp or radiator, in summer before a fan. Never blot thick films. Note: Always clean staining dishes after use. For maximum stain- ing qualities use Giemsa stain only once. c) A long method of staining: It is known that if thick films are allowed to dry for 24 hr, the blood films will be more firmly attached * Methylene blue-phosphate mixture: Methylene blue, 0.5 g; anhydrous disodium phosphate (Na2HPOs4), 1.5 g; sodium dihydrogen phosphate (NaH:2P0O4+.H:20), 0.5 g; distilled water, 400 ml; pour the quantity necessary to cover a slide into a Coplin jar. Even with the Coplin jar well covered the mixture loses much of its efficacy after about 5 days. Discard the used mixture and start with a fresh lot instead of trying to boost it by adding fresh stain to it. f+ Adapted from a technic published by Field in 194012 and one used by Walker.13,14 MALARIA 749 to the slides and in staining the breaking up of parasite and white cell material will be greatly reduced. When thick films are dried this long or longer for any reason, they will not as a rule stain well by a quick method. Extra time is essential to permit thorough penetration of the stain into the parasite and cell elements. The following stain- ing method is recommended for such slides. It can be used on fresh films, too, if the technician is very careful in handling the slides and is familiar with the distortion produced in the cells and parasites. The detail in the thin-film parasites will be far better than that obtained using the quicker staining method. (The thin film will not be good for differential cell count. It is recommended that a separate thin film be made and stained by an accepted method for this purpose.) 1) Fix the thin film only as in steps (1) and (2) under the shorter method of staining given above. 2) Completely immerse the slide for 45 min in a solution of 1 part Giemsa stain solution to 50 parts distilled water pH 7.0 to 7.2 (see tabulation which follows). 3) Remove slide from stain, dip the entire slide quickly in and out of distilled water pH 7.0 to 7.2, then immerse the thick film only for 3 min. 4) Follow directions as given in Paragraph (8) preceding under the shorter method of staining (Section 5 (b)). d) Buffer solutions: Blood films stained with water that is too acid will frequently show no malaria parasite cytoplasm, while those stained with water too alkaline will show little contrast between reds and blues, giving a muddy monotone. Water buffered to pH 7.0 or 7.2 for staining and washing blood films ensures that blue and red elements in the stain will be taken up in correct degree by the blood and the parasites for maximum color contrast and easier diagnosis. Prepare stock buffer solutions of disodium phosphate and either sodium or potassium acid phosphate in M/15 solutions by dissolving the salts in boiled distilled water as follows: Na,HPO, (anhydrous), 9.5 g per liter Either NaH,PO4H,0O, 9.2 g per liter or KH,POy, 9.07 g per liter Store the solutions in separate sterile glass-stoppered pyrex bottles. These precautions prevent the growth of molds or bacteria and thus make it possible to keep the stock solutions indefinitely. Use the 750 MALARIA following amounts of the stock solution to buffer the indicated amount of distilled water for staining: M/15 NaH,POy4 or M/15 Na,HPO, M/15 KH,PO, Distilled pH (ml) (ml) H.O (ml) 7.0 61.1 38.9 900 7.2 72.0 28.0 900 Test the pH of the buffered water and adjust if necessary. Bromthymol blue indicator solution and a set of bromthymol blue color standards are convenient for testing. Prepare buffered water fresh each week or keep it under the follow- ing conditions: Put the buffered water (prepared after boiling) into a large, sterile bottle or flask provided with a stopper and an inverted U-shaped outlet tube, the end of which reaches almost to the bottom inside the bottle. The other end should reach about to the shoulder of the bottle on the outside. To this shorter end attach a series of short sections of glass tubing joined by very short sections of rubber tubing. On the last of the rubber joints put a pinchcock. (As little rubber as possible is used because the pH of water standing in rubber is changed. If used infrequently, discard the water in the tube each time.) Over the surface of the buffered water, run a layer of mineral oil about 4 in. deep to keep out the air. The water would absorb carbon dioxide from the atmosphere and the pH would be changed otherwise. Covered with the oil, it will retain its pH indefinitely. Replenish the buffered water before it reaches the bottom of the inside glass tube. e) Stain: For staining blood films for malaria, use Giemsa stain®* Azure B type, which has been certified by the Biological Stain Commission. This can be bought in solution (specify “for malaria” in ordering), or it can be made from stain powder. When made ac- cording to the following directions the powder gives results superior to those with the ready-made solution : Stain DOWEL ivunsvisisovssmesvsss does wns oo Se samniins so sae stim 600 mg Acetone-free methyl alcohol, reagent quality ............ccovvivn.... 50 ml Glycerol reagent quality (from a freshly opened bottle) .............. 50 ml * National Aniline Div., Allied Chemical & Dye Corp. MALARIA . 751 Grind the powder in a mortar before weighing, and weigh. Grind powder with part of the measured glycerol in a chemically clean mortar, pour off the top third into a chemically clean flask, add more glycerol and grind again. Repeat until most of the powder has been mixed with the glycerol and the mixture poured into the flask. Scrape out the last of the glycerol and powder from the mortar with a chemically clean spatula. Stopper the flask with a cotton plug and cover the plug with a heavy paper cap bound with rubber bands. Place the flask in a water bath and let stand for 2 hr at 55° to 60° C. Shake gently at intervals of I; hr. Remove the mixture from the water bath, allow to come to room temperature, then add the measured methyl alcohol. The stain can be filtered and used immediately, but preferably let it stand about 2 weeks with intermittent shaking, then filter for use. Keep a small bottle of the stain separate for current use. Never put a wet or soiled pipette into the stain. 6. Making permanent preparations using diaphane When teaching material is to be preserved, the following technic will be valuable. Tt is better to cover slides before examining them with oil. If slides have been examined with oil, clean them thoroughly of all oil with xylol and let them dry. Grasp the tip of one end of the slide firmly between thumb and forefinger of the left hand, the free end of the slide pointing toward one’s right. With a medicine dropper or smooth glass rod place about three drops of diaphane* at the left end of the slide. With the side of the dropper or rod spread the diaphane across the width of the slide. Then with gentle, even pres- sure quickly push the diaphane ahead of the dropper or rod toward the opposite end of the slide. This will spread a thin, even film over the length of the slide. There should be no excess. Place the covered slides in a dust-free slide box and let dry for at least 24 hr before using. Diaphane solvent® may be used to wipe away any excess at the end of the slide, or for thinning the diaphane when it becomes viscous. Diaphane is neutral and should be kept that way by making sure that nothing acid or alkaline comes in contact with it. Use either Shillaber’st or Crown} immersion oil rather than cedar * Will Corporation, Rochester, N.Y. + Eimer & Amend, New York, N. Y.; or Fisher Scientific Co., Pittsburgh, Pa. + Techni-Products Co., 5 Woodette Place, Buffalo, N. Y. 752 MALARIA oil on slides covered with diaphane, since the xylol necessary to remove cedar oil will give the covered film a milky cast which makes it unfit for further use. The oils recommended may be gently absorbed with facial tissue. Do not scrub or rub the diaphaned surface with tissue. Ill. MICROSCOPICAL EXAMINATION OF THE BLOOD FILM A. Equipment A good microscope, preferably a binocular, and a microscope lamp giving sufficient blue-white light for the scope in use, are essential. Most substage lamps give enough light for a monocular but not enough for a binocular microscope. A Corning “Daylite” glass in a strong microscope lamp gives a pleasing light for the binocular. Use the flat side of the microscope mirror. Keep microscope lenses clean. Lens paper and a small camel’s hair brush may be used. The low power ocular, 5X, 6X or 7.5X, gives a sense of distance and hence less eye fatigue when used for long periods. The 10X or 12.5X oculars should be kept at hand for close study of questionable objects; with wide-field lens, they are splendid for use with glasses. The fluorite oil- immersion lens has better color correction than the achromatic lens and is worth the extra expense. Always use a mechanical stage when ex- amining for malaria. A mechanical ringer for marking any parasites found saves time and sometimes argument. B. Identification of the Species For proper diagnosis of malaria, in addition to demonstrating the plasmodia in the blood one should identify the species of plasmodia present. The best reasons for this statement are: the fact that P. falciparum, if untreated, is often severe and may terminate rapidly in death, making proper, immediate treatment imperative ; that P. vivax, unless treated with the proper drug, has a tendency to relapse months later ; and that P. malariae may remain latent over a period of many years and can cause an attack in the recipient of transfused blood from a donor with such a latent infection. The present use of blood banks makes it imperative that anyone who has ever had malaria know what species was involved if he anticipates giving blood at any future time. I. In the thin film Each of the species has distinctive morphology and only a careful study of this differential morphology of the species in the thin film, MALARIA 753 where outlines and details of cells and parasites are well preserved, will give the student accurate information on species differentiation. With this knowledge the less distinct appearances in thick films usually can be specifically interpreted. a) P. vivax (benign tertian malaria)—The youngest form of the malaria parasite is a ring shape consisting, in the stained film, of one red chromatin dot, possibly more, in or on a circle of blue cyto- plasm. In P. vivax the dot is rather heavy and the diameter of the ring about one-third that of the red cell. Not infrequently the vivax ring may have more than one chromatin dot. Rings may at times be found in polychromatophilic cells or in cells that are full of basophilic stippling. After a few hours Schiiffner’s dots may be demonstrated in some of the parasitized cells and are present in correctly stained films with all stages of the parasite after this period.* As the young ring form grows, it may continue to have a ring-like appearance with much thickened cytoplasm and enlarged chromatin mass, or, as is more likely, it may exhibit pseudopodial processes indicative of ameboid activity, a characteristic very pronounced in this species. After 5 or 6 hr there appear in the cytoplasm small yellowish brown, most often rod-like granules called pigment. These increase in number with the growth of the trophozoite. In young forms they frequently cannot be distinguished as separate granules but give a yellowish or tannish tinge to portions of the cytoplasm. The red cell infected with P. vivax enlarges as the parasite grows. The growing trophozoite may assume practically any shape within the enlarged cell, with tenuous cytoplasmic projections and one or several vacuoles. Meanwhile cytoplasm, chromatin and pigment increase in amounts. This stage of P. vivax is larger than in the other species. In 36 to 40 hr the parasite almost fills the enlarged cell. It has now completed its vegetative growth and prepares for asexual reproduc- tion. It draws in its pseudopodia, assumes a rather compact form, usually irregular in outline, with cytoplasm only slightly vacuolated or mottled as though unevenly massed. It still has a single nucleus. Due to the compactness, the staining of mature trophozoites may be quite dark. (See Table 1 following.) * Schiiffner’s dots are pink granules which appear evenly distributed through- out that part of the parasitized cell not occupied by the parasite. The dots are rather consistently fine and uniform in size. As the parasite grows, the dots often become more pronounced, and take a somewhat deeper stain. This stippling is peculiar to P. vivax and P. ovale, hence is of diagnostic value. Careful stain- ing is necessary to demonstrate the dots in the maximum number of infected cells and prolonged washing can obliterate them, Their exact nature is unknown. Table 1—Summary of Malaria Parasite Differentiation in Stained Thin Film Stage of Development Plasmodium vivax Plasmodium malariae Plasmodium falciparum Plasmodium ovale Infected cell Small trophozoite (early rings) Growing trophozo- ite Large trophozoite Larger than normal, pale, often bizarre in shape. Schuffner’s stippling very often present, Litiouadly beyond ring stage. Tultiple infection of erythro- cyte not uncommon. Ring about 1/3 diameter of red cell, with heavy chromatin dot and large cytoplasmic circle, possibly with fine pseudopodia. Same as small trophozoites, with gradual increase in amount of cytoplasm and chromatin. Often has tenuous pseudopo- dial processes and large va- cuoles. Small yellowish brown pigment rodlets in cytoplasm, number increasing with age of parasite. One abundant mass of chroma- tin. Loose, irregular or close, compact cytoplasm, with in- creased amounts of fine brown pigment. Parasite practically fills enlarged cell by end of 36 to 40 hr. About normal or slightly smaller, sometimes darker in early stages. Ziemann’s stippling rarely seen. Multiple infec- tion of erythrocyte rare. Ring form with heavy chromatin dot and cytoplasmic circle which is often smaller, thicker and heavier than that of P. vivax, but not always dis- tinguishable from it. Chromatin rounded or elongated, cytoplasm in a compact form, with little or no vacuole or in a narrow-band form across the cell. Round, dark brown pig- ment granules may have peri- pheral arrangement. One mass of chromatin, often elongated, frequently less defi- nite in outline than that of P. vivax. Cytoplasm dense, com- pact, with few irregularities of outlines; in rounded, oval or sometimes band shape. Round pigment granules, larger, darker than in P. vivax, with a great tendency toward eripheral arrange- ment. ills or almost fills normal cell. Normal in size. Multiple infec- tion of erythrocytes more fre- quent than in the other species. Maurer’s spots some- times seen with older rings and stages beyond (in over- stained films or when pH of water is on alkaline side). Ring 1/5 diameter of red cell, with small thread-like cyto- plasmic circle and one or two small chromatin dots (double chromatin dots more frequent than in other species). Mar- ginal and bridge forms are frequent. May fisopap in this stage from peripheral cir- culation and return to internal organs for development. This stage remains in the ring form but chromatin and cyto- plasm increase to the extent that in size the parasite re- sembles closely the small tro- phozoite of P. wivar. A few pigment granules give a yel- lowish tinge to the cytoplasm. This is usually the oldest asexual stage seen in peri- pheral circulation. Stage seldom seen in peripheral blood. Very small, usually solid, with one small mass of chromatin. Lightly staining, compact cytoplasm; with haze of pigment scattered through the cytoplasm or with very dark pigment collected in one small, dense block on clear cytoplasm. Somewhat larger than normal, often with fringed or irregu- lar edge and oval in shape. Schuffner’s stippling present even with many rings. Small, darker in color and more solid, as a rule, than those of P. falciparum. Schuffner’s stippling, with greater per- centage of ring forms than in P. vivar (Giemsa stain). Compact, with little vacuolation —resembles closely the same stage of P. malariae but is somewhat larger and is in enlarged, possibly oval cell, usually with Schuffner’s stip- pling. Pigment is lighter in color and less conspicuous than in P. malariae, similar to that of P. wivaxr. bSL VIIVIVIA Immature (preseg- menting) schizont Mature schizont (segmenter) Macrogametocyte Microgametocyte Length of asexual cycle Stages in periph- eral blood Remarks Chromatin divided into 2 or more masses. Cytoplasm shows varying degrees of separation into strands and particles. Pigment shows tendency to collect in parts of the parasite. 12 to 24 divisions or merozoites, each composed of a dot of chromatin and a small mass or circle of cytoplasm. The pigment is in one or two clumps. Parasite practically fills enlarged cell. Rounded or oval, usually regular in outline, Dark blue homo- geneous cytoplasm with no vacuoles, Small, compact, dark red, usually eccentric chroma- tin. Abundant brown pigment scattered through SYiileens. When grown, usually fills or nearly fills enlarged cell. Small amount of light blue, gray, pink or almost colorless cytoplasm containing large, diffuse mass of light red or pink chromatin—usually cen- trally placed, often surrounded by a vesicular area. Abundant dark pigment throughout cyto- plasm. When grown, about size of a normal cell. Usually circular in outline. 43 to 48 hr. All More stages of growth likely to be seen in one film than in other common species. Game- tocytes appear early in cycle. Same as P. vivax except that the parasite is smaller and shows fewer divisions of chro- matin, in irregular shapes and sizes, as it approaches segmen- tation. There is more delayed clumping of pigment. 6 to 12, usually 8 or 10 mero- zoites in a rosette or irregu- lar cluster. Practically flls normal-sized cell. Merozoites, being fewer in number, ap- pear more husky than those of P. vivax. Cytoplasm and chromatin same as vivax. Abundant round, dark brown pigment, granules coarser than in P. vivax. when grown, usually fills the normal sized cell. Outline cir- cular or ovoid. Frequently confused with mature tro- phozoites of same species. Same as vivax except smaller in size. hen grown, fills or almost fills normal-sized cell. 72 hr. All Parasites usually compact, with heavy pigment and hence ap- pear more intensely stained than those of other common species. ~~ Gametocytes rarer fos, in other species, appear ate. When found in peripheral blood this stage resembles the same stage of P. malariae but is smaller and the pigment is likely to be completely clumped in one small dark mass. oo to 24 or more merozoites, very small cempared to those of other species. Bocely found in peripheral blood. Fills about two-thirds of normal-sized cell. Crescentic or sausage-shaped) about 114 times diameter of erythrocyte in length, possibly longer and more slender than microgametocyte, ~~ Cytoplasm possibly a deeper blue than in microgametocyte. Usually a single dark red chromatin mass near center associated with concentrated aggregation of pigment, darker than in microgametocyte. Often cytoplasm is paler than in macrogametocyte—grayish blue or pink. Loose, diffuse light-staining granules or threads of chromatin scattered with numerous granules of pigment throughout central half or more of parasite. Para- site possibly broader, shorter and with more rounded ends than those of macrogameto- cyte. v 48 hr. Usually ring form trophozoites and gametocytes. Other stages rarely found except in severe cases. Parasites frequently more nu- merous than in other infec- tions. Unlike other species, growth of asexual forms, fol- lowing the ring stage, takes place in internal organs. Many of the infected cells are definitely of oval shape. Pic- ture is often that of a round parasite in center of an oval cell. Many cells with indefi- nite fringed outline, Pigment lighter in color and less coarse than in P. malariae, more like P. vivax. 6 to 12, usually 8 merozoites in rosette or irregular cluster around mass of pigment. Cell enlarged, possibly oval, con- tains Schuffner’s stippling. Smaller than in P. vivax. Distinguished from P. malariae by size of infected cells and by Schuffner’s stippling. Not easily differentiated from vivax, although somewhat smaller. Average, 49.5 hr. All Rarest of the species. Differ- entiation not possible in thick films except by those compar- ing it daily with other species. One-slide diagnosis Supossiils, because of its resemblance to aberrant forms of P. vivax. VIIVIVHA SSL 756 MALARIA The division of chromatin begins and the parasite becomes an immature schizont (presegmenter). First, there are two divisions, and then successive divisions until usually 12 to 24 segments are formed. At first, these are irregular in shape but as division ap- proaches completion the segments appear more regular in size and shape and rather small as compared to earlier appearances. Coinci- dentally with the dividing of the chromatin, the cytoplasm breaks up, and ultimately one portion of the cytoplasm surrounds or adheres to each compact dot of chromatin, forming individual parasites. This stage is the mature schizont (segmenter). The number of merozoites may vary within a single strain and it has been shown that they vary considerably among different strains, although 16 is a rather common number. There are usually fewer mature schizonts of P. vivax in the peripheral blood than any of the previous stages, for many of the parasites seem to disappear from the peripheral blood just before segmentation. The time required for the complete development of the P. vivax parasite from one paroxysm to the next varies with the strain and is usually under 48 hr.'® The majority of the parasites of a single brood attain maturity at the same time. Some may lag in development how- ever, or there may be two broods of parasites maturing on alternate succeeding days. Hence, there are often several stages of the parasite in the peripheral blood at the same time. Gametocytes of P. vivax are sometimes found with first parasites in the peripheral blood, but more frequently they appear when schizogony has continued through several generations!” The shape of the growing gametocyte changes very little from the compact merozoite form. This is said to be due to the lack of ameboid activity which characterizes the trophozoite. The mature microgametocyte is often about the size of a normal red cell; the macrogametocyte is often distinctly larger. Both are in enlarged cells usually accompanied by Schiiffner’s stippling, like the other older forms of P. vivax. There is considerable pigment in both, usually scattered rather evenly throughout the cytoplasm, though in the male there are at times accretions. The female possesses a densely blue-staining, homogeneous cytoplasm; its nucleus is comparatively small and compact, is stained a deep magenta, and is situated usually at the edge of the parasite. The smaller male form has less cytoplasm, which stains lightly—gray-blue, greenish blue, or even pink through- out. The nuclear system is larger, loose, light-staining, with a net- like distribution of chromatin, and is very often centrally placed. There may be in cither male or female a colorless or very lightly PLATES FOR CHAPTER 25—MALARIA Growth Stages of the Three Dominant Malaria Species Infecting Man Plate I—Plasmodium vivax 1. Normal-sized red cell with marginal ring-form trophozoite. 2. Young signet ring form of trophozoite in a macrocyte. 3. Slightly older ring-form trophozoite in red cell showing basophilic stippling. 4. Polychromatophilic red cell containing young tertian parasite with pseu- dopodia. 5. Ring-form trophozoite showing pigment in cytoplasm in an enlarged cell containing Schiiffner’s stippling.* 6-7. Very tenuous medium trophozoite forms. 8. Three ameboid trophozoites with fused cytoplasm. 9 and 11-13. Older amebiod trophozoites in process of development. 10. Two ameboid trophozoites in one cell. 14. Mature trophozoite. 15. Mature trophozoite with chromatin, apparently in process of division. 16-19. Schizonts showing progressive steps in division (presegmenting schiz- onts). 20. Mature schizont. 21-22. Developing gametocytes. 23. Mature microgametocyte. 24. Mature macrogametocyte. * Schuffner’s stippling does not appear in all cells containing the growing and older forms of P. vivax, as would be indicated by these illustrations, but it can be found with any stage from the fairly young ring form onward. INEZ DEMONET Plate II—Plasmodium malariae 1. Young ring-form trophozoite of quartan malaria. 2-4. Young trophozoite forms of the parasite, showing gradual increase of chromatin and cytoplasm. 5. Developing ring-form trophozoite showing pigment granule. 6. Early band-form trophozoite—elongated chromatin, some pigment apparent. 7-12. Some forms which the developing trophozoite of quartan may take. 13-14. Mature trophozoites—one a band form. 15-19. Phases in the development of the schizont (presegmenting schizonts). 20. Mature schizont. 21. Immature microgametocyte. 22. Immature macrogametocyte. 23. Mature microgametocyte. 24. Mature macrogametocyte. 24 INET DEMONET Plate I1I—Plasmodium falciparum 1. Very young ring-form trophozoite. 2. Double infection of single cell with young trophozoites, one a “marginal” form, the other “signet ring” form. 3-4. Young trophozoites showing double chromatin dots. 5-7. Developing trophozoite forms. 8. Three medium trophozoites in one cell. 9. Trophozoite showing pigment, in a cell containing Maurer’s spots. 10-11. Two trophozoites in each of two cells, showing variation of forms which parasites may assume. 12. Almost mature trophozoite showing haze of pigment throughout cyto- plasm. Maurer’s spots in the cell. 13. Estivo-autumnal slender forms. 14. Mature trophozoite showing clumped pigment. 15. Parasite in process of initial chromatin division. 16-19. Various phases of development of the schizont (presegmenting schizonts). 20. Mature schizont. 21-24. Successive forms in development of the gametocyte—usually not found in the peripheral circulation. 25. Immature macrogametocyte. 26. Mature macrogametocyte. 27. Immature microgametocyte. 28. Mature microgametocyte. MALARIA 757 colored vesicular area around the nuclear mass. This is said to be the nonstaining part of the nucleus. Particularly noticeable in some males, it may make their entire nuclear areas occupy a third to a half of the parasites—a much greater area than in any other stage. It is difficult at times to differentiate between the full-grown trophozoite and the macrogametocyte. These points can help: The cytoplasm of the trophozoite may be mottled or may contain vacuoles, while that of the female gametocyte is rather homogeneous and has no vacuoles. The outline of the trophozoite may be slightly irregular and may show indentations from previous ameboid activity, while the outline of the macrogametocyte is regularly circular or ovoid. The full-grown macrogametocyte is often larger than: the full-grown trophozoite and ‘its pigment frequently heavier. b) P. malariae (quartan malaria)—The young trophozoites or rings of P. malariae are about the size of or slightly smaller than those of P. vivax, although they sometimes seem to. have a broader circle of cytoplasm and a slightly heavier chromatin dot: Double chromatin dots are rare. The vacuole closes in partially or completely soon after the growth begins, making a compact-appearing parasite. The growing trophozoites of P. malariae may assume band forms which stretch across the red cell, but much more often they are angular, round or ovoid in outline, often without. vacuoles, . The chromatin mass may be rounded, elongate, or semicircular in shape. The pigment appears early in the growth, is usually a darker brown than that of the trophozoite of P. vivax, and the round individual granules are usually larger and more prominent than in P. vivax. The pigment is frequently peripheral in arrangement. - The cell parasitized with P. malariae does not enlarge—in fact, it may appear smaller than surrounding cells. The activity of the trophozoite is sluggish and its growth slow, so that one seldom finds the irregular, tenuous ameboid forms common in P. vivax. The malariae parasite spends nearly two- thirds of its 72 hr cycle in the trophozoite stages.!’® As the tropho- zoite develops, the band form may become wider and denser, the round or oval forms larger and more compact, with a greater amount of dark pigment. This dark pigment, illumined by the transmitted light of the microscope lamp, frequently gives a greenish effect to the dense cytoplasm. The mature trophozoite of P. malariae fills the red blood cell, which in this infection is normal in size, and it may be rounded, oval, or a wide band in shape, with rounded or elongated chromatin mass, dark compact cytoplasm, and much heavy, dark pigment. 758 MALARIA The divisions of chromatin in the immature schizonts are uneven in size and shape. Sometimes the dark red masses are difficult to dis- tinguish or to count in the dark compact cytoplasm with the heavy dark pigment. In the mature schizont of P. malariae there are from 6 to 12 merozoites (usually 8), sometimes arranged peripherally around the centrally clumped pigment (rosette formation) but more often in an irregular cluster which practically fills the normal-sized host cell. Individual merozoites seem a little bulkier than individuals of P. vivax. P. malariae has fewer gametocytes than the other two common species. These are frequently very difficult to find. The young gametocytes cannot be distinguished definitely from some of the com- pact growing trophozoites; and mature macrogametocytes are very difficult to differentiate from mature trophozoites, which may be numerous in the film. The definite presence of gametocytes fre- quently depends on identification of the microgametocyte with the large nucleus and light stain. The sexual differences and general appear- ance of the gametocytes of P. malariae are like those of P. vivax, except that they are smaller in normal-sized cells and have no Schiiffner’s stippling. They are spherical or oval in shape and the pigment is abundant, dark brown, usually in coarse, round granules. On occasions there have been demonstrated in Giemsa-stained cells containing P. malariae parasites certain pale pink dots which are round, irregular in size, less numerous and less distinct than are Schiiffner’s dots in P. vivax. These dots seem to be brought out by intensive staining or by a pH of about 7.5. They have been named “Ziemann’s stippling.” They are less developed than are the Maurer’s spots found in P. falciparum. c) P. falciparum (estivo-autumnal malaria)—The youngest trophozoites or rings of P. falciparum are usually much more deli- cate and much smaller than the equivalent stage of the other species of human malaria, being only about one-fifth the diameter of a normal red cell and having a fine thread-like line of cytoplasm and one or more small dots of chromatin. At this stage this small size and delicacy are leading diagnostic characteristics. A high percentage of double-chromatin dots and/or marginal (appliqué) ring forms (Plate III, Fig 2) may also be helpful in species diagnosis; as may be a high percentage of multiple infections of the host cell. None of these characteristics are confined to P. falciparum but are usually more numerous and noticeable in this species. A combination of any two of them in high percentages in a blood film, even when rings are not MALARIA 759 numerous, should suggest a falciparum infection. The cell containing the falciparum parasite does not enlarge. P. falciparum usually retains a ring formation through its growing trophozoite stage. The older ring differs from the very young one in the increased size and increased amount of cytoplasm and chromatin; also in the fact that traces of pigment, as occasional, minute dark granules in the cytoplasmic line or as a yellowish haze over part of the cytoplasm, are present. These parasites, which correspond in age to the large ameboid forms of P. vivax, may be confused, in films containing few parasites, with the younger ring forms of P. vivax, since they are about the same size. This is a pitfall in diagnosis. Because falciparum parasites do retain the form of rings during much of their trophozoite growth, because falciparum infections are fre- quently not synchronous, giving some stage of the ring in the blood at nearly any time, and because great numbers of parasites are pro- duced in P. falciparum infections, there are likely to be a greater num- ber of rings (of one or more ages) of this species in the peripheral blood at one time than in any of the other species. Also, unlike P. vivax or P. malariae, it is the tendency of P. falciparum parasites to disappear from the peripheral circulation in the ring stage and com- plete their asexual growth in the internal organs. Hence, usually only ring forms and/or gametocytes are found in the peripheral blood. The exact stage at which a brood of rings disappears from the circulation varies: Sometimes they disappear soon after they enter the red blood cells; at other times they remain until the rings are quite large and heavy. With the foregoing facts it may be stated that when only a large number of rings are seen and no older trophozoites or schizonts can be found, the infection is in all probability one of P. falciparum. The mature trophozoites and schizonts of falciparum are, as a rule, not found in the peripheral blood. However, in some heavy or in- tense infections rare ones may occur, usually along with large num- bers of ring forms. The old trophozoite consists of a small area of solid, light-staining, clear blue cytoplasm containing a granule of chromatin somewhat larger than in the ring and usually a dull blur or dense, almost black, block of clumped pigment. This clumping of pigment before complete schizogony takes place is peculiar to falci- parum malaria. Chromatin and pigment masses are frequently about the same size. The parasite at this stage is sometimes no larger or not much larger in circumference than a large ring. The immature and mature schizonts of P. falciparum resemble those of P. malariae somewhat, but the respective stages of P. falciparum 760 MALARIA are smaller and contain less pigment, which is clumped in a small, dark block, and even the oldest schizont does not fill the normal-sized host cell. The mature schizont contains 8 to 24 merozoites (usually around 20). These merozoites are quite small when compared with those of other species. The mature schizont of P. falciparum is the stage of malaria parasite least often seen. ‘Sometimes in red blood cells containing P. falciparum, particularly the older stages beginning with the very large, heavy ring, there may be found pink-staining dots, spots or clefts, called Maurer’s spots. These are irregular in shape and size, generally much coarser than the Schiiffner’s dots, and not nearly so numerous. They are brought out in some slides by overstaining or with staining solution on the alkaline side. Some films show them far better than others and it is possible that they are peculiar to certain strains of the species. The gametocytes of P. falciparum are quite different from those of the other species. In very severe cases the young sexual forms may appear in the peripheral blood. The youngest ones seen are small com- pact bodies, very like the older trophozoites, except that they may be more angular, the chromatin is more likely to be elongated, and the pigment is scattered through the cytoplasm. When immature gameto- cytes are present, the older immature forms, which are long, often thin, pointed at the ends, and show pigment scattered the full length of the body, are the ones most often seen. The mature gametocyte (crescent) assumes an eliigtiodly crescentic or sausage shape and the pigment and chromatin are more or less centrally placed, leaving the cytoplasmic ends of the gametocyte clear of pigment. It is the most distinctive and easily recognized stage of any malaria parasite. In the macrogametocyte the chromatin is aggregated in a small red mass near the center and the pigment lies close to.it; around it, or even over it. In, the microgametocyte the separate chromatin granules are scattered with the pigment granules through the middle half or two-thirds of the parasite. The macro- gametocyte is often more slender, slightly longer and more deeply blue than the microgametocyte. The microgametocyte, by comparison, is likely to be broader, shorter and more sausage-shaped, with lighter staining qualities throughout, its cytoplasm being pale or grayish blue, or at times pink. As a rule mostly mature gametocytes are seen. The red blood cell containing the crescent form stretches as the longitudinal growth of the parasite proceeds and sometimes can be seen as a narrow, pink-stained zone around the gametocyte. At other times, part of the cell may be seen on the concave side of the parasite :as a faint bow-shaped rim reaching from one crescent tip. to the other. MALARIA 761 d) P. ovale (ovale malaria)—Most of the cases of malaria caused by P. ovale have been reported from Africa, though there are records of individual cases from scattered parts of the world. The parasite was probably best described by James, Nicol, and Shute,® who established that P. ovale is a separate species.?® It has also been proved that a strain of the parasite was brought to the United States from the Pacific, probably from the Philippines, by a service- man of World War 11.21:22 This Pacific (Donaldson) strain has some minor differences from that described by James, Nicol, and Shute, but resembles it in all major respects, clinically as well as morphologically. Temperature charts on over 100 patients have shown that the periodicity of this parasite is a little less than 50 hr, as compared with 43 to 48 hr?! in strains of P. vivax and 72 hr in P. malariae. P. ovale has resemblances to both P. vivax and P. malariae, a fact that makes its identification in thick films an impossibility except by those who see it constantly in comparison with the other species. Microscopical comparison of well-stained thin films of the other species with those of P. ovale are usually necessary to ascertain the characteristics which distinguish it. As in P. vivax, the infected cell shows Schiiffner’s dots and enlarges as the parasite grows older,® but not so much as it does with P. vivax (by average measurement). As in both P. vivax and P. malariae, all stages of the parasite appear in the peripheral blood. The parasite itself is smaller (by measure- ment) than the respective stage of P. vivax'® but usually larger than that of P. malariae.® It has the general compactness of the malariae parasite and like it has a low number of merozoites. The pigment, however, is more like that of P. vivax, lighter than in P. malariae, smaller and more likely to be rod-shaped than round. In the Pacific strain about 60 per cent of red cells containing rings showed Schiiffner’s dots when stained with dilute Giemsa. James et al'® reported 100 per cent of such cells showing dots in the African strain, using their own staining procedure. In P. vivax only 37 to 41 per cent of parasitized cells showed Schiiffner’s dots.’ With the Giemsa method of staining, the Schiiffner’s dots seemed no larger in granules in P. ovale than in P. vivax.*® The rings of P. ovale are often thicker than in P. vivax and at times filled in, but they are not diagnostic in themselves. The older trophozoite stages of P. ovale, although they are larger and have less conspicuous pigment, bear a striking resemblance to those of P. malariae—filled in, compact, with little evidence of ameboid 762 MALARIA activity. Fifty per cent or more of the infected cells are round but a large proportion containing this stage and succeeding stages are rather longer than wide and frequently have irregular, fringed, not well- defined margins drawn out into ragged points. While such cells may be found upon long search in P. vivax, they are frequent enough in P. ovale to be characteristic of that type of infection. The immature schizonts resemble closely the same stages of P. malariae, but they are rounded parasites often found in a definitely oval-shaped, somewhat enlarged cell which may have the fringed edge noted above and, in well-stained films, have heavy Schiiffner’s dots. The mature schizont, as in P. malariae, has 6 to 12 (most often around 8) merozoites, at times arranged in rosette form ; the infected cell is enlarged, it may be oval and fringed, or round, and in well- stained films always has heavy Schiiffner’s dots. Though as a rule a little smaller, the ovale gametocytes resemble those of P. vivax and are difficult to distinguish from them. They differ from those of P. malariae in that they have the enlarged host cell and Schiiffner’s stippling. Not infrequently aberrant forms of P. vivax may be found which resemble P. ovale. Sometimes one can make certain of the species by following the parasite through several days’ growth and noticing that these forms do not persist. It is unwise to make a diagnosis of P. ovale on one slide. e) Mixed infections—With the present low incidence of malaria in the United States, there is not so much likelihood of the existence of mixed infections as there was once. It is well, however, to keep in mind the fact that a person from a highly endemic area of the world may carry more than one species of the parasite. There is a tendency in mixed infections for one species to predominate,® so that the one is easily found, the other easily missed. Typical and characteristic forms of the respective species must be found to determine a mixed infection. In the thin film these forms of P. falciparum are the crescent-shaped gametocytes, or possibly the very small and delicate rings—too small to be those of any other species. To the in- experienced, ring forms alone are not very definite stages on which to base diagnosis of a mixed infection. In P. vivax the forms upon which reliance is placed are the large, distinctly ameboid trophozoites or any stage of the parasite in an enlarged red blood cell containing Schiiffner’s dots, barring the unlikely possibility of P. ovale. In P. malariae they are the heavily pigmented forms beyond the ring stage in unenlarged cells. MALARIA 763 Accompanying blood picture in the thin film—In malaria the total number of white cells is below normal as a rule, although the count may rise during the febrile period, particularly in P. falciparum infection or with some complicating factor such as pneumonia. It takes about a week or so for the maximum development of leukopenia. In the stained film the blood picture is one of a simple hypochromic type of anemia—increased polychromatophilia and basophilic stip- pling, central achromia of the red cells, variation in size and shape of red cells and, in severe cases, nucleated red cells. “There is not uni- versal agreement as to the leucocyte reaction during the paroxysm,”’?® even though workers agree that monocytes and lymphocytes manifest a transitory numerical decrease during the paroxysm and a subsequent increase. The monocytes, at times neutrophils, play a part in com- bating the infection by ingesting the pigment and less frequently the parasites or infected cells. Clumps of pigment in white cells are almost as certain proof of malaria as the parasites themselves, but when this pigment is present, one usually finds many malaria parasites, provided treatment has not yet been given. 2. In the thick film General appearance of blood in the thick film: In the thick film the blood cells are piled upon each other and removal of the hemoglobin from the cells is necessary to make microscopical ex- amination possible. Dehemoglobinization takes place in the process of staining the unfixed film with an aqueous solution of Giemsa stain. The outlines of the red cells are thus obliterated and a microscopical picture is presented which differs considerably from that of the thin film. One must learn to think of the parasite separate from its con- taining cell. In the thicker portion of the thick film the background varies in color from a clear light blue to a mottled gray-blue, depending upon stain factors, age of the film, and individual blood variations; while at the edge the thinner portion of the film (often only one cell in depth and about the width of one or two microscopic fields) often takes a pinkish color. This effect is frequently macroscopical as well as microscopical. Against the background of the laked red cells the familiar purple nuclei of the white cells, in varying states of preserva- tion, stand out clearly. The eosinophilic and often neutrophilic granules show rather distinctly in their characteristic color, The cytoplasmic outlines of leukocytes in very fresh films may be quite ragged unless special steps are taken to preserve them (see Section IT B5(b) preceding). Table 2—Summary of Malaria Parasite Differentiation in Stained Thick Film Stage of Development Plasmodium vivax Plasmodium malariae Plasmodium falciparum Additional Comments Small trophozoite (early ring) Growing trophozo- ite Large trophozoite Immature (preseg- menting) schizont Larger, heavier ring form than the same stage in P. falci- parum, often with a variety of cytoplasmic patterns and irregularities in shape. Usually older stages of the parasite can also be found. Stage usually ameboid in ap- pearance, with large variety of shapes. Cytoplasm fre- quently fragmented and arranged irregularly in cluster of varying-sized pieces or streamers, about or close to a large chromatin mass. Small yellowish brown pigment gran- ules scattered through parts of the cytoplasm. This stage of P. vivax is the most char- acteristic; frequently younger or older stages accompany it. Frequently quite solid and dark staining. More or less irregu- lar in outline, possibly with one or more vacuoles. Fine brown pigment scattered throughout the cytoplasm. Can be confused with macrogame- tocyte. Irregular or compact clusters of chromatin divisions, often dark reddish purple in color. Cytoplasm in irregular broken masses and wisps, containing light brown pigment granules which are clumping in spots. Usually accompanied by other stages; may be confused with same stage of P. malariae. Compact, dark, Ring likely to be heavy, with large dot of chromatin and small amount of cytoplasm, which is often “filled in,” with no vacuole. Pigment forms early and may appear as a single bead or as a haze in cytoplasm. Rings prac- tically always associated with older forms. Ring phase is brief, so that stage is not found as often as older stages. Small, usually rounded, mnon- vacuolated forms “like mar- bles in a ring.” Profuse, round, dark large-grained pigment. Forms frequently so solid that chromatin seems buried in the mass. This stage and the one that follows are the commonest forms of P. malariae seen. larger than “growing’’ stages. Sometimes in thinner portion of the smear, spreads to normal size. Profuse, fairly coarse, dark brown pigment—often mask- ing the chromatin. May be confused with “rounded up” P. falciparum gametocytes or with gametocytes of P. ma- lariae. Much like P. vivax of the same stage except that parasites are often smaller, with darker, larger pigment granules. Often so compact that internal structure is difficult to define. Usually accompanied by other stages. May be confused with presegmenting schizonts of P. vivax. Small size rings, with small chromatin dot and delicate, scanty cytoplasm. Frequently rings have double chromatin dots. Tendency toward large number of rings. Many ring forms with no older stages— practically certain to be falciparum infection. Diag- nosis on basis of small num- ber of rings often assisted by finding distinguishing gameto- cyte, although this stage is not necessarily present. Heavy large ring forms—re- semble young rings of vivax. Sometimes show pig- ment grains or haze rather clearly in cytoplasm. Ring vacuole lost or almost lost. Parasite quite small and com- pact; cytoplasm often quite pale, irregularly circular or oval. One large chromatin dot. Pigment in blurred mass or very dark clump about size of chromatin. Stage is usually found only when the infection is intense and usually ac- companied by numbers of ring-form trophozoites. Stage, not often seen, is usually accompanied by large num- bers of growing trophozoites when present. Parasite very small. Contains 2 or more di- visions of chromatin and very little cytoplasm (often pale) in which there is located one or more small, dense blocks of very dark pigment. Ring forms of all species often not complete circle—may “swallow” forms, “exclama- tion mark,” “comma” forms, or “interrupted rings.” When rings only are present and the number is small, it is practi- cally impossible to differen- tiate species. In well-stained films and in Ims which have been kept for several days before stain- ing, the “ghost” of the en- larged host cell and persist- ence of Schuffner’s stippling, or a pink cell area remaining from the stippling, may assist in diagnosis of P. vivax para- sites of any stage except very young rings. On rare occasions evidence of Maurer’s spots has been ob- served in thick films of P. falciparum. The infrequently found stages of P. falciparum are, of course, more readily found in thick films. Band forms have tendency to be- come rounded in‘thick films of P. malariae—except pothaps in very thin edge of film. Schizonts are much like thin film forms of same stages— more compact, smaller in thicker portions of film. This is the most difficult stage {except infrequent ring orms) on which to diagnose species. Schuffner’s stippling or a_ pink infected cell area may identify P. vivax. y9L VIdVIVHA Mature schizont (segmenter) Young gametocyte Mature gametocyte Usually contains around 16 merozoites (which are indi- vidually larger than those of P. falciparum) accompanied by a clump of pigment. Stage usually relatively larger than in other species. Nearly al- ways associated with other stages; not so often found as other stages of P. vivax. Not easily differentiated from compact growing trophozoites in thick films. When found, is a small, compact, usually rounded parasite, with one chromatin mass, often in the center of cytoplasm, and fre- quently has unstained area around chromatin mass. Sex impossible to determine. Macrogametocyte is larger as a rule than in other species. Pigment is light, plentiful, well dispersed through non- vacuolated cytoplasm, Except in thin edge of film, cannot be differentiated from some mature trophozoites of same Perks, Microgametocyte often istinguishable as large, dense blob of chromatin (varying from pink to purplish red) surrounded by or close to a scant amount of pale or color- less cytoplasm in which many igment granules are more or ess evenly dispersed. The chromatin mass is larger than in any other stage, mak- ing this form easily distin- guished. Other stages of the parasite can usually be found. Most distinctive stage of P. ma- lariae in thick film. Often found in large numbers, usually with trophozoites or presegmenting forms or both. About 8 merozoites—each with large chromatin dot and small amount of cytoplasm— may be compact or clearly separated. requently the chromatin and pigment only are seen, the chromatin dots being bare and well separated. The dark, heavy pigment is most often concentrated, al- though sometimes dispersed. Same as P. vivax except that P. malariae is even less fre- quently found and resembles compact trophozoite so closely that positive differentiation is impossible. As a rule, few in number, some- what smaller than P. vivax, otherwise having the same distinguishing features, except that pigment is coarser and darker. May resemble rounded P, falciparum gametocytes. Seldom seen except in severe cases. Always associated with many small trophozoites. Usually contains 8 to 24 or more tiny merozoites clustered around a small, very dark pig- ment mass. Sometimes long, slender and pointed, with pigment scat- tered to the A Usually as- sociated with many tropho- zoites. Differentiation of sex difficult or impossible. As ‘‘crescent” or ‘sausage’ shapes, may be quite diagnostic of species. In thicker portions of film, may take an oval, rounded or somewhat eroded appearance which can be confused with P. malariae trophozoites or gametocytes. ttimes may be distinguished by difference in amount and appearance of pigment or by pink or red “flag” protruding from edge of parasite. May be accom- panied by ring-form tropho- zoites or appear alone and infrequently. Often appears in “showers.” Usually smaller than same stage in thin film, When typical, segmenters are among the better stages for species diagnosis. In treated cases the gametocytes often persist beyond the asex- ual forms. In old cases, rare gametocytes of P. falciparum may be found when no other parasites are present. VIdVIVH S9L 766 MALARIA Blood platelets are pinkish violet in color, finely granular in texture, and hazy in outline. They may lie singly or in groups, and because of their distinctive granular appearance are not likely to be confused with parasites once one recognizes them in the thick film. If the blood is taken from a free-flowing puncture, there will be little likelihood of excessive clumping of platelets. In very fresh films there may be an interlacing of fine pink lines of fibrin. Very often, particularly in anemic bloods, after the disease has run its course for a while, there may be in the background of the thick film (the thicker the film, the more evident it is) reticular remains of immature red cells, or cellular debris. These may lie singly, often filling a space the exact size and shape of the laked cell, or they may appear in blue clouds or in fine skein-like masses or as stippling, particularly notice- able in quick stains on fresh blood. Barber claimed that the degree of anemia can be estimated by the degree or density of these blue clouds.?¢ In the thin edge, the red cells of the thick film frequently retain a ghost-like outline and one finds characteristics of infected cells and of parasites duplicating those in the thin film—for example, the en- larged infected cells and distinct Schiiffner’s dots of P. vivax; band forms of P. malariae; marginal ring forms, doubly infected cells and perfectly formed gametocytes of P. falciparum. It is well in learning thick films to start in this thin edge and work toward the thicker por- tion, comparing the typical forms with the less characteristic ones. With good staining of the film and a short washing period, Schiiffner’s dots may frequently be seen as individual granules in the infected cells at the edge of the film, whereas in the thicker portion one some- times sees a delicate pink area of enlarged cell size around the parasite. This can be interpreted as the color remaining from Schiiffner’s dots, for the infected cells are hemolyzed just as are uninfected cells. These pink cells are among the most diagnostic characteristics of the thick films. For size they may be compared with the white cell nuclei, the only “landmarks” left in the thick film. Maurer’s and Ziemann’s spots have not been observed in the thick film, but pink cell areas have been seen infrequently when these spots were present in the adjoining thin film. Except in the very thin edge, the parasites usually appear without the definite outline of their host cells, the only exceptions being the pink infected cell area in P. vivax or P. ovale. The chromatin and cytoplasm stain characteristically and the pigment retains its usual appearance, although it is often more prominent than in the thin film. In the thicker portion of the film the parasites seem smaller and more MALARIA 767 shrunken or compact. This may be due in part to the destructive effects of lysis and also to the heaping and crowding of the red cells in the thick film, which prevents parasites from flattening and spreading out as they do in the thin film. Also, in a slow-drying thick film the parasites have an opportunity to draw in their pseudopodia, which gives them this denser, smaller appeararice. Almost constant focusing is necessary to distinguish all the parasites in the thicker fields, as the depth of the blood gives varying ocular planes. As one scans a field in which there are young parasites, the red or purplish red of the chromatin will strike the eye first, then the accompanying light blue cytoplasmic outline can be distinguished. If older forms are present, the pigmented darker cytoplasm will be the most prominent feature. Always examine closely anything that seems to contain pigment. In making species identification take into con- sideration the composite picture of all the parasites found and do not make diagnosis on the appearance of one parasite alone unless it is beyond question. Small trophozoites (rings): In the thick film all ring forms, like other stages of the parasite, are smaller than in the thin film. (If the pH of the stain is on the acid side the cytoplasmic circle may be completely lost in staining, leaving only red dots, on which diagnosis cannot be made.) The rings may appear as complete circles, but more often only a portion of the cytoplasmic outline is apparent, in one of the following patterns. 1. “Swallow” form—a dot of chromatin with curved or straight wings or dashes of cytoplasm joining the dot on opposite sides. 2. “Exclamation mark” or “comma” form—a dot of chromatin joined on one side by a straight or curved portion of cytoplasm, giving the appearance which the names designate. 3. “Broken or interrupted ring” forms—disconnected small piece or pieces of cytoplasm following the outline of a circle on which lies the chromatin dot. They may be opposite the dot or irregularly spaced around the circle. While a diagnosis of malaria can be made on rings alone, species diagnosis, particularly when parasites are scarce, may be impossible. Some aids in the use of this stage may be noted : Falciparum diagnosis may be made on the extremely small size of very young rings (here a comparative knowledge of size is neces- sary) ; on numbers (falciparum rings may be present in great num- bers, perhaps hundreds to a field, with no older stages present); on accompanying characteristically shaped gametocytes (often not present). Vivax rings, larger than the young rings of P. falciparum, if rare or few in number may be confused with older rings of P. falciparum 768 MALARIA or with young rings of P. malariae unless they are accompanied by older, more diagnostic stages of the parasite. Irregularity of the cyto- plasmic pattern often appears early in P. vivax, and with older rings the pink area of cell size around the parasite (indicating Schiiffner’s stippling) may be found. Malariae rings, not very different from vivax rings when young, last only a short time as open rings. Their tendency to fill in early, giving among the rings some very small, rounded, compact forms with a little pigment and a rather large chromatin dot, is sometimes helpful. Malariae rings are rarely, if ever, found unaccompanied by at least an occasional older stage of the parasite, frequently by some lagging mature schizonts. Growing trophozoites—This designation covers the parasite through its period of growth following the ring and preceding the brief stage where growth is complete and the parasite ready for divi- sion. Both P. vivax and P. malariae change considerably from the ring form. P. vivax particularly shows many shapes but P. falciparum retains a ring form. In the ring-shaped growing trophozoite of P. falciparum, the chromatin dot becomes larger and the cytoplasmic circle larger and heavier, with possible minute grains of pigment. This is the stage that may be confused with young vivax rings. In a synchronous infec- tion practically all the rings of P. falciparum present may be of this stage, or, if not synchronous, they may be mixed with younger, more delicate rings of another brood. Great numbers of rings, without older stages, or the presence of falciparum gametocytes may identify the species. The falciparum parasite usually disappears from the peripheral blood in this form and continues growth in the internal organs. There is great variation in the cytoplasmic pattern of the growing vivax trophozoite. In the thick film there is a decided tendency for the tenuous, ameboid cytoplasm of the individual parasite to be fragmented and to be arranged irregularly in a cluster of varying- sized wisps, strands and blobs having no visible connection and as- sociated with a large round or irregularly shaped red or magenta mass of chromatin. The pigment increases in amount with the age of the parasite and appears as small, light brown granules or rodlets in the cytoplasm, or even at times in separate granules free of the cytoplasm but as part of the general outline pattern of the parasite. _ On occasion in slow-drying films or in blood from treated cases some of the parasites of the growing trophozoite stage of P. vivax MALARIA 769 will assume a dense, compact form easily confused with P. malariae of the same or a slightly older stage. Usually more diagnostic ameboid forms will be found, as well. In the thick film the vivax parasites of this stage may be accompanied by Schiiffner’s stippling or surrounded by the pink cell area mentioned. The combination of sprawling irregularity in shape and surrounding pink cell makes this the most diagnostic stage of P. vivax in the thick film. Growing trophozoites of P. malariae are even smaller and more compact and have even fewer vacuoles than in the thin film. They vary in size from the tiny filled-in ring to the late-growing trophozoite, but there is little variety of form. In a majority of parasites the chromatin is embedded in or surrounded by the tight, more or less regularly shaped, heavily pigmented mass of cytoplasm—so solid in texture that the internal form of chromatin, cytoplasm and pigment is often difficult to define. The picture presented in the thick-film microscopic field is that of “marbles in a ring.”?® One cannot identify band forms in the thick film for these, too, round up. When these compact trophozoites of P. malariae are present in sufficient number, they are so distinctive that diagnosis of species is simple. They may be confused, when the number is not great, with the aberrant compact forms of P. vivax, or the larger ones may be mistaken for the rounded gametocytes of P. falciparum. This stage may be accompanied by rings or by schizonts. Mature trophozoites—The seldom-found mature trophozoite of P. falciparum may be easily overlooked among rings in a thick film, The small, very dark block of concentrated pigment and the dot of chromatin are about the same size and lie in a small (hardly larger than a big ring) clear blue, sometimes slightly ragged mass of cytoplasm. They can be a great help in diagnosis, where found, because no other species has clumped pigment in this stage. Do not confuse this form with the growing trophozoites of P. malariae, some of which may be the same size, but in which the pigment, though heavy, is scattered throughout the cytoplasm. The mature trophozoite of P. vivax cannot be differentiated in a thick film, with any certainty, from the macrogametocytes of P. vivax. Both have large, dark, com- pact masses of cytoplasm, usually with no vacuoles or pseudopodia, having one large compact nucleus and much pigment scattered throughout the cytoplasm. They are usually surrounded by Schiiff- ner’s dots or by a pink cell area. Mature trophozoites of P. malariae are very much like the growing trophozoites of P. vivax, except that the gametocytes are larger and have more pigment. Sometimes the 770 MALARIA chromatin is elongate, lying along the edge of the more or less rounded cytoplasmic mass; sometimes it can be noted that the pigment is peripherally arranged. These, like certain of the older growing forms, may be confused with compact stages of P. vivax or “rolled up” gametocytes of P. falciparum. Schizonts (immature presegmenters)—These forms in P. falciparum, like the mature trophozoites, are found usually only in severe cases and may be easily overlooked among the rings. They resemble the mature trophozoites except that they are a little larger and have more than one division of chromatin, together with the block of clumped pigment in the clear blue cytoplasm. The immature schizonts of P. vivax and P. malariae are often not diagnostic for species, particularly in forms with few divisions. If the film is stained to show Schiiffner’s stippling or pink parasitized cells, or if the stage is late enough to show too many divisions for P. malariae, then P. vivax can be diagnosed. Presegmenting forms of P. vivax are usually associated either with distinctive ameboid tro- phozoites or with mature schizonts, which are more easily diagnosed. Presegmenting forms of P. malariae may be associated with their compact, round, heavily pigmented trophozoites or with the mature schizonts containing fewer merozoites than P. vivax and with no Schiiffner’s stippling. The heavy, round granules of pigment in P. malariae may help to differentiate this species. The pigment in P. vivax has a tendency to collect in fewer and fewer masses as division progresses, while in P. malariae it clumps late and is often well scattered in quite advanced presegmenters. Size of the parasite is not a conclusive diagnostic feature in the thick film, since parasites that lie in the top layer of blood or in the thinner part of the film are frequently larger than the same stage which is buried more deeply in the film. Mature schizont—This stage of the three species of malaria in the thick film resembles closely the same stage in the thin film, allow- ing for the absence of cell outline and possible shrinkage of the para- site in the thick film. At times individual merozoites are obscured by others lying over them, making a count of their number a little diffi- cult. Pigment is nearly always clumped in one mass. For P. zivax, in addition to the large number of merozoites, there is the possible Schiiffner’s stippling or pink cell to assist differentiation. P. malariae may show great numbers of mature schizonts in which merozoites are usually grouped around a tight collection of pigment granules. In some instances they may be dispersed over an area larger than that MALARIA mm which a mature schizont of P. malariae would normally cover. In the latter, particularly, the chromatin dots are frequently devoid of cyto- plasm—bald chromatin dots with a group of concentrated or scattered pigment granules, an appearance rarely found in P. vivax. This stage of P. malariae is the one most easily diagnosed as to species. In P. vivax or P. malariae, mature schizonts are accompanied by immature schizonts or by ring stages. The mature schizont of P. falciparum is the least often found of any stage in the four human malaria species and is always accompanied by large numbers of ring forms. Since the merozoites form the tiny, delicate rings of P. falciparum, it is readily seen that they are much smaller than the merozoites of the other species. Like younger schizonts, the mature schizont has the small, very dark block of pigment. Gametocytes—In the thick film the young gametocytes of P. vivax and P. malariae are too easily confused with compact, shrunken trophozoites to permit certainty of differentiation. In P. falciparum, however, the immature gametocytes are frequently seen as long “lanceolate”?® or pointed forms, with pigment scattered the entire length of the parasite. They are usually accompanied by a great many ring forms. The mature macrogametocytes of P. vivax and P. malariae cannot be definitely differentiated in the thick film from mature trophozoites. The microgametocytes, on the other hand, are often easily spotted by an appearance different from any other form seen in the thick film— a large, dark red, round, or stellate nucleus (larger than that of any other stage) surrounded by a halo of pigment granules or associated with a mass of pigment granules. The cytoplasm in which the pig- ment lies may be a very light blue or may not show at all. At other times microgametocytes retain a thin-film appearance but have a pink- ish color throughout the parasite, including chromatin and cytoplasm. Vivax gametocytes may be surrounded by Schiiffner’s dots or pink cells. The mature gametocytes of P. falciparum (crescents) are easily determined in the thick film so long as they retain their characteristic crescent or sausage-like shape, although it is nearly always impossible in thick films to differentiate the sexes, as most of the gametocytes look like females. In heavier portions of thick films, particularly when the blood dries slowly, mature gametocytes of P. falciparum assume a rounded shape, a change that would take place normally in the mosquito during the early stage of maturation. These forms may be confused with older trophozoites or gametocytes of P. malariae, 772 MALARIA but the stages with which they are associated in the blood will aid in diagnosis as will certain characteristics of the form. Often the pig- ment will be arranged compactly with a clear halo or fringe of clear blue cytoplasm. Frequently also there is found lying, somewhere along the edge of the parasite mass, one or more projections, tongues, or flags of bright pink or red-staining material, the nature of which is not known. TItis possibly the cell wall which contained the parasite, crinkled around the shrunken parasite. This pink projection is often found in thick films with normally shaped falciparum crescents as well as with the rounded or “balled up” forms, and is seen with no other stage or species of malaria parasite. Usually typical crescents will be found in the thin edge of the thick films containing gametocytes, so that species identifi- cation is not as a rule dependent on the rounded forms. Gametocytes of P. falciparum may be found with or without ring stages. Except in P. falciparum one rarely finds gametocytes alone in a film. They will be accompanied by other stages of the parasite. Exflagellation of the microgametocyte, a process that normally takes place in the mosquito’s stomach before fertilization of the female gametocyte, may on some occasions take place in a slow-drying thick film. The remains of the parasite form a small dark mass containing all the pigment, and extending from this mass will be the 4 to 8 thread- like flagella which may or may not have small masses of chromatin in each one. 3. Sources of confusion or error in stained films In examining thin films for malaria the inexperienced observer may mistake a platelet which lies over a red cell for a young trophozoite, or a group of platelets for a group of merozoites. The granular texture of the platelets, as contrasted with the rather solid appearance of the chromatin dot of the ring or the merozoite, should be of help here. Basophilic stippling, Howell-Jolly bodies and Cabot’s rings are some- times confusing. In either type of film, precipitated stain or scratches in the slide that take the stain are often misleading. One may have to rule out bacteria or dirt from the skin, particularly when the blood has run under the fingernail ; dust particles, plant spores, yeast cells or fungi from the air; bacteria, molds, protozoa or other contaminants from old dis- tilled water or from water which was collected in an unclean, unsterile container. If cleanliness in every respect is observed, these disturbing factors are reduced to a negligible minimum. Artifacts which may deceive the inexperienced technician will be found frequently, upon MALARIA 773 focusing, to lie above the blood plane or may be refractile or focus out of the field unevenly. In the thick film red dots sometimes appear in the preparation with- out any visible cytoplasm. It is possible that some of these dots are remnants of parasites, but one should never call a slide positive on the basis of these dots alone. The dots may be cocci or they may be products of degeneration of the red cells. Where the dots are evenly distributed throughout the film, they are more likely to be associated with the red cells than where they are found in clumps or in only a part of the film. If a red-stained coccus from the skin or a small nuclear remnant lies adjacent to a blue-stained particle of reticulum or other cellular substance, there may be a resemblance to a parasite. This rarely occurs more than once or twice on a single slide and, when the objects are cocci, numbers of free cocci will usually be found, whereas in well-stained films free chromatin dots are rarely noted. Remember, too, that growing and older parasites contain pigment. According to Barber and Komp,®® a good general rule is not to con- sider anything a parasite which may be interpreted as an artifact, C. Enumeration of Parasites Boyd?! says, “The quartan parasitemias seldom exceed 10,000 per cubic mm and those of vivax seldom exceed 50,000 per cubic mm. On the other hand the falciparum parasitemia has no potential limits and it is important to note that the prognosis is definitely bad if the count attains or exceeds 500,000 per cubic mm.” Sometimes a count of 70,000 or 100,000 in P. falciparum infections may jump to 500,000 in a matter of a few hours. A simple method of enumerating parasites which may not be ac- curate but which will give an acceptable estimation of the number of parasites without any special equipment is as follows: A thick film of the blood is made at the same time that a white cell count is taken. One hundred (or multiples of 100) white cells are counted on the thick film. In the same microscopic fields with these cells the malaria para- sites are counted also. The parasites per cubic mm are calculated by the following formula: No. of parasites counted in the No. of parasites per cu mm same fields with 100 white cells White cell count per cu mm = No. of white cells counted (100 in this case) Example: X 1,200 , so that 100X =4,800,000 and X ==48,000. 774 MALARIA IV. REPORTING RESULTS OF EXAMINATION In reporting results of the examination of blood films for malaria one should not only specify, if possible, the species of parasites found, but should give some indication of the stages of the parasite and the number found. Thus one may report P. falciparum, P. vivax, or P. malariae, showing (many, few, or rare) (1) trophozoites (small, large) ; (2) schizonts (presegmenting, mature) ; and (3) gametocytes (young, mature). In the thick film we use the word “many” to designate the number of parasites when they are found in every microscopic field; “few” when they are found in every third to tenth field; and “rare” when they have to be searched for. A positive report can be made on one unmistakable parasite, but it is wise in cases of rare parasites to search carefully for additional forms to confirm the report and, if possible, to examine additional films. When it is impossible to make species identification because of a scarcity of parasites, report “Malaria— species unidentified” and try to get additional films for examination. When P. falciparum is diagnosed, the report should reach the physi- cian immediately, by telegram if necessary, since it can be a highly dangerous infection and requires immediate and effective therapy. V. COMMENTS It should be borne in mind that a large proportion of cases of recent malarial activity will give positive serological reactions with most tests for the diagnosis of syphilis. Many diagnostic methods other than blood films have been tried, among them cultivation of parasites, precipitin or flocculation tests, biochemical colorimetric tests and complement-fixation tests, but because of technical difficulties encountered, lack of specificity, un- availability of commercial antigen, or for other reasons, none has yet proved practical for the diagnostic laboratory. In suspected malaria cases some workers have reported good results using provocatives, such as adrenalin or ephedrine, particularly in cases of P. falciparum, to force the parasites from the tissues into the circulating blood. It is the opinion of the authors that best results will be achieved if several successive thick films are examined with- out regard to whether adrenalin is given. Sternal puncture may reveal hidden parasites of latent infections, particularly of P. falciparum, though seldom is such a procedure necessary. Splenic or liver punctures may be hazardous and are not MALARIA 775 recommended unless unusual circumstances justify their use in an individual patient. The resort to therapeutic trials with antimalarial drugs in the absence of a positive film is poor clinical practice under most cir- cumstances, although there may in rare instances be some compelling reason why this should be done, perhaps to save the life of a patient at a time when laboratory examination is unavailable, or when cerebral malaria is strongly suspected and parasites cannot be found. From a scientific and medicolegal point of view, such treatment does not per- mit exact diagnosis, which depends upon demonstration of the etiologi- cal agent. The Parasitology Laboratories of the Communicable Disease Center, Public Health Service, P. O. Box 185, Chamblee, Ga., has been designated National Depository for Malaria Slides. Slides be- lieved to be positive or those on which consultation is desired should be sent to this laboratory. Avie Wircox, Chapter Cha'rman Victor H. Haas, M.D. E. Harorp Hinman, M.D., Pa.D. Harry Most, M.D, D.M.S., D.T.M.H. REFERENCES 1. Anprews, J. M. Malaria Eradication in the U. S. A. Abstr. 6th Int'l Cong. Trop. Med. & Mal., p. 331. Instituto de Medicina Tropical, Lisbon, Portugal. 2. ArcumamseauLt, C. P. Mass Antimalarial Therapy in Veterans Returning from Korea. J. A.M. A. 154 (17) :1411-1415, 1954. 3. Famriey, N. H. Sidelights on Malaria in Man Obtained by Subinoculation Experiments. Tr. Roy. Soc. Trop. Med. & Hyg. 40:621, 1947. 4, SHortr, H. E,, and GarNHAM, P. C. C. The Pre-erythrocytic Development of Plasmodium cynomolgi and Plasmodium vivax. Tr. Roy. Soc. Trop. Med. & Hyg. 41(6) :785, 1948. 5. Smorrt, H. E, et al. The Pre-erythrocytic Stages of Plasmodium falci- parum. Tr. Roy. Soc. Trop. Med. & Hyg. 44 (4) :405-419, 1951. 6. GarnaAM, P. C. C, et al. The Pre-erythrocytic Stages of Plasmodium ovale. Tr. Roy. Soc. Trop. Med. & Hyg. 49(2) :158-167, 1955. 7. Suorrt, H. E., and GarnHAM, P. C. C. Demonstration of a Persisting Exo-erythrocytic Cycle in Plasmodium cynomolgi and Its Bearing on the Production of Relapses. Brit. M. J. 1:1225-1228, 1948. 8. Young, M. D. A Rapid Method of Drying Thick Blood Films. Pub. Health Rep. 53:1256, 1938. 9. WiLcox, A. Manual for the Microscopical Diagnosis of Malaria in Man. N. I. H. Bull. No. 180 (rev.). Washington, D. C.: Gov. Ptg. Off., 1960, page 31. 10. Diagnostic Procedures and Reagents (3rd ed.). New York: American Public Health Assn., 1950, page 383. 11. Downavrpson, A. W., and Brooke, M. M. The Effects of Various Modifica- tions of a Mass Staining Procedure on the Transfer of Malarial Parasites Between Blood Films, J. Nat. Malaria Soc. 9:239-247, 1950. 776 MALARIA 12. Fiewp, J. W. A Simple Method of Preserving the Outlines of Leucocytes and Malarial Parasites in Giema-Stained Thick Blood Films. Tr. Roy. Soc. Trop. Med. & Hyg. 33:635-638, 1940. 13. WALKER, A. J. A Blood Staining Technique for Malaria. Medical Depart- ment, Sierra Leone. Freetown: Gov. Printer, 1940. 14. — Laboratory Diagnosis of Malaria. Am. J. Clin. Path. 22:495, 1952. 15. Wircox, A. Jerrery, G. M., and Young, M. D. The Donaldson Strain of Malaria. 2. Morphology of the Erythrocytic Parasites. Am. J. Trop. Med. 3(4) :638-645, 1954. 16. Young, M. D., Eruis, J. M,, and Stusss, T. H. Some Characteristics of Foreign Vivax Malaria Induced in Neurosyphilitic Patients. Am. J. Trop. Med. 27(5) :585-596, 1947. 17. Boyp, M. F., StratMAN-THOMAS, W. K., and MuencH, Huco. The Occur- rence of Gametocytes of Plasmodium vivax during the Primary Attack. Am. J. Trop. Med. 16:133, 1936. 18. Young, M. D., Stusss, T. H.,, and CoarnEY, G. R. Studies on Induced Quartan Malaria in Negro Paretics. Am. J. Hyg. 31c:51, 1940. 19. Jawmss, S. P., Nico, W. D., and SHUTE, P. G. Plasmodium ovale Stephens, 1922. Parasitology (London) 25:87, 1933. 20. ——————. Plasmodium ovale, Stephens; Passage of the Parasite through Mosquitoes and Successful Transmission by Their Bites. Ann. Trop. Med. & Parasit. 26:139-145, 1932. 21. Jerrery, G. M,, Young, M.D, and Wircox, A. The Donaldson Strain of Malaria. 1. History and Characteristics of the Infection in Man. Am. J. Trop. Med. & Hyg. 3(4) :628-637, 1954. 22. Young, M. D,, Evies, D. E., and Burcess, R. W, Studies on Imported Malarias: 10. An Evaluation of the Foreign Malarias Introduced into the United States by Returning Troops. J. Nat. Malaria Soc. 7(3) :171-185, 1948. 23. Young, M. D., and EviEs, D. E. Parasites Resembling Plasmodium ovale in Strains of Plasmodium vivax. J. Nat. Malaria Soc. 8:219-223, 1949. 24. MAYNE, Bruck, and Youn, M. D. Antagonism between Species of Malaria Parasites in Induced Mixed Infections. Pub. Health Rep. 53:1289, 1938. 25. Boyp, M. F. Malariology. Philadelphia, Pa.: W. B. Saunders, 1949, Vol. 2, p. 986. 26. BarBEr, M. A. The Time Required for the Examination of Thick Blood Films in Malaria Studies, and the Use of “yPolychromatophilia as an Index of Anemia. Am. J. Hyg. 24:25, 1936. ‘ 27. Fiewp, J. W,, and Le FrLEminG, H. Morphology of Malaria Parasites in Thick Blood Films. Part I. The Thick Film of Plasmodium vivax. Tr. Roy. Soc. Trop. Med. & Hyg. 32:467-480, 1939. 28. ———— Morphology of Malaria Parasites in Thick Blood Films. Part III. Plasmodium malariae. Tr. Roy. Soc. Trop. Med. & Hyg. 34:297-304, 1941. 29. ————————— Morphology of Malaria Parasites in Thick Blood Films. Part II. Plasmodium falciparum. Tr. Roy. Soc. Trop. Med. & Hyg. 33:507-520, 1940. 30. Barser, M. A,, and Komp, W. H. W. Method for Preparing and Examining Thick Films for the Diagnosis of Malaria. Pub. Health Rep. 44:2330, 1929. 31. Boyp, M. F. Present-Day Problems of Malaria Mortality. J. A. M.A. 124: 1179, 1944. CHAPTER 26 HELMINTHIASIS AND INTESTINAL PROTOZOIASIS I. Helminths A. Introduction B. Materials and Procedures 1. Examination of Feces a. Macroscopical Examination for Adult Worms b. Microscopical Examination for Protozoa, Eggs and Larvae 1) Direct Fecal Film 2) Concentration Methods 3) Egg Count for Worm Burden Estimates . Examination of Perianal Secretions and Fecal Debris Examination of Urine for Schistosoma hematobium eggs Examination of Duodenal or Biliary Aspirates Examination of Sputum Examination of Blood and Skin a. Fresh Blood b. Stained Blood Films c. Concentration of Microfilariae by Centrifugation 7. Examination of Tissues a. Direct Examination of Pressed Tissue b. Concentration by Digestion 8. The Bentonite Flocculation Test Suh wn II. Intestinal Protozoa A. Introduction B. Materials and Procedures 1. Collection and Preservation of Fecal Specimens a. Refrigeration b. Formalin c¢. PVA (Polyvinyl Alcohol) Fixative d. MIF (Merthiolate-Iodine-Formalin) e. Fixed Films in Schaudinn’s Solution 2. Macroscocical Examination of Feces 3. Microscopical Examination of Feces Direct Films Concentration Methods Permanently Stained Fecal Films Fixed Fecal Films, Trichrome-stained PVA Films, Hematoxylin-stained f. MIF Temporary Mounts 4. Culture Methods 5. Serological Tests crop References 777 778 HELMINTHIASIS, PROTOZOIASIS I. HELMINTHS A. Introduction Each helminth that infects man usually lives in a specific organ or tissue and always, when mature, produces eggs or larvae of charac- teristic size and form. Depending on the parasite’s location in the body, its products of reproduction will be found in the feces, sputum, urine, blood or tissues. Some species of worms produce eggs or larvae abundantly, making it possible even in light infections to detect their presence by simple, direct microscopic examination. Others produce eggs or larvae so sparingly that unless the infection is heavy, special concentration methods must be employed to detect them. The eggs and larvae are large enough to be seen readily under low magnification (7X or 10X eyepiece and 16 mm objective), although greater magni- fication may be required to permit recognition of specific characteris- tics. Frequently the significance of an infection depends upon the number as well as the kind of worms present and, in general, the worm burden can be estimated by determining the abundance of their repro- ductive products. In some instances, the worms themselves are passed through the rectum or are removed surgically. In either case, diag- nosis can be based upon accurate identification of the adult stages. There are therefore four general types of examination that may be made: (1) recovery and examination of macroscopic adult worms, (2) microscopic examination of unconcentrated material in a simple, direct film, (3) microscopic examination of concentrates produced by sedimentation or flotation technics, and (4) quantitative determina- tion of the output of larvae or eggs. It is impractical in the routine work-up of patients to attempt to find or to rule out parasitic infec- tions of all possible kinds by examining in every known manner the feces, sputum, urine and blood. On the other hand, when specimens of any of these materials are being studied, the discovery of an un- suspected parasitic infection is an ever-existing possibility. Usually clinical observations and a history of possible exposure to infection suggest certain parasites, which in turn suggest the particular diag- nostic technics to be employed. The following table lists the important helminths of man, their usual location in the body, the kind of reproductive products to be looked for in making a diagnosis, and the material in which they usually occur. (See also Fig 1.) Table 1—Important Helminths of Man Species Common Name Body Site Description Specimen of Choice Nematoda (Roundworm) Necator americanus Ancylostoma duodenale Ascaris lumbricoides Trichuris trichiura Enterobius vermicularis Strongyloides stercoralis Trichinella spiralis Wuchereria bancroftii Wuchereria malayi Acanthochetlonema perstans Mansonella ozzardii Loa loa Onchocerca volvulus New World hookworm Old World hookworm Large roundworm Whipworm Pinworm Threadworm Trichina worm Bancroft’s filaria Malayan filaria Persistent filaria Ozzard’s filaria Eye worm Convoluted filaria Small intestine Small intestine Small intestine Large intestine Large intestine Small intestine Small intestine, skeletal muscle Lymphatics Lymphatics Body cavities Body cavities Subcutaneous tissue Subcutaneous tissue Ovoid eggs, with thin, colorless shell Eggs similar to N. americanus Ovoid eggs, with rough, thick yellow-brown shell Spindle-shaped eggs, thick yellow-brown shell with polar plugs Elliptical-ovoid eggs, with thick, smooth, colorless shell Motile larvae Larvae Microfilariae (motile embryos) Microfilariae Microfilariae Microfilariae Microfilariae Microfilariae Feces Feces Feces Feces Perianal secretions, occasionally feces Feces or duodenal aspirates Skeletal muscle, occasionally feces Blood Blood Blood Blood Blood Skin 'SISVIHLNIW13H SISVIOZO10Odd 6LL Table 1—Important Helminths of Man—Continued Species Common Name Body Site Description Specimen of Choice Cestoda (Tapeworm)* Taenia saginata Taenia solium Diphyllobothrium latum Hymenolepis nana Hymenolepis diminuta Dipylidium caninum Echinococcus granulosis Beef tapeworm Pork tapeworm Fish tapeworm Dwarf tapeworm Rat tapeworm Dog tapeworm Hydatid cyst Small intestine Small intestine Small intestine Small intestine Small intestine Small intestine Small intestine Spherical eggs with thick, striated dark brown shell. Frequently eliminated in unruptured proglottids Eggs similar to 7. saginata. Frequently eliminated in unruptured proglottids Ovoid eggs, with thin, brownish operculate shell Spherical eggs, with polar filaments on inner shell Spherical eggs, with widely separated, inner and outer shell membranes, inner shell lacking filaments, outer shell yellow Spherical eggs, with color- less shell, clustered in membranous capsule. Frequently eliminated in unruptured proglottids Of varying size, outer laminated membrane and inner germinal layer, daughter cysts and free scolices Feces Feces Feces Feces Feces Feces Feces 08L 'SISVIHLNIW13H SISVIOZOLlOW¥d. Trematoda (Flukes)t Schistosoma hematobium Schistosoma mansoni Schistosoma japonicum Clonorchis sinensis Opisthorchis felineus Fasciola hepatica Fasciolopsis buskii Heterophyes heterophyes Metagonimus yokogawai Paragonimus westermanit Bilharzia vesical blood fluke Manson's blood fluke Oriental blood fluke Oriental liver fluke Cat liver fluke Sheep liver fluke Giant intestinal fluke Minute intestinal fluke Yokogawa’s fluke Lung fluke Genitourinary tract Circulatory system Circulatory system Liver Liver Liver Small intestine Small intestine Small intestine Lungs Eggs with terminal spine Eggs with large lateral spine Thin-shelled eggs, with minute lateral spine Small urn-shaped eggs, with seated operculum Eggs similar to C. sinensis, but narrower Large ovoid eggs, with relatively small operculum Eggs similar to F. hepatica Eggs similar to C. sinensis Eggs similar to C. sinensis Large ovoid eggs, with seated operculum Urine, occasionally feces Feces Feces Feces Feces Feces Feces Feces Feces Sputum and feces * Adult tapeworms all live in the small intestine and eliminate their eggs or proglottids in the feces. Larval tapeworms of several species are found in various organs and tissues and can be identified on surgical removal. + Trematodes live in various organs, and their eggs, usually yellow-colored, are passed in the stool, urine or sputum. 'SISVIHLNIWT3IH SISVIOZOL1lOYdd 18L HELMINTHIASIS, PROTOZOIASIS 782 Strongyloides stercoralis Lo rhabditiform larva Figure 1—Eggs of helminths found in man HELMINTHIASIS, PROTOZOIASIS 783 H. W. BROWN, COLUMBIA UNIVERSITY Figure 1 (Continued) 784 HELMINTHIASIS, PROTOZOIASIS Figure 1 (Continued) HELMINTHIASIS, PROTOZOIASIS 785 Figure 1 (Continued) B. Materials and Procedures I. Examination of feces Fecal examination may reveal worm infections of the intestine, liver, lungs or blood. Specimens should be collected in. clean re- ceptacles and examined as soon as possible after collection. A large container is not usually necessary, since routine examination requires only a few grams of feces, but the large container has the advantage of permitting defecation directly into it. Heavy glass, wide-mouth, screw-capped vials or 1 oz bottles are suitable for shipment in ap- proved types of mailing tubes. Identification data accompanying the specimen should include date of collection, name and address of pa- tient, name and address of the person to receive the report, and some indication of what parasites are suspected. - In: reporting results all information of possiblevalue todd oe given, such as date of examination, species of parasites found or 786 HELMINTHIASIS, PROTOZOIASIS “parasites not found,” and the methods of examination employed. Since the person receiving the report usually will not have seen the specimen, it is important to give a description of the material ex- amined, noting especially any unusual characteristic or elements such as Charcot-Leyden crystals, or unusual quantities of normal elements such as neutral fats. If a specimen is of insufficient quantity or is otherwise unsatisfactory for reliable examination, that fact should be reported, especially when findings have been negative. Egg counts may be reported as eggs per film, in mm or ml quanta. Egg-count interpretation, whether in terms of the estimated number of worms or the relative worm burden indicated by the egg count, should be re- ported only when specifically requested. a. Macroscopical examination for adult worms—Two methods are generally used for separating macroscopic worms from feces: (1) straining through wire sieves and (2) sedimentation-decantation. Because straining is reliable and requires less time, it is preferred when sieves of proper mesh are available. Two sieves are generally used : one with mesh coarse enough to pass the medium-size worms but fine enough to stop larger fecal rubbish (10-20 mesh) ; another small enough to stop the smaller worms and scolices of tapeworms (40-50 mesh). The stool is stirred into a watery suspension, diluted, and poured through the nested sieves, which are then washed under a stream of water. Sedimentation-decantation requires only such equipment as will in general be readily available. These technics are of special value in following the effectiveness of chemotherapy. Equipment: Two large vessels of equal capacity (2-5 liters) Flat-bottomed dish or pan Medicine-dropper pipette Straight dissecting needle with the tip bent 90° Small forceps Formalin (10%) or other fixative-preservative if preferred Procedure: 1) Fill one vessel with tap water. 2) In another vessel of equal size stir fecal mass into a water suspen- sion and allow to stand with frequent gentle stirring of the surface film for 30 min. HELMINTHIASIS, PROTOZOIASIS 787 3) Decant and discard supernatant fluid. 4) Holding the first vessel high to produce a brisk stirring effect, pour its contents onto the sediment. 5) Repeat steps 1 and 2 until supernatant fluid is clear. 6) Transfer portion of washed sediment into a shallow dish or pan and under good light recover worms with a pipette, curved pointed needle, or forceps. Preserve worms in 10% formalin. b. Microscopical examination for protozoa, eggs and larvae 1. Direct Fecal Film Equipment: Wooden applicator Glass microscope slide, 1X3 in. Cover glass, 22X22 mm, No. 1 or No. 2 Physiological salt solution (0.85% sodium chloride) Dobell and O’Connor’s iodine solution: Todine (powdered Crystals) «cous. ssenmmvasy sas snseons sos sommms 15435 Potassium. J0AIE «vo ve summmtmss 3 suede se « 3 3 woman £3 PETREEE ES 1 2 Distilled Water .....covvviinererirtnrnnersvnnranessersrssannssnnens 100 cc Filter or decant. Prepare fresh about every 2 weeks. Procedure: 1) Place a drop of salt solution on center of slide. 2) With applicator select a 1-2 mg sample of feces, carefully avoid- ing nonfecal elements (unless schistosome eggs or amebas are especially indicated, in which case flecks of mucus and blood should be selected). One milligram of feces is about 1 cubic mm in size. 3) Stir into the salt solution, making an even suspension. 4) Remove coarse fiber, seeds, sand, etc. 5) Cover with 22X22 mm cover glass. 6) If the preparation is satisfactory in all respects, examine it; if not, discard it. No time should be wasted on a preparation that can only yield doubtful results if negative. 7) Examine the preparations systematically, using the low power of the microscope. 8) If protozoa or larvae are found requiring stain for identification, run iodine stain under the cover glass or lift the cover glass with an applicator, add stain to the film, and mix by repeatedly lifting the cover glass until evenly spread. 788 HELMINTHIASIS, PROTOZOIASIS 2. Concentration Methods: Feces normally contain a great variety of materials, most of which are either lighter or denser, smaller or larger than the cysts, eggs and larvae of parasites. The purpose in using a concentration method is to separate as completely as possible the cysts, eggs and larvae of parasites from all other elements of the stool. In general this is accomplished, somewhat imperfectly of course, by screening (which removes the larger objects), by sedimen- tation (which removes the lighter elements in the supernatant fluid), and by flotation (which lifts the parasitic elements out of the mass of denser material). Several of the established concentration methods in- corporate all three of these principles, usually in the order listed. An important requirement for efficiency of any concentration method is met through comminution of the feces in a diluent, An efficient, con- venient and inexpensive instrument for this purpose is an adapted small electric hand drill.? Zinc sulfate centrifugal flotation: This method? is one of the most widely used methods of concentration. = It is unsuitable for fatty stools and like all other flotation methods it does not concentrate the eggs of most species of trematodes. It has the special merit of being suit- able for routine examination both for cysts of protozoa and for eggs of most helminths. Eggs of the flukes and large tapeworms are better concentrated by one of the sedimentation technics. Many modifica- tions have been proposed. The one here described is designed for laboratories in which examination of stools is Seiad frequently but notasa daily routine. nah 3 : Equi TI sd M aterials: 1. Zinc sulfate solution, specific glovity 1.18 (331 g of crystals in 100 ml solution). A hydrometer should be used. Todine stain of Dobell and O’Connor Wassermann tube, or test tube about 12X 100 mm Paper cup, small (about 50 ml), unwaxed Surgical ay 4-ply, cut nth 3 in. squares Wire loop, about 28 gauge, 5-6 mm diameter Glass slide, 1X3 in., free of wax xand oil Wood applicator al OI, Nox nad BN Centrifuge with viet cups s siable for Wesstaion tubes, giving at least 2,000 rpm at top speed. y HELMINTHIASIS, PROTOZOIASIS 789 Procedure: 1) With applicator transfer about 25 ml of feces (more if stool is very fibrous) to paper cup. 2) Add 1 ml of water and comminute completely. 3) Fill Wassermann tube with water and add all of it to fecal sus- pension in cup. 4) Mix and fill tube with suspension, leaving coarse debris in cup by crimping its lip and pouring through the slit thus formed. 5) Centrifuge 1 min at approximately 2,000 rpm. 6) Pour off completely and discard supernatant fluid. 7) Add 1 ml zinc sulfate solution to sediment and thoroughly break it up by shaking tube or stirring with an applicator. 8) Fill tube to within 2-3 mm of rim with additional zinc sulfate solution. 9) If suspension contains much coarse debris or flock, strain through gauze held across the brim of a paper cup and slightly de- pressed (the cup used in step 1 could be rinsed and reused in this step). After straining, return suspension to tube and add enough zinc sulfate to fill within 2-3 mm of top. 10) If suspension is relatively clear, omit step 9 and proceed to 11. 11) Centrifuge 1 min at 2,000 rpm and permit centrifuge to come to a stop without interference or vibration; balance must be nearly perfect. 12) Prepare a slide by placing on its center 75 to ¥ drop of iodine stain ; this requires touching slide with dropper pipette. 13) Without removing the tube from the centrifuge, and using a freshly flamed wire loop, remove 1 or 2 loopfuls from the surface film and mix into iodine stain on a glass slide without spreading it more than necessary to make a thin film. 14) Examine with or without a cover glass under low power. If “positive,” and prolonged study with a higher power is indicated, use cover glass. Laboratories in which several or many stool examinations are re- quired daily should be provided with a mechanical mixer and elevated supply bottles for water, zinc sulfate solution and 10 per cent formalin, each delivered through dropper-sized spouts so that the force of gravity will produce jet action sufficient to stir as the fluid is added to fecal suspensions in tubes or cups. Any small high-speed electric hand 790 HELMINTHIASIS, PROTOZOIASIS drill can be adapted to serve as a mechanical mixer of suspensions in Wassermann tubes. It need only be anchored in a vertical position (best over a sink) and have inserted into the drill chuck a hollow- centered rubber stopper (serum-bottle type, with 6 mm plug, sleeve removed) punctured at its base to permit deep insertion of a wood ap- plicator. The stopper and applicator both are readily replaceable, the latter being used only once. With these facilities, the zinc sulfate flotation procedure is more efficient and can be very rapidly carried out as follows: 1) Comminute about 25 ml of feces in 2 ml of water in a Wassermann tube, using a motor-propelled wood applicator. Fill tube, then dis- card the applicator. 2) Centrifuge 1 min and discard supernatant fluid. 3) Add 1 ml of ZnSOy solution, flick sediment free, and with a strong jet stream add ZnSO, solution to fill tube, at the same time stirring its contents. 4) Strain through gauze into paper cup, return filtrate to tube, and add ZnSO solution nearly to fill. 5) Centrifuge, collect the surface film with the loop, and examine as described above in paragraph 13. Brine gravity flotation: First described by Willis in 1921,® brine flotation may be preferred to the zinc sulfate flotation when interest is limited to nematodes and certain tapeworms and a centrifuge is not available. Equipment: Applicator or tongue depressor Saturated aqueous solution of sodium chloride Paper cup, unwaxed (may not be needed) Lipless test tube about 1.5X 12 ecm (may not be needed) Wire loop, freshly flamed Microscope slide, 1X3 in. Procedure: If fecal specimen has been received in a small tin or glass container and is less than half filled with feces: 1) Add 1 or 2 ml of brine to the feces and stir to a pasty consistency. 2) Add brine to fill container and stir to produce an even suspension. 3) Allow to stand entirely undisturbed 30 to 60 min. HELMINTHIASIS, PROTOZOIASIS M1 4) Using a wire loop or the brim of a lipless test tube, transfer some of the surface film to a clean slide. 5) Examine under low power of microscope, focusing on upper surface of sample. Prolonged study of puzzling objects may re- quire use of a cover glass. If the specimen container is unsuitable for this procedure: 1) Add about 1 ml of feces to 2 ml of brine, using applicator, in a paper cup and comminute, 2) Fill test tube with brine, empty it onto fecal suspension, and mix thoroughly. 3) Pouring through a crimp in the cup’s brim, fill test tube with suspension. 4) Allow suspension to stand 30 to 60 min undisturbed. 5) Using a freshly flamed wire loop, transfer material in surface film from several locations onto a clean slide. 6) Examine upper surface of sample, using low power of microscope. Formalin-ether centrifugal sedimentation: This method was devised by Ritchie in 1948.# Like the zinc sulfate flotation method, it is best used to concentrate the eggs and larvae of helminths and the cysts of protozoa in stools and is especially effective when excessive amounts of fats and fatty acids are present in the stool. It is used also when circumstances require preservation of fecal specimens that are to be examined later, usually after shipment to a distant laboratory. It may be used for concentrating the eggs of schistosomes and of other trematodes, although it appears to be less efficient for this purpose than the sodium sulfate-acid-ether or gravity sedimentation technics, Equipment and Materials: Formalin (10% solution) Ether, any standard grade Centrifuge tube, conical, 15 ml Gauze, 4-ply Unwaxed paper cup or small beaker Wood applicator Iodine stain Pipette, 15 cm or longer, with rubber bulb Slide (3X1 in.) and cover glass (22X22 mm or larger) 792 HELMINTHIASIS, PROTOZOIASIS Procedure: 1) With applicator transfer 1 ml or less of feces to Wassermann tube, add about 3 ml of water, and comminute, using the same appli- cator in a mechanical mixer if available. 2) Add water to fill tube, mix, and strain into paper cup (or beaker) through gauze. Add water through the gauze to make ap- proximately 15 ml of filtrate. 3) Transfer to 15 ml conical tube, centrifuge 1 min at about 2,000 rpm and discard supernatant fluid. 4) Add 10 ml of 10 per cent formalin and allow suspension to stand 10 min or longer for fixation. 5) Add 3 ml of ether, stopper tube, and shake vigorously for 20-30 sec. 6) Centrifuge 2 min at about 1,500 rpm. 7) Decant supernatant and transfer sediment by pipette to a slide. 8) Add a drop of iodine stain if protozoa are to be identified, cover with cover glass and search the entire preparation under the low power of the microscope. Note: 1f the specimen has been preserved in 10 per cent formalin, take a sample that contains about 0.5 ml of sediment, dilute to 15 ml, strain through gauze, centrifuge, decant, add 10 ml of 10 per cent formalin and 3 ml of ether, then proceed as above from step 5. Sodium sulfate-acid-Triton-ether centrifugal sedimentation: This is one of the most efficient, rapid methods of concentrating schisto- some eggs in feces. Eggs and larvae of other helminths also are concentrated by this method, but protozoan cysts generally are con- centrated poorly and are somewhat damaged. It may be noted that methods employing various combinations of acid, ether and other reagents have been devised and variously modified. Comparative studies by Maldonado and Acosta-Matienzo® indicate that the method described here, first used by Hunter et al.® for concentrating S. japonicum eggs, is very effective also when used for the diagnosis of S. mansonii infections. Equipment and Materials: Sodium sulfate-hydrochloric acid mixture, prepared as follows: Combine one part concentrated HCl with one part distilled water, then mix the diluted HCl with an equal amount of 10 per cent NasSOj. Triton NE.* * Rohm & Haas, Philadelphia, Pa. HELMINTHIASIS, PROTOZOIASIS 793 Other materials are the same as for formalin-ether, except iodine stain, which is not generally used here. Procedure: 1) Transfer 1 ml or less of feces to Wassermann tube, add about 3 ml of water, and comminute (with motor-propelled applicator if available). 2) Add water to fill tube, mix and strain through gauze into a paper cup; rinse gauze with additional water to make approximately 15 ml of filtrate. 3) Transfer to 15 ml conical tube, centrifuge 1 min at about 2,000 rpm and discard supernatant fluid. 4) Add 10 ml of the Na»SO4-HCl mixture, 3 drops of Triton NE, and 3 ml of ether; stopper and shake vigorously for 20-30 sec. 5) Centrifuge for 1 min at about 2,000 rpm. 6) Insert a pipette along tube wall, cautiously and slowly to permit ether to vaporize and escape at tip of pipette while breaking through the plug at the interface; yield the sediment and after withdrawing pipette, draw it horizontally through a fold of gauze or absorbent paper to remove fluid and debris from outside surface. 7) Deliver 1 drop of sediment to a slide, cover, and search entire preparation under low magnification. 8) There will generally be enough sediment for one or more addi- tional preparations, which should be examined if eggs are not found in the first. Gravity sedimentation: Gravity sedimentation as described by Faust and Ingalls” is more efficient than other methods for concentrating schistosome eggs but it has the disadvantage of being time-consuming and of requiring considerable space when numerous specimens are being processed simultaneously. One of its advantages is that it in- volves simple procedures and very little equipment. Equipment and Materials: One part pure glycerol in 200 parts of tap water Conical sedimentation flask, 200-350 ml capacity Gauze, 4-ply Paper cup or small beaker Wood applicator Pipette of suitable size for sedimentation flask Slide and cover glass 794 HELMINTHIASIS, PROTOZOIASIS Procedure: 1) Comminute about 5 ml of feces in 10 ml of glycerolated water; 25-50 ml of feces with adequate amounts of glycerolated water are required in light infections. 2) Dilute to capacity of sedimentation flask using glycerolated water and strain suspension through four layers of gauze into flask. 3) Sediment 1 hr and decant. 4) Refill the flask with glycerolated water, stir, and sediment 45 min, then decant. 5) Again refill flask, stir, sediment 30 min, and decant. 6) If supernatant fluid remains cloudy, repeat step 5 preceding. 7) After last sedimentation, decant very slowly, leaving sediment rela- tively undisturbed. 8) Remove 1 or 2 drops of sediment (separately) from middle and bottom layers, transfer to a slide, add cover glass, and examine all of each preparation. 3. Egg Count for Worm Burden Estimates: In general, the damage produced by helminths varies directly with their numbers. Since the output of eggs in the feces is proportional to the number of egg- laying females, egg counts aid in the clinical evaluation of worm infections. They may be used also to determine the efficacy of anti- helminthic medication. Two methods of egg counting are used: the dilution method of Stoll and Hausheer® and the direct film of Beaver.?1t The dilution method will be presented here: Equipment and Materials: 1. N/10 (0.4%) NaOH solution 2. Erlenmeyer-like flasks (Stoll*) marked at 60 ml and 56 ml levels and fitted with rubber stopper 3. Glass beads or BB shot 4. Slide and 22X30 mm cover glass 5. Pipette* calibrated to deliver 0.075 ml 6. Wood applicator and soft paper towel or cellulose wipes * Stoll flasks and pipettes may be secured from A. H. Thomas Co., Phila- delphia, Pa. HELMINTHIASIS, PROTOZOIASIS 795 Procedure: 1) Fill flask to the 56 ml mark with N/10 NaOH solution. 2) With an applicator carefully (not soiling neck of flask) add feces to bring contents up to the 60 ml mark. 3) Add 10-15 beads or BB shot, stopper, and shake vigorously. 4) Allow to stand 12-24 hr with occasional shaking. 5) Shake vertically, throwing beads briskly against rubber stopper for 20-30 sec and immediately withdraw exactly 0.075 ml. 6) Wipe outside of pipette to avoid adding excess suspension to sample and transfer 0.075 ml sample to a slide. 7) Cover with 22X30 mm cover glass and count eggs in entire preparation. 8) Multiply the count by 200 and record as eggs per ml, uncorrected. 9) Correct for stool consistency by multiplying the uncorrected count as follows: hard-formed (difficult to puncture with applicator) X1; mushy-formed (can be cut with applicator) X2; mushy- diarrheic (nonliquid but takes shape of container) X3; diarrheic (liquid) X 4. 10) Report corrected count as eggs per ml, formed stool basis (£.s.b.). Interpretation of egg counts: It has been estimated that each female Ascaris produces on the average about 2,000 eggs per ml or g of feces and each female Necator and whipworm produce approximately 50 per ml. Although the interpretation of egg counts must take into account such conditions as age, health and diet, infections giving counts of less than 20 Ascaris, 5 Necator or 5 whipworm eggs per mg may be regarded as light, while those above 50 Ascaris, 25 hookworm or 25 whipworm eggs may be considered as relatively heavy. Standard direct film egg count: Films containing 1 or 2 mg of feces can be used both in the routine examination for protozoa and hel- minths and for making counts of eggs if present. The 1 mg film is adequate in most instances, especially if a concentration method has been used to determine the presence of eggs in the stool sample and the purpose of egg counting is merely to determine whether the infec- tion in question is heavy, moderate or light. If the apparatus for making standard films is not available, or if only rough estimates of egg output are required, egg counts can be made on ordinary direct fecal films. They should be recorded as eggs per film although they can be interpreted as being roughly equivalent 796 HELMINTHIASIS, PROTOZOIASIS to counts in 1 to 2 mg preparations. Films prepared by experienced technicians seldom contain less than 1 mg or more than 2 mg of formed feces. Films containing 3 mg of feces are too dense for accurate examination for protozoa. Equipment: A photoelectric light meter, adapted and calibrated. Note: To adapt the meter, (1) fit to the photoelectric cell's window a wood or other solid platform 18 mm thick with a central circular hole 16 mm in diameter. (2) Directly above the platform suspend an adjustable electric lamp that provides sufficient light to give a reading of 20 foot-candles (ft-c) through the 16 mm aperture from a distance of 8 in. or more; a 60 watt bulb in a goose- neck lamp is adequate. To calibrate the meter, (1) prepare 2N Na,SO, and N/1 BaCl, solution. (2) To each solution add pure glycerol, two parts solution to one part glycerol. (3) For a 1 mg standard suspension combine one part of the glycerolated BaCl, with six parts of glycerolated Na,SO,; and for 2 mg standard suspension, com- bine one part of glycerolated BaCl, with three parts of glycerolated Na,SOy,, giving in each case a white suspension of BaSO, precipitate. (4) With a clean microscope slide on the platform and centered over the 16 mm window, adjust the light to give a whole-number ft-c reading. (5) Place 1 drop (149 ml) of the 1 mg standard BaSO, suspension on the slide above the window and spread just to cover the window. Note and record the reduction of the meter reading. Repeat until a reliable average is obtained, starting each time with the same arbitrarily selected whole-number reading through the clear slide. The average amount of light reduction produced by the standard suspension is the same as that produced by an even suspension of 1 mg of formed feces in 1 drop of water spread evenly over the 16 mm window. (6) Using the 2 mg standard BaSO, suspension, repeat the procedure outlined in step 5 to calibrate the adapted meter for making 2 mg fecal films. Procedure: 1) Place a clean slide on the window of the meter platform and adjust the light to give the predetermined “zero point.” 2) Put 1 drop of water (or salt solution if trophozoites are to be looked for) on the slide, spread just to cover the window and, with applicator, stir (do not smear) feces into the water until the fecal suspension reduces the light to equal that of the 1 or 2 mg standard BaSOy suspension. 3) Cover with 22X22 mm cover glass, then systematically count the eggs in the entire preparation. 2. Examination of perianal secretions and fecal debris Microscopical examination of material from around the anus usually is made for the diagnosis of pinworm infection. However, any of the helminth eggs that occur in feces and resist drying (Ascaris, whipworm, Taenia) may also be found there. To transfer the secre- HELMINTHIASIS, PROTOZOIASIS 797 tions and fecal debris from the anus to a slide for microscopical ex- amination, perianal impressions on transparent cellulose adhesive or Scotch tape, introduced by Graham in 1941,'% are generally used in the manner suggested by Jacobs in 194213 (see Fig 2). LODP TAPE OVER END TOUCH GUMMED SURFACE SMOOTH TAPE WITH COTTON OF SLIDE TO EXPOSE SEVERAL TIMES TO PERI- OR GAUZE GUMMED SURFACE ANAL REGION Figure 2—Enterobius vermicularis: Scotch tape diagnosis Equipment: Scotch tape, 3X34 in. strips (cellulose tape) Tongue depressor Paper label, 4X1 in. Microscope slide, 1X3 in. Toluene Procedure: 1) Over one end of a tongue depressor place a 3X34 in. strip of Scotch tape, sticky side out, and hold it in place with thumb and finger. 2) Spread buttocks to expose outer anal canal and press tape against right and left perianal folds, being careful to cover the area between the dry and moist areas. 3) Spread tape smoothly with sticky side down on microscope slide, placing at the same time a paper label bearing identification data between slide and tape at one end. 4) With low power of microscope briefly scan the area bearing epidermal cells and fecal debris; if eggs are not found immediately, mark the “exposed” area (showing evidence of perianal contact), lift tape to include just that area, place 1 drop of toluene on slide, replace tape, and systematically examine it. Note: Pinworm eggs remain uncleared in the toluene, somewhat resembling air bubbles, whereas most other elements become relatively invisible. After a few days in perianal impressions on Scotch tape, pinworm eggs tend to become clear and more difficult to recognize. However, they can still be diagnosed after several weeks. 798 HELMINTHIASIS, PROTOZOIASIS 3. Examination of urine for Schistosoma hematobium eggs Equipment: Sedimentation flask, conical, 250-300 ml capacity Pipette long enough to reach bottom of flask Slide and cover glass Procedure: 1) Collect urine directly in sedimentation flask; the portion voided last is preferred. 2) Sediment for 30 min or longer. 3) With pipette, transfer sediment to a slide. 4) Add cover glass and examine under low power of microscope. The sediment from the flask can be further concentrated, or the urine more rapidly sedimented, by centrifugation. 4. Examination of duodenal or biliary aspirates In a small percentage of Strongyloides infections, larvae can be demonstrated in duodenal aspirates more readily than in feces. Rarely, the same is true for Giardia infections. Also, there may be uncertainty whether large trematode eggs in stools are those of the liver fluke Fasciola or of the intestinal fluke Fasciolopsis. The presence of eggs in aspirates of pure, or nearly pure, bile indicates, of course, that Fasciola is present. Material aspirated from the duodenum should be examined in direct films and in sediment that has been concentrated by centrifugal sedi- mentation. For the latter, normal salt solution (0.85% NaCl) should be used as a diluent. 5. Examination of sputum Paragonimus eggs are eliminated in the sputum somewhat ir- regularly and as a rule their distribution in sputum samples is uneven, Their presence may be suspected on the basis of brown or rusty colored flecks seen grossly, usually but not invariably scattered in areas streaked with blood. In such cases they may be identified in direct films of samples taken from selected areas; when scanty, their detection may require concentration from larger quantities of sputum collected over a 24-48 hr period. Patients harboring Paragonimus frequently swallow their sputum, and the eggs of the parasite are found in the stool. HELMINTHIASIS, PROTOZOIASIS 799 a. Direct examination Equipment and Materials: Wood applicator Physiological salt solution (0.85% NaCl) Slide and cover glass Procedure: 1) Select samples with applicator from bloody or brownish areas and transfer to a slide in a drop of salt solution. 2) By teasing and rolling the applicator, break up the sample and apply cover glass. 3) Distribute material by putting light pressure on cover glass and examine under low power of microscope. b. Concentration Equipment and Materials: 1. Sodium hydroxide (5% solution) 2. Beaker or glass jar, at least five times greater in volume than that of sputum to be examined 3. Centrifuge tubes, conical, 15 ml capacity 4. Pipette suitable for transferring from centrifuge tubes 5. Slide and cover glass Procedure: 1) Transfer sputum sample to beaker or glass jar containing 5 ml or more of 5 per cent NaOH for each ml of sputum. 2) Stir mixture occasionally until mucus is completely digested, usually 2 or 3 hr at room temperature. 3) Pour some or all of digested sample into conical centrifuge tube (s) and centrifuge 1 min at 2,000 rpm. 4) Transfer sediment to a slide and examine under low-power micro- scope ; a cover glass may not be required. 6. Examination of blood and skin Microfilariae of W. bancroftii and W. malayi occur most abundantly in night blood; blood drawn during the daytime is satisfactory for other filarial species and for the nonperiodic WW. bancroftii. Micro- filariae of O. volvulus normally do not occur in the blood but are 800 HELMINTHIASIS, PROTOZOIASIS most readily found in material teased out of snips of skin taken from the area near nodules containing the parent worm, or in serum from an incision too shallow to reach the blood capillary. In either case the microfilariae are fixed, stained and identified in the same manner as though they were in thin blood films. Identification of species is based on the presence or absence of a sheath and the distribution of nuclei, especially those of the tail (Fig 3). WUCHERERIA BANCROF TI LOA LOA ACANTHOCHEILONEMA PERSTANS < | Z| fk | = 1% % EE JE S Ie 4 | { OQ. IY Ss 4) il 7 ( x500 a 2. B= o> a= S x - i oo|Eoowl | 42 B= = | —— 72} i = %2000 X2000 X 2000 ONCHOCERCA VOLVULUS MANSONE LLA OZZARDI | MICROFILARIA MALAY $ $ : aS 8: 7 ry 8 < fi g g : . = 4 Le ii S i a i Q 3 s a) nr: e.L, x500 x 500 a = ewig oO - ie g Soo | OC El \ Sooo 2 Xx 2000 x 2000 x2000 FROM BELDING’S Clinical Parasitology, 1942 Figure 3—Microfilaria of man HELMINTHIASIS, PROTOZOIASIS 801 a. Fresh blood: By skin puncture in the usual manner, collect a drop of blood on a slide and spread evenly under a large (22X40 mm) cover glass. Microfilariae if present will be active and can be readily detected under low magnification. b. Stained blood films: Using Giemsa stain, microfilariae may be recognized and sometimes diagnosed in blood films prepared for, or in the same manner as for, the diagnosis of malaria. Preparations are more satisfactory, however, when stained with hematoxylin. By varying the procedure somewhat, any of the standard hematoxylin stains can be used, although Delafield’s hematoxylin is a good choice if available. Equipment and Materials: Items necessary for making blood preparations Ether and absolute methyl alcohol, 1:1 mixture Delafield’s hematoxylin Hydrochloric acid, 0.05 per cent (1 drop concentrated HCI in 100 cc distilled water) Procedure: 1) Make thick films in the same manner as recommended for ex- amination for malaria parasites (see Chapter 25). 2) Air-dry thoroughly but not for more than 24 hr if possible; use 50° C heat in humid climate. 3) Immerse in distilled water for 5-10 min or until blood is com- pletely laked. 4) Dry thoroughly. 5) Immerse in ether-alcohol mixture 5-10 min. 6) Dry thoroughly. 7) Stain in undiluted Delafield’s hematoxylin 10 min. 8) Immerse in 0.05 per cent HCI to remove excess stain, usually 5-10 min. 9) Wash in running tap water 5 min. 10) Dry thoroughly. 11) Cover with a film of immersion oil for immediate examination under low or higher powers of the microscope, or add mounting medium and cover glass directly to the dry film (Fig 3). 802 HELMINTHIASIS, PROTOZOIASIS c. Concentration of microfilariae by centrifugation: Micro- filariae are often too scanty to be detected in ordinary films. They may be concentrated by centrifugation of citrated blood or of laked blood. The former offers the advantage of preserving motility of the organisms, whereas the latter gives greater concentration and may be used to provide permanent mounts of well-stained microfilariae, as demonstrated by Knott in 1939.14 Citrated Blood Equipment and Materials: Sodium citrate solution (2.0% sodium citrate in 0.85% sodium chlo- ride solution) Centrifuge tube, conical, 10-15 ml capacity Capillary pipette Microscope slide Procedure: 1) Place 1 ml of sodium citrate solution in a centrifuge tube. 2) Draw 5 ml of venous blood, immediately add it to the citrate solu- tion, and mix thoroughly. 3) Centrifuge at low speed (about 1,000 rpm) for 10-15 min. 4) With a capillary pipette, transfer 1 drop of sediment from the extreme bottom of the tube to a slide, spread to proper thickness, and examine under low magnification for motile microfilariae. Laked Blood Equipment and Materials: Formalin, 2% solution Centrifuge tube, conical, 15 ml capacity Capillary pipette Microscope slide Giemsa stain Procedure: 1) Mix 1 ml of fresh venous blood with 10 ml of 2% formalin in a 15 ml conical centrifuge tube. 2) Allow to stand for at least 10 min; red cells will be laked and microfilariae will be fixed by the formalin, 3) Centrifuge at 1,500 to 2,000 rpm for 2 min. HELMINTHIASIS, PROTOZOIASIS 803 4) With capillary pipette transfer sediment in 1 or 2 drops of fluid to a slide. 5) Spread and examine wet film or spread sediment uniformly with needle or applicator and dry in air. 6) When thoroughly dry, fix briefly in ether-alcohol mixture, dry for 1 or 2 min and immediately stain in Giemsa or hematoxylin as out- lined for thick films above. 7. Examination of tissues Two of the parasitic diseases of man are of such a nature as to make ordinary methods of diagnosis valueless for their detection. Trichinosis, caused by Trichinella spiralis, is known to be con- tracted by ingestion of uncooked parasitized pork (rarely bear or walrus meat) and characterized by a complex of symptoms which result chiefly from damage produced by larvae in the skeletal muscles. Infection of man by larval ascarids of the dog, cat and possibly other mammals produces a condition known as visceral larva migrans. The larvae have been found in the liver, lungs, kidneys, brain, muscle and other organs. S. mansonii eggs may be found in rectal biopsy material when they cannot be found by stool examination.’ The small piece of biopsied rectal mucosa (unfixed) is pressed between two slides or under a No. 2 cover glass and examined under the low power of the microscope. Three methods are commonly used to demonstrate the presence of nematode larvae in biopsy or autopsy specimens of human tissue or in specimens of pork suspected of being the source of trichinosis: (a) direct microscopic examination of material pressed between glass slides; (b) digestion of the tissues, followed by microscopic examina- tion of the concentrated sediment; (c¢) microsectioning, as for histo- pathological study. Histopathological procedures are not described here. a. Direct examination of pressed tissue—Biopsy or autopsy specimens of rectal mucosa, muscle, liver, brain and other soft tissues can be pressed thinly enough between glass slides or under a heavy cover glass to give clear visibility under low power of the microscope. Equipment and Materials: Glass slides or a trichinoscope or compressor Physiological salt solution (0.85% NaCl) Dissecting needles and scalpel 804 HELMINTHIASIS, PROTOZOIASIS Procedure: 1) Avoiding fat and connective tissues as carefully as possible, place a small portion of the tissue, sliced into thin pieces, on a slide in 1 or 2 drops of salt solution. 2) With dissecting needles, tease fibrous samples into small strands and cut nonfibrous samples into minute cubes. 3) Spread material linearly along the middle third of the slide, cover with slide, compress as much as possible, and examine under low magnification. A trichinoscope can be used in place of slides. b. Concentration by digestion—Except when in bone, fat or nervous tissues, nematode larvae can be concentrated by pepsin diges- tion and sedimentation. Equipment and Materials: 1. Physiological salt solution (0.85% NaCl) 2. Digestion fluid: Physiological salt solution containing 0.5 per cent pepsin (dry granular pepsin 5 g per liter) and 0.7 per cent hydro- chloric acid (7 ml concentrated HCI per liter) 3. Incubator or water bath regulated to 35°-37° C 4. Meat grinder, Waring blendor, tissue grinder or other chopping device (scissors and scalpel can be used) 5. Flask, 250 ml capacity or larger if tissue sample exceeds 10 g 6. Centrifuge tubes, conical, 15 ml capacity 7. Surgical gauze, 4-ply 8. Sedimentation flask, 250 ml capacity or larger 9. Pipettes suitable for use in centrifuge tube and sedimentation flask Procedure: 1) Grind or chop tissue into bits not more than 2-3 mm thick. 2) Transfer to flask of suitable size and for each gram of ground tissue add 10 ml or more of digestion fluid. 3) Incubate 4 to 12 hr at 35° C with occasional stirring. 4) Strain through gauze into sedimentation flask. 5) After stirring, fill one or two centrifuge tubes immediately, centri- fuge 1 min at 2,000 rpm, and decant. HELMINTHIASIS, PROTOZOIASIS 805 6) Resuspend sediment in salt solution, recentrifuge, and decant. 7) Add just enough salt solution to suspend sediment in a pipette, transfer to 1.5X3 in. slide, and examine for motile larvae under low power of microscope. 8) If larvae are not found in centrifuged sample (s), allow remainder of digested suspension to stand in sedimentation flask for 1 hr and repeat steps 5, 6, and 7, using samples drawn from bottom of flask. 9) To quantitate yield of larvae, measure suspension obtained in step 4, count all larvae obtained in an aliquot of step 7, and compute in terms of larvae per gram of original tissue. 8. The bentonite flocculation test Suessenguth and Kline in 194416 described a flocculation test for trichinosis. These authors sensitized cholesterol particles with an alkaline extract of powdered trichinella larvae. Later a flocculation test employing bentonite coated with trichinella extract was reported by Bozicevich (1951).17 The test herein described is rapid and specific. Materials and Methods: Preparation of trichinella extract'8 1) Inoculate rats intragastrically with T. spiralis larvae; 4 to 6 weeks later, sacrifice the animals. 2) Skin and eviscerate animals and put carcasses through a meat grinder. 3) Place about 70 g of the ground carcasses in a battery jar con- taining 3 liters of artificial gastric juice. The digestive fluid is pre- pared by adding 21 ml of HCI (sp gr 1.10-1.19) to 3 liters of tap water (37°-40° C), then adding 15 g of pepsin. 4) Incubate at 35° C and stir slowly with stirring apparatus for 6 hr. 5) Pour the digest through one layer of surgical gauze (35-40 mesh per in.) into a 3 liter funnel, the stem of which is fitted with a 15 ml conical centrifuge tube. 6) Allow mixture to stand for 17% hr so that larvae may gravitate to bottom of centrifuge tube. With a pinchcock, close the rubber tube between funnel and centrifuge tube. Remove tube containing larvae. 7) Discard supernatant and rat tissue above the larvae. Refill tube with physiological salt solution. Shake thoroughly and allow larvae 806 HELMINTHIASIS, PROTOZOIASIS to settle for 20 min. Repeat washing procedure five times until larvae are free of rat tissue. 8) Suspend 2 ml of settled larvae in 8 ml of physiological salt solution and transfer to a Ten Broeck tissue grinder or one of similar type. Grind larvae thoroughly and add suspension to 90 ml of physio- logical salt solution. 9) Allow to extract for 24 hr at 4° C. Centrifuge suspension at 3,000 rpm for 15 min. Discard sediment and centrifuge supernatant at 15,000 rpm for 15 min. Discard sediment. The supernatant con- stitutes the extract used for sensitizing the bentonite. Preparation of bentonite particles 1) Suspend 0.5 g of standard Volclay (Wyoming bentonite*) in 100 ml of distilled water. 2) Homogenize in a blender for 1 min, repeat after 5 min for another min. 3) Transfer bentonite suspension into a 500 ml glass-stoppered graduate and add distilled water to make 500 ml. 4) Shake thoroughly and allow to settle for'1 hr. 5) Pour off and save supernatant, discard sediment. 6) Pour supernatant into six 100 ml centrifuge tubes (heavy-duty, pyrex) and centrifuge at 1,300 rpm (by tachometer) for 15 min. Usé International Centrifuge Size 2 with No. 240 head. 7) Pour off supernatant. Discard sediment. 8) Centrifuge supernatant in 100 ml tubes at 1,600 rpm (by tacho- meter) for 15 min. 9) Pour off supernatant and discard it. - 10) Resuspend accumulated sediment from the six tubes in 100 ml distilled water and homogenize in a blender for 1 min. This is the stock bentonite suspension. In our experience, it has remained stable for as long as 6 months without losing its absorptive properties. It is important to have colloidal particles of the size obtained by centrifugation at 1,300-1,600 rpm (relative centrifugal force of ap- proximately 500-750). The larger colloidal particles obtained by centrifugation at speeds of less than 1,300 rpm tend to flocculate spontaneously and thus to give false-positive reactions in the test, * Obtained from the American Colloid Co., Merchandise Mart Plaza, Chicago 54, TIL. HELMINTHIASIS, PROTOZOIASIS 807 while the smaller particles, obtained by centrifugation at speeds greater than 1,600 rpm, do not tend to flocculate as readily in the presence of a positive serum, thus giving false-negative reaction. It has been noted that if the stock bentonite, which has been suspended in 100 ml of fresh distilled water after the 1,600 rpm centrifugation, is now centrifuged at 1,300 rpm, practically all the particles are thrown down. Preparation of sensitized bentonite particles 1) Thoroughly shake the stock bentonite suspension to distribute particles evenly. Remove 10 ml of the uniformly dispersed particles and place in a 15X200 mm culture tube. 2) Add 2 ml of the trichinella extract and shake. Allow suspension to remain at room temperature for 30 min. 3) Add 0.10 ml of 1 per cent Tween 80 and shake. Allow to stand for 5 min. 4) Add 1 ml of 0.1 per cent methylene blue dye and shake. 5) Centrifuge at 2,000 rpm for 5 min. Discard supernatant. 6) Add 5 ml of distilled water to the sensitized bentonite. Shake vigorously and add an additional 10 ml of distilled water. Shake thoroughly. Centrifuge for 5 min at 2,000 rpm. 7) Repeat step 6 two more times in order to remove all free Trichinella antigen. 8) After final centrifuging, the sensitized bentonite particles are resuspended in 5 ml of salt solution. This constitutes the sensitized bentonite particles. The suspension should be thoroughly shaken before use. Method of conducting the test 1) Inactivate for 30 min at 56° C all sera used in the flocculation test. 2) Prepare twofold serial dilutions of the serum in physiological salt solution. 3) Place 0.10 ml of each dilution onto wax-ringed slides (micro- scopic slides, 3X2 in. with 12 rings, designed for serological floccula- tion tests). 4) Shake sensitized bentonite suspension and add 1 drop to each ring. The drop is of such size that a capillary pipette should dispense about 40 drops per ml. 808 HELMINTHIASIS, PROTOZOIASIS 5) Place slide on a Boerner-type rotating machine and rotate 100- 120 times per min for 20 min. Read immediately under a microscope at low-power magnification (50X to 60X). Avoid drying. A reaction is regarded as 4 plus when all the sensitized particles are clumped in separate masses. There may be a few large clumps or a number of small ones, depending upon the titer of the serum. Never- theless, the fields between the flocs are almost clear. A reaction is re- garded as 3 plus when approximately three-fourths of the sensitized particles have clumped. A 2 plus reaction is one in which half the particles are clumped and half still remain in colloidal suspension. In a 1 plus reaction only one-fourth of the particles are clumped. In a negative reaction the sensitized bentonite particles are in colloidal suspension. The flocculation test is considered positive when a 2 plus or stronger clumping occurs in a 1:4 or higher serum dilution. Every time the test is conducted it is essential to test known positive and negative sera. This will detect any change in the sensitized bentonite particles. With experience, the spurious aggregates can readily be distinguished from true flocculation. When the test is positive, the flocs are of uniform density, evenly distributed in the microscopic field, and free of hyaline-like material or other debris. In our experience the sensitized bentonite particles have remained stable over 2 months when kept in the refrigerator. Il. INTESTINAL PROTOZOA A. Introduction The intestinal protozoa occur in two common forms—trophozoites and cysts. The former is the vegetative, motile stage, which in most species normally becomes encysted before passage in the feces. Both stages are small and delicate, making them much more difficult to de- tect and identify than the helminth eggs. However, with a 10X eye- piece even the smallest of the intestinal protozoa can be detected with a 16 mm objective, although their identification often requires an oil-immersion lens. The intestinal protozoa of man (see Figs 4 and 5) include the fol- lowing species : 1. Amebas: Entamoeba histolytica Entamoeba coli Endolimax nana Todamoeba butschlii Dientamoeba fragilis HELMINTHIASIS, PROTOZOIASIS 809 DRAWN BY B. FERRELL Figure 4—Intestinal protozoa of man 2. Flagellates: Trichomonas hominis Giardia lamblia Chilomastix mesnili Embadomonas intestinalis Enteromonas hominis 3. Ciliates: Balantidium coli 4. Sporozoa: Isospora hominis Isospora belli The last five species listed are rarely reported in North America. Most of the intestinal protozoa of man inhabit the colon. Giardia and Tsospora live in the small intestine. All may be diagnosed by finding cysts or trophozoites in the feces. Dientamoeba and Trichomonas are 810 HELMINTHIASIS, PROTOZOIASIS found only in the trophozoite stage. Although Isospora has many stages in its life cycle, the o6cyst usually is the stage seen in fresh feces. 17 oo FROM BELDING’S Clinical Parasitology, 1942 Figure 5—Cysts of intestinal protozoa treated with iodine: 1 and 2, En- dolimax mana; 3 and 4, Iodamocba butschlii; 5-7, Entamoeba histolytica; 7-10, Entamocba coli; 11 and 12, Chilomastix mesnili; 13 and 14, Embadomonas intestinalis; 15 and 16, Enteromonas hominis; 17, Giardia lamblia; 18, Blasto- cystis hominis, a yeast resembling a protozoan cyst. HELMINTHIASIS, PROTOZOIASIS 811 E. histolytica, Bal. coli and I. hominis are definitely pathogenic but do not always produce apparent disease. The pathogenicity of Dientamoeba and Giardia is in question ; the remainder are considered to be harmless. Regardless of their probable role in disease, all species observed in specimens submitted to the laboratory should be identified as to species and reported by scientific name. It is the usual practice to report whether cysts or trophozoites or both were observed. In view of the major importance of amebiasis, the laboratory diagnosis of this infection will be emphasized. Negative findings on examination for E. histolytica and other pro- tozoa are far less reliable than for the helminths. Since the intensity of the protozoan population in the intestinal tract changes from day to day and the character of the feces is subject to great variation, the organisms eliminated in the feces are at times extremely scarce and they may disintegrate before the specimen is examined. It is therefore common practice to examine several, usually at least three, fresh normal specimens. If these fail to reveal the organisms and clinical evidence suggests amebiasis or balantidiasis, a saline cathartic should be administered and two more postcathartic specimens examined. Examination of material aspirated from the lower colon during sigmoidoscopy or from the duodenum occasionally reveals protozoa that were not detected in stools.!® (See also Section I B 4 of this chapter.) The variety of fecal specimens bearing numerous possible species and stages of protozoa require special types of diagnostic technics. Thus several of the more commonly employed technics are described. Routine procedures generally include : For fresh specimens—Direct film in salt solution and concen- tration by zinc sulfate flotation or formalin-ether sedimentation. Hematoxylin-stained permanent films of positive specimens may be included as a routine. For old specimens, not likely to contain trophozoites—Zinc sulfate flotation or formalin-ether sedimentation. For preserved specimens—(1) in formalin: formalin-ether sedi- mentations; (2) in PVA: hematoxylin-stained dry films; (3) in MIF : direct moist films. For aspirated material from colon or duodenum—Direct films in salt solution and hematoxylin-stained films. Supplementary technics include in vitro cultivation in various media and the complement-fixation test. 812 HELMINTHIASIS, PROTOZOIASIS B. Materials and Procedures 1. Collection and preservation of fecal specimens—When pos- sible, examination of feces should be carried out immediately, If delay of over 30 min is necessary, the specimen should be cooled or preserved chemically. Keeping feces at body temperature after pas- sage rapidly destroys trophozoites of amebas and other protozoa as reported by Tsuchiya in 1945.2° Stools to be examined for protozoa must not be mixed with urine or water. a. Refrigeration at 3°-5° C will preserve trophozoites for several days in dysenteric stools, and cysts in normal feces may remain viable for more than a month. Storage in a closed container is essential to prevent desiccation. Freezing should be avoided. b. Formalin is a good preservative for protozoan cysts (not tropho- zoites) and helminth eggs and larvae. Comminute feces thoroughly in as little water as possible and for each part of the suspension add at least 5 parts of 10 per cent formalin. Preservation generally is good for many months. c. PVA fixative is especially good for trophozoites. It is therefore a good choice for preserving watery or dysenteric stools and material aspirated at sigmoidoscopy. To 8-10 ml of the fixative add 1-2 ml of the specimen, mix thoroughly, and stopper. Trophozoites are pre- served indefinitely; thus films on slides may be prepared immediately or later. Preparation of the PVA fixative is as follows :2! Materials: Mercuric chloride, saturated aqueous solution ............covuivnn... 62.5 ml Eth). alooliol (0890). +. ssaiimmss ss sma smaisiny saan gs soa ames ne 31.0 ml Glacial acetic aid ..ovvvvrvvirrvtivsvnserssnsvsnrrsnsersnrnrnses oe 5.0 ml CINOSEOL" | comin v4.05 SAEs SPDR Hh Sh BR Ae Th SAE 5 I 1.5 ml Polyvinyl alcohol (PVA) powder (Elvanol 90-26)* ................. 5 gz Procedure: 1) Combine materials 1-4, in order. 2) Stir PVA powder into mixture at room temperature, then heat to 75° C or higher until suspension clears. d. MIF (merthiolate-iodine-formalin) stain-preservative is suitable for all kinds and stages of parasites found in stools.?? A disadvantage * Produced by E. I. Dupont de Nemours & Co., Electrochemical Dept., Wil- mington 98, Del. In addition to the original grade, Elvanol 90-26, other grades of high-hydrolysis, medium-viscosity PVA (71-24 and 71-30) have proved satis- factory. PVA powder in small quantities may be purchased from Delkote, Inc., P. O. Box 1335, Wilmington, Del. HELMINTHIASIS, PROTOZOIASIS 813 is that one part of the fixative (iodine) is unstable. MIE gives ex- cellent preservation, and all stages of protozoa as well as eggs and larvae are diagnosable without further staining in temporary films made immediately after fixation or many weeks later. Furthermore it is useful for all common types of stools and aspirates. Two solutions are prepared, stored separately, and combined im- mediately before use. Solution I is 40 parts tincture merthiolate (Lilly), 5 parts formaldehyde (U.S.P.) and 1 part glycerol. Solution 2 is freshly prepared (good for several weeks if stored in dark, well- stoppered bottle) Lugol's solution (5% iodine in 10% aqueous potassium iodide solution). For use, combine 15 parts of solution I with 1 part of solution 2. One ml of feces in 8-10 ml of the stain- preservative is an adequate sample. On reaching the laboratory, moist films of the mixed specimen, or of the sediment if scanty, are ex- amined microscopically without additional staining. e. Films fixed in Schaudinn’s solution and either stored unstained in 70 per cent alcohol or stained and permanently mounted under cover glasses are useful for preserving specimens that are collected for examination later. The stained permanent-slide preparation serves especially well where species diagnosis is difficult and as a record of positive findings. 2. Macroscopical examination of feces—Since perishable tro- phozoites are more likely to be present in the more liquid specimens, these should be examined before formed specimens. Unpreserved specimens should be examined grossly, for color, presence of blood, pus or helminths, and the consistency of the stool noted and recorded. White and red blood cells should be confirmed microscopically. Normal elements such as neutral fats, soaps and fatty acids when present in abnormal proportions also should be noted (see Section I B 1 of this chapter). 3. Microscopical examination of feces— (a) Direct films made from fresh stool specimens are a routine procedure. Unstained films in salt solution examined systematically under low magnification serve for the detection of protozoa. Their identification usually re- quires staining with iodine. Trophozoites of the amebas and Balantid- ium tend to overstain in full-strength iodine stain. Therefore, when trophozoites have been observed in the direct film, it is better to use the iodine conservatively or in diluted form (Dobell and O’Connor, 192123), or to choose another stain such as Velat’s,?* which possibly demands less skill for satisfactory results. 814 HELMINTHIASIS, PROTOZOIASIS (b) Concentration methods are useful in revealing light infections when cysts are present but as yet there is no satisfactory method for concentrating trophozoites. Both protozoan cysts and helminth eggs are concentrated effectively by the zinc sulfate centrifugal flotation and by the formalin-ether centrifugal sedimentation technics. The former is more generally used for fresh specimens. The latter also can be used for fresh specimens but its outstanding advantages are that it may be used after formalin preservation of specimens collected at a distance from the laboratory and it is suitable for certain helminth eggs (trematodes) that cannot easily be concentrated by zinc sulfate flotation. (c) Permanently stained fecal films: Heidenhain's iron hema- toxylin is a standard stain for fecal films. Many different modifi- cations have been proposed, all of which require considerable practice before uniformly good results will be obtained. Important factors in getting good preparations are: freshness of the fecal specimen ; proper thickness of the films; care not to permit drying of the film; good fixation; good quality, fresh mordant; well-ripened stain; complete dehydration and clearing. Heidenhain’s technic follows. Equipment and Materials: 1. Microscope slide and cover slip 2. Applicator stick or small camel’s hair brush 3. Schaudinn’s fixing solution: Prepare 200 ml of a saturated solu- tion of mercuric chloride in distilled water. Add to this solution 100 ml of 95 per cent alcohol. This solution will keep indefinitely. Add 5 ml of glacial acetic acid to 100 ml of solution just before using. 4. Iodine alcohol: Prepare a stock solution by adding enough iodine crystals to 70 per cent alcohol to make a dark, concentrated solu- tion. For use, add drop by drop to 70 per cent alcohol until an orange-colored solution is obtained. The exact concentration of this solution is not important. 5. Mordant: Prepare fresh just before use a solution of ferric ammonium sulfate that has the color of dark urine (about 4%). To distilled water add a few clear purple crystals of the salt, stir until the color is proper, and either remove the crystals or pour off the solution to another container. 6. Stain: 5.0 ml stock solution of ripened 10 per cent hematoxylin in 95 per cent alcohol and distilled water to make 100.0 ml; or 0.5 HELMINTHIASIS, PROTOZOIASIS 815 g of hematoxylin crystals and distilled water to make 100.0 ml. The staining qualities are enhanced by ripening the stain for several weeks in a flask plugged with cotton and stored in a warm place. 7. Destaining solution: Dilute mordant or prepare a fresh solution having the color of pale urine (about 1%) Alcohol (50,70, 95 and 100% ) Carbolxylene (phenol 25 parts, xylene 75) 10. Xylene 11. Permount Procedure: 1) Using applicator or soft brush, make a thin film of feces on clean 1X3 in, slide. Note: If necessary, dilute feces to a pasty consistency with physiological salt solution. In following the steps below, do not permit drying of film; hold closely to the time schedule except in steps 2, 11 and 14, where overnight or longer delay does not matter. 2) Fix films in Schaudinn’s solution, 5 min at 50° C or 1 hr at room temperature. 3) Alcoholic iodine, 5 min. 4) 50 per cent alcohol, 3 min. 5) Tap water, 3 min. 6) Mordant solution 10 to 20 min at 40°-50° C, or 2 to 4 hr at room temperature. 7) Distilled water, two changes, 3 min total. 8) Hematoxylin stain, 5 to 10 min at 40°-50° C, or 12 to 24 hr at room temperature. 9) Tap water, two changes, 3 min total. 10) Iron alum. Destain (solution 7 above) by observation under microscope. Here is the critical step in this technic. The time may vary from 5 to 30 min, depending on the film, the organisms, and other factors. 11) Running tap water, at least 5 min, preferably 30 min or longer. 12) 70 per cent alcohol, 3 min. 13) 95 per cent and absolute alcohol, 3 min each. 816 HELMINTHIASIS, PROTOZOIASIS 14) Carbolxylene, 5 min. (Two changes of unadulterated absolute alcohol can be used, making the carbolxylene in this step unnecessary.) 15) Xylene, 3 min. 16) Mount in Permount. 17) Examine for at least 15 min, using the oil-immersion objective. Tompkins and Miller®® have modified the Heidenhain technic, which permits automatic destaining in a 2 per cent aqueous solution of phos- photungstic acid. Other steps are the same as outlined above, except that in step 9 the phosphotungstic acid is substituted for iron alum and the time allowed for destaining is not critical—merely 2 min or longer. d) Fixed fecal films, trichrome-stained: The trichrome staining procedure, slightly modified as given by Wheatley,?® offers a rapid method for the staining of fixed fecal films. Equipment and Materials: 1. Items 1, 2, 4, 9 and 10 as given for the Heidenhain iron-hema- toxylin stain. 2. Trichrome stain: Add 1 ml glacial acetic acid to dry stain com- ponents (0.6 g Chromotrope 2R, C.I. No. 16570; 0.15 g Light Green SF Yellowish, C.I. No. 42095; 0.15 g Fast Green FCF, C.I. No. 42053) ; mix and allow to stand for 30 min, after which add 100 ml distilled water. Since the stain solution is quite stable, 10X quantities may be prepared and used as needed. 3. Destaining solution: 90 per cent acidified alcohol (1 drop glacial acetic acid in 10 ml alcohol). 4. Alcohol (70, 95 and 100%). Procedure: 1) Follow steps 1, 2 and 3 given for the Heidenhain iron-hema- toxylin staining procedure. 2) 70 per cent alcohol, two changes, 2 min each. 3) Trichrome stain, 10 min. 4) 90 per cent acidified alcohol, 10-20 sec, or until stain barely runs from film. 5) 90 per cent alcohol, 2 min. 6) 100 per cent alcohol, 2 min. 7) Xylene, 2 min. 8) Mount in permount. HELMINTHIASIS, PROTOZOIASIS 817 e) PVA films, hematoxylin-stained: 1f the fecal specimen has been preserved in PVA fixative, 1 or 2 drops of the mixture should be placed on the center of a microscope slide and spread over an area 1 in. square, using an applicator stick. The PVA films are allowed to dry thoroughly (preferably overnight in a 35° C incubator). The dried films can be stained with a rapid iron-hematoxylin technic, for example, the phosphotungstic acid procedure which requires no con- trolled destaining. Later, if more critical results are needed, a second slide from the specimen can be stained by the Heidenhain iron-hema- toxylin technic. f) MIF temporary mounts: Fecal samples in MIF stain-preserva- tive are examined in temporary wet mounts. Shake well and after permitting the gross, heavier elements to settle (3-5 min), transfer a drop of the sample to a slide, cover, and examine under low and high magnification as for direct films. 4, Culture methods—More for E. histolytica than for other pro- tozoa, cultivation can be employed to advantage with fresh speci- mens, given adequate facilities, time and interest; but it is not a recommended procedure for public health laboratories or any labora- tory which receives specimens by mail. The examination of cultures should include not only direct films of the sediment but also stained films prepared from sediments preserved in PVA fixative on at least those cultures exhibiting organisms during examination of direct film.27 Several media are available, such as Cleveland and Collier’s liver extract,?* modified Boeck and Drbohlov’s LES ?® egg yolk in- fusion,®® or alcohol extract? Rather than attempt to use several media, however, it is generally best to select a single medium and to become proficient with its use. Only the Cleveland and Collier medium will be presented. Equipment and Materials: 1. Standard bacteriological test tubes 2. Cotton 3. Glass slide 4. Cover glass 5. Entamoeba medium (Cleveland and Collier) * Available commercially as Endameba Medium from Digestive Ferments Co., Detroit, Mich. 818 HELMINTHIASIS, PROTOZOIASIS 6. Fresh horse serum sterilized by filtration and diluted sixfold with sterile salt solution 7. Sterile rice powder Preparation of medium 1) Suspend 33 g of the dehydrated medium in 1,000 ml of cold dis- tilled water and heat to boiling to dissolve completely. 2) Pour into test tubes to a height of about 4 cm, slant, and sterilize for 15 min at 15 Ib pressure (121° C). 3) Cover slant with horse serum. 4) Adda 5 mm loop of sterile rice powder. Procedure: A sample of feces the size of a pea (about 50 mg) is thoroughly mixed with the medium. If the feces are fluid or semiformed, 0.2-0.5 ml of this material should be introduced into the medium with a pipette. The tubes should be incubated at 35° C and examined microscopically at the end of 24 and 48 hr. To maintain E. histolytica cultures in Cleveland-Collier medium, it is necessary to make transfers to fresh medium every 2 or 3 days. In transferring, it is necessary to observe aseptic precautions, as it has been found that the introduc- tion of new bacteria into the culture medium may result in the death of the amebas. Examination of culture: Using pipette, secure 1 drop of material from bottom of culture, place on glass slide, and cover with clean cover glass. The amebas may be located with low power of micro- scope and studied under high dry objective. At least six preparations from the culture must be fully and carefully examined before it can be considered negative: three from the 24 hr culture and three addi- tional preparations made after 48 hr if the 24 hr examinations were negative. The morphology of E. histolytica in culture is essentially the same as in the stools. Cultured specimens may be a little larger than amebas found in stools and the food particles within them will be bacteria and rice starch particles rather than red blood cells. These amebas from the culture exhibit sluggish movement and they tend to drag detritus along with them. The cysts of E. histolytica usually are not present in cultures. 5. Serological tests—The complement-fixation test is of doubt- ful value in the diagnosis of intestinal amebiasis. However, it may HELMINTHIASIS, PROTOZOIASIS 819 be helpful in suspected cases of amebic hepatitis and abscess.?? Since a reliable antigen is not available commercially, serum specimens must be sent to a laboratory known to be currently offering this specialized service. Harop W. BrowN, M.D., Chapter Chairman Paul C. BEAVER, PH.D. JouN BozicevicH M. M. Brooke, Sc.D. T. W. M. Cameron, M.D., Pu.D., Sc.D. HerBert G. JounsTONE, M.D. PH.D. Howarp B. SHookHOFF, M.D. ALLEN YARINSKY, PH.D. REFERENCES 1. BEAVER, P. C. The Detection and Identification of Some Common Nematode 2 10. 11. 12. 13. 14. 15. Parasites of Man. Am. J. Clin. Path. 22:481-494, 1952. Faust, E. C, et al. Comparative Efficiency of Various Technics for the Diagnosis of Protozoa and Helminths in Feces. J. Parasitol. 25:241-262, 1939. Wires, H. H. A Simple Levitation Method for the Detection of Hook- worm Ova. M. J. Australia 2:375-376, 1921. Rrrcuie, L. S. An Ether Sedimentation Technique for Routine Stool Ex- aminations. Bull. U. S. Army Med. Dept. 8:326, 1948. . Marbonapo, J., and Acosta-MATiENZO, J. A Comparison of Fecal Ex- amination Procedures in the Diagnosis of Schistosomiasis Mansoni. Exper. Parasitol. 2:294-310, 1953. Hunter, G. W,, III, IncaLLs, J., and CoHEN, M. Comparison of Methods for Recovery of Eggs of Schistosoma faporicum from Feces. Am. J. Clin. Path. 16:721-724, 1946. Faust, E. C, and IncaLLs, J. W. The Diagnosis of Schistosomiasis Japonica. III. Technics for the Recovery of the Eggs of Schistosoma japonicum. Am. J. Trop. Med. 26 :559-584, 1946. Storr, N. R., and HausHEier, W. C. Concerning Two Options in Dilution Egg Counting: Small Drop and Displacement. Am. J. Hyg. 6:134-345, Mar. Suppl., 1926. Beaver, P. C. Quantitative Hookworm Diagnosis by Direct Smear. J. Parasitol. 35:125-135, 1949. —————— Methods of Pinworm Diagnosis. Am. J. Trop. Med. 29:577- 587, 1949. ————————— The Standardization of Fecal Smears for Estimating Egg Production and Worm Burden. J. Parasitol. 36 :451-456, 1950. GraHAM, C. F. A Device for the Diagnosis of Enterobius Infection. Am. J. Trop. Med. 21:159-161, 1941. Jacoss, A. H. Enterobiasis in Children. Incidence, Symptomatology, and Diagnosis, with a Simplified Scotch Cellulose Tape Technique. J. Pediat. 21:497-503, 1942. Knorr, J. I. A Method for Making Microfilarial Surveys on Day Blood. Tr. Roy. Soc. Trop. Med. & Hyg. 33:191-196, 1939. HEerNANDEZ-MoRALES, F., and MarpoNapo, J. F. The Diagnosis of Schisto- somiasis Mansoni by a Rectal Biopsy Technique. Am. J. Trop. Med. 26 :811-820, 1946. 820 HELMINTHIASIS, PROTOZOIASIS 16. SurssencutH, H., and Kung, B. S. A Simple Rapid Flocculation Slide Test for Trichinosis in Man and in Swine, Am. J. Clin. Path. 14:471-484, 1944, 17. BozicevicH, J. J., et al. A Rapid Flocculation Test for the Diagnosis of Trichinosis. Pub. Health Rep. 66 :806-814, 1951. 18. BozicevicH, J. Studies on Trichinosis. XII. The Preparation and Use of an Improved Trichina Antigen. Pub. Health Rep. 53:2130-2138, 1938. 19. Hoop, M., SopemaN, W. A. and AkeNHEAD, W. R. Comparison of the Effectiveness of the Examination of Multiple Stools and Proctoscopic Ma- terial for the Detection of Amebiasis. Am. J. Trop. Med. & Hyg. 1:539- 542, 1952. 20. TsucHivA, H. Survival Time of Trophozoites of Endamoeba histolytica and Its Practical Significance in Diagnosis. Am. J. Trop. Med. 25:277- 279, 1945. 21. GorpMmAN, M., and Brooke, M. M. Protozoans in Stools Unpreserved and Preserved in PVA-Fixative. Pub. Health Rep. 68:703-706, 1953. 22. SAPERO, J. J., and LawLess, D. K. The “MIF” Stain-Preservation Technic for the Identification of Intestinal Protozoa. Am. J. Trop. Med. & Hyg. 2:613-619, 1953. 23. DoseLL, C., and O'Connor, J. The Intestinal Protozoa of Man. London: John Bale & Sons & Danielson, 1921. 24. VELat, C. A, WrINstEIN, P. P., and Otro, G. F. A Stain for the Rapid Differentiation of the Trophozoites of the Intestinal Amoebae in Fresh, Wet Preparations. Am. J. Trop. Med. 30:43-51, 1950. 25. Tompkins, V. N,, and Mitre, J. K. Staining Intestinal Protozoa with Iron-Hematoxylin-Phosphotungstic Acid. Am. J. Clin. Path. 17:755-758, 1947. 26. WHEATLEY, J. B. A Rapid Staining Procedure for Amoebae and Flagel- lates. Am. J. Clin. Path. 21:990-991, 1951. 27. NorMAN, L., and Brooke, M. M. The Effectiveness of the PVA-Fixative Technique in Revealing Intestinal Amebae in Diagnostic Cultures. Am. J. Trop. Med. & Hyg. 4:479-482, 1955. 28. CreveLAND, L. R., and CoLLIER, J. Various Improvements in the Cultivation of Entamoeba histolytica. Am. J. Hyg. 12:606-613, 1930. 29. RearooN, L. V., and Rees, C. W. The Cultivation of Endamoeba his- tolytica without Serum. J. Parasitol. 25(Suppl.) :13-14, 1939. 30. BaramurH, W. Improved Egg Yolk Infusion for Cultivation of Entamoeba histolytica and Other Intestinal Protozoa. Am. J. Clin. Path. 16:380-384, 1946. 31. Nersown, E. C. Alcoholic Extraction Medium for the Diagnosis and Culti- vation of Endamoeba- histolytica. Am. J. Trop. Med. 27:545-552, 1947. 32. Hussey, K. L., and Brown, H. W. The Complement Fixation Test for Hepatic Amebiasis. Am. J. Trop. Med. 30:147-157, 1950. CHAPTER 27 ANTIMICROBIAL SUSCEPTIBILITY TESTS I. Introduction II. Antibiotic Susceptibility Tests A. Culture Media B. Time and Temperature of Incubation C. Antibiotic Stock Solutions D. Methods 1. Agar Diffusion (Medicated Disk) Method 2. Dilution Method 3. Testing Susceptibility of M. tuberculosis Strains 4. Measuring the Combined Action of Antimicrobial Drugs E. Apparatus References I. INTRODUCTION It is the purpose of this chapter to define the function of the routine diagnostic clinical, hospital or public health laboratory in providing assistance to the physician using antimicrobial agents in the treatment of disease. Methods for determining the susceptibility (or sensitivity, as it is often referred to) of microorganisms to these agents will also be described. The second report of the Expert Committee of the World Health Organization’ has pointed out that microbial resistance to anti- microbial drugs is perhaps the principal obstacle to their successful therapeutic use. This resistance may deprive an antimicrobial agent of its proper therapeutic effect in the individual ; or, more importantly, it may have widespread effect within a community, especially if the organisms spread from person to person. Knowledge of the degree of susceptibility or resistance of pathogenic microorganisms to the antimicrobial agents in use today is thus of prime importance, and for this reason estimation of the susceptibility or resistance of the individual infecting strains likewise assumes importance. To have a proper understanding of the problem, one must have an appreciation of both in vitro and in vivo action of these drugs. They are unique among therapeutic substances in that they act against the organism itself. They do not, so far as we know, stimulate or enhance any of the body’s defense mechanisms. Their action may be bac- tericidal or bacteriostatic. Furthermore, it will matter little that the causative organism is susceptible to a given antimicrobial agent in vitro 821 822 ANTIMICROBIAL TESTS if the antimicrobial agent, due to pharmacodynamic activity or for any other reason, is unable to reach the site of the infection in sufficient quantity to be effective. The tests are usually standardized, so that in vitro susceptibility will indicate potential benefit from therapeutic use of the drug. How- ever, since many considerations other than in vitro activity determine the selection of drugs, great care must be taken to avoid recommend- ing treatment on the basis of laboratory tests alone. Indeed, labora- tory reports should include only a statement of the drug concen- trations tested and observed to be capable or incapable of inhibiting the growth of the organism in question. In no instance should re- ports include recommendations for treatment. Test procedures, more- over, should be changed as infrequently as possible in any given laboratory, since it is important that the physician responsible for treatment be familiar with the test and capable of interpreting its results. The ever-increasing number and availability of antimicrobial agents add materially to the problems confronting the laboratory. At the present time well over 20 such drugs are available in Canada and the United States. In addition to the antibiotics, there are also such agents as the sulfonamides, the nitrofurans, and agents such as para- amino salicylic acid (PAS) and isoniazid. The action of each drug is selective, and with few exceptions among the species not all members of a single species will be resistant or susceptible to an individual drug. It is common practice to test microbial cultures against several antimicrobial drugs. Resultant information often provides the physi- cian with knowledge that is useful in characterizing the infecting microorganisms and that may also provide a basis for the selection of alternate drug regimens where drug allergy is known or toxicity is a hazard. The WHO report! points out that to simplify routine tests it is necessary to include only one representative of any group of antibiotics with closely similar actions between which there is cross- resistance. The results, however, should always be reported in rela- tion to the antibiotic actually used. This report also suggests that tests performed with penicillin G are adequate for most clinical purposes, although it should be remembered that the activity of peni- cillin V differs from that of penicillin G against some species and that other penicillin drugs are being introduced into therapeutics which differ more widely in activity than those already in common use. In certain circumstances, therefore, it may be advisable to perform a test with a penicillin other than penicillin G. Streptomycin and ANTIMICROBIAL TESTS 823 dihydrostreptomycin are identical in antibacterial action, so that only one need be used. Although there are some differences among mem- bers of the tetracycline group in activity against individual bacterial species, >? cross-resistance between them is almost complete when resistance is considerably increased above the normal level and only one need be used in the test for susceptibility to all members of the group. Allowance should also be made for the instability of chlor- tetracycline in vitro. Members of the neomycin group are closely related and for most purposes not more than one of them need be used, but the test should be performed preferably with the member of the group which is to be used in treatment . Organisms resistant to erythromycin are some- times also resistant to oleandomycin and spiromycin* and sometimes not ; both may have to be included in the susceptibility test when their therapeutic use as alternatives to erythromycin is contemplated. A similar situation exists with reference to vancomycin and ristocetin. The choice of antibiotics within these groups for laboratory tests should be determined by consultation between the bacteriologist and the clinician. New antimicrobial drugs should not be introduced casually into the list of drugs tested. Inclusion of a new drug among those agents to which an organism is sensitive may imply recommendation of the compound, which in turn may at times lead to the unjustified accept- ance of the drug by physicians. Not all antimicrobial agents that perform well in vitro are therapeutically safe and effective. Acceptance of such drugs should not be encouraged without responsible medical authorization. It is of value to the technician and to the physician submitting specimens to know the susceptibility pattern of the various pathogenic bacteria to the antimicrobials in common use. Such knowledge will enable the physician to initiate treatment with greater confidence during the period when he is awaiting results of specific tests. In fact, this knowledge may make it unnecessary to perform the tests. It will also permit early detection of the emergence of drug-resistant strains—a matter of particular importance in the case of staphylococci. It is therefore assumed that the tests will be carried out with a pure culture or that the probable identity of the infecting organism is known from the examination of a stained film when the test is per- formed with a primary culture. : The degree of resistance or susceptibility of a microbial strain to an antimicrobial drug is dependent in large part upon the number of microorganisms present and the concentration of drug in contact with 824 ANTIMICROBIAL TESTS those organisms. This fact is of the utmost importance in the interpretation of laboratory drug-susceptibility data, for rarely if ever can one even approximate under in vitro conditions the number of infecting organisms or the exact concentration of the drug existing at the appropriate site within an infected host. All microbial strains are heterogeneous in nature and contain cells of widely varying degrees of drug susceptibility. The results of any in vitro drug susceptibility tests thus indicate only the degree of susceptibility of the most resistant cells within the population. An insufficient inoculum or environmental conditions preventing optimum growth may mask drug resistance. Too large an inoculum, on the other hand, may lead to results which overemphasize the importance of those resistant cells present within the total population. In a like manner, the use of high concentrations of the antimicrobial agent under test may prevent the detection of significant numbers of resistant organisms, while low concentrations of the drug may suggest a totally resistant population. Il. ANTIBIOTIC SUSCEPTIBILITY TESTS The standardization of methods for conducting microbial drug susceptibility tests has been discussed in detail in a recent report from the World Health Organization.! Such standardization is essential if laboratories are to provide information that will serve as a reliable guide to treatment. It is essential if data obtained sequentially are to be compared. The information contained in this report and the methods described are vital to any laboratory concerned with the test- ing of microorganisms for their susceptibility or resistance to anti- microbial drugs and are a requisite for any physician charged with the responsibility of interpreting laboratory results of drug sus- ceptibility tests. Two methods are commonly used to determine the susceptibility of a given microbial strain to an antimicrobial drug: (1) the diffusion method and (2) the dilution method. In the diffusion method, the antibiotic is allowed to diffuse in solid medium, which is subsequently inoculated with the organism in question, and the activity of the drug is measured by the presence of a zone of growth inhibition surround- ing the site of inoculation of the antimicrobial drug. In the dilution method, varying concentrations of antibiotic are added to a series of tubes or other containers of a liquid or solid culture medium, each of which is then inoculated with the microorganism to be tested, and the presence or absence of growth is recorded after a suitable incubation period, with the lowest concentration of drug capable of completely ANTIMICROBIAL TESTS 825 inhibiting growth accepted as the minimal inhibitory concentration of that drug for the particular microbial strain tested. Both technics require careful standardization of culture inoculum and both are dependent on the length of time required for growth of the microorganism being tested. Since by definition the ability of the antimicrobial agent to inhibit microbial growth is the property of the agent that is of prime interest, all procedures designed to measure this activity require an incubation period for the growth of the organism. No rapid method for estimating their activity has been developed. Only by directly testing the susceptibility of organisms in clinical specimens without prior isolation of the significant pathogen(s) in pure culture is it possible to speed the process. Such direct test methods suffer, however, from the inherent fallacy associated with an uncontrolled and often inadequate inoculum. Although rapid and at times helpful, they frequently lead to erroneous conclusions. They are of value primarily in testing the susceptibility of tubercle bacilli, whose rate of growth is so slow that the clinician must decide upon a suitable therapeutic regimen before laboratory reports are available. A. Culture Media When a liquid medium is employed, any nutrient broth that allows good and rapid growth of the pathogen in question may be employed. A buffered yeast extract medium (CM No. 116) has been used for years by the New York City Department of Health for this purpose. It is also important to recognize the fact that media containing thioglycolate may inactivate certain antimicrobial drugs, particularly penicillin. The preparation of solid media containing standardized quantities of drug may at times prove difficult, in which event it may be desir- able to use a commercially prepared medium. Many of the anti- microbial agents are partially degraded on autoclaving, and drug solutions should be added only to previously sterilized media shortly before use. Laboratories employing inspissated media in particular may wish to use commercially prepared media, since standardization of the concentration of drug in inspissated media is especially difficult. Antibiotics in culture media are usually no more stable than they are in aqueous solution. Media containing antimicrobial drugs should therefore be stored under the same conditions as specified for anti- biotic stock solutions in the following paragraph. B. Time and Temperature of Incubation A temperature of 34° to 36° C and an incubation time of 16 to 18 hr are satisfactory for conducting most susceptibility tests. Most 826 ANTIMICROBIAL TESTS bacteria grow well under these conditions, and currently used anti- biotics do not suffer marked loss of potency. However, this time must be extended in some instances: Certain fungi and other higher bacteria must be incubated for at least 48 hr, while Mycobacterium tuberculosis requires 14 days for incubation. C. Antibiotic Stock Solutions® Dilute a weighed quantity of antibiotic* in sterile distilled water (erythromycin should first be put in solution with a minimal amount of methyl alcohol and then diluted with water) to make a concentra- tion of 100 u. or pg per ml or higher when resistant organisms are anticipated. Sterilize the antibiotic solutions by passing through a sintered-glass bacteriologic filter. Dispense small amounts (1-2 ml) into 13X100 mm test tubes or other suitable containers. Store these in a deep freeze at —20° C or in a carbon dioxide chest until needed. When needed, remove an amount sufficient for the day’s test from the freezer and thaw. After thawing, mix thoroughly. If a freezer or carbon dioxide box is not available, use the freezing compartment of an ordinary refrigerator. As an additional check on the proper strength of the antibiotic, a standard organism may be included for each antibiotic used. D. Methods 1. Agar diffusion (medicated disk) method—The medicated disk method is the most widely used technic in routine laboratory tests for determination of the susceptibility of microorganisms to anti- microbial agents. It is a simple, rapid method that when used properly will yield results comparing favorably with those obtained by the broth tube dilution method. The concentration of antibiotic in the disk should be such that it will give an indication of the susceptibility or resistance of the organ- ism to the particular drug in terms of the anticipated clinical end result. The selection of concentrations for this purpose can be very difficult, since the technics used in different laboratories are rarely identical and disks with potencies satisfactory for one laboratory may not be equally satisfactory for another. In some European countries, levels up to 1,000 u. or pg per disk are used.® On this continent we prefer levels which will yield on * Antibiotic diagnostic kits containing buffered preweighed small amounts of all commonly used antibiotics are available. They may be obtained from the Laboratory of Hygiene in Ottawa, Canada, or without charge to any requesting laboratory from Chas. Pfizer & Co., Inc, New York, N. Y. ANTIMICROBIAL TESTS 827 diffusion, in theory at least, levels of antibiotic similar to those which have been obtained in blood and urine during treatment. To measure satisfactory performance, special regulations have been put in force in Canada” and similar regulations have been proposed for the United States.® In Canada, before a manufacturer can distribute his disks he is required to show that his methods of manufacture and control are capable of producing uniform and satisfactory disks. Prior to their release each lot is tested for potency (by an extraction technic) and for performance as well. The individual disks may not vary more than == 40 per cent of the labeled potency and, for satisfactory performance, must release the antibiotic within 5 min after being placed on an agar plate. In the United States similar requirements have been set for disk manufacture and certification. a. Preparation of plates: Suitable medium, such as beef heart in- fusion agar (CM No. 22), trypticase soy agar, or heart infusion agar with blood (CM No. 25), is measured into a petri plate. It is important that the same amount of agar be added to each petri dish so that the depth of the medium will be constant. The entire surface of the plate should be seeded evenly with the specimen. Any one of several methods may be used for this purpose. A suspension of the isolated culture, preferably standardized for density, may be spread evenly over the surface of the plate with a sterile cotton swab or a sterile nichrome spatula; or 0.5 ml of broth culture can be added to 5 ml of suitably cooled agar and poured over the base layer of 5 ml of the same medium which was poured previously and allowed to harden. These procedures may be carried out before isolation and identification of the infecting agent. However, whenever possible a pure culture should be used, since mixed cultures do not always reflect the true susceptibility of the specific infecting microorganism. Greater uniformity of inoculum will be obtained if a young culture of standard turbidity is used. Only fresh agar plates should be used, for very dry plates may interfere with the release of antibiotic from the disk. The disks are then placed on the inoculated agar surface with a sterile forceps or are dropped simultaneously from an automatic dispenser, pressed gently, and incubated overnight. b. Reading the plates: The zones of inhibition surrounding the disks are recorded following incubation. These will vary in size, de- pending on a number of factors. Antibiotics vary in their ability to diffuse in an agar medium and the size of the zone may, in some in- stances at least, be more of a measure of the diffusion rate than of the susceptibility of the bacteria. 828 ANTIMICROBIAL TESTS Since the apparent rate of diffusion alone is not necessarily a sign of relative therapeutic efficiency, great caution should be exercised in the interpretation of zone size. The zone size will vary with the age and thickness of the agar medium, the density of the inoculum, and the culture medium used. In general it has been conceded that zone sizes should not be reported as such and only the presence or absence of a clear zone should be considered of significance. In prac- tice, some have found that small zones of 4 mm or less may be an indication of susceptibility. Sometimes a very small zone or no zone at all may be observed with organisms that are highly susceptible to the same antibiotic by the tube dilution method. Penicillin disks, for example, usually have large inhibition zones with susceptible strains of Staphylococcus aureus. Oc- casionally, however, fast-growing strains will grow to the edge of the disk, giving the appearance of resistance, even though they can be shown to be highly susceptible by the tube dilution test. Personal experience would therefore appear to be the best guide to interpretation of test results and results preferably should be re- ported only as “susceptible” or “resistant.” Findings have been re- ported according to the various degrees of susceptibility or resistance when more than one disk level has been used for the test. It is recommended that susceptibility test results be reported according to the directions supplied with the disk being used. The single-disk susceptibility tests of Kirby® and of Anderson!® are commendable approaches to the standardization of susceptibility testing in clinical laboratories. The reader is also referred to the collective views of an international group of experts on the matter of standardization. 2. Dilution method—Place 10 sterile 16X100 mm test tubes in a rack. To each of the last 9 tubes add 0.9 ml of the broth employed in the test. To the first tube (empty) add 1.8 ml of the same broth. Then add 0.2 ml of stock drug solution containing 100 or 1,000 u. or pg of active drug per ml of solution to the first of the series. This tube will then contain a broth solution with the equivalent of 10 or 100 u. or pg of the antimicrobial agent per ml of solution. Mix well and transfer 0.9 ml of drug containing broth from tube 1 to tube 2. Again mix well and transfer 0.9 ml from tube 2 to tube 3. Continue this process until the ninth tube is reached. Discard 0.9 ml of the drug-containing broth from the ninth tube. The last (tenth) tube will contain broth only and will serve as a culture control. Care must be taken that the contents of each of the tubes are well mixed before transfer of the solution to the next tube. In addition, ANTIMICROBIAL TESTS 829 care must be taken to avoid depositing any portion of the transferred solution onto the upper wall of the tube, as the resultant loss of solu- tion causes significant discrepancies in the concentration of drug in the broth. A separate sterile pipette should be used for each individual transfer, since the quantity of the drug adhering to the wall of the pipette likewise increases markedly the error of the test. Use a 6 hr plain broth culture prepared from a freshly cultivated 16-18 hr broth culture. A beef infusion medium buffered at a pH between 7.4 and 7.8 is sufficiently rich to support the growth of most pathogenic organisms. The 6 hr cultures prepared as described should be diluted with broth to a constant density before use. A density equivalent to a MacFarland barium sulfate No. 1 Standard and allowing 70 to 80 per cent transmission on a Photovolt Lumetron #400 is satisfactory for most bacterial species. Add 0.1 ml of suitably diluted culture to each of the drug-contain- ing tubes prepared as above and again mix well. Incubate at 35° C for 18-24 hr, or until the control tube containing no antimicrobial drug shows heavy growth. The susceptibility of an organism may be accepted as the least amount of drug causing complete inhibition of growth as evidenced by absence of gross turbidity upon completion of the incubation period. It is important to remember that this test is a measure of bacteriostatic effects only. If a bactericidal end point is desired, each tube showing no growth at the end of the incubation period must be subcultured (preferably onto a solid medium) to determine the lowest concentration capable of actually killing all organisms. The procedure may be modified for use with a solid medium when indicated. In such instances a series of standardized drug solution containing graded concentrations of the drug in question must be prepared in advance. Prior to the test, pipette 1 ml of each such solu- tion into a sterile test tube, add to each 9 ml of the desired medium, making certain that it has been cooled to below 50° C. Mix well, slant, and allow to harden. A 2 mm platinum loopful of culture grown as described above provides an adequate inoculum, although the number of organisms likely to exist in such an inoculum may vary to some extent. Incubate at 37° C. The least concentration of drug capable of inhibiting growth, as evidenced by the absence of colonial growth at the end of the incubation period, may be accepted as the minimal inhibitory concentration of the antimicrobial agent, or the susceptibility of the organism. 3. Testing susceptibility of M. tuberculosis strains—A modi- fication of the dilution method may be used to test the susceptibility of 830 ANTIMICROBIAL TESTS strains of M. tuberculosis to streptomycin, isoniazid, viomycin, or para-aminosalicylic acid (PAS). This method may be carried out satisfactorily on Lowenstein-Jensen (CM No. 101) or on ATS medium (CM No. 100). Middlebrook’s 7-H-10 medium (CM No. 104) is also satisfactory. Special media for testing the effects of other antimicrobial agents are described in detail in the Manual of Laboratory Methods of the V.A-Armed Forces Cooperative Study on the Chemotherapy of Tuberculosis. tt Solutions of the antimicrobial drugs should be prepared quanti- tatively, using pure sterile drugs containing no preservatives or other additives. Do not use tablets or parenteral products dispensed for other purposes and containing substances other than the antimicrobial agent in question. Pure antimicrobial drugs dispensed for laboratory purposes are labeled with the information indicating the quantity of solvent required to give a solution containing a specific concentration of drug. Appropriate dilutions should be made from such a stock solution. All solutions should be stored in the refrigerator. To avoid possible contamination or evaporation, as well as decomposition of the drug itself, the use of freshly prepared drug solution is recom- mended. This is mandatory for cycloserine, which is unstable in solu- tion. To prepare drug-containing media, divide liquid egg medium before inspissation into as many parts as the number of drug concentrations desired. Add solutions of the antimicrobial drug to each portion of egg medium in sufficient quantity to give the desired resultant drug concentration. Mix the drug thoroughly with the liquid egg medium, subdivide into tubes or bottles as desired, and inspissate for 1 hr at 90° C. Store in refrigerator until used. Cycloserine media should not be stored longer than 2 weeks. Several concentrations for testing the susceptibility of strains of M. tuberculosis to the standard antituberculosis agents are recom- mended : Streptomycin* 0, 3.8, 10, 100 pug per ml Isoniazid 0, 0.2, 1.0, 5 ug per ml Viomycin* 0, 10, 100, ug per ml PAS 0, 5 10, 100 pg per ml Cycloserine 0, 3 10, 20, 50 pg per ml Pyrazinamide 0, 10, 20, 100, 500 ug per ml * As a result of heat lability, absorption and other processes, loss of activity of some drugs occurs on incorporation into the medium or on subsequent inspissation. To compensate for this loss, it is necessary that the concentrations of streptomycin and of viomycin prior to inspissation be adjusted to 0, 10, 30, 300 ug per ml and 0, 12.5, 125 ug per ml of medium, respectively. ANTIMICROBIAL TESTS 831 Drug-containing and control (no drug) tubes of media should be inoculated in the following manner : Remove portions of all colonies or widely representative portions of the culture surface with a platinum spade and place in a sterile tube or mortar. Add a few drops of diluent to the bacillary mass and grind ; add diluent gradually to produce a heavily turbid suspension (3-6 ml). Allow suspension to stand for 60 min, so that the undispersed clumps will settle. Transfer supernatant to a sterile tube, add sufficient diluent to bring turbidity to the equivalent of a MacFarland barium sulfate No. 1 Standard and allowing 70 to 80 per cent transmission on a Photovolt Lumetron #400. Then dilute the suspension 1:10 with diluent, Inoculate one drug-free control tube (containing 0 pg per ml) with 0.1 ml of this suspension, taking care to distribute the inoculum evenly over the whole surface of the medium. Similarly inoculate all tubes of the drug-containing media. Incubate at 35° C positioning the cultures so that the surface of the medium is hori- zontal, Examine cultures weekly and record amount of growth as follows: Confluent FIOWANL + .cormmumsnes oes bumpin someon amma sabe 43 wa + 4408 44 Heavy growth, but not confluent .........c.coiiiieriineiineenenennnnnn 3+ Intermediate growth (more than 200 colonies) .........ccovveivennnnnn. 24 S0:-200 COIOHIBE . vusvcrvnis vos SHEATH ERE + obo wan warms a eames SRE TER SHES 1+ If less than 50 colonies, record actual number of colonies. Report results after 3 or 4 weeks, or when growth on the drug-free medium is 3+ or 4+. Report the extent of growth on the drug-free medium, as well as the extent of growth on the drug-containing medium. Do not report the strain as susceptible or resistant, for in- formation is not yet available to indicate the threshold of resistance. 4, Measuring the combined action of antimicrobial drugs—No reliable method exists whereby one may estimate the susceptibility of a microbial strain to two or more antimicrobial drugs used in com- bination. The susceptibility of any organism to an antimicrobial drug is dependent upon the number of cells present and the concentra- tion of the drug in contact with those cells. One cannot approximate in vitro the number of cells that will exist at any given site within the infected host. Moreover, no two drugs are absorbed at the same rates following administration, nor are any two drugs distributed identically within the body. Thus one cannot estimate the relative concentrations of two or more drugs that will exist at any given site within the body. 832 ANTIMICROBIAL TESTS It is recommended in all instances that the susceptibility of the in- fecting microbial strains be tested against each individual antimicrobial drug separately and that the results of such tests be reported indi- vidually to the physician, who then decides which agent or combina- tion of agents may be most effective for the patient under treatment. Antagonism between antimicrobial agents has rarely if ever been observed clinically and should not be of concern to the physician. E. Apparatus Special petri dishes—When it is essential that there be little varia- tion in the thickness of the medium, specially made flat-bottom petri dishes should be used. These may be obtained from various ap- paratus supply firms and are listed in the Corning catalog as item No. 3162. Petri dish covers—These may be either Coors porcelain, glazed on the outside and unglazed on the inside, to fit 100 mm glass petri dish bottoms ; or aluminum covers containing an absorbent cardboard inner liner. The porcelain covers are listed as a stock item in most ap- paratus suppliers’ catalogs. The aluminum covers may be obtained from the Baltimore Biological Laboratories, Baltimore, Md. Instruments for reading zone diameters—The Fisher-Lilly zone reader is available from the Fisher Scientific Co. and is described in the company’s catalog under item No. 7-907. Colorimeters and other instruments—Many such instruments are available that are satisfactory for measuring the turbidity of cultures used in the tests. The photovolt Lumetron #400 is used extensively. CaroLyN R. FALk, Chapter Chairman Freperick C. Fink, Pu.D. Lours GREENBERG, PH.D, Grapnys L. Hoey, Pu.D. VeErNoN KnicuT, M.D. REFERENCES 1. Standardization of Methods for Conducting Microbic Sensitivity Tests. WHO Tech. Rep. Ser. No. 210. Geneva, Switz.: WHO, 1961. 2. WeLcH, H.; Ranparr, W. A.; Reeny, R. J.; and OswaLrp, E. J. Varia- tions in the Antimicrobial Activity of the Tetracyclines. Antib. & Chemo- therap. 4:741-745, 1954. 3. Reeny, R. J, Ranparr, W. A, and WeLcH, H. Variations in the Anti- microbial Activity of the Tetracyclines, IT. Antib. & Chemotherap. 5:115- 123, 1955. ANTIMICROBIAL TESTS 833 4. 10. Al. MANIAR, A. C,, Emus, L., and GREENBERG, L.. A Comparison of the in vitro and in vivo Activity of Erythromycin and Spiramycin. Antib. & Chemo- therap. 10:726-730, 1960. Grove, D. C., and Ranparr, W. A. Assay Methods of Antibiotics. Anti- biotics Monographs No. 2. New York: Medical Encyclopedia, Inc., 1955. Lunp, Erna. Resistance Determinations with Tablets Containing Various Amounts of Active Substances. Acta path. et microbiol. scandinav. 33:278- 284, 1953. Food and Drug Regulations (Canada). Issued by the Department of Na- tional Health and Welfare, Sept. 18, 1958. Antibiotic Drugs. Intended for Use in the Laboratory Diagnosis of Disease. 21 CFR, Parts 146-147. Notice of Proposed Rule-Making. Fed. Register, Apr. 9, 1960. Kimrey, W. M. M,, et al. “Clinical Usefulness of a Single Disc Method for Antibiotic Sensitivity Testing,” in Antibiotics Annual, 1956-1957. New York: Medical Encyclopedia, Inc., 1957, pp. 892-897. AnDErRsON, T. G., and TrovaNosky, A. “Antibiotic Susceptibility Testing by the Disc Method,” in Antibiotics Annual, 1959-1960. New York: Medical Encyclopedia, Inc., 1960, pp. 587-595. Manual of Laboratory Methods of the VA-Armed Forces Cooperative Study on the Chemotherapy of Tuberculosis. Washington 25, D. C.: VA Dept. of Med. & Surg., Central Office, July 1960. I. IL III. IV. VIL VIL CHAPTER 28 BLOOD GROUPING AND Rh TYPING Blood Grouping A. Minimum Requirements of Anti-A and Anti-B Blood Grouping Sera B. Technical Procedures 1. Test Tube Method—Preparation of Cell Suspension 2. The Test 3. The Well Slide Method 4. The Flat Slide Method 5. Confirmation of Blood Grouping or Reverse Blood Grouping C. Subgroups of Groups A and AB Rh Typing A. Introduction B. Complexity of the Rh-Hr System C. Rh-Hr Antibodies D. Rh Typing with Anti-Rh,(D) Saline Agglutinins E. Method of Testing F. Rh Typing with Albumin Agglutinins 1. Technic of Slide Test 2. Sources of Error . Test Tube Typing with Albumin Antibodies . The Weakly Reacting Variety D* The Subtypes of the Rh-Hr System A. Method for Determining Subtypes B. Selection of Donors C. Differentiation of Homozygous and Heterozygous Rh-Positive Individuals D. Presumptive Method IQ Isoimmunization of Rh-Positive Individuals by Pregnancy and Transfusion . Detection of Intragroup Isoimmunization A. Introduction 1. Selection of the Test Blood 2. The Test B. The Indirect Antiglobulin Test for Detection of Intragroup Isoimmunization 1. Titrations 2. Diluents, Suspending Media and Test Cells Tests To Determine the Specificity of Rh-Hr and Other Antibodies The Antihuman Globulin Test (Coombs Test) A. Introduction 1. In vivo Sensitization: The Direct Test 2. In vitro: The Indirect Test B. The Direct Coombs Test 834 BLOOD GROUPING, RH TYPING 835 VIII. The Compatibility Test A. The Major Compatibility Test B. The Minor Compatibility Test With the increasing use of blood transfusion and the importance of intragroup isoimmunization, mainly by the Rh, or D factor, in the pathogenesis of hemolytic disease of the newborn and intragroup hemolytic transfusion reactions, it becomes essential to employ ac- curate methods for determinations of the ABO blood group and Rh type (Rh+ or Rh—) and for compatibility tests. These are the tests required for selection of a compatible donor for blood transfusion. In prenatal testing, the same procedures are required but instead of a compatibility test, itis necessary to detect those patients, Rh-negative or Rh-positive, who may have been immunized to one or more blood factors as a result of previous transfusions and/or pregnancies. In addition, husbands of immunized women should be tested in order to determine their most likely genotype as homozygous or hetero- zygous for Rh, (D) factor or, exceptionally, for other blood factors. If a patient to be given a blood transfusion or a pregnant woman is found to be immunized, the serum is then tested to identify the one or more antibodies present. I. BLOOD GROUPING The well-known scheme of the four blood groups, based on the presence of two main agglutinogens or factors, A and B, and their corresponding antibodies, anti-A and anti-B, are given in Table 1. Table 1—Blood Group Distribution Based on Agglutinogens A and B Per cent Antigens in Antibodies Incidence, Group Red Cells in Serum U. S. Caucasoids Oo - anti-A, anti-B 45 A A anti-B 40 B B anti-A 11 AB A+B PY 4 Reactions with two sera, anti-A (obtained from individuals of group B) and anti-B (obtained from individuals of group A), yield the four blood groups. The normal concentration of anti-A and anti-B 836 BLOOD GROUPING, RH TYPING varies from individual to individual. As diagnostic reagents they are frequently obtained by immunizing volunteers with chemically pre- pared extracts from animal sources containing A or B activity. As a rule, the A substance is extracted from the gastric mucosa of hogs and the B substance from the gastric mucosa of horses. As a result of the injection of volunteers with these substances (group A donors injected with B substance and group B donors with A substance), powerful diagnostic reagents are now available. These are particularly valuable for detection of the weakly agglutinable variants of the A factor. The scheme in Table 1 represents an oversimplification and does not include the variants in the A factor, that is, subgroups A, the less agglutinable Ao, and the still less sensitive Az, A4 and A,. By and large, such striking variation in agglutination does not exist in group B individuals, although the presence of B in A.B diminishes the agglutinability of A» with anti-A. There is a similar but less strik- ing effect of A; on agglutinogen B in blood group A;B. The scheme of arrangement of the isoagglutinins is also oversimpli- fied because the two antibodies in group O do not represent a separate anti-A plus a separate anti-B. In point of fact, the anti-A of some O sera has slight to distinct affinity for B and, similarly, anti-B has some affinity for A as indicated by absorption and elution experiments. These have been referred to as cross-reacting antibodies. Further- more, the antibodies of group O mothers more readily pass the placenta than the antibodies of group A and group B mothers. These recently established facts have a practical bearing on the pathogenesis of hemolytic disease due to ABO incompatibility. Table 1 does not take into account the occasional presence of (1) a weak anti-A; in the sera of group A: and especially AB individuals, and (2) a weak anti-Az (more frequently called anti-H or anti-O) in the sera of group A; but more frequently in the sera of A;B indi- viduals. Still more rarely, other atypical antibodies independent of the four blood groups may be present in normal human serum, such as anti-Lewis, anti-P or anti-M. Fortunately, when these are present, they are often only in low titer and their activity is limited mainly to temperatures below 37° C. A. Minimum Requirements of anti-A and anti-B Blood Grouping Sera The National Institutes of Health requires that all blood grouping sera satisfy certain requirements of avidity, titer and specificity. The avidity values, as determined in tests on the open slide of 10 per cent BLOOD GROUPING, RH TYPING 837 suspensions of the various red cells, are given in the following tabula- tion: Time Beginning Serum Cell Suspension Agglutination (seconds) anti-A Ay 15 A, 30 A,B 30 5B 45 anti-B B 15 The titer requirements of a potent diagnostic serum as given by the National Institutes of Health for the test tube technic using a 2 per cent suspension are as follows: Minimal Acceptance Serum Cell Suspension Titer or Unit Value anti-A A, 256 A, 128 AB 128 A,B 64 anti-B B 256 The sera must be specific, that is, they must not contain atypical agglutinins, and they must be free from undesirable qualities which may serve as sources of error—for example, isohemolysis, rouleaux formation, or autoagglutination. Technic of blood grouping—Blood grouping may be carried out by any one of the several recognized procedures. The test tube and the flat slide methods are more frequently employed for large- scale testing. The two reagents are identified not only by proper label- ing but in addition anti-A contains a blue or green dye, while anti-B contains a yellow dye. In selecting a method for blood grouping and simultaneous Rh typ- ing, much will depend upon the variety of anti-Rh reagents available, the type of equipment, such as test tubes and centrifuges, and the volume of work. B. Technical Procedures 1. Test tube method: preparation of cell suspension—A 2 per cent cell suspension (in terms of sediment) in 0.9 per cent NaCl 838 BLOOD GROUPING, RH TYPING solution is prepared from clotted, oxalated or citrated blood. It is preferable to obtain blood by venipuncture so that either serum or plasma may be available for confirmation of the blood grouping with known red cells of groups A; and B. In testing freshly drawn clotted blood, washing of the red cells is not necessary, but red cells obtained from oxalated or citrated blood should be washed. 2. The test a) Place two properly labeled test tubes (10X75 mm, inside di- ameter 7 mm) in a rack. b) Add 1 drop of anti-A serum to one of the tubes and 1 drop of anti-B serum to the other, ¢) Add 1 drop of the blood suspension to be tested to each of the two tubes. Shake the tubes thoroughly for proper mixing of reagents. In testing numerous bloods, only one pipette need be used for delivery of the cell suspensions provided that the pipette is rinsed several times with salt solution between deliveries. d) Readings may be made without incubation if the tubes are cen- trifuged at low speed (500-1,000 rpm) to avoid overpacking. As an alternative procedure, the mixture may be allowed to remain at room temperature for 1 hr and read without centrifuging. In both in- stances, readings are made after gentle shaking to dislodge the sedi- mented cells, and the presence or absence of agglutination is recorded. Readings may be made with the naked eye, but all negative or doubt- ful readings should be confirmed with the aid of a 6X hand lens or concave mirror, or by microscopic examination. e) The stick test method as described in Section II of this chapter may also be used. 3. The well slide method—Add 1 drop of anti-A serum to one well and 1 drop of anti-B serum to an adjacent well. Add 2 drops of the 2 per cent suspension in salt solution of the red cells to be tested. Rotate the well slides either by hand or preferably on a rotat- ing machine for 10 min at room temperature. Read macroscopically over an illuminated glass background. 4. The flat slide method a) Preparation of the cell suspension: With the flat slide method of testing donors, use whole oxalated blood or a 10 per cent suspension of cells in serum or salt solution. In testing recipients it is preferable to use 10 per cent suspensions in salt solution in order to avoid errors caused by rouleaux formation or autoagglutination, BLOOD GROUPING, RH TYPING 839 b) The test: Divide the slide in the center with a crayon. Place 1 drop of anti-A serum on one side and 1 drop of anti-B serum on the other. Add, to each, 1 drop of the 10 per cent salt solution suspension of the blood cells to be tested. Mix the suspensions on each side with different wooden applicator sticks, spreading the mixture over an area about 1 in. in diameter. On tilting the slide back and forth, beginning agglutination should be visible in less than 1 min. Nega- tive readings should be under observation no longer than 2 min. In these tests care must be taken not to interpret drying at the periphery as agglutination. 5. Confirmation of blood grouping or reverse blood grouping— The blood group should always be confirmed by testing the reactions of the serum or plasma with known group A and group B red cells. The A and B test cells should be freshly drawn or preserved in a Rein and Bukantz modification of Alsever’s solution or in ACD mixture for periods not exceeding 4 weeks. For each day's run, fresh cell suspensions should be prepared. The A test cells should be of subgroup A; or a pool of five or more A bloods and the B test cells should be a pool of two or more B bloods. These tests are preferably carried out in test tubes with 2 per cent red cell suspensions. The serum should be inactivated at 56° C for 5-10 min to prevent a source of error due to very weak agglutination from partial hemolysis. In testing plasma, agglutination only will be observed. Except for the blood of infants and rarely in adult patients or donors, the isoagglutinin content of the serum will correspond perfectly with the ABO factors in the red cells, as shown in Table 1. When the tests of red cells and serum fail to agree, further tests are required. Unexpected reactions with a particular serum may be caused by atypical agglutinins, which in the case of patients may cause difficulty in compatibility tests. In donors these are rarely of importance. Sera giving atypical reactions should be set aside for later studies, as described under identification of atypical antibodies. The value of reverse blood grouping is seen also in the studies of a type of blood superficially resembling group O but which should be classified as group A. The A factor in this type of blood is poorly agglutinable or entirely inagglutinable with the most potent anti-A of group B. These bloods, called A, bloods, include Am (genetically de- termined) and Ag (acquired in some blood disorders). They contain the usual anti-B and may, in addition, contain anti-A,, so that super- ficially they may resemble group O. This error can be detected if all 840 BLOOD GROUPING, RH TYPING bloods initially screened as group O are tested with diagnostic group O sera whose anti-A may agglutinate weakly reacting bloods of this phenotype. The principal importance of serum antibody tests (reverse blood grouping) is for the detection of clerical errors in records and reports, C. Subgroups of Groups A and AB By and large, the differentiation of A; and A has no importance in the transfusion of group A recipients with group A blood. In short, A; blood may be given to Az recipients and Az blood may be given to A, recipients. In rare instances the serum of individuals of subgroup A; or A,B contains the atypical anti-Az (also known as anti-O or anti-H), or the serum of individuals of Az or A2B contains the atypical anti-A;. These atypical agglutinins are of low titer but may react under the conditions of compatibility testing. They do not cause any serious blood destruction in vivo but may diminish survival of the transfused red cells, The A;— A, differentiation becomes important in the use of group O universal donors because the ill effects of the high titer anti-A are specifically directed toward the A; recipient. More recently it was shown that in hemolytic disease due to AXO incompatibility, only A; infants are affected, so that the disease in the infant is limited to those derived from matings A;XO or A;BXO and not from AsXO or AsBXO. The A; — A, differentiation, how- ever, is not clear-cut in infants. Many infants serologically A. at birth are later found to be in subgroup A,. The classification of bloods as A; or Az may be made with the aid of an absorbed anti-A serum or an extract of Dolichos biflorus seeds. Absorbed anti-A is a serum from a group B individual absorbed with A blood cells. The reactions obtained with this reagent are consider- ably weaker than those obtained with anti-A serum. There is also a type of blood which is intermediate between A; and A, occurring more frequently in Negroes than in Caucasoids. 1. Test tube method a) Add 1 drop (0.05 ml) of the anti-A, reagent to a small test tube (10X75 mm). b) Add 1 or 2 drops of a 2 per cent suspension in salt solution of the red cells to be tested. c) Allow to stand for no longer than 15 min and read, preferably without centrifugation. BLOOD GROUPING, RH TYPING 841 d) Group A; (or A,B) cells will show gross agglutination, while Az (or AzB) cells will be negative. Cells of the intermediate type may show weak agglutination, which may become more distinct only after centrifugation. e) Known A; and A; bloods should always be included as controls. 2. Slide tests a) Using a 10 per cent suspension of cells from freshly drawn blood washed in salt solution, add 1 drop of the serum and mix with the aid of an applicator. b) Rotate the slide slowly. ¢) Group A, blood will show beginning agglutination before 1 min and gross agglutination will be visible within 2 min. A,B cells may react somewhat more slowly. Do not read after 2 min. Cells of the intermediate type may be missed when the slide method is used. The positive identification of As bloods can be made with the aid of an agglutinin known as anti-H (or anti-O) which reacts with bloods of group O, Az, and A, but not with A;. Potent anti-H is fre- quently found in normal chicken serum, eel serum and extracts of certain lectins—for example, Ulex europaeus. Weakly reacting anti-H may also be found in the serum of A;B and more rarely in A, individuals.?* Il. Rh TYPING A. Introduction Rh typing is now routine for all candidates for blood transfusions and for all bloods collected in the blood banks. Tt is also routine for the pregnant woman and in certain instances for her husband. Clinically, the most important differentiation is that of Rh positive and Rh negative as determined in tests with human anti-Rh, (anti-D) serum. It is now generally accepted that candidates for blood transfusions, and particularly all females, found to be Rh negative must receive Rh-negative blood. The application of this simple measure will sharply reduce the incidence of intragroup hemolytic transfusion accidents, and, for the female Rh-negative population, it will also reduce the incidence of hemolytic disease of the newborn. From the point of view of preventing transfusion accidents in general, it is preferable to think in terms of at least eight blood types rather than the classic four blood groups. Since the scheme of the * No attempt has been made to key all bibliographical listings to text. 842 BLOOD GROUPING, RH TYPING four blood groups is genetically independent of the Rh factor, the frequency of Rh-positive and Rh-negative individuals within any of the four blood groups is consistently 85 per cent and 15 per cent respectively, These relationships for a Caucasoid population of the United States and Western Europe are given in Table 2. Table 2—Frequency of Rh-Positive and Rh-Negative Individuals in Relation to Blood Groups in the Caucasoid Population of the U. S. and Western Europe Antiodies Reactions of Red Blood Cells of Group Group in Serum oO A B AB A anti-B 0 0 + 4 B anti-A 0 + 0 + Incidence in population 45% 41% 10 % 4 % Incidence of Rh factor 38% 35% 8.5% 3.4% Incidence of Rh— factor 7 % 6 % 1.5% 0.6% B. Complexity of the Rh-Hr System There are a large number of Rh-Hr antigens, each of which is inherited and is detectable in the heterozygote. The most important antigen is Rho (D). This antigen is rarely if ever inherited alone. Two contrasting pairs of antigens, rh’(C) or hr’(c¢) and rh” (E) or hr” (e), are also important, and one antigen of each pair is almost always in- herited along with Rh,(D), which has no known contrasting antigen. The five factors are identified with the aid of selected and standard- ized human sera containing specific immune isoagglutinins, The in- cidence of positive and negative reactions in a Caucasoid population is given in Table 3. Table 3—Incidence of Positive and Negative Reactions in a Caucasoid Population Per cent Per cent Alternate Terminologies Positive Negative Anti-D anti-Rh, 85 15 Anti-C anti-rh’ 73 27 Anti-c anti-hr’ 80 20 Anti-E anti-rh” 30 70 Anti-e anti-hr” 97 3 BLOOD GROUPING, RH TYPING 843 An Rh-positive individual is one whose red cells react with anti- Rh,(D) irrespective of their reactions with other Rh-Hr antisera. Similarly, an Rh-negative individual is one whose red cells fail to react with anti-Rh,(D) irrespective of their behavior with other Rh- Hr antisera (see Section III of this chapter). C. Rh-Hr Antibodies Rh-Hr antibodies are derived from human sources and, except for anti-Rh,(D), all others are produced only infrequently. Each of the antibodies may occur in at least two forms, as indicated by their be- havior in tests with suspended cells in salt solution. One variety, and the less frequent one, will agglutinate red cells suspended in salt solution; in this chapter these will be referred to as “saline agglutin- ins.” The more frequent variety will combine and sensitize cells sus- pended in salt solution without causing visible agglutination. This variety, however, will agglutinate directly red cells suspended in many hydrophilic colloids (human plasma or serum, bovine or human albumin, gelatin, acacia, pectin, methyl cellulose, dextran, and poly- vinyl alcohol and some of its derivatives). At present the reagent of choice is 20-30 per cent bovine albumin. These antibodies will be designated as “albumin agglutinins” (see Table 4). Table 4—Comparison of Human Rh-Hr Saline and Albumin Agglutinins Saline Agglutinins Albumin Agglutinins Other terms Suspending media for test cells Typing technic Indications for use Stability Available supply For Rh, (D) For others Agglutinins, early immune, complete, bivalent Saline Test tube incubated at 37°-40° C, capillary tube Large-scale work generally more accurate Destroyed above 65° C Low Low Agglutinoids (glutinins), late hyperimmune, incom- plate univalent blocking, inhibiting, coating Bovine or human albumin, serum or plasma, other hydrophilic colloids Warm slides, modified test tube (stick test) Rapid results in small scale on slides, large scale with modified tube method Generally very stable High Low 844 BLOOD GROUPING, RH TYPING The sensitizing property of albumin agglutinins on the red cells in salt solution results in the coating of the surface of the unagglutin- ated red cells with a layer of human antibody globulin. Such coated red cells will then be agglutinable with antihuman globulin from rabbits or other animals. Albumin agglutinins will react directly with red cells in salt solu- tion which have been treated with a suitable proteolytic enzyme, say, trypsin, papain, ficin or bromelin. The mechanism of the enzyme action on the red cells is not understood. D. Rh Typing with Anti-Rh,(D) Saline Agglutinins Saline agglutinins for routine Rh typing are sometimes preferred because the same cell suspension may be used for blood grouping, for Rh typing, and for subtyping. In the initial screening of all bloods as Rh positive or Rh negative, it is important to consider the nonreacting type as presumably Rh negative. These bloods should be tested further to exclude from the final list of Rh-negative donors the weakly reacting variety of Rh positives known as D" described in Section H following. Anti-Rh,(D) saline agglutinin typing serum should meet the fol- lowing requirements of the National Institutes of Health: 1. It must give a clear-cut differentiation of Rh-negative and Rh-positive bloods, without the undesirable properties of rouleaux formation or hemolysis. 2. Specificity: It must react with the Rho, (D) factor only. The reagent must not contain anti-A or anti-B agglutinins and must be free of atypical agglu- tinins. Titer: It must have a titer of at least 1:32. Protein content: The final protein content of a diluted serum must not be below 25 per cent of its original protein content; 6 per cent bovine albumin or normal serum of group AB may be used as diluent. 5. The serum should be sterile and when stored at 5° C or lower should retain its potency for at least one year. os E. Method of Testing 1. Preparation of cell suspension—See Section I B (1) of this chapter. 2. Performance of test—Add 1 drop of antiserum to a small test tube (10X75 mm) and then 1 drop of the washed cell suspen- sion. Shake the tube thoroughly and incubate in a water bath at 37°- 40° C for 1 hr. Agglutination is enhanced by occasional shaking during the course of the incubation. 3. Readings—The presence or absence of agglutination is noted. Before disturbing the contents of the tube, examine the BLOOD GROUPING, RH TYPING 845 character of the sedimented cells with the aid of a small 6X hand lens. A round, smooth, firm sediment indicates the absence of agglutination, while an irregular, loose sediment signifies the presence of some ag- glutination, Readings are further facilitated by gentle rotation of the tube just sufficient to disturb the sediment so that the quality of the resuspended cells may be noted. Those mixtures in which no agglutination is visible may be examined microscopically. Readings may be taken after shorter incubation (15-30 min) but negative or weak reactions should be confirmed by light centrifuga- tion. The character of the sediment is noted and the cells are gently resuspended as described above. Blood showing no agglutination is considered presumably Rh negative. These cannot be finally classified as Rh negative without a test for weakly reacting factors. 4. Sources of error a. Hemolysis or contamination of the red cells. b. Contaminated, outdated serum and serum not stored at 5° C or lower, which may give false readings. It is well to plan the tests so that numerous bloods are tested at one time. In all cases, known Rh+ and Rh— blood should be in- cluded as controls. F. Rh Typing with Albumin Agglutinins In contrast to saline agglutinins, albumin agglutinins fail to agglu- tinate red cells suspended in salt solution. Typing with albumin agglutinins may be carried out either on slides or in test tubes. The slide test is sometimes preferred for small-scale work because agglu- tination is obtained within 1 min. The diagnostic serum should meet the following requirements of the National Institutes of Health: 1. Tt must give sharp, clear-cut differentiation of Rh-positive and Rh-negative bloods without the undesirable properties of rouleaux formation or hemolysis. 2. Specificity: The serum must react with the Rh, (D) factor only; the reagent must not contain anti-A or anti-B agglutinins and must be free of atypical agglutinins. 3. Titer: The serum should have a titer of at least 1:32. 4. Avidity: When used in the slide test, beginning agglutination on warmed slides should be seen within 1 min and clumps the size of 1 sq mm should appear within 2 min. 5. Protein content: Albumin agglutinins may be diluted with 20-30 per cent bovine (or human) albumin or with normal human serum. 6. The reagent should be sterile and, when stored at 5° C or lower, should retain its potency for at least one year. 846 BLOOD GROUPING, RH TYPING 1. Technic of slide test a) Preparation of cell suspension—Best results are obtained in the slide test only with heavy (40-50%) suspensions of cells in their own plasma or serum. The most convenient preparation is oxalated blood, since this usually represents approximately a 40 per cent suspension of cells, which can be tested without further manipulation. The dried potas- sium and ammonium oxalate may be prepared according to the Heller-Paul formula (6 g ammonium oxalate and 4 g potassium oxa- late diluted with distilled water to make 500 ml. Add 0.5 ml to test tube and evaporate to dryness. The tube is now suitable for 5 ml of blood). Clotted blood may be used if a sufficient number of free cells are obtained and suspended in their own or in group-compatible serum. Again, the concentration of red cells must be 40 to 50 per cent. Al- though not recommended as a routine procedure, blood obtained by finger puncture may be tested directly provided large drops are obtained and are mixed quickly with the test serum. An anticoagu- lant should not be added to the mixture. b) Performance of the test—The most rapid and clear-cut reactions are obtained when the test is performed on a prewarmed slide. An illuminated view box with a surface temperature between 40° and 50° C provides a satisfactory warming surface. Place 2 drops of the blood to be tested on the warm slide. Add 1 drop of the typing serum. Mix thoroughly with the aid of an appli- cator, spreading the mixture over about a third of the slide area. Rock the slide slowly from side to side. Readings: Positive blood will show agglutination in 30 sec or less. At the end of 2 min all red cells will be clumped, with many large masses visible. Negative blood will show no agglutination, so that at the end of the 2 min the cells remain smooth and uniform in appearance to the naked eye. Do not read microscopically or after drying has set in. Tt is well to discard the slide at the end of the 2 min reading. In order to differentiate true agglutination from pseudoagglutination, it is recommended that at the end of the 2 min reading, 1 or 2 large drops of salt solution be added and final readings made after a few rotations for mixture of the reagents. In any single run of tests, Rh-positive and Rh-negative control bloods should always be included. For controls to detect false-posi- tive reactions see the following: BLOOD GROUPING, RH TYPING 847 2. Sources of error a. False-positive results may be obtained in those rare cases where an antibody already coats the cells being tested (for example, in acquired hemolytic anemia and in hemolytic disease of the newborn). Such sensitized cells frequently exhibit nonspecific agglutination in protein media. As a control of this source of error, the test cells should be tested with a drop of plain albumin solution (20-30 per cent) in place of the typing serum. Blood cells which are agglutin- ated by the albumin solution alone may be typed with a test tube serum containing saline agglutinins. b. Satisfactory results are obtained with freshly drawn blood either oxalated or clotted. It is important to use only the specified amounts of oxalate, since the reaction is inhibited as the salt concen- tration is increased. c. The reactions are less distinct with stored blood and are entirely unreliable with contaminated blood. d. Pseudoagglutination may be inherent in the blood of many pa- tients. Such blood is not suitable for this technic of typing. A control consisting of the test blood without antiserum is required to detect the false clumping. e. Confusing effects may be produced by drying. This source of error can be eliminated if the slides are discarded at the end of 2 min. f. False-negative reactions may result from the use of too dilute a cell suspension, too low a temperature, or excessive amounts of oxa- late or some other anticoagulant . g. All bloods giving doubtful reactions or bloods screened as nega- tive should be submitted to tests for weak variants as described subsequently under heading “H” in this section. G. Test Tube Typing with Albumin Antibodies Rh typing with albumin antibodies may also be carried out in test tubes, but the blood cell suspension, as in the case of the slide test, must not be in salt solution. The suspending medium for the red cells may be bovine albumin, although in practice serum is employed— either the serum of the blood to be tested or normal group AB serum. Two testing methods are in common use, (1) the modified tube test and (2) the stick test. 848 BLOOD GROUPING, RH TYPING 1. The modified tube test a) Preparation of cell suspension—Prepare a 2-3 per cent suspen- sion of the red cells in their own serum, or preferably in group AB serum. The serum from a clotted specimen may be poured into an- other test tube and cells added to make a light suspension. b) Performance of the test—Place 1 drop of the typing serum in a small test tube (10X75 mm) and add 1 drop of the serum-cell suspension. Mix the tubes thoroughly and incubate at 37°-40° C for 15-60 min after which centrifuge lightly. ¢) Readings—Rh-positive blood will show large, solid clumps readily visible to the naked eye when resuspended. Rh-negative blood may appear to be clumped on first appearance, but on gentle shaking the sediment will resuspend smoothly. Great care must be taken in making the reading because the sediment in negative re- actions differs from that in tests using saline agglutinins, Less emphasis should be placed on the appearance of the sediment and more emphasis on the presence of clumps of agglutinated cells which persist after more vigorous resuspension. If centrifugation is em- ployed after short incubation in order to accelerate the reaction, higher speeds are required because of the greater viscosity of the reagents. At all times suitable control bloods should be included. The mixtures in tubes showing no agglutination are then subjected to the test for weak variants as described subsequently under head- ing “H” which follows. 2. Sources of error—Sources of error in performing the modi- fied tube test are much the same as those obtaining for the stick test described in the following, with especial reference to the confusing effects of abnormal proteins. 3. The stick test—All blood specimens are suitable for the stick test (clotted, oxalated, citrated and heparinized). Serum or plasma should be removed to facilitate the transfer of red cells with the ap- plicator stick. Performance of the test and readings: Add 1 drop of albumin anti- serum to a small test tube. With a clean wooden applicator stick, transfer enough red cells from the specimen to be tested to constitute a 2-4 per cent suspension in the typing serum. Centrifuge sufficiently to pack the red cells into a firm “button.” Add 2 drops (0.1 ml) of fresh salt solution to reduce viscosity and then rotate the tube to dis- lodge the red cells from the bottom. As an alternative procedure the mixtures may be incubated at 37° C for 15 min or longer prior to BLOOD GROUPING, RH TYPING 849 centrifugation. All bloods strongly agglutinated are classified as Rh positive. All doubtful and negative reactions on immediate centrifu- gation are resuspended and are further incubated for 1 hr at 35° C so that the antiglobulin test for weak variants may be performed. 4. Sources of error a. Prozone—Some potent albumin antisera fail to agglutinate after prolonged incubation, although without incubation they agglutinate very strongly. b. Weak antisera—Weak antisera require incubation for suitable agglutination, c. False-positive results—In rare cases with an autoantibody coat- ing the red cells, nonspecific agglutination may be observed in protein media. As control, another test is required using the same cell suspension in plain albumin rather than in antiserum. Bloods agglu- tinated as strongly in the control tube as in the tube containing the typing serum can usually be tested accurately with saline agglutinins. d. Confusing effects from abnormal proteins and rouleaux may be avoided by thorough removal of serum or plasma from the red cells to be tested. H. The Weakly Reacting Variety D* All bloods screened as presumably Rh negative should be tested further in order to exclude the weakly reacting varieties of those Rh factors termed D". Such bloods may give weak direct reactions with some antisera (either saline or albumin agglutinin) but will fail to react with others. They do not constitute a single entity but rather many varieties, the extremes of which are designated as “high grade” or “low grade,” depending upon the degree of their agglutinability. An antibody specific for these bloods does not exist and they can be recognized only by their capacity to adsorb Rh albumin aggluti- nins. The resulting sensitization or coating can then be detected by agglutination with antihuman globulin serum as described in the pro- cedure following. Weak variant Rh-positive bloods have been shown to be anti- genic to Rh-negative individuals either by pregnancies with hemolytic disease or by transfusions with hemolytic reactions. Weak Rh variants are more frequent in Negroids than in Caucasoids. 1. Routine procedure for detection of weak variants—All bloods presumed to be Rh negative in initial typing with either saline or albumin agglutinins should be investigated further as follows: 850 BLOOD GROUPING, RH TYPING a) Prepare a 2 per cent suspension in salt solution of the test red cells. b) Add 1 drop of albumin antiserum to a small test tube (10X75 mm) and 1 drop of 20-30 per cent albumin to another tube to serve as a control. c¢) To each tube add 1 drop of the test red cell suspension. d) Mix well and incubate both tubes for 60 min at 35° C. If both tubes are negative, then proceed to: e) Wash cells in each tube three times with tubefuls of fresh salt solution. Decant completely after each washing, taking special care in doing so after the last washing. f) Add 1 drop of antihuman globulin serum to each tube. Shake well and centrifuge lightly. g) Resuspend the sedimented cells by gentle agitation and examine for agglutination. 2. Interpretation of results a. If there is no agglutination in either tube, the blood can be classified as Rh negative. b. If agglutination occurs only in the tube which received the anti- serum, the blood is of the weak variety and should be classified as Rh positive, c. If the red cells of both tubes are agglutinated, the test cells were nonspecifically coated and cannot be tested for the Rh factor by this technic. Weak Rh variants should always be classified and registered as “Rh, (D) positive” with the notation, “weak Rh (D") variant,” added to cards and files for recording the blood group and Rh status. This applies almost equally well for patients and donors because Rh im- munization in weak variants is exceedingly rare and occurs less often than immunization to other blood factors. Il. THE SUBTYPES OF THE Rh-Hr SYSTEM A study of the Rh-Hr subtypes is necessary (1) in a comparatively small number of persons in whom isoimmunization is directed to factors other than Rh,(D) and (2) in the differentiation of Rh- positive husbands of Rh-negative women as presumably homozygous or heterozygous. BLOOD GROUPING, RH TYPING 851 A. Method for Determining Subtypes When Rh-positive and Rh-negative individuals are tested with anti- rh’ (C) serum, they are divided into two unequal subtypes, and these in turn are each further subdivided when tested with anti-rh” (E) antibodies, so that a total of eight subtypes results. These relation- ships, without reference to further subdivisions from tests with avail- able Hr antisera, are given in Table 5. The subtypes can be demon- strated with the aid of either saline agglutinins or albumin agglutinins. As in the case of Rh saline and albumin agglutinins, the sera required for a more complete Rh-Hr phenotype should be properly standard- ized with reference to titer and specificity as already given. Sera containing anti-rh’(C) antibodies may be produced by Rh- positive individuals of phenotypes Rh. (¢cDE) or Rh, (cDe), but these sera are exceedingly rare. Most frequently, these saline agglu- tinins will be found in the sera of Rh-negative patients along with Rh,(D) albumin agglutinins, Such sera may be used with cells suspended in salt solution in the tube tests, since the accompanying albumin agglutinins will not react under these conditions. These sera should not be used by antiglobulin or enzyme technics. Clinical data indicate that Rh,(D) is usually far more antigenic than the combined total of the other Rh-Hr factors and accounts for about 98 per cent of all intragroup isoimmunization, either alone or in combination with other antibodies. The remaining 2 per cent are usually Rh positive and constitute the principal source of other anti- sera. The supply of such reagents is thus limited. B. Selection of Donors For transfusion purposes it is not necessary to obtain a complete identity of antigenic structure in the erythrocytes of patient and donor. The main objective is to prevent isoimmunization and hemo- lytic transfusion reactions attributable to the clinically important Rh, (D) antigen. Provided that a suitable compatibility test for detection of intragroup antibodies is carried out, it suffices in almost all in- stances to select Rh-negative donors for Rh-negative patients and Rh- positive donors for Rh-positive patients. The routine practice in most blood banks is first to identify the 15 per cent of bloods which are presumably Rh negative and then to eliminate weak variants by direct tests. Rh-negative bloods may then be tested for the presence of the rh’(C) and rh” (E) antigens with suitable antisera which may also contain Rh,(D) agglutinins. This procedure will select about 1.5 per cent of all individuals whose red cells, although Rh negative, contain either or both additional sub- Table 5—Relationships and Frequency of Blood Group Subtypes 258 Reaction Reaction Phenotype Reaction with Frequency with with Rh-Hr CDE Frequency Anti-Rh, (D) Phenotype Percentage anti-rh’ (C) anti-rh” (E) Notation Notation Percentage + Rh; Rh, CDE 15 $i re Rh, CDe 51 + Rh 85 Rh+ + Rh, cDE 16.5 — Rh, cDe 23 + rh’rh” CdE Rare + — rh’ Cde 1 — rh 15 Rh— + rh” cdE 0.5 — rh cde 13.5 'ONIdNOY¥YD aoo018 ONIdAL HY BLOOD GROUPING, RH TYPING 853 factors. Such Rh-negative bloods—phenotypes rh’(Cde), rh” (cdE) or rth’rh” (CdE)—may be reserved for Rh-negative men, or for Rh- negative women beyond their childbearing period. In this manner, the supply of Rh-negative bloods of type rh(cde) will be conserved for more urgent cases. For a discussion of weakly reacting variants see Section IT H pre- ceding. C. Differentiation of Homozygous and Heterozygous Rh-Positive Individuals From a clinical viewpoint it is helpful to determine the genotype of the Rh-positive husband of an Rh-negative woman (genotype 77). If he is homozygous (RR), he inherited from each of his parents a gene bearing the Rh factor and all offspring must be genetically R7 and phenotypically Rh positive. Each pregnancy then offers an op- portunity for immunization. If the husband is heterozygous (Rr), having inherited one gene not bearing the Rh factor, there will be a 50 per cent chance that any offspring will be Rh negative. These two contrasting matings are shown in Table 6. Obviously, family studies can be very helpful because an Rh-negative grandparent or child immediately establishes heterozygosity of the Rh-positive parent. Table 6—Matings Incompatible for Rh Homozygous Heterozygous Father Mother Father Mother Genotypes RR X rr Rr X rr Genes in gametes Be rst ae 7 R OF Punouravsmmmns r 7 Offspring 100% Rr Rh+ 50% Rr Rh+ 50% rr Rh— D. Presumptive Method By blood typing alone it is not possible to define the Rh genotype of Rh-positive persons because there is no way of determining the presence or absence of a factor which substitutes for Rh,(D). The 854 BLOOD GROUPING, RH TYPING use of suitable antisera for Rh-Hr subfactors establishes a more rational phenotype and restricts the number of possible genotypes. An exact genotype is possible only with family data and phenotype in- formation, The presence or absence of the Rh,(D) factor is determined by chromosomes which also determine the presence of rh’(C) or hr’(¢<) and of rh” (E) or hr” (e). The presence or absence of contrasting Rh subfactors is therefore related to the Rh genotype. The combinations of familiar Rh-Hr antigens that are transmitted by a single gene or chromosome are shown in Table 7. Table 7—Frequency of Rh-Hr Antigens Transmitted by a Single Gene or Chromosome Designation of Presence or Absence of Familiar Antigens pear cent Rh Chromosome in the Gene Product* Frequency in Rho rh’ hr’ rh” hr” Caucasoids, Rh-Hr CDE D C g E e U.S.A. r cde — — 4 —- + 36.6 7 Cde _— He _— — Rs 1.4 ” cdE — - + + — 0.5 r CdE —- + — + — 0.01 Re cDe + = + — + 33 RB: CDe #2 + — ss + 41.1 R? ¢DE + = + + wt 17.0 BR CDE + + -_— + £= 0.1 * Sometimes called the properties of the ‘“‘agglutinogen.” The most common genotypes result from combinations of the most frequent chromosomes. These are R1RY, Rr, R'R?, R%, rr and R?R?, It is well to remember that the frequency of a heterozygote such as Rr is determined by the formula 2 X frequency of chromosome R® X frequency of chromosome » (because the combination may be Rr or rR'), whereas the frequency of a homozygote such as RR? is determined by the frequency of the R! chromosome squared. The serologic characteristics of the genotype are determined by the sum of the gene products of both somatic chromosomes. Because of this, more than a single genotype may have similar serologic characteristics and further distinction is not possible without family data. A number of examples are given in Table 8. BLOOD GROUPING, RH TYPING 855 From Table 8 it is apparent that blood typing with multiple Rh-Hr antisera fails to identify the exact genotype, although the “probable” or “most likely” genotype might be estimated from the frequency values. Unfortunately these frequency values vary in different popu- lations and the error of guessing can be considerably greater than indicated. On the other hand, the true genotype may often be determined by family studies. In the important mating of an Rh-positive man and an Rh-negative woman, an Rh-negative parental grandparent or an Rh- negative child establishes that the Rh-positive husband is heterozy- gous. Further opportunities to establish Rh zygosity are available with more complete blood typing. Thus, in the same mating, one child of type Rh;(CDe) and another of type Rh,(cDe) reveals that the genetic patterns of both paternal chromosomes bear the Rh,(D) factor. The Rh-Hr picture is still further complicated by the existence of additional antigens [rh™' (CV), th*(C¥), th"*(EY), hr(f), hr" (V), and rh; (Ce)] and Rh chromosomes which bear patterns of antigens differing from those shown in Table 8 [R°(-D-)]. IV. ISOIMMUNIZATION OF Rh-POSITIVE INDIVIDUALS BY PREGNANCY AND TRANSFUSION A finer serologic analysis is indicated in the study of bloods of Rh-positive mothers of affected infants and in Rh-positive patients who suffer from hemolytic transfusion reactions. In hemolytic disease of the newborn, the specific blood factor involved will be re- vealed in a comparative study of the antigens present in the red cells of the father, mother and affected infant. In patients immunized by multiple transfusions, identification of antibodies is facilitated by determining those antigens not present in the red cells of the recipi- ent or in the surviving transfused red cells but which may be present in the blood of one or more of the donors. The factors most frequently responsible for production of intra- group antibodies are hr’(c), rh” (E), and Kell. The sequence given is the order of frequency in pregnancy, whereas the order of fre- quency in transfusion reactions is reversed. More rarely a large variety of blood factors may induce isoimmunization either by preg- nancy or transfusion. The situation is complicated by the existence of blood factors with exceedingly high incidence, as well as blood factors with very low incidence, in the general population. Table 8—Examples of Serologic Characteristics of Genotypes Determined by the Sum of Both Gene or Chromosome Products Serologic Properties Per cent Frequency in Zygosity in Rh, rh’ hr’ rh” hr” Possible Caucasoids, Terms of D Cc c E e Genotypes Phenotypes U.S.A. Rh, (D) Rly (CDe/cde) 30.1 Heterozygous + + + — + R1R° (CDe/cDe) Rhirh (CcDee) 2.7 Homozygous R°r" (cDe/Cde) 0.1 Heterozygous R1R1 (CDe/CDe) 16.9 Homozygous + = —- - + { Rly" (CDe/Cde) RhiRh1 (CCDee) { 12 Heterozygous R2r (cDE/cde) 124 Heterozygous 2 - wo + ie R%" (cDe/cdE) Rherh (ceDEe) .03 Heterozygous R2R° (cDE/cDe) 1.1 Homozygous R2R? (cDE/cDE) Sg 29 Homozygous % = + + = { R2r" (cDE/cdE) RhzRhz (ccDEE) { 017 Heterazygons R°r (cDe/cde) 24 Heterozygous + - + - + { R°R° (cDe/cDe) Rhorh (ccDee) { 0.11 Homozygous R1R2?2 (CDe/cDE) 14.0 Homozygous RY" (CDe/cdE) 4 Heterozygous R2%y' (cDE/Cde) 5 Heterozygous + + f + + R*r (CDE/cde) RhiRh2 (CcDEe) .07 Heterozygous R?R° (CDE/cDe) < .01 Homozygous RR» (cDe/CdE) < 01 Heterozygous R2y (cDE/CdE) < 01 Heterozygous R2R* (cDE/CDE) { 03 Homozygous + + + + =% { R*»" (CDE/cdE) Rh.Rhz (CeDEE)* < 01 Heterozygous * Information is often not available, so that a total of nine genotypes must be considered to comprise the RhaRh: (CcDEe) phenotype. 958 'ONIdNOYD Aa0018 ONIdAL HY BLOOD GROUPING, RH TYPING 857 Atypical antibodies found in human serum may be broadly clas- sified: 1. The so-called “naturally occurring” antibodies, which are usually inactive at 37° C and may not be detected in the compatibility test. Although not re- sponsible for hemolytic disease or transfusion reactions, survival of the transfused red cells may be diminished. 2. “Warm” immune antibodies, which are often responsible for hemolytic y disease and transfusion reactions. In a number of instances the naturally occurring antibodies, as a result of the antigenic stimulus of a transfusion and/or pregnancy, may increase in titer and in thermal amplitude. Occasionally these antibodies assume the properties of the warm immune variety, but more frequently they remain intermediate. Thus, examples of anti-M, anti-Le* or anti-S have been observed which belong to each of the three categories shown in Table 9. This consideration alone illustrates the difficulty in applying rigid criteria for any classification. Table 9—Characteristics and Classification of Intragroup Isoantibodies Naturally Inter- Occurring mediate Warm Immune Optimum temperature 18°-20° C 20°-37° C 37° C or lower Titer Low Inter- May be very high mediate Antigenic response Weak, rare Occasional ~~ Distinct Positive antiglobulin Negative as Occasional ~~ Always* test a rule Clinical importance: Transfusion None Occasional Important Hemolytic disease None Not proved Important} Use in paternity tests None Selected Very usefult sera only Blood factors Ai-A., P, Le'Le®, S-s, Rh-Hr system, K-k, LeLe®, M-N, Tj, Kp*-Kp®* Fy*-Fy", M-N, Lu*-Luo* JJ? , S-s, Js, Lat, S Le*-Le®, high- and low-incidence blood factors * With albumin agglutinins. + For most factors of the group. 1 With properly standardized reagents. 858 BLOOD GROUPING, RH TYPING A list of known blood factors other than A and B, antibodies for which may be found in human serum, is given in Table 10, along with the incidence of positive and negative reactions, The antigens of the Rh-Hr system, which are given elsewhere, are not included. The factors are listed roughly according to the frequency of occurrence of their antibodies in human sera. Identification of one or more anti- bodies in a particular serum can be made only with the aid of a panel of red cells so selected as to contain the more important blood factors, including those of the Rh-Hr system. Table 10—Incidence of Positive Reactions in a Caucasoid Population Reaction Per cent Per cent Symbol Name Positive Negative K Kell 9 91 Fy* Duffy 66 34 Le® Lewis 23 77 Leb 7 71 29 P 79 21 S 55 45 Jk» Kidd 75 235 k Cellano 99.8 0.2 s 89 11 M 78 2 N 72 28 Tj (P14P2) Jay 99.9 + Very rare Lu® Lutheran 3 92 Lo ” 99.8 0.2 Ji® 75 25 Js Sutter 0 100 Fy* 83 17 Ve* 99.9 + Very rare U 99.9 4+ Very rare Yt 99.6 0.4 “Private” blood factors* Very rare 999+ * For a list of private or low-incidsnce blood factors, the reader is referred to Blood Groups in Man,® page 235. V. DETECTION OF INTRAGROUP ISOIMMUNIZATION A. Introduction With the selection of proper test cells, it is now possible to perform qualitative tests which will screen the sera of Rh-negative and Rh- positive individuals to determine which contain atypical antibodies. In BLOOD GROUPING, RH TYPING 859 many cases, the antibody will be anti-Rh, (D) produced by an Rh- negative individual. Once an atypical antibody is found, its specificity can be determined in tests with a panel of selected cells. 1. Selection of the test blood—ZFresh or sterile blood in citrate preservative (Section I B) should be used. It is preferable to mix in equal quantities two group O bloods of types Rhy (CDe) and Rhy (cDE) one of which is Kell+. This pool will contain the principal Rh-Hr and Kell antigens. The pool of the two bloods recommended should be so selected, if possible, as to contain also antigens Fy®, S and Jk*., This will permit the identification of a larger number of immunized individuals by means of the qualitative test. 2. The test a. Saline agglutinins—See directions, Section II, Heading “D,” of this chapter. b. Albumin agglutinins—See directions, Section II, Heading “IF,” of this chapter. c. Enzyme technic—Almost all antibodies will be detected using steps a. and b. Enzyme-treated cells for the detection of antibodies is a popular and efficient technic. It is not recommended as the sole test because of some nonspecific agglutination and failure to detect all isoantibodies. The enzymes most frequently employed are trypsin, papain, ficin and bromelin. The routine use of ficin requires caution against the hazard of hypersensitivity in the technician. A description of the preparation of one of the enzymes, trypsin solution and trypsinization of red cells, will suffice as an example of the group: Prepare a 1 per cent stock solution of trypsin powder (1:250) in phosphate buffer, pH 7.4; clarify by filtration through Whatman No. 1 filter paper. Distribute the solution into small tubes and store by freezing. To trypsinize cells, add 1 part of the stock trypsin solution to 10 parts of a 2 per cent suspen- sion of cells in salt solution. Incubate at 37° C for 15 min. Wash at least once with salt solution and reconstitute to original volume. Technic: Performance of the test is the same as the tube test with saline agglutinins given in Section II of this chapter under Heading “D,” except that enzyme-treated cells in salt solution are substituted. These tests must be incubated at 37° C because enzyme- treated cells are sensitive to cold agglutination. B. Indirect Antiglobulin Test for Detection of Intragroup Isoimmunization Where the test for saline agglutinins is negative after incubation at 37° C, the same test tube mixtures should be saved and tested by the 860 BLOOD GROUPING, RH TYPING indirect antiglobulin technic. The incubation thus constitutes the first stage of this test. In the second stage, the unagglutinated cells suspended in salt solution which have been exposed to the patient’s serum are washed three times by filling the tube with fresh salt solution, centrifuging and decanting. After the last wash, the salt solution is poured off as completely as possible and 1 drop of the antiglobulin serum is added to the mixture. After shaking to mix, the tube is lightly centrifuged without further incubation. Readings are then made as given sub- sequently in Section VII of this chapter for the direct antiglobulin test. Except for antibodies demonstrable only with the indirect anti- globulin method described also in Section VII, this test will confirm the results of the tests with albumin-suspended cells and with enzyme- treated cells, giving in some instances stronger, but rarely weaker, results. A suitable antihuman globulin reagent will detect antibodies in sera which give a prozone in the qualitative test with albumin-suspended red cells. (Prozone-containing sera give negative or weak reactions when tested undiluted or in low dilution, but they show strong agglu- tination in higher dilutions upon titration.) The antihuman globulin serum reagent will regularly detect the presence of such antibodies as anti-Fy*, anti-Jk% and anti-K. These antibodies usually fail to agglu- tinate albumin-suspended or enzyme-treated red cells. Some sera containing anti-Lewis or anti-Kidd will fail to react in the indirect antiglobulin test carried out on cells suspended in salt solution but may react if the test is performed on enzyme-treated cells. 1. Titrations—In all instances in which any one of the several qualitative tests has been found positive, serial double dilutions of the patient’s serum may be tested to determine the antibody titer. One method of performing titrations may be given here: The dilutions are made by using a row of ten 10X75 mm test tubes. Each tube except the first receives 0.1 ml of a suitable diluent as indicated below. With the aid of a 0.2 ml pipette, 0.1 ml of the patient’s serum is delivered to the 1st tube and 0.1 ml to the 2nd tube. The contents of the second tube are mixed, and 0.1 ml of the mixture is delivered to the next tube, etc. Irom the 9th tube, 0.1 ml is dis- carded. The titration values will run from 1:1 (undiluted), 1:2, 1:4, etc., to 1:256, with the 10th tube serving as an antigen control, After performing the titration, each tube then receives 0.1 ml of the suitable test cell suspension as indicated. BLOOD GROUPING, RH TYPING 861 2. Diluents, suspending media, and test cells a. Saline agglutinins: For the titrations, prepare serum dilutions and test cell suspensions in salt solution. b. Albumin agglutinins: For the titrations, prepare serum dilutions in normal male group AB serum and add to each dilution 2 drops of albumin-suspended cells. In those cases where the indirect antiglobulin was the only positive qualitative test, perform a salt solution titration and follow with an indirect antiglobulin test on each tube. Incubation and readings in these tests are made as in the qualitative procedures given in this chapter. Shaking once or twice during incu- bation enhances the reaction. The titer is the reciprocal of the highest dilution in which agglutination is still present. VI. TESTS TO DETERMINE THE SPECIFICITY OF Rh-Hr AND OTHER ANTIBODIES Once a serum containing antibodies is found in the qualitative test, it should be subjected to further analysis in order to determine specificity. For this purpose a panel of carefully selected group O test cells should be available. The panel should be so chosen as to contain all blood factors known to be antigenic either by pregnancy or transfu- sion. In addition, selected bloods should be included for the identifi- cation of antibodies which are more active at room temperature than at 37° C, such as anti-P, anti-Lu®*, and anti-M. Approximately 98 per cent of all sera that react in the qualitative test will contain anti-Rh,(D) either alone or in association with anti- rh’ (C) or anti-rh” (E); others from Rh-negative or Rh-positive persons may more infrequently contain antibody of another specificity, such as anti-K or anti-Fy®". Accordingly, two cell suspensions in the panel should be selected so that one will be specific for factor rh’ (C) (Cde) and the other for rh” (E) (cdE). It is desirable to have two Rh-negative cell suspensions, one containing K and the other Fy". In cases of suspected hemolytic disease due to factors other than Rh, (D), the husband’s blood should always be included if it is ABO- compatible, In cases of hemolytic transfusion reactions, the bloods of one or more donors should be added to the panel. A representative panel of test cells is given in Table 11. The choice of test cells in the panel is not binding and depends in large measure upon available bloods, preferably those available from the personnel of the institution. Such panels are now obtainable commercially. 862 BLOOD GROUPING, RH TYPING Table 11—Antigens in Suggested Panel of Group O Test Cells for Identification of Antibodies Rh Antigens Other Antigens Most Likely Genotype MN Ss Ke Fy Jk 1. P La 1. RR CDe/CDe MN ++ 04+ 0 O 0 0 0 2 R= C¥De/cde N 0+ 04+ + + + + + 3. RR? cDE/cDE N 0+ 0+ + O 0 0 0 4 rr Cde/cde M 04+ 0+ + + 0 + + 5 rr cdE/cde MN ++ 04+ + O 0 RN 0 6. rr cde/cde M 0+ ++ + 0 4 0 0 7. rr cde/cde M +0 04+ + O + 0 0 8. husband or donor (s) Salt solution suspensions are employed and after 1 hr incubation, preferably at 37° C, reactions specific for saline antibodies are re- corded. In the absence of direct reactions the second stage of the antiglobulin test is carried out for detection of antibodies other than saline agglutinins. At the end of the first stage, however, it is important to note the occurrence of hemolysis, which is exhibited by some sera (fresh and therefore complement-containing) in which anti-Le*, anti-Tj* and, rarely, others may be present. Occasionally, sera giving reactions in the first stage may contain antibodies which are not “warm” agglu- tinins. In these cases no agglutination will occur in tests with pre- warmed reagents incubated at about 40° C. Identification of single antibodies in a particular serum is readily made in the vast majority of cases. It can be confirmed if the panel is enlarged to include selected test cells. Difficulties may arise in the identification of rare antibodies or multiple antibodies when more than one antibody results from multiple transfusions, such as anti k, anti- Fy’, anti-Jk" and anti-Kp®. In these cases the specimens should be re- ferred to larger centers where very rare test bloods are available. VII. THE ANTIHUMAN GLOBULIN TEST (COOMBS TEST) A. Introduction This test is a valuable aid in the detection of (1) in vivo sensitiza- tion of red cells, as in hemolytic disease of the newborn and in acquired hemolytic anemia; (2) in vitro sensitization to indicate the presence of coating antibodies in a particular serum. The in vivo sensitization is referred to as the “direct antihuman globulin test” or BLOOD GROUPING, RH TYPING 863 “direct Coombs test,” while the in vitro sensitization is the “indirect antihuman globulin” or “indirect Coombs.” The test is based on the specific reaction of a processed rabbit (goat or other animal) antihuman globulin serum with red cells in salt solution which have absorbed on their surface, either in vivo or in vitro, a human-immune globulin fraction. The globulin fraction in- volved is almost always the so-called albumin agglutinin or incom- plete agglutinin, which, although combined with its specific antigen on the red cells, fails to produce clumping in a protein-poor medium. Such sensitized red cells, after thorough washing in order to remove uncombined or mechanically adherent globulin, react with the anti- globulin serum to produce the visible effect of agglutination. Thus the reaction is a globulin-antiglobulin reaction in which the layer of anti- body globulin on the surface of the red cell serves as antigen. The antiglobulin reaction is now known to have at least two distinct serologic specificities. The most common and valuable is human gamma globulin of low molecular weight. It should be noted that directions given here apply to the use of animal antisera to human gamma globulin, or animal antisera to whole human serum diluted so that serological activity against human gamma globulin represents the major serologic specificity. A different specificity directed against bound human complement is obtained in several ways, the usual being concentrated antiserum against whole human serum inhibited by the addition of human gamma globulin. This is termed “complement anti- globulin serum” or “nongamma antiglobulin serum.” Serum of the latter specificity agglutinates red cells sensitized by antibody plus human complement, and the sensitizing antibodies often tend to be hemolytic in vitro (for example, ABO, Lewis, P, Kidd and some- times the Duffy and Kell blood group systems). Unfortunately, complement antiglobulin reagents of adequate potency cannot be used with test red cells which have been refriger- ated due to an autoantibody of H specificity, which is present in al- most all sera so stored. This source of error can be controlled if all blood specimens for these tests, such as donor pilot tubes, are pre- served in a suitable anticoagulant which is anticomplementary. The numerous applications of the antiglobulin test may be sum- marized as follows: 1. In vivo sensitization: The direct test a. Diagnosis of intragroup hemolytic disease of newborn, in tests of the blood of newborn, preferably cord blood. This test is generally negative or weakly positive in ABO hemolytic disease. The test is 864 BLOOD GROUPING, RH TYPING also used in follow-up studies of infants receiving replacement therapy. b. Diagnosis of acquired hemolytic anemia. c. The study of hemolytic transfusion reaction—most frequently a positive direct test of the donor’s surviving incompatible red cells; more rarely, a positive direct Coombs test on the recipient's own red cells, for example, A; recipients of group O blood containing high- titered anti-A;. 2. In vitro sensitization: The indirect test a. Detection (direct compatibility test) and identification of atypical antibodies. b. Identification of antigens in red cells with the aid of known anti- bodies, as, for example, in the detection of weak variants, D*, E* and C"; and detection of such groups as Kell, Duffy or Kidd with known specific antibodies B. The Direct Coombs Test 1. Function—Almost all cases of intragroup hemolytic disease of the newborn are due to albumin agglutinins which coat or sensitize the fetal blood in utero. This sensitization can be detected by a positive direct Coombs reaction on the washed cord red blood cells. Sensitiza- tion does not occur when the mother’s serum contains only saline agglutinins. In acquired hemolytic anemia a circulating antibody, either non- specific or of varying specificity, coats the red cells in vivo with a layer of globulin in a way similar to passive sensitization in hemolytic disease of the newborn. A positive direct Coombs test differentiates this condition from other hemolytic anemias due to hereditary ab- normalities (congenital spherocytic anemia, sickle cell anemia, thalas- semia, etc.). 2. Test tube method for the direct test* a) Prepare a 2 per cent suspension in salt solution of the cells to be tested for in vivo sensitization. b) Wash the cells three times with tubefuls of fresh salt solution in order to remove all traces of free serum. * When commercial antiglobulin preparations are used, the manufacturer's directions should always be followed. BLOOD GROUPING, RH TYPING 865 c) After the last wash, reconstitute to the 2 per cent cell concen- tration. d) Using a clean pipette, place 2 drops of the antihuman globulin serum into a small test tube (10X75 mm, inside diameter 7 mm), followed by 2 drops of the washed cell suspension. e) Centrifuge immediately at low speed. Incubation is not recom- mended. f) Readings: Strongly coated red cells will show maximal agglu- tination. Readings are made by gently resuspending the sediment and examining with the aid of a 6X hand lens. 3. Positive and negative controls—A positive control for the antihuman globulin serum may be prepared by adding 2 drops of a 2 per cent suspension of fresh normal Rh-positive cells in salt solution to an equal volume of anti-Rh,(D) albumin agglutinin. A convenient source of such an antibody is the anti-Rh,(D) reagent used in screen- ing Rh-positive and Rh-negative bloods. For the negative control the same procedure should be repeated with a fresh normal Rh-negative cell suspension. Another negative control is the simple suspension in salt solution of the red cells under test. In the direct antiglobulin test the positive and negative controls should be treated in the same manner as the bloods to be tested. 4. Sources of error a. It is important to employ clean glassware and fresh salt solution. Contamination with traces of fresh human serum will diminish the potency of the antihuman globulin serum. b. False-positive reactions may result from centrifugation at too high speed and improper handling of the sedimented cells. c. Bloods submitted for direct antiglobulin reactions should be tested immediately without previous refrigeration, 5. The slide method: direct test a) The blood to be tested is washed at least three times, as in the test tube method, and the washed red cells are brought to a concen- tration of about 25 per cent. b) Two drops of this suspension are now placed on a glass slide or surface and mixed with 2 drops of the antihuman globulin serum. The cells and reagent are mixed with the edge of a wooden appli- 866 BLOOD GROUPING, RH TYPING cator and the slide, kept in a moist chamber, is gently rocked over a lighted surface, The presence of agglutination and its intensity are noted, with final readings made at the end of 10 min. c) For a control, 2 drops of salt solution are added, in place of the reagent, to 2 drops of the cell suspension under test. For a further control, 2 drops of the antihuman globulin serum may be added to a known sensitized and a normal nonsensitized red cell suspension prepared in the same manner as the cells to be tested. The indirect antiglobulin test is described in Section V, Heading B, of this chapter. Viil. THE COMPATIBILITY TEST The successful operation of a transfusion service is largely de- pendent on, first, the efficiency of the blood grouping and Rh typing procedures; second, the use of satisfactory compatibility tests on bloods of recipient and donor. Other important considerations are: adequately trained personnel available around the clock and proper liaison between hospital staff and members of the blood transfusion service. In recent years there has developed a tendency toward uniformity in technics for blood grouping and Rh typing, but a review of the compatibility tests submitted by several experts reveals that in some important respects no two laboratories use quite identical procedures. This lack of uniformity is largely attributable to the existence of several methods for the detection of a variety of intragroup isoanti- bodies. There is an ever-increasing tendency to employ for the compati- bility test the indirect antiglobulin (Coombs) reaction of the re- cipient’s fresh serum and the donor’s ABO group-compatible red cells because this reaction will reveal the maximum number of clinically important antibodies, including some specimens of anti-Fy*, anti-Jk* and anti-k, which may not react with albumin-(or serum-) suspended or enzyme-treated red cells. Very rarely antibodies are found which will fail to react in the indirect antiglobulin test but will react with enzyme-treated red cells or, less frequently, with protein-suspended cells. As a rule their titer is very low. Most thoughtful transfusionists recommend the indirect Coombs test for all transfusions. In the procedures to be outlined here, an attempt has been made to incorporate the most desirable features of the procedures submitted by responsible workers in charge of large transfusion services. BLOOD GROUPING, RH TYPING 867 The objection that the indirect Coombs test is too time-consuming has been met by reducing the incubation period during the first stage of the reaction. Where time permits, however, there is no real need to shorten this period. A. The Major Compatibility Test Patient’s serum and donor’s salt solution (or serum) suspended cells are being tested. The object of this test is twofold: It will detect gross errors in blood grouping, and it constitutes the first stage of the indirect antiglobulin test, which should not be omitted. In addi- tion to the indirect antiglobulin reaction for compatibility test, some transfusionists use (1) a high-protein bovine albumin test (see Sec- tion IT G and H of this chapter); (2) or enzyme-screening test (see Section VA 2 c¢ of this chapter). However, these additional tests are not adequate substitutes for the indirect antiglobulin test. B. The Minor Compatibility Test The minor compatibility test is not necessary where donor sera are screened for the occurrence of atypical antibodies. When carried out without prior screening, procedures should consist of a saline agglu- tinin test between donor serum and patient’s red cells, followed by the indirect antiglobulin reaction. In cases of acquired autoimmune hemolytic anemia, the minor compatibility test may be positive due to the coating of the recipient’s red cells with an autoantibody. In the transfusion therapy of infants, donors should be selected whose blood is compatible with the maternal serum. Pure Levine, M.D., Chapter Chairman M. Grove-Rasmussen, M.D. AaroN KeLLNER, M.D. RoBert T. McGEE James F. Morn, M.D. R. E. RosenrFieLp, M.D. Kurt Stern, M.D. BIBLIOGRAPHY No attempt has been made to give a complete list of references which are available in the works listed. 1. Awien, F., and Diamonp, L. K. Erythroblastosis fetalis. Boston, Mass. : Little, Brown, 1958. 2. American Association of Blood Banks. Technical Methods and Procedures. Chicago 2, Ill. : Burgess Publishing Co., 1956. 3. BoormaN, K. E, and Dopp, B. E. Introduction to Blood Group Serology. (2nd ed.). Boston, Mass. : Little, Brown, 1961. 868 BLOOD GROUPING, RH TYPING 4. Duxsrorp, I., and BowLEy, C. C. Techniques of Blood Grouping. London, England : Oliver & Boyd, 1955. 5. Dunsrorp, I, and GraNT, J. The Anti-globulin (Coombs) Test in Labora- tory Practice. London, England : Oliver & Boyd, 1959. MorrisoN, P. L. Blood Transfusion in Clinical Medicine (3rd ed.). Spring- field, Ill. : Charles C Thomas, 1961. MouranT, A. E. The Distribution of the Human Blood Groups. Spring- field, Ill.: Charles C Thomas, 1954. RACE, R., and SANGER, R. Blood Groups in Man (3rd ed.). Springfield, IIL: Charles C Thomas, 1962. 9. StrarrON, F., and RENTON, P. H. Practical Blood Grouping. Oxford, England : Blackwood Scientific Publications, 1958. 10. WIENER, A. S. Heredity of the Blood Groups. New York and London: Grune & Stratton, 1958. go NS A ABO antigens and antibodies, 835, 840 Absidia corymbifera, 735 Acanthocheilonema perstans, 779 Accidents (see Laboratory Infections and, Accidents), 89-104 Acetamide confirmatory medium, for Pseudomonas, 637 Achromobacter lwoffi, 621 Acid poisoning, treatment for, 33 Acid-fast, bacteria, 262 stains, 40-42 Acinetobacter anitratum, 621 Actinobacillus mallei (see Malleo- myces mallet), 592-598 Actinomyces, 690, 730 in throat cultures, 235 Actinomyces baudetii, 690 A. bovis, 690, 696, 721, 727, 734 incubation of, 38 A. israelii, 690, 696, 721, 727, 734, 736 Actinomycosis, 690, 720 Administration (laboratory), 5-6 Aerobacter aerogenes, 606 Aerobacter group, in meningitis, 429 Agar, for culture media, 118 Agents of laboratory infections, 90 Agglutination tests for identification of Bordetella pertussis, 404 of Brucella, 342 of Escherichia coli, 308 of Hemophilus influenzae, 422 of Klebsiella, 609-612 of meningococci, 451, 458 of Pasteurella multocida, 368 of P. pestis, 386, 387 of P. pseudotuberculosis, 358-360 of P. tularensis, 372 of Salmonella, 299 INDEX Agglutination tests—continued of Shigella, 299 of staphylococci, 219 of Vibrio comma, 315, 316 Agglutination-lysis test for spirosis, 545, 558, 560, 566 Albert's stain, for diphtheria, 237 Albimi media, for Brucella, 339, 341 Albumin agglutinins, in Rh typing, 843, 845 Alcaligenes, 295 in meningitis, 429-430 Alkalescens-dispar groups of Entero- bacteriaceae, 293, 295 Alkali poisoning, treatment for, 33 Alkaline methylene blue stain, for diphtheria, 236 for Toxoplasma, 655 Allescheria boydii, 724, 733 Amebas, 808 American Trudeau Society, egg yolk- potato flour medium for M. tu- berculosis, 171, 273 Amino acids in culture media, 109 Anaerobes, atmospheric requirements, 110 Anaerobic, bacilli, nonsporulating, 682 cocci, 679-682 examination technics, 37-38, 664-666 ANAEROBIC INFECTIONS, 662-698 organisms causing, 662 Ancylostoma duodenale, 779 Andrade’s indicator, 148 Animal inoculation, 50 for anthrax, 585 for botulinum toxin, 325 for Erysipelothrix, 601 for fungi, 702 for glanders, 594 for leptospirosis, 554 for Listeria, 617 for Mima and Herellea, 626 lepto- Nore—The outline of chapter contents, as given at the beginning of each chapter, is not repeated in this index. Please refer to it for complete informa- tion on chapter contents. 869 870 Animal inoculation—continued for Pasteurella multocida, 366 for P. pseudotuberculosis, 362 for plague, 384 for pneumococcus infections, 225 for tuberculosis, 278 for tularemia, 371 for Spirillum minus, 640 for Streptobacillus moniliformis, 650 for Vibrio fetus, 660 in meningitis, 445 of Bordetella pertussis, 408-410 of Brucella, 344 of clostridia, 693 Animals, autopsy methods, 51 injection of, 50 Pasteurella infections of, 364 safety precautions with, 98 sources of laboratory infections, 98 ANTHRAX (Bacillus anthracis), 578- 501 Antibiotic assays, extract broth for, 179, 825 Antibiotic sensitivity (see also Anti- microbial-susceptibility tests) of E. coli, 312 of gonococci, 484 of meningitis-causing organisms, 456 of Mima and Herellea, 624-625 of M. tuberculosis, 275, 829 of Pasteurella, 367, 391 of staphylococci, 214, 219 of streptococci, 190 Antibiotics, choice for sensitivity tests, 822 stock solutions of, 826 Anticomplementary reactions in tests for syphilis, 508, 535 Antidotes for poisoning, 33 Antigen, for serologic tests for Bordetella pertussis, 407 for Brucella, 347-351 for Erysipelothrix, 604 for Klebsiella, 609 for Kolmer tests for spyhilis, 525, 528 for leptospiral tests, 559 for Pasteurella pestis, 386 for P. pseudotuberculosis, 359 for P. tularensis, 374 for Streptobacillus 650 for syphilis, 512, 525, 528 moniliformis, INDEX Antigen for skin tests for chancroid, 49 for fungus infections, 702 for glanders, 595 Antiglobulin tests for isoimmuniza- tions, 859 Antihuman globulin (Coombs) test, 862 ANTIMICROBIAL SUSCEPTIBILITY Tests, 821-833 Antisera (see specific diseases or microorganisms) Antitoxin, for clostridia, 697 for diphtheria virulence tests, 252 Anton ophthalmic reaction for listeri- osis, 617 Arizona Group of Enterobacteriaceae, 293, 295, 299 Arthropods, plague in, 378, 380 Ascaris lumbricoides, 779, 783 Ascoli test, for anthrax, 589 for tularemia, 375 Asparagine enrichment medium for Pseudomonas, 637 Aspergillus fumigatus, 735 Autoclave sterilization, 113 of instruments for collection of blood, 4 Autopsy of animals (see also specific diseases or microorganisms), 51 in diphtheria virulence tests, 248 Autotrophs, 106 Avian tuberculosis, 280 B5W, a group of Herellea, 620 Bacillus anthracis, 578-591 in meningitis, 430 B. cereus, 586 in food poisoning, 320, 333 B. mesentericus, B. mycoides, B. siamensis, B. subtilis, B. trop- icus, 586 Bacterial Foon PorsoNing, 319-336 BACTERIAL INFECTIONS, OF THE GAs- TROINTESTINAL TRACT, 287-318 laboratory-acquired, 92 BACTERIAL MENINGITIS, 426-461 Bacteriophage, for identification of P. pestis, 384 typing, of Pseudomonas, 638 of S. typhosa, 301 of staphylococci, 217 INDEX Bacterium anitratum, 620 Bact. lactis aerogenes, 606 Bacteroides, 687-690 in meningitis, 429 Bacteroides fragilis, 689 B. funduliformis, 689, 695 B. melaninogenicus, 687 Bacto supplements for gonococcus cul- tures, 473 Balantidium coli, 809 Barnes, medium for enterococci, 331 thallous acetate-tetrazolium-glucose agar, 138 Basic fuchsin tryptose agar for Bru- cella, 345 BCG vaccination of laboratory work- ers, 102 Beef heart, infusion for streptococcal media (CM No. 23), 137 broth with blood (CM No. 29), 139 Beeson and Heyman, cultivation of H. ducreyi, 489 Bentonite flocculation test for trichi- nosis, 805 Bethesda-Ballerup group, 299 Bile, examination for Salmonella, 290 solubility tests, for pneumococci, 226 Biochemical reactions, of Alkalescens- dispar group, 298 of Bordetella, 405 of clostridia, 677-679 of Corynebacterium, 255, 256 of Hemophilus, 418 of Klebsiella, 609 of Listeria, 617 of Mima and Herellea, 622-624 of M. tuberculosis, 274 of Neisseria, 450, 483 of Pasteurella, 361, 367, 383 of Salmonella and Shigella, 297, 298 of Streptobacillus moniliformis, 648 of Vibrio fetus, 660 Biochemical tests, 38, 45-49 Catalase, 48 Coagulase, 48 Hydrogen sulfide production, 45 Indole, 45 Methyl red, 46 Nitrate reduction, 47 Oxidase, 48 Voges-Proskauer, 46 Biologic false-positive reactions for syphilis, 506 871 Biopsy (see Tissue examinations) Bismuth sulfite agar, Hajna and Da- mon (CM No. 58b), 154 Wilson and Blair (CM No. 58a), 153, 291 Blastocystis hominis, 810 Blastomyces brasiliensis, 725, 732 B. dermatitidis, 725, 732, 735, 736 Blastomycosis, 720 intradermal tests, 702 Blood agar (CM No. 16), 133 for Cl. botulinum, 324 for Hemophilus, 417-419 for pertussis, 401 for staphylococci, 213 for streptococci, 195 Blood, collection of specimens, 5 counts in whooping cough, 400 Blood digest enrichment broth, Fildes (CM No. 31), 139 Blood donors, selection of, 851, 866 Blood examinations, for anaerobic infections, 664 for anthrax, 580 for botulinum toxin, 325 for brucellosis, 339-341 for filariasis, 799-803 for fungi, 737 for gastrointestinal infections, 290 for glanders, 593 for gonorrhea, 470 for Hemophilus infections, 418 for leptospirosis, 546-550 for malaria, 739-775 for meningitis, 435, 445 for Pasteurella infections, 370, 379 for pneumococcus infections, 229 for ratbite fever, 643 for sodoku, 640 for staphylococcus infections, 211, 214 for streptococcus infections, 192 for syphilis, 502-537 Blood film, stains, 39-40, 747-752 in malaria, 743 Blood glucose-cystine agar, (CM No, 115), 178 Broop GrourING AND Rh TYPING, 834-868 Body fluids, collection for meningitis, 435 cultures 664 Boeck and Drbohlov’s LES medium for intestinal parasites, 817 Francis for anaerobic infections, 872 Bone marrow, for brucellosis, 344 for fungi, 737 for plague, 379 Bordet-Gengou agar (CM No. 18), 134, 234 for Bordetella pertussis, 401 Bordetella bronchiseptica, 398 B. parapertussis, 398 B. pertussis, 398-413 fluorescent-antibody tests, 62 Botulism, (see also Clostridium botu- linum), 321, 322-326 Bovine albumin agar for gonococci, 148, 468 Brain-heart infusion, agar, dehydrated for Cl. botulinum (CM No. 22), 136, 324 broth, dehydrated (CM No. 21), 136 Brewer, anaerobic jar, 37, 665 and Lilley phenylethylalcohol agar (CM No. 7), 130 Brilliant green agar for Salmonella (CM No. 61), 156, 291 Broquet’s fluid, 380 Brucella, 337-356 CO, requirements, 112, 341, 345 in meningitis, 430 species differentiation, 344 yeast autolysate broth and agar for, 142 Brucella abortus, 337 Br. bronchiseptica, 398 Br. melitensis, 337 Br. suis, 337 Brucerrosts, 337-356 Buboes, in chancroid, 487 in plague, 379 Buffer solutions, for malaria stains, 749 saline, for VDRL test, 516 Sorensen’s, 49 Burke's iodine solution, 42 Cc C-carbohydrates, in pneumococci, 227 Candida, 724, 727, 731 Candida albicans, 717, 731, 736 C. guilliermondii, C. krusei, C. parapsilosis, C. pseudotropicalis, C. stellatoidea, C. tropicalis, 731 INDEX Candidiasis, 720 Canicola fever, 545 Capsular antigens of Klebsiella, 607 Capsular swelling tests for Hemophilus, 422 for Klebsiella, 612 for meningocccci, 441, 453 for Mima and Herellea, 626 for Pasteurella, 368 for pneumococci, 224 Carbohydrate, broth base for Erysip-- elothrix (CM No. 98), 171 fermentations (see Fermentation re- actions) media, 10 per cent agar base for, 159 solutions, sterilization of, 117 Carbon dioxide, freezer chests, 15 requirements, for Brucella, 345 tension for incubation, 36, 112, 476 Cardiolipin antigen for Kolmer tests for syphilis, 525, 528 Carriers, of Corynebacterium diph- theriae, 232 of Salmonella typhosa, 288 Casein-soy digest, agar (CM No. 12), 132 broth (CM No. 11), 131 Catalase test, 48, 201 for M. tuberculosis, 274, 278 Cats as test animals for food poison- ing, 329 Celebes type of Vibrio cholerae, 314 Cell, counts, of spinal fluid in menin- gitis, 440 suspensions, for blood grouping, 837 for Kolmer tests for syphilis, 526 Centrifuges, 16 Centrifuging, safety precautions dur- ing, 97 Cerebrospinal fluid examinations, for brucellosis, 343 for fungus infections, 736 for globulin (Pandy test), 614 for gonococcus infections, 470 for Hemophilus infections, 418 for leptospirosis, 546 for listeriosis, 614 for meningitis, chemical, 435, 442, 444 cultural, 443 microscopic, 440 for pneumococcus infections, 229 for protein content, 536 for streptococcus infections, 194 INDEX Cerebrospinal fluid—continued for syphilis, 509, 510, 522, 526 for toxoplasmosis, 654 for tuberculosis, 265 culture, 273 Cervical specimens, for gonococci, 469 fluorescent antibody tests, 62 for listeriosis, 615 Chancroid (see Hemophilus ducreyi), 485-491 Chang’s broth and semisolid agar for Leptospira (CM No. 91), 168 Chapman, mannitol salt agar (CM No. 9), 131 -Stone medium for staphylococci in food poisoning, 327 Chemical, hazards, 31 sterilization, 28 tests, of spinal fluid in meningitis, 444 Chemicals, for culture media, 117 Chemotherapy of tuberculosis, 280 Chick tests, for diphtheria virulence, 249 for leptospirosis, 555 Chicken as test animal for avian tuberculosis, 280 Chilomastiz mesnili, 809, 810 Chloral hydrate-sodium azide blood agar for anaerobes, 669 Chocolate agar (CM No. 17), 134 for Hemophilus, 419 Cholera (see also Vibrio comma), 313 epidemiology of, 313 red reaction, 314 Cholera vibrio infections of the gas- trointestinal tract, 313 Chopped meat medium (CM No. 114), 178 Christensen urea agar, 158 Chromoblastomycosis, 720 Ciliates, 809 Citrate, agar, Simmons (CM No. 66), 158 utilization by Enterobacteriaceae, 297, 298 Cladosporium werneckii, 716 Cleaning fluid, 24 Cleveland and Collier's liver extract for intestinal parasites, 817 Clonorchis sinensis, 781, 784 Clostridia, 670-679 antitoxins of, 697 key to, 677 toxins of, 696 873 Clostridium bifermentans, 671, 674, 692, 696 Cl. botulinum, 674, 692, 696 in food poisoning, 320 Cl. capitovale, 674 Cl. carnis, 693 Cl. chauvei, 674, 693 Cl. cochlearium, 674 Cl. fallax, 675 Cl. hemolyticum, 674, 693, 694 Cl. histolyticum, 674, 696 Cl. novyi, 674, 694, 696 Cl. parabotulinum, 674, 692 Cl. perfringens, 671, 673, 674, 675, 694, 695, 696 in food poisoning, 320, 333 Cl. septicum, 671, 674, 695 Cl. sordellii, 674, 692 Cl. sporogenes, 671, 673, 674 Cl. tertium, 674 Cl. tetami, 671, 674, 695, 696 Clot cultures, for brucellosis, 339-340 for enteric organisms, 290 Coagulase test, 48 for staphylococci, 215 Coccidioides, safety precautions with, 96 Coccidioides immitis, 724, 725, 732. 736 Coccidioidomycosis, 720 intradermal tests, 702 Cole and Onslow, pancreatic extract, 147 Collection of specimens (see also spe- cific diseases or microorganisms), 4, 74-77 Colloidal gold test, value in syphilis, 511 Colloides anoxydana, 620 Communicable Disease Center Labo- ratories, 86, 217, 301 Compatibility tests, for blood donors, 866 Complement-fixation tests, complement for, 529 for glanders, 595 for gonococcal infections, 464 for leptospirosis, 545 for mycoses, 703 for Pasteurella, 368, 390 for syphilis (Kolmer) 525-536 for toxoplasmosis, 658 for whooping cough, 410 Concentration methods, for intestinal parasites, 788-794 for microfilariae, 802 874 Concentration methods—continued for nematode larvae, 804 for protozoan cysts, 814 for tubercle bacilli, 263, 270 Conjunctival scrapings, for Pasteur- ella tularensis, 370 Conjunctival swabs, 470 for Hemophilus, 418 Containers, for refrigerated speci- mens, 73 for tuberculosis specimens, 270 mailing, 72 Contaminated material, 101 CoNTRIBUTORS to this edition, vii Coombs test, direct, 863 indirect, 864 Cornmeal agar (CM No. 106), 176 Corynebacterium diphtheriae, 231-260 test for virulence, 245-254 in animals, 245-251 in vitro, 165, 251-254 type determination, 254 Cough plates, in whooping cough, 400, 401 Cryptococcosis, 720 Cryptococcus hominis, 736 C. neoformans, 721, 725, 727, 734 in meningitis, 430, 736 Crystal violet hormone agar, Meyer and Batchelder (CM No. 75), 161 Curture Mep1ia, 105-186 for Actinomyces, 691 for anaerobes, 664-668 differential, 669 for anthrax, 583 for antibiotic-sensitivity tests, 179, 825, 827 for M. tuberculosis, 830 for Bordetella pertussis, 124, 134, 401 for Brucella, 142, 341, 345 for Clostridium botulinum, 324 for Corynebacterium diphtheriae, 234 toxigenicity tests, 251 for enterococci in food poisoning, 33 for enteropathogenic coli, 310 for gonococci, disposal of, Escherichia INDEX Culture Media—continued for Erysipelothrix, 157, 169-171, 602 for fungi, 176, 701, 705, 714, 725, 735, 736, 737 for gastrointestinal infections, 290 310 for glanders, 594 for gonococci, 142, 143, 148, 473, 475 for Hemophilus, 139, 140, 417-419 H. ducreyi, 487 for histoplasmin, 177, 702 for intestinal protozoa, 817 for Klebsiella, 608 for Leptospira, 167-169, 553, 557 for meningococci, 143, 437, 447 for methyl red, Voges-Proskauer tests, 177 for Mima and Herellea, 622 for motility test, semisolid agar, 159 for nitrate reduction test, 157 for nonsporulating anaerobes, 683 for Pasteurella multocida, 365 for P. pestis, 381 for P. pseudotuberculosis, 360 for P. tularensis, 371 for plague vaccine, 161 for pleuropneumonia-like organisms, 162, 630 for pneumococci, 229 for Pseudomonas, 178, 637 for Salmonella and Shigella, 290, 291 for staphylococci, 213 for Streptobacillus momniliformis, 646 for streptococci, 190-201 for Trichomonas vaginalis, 496 for tuberculin, 177 for tuberculosis, 160, 171, 173, 273 for Vibrio cholerae, 315 for VV. fetus, 142, 166, 659 for Voges-Proskauer methyl red tests, 177 Culture media, formulas, listing, 125-127 inoculation of, 34-36 Culture media formulas acetamide confirmatory medium for Pseudomonas, 637 agar base for carbohydrate medium, 159 agar for nitrate reduction test, 157 American Trudeau Society egg yolk-potato flour medium, 171 numerical INDEX Culture media formulas—continued asparagine enrichment medium, for Pseudomonas, 637 Barnes thallous acetate-tetrazolium- glucose agar, 138 beef heart infusion for streptococcal media, 137 beef heart infusion broth with blood, 139 bismuth sulfite agar, 153-154 blood agar, 133, 191 blood digest enrichment broth, Fildes, 139 blood-glucose-cystine agar, Francis, 178 Bordet-Gengou agar, 134 brain-heart infusion agar, 136 brain-heart infusion broth, 136 brilliant green agar, 156 broth for fermentation studies, 128 broth for Leptospira, 167-168 broth for nitrate reduction test, 157 broth, glucose-asparagine for histo- plasmin and tuberculin, 177 buffered glycerol-sodium chloride solution, 160 carbohydrate broth base for Erysip- elothrix, 171 carbohydrate media, agar base for, 159 casein-soy digest agar, 132 casein-soy digest broth, 131 Chang’s broth and semisolid agar for Leptospira, 168 chloral hydrate-sodium azide blood agar for anaerobes, 669 chocolate agar, 134 chopped meat medium, 178 Christensen urea agar, 158 citrate agar, Simmons, 158 Cole and Onslow, pancreatic ex- tract, 147 cornmeal agar, 176 crystal violet hormone agar, Meyer and Batchelder, 161 cystine-tellurite-blood agar, Fro- bisher and Parsons, 163 Dorset’s egg medium, 160 Douglas agar with ascitic fluid and carbohydrate, 148 Douglas tryptic digest agar, 147 Douglas tryptic digest broth, 147 Edwards and Bruner semisolid agar for motility test, 159 Edwards’ extract agar with crystal violet for Erysipelothrix, 170 875 Culture media formulas—continued egg medium, Dorset’s, 160 egg medium, Pai’s, 163 egg-potato flour medium, Lowen- stein-Jensen, 172 egg-potato starch medium, Petrag- nani, 173 egg yolk or lecithovitellin agar for anaerobes, 668 egg yolk-potato flour medium, American Trudeau Society, 171 Endo’s agar, 151 enrichment broth, tetrathionate, 156 eosin-methylene blue agar, Levine's, 151 extract agar, 129 extract agar with crystal violet for Erysipelothrix, Edwards’, 170 extract broth for antibiotic assays, 179 extract broth for Erysipelothrix, Packer’s, 169 extract gelatin, 132 for Erysipelothrix, 170 Fildes’ blood digest enrichment broth, 139 Fletcher's semisolid agar for Lepto- spira, 169 “Flo” medium for Pseudomonas, 637 ’ Francis, blood-glucose-cystine agar, 178 Frobisher and Parsons cystine-tel- lurite-blood agar, 163 Frobisher et al. in vitro virulence test agar, 165, 251 GC medium base agar with hemo- globin, 142 gelatin-blood agar, 145 gelatin-egg albumin agar, 146 glucose-asparagine broth for histo- plasmin and tuberculin, 177 glucose-peptone broth for MR-VP tests, 177 glucose-serum-tellurite agar, Whit- ley and Damon, 165 glycerol-asparagine medium, Pros- kauer and Beck, 171 glycerol-sodium chloride solution, buffered, 160 Hajna and Damon bismuth sulfite agar, 154 Hardy, modification of Kligler’s iron agar, 149 heated blood-tellurite agar, Kellogg and Wende, 165 876 Culture media formulas—continued hormone agar for plague vaccine, 161 Hugh and Leifson, O F agar, 161 im vitro virulence test agar, Fro- bisher et al., 165, 251 Kellogg and Wende, heated blood- tellurite agar, 165 Kliglers’ iron agar, 149 Korthof’s broth for Leptospira, 167 lead acetate agar, 141 lead acetate for HoS production by Erysipelothrix, 170 lecithovitellin (egg-yolk) agar, 668 Levine's eosin-methylene blue agar, 151 Levinthal agar, 140 Levinthal broth, 140 Littman’s oxgall agar, 176 liver infusion broth, for Iibrio fetus, Plastridge, 166 Loeffler’s coagulated serum, 163 Lowenstein-Jensen, egg-potato flour medium, 172 MacConkey agar, 155 maltose agar, Sabouraud’s, 178 mannitol salt agar, 131 McLeod's agar for Corynebacterium diphtheriae, 163 McLeod’s agar with plasma and hemoglobin, 143 meat infusion agar, plain, 130 meat infusion broth, plain, 129 Meyer and Batchelder, crystal vio- let hormone agar, 161 Middlebrook and Cohn, 7H-10 me- dium, 174 milk agar, for staphylococci, 213 milk with indicator, 132 mycophil or mycological agar, 176 O F agar, Hugh and Leifson, 161 oxgall agar, Littman’s, 176 Packer’s extract broth for Erysip- . elothrix, 169 Packer’s sodium azide-crystal violet extract agar for Erysipelothrix, 169 Pai’s egg medium, 163 pancreatic extract, Cole and Ons- low, 147 Peizer, Steffen, Klein, transport agar medium for gonococcus, 148 peptone agar with plasma and hemoglobin, 144 INDEX Culture media formulas—continued peptone solution for indole produc- tion, 129 Petragnani egg-potato starch me- dium, 173 phenolphthalein phosphate agar, 130 phenylethylalcohol agar, 130 Pike sodium azide-crystal broth, 139 Plastridge liver infusion broth, 166 potassium gluconate medium for Pseudomonas, 637 potato glucose agar for fungi, 176 PPLO agar, 162 PPLO broth, 162 Proskauer and Beck's glycerol- asparagine medium, 171 raffinose-serum-tellurite agar, Whit- ley and Damon, 164 rice-Tween 80 agar, 177 Rustigian and Stuart urea broth, 158 Sabouraud’s agar, 175 Sabouraud’s maltose agar, 178 Salmonella-Shigella (SS) agar, 155 selenite (F) broth, 157 semisolid agar, 132 for Leptospira, 168-169 for motility test, Edwards and Bruner, 159 semisolid fermentation media, 128 serum substitute for virulence test agar, 166, 252 7H-10 medium, Middlebrook and Cohn, 174 Simmons citrate agar, 158 sodium azide-crystal violet broth, Pike, 139 sodium azide-crystal violet extract agar, Packer’s, 169 sodium desoxycholate agar, 152 sodium desoxycholate citrate agar, 152 sorbic acid-polymyxin thioglycolate broth for anaerobes, 669 SS agar, 155 staphylococcus enterotoxin agar, 131 starch agar for gonococci and men- ingococci, 143 Stuart et al. transport agar medium for gonococcus, 149 Stuart’s broth for Leptospira, 168 “Tech” medium for Pseudomonas, 637 tetrathionate enrichment broth, 156 violet INDEX Culture media formulas—continued thallous acetate-tetrazolium-glucose agar, Barnes, 138 thioglycolate broth, 135-136 Thiol broth with 0.1% agar, 166 Thjotta and Avery yeast extract, 140 Todd-Hewitt agar with sheep blood, 137 transport agar media for gonococci, 148-149 triple-sugar-iron agar, 150 tryptic digest agar, Douglas, 147 tryptic digest broth, Douglas, 147 tryptose dextrose vitamin B, agar, 141 broth, 141 tryptose extract agar with sheep blood, 138 tryptose infusion agar with sheep blood, 138 tryptose phosphate semisolid agar, 141 Tween-albumin medium, 173 urea agar, Christensen, 158 urea broth, Rustigian and Stuart, 158 Verwoort-Wolff broth for Lepto- spira, 167 White and Sherman medium enterococci, 331 Whitley and Damon glucose-serum- tellurite agar, 165 Whitley and Damon raffinose- serum-tellurite agar, 164 Wilson and Blair bismuth agar, 153 yeast autolysate, agar for Brucella, 142 broth for Vibrio fetus, 142 yeast extract, Thjotta and Avery, 140 Culture plates, fishing, 34 Cycloserine-sensitivity tests, 830 Cystine-tellurite-blood agar, Frobisher and Parsons (CM No. 79), 163 Cultural characteristics (see specific microorganisms) for sulfite Dairy products, examination for Bru- cella, 339 in food poisoning, 321 Dark-field examinations, spira, 551 for Lepto- 877 Dark-field examinations—continued for Spirillum minus, 640 for Treponema pallidum, 538 Deacon, cultural methods for H. du- creyi, 488 Dehydrated culture media, 122-124 Demineralizers, water, 21 Desoxycholate citrate agar for Pas- teurella, 360 for Shigella, 291 Dextrose-serum-tellurite diphtheria, 235 Dialister pneumosintes, 685 Diaphane, preservative for malaria films, 751 Diarrhea, infantile, 304 staphylococcal, 214 Dienes stain, 44, 633 Dientamoeba fragilis, 808, 809 Differential characteristics (see spe- cific microorganisms) DipaTHERIA (see Corynebacterium diphtheriae), 231-260 Diphtheroids, in diphtheria, 241, 243 in nongonococcal urethritis, 495, 497 Diphyllobothrium latum, 780, 783 Diplococcus mucosus, 620 D. pneumoniae (see Pneumococcus), 222-230 Dipylidium caninum, 780 Disposal of contaminated material, 101 plague specimens and cultures, 385 Dobell and O’Connor’s iodine solution, 787 Dolichos biflorus seeds, use in blood grouping, 840 Dolman and Wilson, medium for staphylococcus enterotoxin (CM No. 10), 131, 328 Donovan bodies, 491 Donovania granulomatis, 491 Dorset’s egg medium (CM No. 72), 160 Douglas’ agar, for gonococci No. 49), 148, 475 Douglas’ tryptic digest agar and broth (CM Nos. 48 and 47), 147 Dubos’ medium, 277 Duodenal aspirates for intestinal para- sites, 798 for Salmonella, 290 Dye plate differentiation of Brucella, 345 Dye test for toxoplasmosis, 655 agar for (CM 878 E Echinococcus granulosis, 780 EDTA solution, 74 Edwards and Bruner semisolid agar for motility test, 159 Edwards’ extract agar with crystal violet for Erysipelothrix (CM No. 97), 170 Egg, albumin for culture media, 146 medium, Dorset’s, 160 Pai’s, 163 -potato fluor medium, Lowenstein- Jensen (CM No. 101), 172 starch medium, Petragnani (CM No. 103), 173 lecithovitellin (yolk) agar for anaerobes, 668 yolk-potato flour medium, Amer- ican Trudeau Society (CM No. 100), 171 El Tor type of Vibrio cholerae, 314 Ewmbadomonas intestinalis, 809, 810 Endolimax nana, 808, 809, 810 Endo’s agar (CM No. 55), 151 Enrichment broth, for enteric patho- gens, 156, 157, 291 Enrichment, cultures for anaerobes, 664 media for Cl. botulinum, 325 Entamoeba coli, 808-810 E. histolytica, 801-810 Enteric diseases, bacterial, 287-318 Enterobius vermicularis, 779, 782, 797 Enterococci, in food poisoning, 330 in meningitis, 430 Enteromonas hominis, 809-810 Enteropathogenic ~~ Escherichia 304-313 Enzyme technic for isoantibody detec- tion, 859 Eosin-methylene blue agar, Levine's (CM No. 54), 151 Epidermophyton floccosum, 713, 717 Equipment, laboratory, 6-30 Erysipelothrix, culture media for, 157, 169-171 infections, 599-605 Erysipelothrix rhusiopathiae, 599-600 Erythrasma, 713 Escherichia coli, enteropathogenic, 304-313 identification by fluorescence tech- nics, 60, 312 in meningitis, 429-430, 433, 456 methyl red, Voges-Proskauer tests of, 46 coli, INDEX Escherichia coli—continued relation to Klebsiella, 606 E. freundii, cultural characteristics, 293 Exudates, examination for (see spe- cific diseases or microorganisms) Eye worm, 779 Farcy, 592 Fasciola hepatica, 781 Fasciolopsis buskii, 781, 785 Feces examination, for anaerobic in- fections, 664 for cholera, 315 for enteropathogenic Escherichia coli, 309 by fluorescence technics, 60 for food poisoning, 321 for gastrointestinal infections, 289 for intestinal parasites, 785-797 for staphylococci, 212 Fermentation reactions (see Biochem- ical reactions) Filaria, 779 Fildes’ blood digest enrichment broth, 139 Filtration sterilization, 29 Fishing culture plates, 34 Flagellates, 809 Fleas, vectors of plague, 377, 380 Fletcher's semisolid agar for Lepto- spira (CM No. 92), 169 “Flo” medium for Pseudomonas, 637 Flocculation test, bentonite, for Trich- inella, 805 VDRL, for syphilis, 514-525 Flotation methods, sputum, for tuber- culosis, 264 stools, for cysts and ova, 788-791 Flukes, 781 Fluorescein production by monas, 636 Fluorescence, microscopy, 11, 52-55 stain, Richards and Miller, 41 staining for acid-fast bacilli, 267 Fluorescent-antibody (FA), technics, 52-63 tests, for Bordetella pertussis, 402 for enteropathogenic E. coli, 60, 312 for N. gonorrheae, 62 for Pasteurclla pestis, 390 for streptococci, 58, 203 treponemal antibody test for syphilis (FTA), 506 Pseudo- INDEX Fluorescein isothiocyanate, 54 Food handlers, routine examination of, 3 Food poisoning, bacterial, 319-336 FOREWORD, Vv Formaldehyde sterilization, 29 Formalin preservative for protozoan cysts, 812 Formulas of culture media (see also Culture media formulas), 127-179 Francis, blood-glucose-cystine agar, 178 Freezers, 15 Friedlander’s bacillus (see Klebsiella pneumoniae), 223, 606 Frobisher, in vitro virulence test agar, 165 and Parsons cystine-tellurite-blood agar, 163 Fungal infections, laboratory-acquired, 93 Fungi, keys to, 704, 708, 713, 717, 721, 726, 734 Funcus INFECTIONS, 669-738 in food poisoning, 319 Fusobacteria, 686 G Gas sterilization, 29 Gastric lavage, for fungi, 734 for tubercle bacilli, 272 Gastrointestinal tract, bacterial infec- tions of, 287-318 GC medium, base agar with hemo- globin (CM No. 41), 142 Gelatin, blood agar (CM No. 45), 145 egg albumin agar 146 for culture media, 118 liquefaction by enteric organisms, 299 GENERAL 1-70 Genotypes, Rh-Hr, 854, 856 Geotrichum, 724, 727 Germicides, 28 Giardia lamblia, 809, 810 Giemsa stain, 40 GLANDERS AND MELI0IDOSIS, 592-598 (Malleomyces mallet and Mal- leomyces pseudomaller) (CM No. 46), Procepures (laboratory), 879 Glassware, 19 washing and handling, 23-25 Glucose, asparagine broth, for histo- plasmin and tuberculin, 177 peptone broth for MR-VP tests (CM No. 112), 177 serum-tellurite agar, Whitley and Damon (CM No. 83), 165 “Glucose evolue,” preparation of, 111 Glycerol-asparagine medium, Pros- kauer and Beck (CM No. 99), 171 -sodium chloride solution, buffered (CM No. 71), 160 Gonococcal infections, 463-485 Gonococcus (see N. gonorrheae) starch agar for, 143 transport media for, 148 Gram stain, 42 Granuloma inguinale (see Donovania granulomatis) 485, 491-494 Gravis type of diphtheria bacilli, 239- 242 Gravity sedimentation for intestinal parasites, 793 Greig test for Vibrio comma, 314 Ground meat medium (CM No. 114), 178 Grouping, ABO, of blood (see also specific microorganisms), 834-841 Guinea pig inoculations (see also Ani- mal inoculation), 50 H Hajna and Damon, bismuth sulfite agar, 154 Halo reaction of meningococci, 460 Hamsters (see Animal inoculation) Hardy, modification of Kligler’s iron agar, 149 Haverhill fever (see Streptobacillus moniliformis), 642 Hazards, laboratory, 30 Heidenhain’s iron hematoxylin stain, 814 HELMINTHIASIS AND INTESTINAL Protozoiasts, 777-820 Helminths, 777-808 egg counts, 794 Hemagglutination tests, 302 with Pasteurella pestis, 389 with Salmonella and Shigella, 302 with Toxoplasma, 657 Hemolysis in blood specimens, 5 HEMOPHILUS INFECTIONS, 414-425 Hemophilus aegyptius, 414, 416, 418 H. bronchisepticus (Bordetella bron- chiseptica), 398 H. ducreyi, 485-491 H. hemolyticus, 191, 201, 414, 416, 418 H. influenzae, 414, 418 in meningitis, 429-430, 432, 441, 454 Levinthal broth medium for, 140 H. parapertussis (Bordetella para- pertussis), 398 H. pertussis (Bordetella pertussis), 398-413 H. vaginalis, 417, 496 Hemorrhagic septicemia, 363 (Pasteurella multocida) Hepatitis, laboratory infections, 4, 95, 102 Herellea vaginicola, 620 Heterophyes heterophyes, 781 Heterotrophs, 106, 109 Heterozygous Rh individuals, 853 High-salt agar for staphylococci, 213 Histoplasma capsulatum, 724,725, 727 732, 736, 737 H. duboisti, 724 Histoplasma staining, 701 Histoplasmin, glucose-asparagine broth for, 177 Histoplasmosis, 720 intradermal tests, 702 History slips, 71 for gonorrhea specimens, 465 Homogenizing, safety precautions dur- ing, 98 Homozygous Rh individuals, 853 Hookworms, 779 Hormodendrum pedrosot, 733 Hormone agar for plague vaccine (CM No. 74), 161 Hospital infections, by Pseudomonas, 638 by staphylococci, 207 Hot-air sterilization, 25 Hucker’s crystal violet, 42 Hugh and Leifson, O F agar, 161 Humidifying equipment for incu- bators, 12, 13 Hydatid cyst, 780 Hydrogen sulfide production, 45 by Brucella, 345 by enteric organisms, 295 INDEX Hymenolepis diminuta, 780, 783 Hymenolepis nana, 780, 782 Immunization, prophylactic, of labora- tory workers, 102 Inaba type of Vibrio cholerae, 315 Incubation (see also specific or- ganisms) anaerobic, 37, 665, 672 increased carbon dioxide tension, 36, 477 Incubators, 12-14 India ink preparations, 199, 721 Indicator, Andrade’s, 148 Indole production, test for, 45, 129 by anaerobes, 669 by enteric organisms, 291, 294, 298 Infantile diarrhea, Escherichia coli in, 304 Infections (see Laboratory Infections and Accidents) 89-104 INH (isoniazid) sensitivity tests, 278 Inoculation, of animals (see also Ani- mal inoculations), 50 of media, 34-36 Intermedius type of diphtheria bacilli, 239-242 Interpretation of laboratory results (see also individual chapters), 63 66, 76 Intradermal tests for chancroid, 490 for diphtheria virulence, 245 for fungus infections, 702 for glanders, 595 for Pasteurella multocida infec- tions, 369 for toxoplasmosis, 658 in animals for Bordetella identifi- cation, 408 lTodamoeba butschlit, 808-810 Iodine stain for direct fecal films, 787 Ionizing radiation sterilization, 30 Isoimmunization of Rh-positive indi- viduals, 855-858 Isoniazid (INH) sensitivity tests, 278, 830 Isospora belli, 809 Isospora hominis, 809 Ito-Reenstierna reaction for chanc- roid, 490 INDEX Jenner’s stain, 39 Joint fluid, for brucellosis, 343 for gonococcus, 470 for Streptobacillus 643 moniliformis, K Kahn-type shakers, 19 KCN medium, for enteric organisms, 298, 299 Kellogg and Wende, heated blood- tellurite agar, 165 Key, to clostridia, 677-679 to fungi, 704-734 to peptococci, 682 to peptostreptococci, 681 Kinyoun’s stain, 42, 267 Klebsiella-Aerobacter group, 293, 295, 299 Klebsiella infections, 606-613 in meningitis, 429 Klebsiella pneumoniae, 223, 606 Kligler’s iron agar (CM No. 52), 149 Koch-Weeks bacillus (see Hemophilus aegyptius), 416 Kolmer, complement-fixation test for syphilis, 525-537 Reiter protein test for syphilis (KRP), 505 Korthof’s broth for Leptospira (CM No. 89), 167 Kovacs’ reagent, 45, 294 L L, colonies of Streptobacillus monili- formis, 647 Labeling antibodies, fluorescent, 54 Laboratory animals, 50-52 Laboratory, hazards, 30-34 LABORATORY INFECTIONS AND AccCI- DENTS, 89-104 Lactophenol-cotton blue staining of fungi, 701 Lead acetate agar (CM No. 38), 141 for H,S production by Erysipelo- thrix (CM No. 96), 170 Lecithovitellin agar for anaerobes, 668 Leifson’s desoxycholate citrate agar, 152, 360 Leprosy (see Mycobacterium leprae), 262, 283-285 Leptospira species, 544, 545, 552, 563 881 LEepProspPIrROSIS, 544-577 Levine's eosin-methylene blue agar, 151 Levinthal, agar (CM No. 34), 140 broth (CM No. 33), 140 Lindberg, cultivation of H. ducreyi, 489 Lissamine rhodamine, 54 Listeria infections, 614-619 Listeria monocytogenes, 617 in meningitis, 429 Littman’s Oxgall Agar 109), 176 Liver infusion broth for Vibrio fetus Plastridge (CM No. 87), 166 Ljubinski’s stain for Corynebacterium diphtheriae, 237 Loa loa, 779 Loeffler’s, coagulated serum (CM No. 81), 115, 163 methylene blue stain, 43 Lowenstein-Jensen, egg-potato flour medium, 172 Lymph nodes, for Pasteurella, 370 for Spirillum minus, 640 (CM No. Macchiavello stain, 45 MacConkey agar (CM No. 60), 155 Madurella, 724 Maduromycosis, 720 MAILING, RECEIVING AND PROCESSING SpeciMENS, 71-88 Malachite green stain, 44 MALARIA, 739-776 Malassezia furfur, 716 Malleomyces mallei, 592-595 M. pseudomallei, 595-597 Maltose agar, Sabouraud’s, 178 Mannitol salt agar, Chapman (CM No. 9), 131 Mansonella ozzardii, 779, 800 May-Gruenwald stain, 39 McLeod's agar, for Corynebacterium diphtheriae (CM No. 80), 163 with plasma and hemoglobin (CM No. 43), 143 Meat infusion, agar, plain (CM No. 6), 130 broth, plain (CM No. 5), 129 Media (see Culture media) Melioidosis (pseudoglanders), 595-597 Meningitis (see Bacterial Meningitis), 426-461 organisms causing, 415, 429-430, 736 882 Meningococci, classification of, 431 Meriones (field rodents) for lepto- spirosis, 554 Merthiolate-iodine-formalin ~~ (MIF) fixative for ova and cysts in feces, 812, 817 Methyl red, test, 46 Meyer and Batchelder, crystal violet hormone agar, 161 M’Faydean reaction for anthrax, 581 Mice, inoculation of (see also Animal inoculation), 50 Microaerophilic bacteria, oxygen re- quirements, 110 “Microbincinerator,” 96 Micrococcus pyogenes (Staph. aur- eus), 210 Microfilaria, 800 Microscopes, 7-12 Microscopic examination (see spe- cific microorganisms) Microsporum audowinii, 703, 708 M. canis, 703, 708 M. gypsewm, 708 M. lanosum, 708 Middlebrook and Cohn, 7H-10 me- dium, 174 MIF fixative, 812, 817 Milk, agar for staphylococci, 213 examination for streptococci, 194 sickness, a food poisoning, 319 with indicator (CM No. 14), 132 Mima polymorpha, 620 Mimeae, infections, 620-628 species in meningitis, 429 Minimus type of diphtheria bacilli, 240, 242 MISCELLANEOUS INFECTIONS, 599-661 Mitis type of diphtheria bacilli, 239 242 Monilia (Candida), 724 Monkey feeding tests for food poison- ing, 327 Monosporium apiospermum (Al- lescheria boydii), 724, 733 Moraxella lwoffi, 620 Motility test, semisolid agar for, 159 Mushrooms in food poisoning, 319 Mycobacterium leprae, 263, 283-285 M. smegmatis, 265 M. tuberculosis (see Tuberculosis and Leprosy), 261-286 antibiotic-sensitivity tests, 278, 829 in meningitis, 427, 429-430, 433 275- INDEX “Mycophil” or mycological agar (CM No. 108), 176 Nasal scrapings for leprosy, 284 Nasopharyngeal cultures in diphtheria, 232 in infantile diarrhea, 310 in meningitis, 436, 446 in streptococcus infections, 193 in whooping cough, 400, 402 Nasopharyngeal swabs fluorescent-antibody tests for Borde- tella pertussis, 62, 402 enteropathogenic Escherichia coli, 310 for streptococci, 58-59, 203 Necator americanus, 779 Neisseria, fermentation reactions, 450 oxidase test for, 48, 460 N. gonorrheae, 463-485 base agar medium with hemo- globin for, 142 collection of specimens, 74 identification by fluorescence tech- nics, 62 in meningitis, 430 in venereal infections, 463-485 starch agar for, 143 transport media for, 148 N. meningitidis, 428-430 (see also Bacterial Meningitis, 426-462) collection of specimens, 74 in venereal infections, 464 Nitrate reduction test, 47 media for, 157 Noble, agglutination technic, 451 Nocardia asteroides, 724, 727, 730, 734 in meningitis, 429-430, 736 staining, 701 N. brasiliensis, 721, 730 N. minutissima, 716 Nutrition of microorganisms, 106-109 for o O F agar, Hugh and Leifson (CM No. 73), 161 Ogawa type of Vibrio cholerae, 315 Onchocerca volvulus, 779, 800 Ophthalmic reaction for listeriosis, 617 Opsonocytophagic tests in whooping cough, 411 Optochin as inhibitor of pneumococci, 227 INDEX Oxgall agar, Littman’s, 176 Oxidase test, 48 for gonococci, 478 for meningococci, 460 Oxidation-reduction (O-R) tials, 111 Oxygen requirements of microorgan- isms, 110-112 poten- P Packer’s, extract broth for Erysipelo- thrix (CM No. 93), 169 sodium azide-crystal violet extract agar for Erysipelothrix (CM No. 94), 169 Pai’s egg medium (CM No. 78), 163 Pancreatic extract, Cole and Ons- low, 147 Pandy test for globulin, 614 Para-amino salicylic acid sensitivity tests, 277, 830 Paracoccidioides brasiliensis, 725, 732, 736 Paragonimus westermanii, 781, 785 Parameningococcus, 431 Parasitic infections, quired, 93 PAS sensitivity tests, 277, 830 PASTEURELLA INFECTIONS, 357-397 Pasteurella hemolytica, 366 P. multocida, 363-369 in meningitis, 430 P. muricida, 363 P. pestis, 377-391 P. pseudotuberculosis, 358-363, 382 P. tularensis, 369-377 cross agglutination with Brucella, 354 Pathogenicity for animals (see spe- cific microorganisms) Peizer, Steffen, Klein transport agar medium for gonococcus (CM No. 50), 148 Peptococci, 681-682 Peptone, agar with plasma and hemo- globin (CM No. 44), 144 for culture media, 108, 118 solution for indole production (CM No. 2), 129 Peptostreptococci, 679-681 Perianal secretions, examination for parasites, 796-797 Peroxidase reactions, 111 Petechiae, in meningitis, 435, 446 Petragnani’s egg-potato starch me- dium, 173 (PAS) laboratory-ac- 883 pH, buffer solutions, 49 determination of, in culture media, 120 Phenolphthalein phosphate agar, modi- fied (CM No. 8), 130 Phenotypes, Rh-Hr, 856 Phenylethylalcohol agar, Brewer and Lilley (CM No. 7), 130 Phialophora compacta, Ph. pedrosot, Ph. verrucosa, 724, 733, 734 Photography, fluorescent, 53 Piedraia hortai, 705 Pigeons, inoculation of, for Erysipelo- thrix, 601 for Pasteurella, 368 Pike's sodium azide-crystal broth, 139 Pinworms, 779, 797 Pipettes, 20 washers for, 25 Pipetting, safety precautions in, 97 Plague (see Pasteurella pestis), 377- 391 antibodies in FA tests, 54, 387 hormone agar for vaccine, 161 Plasmodium falciparum, P. malariae, P. ovale, P. vivax, 739 through 776 Plastridge, liver infusion broth, 166 Pleuropneumonia-like organisms (PPLO), in urethritis, 495 infections with, 629-634 media for, 162 PNxEuMococcus INFECTIONS, 222-230 in meningitis, 429-430, 432, 454 Polyethylene ware, laboratory appa- ratus, 20 Polyhydric alcohols, sterilization of, 117 Polyvinal alcohol (PVA) fixative, 75, 812, 817 Ponder’s stain for diphtheria, 237 Pork, examination for Trichinella, 803 Post-mortem examination of animals, 51 Postal regulations, 71, 86-88 Potassium gluconate medium for Pseudomonas, 637 Potato glucose agar for fungi (CM No. 107), 176 Pottenger’s stain for acid-fast or- ganisms, 266 PPLO, agar (CM No. 77), 162 broth (CM No. 76), 162 violet 884 Precipitin tests (see specific diseases or microorganisms) Pregnancy, Rh immunizations in, 841 Preservation of fecal specimens, 74, 812 Processing specimens, 71-83 Propionibacterium acne, 691 Proskauer and Beck, glycerol-aspara- gine medium, 171 Protein, determination in spinal fluid, 536 Proteose No. 3 broth with chloride, 467 Proteus group, cultural characteristics, 293, 295 in meningitis, 429-430, 433, 455 Protozoa, intestinal, 808-819 Providence group of Enterobac- teriaceae, 299 Pseudoglanders (melioidosis), 595-597 Pseudomonas infections, 635-639 cultural characteristics, 293, 295, 638 in meningitis, 420-430, 433, 455 in urethritis, 495, 497 media for, 178, 637 Pseudomonas aeruginosa, 635 media for, 178, 637 P. (Malleomyces) pseudomallet, 596 Pseudotuberculosis (see also Pasteur- ella pseudotuberculosis), 358 Pulmonary infections (see specific dis- eases or microorganisms) Pus specimens (see specifiic diseases or microorganisms) PVA fixative, 812, 817 Pyrazinamidé-susceptibility tests, 830 @ Quebec colony counter, 292 Quellung reaction (see also capsular swelling), 224 Rabbits, inoculation of (see also Ani- mal inoculation), 51 Raffinose-serum-tellurite, agar, Whit- ley and Damon (CM No. 82), 164 Ratbite fever, 640, 642 Rats, inoculation (see Animal inocula- sodium tion) vectors of melioidosis, 596 plague, 377 Reagin tests for syphilis, 503, 511-536 Rectal swabs for gastrointestinal in- fections, 289 INDEX Reference laboratories, 85 for plague, 379 for typing, Salmonella, 301 staphylococci, 217 Refrigerators, 15 Reilly skin test for Pasteurella, 369 Reiter protein tests for syphilis (RPCF), 505, 525 Reporting, laboratory infections and accidents, 103 plague cases, 379 results of examinations, 83-85 (see also specific diseases or micro- organisms) for antimicrobial-susceptibility tests, 822 Rh typing, 841-867 Rhinosporidiosis, 720 Rhinosporidium seebert, 724, 725, 734 Rhodamine isothiocyanate, 54 Rice-Tween 80 agar (CM No. 110), 177 Richards and Miller fluorescence stain, 41 Rickettsial stain, 45 Ringer's solution, 167 Rotators, 19 Roundworms, 779 Rustigian and Stuart urea broth, 158 S Sabouraud’s agar (CM No. 105), 175 maltose (CM No. 113), 178 Safety precautions, 94 in lyophilization, 100 in plague laboratory, 385 in transferring cultures, 95 in tuberculosis laboratory, 285 with animals, 98 with botulinum toxin, 322 with centrifuges, 17 with chemicals, 32 with homogenizers, 98 with syringe and needle, 98 Saline agglutinins in Rh typing, 843 Salmonella group, of Enterobacteri- acae, 288-304 (see Bacterial In- fections of the Gastrointestinal Tract) in food poisoning, 320 in gastrointestinal infections, 288- 304 in meningitis, 429-430 Salmonella-Shigella agar 59), 155 (CM No. INDEX Sanderson and Greenblatt, cultivation of H. ducreyi, 489 Schaudinn’s solution, 814 Schistosoma hematobium, 781, 784 S. japonicum, 781, 784 S. mansonii, 781, 784, 803 Schiiffner and Mochtar (agglutina- tion-lysis) test for leptospirosis, 545 Selenite (F) broth (CM No. 63), 157 Semisolid agar (CM No. 15), 132 for Leptospira, 168-169 for motility test, Edwards Bruner (CM No. 69), 159 Semisolid fermentation media, 128 Sensitivity tests, antibiotic, 824 of tubercle bacilli, 275, 829 Serological examinations as aids in diagnosis (see specific diseases or microorganisms) Serological examinations for identification (see specific mi- croorganisms) Serotypes, of enteropathogenic Es- cherichia coli, 305-307 of Hemophilus, 422 of Herellea, 626 of Klebsiella, 609-612 of Leptospira, 563 of Listeria, 618 of meningococci, 452-453 of Pasteurella multocida, 367-368 of P. pseudotuberculosis, 358-360 of pneumococci, 224 of Salmonella, 297, 301 of Shigella, 297, 299 of staphylococci, 219 of streptococci, 189, 203 SF medium for enterococci, 332 Shaking machines, 19 Sheep cell suspensions for Kolmer syphilis tests, 526 Shigella group of Enterobacteriaceae, 288-304 (see Bacterial Infections of the Gastrointestinal Tract) Shigella ambigua, Sh. boydii, Sh. dysenteriae, Sh. flexneri, Sh. son- net, 296 and 298 Simmons’ citrate agar, 158 ‘Skin, examination of, for microfilaria, 799 test (see Intradermal tests) scrapings for leprosy, 283 and 885 Slide agglutination tests (see specific microorganisms) in blood grouping, 838, 841 in Rh typing, 846, 865 Solutions (see also Stains) Andrade’s indicator, 148 Broquet’s fluid, 380 buffer, for malaria stains, 749 Sorensen’s, 49 Dobell and O’Connor’s iodine, 787 egg albumin for culture media, 146 hemoglobin for GC medium base agar, 142 heparin, for Toxoplasma dye test, 655 indicator for anaerobiosis, 665 methylene blue-phosphate mixture, for malaria, 748 PVA fixative, 812, 817 resuspending (EDTA), for syphilis antigens, 512 Ringer’s, 167 saline, for VDRL tests for syphilis, 516 Schaudinn’s fixative, 814 Sodium azide-crystal violet, agar, Packer’s, 169 enrichment broth, Pike (CM No. 30), 139 Sodium desoxycholate, agar (CM No. 56), 152 citrate agar (CM No. 57), 152 Sodium oleate in media for Hemo- philus, 419 Sodium sulfate-acid-triton-ether cen- trifugal sedimentation for intes- tinal parasites, 792 Sodoku (see Spirillum minus), 640 Sorbic acid-polymyxin thioglycolate broth for anaerobes, 669 Sorensen’s pH buffer solutions, 49 Spinal fluid (see Cerebrospinal fluid) Spirillum minus, 640 Spores, thermal death time of, 26, 27 Sporotrichosis, 720 Sporotrichum schenckii, 724, 725, 732 Sporozoa, 809 Sputum examination (see specific dis- eases or microorganisms) extract SS agar, 155 Staining (see specific microorgan- isms) Stains alkaline methylene blue, for Toxo- plasma, 665 886 Stains—continued Burke's iodine, 42 Dienes, 44 Giemsa, 40 for malaria, 750 Gram, 42 Heidenhain’s iron hematoxylin stain, 814 Hucker’s crystal violet, 42 iodine stain for direct fecal films, 787 Jenner's, 39 Kinyoun's, 42 Ljubinski’s, 237 . Loeffler’s methylene blue, 43 Macchiavello, 45 May-Gruenwald, 39 Richards and Miller fluorescence, 41 rickettsial, 45 Steenken’s, 267 Thedane blue solution T-5 for Spiril- lum minus, 641 "trichrome, for protozoa specimens, 816 Wayson’s, 44 Wright-Giemsa, 40 Wright's, 39 Ziehl-Neelsen, 40 Staphylococci, anaerobic, 681-682 coagulase-positive, 48, 215, 326 enterotoxin-producing, in food poi- soning, 320, 326-330 STAPHYLOCOCCUS, INFECTIONS, 207-221 enterotoxin agar, Dolman et al. (CM No. 10), 131 Staphylococcus albus, 210 Staph. aureus, 210 in meningitis, 429-430, 433, 455 Staph. citreus, 210 Staph. epidermis, 210 Staph. pyogenes (aureus) 209, 210 in pneumonia, 223 Starch agar for gonococci and menin- gococci (CM No. 42), 143 Steam sterilization, 25 Steenken’s stain for acid-fastness, 267 Sterility tests of culture media, 116 Sterilization, 25-30 of culture media, 112-117 of instruments for collection of blood, 4 Sternal, marrow cultures for Bru- cella, 338 punctures for malaria, 774 in fecal INDEX “Stick test” in Rh typing, 848 Stills, water, 21 Stock cultures, of Hemophilus, 423 of leptospiras, 557 of streptococci, 191 Stomach contents, examination, for fungi, 734 for tubercle bacilli, 272 Straus reaction for glanders, 594 Streptobacillus moniliformis infec- tions, 642-651 Streptococci, anaerobic, 679-681 habitat of, 189 identification by fluorescent-anti- body tests, 58-60, 203 in food poisoning, 320, 330 STREPTOCOCCUS INFECTIONS, 187-206 Streptococcus pyogenes, 188 in meningitis, 429-430, 455 Streptococcus species, 188 Streptomyces species, 721, 730 Streptomycin-sensitivity tests, 277, 830 Strongyloides stercoralis, 779, 782 Stuart et al. transport agar medium for gonococcus (CM No. 51), 149 Stuart's broth for Leptospira (CM No. 90), 168 Sugar in spinal fluid, test for, 442, 444 Swabs (see also Nasopharyngeal swabs) for diphtheria, 233 for gonorrhea specimens, 465 SypHiILIS, 502-543 (see also Treponema pallidum) selection of tests for, 503 T TaBLE oF CONTENTS, Xi Taenia saginata, 780, 783 T. solium, 780, 783 Tapeworm, 780 “Tech” medium for Pseudomonas, 637 Tetanus (see also Clostridium tetani), 662 Tetrathionate enrichment broth (CM No. 62), 156 Thallous acetate-tetrazolium-glucose agar, Barnes (CM No. 28), 138 Thedane blue solution T-5, stain for Spirillum minus, 641 Thermoprecipitation test (Ascoli) for anthrax, 589 Thermoregulators, 21 INDEX Thioglycolate broth, with glucose (CM No. 19), 135 without glucose or indicator (CM No. 20), 136 Thiol broth with 0.1% agar (CM No. 86), 166 Thionin tryptose agar for Brucella, 345 Thjotta and Avery, yeast extract, 140 Threadworms, 779 Throat cultures, for diphtheria, 232 for streptococci, 193 Throat swabs, for enteropathogenic Escherichia coli, 310 for Hemophilus, 418 for streptococci by fluorescence technics, 58-60, 203 Tinea, capitis, 703 corporis, cruris, nigra, pedis, ungu- ium, versicolor, 712 and 713 Tissue examinations for chancroid, 491 for granuloma inguinale, 494 for Leptospira, 551 for listeriosis, 615 for nematode larvae, 803-805 for plague, 379 for toxoplasmosis, 654 for tuberculosis, 266, 267, 273, 282 Todd-Hewitt, broth (CM No. 24), 137 sheep blood agar (CM No. 25), 137 Torula histolytica (Cryptococcus neo- formans), 736 Torulopsis species, 727 Toxoplasma gondii, 652-658 Toxoplasmosis, 652-658 Trematodes, 781 Treponema pallidum, complement-fixa- tion test for syphilis (tpcf 50), 506 dark-field demonstration of, 537-541 immobilization test for syphilis (TPI), 505 Treponemal tests for syphilis, 504 Trichinella spiralis, 779, 803 Trichomonas hominis, 809 T. vaginalis in urethritis, 495 Trichomycosis axillaris, 704 Trichophyton species, 704, 705, 709. 712, 717 Trichosporum beigelii, 705 Trichosporon beigelit (see Tricho- sporum beigelit), 705 Trichuris trichiura, 779, 782 887 Triple sugar iron (TSI) agar (CM No. 53), 150 reactions on, 294, 295 Tryptic digest, agar, Douglas (CM No. 48), 147 broth, Douglas (CM No. 47), 147 Trypticase soy broth for Brucella, 131, 341 Tryptone broth, 129 Tryptose dextrose vitamin B, agar (CM No. 37), 141 broth (CM No. 36), 141 Tryptose, extract agar with sheep blood (CM No. 26), 138 infusion agar with sheep blood (CM No. 27), 138 phosphate semisolid agar (CM No. 35), 141 Tube agglutinations (see Agglutina- tion tests) Tuberculin, glucose-asparagine broth for, 177 TuBERCULOSIS AND LEPROSY, 261-286 Tularemia (see Pasteurella tularen- sis), 369 Typing (see Serotypes) u Ultracentrifuges, 17 Ultraviolet sterilization, 30 Urea, agar, Christensen (CM No. 68), 158 broth, Rustigian and Stuart (CM No. 67), 158 Urease test, 294, 297, 298 Urethral specimens, for gonococci, 469 FA tests, 62 Urethritis, nongonococcal, 495-498 microorganisms found in, 495 Urine examinations (see specific dis- eases or microorganisms) Uterine specimens for listeriosis, 615 Vv Vaginal specimens, for gonococci, 469 FA tests, 62 for listeriosis, 615 Vapor-heat sterilizers, 28 VDRL flocculation tests for syphilis, 514-525 VENEREAL Diseases, EXCLUSIVE OF SypHILIS, 462-501 Verwoort-Wolff, broth for Lepto- spira (CM No. 83), 167 Vi antigens, 302 Vibrio comma (cholerae), 313-316 VV. fetus Infections, 659-661 Viomycin-sensitivity tests, 830 Viral infections, laboratory-acquired, 92 Virulence of Corynebacterium diph- theriae, 245-253 Voges-Proskauer test, 46 w Washing glassware, 23 Water, baths, 14 for culture media, 117 stills and demineralizers, 21 Wayson’s stain, 44 Whipworm, 779 White and Sherman, enterococci, 331 Whitley and Damon, glucose-serum- tellurite agar, 165 raffinose-serum-tellurite agar, 164 WaooriNnGg CoucH (see Bordetella pertussis), 398-413 Wilson and Blair bismuth sulfite agar, 153 Wood's light, 703, 704 medium for INDEX Wound cultures for anaerobic infec- tions, 662 Wright-Giemsa stain, 40 Wright's stain, 39 Wuchereria bancroftii, 779. W. malayi, 779, 800 X Xenopsylla cheopis, vector for melio- idosis, 596 Y Yeast autolysate, agar for Brucella (CM No. 40), 142 broth for Brucella (CM No. 39), 142 Yeast extract, Thjotta and Avery (CM No. 32), 140 z Ziehl-Neelsen stain, 40 for acid-fast organisms, 266 for fungi, 701 Zinc sulfate centrifugal flotation for intestinal protozoa, 783 0 1 A U. C. BERKELEY LIBRARIES CO47249841 Br we Fa Seu te oo Pre 4 +