Sor eee pniaielitetel ote SPEAR TE eae rape eee Neca aes setsebet, C2 ARAN Tris eis aes igi tae Hi Be: i eet rere ret SS RARE San potas eae Ned Sie aes +t F SERS a tiers Se ee i f re Fre a iol. ? sesh ay Baie Be rere Aitet re paciete ae Se ase Sette sane ibe Ane eC GTA ee Sterile inn oa stepireaden * La 4 3 ae eye pigs tate a prt pte es : Ban ies 2 eee eae tf. Ltt ree Bhatignse oly ee rs Cornell Aniversity Library BOUGHT WITH THE INCOME FROM THE SAGE ENDOWMENT FUND THE GIFT OF Henry W. Sane x189Q1 RETURN TO ALBERT R. MANN LIBRARY ITHACA, N. Y. Cornell University Library _ QR 46.W72 1905 - (mi f bi wi 3 201 922 nam 0 A MANUAL OF BACTERIOLOGY WILLIAMS A MANUAL BACTERIOLOGY BY HERBERT U. WILLIAMS, M.D. PROFESSOR OF PATHOLOGY AND BACTERI'LOGY, MEDICAL DEPARTMENT, UNIVERSITY OF BUFFALO REVISED BY B. MEADE BOLTON, M.D. ’ EXPERT, BUREAU OF ANIMAL INDUSTRY, WASHINGYON, wv C. WITH 108 ILLUSTRATIONS FOURTH EDITION, REVISED AND ENLARGED PHILADELPHIA: P, BLAKISTON’S SON & CO. 1or2 WALNUT STREET 1905 + Ek \ \Aq\ ote Copyright, 1905 By Hersert U. WILLIAMS. PRESS OF WM. F. FELL COMPANY PHILADELPHIA PREFACE TO THE FOURTH EDITION. In the present edition the scope and purpose of former editions of the manual will be found unaltered, and, aside from verbal changes where these seemed desirable, only such, modifications as were required to bring the work up to date have been made. The object aimed at in the revision has been to preserve the char- acteristic features of the work, while introducing those modifica- tions and additions which progress demanded. Recent advances made it necessary to enlarge the chapters on bacterial poisons and on immunity, and the additional space given to these subjects is justified by their importance not only to the specialist in bacteriology, but to every well-qualified phy- sician as well. The chapter on immunity in particular will be found to be expanded in the matter of immunity proper, and also in regard to the nearly allied processes of the formation of specific agglutinins, precipitins and cytolysins. In the treat- ment of these subjects the aim has been to present the well- established results of investigations, and to state briefly the conflicting views in yegard to their theoretical explanation. For, although Ehrlich’s side-chain theory seems to have found in America and some other countries more general acceptance than the other explanations that have been offered, those of Bordet and of Arrhenius are deserving of consideration on ac- count of the important experimental work which has been done in their support, if for no other reason. Bacteriology is not a subject which can be learned from books alone or without instructors, and the manual is intended to supplement laboratory work and teaching, but not to supplant v Vi. PREFACE TO THE FOURTH EDITION. these. Still, methods of cultivation and examination are given in some detail. Dr. Williams was unable, for a number of reasons, to revise and correct the book himself. The entire responsibility for the present edition (including the chapters by Dr. Clinton and Dr. Carpenter) rests with the reviser. B. Mreapr BOLton. WasHinctTon, D. C., September, 1905. PREFACE TO THE THIRD EDITION. The plan used in the preceding editions of this manual has been followed in the preparation of the present one. The only departures have been in the insertion of a short historical sketch and the freer use of references to original articles and reviews. It is hoped that these features will assist in arousing the interest of students. As far as possible, reference has been made to articles in American and English journals likely to be easy of access. Besides the ones just named, numerous addi- tions have been made which the recent advances in our knowledge have rendered necessary. Most of the illustrations of apparatus are new. The photomicrographs also are new and original with a few exceptions noted inthe text. Itis probably needless to say that none of them were retouched. The writer is indebted to the Gratwick Laboratory of Buffalo for the use of its facil- ities in making these photographs. BurraLo, New York, August, 1903. vii PREFACE TO THE SECOND EDITION. Although there has been no lack of works on bacteriology, it seemed to the writer that there was still a field open for one which sought to give the portions essential to medical science in a concise manner. It is gratifying, therefore, that the first edition of this little book should have been exhausted so soon. Whether wisely or not, it is a fact that many medical schools require their students to absorb an amount of knowledge that taxes the brain to the utmost. While such conditions remain, the need is urgent for presenting what is taught in the accessory branches in as condensed a form as is consistent with a clear understanding of their great fundamental principles. It is mastery of such principles, after all, which is the object of a course in bacteriology, for they are essential to a correct under- standing of most of the other branches. After that has been accomplished (including the applications of bacteriology to diagnosis), it must be admitted that other branches deserve a larger amount of the student’s time. This may be said without meaning to minimize the importance of bacteriology in the train- ing of a physician. In the opinion of the writer it is neither possible nor desirable that every graduate should be a trained bacteriologist. However, no instructor can hope to bring the principles above mentioned home to his classes except by labo- ratory work. Very little attempt has been made to outline the program of a laboratory course, as that will always need to be planned according to the circumstances under which it is given. The purpose of this book is to give in the smallest possible space the facts which a physician must know, with some of those ix x PREFACE TO THE SECOND EDITION. which it is desirable that he should know, and a little of that which he may learn if his needs or inclinations lead him to go further. It is acknowledged, however, that, in deference to precedent, this purpose has not been carried to its fullest extent. Much time has been spent on the index, in order to make the con- tents quickly accessible.. It is a source of regret to the writer that the additions which the revision seemed to demand have made the present book a little larger than the first edition. BuFrato, NEw York, June, 1901. PREFACE TO THE FIRST EDITION. In this manual the writer has endeavored to describe the laboratory technique which the beginner must follow, and at the same time to give a concise summary of the facts in bacteriology most important to the physician. In preparing a work of this character, which claims to be nothing more than a compilation, the standard text-books were necessarily consulted freely. On account of the need for brevity it has, in most cases, been im- possible to mention authorities. The writer is glad to have this opportunity to acknowledge his obligation to the works of Sternberg, Fliigge, Giinther, Eisenberg, Abbott, W. H. Park, Muir and Ritchie, Vaughan and Novy, and McFarland; and to numerous papers by Pro- fessor Welch and others. It is thought that the chapters on Germicides and Surgical Disinfection, by Drs. Thomas B. Car- penter and Marshall Clinton, will be useful not only for the information presented in them, but especially in correlating that information with the facts of bacteriology. BuFFALo, NEw York, October, 1898. CONTENTS, PAGE Tntroduction soso meke ede dpsed “ees ode eine Seek Gdeae gas eaoeas I Historical Sketehiceiscsece y4 ve'e eybieretare foee-$ asa dialdiala delved tecredaavevevesats 8 PART I. BACTERIOLOGICAL TECHNIQUE. CHAPTER I. Examination of Bacteria with the Microscope, including Methods of TAMING ys. sales 2s Sesan wapentvoulie @ Sa aoe anladom ded a eanraemiale 18 CHAPTER II. Sterilization 20S Saee ec com c tenes eal area oleae bananas 49 CHAPTER III. Culture med ia xpscvae chiens se x tse peas geass ates Sie eave evn bn ansehen els 59 CHAPTER IV. The Cultivation of Bacteria. Tube-cultures; the Incubator; Anaérobic Methods: iccrncccanwanl Sia exept Sead dp eatedete ess segs 72 CHAPTER V. The Cultivation of Bacteria (Continued). Isolation of Bacteria; Plate- CUlEURES 2 ys oe icckd cee os cue chee ses 22 kaesae ele ay sy 84 CHAPTER VI. Inoculation of Animals. Autopsies; Collodion Sacs.................--- 92 CHAPTER VII. Rui vcuus ss ca reteecewes tee es i ase seines bee e eae ew eee sy IOI xiv CONTENTS. PART II. CHAPTER I. PAGE Classification; General Morphology and Physiology of Bacteria........-- 106 CHAPTER It. Products of the Growth of Bacteria........-----20eeeee ee eee reece 117 CHAPTER III. Bacteria of Soil, Air, Water, and of Milk and Other Foods.....--------- 122 CHAPTER IV. Bacteria of the Normal Human Body.......-----------++-+--+ eee ee ee 14¢ " CHAPTER V. Bacteria in Diseases oa 2s ce aentye Soieiiotte ast oem meee eee OBS 146 CHAPTER VI. Bacterial Poisons; Agglutinins; Lysins; Precipitins...........---+-.---- 161 CHAPTER VII. Immunity; Phagocytosis; Antitoxin...............-2-----220 eee eee 169 CHAPTER VIII. Disinfectants and Antiseptics. By Thomas B. Carpenter, M.D. ...-.-...-.- 193 CHAPTER IX. Preparation of Instruments, Ligatures, Dressings, etc., for Surgical Pur- poses. By Marshall Clinton, M.D..........---.. 2-00-20 eee eens 210 PART III. NON-PATHOGENIC. BACTERIA sunsiucevesuencexveerueersease 220 Yeasts and Mouldsi2s20essea ees osecs sandeeesbe sete e sa gaeseee = 220 PART IV. PATHOGENIC BACTERIA. Suppuration and. Allied Conditions... . 2. sccacacccaue sere eeweeecee's 233 Staphylococcus pyogenes aureuss ..cede oc cicicae desis e ne oe ole eistenimere es 241 a Oe “AlbUS. sswciss'ss' vies oemeinateed ys sees exmoreees xs 244 ie épidérmidis:albusieu: = 22: cs sakecebeeens ts4 tetecge ees ¢ 244 StreptGCOceus pyoreneSenaeenik. : cuuseings Fe cae ietare Sod Gee darseuies oS 244 iy Ob Mrysipelasccsciguis fic Gineigaeeiee 2 Se Acne eases 248 Mickococets telragenus: . icine wins oe aan dawineleutdet sat tammnenawne 248 i. lanceolatus (of Pncumonia).................2..22..0220- 249 “ MelteNnslSsccsanceciOe wee acearae oases games oc gels 253 CONTENTS. XV PAGE Diplococcus intracellularis meningitidis...............------00---000-- 254 Micrococcus of Gonorrhea...... 2.2.2.2 22 0.22 e ee ee eee eee e nee e eee 255 Bacillus of Soft Chancre.............222.22.0--002000: oeaeeeweus eee 258 “« pneumonie (of Friedlinder).............2.2.-200--200eeeeeee 258 “of Rhinoscleroma.... 2.0.00... cece ee ee eee eee neces 259 ME” SHAY CORENES < 31cic:s cio’ S04 cigcaamargeeanee en aGaie dannenomaldianddda sae 260 SS SPYOCVANCUS Ls <5 s 22 cscs maaan iedadse ase Hees eameneds «ace. 260 HS APROLEUS Sots aterasa nd dade adalviemeen seas pose nee eee wens 2 262 eS (6h BubOnIC Plague. <.<.:asencrecataw + ccedeinwescces eeeentes se 263 “f jatbogenes capsulatus scicccccccuuee soos. eninge Ses weae ean ct 267 “of Malignant Edema: .. cccueeicins.« N 5 ° be corrected by adding the necessary amount of as sodium hydroxide. It should be boiled again, and again titrated, and any deficiency made good. * A normal solution of any substance contains, in a liter, as many grams of the substance as there are units in its molecular weight, in case it contains a single atom of replaceable hydrogen. If it has two atoms of replaceable hydrogen the number of grams used equals the molecular weight divided by two; and soon. Thus the molecular weight of sodium hydroxide is 4o, and its normal solution contains 40 grams of sodium hydroxide in a liter. It is not expedient to prepare normal solutions of sodium hydroxide by weight. For convenience, crystallized oxalic acid is used as a starting point in making normal solutions. Its molecular weight, including a molecule of water of crystallization, is 126. As it is a dibasic acid (having two atoms of replaceable hydrogen), half of this weight, or 63 grams, per liter, is taken. Any x acid N . solution will exactly neutralize an equal volume of any ae alkaline solution. To make = sodium hydroxide solution, add about 41 grams of pure caustic I soda to a liter of distilled water. Find the amount of this solution needed to exactly neutralize 1 c.c. of —— solution of oxalic acid; this amount contains I the quantity of sodium hydroxide which should be present in 1 c.c. of a normal solution. It is now possible to calculate the amount of distilled water to be added in order that 1 c.c. of the sodium hydroxide solution may neutralize 1 c.c. of the solution of oxalic acid. With an a5. solution of sodium x hydroxide as a standard, an ~— solution of hydrochloric acid may be prepared. IT Twentieth normal solutions have one-twentieth the strength of normal solutions. 62 MANUAL OF BACTERIOLOGY. It is rarely necessary to repeat the process, except to determine that the neutral point has been reached. After neutralizing it is boiled thirty minutes and filtered. Enough eo hydrochloric acid or sodium hydroxide is added to give r the degree of acidity or alkalinity desired. It is then sterilized. An acid reaction may be denoted by +, an alkaline by —. The degree N Fi : of acidity or alkalinity may be indicated by the amount of oe solution required to render the medium neutral to phenolphthalein, thus + 1.5 signifies that a medium is acid, and requires 1.5 per cent. of = sodium hydroxide to neu- tralize it. A reaction of + 1.5 is recommended as the optimum. There is much dis- agreement as to what reaction is most favorable for the growth of the majority of species of bacteria. In any case the degree of reaction should be noted in descriptions. Bouillon may be modified by the addition to it of other substances, the most important of which are glycerine (6 per cent.) and sugars,—as dextrose,* saccharose or lactose (1 per cent.). It is better to sterilize media containing sugars in the steam sterilizer by the fractional method than in the autoclave, where decomposition of the sugars may occur. Dextrose-jree Bouillon.—Ordinary bouillon often contains some muscle- sugar, which is objectionable if fermentation tests with lactose or saccharose are to be made. Muscle sugar must also be removed from the beef-juice in preparing diphtheria for the production of antitoxine. To secure bouillon free of sugar, beéf-infusion is prepared from meat, and is inoculated in the evening with a quantity of bacillus coli communis, and kept in the incubator. Early next morning it is boiled, filtered, peptone and salt added, and the bouillon is prepared as usual. Nutrient Gelatin. BeGi-extTact: Zecnanacinni scree sand teen Sena 3 grams. P@PLONG ied sec ces sine bad ay aces Pete ieals Io grams. Sodium chlorides wicciicnccsecce ytc2ss. oeeelesaekeeese se 5 grams. Gelatin (best:gold label). a uf ea 5 73 er ‘6 i ! Te ge ete “fe ¢ (a ms +” o- we pope Ei ? o % ‘ ' EN A 1 ‘ ee %, / Aa - ; Pe & So : s ,* "4 ¢ - / es y 4 ‘ f . ‘ . fe ‘ Rr ; ie a ’ £ . a “ 1 2 a Vig 4 ~ is ‘ yf by . ss ~ ~ | Fic. 70.—BACILLUS PYOCYANEUS, PURE CULTURE. (%X 1000.) Indol is formed in Dunham’s peptone solution. Coagulated blood-serum is liquefied. The Bacillus pyocyaneus seems to be rather widely dis- tributed in nature; it has been found on the skin, in nor- mal feces, also in diarrheal discharges and in dysentery. It is the cause of the color in blue or green pus. It has fre- quently been demonstrated in pus, but oftenest perhaps, in mixed infections. It has been found in various abscesses, in otitis media, peritonitis, appendicitis and bronchopneu- 262 MANUAL OF BACTERIOLOGY. monia. It has been known to produce general septicemia.* It is pathogenic for guinea-pigs and rabbits, in whom it may produce septicemia. In animals it may lead only to local suppuration, from which they may recover, being made im- mune from subsequent infection with this organism. It ap- pears that an antagonism exists between the products of the Bacillus pyocyaneus and the anthrax bacillus. Rabbits which have been inoculated with cultures of the anthrax bacillus may recover if they are injected shortly after with a culture of the Bacillus pyocyaneus. There appears to be a whole group of fluorescent organisms of slightly different characters which closely resemble. one another, all classed as pyocyaneus. Bacillus proteus.—A bacillus with rounded ends, vary- ing much in length, breadth 0.4 to 0.64; frequently appearing as short ovals like micrococci; sometimes growing out into long filaments, so that it is said to be pleomorphic. Rounded involution forms occur. It is not stained by Gram’s method. It is motile. Spore formation has not been observed. It is aérobic and facultatively anaérobic. It grows rapidly at or- dinary temperatures. This organism was originally described by Hauser as three different species—Proteus vulgaris, which was said to liquefy gelatin rapidly, Proteus mirabilis, which liquefied gelatin slowly, and Proteus Zenkeri, which did not liquefy gelatin. It seems probable that these organisms were, in fact, varieties of the same species, now called Bacillus proteus. Upon gelatin-plates the colonies present a characteristic phe- nomenon, when seen under the low power, in the projection of processes which subsequently change their form and position, and which may become entirely detached from the original * Lartigau. Philadelphia Medical Journal. September 17, 1898. Journal Experimental Medicine. Vol. III. 1898. Perkins. Journal Medical Re- search. Vol. VI. Igo01. PATHOGENIC BACTERIA. 26 3 colony, so that the surface of the gelatin may become covered with so-called ‘‘swarming islands.” The proteus grows on the usual media, tending to produce a foul odor, decomposition and alkaline reaction. In urine it converts urea into ammonium carbonate. This organism is one of those which were formerly described under the name of Bacterium termo. It is among the most common and widely distributed bacteria. It has been found in decomposing animal and vegetable substances, in the feces, Fic. 71.—BAcILLUS OF BuBonic PLAGUE.—(Yersin.) in the urine in cystitis and in the discharges of children suffering from cholera infantum. It appears that this organism may occasionally be pathogenic to man, causing pus formation, peritonitis and even general infection.* Cultures injected in considerable amounts may be pathogenic to animals. Bacillus of Bubonic Plague.—An oval or short rod-shaped bacillus, with rounded ends, sometimes possessing a capsule. It isnot motile. It does not form spores. With the aniline dyes the ends stain more deeply than the middle; this is called polar * Ware. Annals of Surgery. Vol. XXXVI. 1902. 264 MANUAL OF BACTERIOLOGY. staining; by Gram’s method it is decolorized. It is aérobic. It grows at ordinary temperatures, but better in the incubator. It grows on most media. The growths are grayish-white. Gelatin and blood-serum are not liquefied. In bouillon, the medium remains clear, while a granular deposit forms on the sides and bottom of the tube. In bouillon to which a few minute drops of sterile oil, as cocoanut oil, have been added, a growth takes place from the under side of the oil drops. Such growths extend down, and are called stalactite growths. The stalactites break off, with the slightest disturbance. Remarkable involution forms appear on agar containing 3 per cent. of common salt. The stalactite growths and the forms occurring on salt-agar are considered the most charac- teristic cultural peculiarities.* It is sometimes sensitive to drying, but may sometimes survive prolonged drying. When spread in thin layers, it is killed in three to four hours by direct sunlight; in a few minutes by steam at 100° C., and in one hour by one per cent. carbolic acid.t It is pathogenic for rats, mice, guinea-pigs, rabbits and a number of other animals. In man it appears usually to enter through wounds of the skin. Other possible avenues of infection are the air-passages, the mouth and the gastro-intestinal tract. In plague three different clinical forms are to be recognized—the bubonic, the pneumonic and the septicemic. The bubonic form is com- monest. The point in the skin at which the inoculation takes place seems generally to exhibit no inflammatory reaction. The lymph-nodes are generally swollen, especially the deep in- guinal and axillary nodes. The swollen lymph-nodes may suppurate. The suppurating nodes are often infected simul- * Wilson. Journal Medical Research. Vol. VI. 1901. +See Rosenau. Viability of Bacillus pestis. Marine Hospital Service. Hygienic Laboratory Bulletin. No. 4. Igot. PATHOGENIC BACTERIA. 265 taneously with micrococci. The bacilli are numerous in the enlarged lymph-nodes, but may be detected in the other organs of the body and in the blood. The organism is formed in the fluid drawn with a hypodermic needle from bubo during life. It may be cultivated from this fluid, and recovered from rats and guinea-pigs inoculated with it. In the puewmonic or pulmon- ary form the bacilli occur in the sputum, and may be tested in the same manner. This type of the disease is said to be very fatal. In the septicemic form no primary bubo is found; but a bubonic case may become septicemic, and this form is very fatal. During epidemics of plague it has been noted that rats may die in large numbers, and plague bacilli have often been re- covered from the bodies of such rats. The systematic de- struction by health departments of all the rats possible is im- portant where an epidemic is present or is feared. The same applies to mice. The agency of fleas as carriers of the bacilli has been suggested, but has not yet been proved; this is equally true as to flies. The greatest care must be used in working with the bacillus of plague. A number of fatal results have occurred through it in laboratory investigators. Haffkine has devised a method of protective inoculation against plague consisting of the injection of cultures of plague bacilli which have been sterilized by heat, with a little carbolic acid added. An active immunity which is quite lasting, it is maintained, may be secured by this method in some days. The injection is sometimes followed by considerable consti- tutional disturbance. This method seems likely to be of con- siderable value. Yersin and others have prepared protective sera on the same general principles used in making other sera for effect- ing passive immunity. The results so far obtained with these sera are very encouraging. 23 266 MANUAL OF BACTERIOLOGY. An agglutination reaction has been described; ‘but this is not likely to be of great value in diagnosis. The period of incubation in this disease is from two to seven days. It has occasionally appeared in civilized countries during recent times, though not to a very serious extent. Among the localities of importance to us it has recently visited the Philippine Islands, California and Mexico. It has ravaged “ . arr hk eh - 2" + 5 be Fic. 72.—BACILLUS AEROGENES CAPSULATUS, SMEAR PREPARATION FROM Rassit’s LIVER. (X 1000.) the southeastern part of Asia within a few years. In the Middle Ages, and in succeeding centuries, it devastated many of the countries of Europe, where it was one of the most important of the pestilences that went in those days by the name of the “plague.” It appears to have been the disease known in English history as the “black death.” * * For further details concerning plague consult articles by Barker, Novy and Flexner. Transactions of the Association American Physicians. 1902. Calvert. American Medicine. January 24, 1903. PATHOGENIC BACTERIA. 267 Bacillus aérogenes capsula- tus.—A thick bacillus, 3 to 6 4 in length, frequently capsulated, discovered by Welch and Nuttall. The capsules may be found in preparations from animal tissues, but rarely in cultures. It some- times forms spores, chiefly in cultures on blood-serum. The vegetative forms are destroyed at 58° C. moist heat in ten minutes, but the spores withstand boiling nearly eightminutes. It is not mo- tile. Itstains by Gram’s method. It is anaérobic, and is readily cultivated by Buchner’s method for anaérobes. It grows best at the body temperature, but will grow at the room tempera- ture. It may liquefy gelatin slowly or not at all. The growths are whitish. In media containing lactose, dextrose or saccharose it produces an abun- dance of gas; but according to Welch, it is also able to form gas from proteids. Milk is co- agulated, and the reaction be- comes acid. Gas forms upon potato, where the growth is thin and grayish-white. It occurs in the intestine of man and various other animals, in soil, sewage and water. It is Fic. 73.—BACILLUS AERO- GENES CAPSULATUS, CULTURE IN DEXTROSE-AGAR, SHOWING GAS- BUBBLES. 268 MANUAL OF BACTERIOLOGY. not usually pathogenic for rabbits and mice. In guinea-pigs, sparrows and pigeons it may produce “gas phlegmons.”’ It has been found on numerous occasions in the organs of human cadavers in which a development of gas had taken place, producing bubbles or cavities in the tissues, imparting to them a peculiar spongy character (German, Schaumorgane). Probably this is, as a rule, a post-mor- tem invasion, but there is reason to believe that in some cases it enters the circulation during life. It has been found in cases of emphysematous gangrene or cellulitis, in various uter- ine infections, including physometra and emphysema of the uterine wall, in pneumothorax and pneumoperitonitis, and in other pathological conditions where gas occurs in the tissues. Exceptionally it may cause pus-formation.* This bacillus, or the gas formed by it in the organs of human cadavers, appears to have furnished the basis for some of the cases in which death has been ascribed to the entrance of air into the veins during life. It is the same as the organism described by E. Frankel as Bacillus phlegmonis emphysematose. Bacillus cedematis maligni (French, Vibrion septique). —A bacillus about 1 in breadth, 2 to 10 in length, which may form threads, having rounded ends when occurring singly. It is motile, having flagella at the sides and ends. It forms spores, and may bulge at the center in consequence of the spores formed there. It is decolorized by Gram’s method. It is a strict anaérobe and is best cultivated under hydrogen. It grows at ordinary temperatures, but better in the incubator. It liquefies gelatin and blood-serum. ‘The colonies in gelatin are spherical and appear like little bubbles. It grows well upon agar. Gas may be produced in these media. It is found in garden-earth, street dirt and in putrefying organic material. It is pathogenic to rabbits, guinea-pigs, * Welch. Philadelphia Medical Journal. August 4, 1900. PATHOGENIC BACTERIA. - 269 mice, pigeons and various other animals, including man. Inoculation results in the production of swelling and edema, spreading from the point of inoculation. Gas may be pro- duced in the tissue. It may lead to wide-spread septicemia. Bacillus tetani.—A slim, straight bacillus, with rounded ends, which may form in threads. It is slightly motile. Spores form in culture-media at the end of thirty hours in the in- cubator. The spores are located at one end, which is » ‘ cs _ {eee fi Fo 7 Saas s\ Z, ‘ \ A = RY SA ! Vie pd a eg AZ ee 5 \ See ee % ~ a - Fic. 74.—TETANUS BACILLI, SHOWING SPORES. (X 1000.) swollen, so that in this stage the organism has the shape of a drum-stick. The spores are extremely resistant, and in the dry condition remain capable of germinating under favorable conditions for years. They are killed by moist heat at 100° C. in five minutes; by 5 per cent. carbolic acid, in fifteen hours; by bichloride of mercury, 1-1000, in three hours. The tetanus bacillus stains by Gram’s method. It is a strict anaérobe; it grows in an atmosphere of hydrogen, but not of carbon dioxide. It may sometimes be made to grow 270 MANUAL OF BACTERIOLOGY. very well by Buchner’s method. It may be cultivated at the room temperature, but better in the incubator. It grows upon ordinary culture-media, preferably those containing dex- trose. Gelatin is liquefied slowly; the colonies in gelatin pres- ent characteristic radiating filaments and look like a bristle brush. It grows on the other culture-media. Gas formation is not pronounced. This organism appears to be widely spread in external nature, especially in the soil. It is often found in garden-earth and in the feces of herbivorous animals. McFarland claims that it may occur in vaccine virus when this is carelessly prepared, and this would explain those rare cases of tetanus which occur after vaccination.* Tetanus bacilli have been found in gelatin, and it is stated that tetanus has followed the injection of gelatin as a hemostatic. The infection appears usually, if not always, to be introduced through some wound. Clinically, persons having the disease suffer from spasms of the muscles about the neck and the lower jaw (lock-jaw). The spasms finally be- come general. Inoculation with a pure culture produces tetanus in mice; also in rats, guinea-pigs and rabbits. The tetanic spasms begin in the vicinity of the point of inoculation and after- ward become general. The bacilli are not widely scattered through the body; they occur only in the immediate vicinity of the original lesion, and there are no important macro- scopic alterations in the internal viscera. Tetanus is the type of the purely toxic disease. Its symp- toms may be produced in animals by the injection of liquid cultures which have been deprived of their bacteria by fil- tration. The toxic substance appears not to be a ptomaine, as was at first supposed, and its exact nature is not determined. * Journal Medical Research. Vol. VIL. 1902. t+ Wells. Fourth of July Tetanus. American Medicine. June 13, 1903. PATHOGENIC BACTERIA. 271 The poison is tremendously powerful (sce page 164). It acts as an excitant to the motor cells of the central nervous system, especially the spinal cord. Bolton and Fisch have pointed out the possibility that horses used for the preparation of diphtheria antitoxin may be infected with tetanus, and have tetanus toxin in the blood.* The activity of the poison is destroyed by heat and by direct sunlight; various chemicals diminish its intensity. Antitoxin for tetanus has been prepared according to the FIG. 75.—ANTHRAX BACILLI, FROM A PURE CULTURE.{ (X 1000.) principles employed for antitoxins in general. It has not proved very markedly successful as a curative agent; but as a prophylaxis, where all patients are treated who have deep, dirty wounds, and in a similar way in veterinary practice, it has un- doubtedly proved of value. Unfortunately the disease is seldom suspected until a relatively large amount of toxin has * Transactions of the Association American Physicians. 1902. + The culture was derived from a case of malignant pustule in a tanner. The lesion was excised promptly, and the patient recovered. 272 MANUAL OF BACTERIOLOGY. formed and begun to manifest its action in the patient’s body .* Bacillus anthracis.—This is the largest of the patho- genic bacteria with the exception of the spirillum of relaps- ing fever, which is longer but more slender. The bacillus of anthrax is 1.25 4 broad, and from 3 to 10 long. Bacillus aérogenes capsulatus is of about the same size. The anthrax bacillus often forms long threads. A capsule is sometimes Fic. 70.—ANTHRAX BACILLI, SHOWING SPORES. (X 1000.) present. It is not motile. It forms spores, which are placed in the centers of the bacilli. The spores form only in the pres- ence of oxygen; they do not appear in the body of an infected animal during life. Anthrax spores are the most resistant of all pathogenic bacteria; they have been known to with- * Moschkowitz. Studies, Department Pathology. College Physicians and Surgeons. New York. Vol. VIL. t8aqq-1900. Annals of Surgery. P. 442. 1900. PATHOGENIC BACTERIA. 273 stand boiling for twelve minutes,* 5 per cent. carbolic acid for forty days, and 1-1000 bichloride of mercury for nearly three days. The anthrax bacillus is aérobic, although not strictly so. It stains by Gram’s method. It grows at the room temperature, but better in the incubator. It liquefies gelatin and coagulated blood-serum. Colonies in gelatin seen under a low power display numerous, irregular, fine, hair-like pro- jections; stab-cultures in gelatin also present fine projections passing from the needle-puncture into the solid gelatin. It grows on the ordinary culture-media; the growths are usually Fic. 77.—Cotony or ANTHRAX Bacitui (Low Power). whitish. Cultures on potato kept in the incubator are fa- vorable to the development of spores. Milk is coagulated and later peptonized. It is pathogenic for mice and guinea-pigs, less so for rab- bits; it is also pathogenic for sheep and cattle. Rats and pigeons are quite resistant, but not entirely immune; cats, dogs and frogs are not susceptible, or but slightly so. Anthrax is a disease which occurs chiefly in cattle and sheep. It is commoner on the continent of Europe and in Siberia than * More than half an hour. V. A. Moore. Infectious Diseases of Animals. 1902. 274 MANUAL OF BACTERIOLOGY. in America. In susceptible animals inoculated with virulent cultures of the anthrax bacillus septicemia is produced. Large numbers of the bacilli are found in the blood, and may be crowded together in the capillaries of the liver and kidneys. Men are occasionally affected, especially those whose occupation brings them in contact with cattle or with the hides and wool of animals that die of the disease. The infection usually occurs through wounds of the skin, where it produces a localized inflammation known as malignant pustule. Anthrax of the lungs or ‘“‘wool-sorter’s”’ disease may be acquired by inhalation Fic. 78.—-BacILLus OF ANTHRAX. STICK-CULTURE IN GELATIN.— (Gunther.) of material containing the spores of the bacilli. Infection by way of the intestine occurs occasionally but is less common. Laboratory workers engaged in studying the anthrax bacillus have been accidentally infected in a number of instances. The anthrax bacillus, owing to its large size, was the first of the pathogenic bacteria to be recognized, and its study has furnished the basis for much of our knowledge concerning the infectious diseases. It was for anthrax that Pasteur developed the idea of making a protective vaccine, shortly after he had produced a similar vaccine for chicken cholera. There is PATHOGENIC BACTERIA. 275 some danger to the inoculated animals attending the use of anthrax vaccines. In order to obtain material free from spores the blood of an animal which has recently died of anthrax is taken, because anthrax spores do not form in the living body. Cultures made in bouillon are kept at a temperature of from 42° to 43° C. At this temperature spores do not form, while the virulence of the anthrax bacillus becomes gradually diminished. In time the virulence is so far diminished that rabbits will survive Fic. 79.—ANTHRAX BACILLI WITH SQUARE OR SLIGHTLY CONCAVE ENDS SoMETIMES SEEN; FucusiIn STAIN. (XX 1000.) inoculation, and eventually also mice and guinea-pigs, which are extremely susceptible to anthrax. Small doses of a culture of extremely weak virulence are given to the animals which it is desired to protect, like cattle and sheep, and subsequently a somewhat more virulent culture is employed.* The method is never used in human beings. Bacillus influenze.—A small bacillus, 0.2 to 0.3 4 by 0.5 #4, with rounded ends. It does not form spores, is not motile * For details as to the results of this method see V. A. Moore. Infectious Diseases of Animals. 1902. For other and unique researches on immunity for anthrax see Emmerich. Centralblatt fir Bakieriologie. Original. Bd. XXXII. P. 821. 276 MANUAL OF BACTERIOLOGY. and is decolorized by Gram’s method. It is aérobic, grows only in the incubator, and upon media containing hemo- globin. The medium is prepared by smearing sterile blood over the surface of a tube of agar. Fresh, uncoagulated blood may, with care, be mixed with melted agar sufficiently cooled; the mixture may be poured into tubes and slanted; the tubes should be tested in the incubator before using. The blood of some animals, as the pigeon and rabbit, mav be used instead Fic 80.—ANTHRAX BACILLI IN THE CAPILLARIES OF THE LIVER OF A MovsE, SKETCHED FROM A SECTION STAINED WITH FUCHSIN. of human blood.* The colonies are small and transparent, looking like little drops of water, not becoming confluent. Of a large number of bacilli, the majority are destroyed in twenty-four hours or less by drying. They die out in a similar manner in water. Experiments upon animals up to this time are not conclusive. For diagnostic purposes, the sputum should be carefully collected in a sterile bottle. If the particles of sputum are likely to have become contaminated, rinse in * Centralblatt fir Bakteriologie. Bd. XXXII. Original. P. 667. PATHOGENIC BACTERIA. 277 sterile water. Inoculate on ordinary agar and on blood-agar. The influenza bacillus should grow only on the blood-agar and have the other characters above mentioned. Any organism that grows on both the ordinary and the blood-agar must be rejected. As far as is known, this organism attacks spon- taneously only human beings. It probably does not grow outside the body in nature. In cases of influenza’it is found in the mucous discharges and in the bronchi and lungs. It is the predominating organism in some cases of bronchitis.* According to Canon, the bacilli may sometimes be found in the blood. Bacillus diphtherize (Klebs-Léffler)—A straight or slightly curved bacillus, usually 1.2 to 2.5 in length, with rounded or slightly pointed ends, remarkable for showing irregularities of form, sometimes being club-shaped or spindle- shaped; branching forms have been found. It is not motile and does not form spores. It retains the stain after Gram’s method, but it is best stained with watery solutions of the aniline dyes, especially Léffler’s alkaline methylene-blue. Very characteristic pictures are obtained by the method of Neisser: SoLution No. .. Methylene-blue ....-.-----.------ 22-2 - eee eee I Alcohol (96 per cent.) .....-------------2----0- 20 Distilled. waters. sxrescasewees eee esendeesesec 950 Glacial acetic acid........ Saba ve sess setwek nese 50 Bismarck brow! nsss6cecdewes gee ssc ew eee I Boiling distilled water ........--.-------------- 500 Stain the cover-glass preparation which has been fixed in the flame in No. 1 one to three seconds; wash in water; stain in No. 2 three to five seconds; wash in water; mount *See Lord. Boston Medical and Surgical Journal. December 8, 1902. { Hill. Journal Medical Research. Vol. VII. 1902. 278 MANUAL OF BACTERIOLOGY. as usual. The body of the bacillus is stained pale brown, with dark blue spots, especially at the ends (Fig. 82). The diphtheria bacillus is peculiar in staining irregularly; certain spots stain more sharply than other portions, and darkly stained spots are likely to occur at the ends. It is a facultative anaérobe. It grows most rapidly in the incuba- tor, and slowly, or not at all, below 20° C. Gelatin is not liquefied. It may be cultivated on various alkaline culture- Fic. 81.—BaAcILLUS OF DIPHTHERIA. (X“TOOOu)* media, but grows best on Léffler’s blood-serum mixture. On this medium the growth consists of small white or cream- colored, slightly elevated colonies, which may become con- fluent. The morphology of the bacillus is most character- istic when it is cultivated on blood-serum. It also grows upon glycerin-agar. On potato it produces an_ invisible growth (see Bacillus of Typhoid Fever). In alkaline bouillon containing dextrose or muscle-sugar the reaction becomes PATHOGENIC BACTERIA. 279 acid in forty-eight hours. The reaction of the bouillon sub- sequently becomes alkaline. The growth may form a pellicle over the surface of the bouillon. It has also been successfully cultivated on various media to which egg-albumen has been added. It is killed by a moist heat at 58° C. in ten minutes. It resists desiccation well. Bacteriological Diagnosis of Diphtheria.—In many large cities the bacteriological diagnosis of diphtheria is undertaken by ~e . =~ = - 7 “eS Prete ae eer 2s = Fic. 82.—BACILLUS OF DIPHTHERIA Fic. 83.—SwaB AND CUL- STAINED By NEISSER’s METHOD. (X TURE-TUBE USED IN THE 1000.) Diacnosis oF Dipa- THERIA. boards of health. The methods used differ somewhat in detail, but are similar in the main, and are based upon the procedure devised by Biggs and Park for the Board of Health of New York City. Two tubes are furnished in a box. The tubes are like ordinary test-tubes, about three inches in length, rather heavy and without a flange. Both are plugged with cotton. One contains slanted and sterilized Léffler’s blood: 280 MANUAL OF BACTERIOLOGY. serum mixture; the other contains a steel rod, around the lower end of which a pledget of absorbent cotton has been Fic. 84.—Bacittus oF DIpHTHE- RIA, CULTURE ON GLYCERIN- AGAR. wound and the tube afterwards sterilized. The swab is wiped over the suspected region in the throat, taking care that it touches nothing else, and is then rubbed over the surface of the blood- serum mixture. The swab is re- turned to its test-tube and the cotton plugs are returned to their respective tubes. The plugs, of course, are held in the fingers during the operation, and care must be taken that the portion of the plug that goes into the tube touches neither the finger nor any other object. The principles, in fact, are the same as those laid down in general for the inocu- lation of culture-tubes with bac- teria (see page 72). In board of health work these tubes are re- turned to the office. When it is desirable, a second tube may be inoculated from the swab. The tubes are placed in the incuba- tor, where they remain for from twelve to twenty-four hours, and a microscopic examina- tion is then made of smear pre- parations stained with Loffler’s methylene-blue. On Léffler’s blood-serum kept in the incubator the bacillus of diphtheria grows more rapidly than the other organisms which are ordi- PATHOGENIC BACTERIA. 281 narily encountered in the throat, a property which to a cer- tain extent sifts it out, as it were, from them, and makes its recognition with the microscope easy in most cases. The growth, furthermore, is quite characteristic, and its nature can be predicted with considerable accuracy, even without microscopical examination, by one who has had much practice. Colonies of streptococci frequently look very like those of the bacillus of diphtheria, but those two are easily distin- guished from each other with the microscope. The diagnosis of the diphtheria bacillus, then, is made from the character of the growth upon the blood-serum and the microscopical exami- nation, taking into account the size and shape of the bacilli, with the frequent occurrence of irregular forms and the pe- culiar irregularities in staining. In doubtful cases a sec- ond culture should be made from the throat. The very large number of examinations that have been made by various boards of health have shown that the diphtheria bacillus may persist in the throat for a long time—occasionally several weeks after the patient has apparently recovered; also that diphtheria bacilli are occasionally found in the throat when there is an inflammatory condition without any pseudo- membrane, and that they sometimes appear in an apparent healthy throat, especially in children who have been associ- ated with cases of diphtheria. It has been found that bacilli sometimes occur in the throat which have all the morphologi- cal and cultural properties of the diphtheria bacillus, but which are devoid of virulence when tested upon animals. Such diphtheria bacilli have frequently been called pseudodiphtheria bacilli. A bacillus closely resembling the diphtheria bacillus, but without virulence, has been found in xerosis of the con- junctiva. It is called the xerosis bacillus. If not a transformed diphtheria bacillus, it is at least closely related. The diph- theria bacillus is subject to wide variations in morphology, so that, in dealing with unknown cultures where the forms are 24 282 MANUAL OF BACTERIOLOGY. not characteristic and injection into animals is without result, it may be difficult to decide whether or not the organisms are diphtheria bacilli. Consequently another view with regard to pseudodiphtheria bacilli has arisen. While recognizing that virulent diphtheria bacilli occur, it is also claimed that a dis- tinct pseudodiphtheria bacillus exists, different from the diph- theria bacillus, though resembling it. It is shorter, stains more evenly, shows no polar granules by Neisser’s method of staining, does not produce acid in dextrose-bouillon, and is not patho- genic to animals. It is found occasionally in the nose and throat and has no connection with diphtheria, according to this view.* But there are some who hold that there is no pseudodiphtheria bacillus, and that the organism so called is merely a more or less modified form of the diphtheria bacillus. The diphtheria bacillus is pathogenic to animals. When it is injected into them it produces a toxemia. In the guinea- pig, which is especially susceptible, local inflammation: results, and death occurs usually in two or three days. The bacilli are found to be confined to the vicinity of the wound, and not usually to be disseminated throughout the whole body. The death of the animal, therefore, is due to the poisons elaborated by the diphtheria bacilli—either poisons introduced at the original injection, or substances produced by the bacilli which may have multiplied in the animal’s body. The internal vis- cera, especially the liver, often exhibit small areas consisting of necrotic cells; a transudation of serum takes place in the great serous cavities, and the lymph-nodes are swollen. A genuine diphtheritic membrane may be produced on the trachea of a young kitten by rubbing into it a part of a culture of the diphtheria bacillus. * The different sides of this question will be found fully discussed by the following: Wesbrook, Wilson and McDaniel. Transactions of the Association American Physicians. 1900. Gorham. Journal Medical Research. Vol. VI. tgoo. A. Williams. Jbid. Vol. VIII. 1902. Denny. Ibid. Vol. IX. 1903. Alice Hamilton. Journal of Infectious Diseases. Vol. I., No. 4. 1904. Graham Smith. Journal of Hygiene. Vol. IV. 1904. PATHOGENIC BACTERIA. 283 As is well known, the pseudomembranous affection pro- duced by the diphtheria bacillus in man is generally seen in the larynx and pharynx. Membranous rhinitis is also caused by the diphtheria bacillus. On the other hand, pseudomem- branous affections of the larynx and pharynx may be produced by streptococci.* Pseudomembranes occurring in the throat during scarlet fever and measles may be due to the diphtheria bacillus, but are more often caused by streptococci. The affection known as membranous croup is usually diphtheria of the larynx, produced by the diphtheria bacillus. The diph- theria bacillus is a rare cause of puerperal fever. Although the uninjured skin is not attacked by the diphtheria bacillus, it may be present in pseudomembranes on wounded surfaces, usually in connection with diphtheria in the throat. Most *pseudomembranes formed upon wounds of the skin are pro- duced by other bacteria than the diphtheria bacillus, as is also the case with the pseudomembranous inflammations of the intestines and bladder. Although such inflammations are often called ‘‘diphtheritic,” it must be remembered that the ex- pression is used in an anatomical sense, meaning that a fibrin- ous pseudomembrane has formed, extending deeply into the tis- sues, which is not necessarily caused by the diphtheria bacillus. In cases of diphtheria in man,} the diphtheria bacillus is generally found limited to the vicinity of the pseudomem- brane, and at autopsies it is not usually found in the internal viscera, excepting in the lungs, where diphtheria bacilli may or may not be present when diphtheria is complicated with bronchopneumonia. The general symptoms of the disease, including the paralysis which sometimes follows it, are due to the toxins produced by the bacilli in the throat. * Bissell. Medical News. May 31, 1902. American Journal Medical Sciences, February, 1903. Review of Work of Massachusetts Boards of Health. + For a full study of the lesions of diphtheria see the Monograph of Council- man, Mallory and Pearce. Boston. igo1. 284 MANUAL OF BACTERIOLOGY. Diphtheria antitoxin.* It is necessary first to obtain the toxin produced by diphtheria bacilli in a concentrated form. For this purpose virulent diph- theria bacilli are cultivated in alkaline, sugar-free bouillon, in flasks plugged with cotton, exposing a large surface to the air. The bouillon is prepared by leaching out 500 grams of lean chopped beef in 1000 c.c. of water overnight, straining the water off through cheese-cloth, and finally inoculating it with a culture of the colon bacillus. The last-named procedure rids the broth of all muscle-sugar. After adding salt and peptone to the sugar-free beef-juice it is put in wide, flat flasks—Fernbach flasks—and sterilized in the ordinary manner. The cultures are grown in the incubator. After five to ten days they are ready, and are filtered through porcelain.| The filtrate contains the toxin. The toxin is injected into the animal from which the antitoxin is to be obtained in small doses. The dose depends on the strength of the toxin. The animal usually employed is the horse, which should be healthy; the presence of tuberculosis and glanders should have been excluded by testing with tuberculin and mallein; the possible presence of tetanus should also be considered (see page 271). The injection is repeated at intervals of about one week, using larger and larger doses, until the animal is able to tolerate a very large dose indeed— as much as 300 ¢.c., or even more. If the treatment is successful, the general condition of the animal should not suffer. The injections last over a long period—usually about two or three months. The general condition of the animal remaining good, the toleration of these large doses of toxin is presumed to indicate the existence of a concentrated antitoxic substance in the blood. Small quantities of blood may be withdrawn from time to time, and the serum tested for its antitoxic strength. When a satisfactory serum has been attained, the animal may be bled and the serum saved for therapeutic purposes. ‘Through an incision in the skin a trocar is inserted into the jugular vein. The blood is drawn into sterilized flasks with every precaution to insure sterility. The blood is allowed to coagulate and is placed for a time in the ice-chest. The serum is then withdrawn with sterilized pipettes. Small amounts of chemical germicides, as carbolic acid or chloroform, are sometimes added to assist in preserving it. This serum is the so-called antitoxin used in medical practice. Since antitoxin is not obtained as a pure chemical substance, and conse- quently cannot be weighed and measured as other therapeutic preparations, an arbitrary standard to express the potency of the serum, called an immunity unit, has been devised by Behring and modified by Ehrlich. Formerly this * See articles by Park, A. Williams, Atkinson and T. Smith. Journal of Experimental Medicine. Vol. 1., p. 164; Vol. IIL. p. 513; Vol. IV., pp. 373 and 649. Journal Medical Research. Vol. IX., p. 173. t W. H. Park adds 10 per cent. of a 5 per cent. solution of carbolic acid to kill the bacilli, and filters through paper on the following day; after adding carbolic acid the Berkefeld filter may be used with advantage instead of filter- paper. PATHOGENIC BACTERIA. 285 unit was taken to be 10 times that amount of antitoxic serum which just neu- tralized 10 fatal doses of toxin for guinea-pigs weighing 250 grams. In other words, the exact amount of a certain toxin required to kill a guinea-pig weigh- ing 250 grams in four days having been determined by inoculating a number of guinea-pigs, ten times this amount was put into each of a number of test- tubes, and the antitoxin to be tested was added, a slightly different amount to each tube of toxin. The contents of each tube was then injected into a separate guinea-pig. If any of the animals survived, the amounts of antitoxin in the tubes with which they had been inoculated having been noted, the smallest of these amounts —i. é., the smallest amount found necessary to neutralize the toxin—was re- garded as one-tenth of an antitoxic unit. It was naturally assumed that 10 times this amount of antitoxin would neutralize 100 fatal doses. This, how- ever, was found not to be the case (see Immunity, page 187). So the revised standard now employed in Germany, France, America and other countries is the unit recommended by Ehrlich. This consists of comparing the antitoxin to be standardized with antitoxin specially prepared by Ehrlich for the pur- pose. This antitoxin of Ehrlich is supplied to the various public and private institutions where antitoxin is prepared, and is carefully standardized against very fresh toxin, which therefore contains little toxoid. The Ehrlich standard antitoxin is really used in the first place to determine the strength of a given toxin, which in turn is used to determine the value of antitoxin to be standardized. The actual method is to mix varying amounts of the toxin to be tested each with one unit of the standard antitoxin, and that mixture which just suffices as proved by experiment to kill a 250-gram guinea- pig in three or four days is designated L+ (see Immunity, page 188); the mixture which is just neutral is called LO. That amount of antitoxin which just neutralizes L+ contains one antitoxic unit according to this method of standardizing. The injection of guinea-pigs with antitoxin serves the double purpose of determining the potency of the antitoxin and also of determining the presence or absence of pathogenic substances, such as tetanus toxin. It has been found possible to prepare antitoxin of a high degree of concentration, so that 500 to 1500 units may be contained in a quantity of serum which it is practicable to give at a single hypodermic injection. The large volume of statistics that have been collected from hospitals and from physicians in private practice indicates that the use of diphtheria antitoxin has effected a very great reduction in the mortality from diphtheria. 286 MANUAL OF BACTERIOLOGY. Bacillus tuberculosis.—A slim bacillus 1.5 to 4 / in length, which very frequently presents a beaded appearance, owing to its being dotted with bright, shining spots. Branching forms have been described. The tubercle bacillus is con- sidered by some to be a member of the actinomyces group. It is not motile. It has not been proved that spores are formed: nevertheless certain structures, like caseous lymph-nodes, have been shown to be capable of infecting guinea-pigs with tuber- culosis, although tubercle bacilli could not be demonstrated -4 rN “ 2 s al em sie & ae | ee : : . é mig’ eo Pa Yu- 4 = 1 t7N iN? —~ Be’ & So cs r A ? \ VN ter—~l \ e \ 1 \ : = > e ) . See Ny ney e . mt Fic. 85.—BACILLUS TUBERCULOSIS, FROM A PURE CULTURE. (XX I000.) in them with the microscope. This makes it seem possible that the organisms were present as spores which eluded the microscopical examination. The tubercle bacilli stain with the ordinary aniline dyes and by Gram’s method, but they do not take the stains as readily as most other bacteria, and require prolonged exposure to the dye, on warming of the stain. When once stained, however, with aniline-water dyes or carbol- fuchsin, they are not readily decolorized by acids and alcohol, which fact distinguishes them from all other known bacteria PATHOGENIC BACTERIA. 237 excepting the leprosy bacillus, the smegma bacillus, possibly the bacillus of syphilis (Lustgarten), and certain bacilli found in milk, butter and: cow-dung and on various grasses. All of these may resist decolorization by acids or alcohol, and some resist both. They must always be kept in mind in making a diag- nosis of tuberculosis. (See pages 33 and 294.) In examining sputum it is particularly important to bear in mind that acid- proof bacilli, resembling tubercle bacilli, have been found in rare cases in gangrene of the lung. But the organisms found in these cases are longer than tubercle bacilli, as a rule, and branch more often, besides being less resistant to decolorization.* The tu- - a bercle bacilli appear to owe their pecu- liar staining properties to fatty sub- - ZN, stances contained in the bodies of the ~n* Y bacilli. In stained preparations the ~* 1 Xt bacillus usually appears very distinctly : beaded, owing to the presence of as stained areas which alternate with un- HERBS NGHING stained areas; these unstained areas Form or TUBERCLE m BACILLUS FROM A CUL- have been considered by some to be TURE. (X 1000.) spores. The Bacillus tuberculosis is aérobic. It is cultivated with considerable difficulty—best at about 38° C. It does not grow at a temperature below 29° C., and cannot therefore be cul- tivated upon gelatin. It grows well upon blood-serum, where the growth becomes visible in from ten to fourteen days in the incubator. It forms a dry, mealy, scaly mass, elevated above the surface, of a grayish-brown color. It also grows upon glycerin-agar; or glycerin-bouillon, on which it forms a pel- licle; upon potato; upon milk containing 1 per cent. of agar and upon coagulated egg (see page 69). It is impoitant to have the * Ophiils. Journal Medical Research. Vol. VIII. 1902. Ohlmacher. Journal American Medical Association. got. 288 MANUAL OF BACTERIOLOGY. medium moist. It can be cultivated {from tuberculous sputum only with great difficulty. It is best to obtain it from the tis- sues of an animal that has died of tuberculosis, where the tubercle bacilli may be found unmixed with other bacteria. Pieces of tissue should be taken with the precautions necessary to avoid contamination, and should be broken up and rubbed over the surface of the medium. The tubes must be closed with sealing-wax, paraffin or rubber stoppers, or covered with rubber caps, to prevent drying in the incubator. If rubber ma mee ea * : § i i his fe a 4 f +f | ; ; 5 | ae \ “3 oe * f ee > Fic. 87.—BacILtus TUBERCULOSIS IN SPUTUM, STAINED WITH FUCHSIN AND METHYLENE-BLUE. PHOTOMICROGRAPH IN Two CoLors. (X 1000.) caps are used, they should first be left in 1-1000 bichloride of mercury for an hour, and the cotton plug should be burned before putting on the rubber cap. A number of tubes should be inoculated, using rather large particles of the tuberculous material. Among the tubes inoculated, many will fail to present any growth. After the organism has once been grown upon a culture-medium it may be propagated with less difficulty. PATHOGENIC BACTERIA. 289 It is killed by 5 per cent. solution of carbolic acid in a few minutes. In sputum it is destroyed in twenty-four hours by a three per cent. solution of carbolic acid. It resists desiccation for months, but is killed in some hours by direct sunlight. It is destroyed in a few minutes by boiling. Tt is not known to grow, except in artificial cultures, outside of the animal body.- It is the cause of tuberculosis in man. It produces tuberculosis in apes, cows, sheep, horses, rabbits, guinea-pigs, cats, field-mice and occasionally in other animals. Guinea-pigs and rabbits are extremely susceptible. A guinea- pig inoculated with tuberculous sputum (provided it does not die of septicemia, due to the pyogenic micrococci which are fre- quently present in sputum) will present a swelling of the neigh- boring lymph-nodes in the course of two to four weeks, and will die as a rule in from four to eight weeks, although the time may be longer. Tuberculosis in cattle (German, Perlsucht) is characterized by large, nodular lesions, with a marked tendency to become fibrous, caseous and calcified. The tubercle bacilli of cattle differ somewhat from those of human tuberculosis, as was noted by Theobald Smith.* Whether or not men could be infected with bovine tubercle bacilli has been a question that has been warmly debated in recent years. It seems to have been shown that such infection is possible; also that it is possible that cattle may be infected with human tubercle bacilli. Bovine tubercle bacilli are more virulent for some animals, as rabbits, than human tubercle bacilli.t It seems possible that the danger of infection from cattle has been somewhat overrated. The lesion produced by the tubercle bacilli in the tissues of men and the lower animals is called a tubercle, which in the beginning is a grayish-white area about the size of a millet-seed. In secretions of the tissue young tubercles are found to present several different structures. Near the * Journal Experimental Medicine. Vol. III., p. 451. } TheobaldSmith. Medical News. February 22,1902. Salmon. Bureau of Animal Industry. Bulletin. No. 33. Adami. Philadelphia Medical Journal. February 22, 1902. Ravenel. University of Pennsylvania Medical Bulletin. May, 1902. Lartigau. Journal Medical Research, Vol. VI. 1901. 25 290 MANUAL OF BACTERIOLOGY. center, one or more very large cells called giant-cells occur. They contain several or many nuclei which are frequently arranged in a crescentic manner at one side of the cell. Tuber- cle bacilli can sometimes be demonstrated inside of the giant- cell. Except possibly in the very youngest tubercles, a small area of necrotic tissue will always be found at the center of the tubercle. Around the giant-cells and the necrotic area are seen large cells with distinct nuclei which resemble epithelial cells, and are often called epithelioid cells; they are also often termed granu- lation cells, and represent an attempt at the formation of granu- lation tissue. But no new-formed blood-vessels, such as are found in granulation tissue as a rule, occur ‘in the tubercle. Tubercle bacilli may also be found among the epithelioid cells. Outside of these epithelioid cells is another layer of small cells called lymphoid cells, which represent leukocytes that have appeared in this situation as a part of the inflammatory reaction excited by the presence of the tubercles. The zone of lymph- oid cells may be very indistinct or wanting. Frequently it may be very difficult to make out that the cells are arranged in distinct zones at all. The cells are imbedded in a matrix consisting of the connective tissue originally belonging to the part, to which some fibrin may be added. In addition to the fact that no new blood-vessels are formed to maintain the nutrition of these newly formed cells, the small vessels included in the tubercle and around it suffer from inflam- matory changes. Owing to these causes and to a toxic sub- stance formed by or in the tubercle bacilli, degenerative changes and necrosis take place at the central part of the tuber- cle. As a result of these degenerative changes the center of the tubercle becomes converted into a dry, yellowish-white, friable mass, resembling dry cream-cheese. Such material is said to be caseous, and the process is called caseation. Prud- den and Hodenpyl found that the injection of dead tubercle PATHOGENIC BACTERIA. 2gI bacilli into animals produced lesions having the histological characters of tubercles, but caseation did not take place. The small tubercles first formed are called gray or mili- ary tubercles. As they become larger they also frequently become confluent. The larger, confluent, caseous tubercles are oiten called yellow tubercles. Swollen tuberculous lymph- nodes of the neck are among the manifestations of the con- dition formerly known as scrojula. Masses of caseous tubercles sometimes undergo soften- ing. In the lungs the discharge of the softened material results in the formation of a cavity. This formation of a cavity in the lungs is frequently, if not usually, accompanied by secondary infection with pyogenic micrococci. Caseous tuberculous masses may become partly calcified. Very often they may be encapsulated by new formed fibrous or scar tissue. It is possible for tuberculosis to become cured for all practical purposes by means of this process. Autopsies on human subjects have shown that such cures not rarely take place, especially in tuberculosis of the lungs occurring over a localized area. ‘The statistics of autopsies vary widely as to the number of persons that at some time of life suffer from tuberculosis (25 or 30 per cent. and upward). When a tuberculous area has become caseous and encapsulated and apparently quies- cent, it is possible for it to be excited to renewed activity under suitable conditions, and, owing to the softening and the dis- charge of infected material into one of the vessels or cavities of the body, a wide-spreading and rapidly fatal tuberculosis may follow. Tuberculosis may become disseminated throughout the body from a small focus as a starting-point. The tubercle bacilli may travel through the lymph-spaces and affect adja- cent tissues, some of them reaching the nearest group of lymph- nodes. In tuberculosis of the lungs it is usual also to find tubercles in the bronchial lymph-nodes, and in tuberculosis 292 MANUAL OF BACTERIOLOGY. of the intestines there is also tuberculosis of the mesenteric lymph-nodes. ‘The disease may travel along the serous surfaces and become widely scattered throughout a cavity like that of the pleura or peritoneum. The bacilli may be expelled on some mucous surface and be carried along it to infect some point farther on, as happens when the larynx becomes infected in tuberculosis of the lungs, and when in the same disease tuberculous sputum is swallowed and leads to infection of the intestines. Finally, the infectious material may enter the blood-vessels, especially the veins, and be swept along with the blood-current to become scattered generally throughout the body. In such cases we are likely to have general or acute miliary tuberculosis. Almost every organ of the human body may be infected by tuberculosis. Among the most common may be mentioned the lungs, the lymph-nodes, the bones, the intestines, the skin, the meninges, and the serous membranes. Infection, as far as we know, is always to be attributed directly or indirectly to some preéxisting case of tuberculosis in man or the lower animals. The entrance into the body is most commonly by way of the lungs, where also tuberculous disease is commonest in man, going by the name of consump- tion. This is doubtless due to the prevalent habit of expec- torating in public places. Out of fifty-six samples of sputum collected in street cars by Dr. W. G. Bissell, City Bacteriologist in Buffalo, four were tuberculous. In forty-eight samples taken from the floors of a public building by Dr. C. R. Orr, of the pathological laboratory of the University of Buffalo, tuber- cle bacilli were found three times. According to the researches of Nuttall, a person suffering from tuberculosis may expectorate many millions of tubercle bacilli in the course of twenty-four hours. Coughing and similar efforts may serve to disseminate the bacilli (see page 152). Concerning the occurrence of tubercle bacilli in cow’s milk and butter, see pages 137 and 138. PATHOGENIC BACTERIA. 293 Cases have been recorded in which the disease was trans- mitted from the mother to the child in the uterus; how fre- quently this happens is uncertain. It is usual to attribute greater importance to an inherited tendency to tuberculosis than to the inheritance of the tubercle bacilli themselves. A gglutination of the tubercle bacillus is said to occur with the serum of cases of tuberculosis under certain circumstances. The reaction does not seem likely to be of practical value. Tuberculin is made by concentrating a culture of tubercle bacilli grown in glycerin-bouillon to one-tenth of its original volume, over a water-bath, and filtering through an unglazed porcelain filter. It therefore represents the products of tubercle bacilli. It was proposed by Koch as a remedy for tuberculosis, but it has not met with great success, and is little used as a therapeutic agent. It has been found, however, of great value in the diagnosis of tuberculosis, especially in cattle. When tuberculin is injected into a tuberculous animal there results considerable general disturbance, of which the most noticeable evidence is a sudden rise in temperature, while hyperemia is excited around the tuberculous area. In a healthy subject the injection produces no reaction. There is danger attending its use, so that its application in diagnosis is practically con- fined to cattle.* As a diagnostic measure in cattle it has been found accurate in the great majority of cases. Concerning tuberculosis in cows, see page 137. Supposing that some curative principle exists in the bodies of the tubercle bacilli themselves which could not be procured from cultures de- prived of their bacilli by filtration through porcelain, Koch has recently proposed a new form of tuberculin called “tuber- culin R,” which consists of an extract made from dried and pulverized living tubercle bacilli. The value of this new tuberculin as a remedy is at least doubtful, and physicians are disposed to regard it with a great deal of skepticism. * For details as to its use in cattle see V. A. Moore. Infectious Diseases of Animals. P. 151. 1902. 294 MANUAL OF BACTERIOLOGY. Immunity from tuberculosis has been attained experimentally to a certain degree. In very old cultures the virulence of tubercle bacilli sometimes be- comes greatly diminished, Animals which survive injections of such bacilli may afterwards withstand large doses of virulent bacilli.* Acid-proof bacilli resembling tubercle bacilli have been alluded to a num- ber of times (pages 138, 143 and 287). A number of such bacilli have been cultivated, such as those of butter and grass. Injected into animals they may produce nodules more or less like tubercles. In these nodules they sometimes assume forms resembling the fungus of actinomycosis. The tubercle bacillus rarely shows similar forms. All the bacilli of this class, including the tubercle bacillus, sometimes show branching. It is probable that the bacilli of this group are related to the fungus of actinomycosis.t Similar organisms have been found in fishes, in whom they produce nodules resembling tubercles; it is quite possible that the latter organisms are tubercle bacilli, which have been modified by an altered environment. Another acid-proof bacillus has been found which is pathogenic to rats, producing lesions of the skin with nodules; the disease appears in wild rats in certain localities. Tuberculosis of Birds.—Fowls, ducks and other birds sometimes suffer from tuberculosis due to a bacillus closely resembling the tubercle bacillus of mammals. It has similar staining properties. It sometimes grows in long, branching forms. It differs somewhat from the tubercle bacillus of mammals in its cultural properties. The liver is the organ most often affected. Guinea- pigs are much less susceptible to it than to mammalian tuberculosis. Rabbits are somewhat susceptible, though less so than to mammalian tuberculosis. Pseudotuberculosis.—Guinea-pigs and other rodents sometimes present lesions macroscopically very similar to those of tuberculosis, in which, how- ever, the tubercle bacilli cannot be found. These affections appear not to be tuberculosis at all, and their nature is not well understood. Several organisms have been found in them, all of which are entirely unlike the tubercle bacillus. Bacillus lepre (bacillus of leprosy).—A slim bacillus about 4/in length. Itis probably not motile. It is uncertain whether or not it forms spores. It stains by the Gram and the Weigert fibrin method, and it is also colored by the methods used for staining the tubercle bacillus. It takes the dye, however, more readily than the tubercle bacillus. In stained prepara- tions it appears very similar to the tubercle bacillus, and re- * Trudeau. New York Medical Journal. July 18,1903. Salmon. Phila- delphia Medical Journal. June 13, 1903. + Abbott and Gildersleeve. University of Pennsylvania Medical Bulletin. June, 1902 PATHOGENIC BACTERIA. 295 sembles it in having alternate colored and unstained spots. Although several observers have reported success in attempts . to cultivate the bacillus of leprosy, their claims have been dis- puted. Rost* claims to have succeeded in cultivating the lepra bacillus upon a peculiar medium consisting of a distillate of beef-extract. The distillation is performed by a special arrangement in the autoclave as follows: Pieces of pumice stone dried in the sun and sterilized are saturated with a solution of beef-extract and transferred to a wide-mouthed jar. The jar is tightly corked, and has two tubes inserted through the cork, one running to near the bottom and opening just outside the cork: the other tube opens near the top of the jar and the end projecting out of the cork is bent to an elbow and is brought through an opening in the autoclave. The end of this tube which is in the jar terminates in a rectangular a shaped extremity. When the autoclave is set going, the steam passes into the jar through the tube first described, impinges on the saturated pumice stone, extracts certain substances from the latter, and passes out through the other tube, out of the autoclave, where it is condensed by suita- ble arrangements. This distillate forms the basis of Rost’s various media for the lepra bacillus. The organism is said to grow only in the absence of every trace of sodium chloride. Rost has also prepared a substance analogous to tuberculin from cultures of lepra bacilli. This he calls leprolin, and it consists of a glycerin extract of a culture grown for three weeks at body temperature. The culture is first evaporated to one- tenth the original volume in vacuo over sulphuric acid, and equal amounts of glycerin added. Subcutaneous injection of 5 c.c. of leprolin is said to cause a temperature of 104° F. in twenty- four hours in a person affected with leprosy, and is being used for diagnostic purposes and also as a therapeutic agent. Its use, however, for either purpose is yet in the experimental stage. * Indian Medical Gazette. Vol. XXXIX. 1904. 296 MANUAL OF BACTERIOLOGY. The results of inoculation into man and the lower ani- mals of material coming from cases of leprosy have been uncertain. The bacillus of leprosy has been found so con- stantly in the tissues of those having the disease that it is gener- ally admitted to be the specific cause. The skin and the periph- eral nerves are the parts most affected, although other tissues and the internal viscera may be involved. A granulation tissue, forming nodules and thickenings, appears in the affected parts. The bacilli are found in large numbers in the nodules, partly outside of the cells, but mostly within the cells. It is still uncertain whether or not the disease can be transmitted directly from one individual to another, in extra-uterine life, or whether it can be inherited from the parents. However, no ex- planation can be given for the appearance of the infection in any patient, except communication with some other case. Trans- mission by contact seems at any rate not to take place easily. Bacillus mallei (bacillus of glanders).—A slim bacillus with round or pointed ends, which often shows alternate light and dark spots in stained preparations. Branching forms have been described. It is not motile. It probably does not form spores. It does not retain the stain by Gram’s method. After staining with the ordinary aniline dyes it is easily decolor- ized, and on that account it is difficult to demonstrate in sections of tissues. It is facultative anaérobic. It grows at the room temperature, but better in the incubator. It grows slowly on gelatin, and does not liquefy it, or only after a long time. On agar it produces a moist, white growth; on blood-serum, a yel- lowish or brownish growth; blood-serum is not liquefied. Milk is coagulated slowly, and the reaction becomes acid. On potato the growth is characteristic in one or two days in the incubator, becoming translucent amber-yellow, later a reddish brown, while the surface of the potato becomes discolored. It is killed in five minutes by a 5 per cent. solution of carbolic acid; in two minutes by 1-so00o0 bichloride of mercury. It may survive drying for a number of weeks. PATHOGENIC BACTERIA. 207 In the horse and ass it produces the disease known as glanders, which affects the mucous membrane of the nasal cavity. When the skin is involved, the disease goes by the name of farcy. In the nose, nodules appear in the mucous membrane which become necrotic, forming ulcers. They may become con- fluent, and may extend along the adjacent surfaces as far as the lungs. There is a profuse discharge from the nose. The neighboring lymph-nodes become involved and are swollen, and nodules may be present in the internal viscera. In the skin the nodes lying underneath the skin are called farcy-buds. Histologically the nodules consist of a granulation tissue, but they tend to break down rapidly, and the process in some re- spects is very like ordinary suppuration. This bacillus is pathogenic* for guinea-pigs, field-mice and cats; rabbits, sheep and dogs are less susceptible or only slightly so; also white and house-mice, and hogs; cattle are immune. Men are occasionally infected, especially those who come much in contact with horses. The mucous mem- branes of the nasal cavity may be the part involved, or the skin or the internal viscera. Ina number of instances, workers in the laboratory have been accidentally infected. The diagnosis of the disease is best effected by the inoculation of a male guinea-pig with the material from a case suspected of being glanders, introducing it into the peritoneal cavity (method of Straus). In about two to three days after an inocu- lation of this kind there appears a characteristic swelling of the testicle, indicating the beginning of suppuration, which pres- ently takes place; the animal usually dies after two or more weeks. At least two guinea-pigs should be inoculated; and the test may sometimes fail, when it should be repeated on other guinea-pigs.f * The statements of different writers differ considerably with regard to some of these animals. + Frothingham. Journal Medical Research. Vol. VI. 1901. 298 MANUAL OF BACTERIOLOGY. Mallein is a product obtained from an old glycerin-bouillon culture of the Bacillus mallei. The cultures are placed in a steam sterilizer for several hours, and are filtered through un- glazed portion. The filtrate contains the products of the growth of the Bacillus mallei and is of much the same character as tuberculin. Injected into animals suspected of having glanders, if it produces a local and febrile reaction, the existence of glan- ders is indicated. This reaction is of use in the diagnosis of the disease in lower animals, especially in horses, where it has been largely em- ployed, though it some- times fails. An aggluti- nation reaction has been described for the bacillus of glanders. Actinomyces bovis* (Streptothrix actinomyces; Ray-fungus of Actinomy- cosis)—The morphology Fic. 88,—Ray-roncus or Actinomyco- Of this organism is quite SIS. FRESH, UNSTAINED Prepara- different from that of TION FROM A CASE OF LUMP-JAW IN ‘ A Cow. (DIAGRAMMATIC.) most of the bacteria. It is sometimes considered to be a bacterium of a higher type. The organism appears in the form of threads which show genuine branching. These threads make radiating, interlacing masses. Their external ends are swollen and bulbous under certain conditions. Colonies formed in this manner, seen under moderate mag- nification, have a radiating appearance which has given rise to the name, ray-fungus. The club-shaped external ends are readily distinguished and the growth possesses a very distinc- * Hektoen. Philadelphia Monthly Medical Journal. November, 1899. Ewing. Bulletin Johns Hopkins Hospital. November, 1902. PATHOGENIC BACTERIA. 299 tive form. This is the shape which the organism presents as it grows in the animal body. The club-shaped ends are gen- erally regarded as a degenerative or involution form. Trans- verse divisions may sometimes be distinguished upon the threads. Spherical forms resembling micrococci may appear which may possibly be spores. In some members of this group spores—conidia—form in cultures on the ends of the filaments. The organism stains with the ordinary aniline dyes, by Gram’s method or the Weigert fibrin stain. The fungus may be cultivated upon the usual culture- media, though not easily. It is facultative anaérobic. It grows both at ordinary temperatures and in the incubator. The growth is not rapid. The colonies are fine, dry, elevated, irregular in form, becoming opaque. Bulbous ends upon the threads do not usually appear in cultures. The results of the injection of these cultures into the lower animals are as yet uncertain. The disease produced by the ray-fungus is called actino- mycosis. It occurs in cattle chiefly, seldom in swine and horses, and occasionally in man. Infection appears to be carried by grain or particles of vegetable fiber which pene- trate the tissue. The presence of such foreign particles as well as the organisms appears to favor infection. The infec- tious material frequently enters through the mouth, espe- cially in the vicinity of the teeth, but it may also occur through the skin or the mucous membranes. It leads to the formation of inflammatory, tumor-like nodules, hence the name “‘lump- jaw” given to the disease in cattle. Necrosis of the tissue takes place with the formation of an abscess. The pus is peculiar in containing small yellowish-white particles—so-called “sulphur granules ”—which consist of little clumps of the ray-fungus, and which readily permit the disease to be diagnosed by the micro- scope. The material may be examined in the perfectly fresh condition without any staining. The jaw or its neighborhood 300 MANUAL OF BACTERIOLOGY. is very frequently affected, or the disease may be present in other situations about the head and neck, and may involve the lungs, the intestines and the vertebra, ribs and other bones. The disease is usually localized, but a number of areas may be affected simultaneously. Besides the common actinomyces, there are numerous other ray-fungi, more or less closely related, and whose pathogenic properties are not fully deter- mined. Generally speaking, they appear to be essentially saprophytes, which occasionally become parasitic and pathogenic under especially favorable con- Fic. 89.—ACTINOMYCES BOVIS, SMEAR PREPARATION FROM A PURE yguL- TURE, STAINED BY GRAM’S METHOD. (X 1000.) a ditions. A number of species have been found in air, dust, etc., some of them chromogenic. Wolff and Israel described an anaérobic species, pathogenic to man and animals. Madura disease, Madura foot, or mycetoma is a disease occurring in India (rarely elsewhere), affecting one of the extremities, character- ized by swellings, nodular deposits and abscesses. Some cases are certainly due to a member of the actinomyces group.* Other branching organisms, some of them acid-proof, have been described * Compare Wright. Journal Experimental Medicine. Vol. III., p. 421. PATHOGENIC BACTERIA. 301 chiefly under the name of streptothrix. In man they have been found in a variety-of suppurative and necrotic lesions, in particular, bronchopneumonias.* Bacillus typhosus (Bacillus of Eberth).—A bacillus with rounded ends, varying in length, sometimes making very short, oval forms, sometimes growing out into long threads. It is very actively motile, and possesses numerous flagella which arise from all parts of the surface. It does not form spores. It is not stained by Gram’s method, but it may be colored with the ordinary aniline dyes, when the stain will frequently be A eh ~ a’ | ‘ 4S 4 1 S = we as te : yah { soe La f ’ 71 hey i ' | > ames \ KRG eN 2 4 4 } “ Ne ~~ ~ a a ea Oe ‘ ood Late é wy An 4 4 ¥ Fic. 93.— BACILLUS COLI COMMUNIS. (X 1000.) when it may be difficult to distinguish from micrococci; often longer, even forming threads. It is slightly motile, hav- ing several flagella. It does not form spores. It stains with the ordinary aniline dyes, but not by Gram’s method. It is a facultative anaérobe. It grows well at the room-temperature, but more rapidly in the incubator. It does not liquefy gelatin. In gelatin plates the surface colonies are of a bluish-white color; the centers are denser than the borders, which are translucent. It usually grows more rapidly in gelatin than the bacillus of PATHOGENIC BACTERIA. 311 typhoid fever. Its growths in other media are mostly whitish. Bouillon becomes clouded. Nitrates are reduced to nitrites. In peptone solution it forms indol. On potato it forms an abundant visible growth from cream color to pale brown. Milk becomes acid and is usually, but not always, coagu- lated slowly. It causes the development of gas and acid in media containing dextrose or lactose. In media containing neutral red it is stated that the colon bacillus produces a yellow color with a green fluorescence. Differential points between “ = ‘ Z - ? * 34 . wees ip . a he 4 \ : a + oe Ae EE & a‘? qc hes Ae. gic y ce on : % Le , ; 2 % nna « : . ‘ é ea ae £: s? ite toe = to SS tag ' Ve one > . 1 Ck - * ¥ i * <. e o- FIG. 94.—BACILLUS COLI COMMUNIS WITH FLAGELLA, STAINED BY VAN ERMENGEM’S METHOD. (X I000.) the bacillus of typhoid fever and the Bacillus coli communis are as follows: ist. The typhoid bacillus is actively motile; the colon bacil- lus less actively motile. 2d. The typhoid bacillus has numerous flagella which rise {rom all parts of the surface; the colon bacillus has a smaller number of flagella. 312 MANUAL OF BACTERIOLOGY. 3d. The colonies of the typhoid bacillus in gelatin develop more slowly than those of the colon bacillus. 4th. The superficial colonies of the typhoid bacillus on gela- tin plates are less dense than those of the colon bacillus. 5th. In media containing dextrose or lactose the typhoid bacillus does not produce fermentation with gas and the colon bacillus does produce gas in such media. 6th. The typhoid bacillus produces an acid reaction with- out coagulation in milk, and the colon bacillus produces an acid reaction with coagulation. 7th. In peptone solution the typhoid bacillus, as a rule, produces no indol, and the colon bacillus produces indol. 8th. The typhoid bacillus usually produces an invisible growth on potato, the colon bacillus a visible growth. gth. The typhoid bacillus is said not to reduce neutral red in media, and the colon bacillus to change it to a yellow color. To these may be added the growth of the two organisms on special media like those of Wurtz, of Elsner, of Hiss and of Drigalsky and Conradi and the application of the serum- reaction. Injections of cultures of the Bacillus coli communis into ani- mals produce variable and uncertain results. Subcutaneous injection may lead to pus-formation; in rabbits and guinea- pigs injections may produce death apparently from poisons introduced. With the blood of immunized animals a serum- reaction, similar to that described for typhoid fever, may be demonstrated. Concerning the occurrence of the Bacillus coli communis in the intestine of man see page 144.* At autopsies on human subjects the great viscera are often found to have been infected by the colon bacillus, usually when * See also Moore and Wright. Bacillus coli communis in the Domesticated Animals. American Medicine. March 29, 1902. PATHOGENIC BACTERIA. 313 some lesion of the intestine existed simultaneously, but in most cases without having produced much apparent damage to the or- gans invaded. The Bacillus coli communis frequently occurs in mixed infections, as in wounds, inflammations and abscesses. It is often found in the peritoneum in peritonitis, in the pus in appendicitis, and in the urine in cystitis; it frequently occurs in the interior of gall-stones with whose formation it may be connected,* as first pointed out by Welch. There is a large number of more or less closely related organisms which go by the name of the “‘colon group.” The limits of the colon group are extremely ill defined. Detection of Bacillus coli communis in Water.—To each of a number of fermentation-tubes containing 1 per cent. dextrose-bouillon add some of the suspected water (0.1 to 1c.c. or more). Place in the incubator. Each day mark the amount of gas that has formed in the closed arm. After two days B. coli communis should render the bouillon strongly acid and produce about 50 per cent. of gas (30 to 7o per cent. according to different writers). The. gas is approximately H two parts, and CO, one part (see page 120). From tubes showing these characters plates may be made and the usual tests for the colon bacillus applied.t (See Part IV.) Stokes recommends adding the water to fermentation tubes containing 1 per cent. lactose-bouillon and neutral red (0 c.c. of a § per cent. solution of neutral red to a liter of bouillon); if the colon bacillus is present, 30 per cent. to 50 per cent. of gas is formed (con- sisting of one part of carbon dioxide and two parts of hydrogen), and the neu- tral red in the closed arm changes to a yellow color.t Paracolon or paratyphoid bacilli are the names applied to certain members of the colon group which have recently been shown to be pathogenic to man. They may produce clinical symptoms resembling typhoid fever of a mild and atypical form. The affection is rarely fatal. Probably they may occur with typhoid fever in mixed and secondary infections. Characteristic lesions have not yet been observed. The bacilli have been found in the blood, spleen, liver, gall-bladder and-urine. Like typhoid and colon bacilli they are motile, have flagella, are not stained by Gram’s method and do not liquefy gelatin. They ferment dextrose and maltose, producing acid and gas. They do not ferment lactose. Milk at first becomes acid, later it becomes alkaline, and is not coagulated. On potato a slight visible growth occurs. Media contain- * Lartigau. Journal American Medical Association. April 12, 1902. + Theobald Smith. American Journal Medical Sciences. Vol. CX. 1895. { Journal of Infectious Diseases. I. 341. 27 314 MANUAL OF BACTERIOLOGY. ing neutral red become yellow, as with B. coli communis, but more slowly, and the red color sometimes returns. In respect to the fermentation of saccharose and the formation of indol reports differ; both are usually negative. The blood of the patient agglutinates the bacilli. But, as among the closely related members of this group mutual reactions are sometimes seen, this test is not to be considered invariable.* Several bacilli allied to the above are known. The Bacillus enteritidis of Gaertner is a related form which has been found in cases of meat-poisoning. Bacillus lactis aérogenes (Bacillus aérogenes).—A_ba- cillus having a form similar to that of the colon bacillus, de- scribed as being larger and plumper. In the main its proper- ties are similar to those of the colon bacillus. Its colonies are more circumscribed and elevated than those of the colon bacillus. It is non-motile. It coagulates milk more rapidly than the colon bacillus. It produces gas upon potato more rapidly than the colon bacillus, and more abundantly. It was first described by Escherich, who discovered the colon bacillus, assigning the Bacillus lactis aérogenes rather to the upper part of the small intestine, and the colon bacillus to the lower portion. According to Kruse, the Bacillus lactis aérogenes and its rela- tives differ from the Bacillus coli communis chiefly in lacking motility. Like the colon bacillus it has been found many times in the urine in cystitis. See also B. acidi lactici, page 225. Bacillus dysenteriz (Shiga).—A bacillus with rounded ends, of the size and shape of typhoid and colon bacilli, seldom forming threads. Most observers have found it non-motile. Vedder and Duval have demonstrated flagella. The bacillus does not form spores. It may be stained with the ordinary aniline dyes; it does not stain by Gram’s method. It is a facul- tative anaérobe. It grows at ordinary temperatures, but better in the incubator. It grows on the usual culture-media, but more * Cushing. Bulletin Johns Hopkins Hospital. July-August, 1900. Strong. Ibid. May, 1902. Johnstone, Hewlett and Longcope. American Journal Medical Sciences. August, 1902. Libman and Buxton. Journal Medical Re- search. Vol. VIII. 1902. PATHOGENIC BACTERIA. 315 slowly than B. coli communis. The growths are whitish. Colonies on gelatin plates resemble those of the typhoid bacillus. Bouillon is diffusely clouded; a precipitate may form, but no pellicle. Indol is not produced. Milk becomes acid and is not coagulated. On potato a thin pale layer forms which may become light brown. No gas is formed in media containing glucose or lactose. : Neutral-red agar is not changed. From the feces the ba- cillus is best cultivated on agar plates, in the incubator. Colo- nies of B. coli communis are often more numerous than those of the dysentery bacillus. The colonies which develop in twenty-four hours are likely to be colonies of B. coli communis. The position of these may be marked on the glass with a pencil. Those which appear later are to be planted’ in dextrose-agar. If gas develops, they are not the bacilli of dysentery; otherwise they are to be studied and identified by the cultural and other tests mentioned above, and by the agglutination reaction. The bacilli are destroyed in a few minutes by boiling, and at 58° C. in half an hour. They appear not to be particu- larly resistant to the influences that are harmful to bacteria in general. They have been found in the intestine and the discharges of acute and epidemic dysentery in various climates and coun- tries, including the United States. Thus far their dissemination in the blood and distant organs has not been demonstrated. The lesion of this form of dysentery consists of a severe acute inflammation of the colon, frequently with necrosis of the sur- face and the formation of pseudomembrane. Ulceration may occur, but is usually superficial. Duval and Bassett found the bacillus of dysentery in the stools of infants having summer diarrhea. The introduction of pure cultures into animals by way of the alimentary canal has sometimes been followed by a cer- tain amount of diarrhea, but it does not appear that dysen- 3 16 MANUAL OF BACTERIOLOGY. tery, as it occurs in man, has been reproduced. Most labor- atory animals are, however, very sensitive to the injection into the tissues or veins of cultures, living or dead. They show the lesions produced by various toxins. The bacillus is agglutinated by the patient’s blood, but often only late in the disease and apparently not in all cases. This test seems to have only a limited value in clinical diag- nosis. Many prefer to secure the reaction in a test-tube. The dilutions used vary greatly (from 1 in 20 to 1 in 100}. Fic. 95.—SPIRILLUM OF CHOLERA (XX 1000.) Immunized animals develop the agglutinins in the blood. Results of experiment made for the production of a curative serum are encouraging. It now seems that the bacillus of Shiga has numerous close congeners, constituting with it a “group.” To what extent the others of the group may be concerned in the causation of diarrheal diseases or may occur in the normal intestine is uncertain. According to W. H. Park, some of these form indol and develop acid from mannite which the bacillus of PATHOGENIC BACTERIA. 317 Shiga does not; they also differ from it in their agglutina- tion reactions.* Spirillum cholerz Asiaticze (Comma Bacillus of Cholera).— A rod-shaped organism, somewhat curved, and with pointed ends, hence the name, ‘‘comma” bacillus. The curved forms, placed end to end, may produce an S-shaped body. The length is from 0.8 to 2 » and the breadth from 0.3 too.4y. In cultures some individuals may develop into genuine spirilla. In the whitish particles found in the stools of cases of cholera the organisms may be present in very large numbers. In these particles they may exhibit a very curious arrangement, lying parallel with one another, and, as remarked by Koch, they resemble a school of fish moving up stream. Involution forms, irregular in outline and staining poorly, are often seen in old cultures. The organism is motile, having a flagellum atoneend. Itdoesnotform spores. It stains with the ordinary aniline dyes, but not by Gram’s method. It is aérobic. It grows at the room-temperature, but better in the incubator. On the ordinary media the growths are whitish. It grows best on neutral or alkaline media, and is very sensitive to a small amount of acid. It liquefies gelatin. The colonies on gelatin plates have a very characteristic appearance. They are nearly round at first, and granular as seen under the low power of the microscope; but at the end of about twenty-four hours the outline is slightly irregular, and the surface looks as though it were covered with finely broken glass. The outline later becomes still more irregular or scalloped. As liquefaction of the gelatin takes place a funnel-shaped depression is formed, into which the colony sinks. Gelatin plates should be kept * Shiga. Centralblatt fir Bakteriologie. Bd. XXIV. 1898. Flexner. Philadelphia Medical Journal. September 1, 1900. Vedder and Duval. Journal Experimental Medicine. Vol. VI. Gay. University of Pennsylvania Medical Bulletin. November, 1902. Duval and Bassett. American Medi- cine. Vol.IV. P. 417. 1902. Park and Carey. Journal Medical Research. Vol. IX. 1903. Strong and Musgrove. Journal American Medical Associa- tion. Vol. XXXV. P. 498. 1900. 318 MANUAL OF BACTERIOLOGY. at a temperature of from 20° to 22° C. In stab-cultures in gelatin a white growth forms around the stab, and at the end of about thirty-six to forty-eight hours a funnel-shaped depression occurs at the surface, owing to the liquefaction of the gelatin. This depression increases in size, and the surface of the liquefied gelatin seems to be surmounted by an air-bubble, which appears to have taken the place of the part of the fluid gelatin which has evaporated. In the deeper portion of the stab liquefaction is less noticeable. ‘The growths on agar are not characteristic. oe S Z A ¢ Ds ,O =o mm t) se fot he ow BR we o O@ : Fic. 96.—INvoLution ForMS OF THE SPIRILLUM OF CHOLERA.—(Van Ermengem.) In bouillon a pellicle forms on the surface. On potato in the incubator the growth is whitish or brownish, not conspicuously elevated. After growing it in Dunham’s peptone solution in the incubator the addition of sulphuric acid develops a red color, owing to the presence of indol and nitrites,—the so-called “cholera red” reaction. Considerable doubt has recently been cast upon the formation of nitrites by the cholera spirillum.* The cholera-red reaction is not confined to this organism, and is said to differ from the nitroso-indol reaction. The spirillum of cholera is said to be very sensitive to dry- * Wherry. Journal of Infectious Diseases. Vol. II. No.3. June 24, 1905. PATHOGENIC BACTERIA. 319 ing, and, provided the drying be complete, is usually killed within twenty-four hours. It is killed in five minutes at a temperature of 65° C. and in one hour at 55°C. It may retain its vitality in water for a long time; observations vary widely in respect to determining how long. In the ordinary food- substances it may survive long enough to allow them to act as carriers of the infection if eaten raw. It is an important fact that the cholera spirillum is not a strict parasite, but under \ , a & ty BF? eo © . e@ a. b. c Fic. 97.—SPIRILLUM OF CHOLERA, COLONIES ON GELATIN PLATES. (X I00 TO 150.)—(Frdankel and Pfeiffer.) a. Twenty-four hours old. 8. Thirty hours old. ce. Forty-eight hours old. favorable conditions it may maintain its vitality for some time outside of the human body. The animals ordinarily used for laboratory experiments are, in their normal condition, not susceptible to infection with the spirillum of cholera through the alimentary canal, and no ani- mal is known which suffers from spontaneous cholera except- ing man, though a disease resembling cholera can be reproduced 320 MANUAL OF BACTERIOLOGY. in animals when certain conditions are complied with. The acid of the gastric juice destroys the organism, and this makes it impossible to infect animals by way of the alimentary tract unless this acidity is overcome with an alkali before the intro- duction of the culture. The following plan was adopted by Koch: The gastric juice was neutralized with a solu- tion of sodium carbonate; the movements of the intestines were quieted by the injection of 1 c.c. of tincture of opium for each 200 grams of the body-weight; and a portion of pure culture of the cholera spirillum was intro- duced into-the stomach. When guinea-pigs are treated in this manner, in most cases a condition closely simulating cholera is pro- duced. The animal dies with symptoms of collapse. The small intestine is more or less filled with a watery, flocculent fluid containing a large number of the spirilla of cholera. The Fic. 98.—SpIRIL- 5 5 4 Lum or CHor- Mucous membrane of the intestine is swollen ERA, STAB-CUL- and reddened. TURE IN GELA- tin, Two Days | When mice or guinea-pigs receive an intra- OLp.—(Frénkel : Piernne and Pfeifer.) | Peritoneal injection from a pure culture, death usually results, apparently from the toxic sub- stances contained in the culture. Pfeiffer was the first to show that an animal may be made immune from cholera by repeated small doses of cultures which have been heated in order to kill the organism. He also showed, in the same connection, that when living comma bacilli are introduced in the peritoneum of an immune animal they first clump together and are then rapidly destroyed and disintegrated (see page 192); furthermore, that a drop of the peritoneal fluid added to a hanging-drop culture of the cholera spirillum produces the same effect. This is now called Pfeiffer’s phe- PATHOGENIC BACTERIA. 321 nomenon, and is the underlying principle of all agglutination reactions, such as the Gruber-Widal typhoid test. It seems probable, from the results so far obtained, that it is practicable to use injections of attenuated cultures upon human beings with safety, and in this way to protect healthy persons from cholera during an epidemic.* Although a positive demonstration that the spirillum of Koch is the cause of cholera is lacking, as far as the exact reproduction of the disease in animals is concerned, the neces- sary proof has been supplied by the accidental or intentional infection of laboratory investigators who were working with cholera, which has happened on several occasions. Bacteriological investigations have shown that the spirilla of cholera are present in very large numbers in the watery contents of the intestine, especially early in the disease. They appear in the lumina of the glands, and they may be seen underneath the epithelial cells. They may occur in the matters vomited. They usually are not found widely spread through the organs of the body. It is probable that the symptonis of the disease result from poisonous substances produced by the spirilla or contained in them. The portal of entry in cholera is probably always the ali- mentary tract, and the infectious agent is usually, though not always, transmitted through drinking-water, and numerous epidemics have been traced to this source. In some cases the origin of the contamination of the water with cholera dejecta has been demonstrated. The organism may, however, be introduced into the alimentary tract upon any and every article of food. It may be conveyed from place to place upon soiled clothing and bedding, and then be brought in contact with food. Flies also probably convey the organisms from cholera stools to articles of food. In order to combat the spread of the disease the excreta and bedding should be thoroughly * Strong. American Medicine. August 15, 1903. 322 MANUAL OF BACTERIOLOGY. sterilized; the hands of the attendants should be carefully disinfected and all food should be cooked. Although com- moner in the summer-time, epidemics of cholera have been known to occur in the winter. Bacteriological Diagnosis of Cholera.—When cases sus- pected of being cholera appear in a community, it becomes a matter of the utmost importance to determine the exact nature of the disease in order that it may not become epidemic. One of the first occasions when bacteriological methods were put into practice in the diagnosis of cholera was at the time of the appearance of that disease in the port of New York in 1887. According to Koch, the diagnosis may be made in twenty- four hours or less. It is important to obtain the discharges from the intestines as early in the course of the disease as possible, and while they are perfectly fresh. It may be neces- sary, however, to examine the moist dejecta on the linen or clothing, when no other material is available. In the first place, one of the small, partly solid particles which may be found in the discharges from the intestines should be smeared upon a cover-glass, fixed in the usual man- ner, stained with one of the aniline dyes, and examined with the microscope. If taken early in the disease, the comma bacilli may be present in large numbers, and they are likely to be arranged in parallel groups, as already described. If comma-shaped bacilli are thus found, a strong probability is created that the disease is Asiatic cholera. The motility of the organisms can be determined by examination in the hang- ing-drop. It is to be remembered that spirilla of various forms are common in the normal mouth, and may appear in the stools (see pages 141 and 226). The diagnosis should be confirmed by the use of culture- methods. Using the small, semisolid particles from the intestinal discharges, gelatin plates in the usual three dilu- PATHOGENIC BACTERIA. 323 tions (see page 85) should be made and kept at a tempera- ture of 20° to 22° C. At the end of twenty-four hours or less the colonies of the spirillum of cholera should have been developed and should present the picture characteristic for these colonies in gelatin plates (Fig. 97), which enables them to be differentiated from colonies of other bacteria. From one of these colonies, preparations may be made for micro- scopic examination, and a set of tubes may be inoculated. The most characicristic growth will be from stick-cultures in gelatin. The growth in Dunham’s peptone solution may be tested for the development of indol and nitrites. At the time that the first smear preparations and gelatin plates are prepared, tubes of peptone solution should be inocu- lated directly from the intestinal contents, and kept in the incubator (Schottelius). After development has occurred, the production of indol may be tested by the addition of sul- phuric acid. These tubes are especially valuable when un- favorable material or when material containing small numbers of the spirilla is used. In the incubator the spirilla may be expected to multiply in the peptone solution rapidly, and to. appear upon the surface of the liquid in large numbers, even forming a visible film in six hours. Smears may be made from the surface part of these tubes, stained, and examined with a microscope. From the same material gelatin plates should be prepared, and examined as soon as the colonies develop. When cultures are obtained, their effects may be tested upon guinea-pigs by injecting them into the peritoneum. The production of Pfeiffer’s phenomenon is an additional means of diagnosis between the cholera spirillum and related forms. This consists in testing the suspected organism with serum from an animal immunized with cultures of cholera bacilli, as already explained above. In examining suspected water for the spirillum of cholera one or more liters of water is taken, and to it is added enough 324 MANUAL OF BACTERIOLOGY. of a 20 per cent. peptone solution to make the water contain I per cent. peptone, and enough of a 10 per cent. sodium chloride solution to make 5 per cent. The water, with the salt and peptone in it, is divided among a number of sterilized flasks. After twelve hours in the incubator, any cholera spirilla which happen to be present are likely to multiply and form a scum on the surface of the medium, and may be identified according to the methods given above. See also page 131. Since Koch’s discovery of the cholera spirillum in 1883-84 a considerable number of bacteria have been described which resemble the cholera spirillum more or less closely, and these have to be taken into account in making examinations of suspected material of any sort. This is particularly necessary in the investigation of water, in which such cholera-like spirilla seem to occur quite frequently. Vibrio Metchnikovii.—A comma-shaped organism, which, though somewhat shorter and thicker than the cholera bacillus, is very similar to the latter in form, and, like this, may some- times form genuine spirilla. It is motile and has a flagellum at one end. It does notform spores. It is aérobic. It stains with the aniline dyes, and is not stained by Gram’s method. It grows at the room-temperature. It liquefies gelatin some- what more rapidly than the spirillum of cholera. The colonies on gelatin plates are not all alike; some of them resemble those of Vibrio proteus, and others are extremely like those of the spirillum of cholera. It grows upon the usual media. Coagulated blood-serum is liquefied by it. The growth on agar_is grayish to yellowish, and abundant. It forms a pel- licle on bouillon. In milk an acid reaction is developed with coagulation. In peptone solution it produces indol and _ni- trates like the spirillum of cholera. It is said to give the nitro- soindol reaction more intensely than the spirillum of cholera. It is killed by a temperature of 50° C. in five minutes. It was discovered in chickens suffering from gastro-enteritis. PATHOGENIC BACTERIA. 325 It is ‘pathogenic for chickens, pigeons and guinea-pigs; less so for mice and for rabbits. The comma-shaped organisms are found in the blood in guinea-pigs, pigeons and young chickens. Vibrio proteus (Finkler and Prior).—A comma-shaped organism somewhat larger than the spirillum of cholera, some- times exhibiting genuine spiral forms, and also, at times, involution forms. It is motile and has a flagellum at one end. The developments of the colonies in gelatin and the lique- E =- << — < My. fo: r a , aL - ~ a ~ ey - > - 4 & s rr - ne ive oe ie ( -_ aaa ‘ a hoe of ¥, a , Sire fg . vii aha - +f - re: FIG. 99.—VIBRIO PROTEUS.* faction of this medium are more rapid than with the cholera spirillum. At the end of twenty-four hours the colonies are all circular, larger than those of the spirillum of cholera, and uniformly granular when slightly magnified. On the other culture-media the growths are usually whitish. On potato it produces an abundant, moist, grayish-yellow deposit, and grows at the room-temperature. It liquefies coagulated blood-serum ; milk becomes acid. In peptone solution it does not form indol. It is less pathogenic to animals than the spirillum of cholera. * The magnification is a little greater than in the other photomicrographs. 326 MANUAL OF BACTERIOLOGY. It was supposed by its discoverers to be the cause of cholera nostras, but it appears to have no relation to that disease. Spirillum Milleri—A comma-shaped organism resem- bling Vibrio proteus in many respects, and probably identical with it. In gelatin it grows more rapidly, and produces lique- faction more rapidly than the spirillum of cholera. On gela- tin plates, at the end of twenty-four hours, the colonies are uni- formly circular and granular, lying in little depressions re- sulting from the liquefaction of the gelatin. Its growths in the other media are not characteristic. It liquefies blood- serum. It does not produce indol. It is less toxic to animals than the spirillum of cholera. It was isolated by Miller from a carious tooth. See also Spirillum sputigenum, Part JIT. Spirillum tyrogenum (Deneke).—A comma-shaped organ- ism, not so large as the spirillum of cholera. It is motile, having a flagellum at one end. It does not form spores. In cultures, genuine spirilla may develop. Gelatin is liquefied more rapidly than by the spirillum of cholera, and the colonies develop more rapidly. The circumference of the colony is round, the surface may appear somewhat granular, and it has a greenish-brown color, seen under the low power. Milk con- taining litmus becomes acid, is subsequently decolorized, and is also coagulated. It liquefies coagulated blood-serum. It does not form indol in Dunham’s peptone solution. No pel- licle forms in cultures upon bouillon. It is less toxic to animals than the spirillum of cholera. It was isolated originally from old cheese. Vibrio Berolinensis.—A comma-shaped organism resem- bling the spirillum of cholera in form and in the position of its flagellum. It does not stain by Gram’s method. It grows at the room-temperature, but more rapidly in the incubator. The colonies upon gelatin, one or two days old, when magnified, are decidedly more finely granular and' more transparent than PATHOGENIC BACTERIA. 327 those of the spirillum of cholera, and the margin is almost abso- lutely smooth and circular. As the colonies become older they assume a more irregular and lobulated appearance, but are still more finely granular than the colonies of the cholera spirillum. Gelatin is very slowly liquefied. Its growth on the other culture-media is not remarkable. It forms indol in pep- tone solution, and it increases in the upper layers of the fluid. When guinea-pigs are inoculated in the peritoneal cavity, death Fic. 100.—SPIRILLUM OF RELAPSING FEVER IN THE BLOOD. SKETCHED FROM A STAINED SPECIMEN. Ss occurs in one to two days. This organism was discovered in the water-supply of Berlin. Other spirilla have been isolated from water by Giinther (Vibrio aquatilis in Spree water); by Dunbar from the Elbe River; by Russell from the Gulf of Naples; by Heider from the water of the Danube Canal; and in America, by Abbott, from the water of the Schuylkill (Vibrio Schuylkiliensis); and many others have been described to which the limits of this work will not permit of further allusion. 328 MANUAL OF BACTERIOLOGY. The Spirillum or Spirochzeta Obermeieri (of Relapsing Fever).—A slim spirillum with numerous turns, 16 to’ 40 /# in length. The ends are pointed. It is actively motile. The spirillum is not stained by Gram’s method, but may be colored by the ordinary aniline dyes. The organism has never been cultivated. It is found abundantly in the blood and in the spleen during the attack of fever. The spleen is enlarged. The disease has been produced in apes by inoculating them with blood taken from men having the disease. It is asserted that the spirillum is transferred by bed-bugs from one person to another.* Spirocheta pallida.—First observed by Schaudinn and Hoffmannt in recent as well as more advanced syphilitic lesions, on the surface and deep in the tissues in chancres, indolent buboes and papules. It has been found by many other observers very recently, and is constantly present in the situ- ations named. The evidence is accumulating rapidly in favor of this organism as the cause of syphilis.{ It is 4 to 14 y long, 4 thick and has6to14turns. It is actively motile. Stained with great difficulty. The following stain was recommended originally, and more recently a variety of stains have also been employed by different observers: (1) Three parts Giemsa’s eosin solution (2.5 c.c. 1 per cent. eosin solution in 500 c.c. water). (2) Three parts asur I solution (1 gram asur in 1000 c.c. water). (3) Three parts asur II solution (0.8 gram in 1000 water). Mix and stain dried cover-glass preparations from 16 to 24 hours; wash, dry and mount in balsam. Spirocheta refringens.—Found less frequently than S. pallida in the same locations as the latter. Is larger and stains more easily than S. pallida. * Karlinski. Centralblatt fiir Bakteriologie. Bd.XXXI. Original. got. {Schaudinn and Hoffmann. Vorliufiger Bericht tiber das Vorkommen von Spirochzten in syphililischen Krankheitsprodukten und bei Papillomen. Arbeiten aus dem Kaiserlichen Gesundheitsamt. Bd. XXII, p. 527, 1905. } Spirobacteria in the Lesions of Syphilis. Journal of the American Medical Association. Vol. XLIV, No. 22, p. 1790. 1905. APPENDIX. PATHOGENIC PROTOZOA. Protozoa are unicellular animal organisms. As they are studied by methods that have much in common with those used for the bacteria they may be considered here briefly. Protozoa are numerous in pond and ditch water, and these species seem to be harmless. However, many diseases of the lower animals are caused by protozoa, such as surra, Texas fever and coccidium disease of rabbits. Birds,* reptiles and frogst may show organisms in the blood resembling the para- sites of malaria. Until recently it has been doubtful whether any pathogenic protozoén has ever been propagated in pure culture outside of the body of the host. This has been accom- plished by Novy and MacNeal for a parasite (Trypanosoma) from the blood of the rat t and from many species of birds § on rabbit-blood-agar. Amceba dysenteriz (Amoeba coli).—Associated with amebic dysentery and believed to be its causative agent is the Ameba dysenterie, more often named Ameba coli. These organisms are found in the intestinal ulcers, the feces, the secondary liver abscesses and the sputum (in the latter only when an amebic liver abscess has perforated into the lung). The lesion in the colon is a severe inflammation accompanied by necrosis chiefly of the submucous layer, and leading to ex- * Opie and MacCallum. Journal Experimental Medicine. Vol. III. + Langmann. New York Medical Journal. January 7, 1899. {Novy and MacNeal. Contributions to Medical Research. Dedicated to Victor C. Vaughan. 1903. § Novy and MacNeal. On the Trypanosomes of Birds. Journal of In- jectious Diseases. Vol. II. No. 2. P. 257. March. 1905. 28 329 330 MANUAL OF BACTERIOLOGY. tensive ulceration.* According to Strong,{ at least two distinct species of amebe have been found in the feces in man, only one of which is pathogenic and the cause of dysentery. Unfortu- nately the designation, Ameba coli, has been applied to both species. The ameba of dysentery should be designated Amaeba dysenterie, limiting the term Ameba coli to the non-pathogenic form or forms. The Ameba dysenterié is a unicellular organism, 20-50 ps in diameter when at rest, consisting of a clear, homogeneous ectosarc and a granular endosarc, with an eccentrically placed nucleus. The endosarc contains a number of vacuoles of variable size and very frequently red blood-corpuscles, as well as other foreign bodies, such as bacteria, pigment granules, etc. Many red blood-corpuscles may be seen crowded together inasingle ameba. The organism is actively ameboid, extending its substance into processes or pseudopodia of varying forms. This ameboid motion assists in making easy the recognition of the parasites under the microscope and in distinguishing them from large, swollen cells found in the feces. The stool should be examined while fresh and still warm. The non-pathogenic ameba (Ameba coli), also occasionally found in the intestinal tract of man, differs from the pathogenic dysenteric organism chiefly in its much smaller size (10-24 /2) and the invariable absence of red corpuscles from its interior. The protoplasmic granules are also, as a rule, smaller and are difficult to recognize. The Amceba dysenterize produces ex- perimentally definite ulceration of the gut of cats, whereas the Amoeba coli is harmless. Both varieties of amebe may be stained by a special stain devised by Mallory.t * Councilman and Lafleur. Johns Hopkins Hospital Reports. Vol. II. Harris. American Journal Medical Sciences. Vol. CXV. 1898. { Strong. Circulars on Tropical Diseases. No.1. Chief Surgeon’s Office, Headquarters, Division of the Philippines, Manila, P. I. February, 1001. Ibid. No.1r. April, r901. (Both reports may be obtained from the United States Government, Washington.) { Mallory. Journal of Experimental Medicine. Vol. II. P. 529. Sep- tember, 1897. ' APPENDIX. ast The Malarial Parasite* (Plasmodium or Hematozoén malariz).—The organisms of malaria consist of at least three different species, each associated with one of the three types of malarial fever: The tertian parasite with benign tertian malarial fever, the parasite reaching maturity in forty-eight hours; the quartan parasite with benign quartan malarial fever, the cycle of development requiring seventy-two hours; and the estivo-autumnal parasite with malignant, estivo-autum- nal fever, developing to maturity in a variable period of from twenty-four to forty-eight hours. The parasites are studied to best advantage in a drop of fresh, fluid blood placed between a cover-glass and slide and examined with an oil-immersion objective. For method of making and staining dry preparations see pages 44 and 97. Tertian Parasite——This appears in its youngest form as a small, round, colorless, hyaline body within the red corpuscle, seen during and just after the chill of the disease. This body may be actively ameboid, suddenly changing its contour into various forms. Its size gradually increases, and fine, dark, actively motile, dancing pigment granules begin to appear at its periphery. The red corpuscle harboring the parasite, with the growth of the latter, becomes gradually paler and expands in size. The parasite as it grows loses its earlier ameboid movement, and the pigment granules, still actively motile, accumulate. Near the end of forty-eight hours the organism finally fills the red corpuscle, only a faint rim indicating the latter. The ripe parasite now divides it into from fifteen to twenty-five small, round, hyaline spores, which are arranged somewhat radially about the pigment granules which have lost their motility and become concentrated in a clump at the center of * Thayer and Hewetson. The Malarial Fevers of Baltimore. Johns Hop- kins Hospital Reports. Vol. V. 1895. Thayer. Lectures on the Malarial Fevers. New York. 1897. 332 MANUAL OF BACTERIOLOGY. the spore-forming organism. The spores finally break apart and scatter, each destined to invade a red corpuscle and start anew the cycle of development. This cycle may be repeated over and over again, producing a corresponding number of malarial paroxysms. Fig. ror. Fig. 102. Fig. 103. Fig. 104. Fics. 101-104.—-MALARIAL PARASITES IN VARIOUS STAGES. (X 1000.) Figs. ror, 102 and 103 are tertian parasites. Fig. 103 shows the comple- tion of segmentation. Fig. 104 is the crescentic form of the estivo-autumnal parasite. Certain full-grown parasites do not complete the cycle of development by sporulation, as described, but, breaking loose from the corpuscle, remain as “extracellular’’ bodies. These are seen chiefly after the paroxysm as large, round, pale bodies containing numerous dancing pigment granules scattered through their substance. They ultimately degenerate and disappear. Some of these extracellular forms may be seen to develop long slender processes, flagella, having a very active whip-like motion. Flagella are never observed APPENDIX. 333 in perfectly fresh blood, but develop only after the blood has been drawn some time, usually fifteen or twenty minutes. The extracellular forms of the parasite, the gametes, incapable of further development in their human intermediate host, can continue their life cycle only when, by chance, they happen to be sucked into the body of a mosquito of the genus Anopheles, the definite host, in which they undergo a second com- plete sexual cycle of development with the ultimate production of spores or sporozoids. When in turn the spores chance to be inoculated into the blood of man by the bite of an infected Anopheles, the man becomes infected, and the cycle of development in the red corpuscle, already outlined, commences. The second or sexual cycle of the parasite in the mosquito, here described for the tertian organism, applies as well to the other varieties of the malarial organism, namely the quartan and the estivo-autumnal forms, in the case of each starting from the extracellular mature forms of the organism found in the blood of the human host.* Quartan Parasite.—This resembles quite closely the ter- tian parasite, but differs from it in certain respects. The young, hyaline, intracorpuscular parasite is more highly refractive, its ameboid motion is less marked and more slug- gish, and the pigment granules are darker, much coarser, and have very slight motility. The infected red corpuscles are usually somewhat contracted instead of swollen, and their color is apt to be darker, assuming a bronzed hue. The full-grown parasite is much smaller than the corresponding form of the tertian, approximating the size of a normal red corpuscle. As segmentation begins, a characteristic appear- ance develops which distinguishes the quartan organism, namely, the coarse pigment granules are drawn toward the center of the parasite in certain converging straight paths, giving a stellate arrangement to the pigment, until finally it becomes clumped entirely at the center in a solid mass. The segmenting forms of the quartan parasite thus present a more symmetrical arrangement of the spores, which often resemble the petals of a ‘‘marguerite.”” These spores are oval and num- ber only from six to twelve, being fewer than those of the ter- * Lyon. The Inoculation of Malaria by the Mosquito. A Review of the Literature. Medical Record. February 17, 1900. 334 MANUAL OF ‘BACTERIOLOGY. tian segmenting parasite. The quartan extracellular forms are smaller than those of the tertian, being about the size of a red corpuscle, and contain coarse pigment granules in active motility until degeneration occurs. Flagella may develop from certain extracellular forms. The entire development of the quartan parasite occupies about seventy-two hours. Estivo-autumnal Parasite——This parasite develops to ma- turity in from twenty-four to forty-eight hours, and is usually regarded as representing a single species, though certain ob- servers claim to distinguish two distinct varieties. The usual description of a single variety is here adopted. The youngest forms (hyaline bodies) resemble those of the tertian and quar- tan organisms, but are distinctly smaller and more highly refractive. They often present a ring-like appearance. They are ameboid. Pigment granules later appear at their per- iphery, but are exceedingly minute and scanty, seldom more than one or two being seen. These granules have little or no motility, and in fact are with difficulty made out. The hyaline bodies remain small, seldom exceeding one-third the diameter of a red corpuscle. The infected corpuscle is apt to be crenated, shrunken and dark. These are the forms seen in the circu- lating blood in early infections; the mature forms, with the exception of the extracellular forms, developing in the spleen and bone-marrow, rarely reach the general circulation. Blood from the spleen shows the full-grown forms in abundance. The segmenting forms resemble those of the tertian parasite both in the numbers of the segments and in their arrangement, but are much smaller in the aggregate, as well as in the indi- vidual segments. After the fever has lasted about one week, extracellular forms make their appearance in the circulating blood. These are crescentic, ovoid or small round bodies, containing coarse pigment granules at their center, generally arranged in a ring. The crescents and ovoid bodies are highly refractive APPENDIX. 335 and are in length about equal to the diameter of a red cor- puscle, sometimes larger. The round forms are smaller than a red corpuscle, with the pigment arranged centrally in a ring. They may become flagellated after the blood has re- mained outside the body for some minutes. Any of the extra- cellular bodies may show remnants of the red corpuscle at- tached to its side, like a bib. The extracellular forms are concerned in the cycle of development of the organism in the mosquito, and are sterile in the human body. They are ex- ceedingly resistant to quinine and may continue in the blood for long periods of time. Melaniferous leukocytes are seen in the blood, being espe- cially abundant after the paroxysm in all forms of malarial infection.* These are phagocytes which have taken up the pigment granules liberated by the disintegration of the erythro- cytes. Small-pox and Vaccinia.—Micrococci of various sorts have been found in the pustules of small-pox and vaccinia, but indicate only a secondary infection. Other microdrgan- isms have been described. The most important are certain bodies often considered protozoa. In both small-pox and vaccinia small, round homogeneous bodies, 2 to 4 » in diam- eter, have been found in the epithelial cells of the vesicles. Inoculation of vaccine lymph into the rabbit’s cornea leads to the production of similar bodies in the epithelial cells of the cornea. W. Reef found small ameboid bodies in the blood in cases of small-pox and vaccinia. Vaccine virus that has been filtered through the Chamberland or Berkefeld filter is no longer active. From this it may be presumed that the organ- ism causing it is not too small to be seen with the microscope. Councilman, Magrath and Brickerhoff,t as a result of * See also Ewing. Journal Experimental Medicine. Vols. V. and VI. + Journal Experimental Medicine. Vol. II. P. 515. See also Anna Wil- liams and Flournoy, and W. H. Park. New York University Bulletin Medical Sciences. Vol. II. October, 1902. { Journal Medical Research. Vol. IX. May, 1903. 336 MANUAL OF BACTERIOLOGY. recent studies, believe that the bodies above mentioned are protozoa. Segmentation of the bodies is described, result- ing in the formation of spore-like bodies. The spore-like bodies undergo a further or second cycle of development within the nucleus. The second cycle also ends in segmenta- tion. The two cycles were seen in small-pox; in vaccinia, only the first or extranuclear bodies were observed. FIc. 105.—TRYPANOSOMES IN THE BLOOD OF THE Rat. ROMANOWSKY STAIN. (X 1000.) YELLOW FEVER. It has already been indicated (page 149) that the study of cases of yellow fever has failed to prove that this disease is caused by bacteria. On the other hand, evidence that it is transmitted by the mosquito, Stegomyia, has been increasing. Trypanosomes, —A number of species of Trypanosoma have been described, which produce diseases in the lower animals; recently one has been stated to be the cause of disease in man.* The trypanosoma is a protozoén belong- * For a full description of the life history and classification of Trypanosoma see Salmon and Stiles. Emergency Report on Surra. United States Bureau Animal Industry. Bulletin No. 42. 1902. See also Francis. Marine Hos- pital Service. Hygienic Laboratory. Bulletin No. rr. 1903. APPENDIX. 337 ing to the flagellata. It is of an elongated, spindle-shaped form, with a nucleus, and has a flagellum at one end, which extends along a thin edge, called the undulating membrane. It is actively motile. It occurs in the blood, between, but not in, the blood-corpuscles. Its length is two to several times the diameter of a red corpuscle. Members of this genus are the cause of surra (a fatal dis- ease of horses and mules occurring in India and the Philippine Islands) and of the tsetse-fly disease of South Africa; while others are found in rats, birds, amphibia and fishes. In the horse the infection is transmitted by the bites of flies. Novy and MacNeil have succeeded in cultivating the trypanosoma of rats and birds on rabbit-blood-agar.* Several cases were reported during 1902 where trypanosomes were found in the blood of individuals from tropical Africa, showing that this group of parasites may occur in man.t The symptomatology of these infections re- quires further study. Still more recently it has been claimed by Castellani that a trypanosoma is the cause of “sleeping sickness,” a disease of the natives of Africa. He states that the parasites may be demonstrated in the cerebro- spinal fluid obtained by lumbar puncture and, with greater difficulty, in the blood, during life. Many cases also show at autopsy streptococcus infection, which is believed to be a secondary invasion.{ * Loc. cit. { British Medical Journal. May 30, 1903. t British Medical Journal and Lancet. June 20, 1903. 29 SURFACE DiviIpED IN SQUARE CENTIMETERS FOR CoUNTING COLONIES. INDEX. ABBE condenser, 20 Abrin, 164, 177 Abscesses, 233, 243 metastatic, 241, 243 Absorbent cotton, 70, 80, 218 Accidental infection of laboratory workers, 104 Acetic acid, 29, 40, 119 Acid, acetic, 29, 40, 119 alcohol, 30, 34. aniline dyes, 28 boric, 206 butyric, 119, 223 carbolic, 118, 197, 200, 219 formic, 119 fuchsin, 28 hydrochloric, 143, 199 g lactic, 119, 134, 225 oxalic, 211 picric, 28 propionic, 119 pyrogallic in cultivating anaé- tobes, 79 rosolic, 67 Acid-proof bacilli, 32, 35, 138, 143, 287, 294, 300 Acids, formation by bacteria, 119 Acquired immunity, 170 Actinomyces, 228, 286, 294, 298 Actinomycosis, 235, 298 Active immunity, 177 Acute miliary tuberculosis, 292 Aérobic bacteria, definition, 116 Aérobioscope, 125 Agar-agar, 64 Age, relation to infections, 155 Agglutinating substances in blood- serum, I91, 306 Agglutinins, 165, 185, 191, 306 Aggressins, 181 Air, bacteria of, 124, 152 bacteria conveyed by, 152 Albumen, culture-media containing, 69 343 Albumen, fixative, 38 precipitins for, 167 Alcohol, acid, 30, 34 fixation of tissues by, 36 relation to infection, 155, 191 Alexins, 191 Alimentary canal, bacteria of, 143 Alum filter, 127 Amboceptor, 186 American filtration system, 127 Public Health Association, di- rections for preparing media, 60 Ameeba coli, 329 of dysentery, 329 Anaérobic bacteria, cultivation, 79 definition, 116 Aniline dyes, 28 alcoholic solutions, 28 as germicides, 200 watery solutions, 28 oil, 30, 41 -water solutions, 29, 35 Animals, autopsies on, 94. care of, 92, 93 inoculation of, 84, 92 Anopheles, 154, 333 Anthrax bacillus (see also Bacillus of anthrax), 272 protective inoculation, 172, 274 symptomatic, 172 virus for, 172 Antiagglutinins, 169 Antibodies, 169 Antilysin, 169, 192 Antiprecipitins, 169 Antiseptic, definition, 193 Antitoxic unit, 285 Antitoxins, 169, 175, 187 for diphtheria, 175, 187, 271, 284 tetanus, 175, 187, 271 Antitoxin-toxin mixture, 187 Argentamin, 200 Argonin, 200 Argyrol,. 200. 344 Arnold steam sterilizer, 52 Arrhenius, 190 Arrow-poisons, bacteria in, 6, 122 Arthritis, 240, 252, 258 Arthrospore, 112 Asiatic cholera (see Cholera) Aspergillus glaucus, 231 Autoclave, 56 Auto-infection, 154 Autopsies on animals, 94 bacteriological examinations at, 94,99. disinfection at, 94, 95, 208 on human subjects, 99 Avian tuberculosis, 294 BABES-ERNST bodies, r10 Bacilli, branching forms, 108 acid-proof, 32, 35, 138, 143, 287, 294, 300 Bacillus acidi lactici, Hueppe, 225 acidophilus, 144 aérogenes, 144, 235, 255, 314 capsulatus, 122, 267 amylobacter, 145, 222 anthracis, 14, 122, 125, 152, 153, 155, 162, 172 bifidus, 144 botulinus, 139 buccalis maximus, 229 butyricus, Hueppe, 223 Prazmowski, 222 capsule, of Pfeiffer, 259 coli communis, 134, 135, 144, 164, 235, 239; 259) 310 comparison with typhoid bacillus, 311 detection of, in water, 313 in water, 132, 133 comma, of cholera (see Spirillum of cholera) cyanogenus, 225 definition, 4, 100, 107 diphtheriz, 104, 152, 277 dysenteriz, 129, 314 edematis maligni, 122, 268 enteritidis, Gartner, 139, 314 erythrosporus, 225 fluorescens liquefaciens, 221 putidus, 221 icteroides, 149 Indicus, 222 influenze, 275 INDEX. Bacillus, Klebs-Léffler (see Bacillus diphtheriz) lactis aérogenes, 144, 259, 314 cyanogenus, 225 leprae, 287, 296 mallei, 166, 296 megaterium, 223 mesentericus vulgatus (see also * Potato bacillus), 223 mucosus capsulatus, 258 mycogenes, 260 mycoides, 224 Neapolitanus, 310 cedematis maligni, 122, 268 of anthrax (see Bacillus anthra- cis) of blue milk, 225 of bubonic plague, 235, 263 of chancroid, 258 of diphtheria (see Bacillus diph- theriz) of Ducrey, 258 of dysentery (see Bacillus dysen- teriz). of Eberth (see Bacillus typhi abdominalis) of Emmerich (see Bacillus coli communis) of Escherich (see Bacillus coli communis) of Friedlander (see Bacillus pneu- moniz) of glanders (see Bacillus mallei) of influenza (see Bacillus influ- enze) of leprosy (see Bacillus lepre) of malignant edema (see Bacillus cedematis maligni) of ozena, 259 of rhinoscleroma, 259 of Shiga (see Bacillus dysen- teriz) of smegma, 33, 143 of soft chancre, 258 of syphilis, Lustgarten, 149 Joseph and Piorkowsky, 149 of tetanus (see Bacillus tetani) of typhoid fever (see Bacillus typhi abdominalis) of Vincent, 228 of xerosis, 281 paracolon, 313 paratyphoid, 313 pestis bubonice, 263 phlegmones emphysematose, 267 INDEX. Bacillus phosphorescens Indicus, 223 pneumonie, Friedlander, 235, 258 prodigiosus, 222 proteus, 139, 235, 262 pseudodiphtherie, 281 pyocyaneus, 260 pyogenes foetidus, 235, 310 Tamosus, 224 subtilis, 224 tetani, 84, 122, 269 tuberculosis, 84, 150, 152, 286 in milk,. 137, 138 staining of, 32, 42, 138, 286 typhi abdominalis (Bacillus ty- phosus), 126, 131, 133, 134, 301 vaginalis, 142 violaceus, 222 Bacteria, acid-proof, 32, 35, 138, 143 aérobic, 116 anaérobic, 116 cultivation of, 79 chlorophyll, relation to, 1, 115, 121, 146 chromogenic, 117 classification, 106 cultivation of, 72 definition, 3 diseases caused by, 148 distribution, 4, 122 examination with the microscope, 18 ferments formed by, 117 fluorescent, 117, 221, 262 forms of, 106, 107 higher, 107, 228 in disease, 146 influence of electricity, 116 of oxygen, 115 of sunlight, 116 microscopic examination, 18 motility, 113 multiplication, 111 non-pathogenic, 110 number of species, 221 nutrition of, 115 of air, 124, 152 of foods, 153 of ice, 125, 133 of milk, 133, 153 of soil, 122, 153 of the alimentary canal, 143 of the cranial sinuses, 140 of the gall-bladder, 140, 308, 313 345 Bacteria of the intestines, 143, 144 of the mouth, 141, 226, 229, 251, 259 ‘ of the nasal cavity, 141, 260 of the normal human body, 140 of the skin, 140 of the stomach, 143 of the urethra, 142 of the vagina, 142 of water, 125, 153 pathogenic, 110, 233 phosphorescent, 117 products of growth, 117, 135, 139, 161 pyogenic, 234 size, 4, 110 staining, 27, 28 - in tissues, 36, 39 transmission of specimens by mail, 100 vegetative forms, III -Bacterial poisons, 161 in meat, 161 in cheese, 161 products, 117, 135, 139, 161 Bacteriolysis, 166, 182, 191 Bacterium coli commune, 134, 135, 139) 145, 259, 310, 313 definition, 109 syncyanum, 225 termo, 121, 263 ures, 226 Zopfhii, 226 Bail, 181 Balsam, Canada, 26, 29, 40 Basic aniline dyes, 28 Basophilic granules, 40, 43 Beef-tea, 59 Beggiatoa, 228 Beri-beri, 148 Berkefeld filter, 57 Bichloride of mercury (see Mercury, bichloride) Biedert’s method for examining spu- tum, 36 Birds, tuberculosis of, 294 Bismarck brown, 28, 30 Black death, 266 -leg, 172 Blastomycetic dermatitis, 232 Blood-agar, 69, 276 Blood, cultures from, 98 -poisoning, 237 -serum-agar, 67 germicidal power, 162 346 Blood-serum, Léffler, 68 Marmorek, 247 preparation, 67 sterilization, 55, 67 -test for typhoid fever, 166, 306 specimens of, 97 staining of, 43 Blue milk, bacillus of, 225 pus, 260 vitriol, 207 Bodily conditions disposing to in- fection, 155 Boiling, sterilization by, 51, 128, 210, 213 Boils, 243 Boéphilus, 154 Bordet, 189, 191 Boric acid, 206 Bouillon, 59 sugar-free, 62 Bovine tuberculosis, 137, 289 Branching forms of bacilli, 108 Bread-paste, 69 Bromine as a germicide, 204 Bronchitis, 239, 277 Brownian movement, 24 Bubonic plague, bacillus, 263 toxin from, 164 virus, 173 Buchner’s method for anaérobes, 79 Butter, tubercle bacilli in, 137, 138 Butyric acid, 119 cultivating CADAVER, care of, in diseases, 208 Calcium compounds as germicides, 204 hypochlorite, 204 Canada balsam, 26, 29, 40 Capaldi’s culture-medium, 303 Capsule bacillus of Pfeiffer, 259 Capsules of bacteria, 45, 111 staining of, 45 Carbol-fuchsin, 34 Carbolic acid, 118, 197, 200, 219 Carbon dioxide, 119 Carbuncles, 243 Carmine, 42, 43 Caries of the teeth, 142 Caseation, 291 Catgut, surgical preparation, 214 Cedar-wood oil, 20 contagious INDEX. Celloidin imbedding, 36 Cells, epithelioid, 290 giant, 290 pus, 234 Cellulitis, 245, 268 2 Cellulose, decomposition by bacteria, 118, 145 Centrifuge for milk separator, 136 Cerebro-spinal meningitis, 254 Chancroid, bacillus of, 258 Charbon (see Anthrax) symptomatique, 172 Cheese-poisoning, 134 Chemotaxis, 114, 179, 235 Chicken-pox, 149 Chloride of lime, 204 Chlorine as a germicide, 204 Chloroform as a preservative, 68 Chlorophyll, relation to bacteria, 1, IIS, 121, 146. Cholera, diagnosis, 322 infantum, 263, 315 nostras, 326 -red reaction, 318 spirillum (see also Spirillum of cholera), 317 Chromicized catgut, 216 Chromogenic bacteria, 117 Cladothrix, 228 Classes in bacteriology, hints for teaching, 101, 102, 103, 104, 105 Classification of bacteria, 106 Cleaning fluid, 25 Climate, influence on infections, 155 Clostridium butyricum, 222 definition, 113 Coal-oil, 206 Coccus, definition, 4, 107 Collodion, 36 capsules, 95 Colon bacillus (see also Bacillus co:i communis), 310 contrasted with typhoid bacillus, 311 group, 311 Colonies of bacteria, 85, 88, 90 Comma bacillus of cholera (see a!so Spirillum of cholera), 317 -shaped bacteria, 107, 109 Complement, 191 effects of heat on, 186 Condenser, Abbé, 20 Conjunctivitis, gonorrheal, 258 Consumption, 292 Contagious disease, definition, 147 INDEX. nee disease, disinfection after, 20 Contrast-stains, 28, 30, 32, 35, 43 Copper sulphate, 207 Copperas, 206 Cornet forceps, 25, 67 Corrosive sublimate (see bichloride) Cotton, absorbent, 70, 80, 217 plugs for tubes, etc., 13, 70, 80 Cover-glass forceps, 25, 26, 27 preparations, 25, 26, 27 Cover-glasses, 25 Cow-pox, Io, 171 Cranial sinuses, bacteria of, 140 Cream, ripening, 137 Creolin, 200 Cresol, 200 Croup, membranous, 283 Cultivation of anaérobic bacteria, 79 of bacteria, 72 Culture-media, definition, 7 neutralization, 59, 60, 61 preparation, 59 reaction of, 59, 62, 115 sterilization, 51, 59, 62, 67, 71 -tubes, 69 inoculation of, 72 sterilization of, 70 Cultures at autopsies, 94, 99 destruction of, 104, 105 from blood, 98 sealing of, 79 Cumol, 215 Cupric sulphate, 207 Cutting of sections, 38 Cystitis, 240, 263 Cytolysis, 166 Mercury DELAFIELD’S hematoxylin, 43 Deneke’s spirillum, 326 Dengue, 149 Dental caries, 142 Deodorizers, 193 Dermatitis, blastomycetic, 232 Dextrose, 62 agar, 65 -bouillon, 62 media for anaérobes, 79 Diagnosis of actinomycosis, 299 of bubonic plague, 263 of cholera, 322 of diphtheria, 246, 277 of dysentery, 315, 329 347 Diagnosis of glanders, 297 of gonorrhea, 255 of influenza, 277 of malaria, 331 of Malta fever, 253 of meningitis, cerebro-spinal, 254 of pneumonia, 252 of tuberculosis, 32, 286, 292 of typhoid fever, 303, 307, 308 Dilution-cultures, 88 Diphtheria, 246, 277, 282 antitoxin, 175, 187, 271, 284 bacillus, 104, 152, 277 diagnosis, 246, 277 toxin, 162, 164, 177, 187, 282 Diphtheritic inflammation (see also Pseudomembranous inflammation), 246, 281, 283 Diplococcus, definition, 108 intracellularis meningitidis, 235, 254 of gonorrhea, 255 of pneumonia (see also Micro- coccus lanceolatus), 249 Disease, bacteria in, 146 Diseases caused by bacteria, 148 by protozoa, 329 probably due to microdrgan- isms, 149 infectious, recovery from, 160, 170 Disinfectant, 193 Disinfection at autopsies, 94, 95, 208 of cultures, 86, 104 of dejecta, 207 of hands, 211 of houses, 201, 203, 204, 208 of sputum, 207 of stools, 207 of test-tubes, 86, 104 of urine, 207 surgical, 210, 218 Distribution of bacteria, 122 Dorset’s egg-medium, 69 Dressings, surgical preparation, 218 Drigalsky-Conradi’s method for de- tecting typhoid bacilli in water, 303 Drinking water, sterilization of, 128 Drying, influence on bacteria, 112, 113, 114 Ducrey’s bacillus, 258 Dunham’s peptone solution, 67 Dyes, aniline, 28 as germicides, 200 Dysentery, 261, 314 348 Dysentery, amebic, 329 bacillus, 314 Ear, middle, bacteria of, 140, 240 Eberth’s bacillus (see also Bacillus of typhoid fever), 301 Edema, malignant, bacillus, 122, 268 Egg-albumen as a culture-medium, 69 Egg-medium of Dorset, 69 Eggs, in cultivating anaérobes, 69, 82 Ehrlich’s side-chain theory, 182, 191 Electricity, influence on bacteria, 116 Elsner’s culture-medium, 303 Emmerich’s bacillus, 310 Emphysematous gangrene, 268 Endocarditis, 239, 243, 246, 252, 258 Endogenous spores, 112 Endotoxins, 177 Enzymes, 117, 163 Eosin, 44 Epithelioid cells, 290 Epitoxoid, 189 Epitoxonoid, 189 Erysipelas, 248 Escherich’s bacillus, 310 Esmarch’s method for anaérobes, 82 toll-tubes, 88, 89 Essential oils as germicides, 2067 Eye-piece, 18, 19, 20 FALLOPIAN tube, bacteria of, 140 Farcy-buds, 297 Fat in culture-media, 69 Fats, decomposition by bacteria, 118 Feces, bacillus of tetanus in, 270 bacteria of, 144 disinfection, 207 typhoid bacilli, examination for, 304, 307 Fermentation, 12, 120 -tube, 120 Ferments, development by bacteria, 117 and toxins, 163 Ferrous sulphate, 206 Fibrin, Weigert’s stain, 42 Ficker-Hoffmann’s method for de- tecting typhoid bacilli, 304 Film-preparations, 26, 27, 28 Filter, alum, 127 American, 127 Berkefeld, 57 infusorial earth, 57 INDEX. Filter, Kitasato, 57 mechanical, 127 Pasteur-Chamberland, 57, 128 sand, 57, 127, 128 unglazed porcelain, 57, 128 — Filtration, sterilization by, 128 of water, 57, 127 Finkler and Prior spirillum, 325 Fishing from colonies, 91 Fission of bacteria, 3 Fixation of cover-glass preparations 26, 27, 28 of slide-preparations, 27, 28 of tissues, 37 Fixative, albumen, 38 Flagella, 113 staining, 46 Flies, bacteria carried by, 153, 309, 321 _ Fluid for cleaning, 25 Fluorescence of bacteria, 117, 260 Focusing the microscope, 21, 24 Fomites, definition, 148 Food used by bacteria, 115 Foods, bacteria of, 133, 139 poisoning by, 134, 139 Foot and mouth disease, 150 Forceps, Cornet, 26 cover-glass, 26 for slides, Kirkbride, 27 Stewart, 26 Formaldehyde as a germicide, 200, 209 catgut, 215 disinfection of rooms, 208 fixation of tissues with, 36 Formalin (see Formaldehyde) Formic acid, 119 Fowl-cholera, protective inoculation, 172 Fowls, tuberculosis of, 294 Fractional sterilization, 51 Frinkel’s method for anaérobes, 80 pneumococcus (see also Micro- coccus lanceolatus), 249 Freeman’s pail for pasteurizing, 55 Freezing, influence on bacteria, 133 Friedlander’s bacillus of pneumonia, 221, 197; 258 Fuchsin, 28 acid, 28 Fiirbringer’s method for disinfecting hands, 211 Fusiform bacillus ot Vincent, 22 INDEX. GaBBETT?’s method for staining tu- bercle bacilli, 34, 143 Gall-bladder, bacteria of, 140, 308, 313 Gangrene, emphysematous, 268 Gas-burner, Koch’s, 78 formation by bacteria, 119 -phlegmons, 268 -regulator, 77 Gastric juice, 143 Gauze, sterilization of, 218 Gelatin, 62 liquefaction, 117 tetanus bacilli in, 270 Gélose (see Agar-agar), 64 Gentian-violet, 28, 30 Geppert’s test for germicides, 195 Germicidal power of blood-serum, 166, 186, 191 Germicide, definition, 193 Germicides, tests for, 194 Germ, use of the word, 3 German measles, 149 Giant-cell, 290 Glanders bacillus, 296 Straus’s method for diagnosing, 297 Glass plates, go Glassware, sterilization of, 49 Gloves,. rubber, 212, 213 Glucose (see also Dextrose), 62 Glycerin-agar, 65 -albumen, 38 -bouillon, 62 Gonococcus of Neisser, 235 Gonorrhea, 238, 255, 257 diagnosis, 256 Gram-Giinther method, 31 Gram’s method, 30, 41 bacteria stained by, 31 not stained by, 31, 32 Gram-Weigert method, 41 Gray tubercle, 291 Green pus, 260 Ground-water, 126 Group agglutinins, 165 lysins, 167 precipitins, 168 Groups of bacteria, 107 Gruber-Widal reaction, 166 Guarnieri’s medium, 69 Gun-cotton, 37 Giinther’s modification of Gram’s method, 31 germicidal power, 349 HAFFKINE’s inoculations for plague 173, 178, 265 Hair-follicles, infection around, 237 Hands, disinfection, 211 Hanging-block, 24 -drop, 22 Haptophore, 183 Hardening of tissues, 36 Hay bacillus, 102, 112, 135, 194, 224 Heat, effect on growth of bacteria, 114 sterilization by, 49, 216 Hematoxylin, 43 Hematozo6én of malaria, 331 Hemolysis, 166 Heterologous serum, 165 Higher bacteria, 228 Hill’s test for germicides, 194 Hiss, medium of, 303 stain for capsules, 46 Historical sketch of bacteriology, 8 Hog cholera, 176 ; Holmes, O. W., 11 Homologous serum, 165 Honing of knives, 39 Horse-hair, surgical preparation, 217 Hot-air sterilizer, 50 Houses, disinfection, 201, 203, 208 Hueppe’s method for anaérobes, 82 Hydrochloric acid, 143, 199 Hydrogen, cultivation of anaérobes under, 80 peroxide, 205 sulphide, 119 Hydrophobia, 149, 173 preventive inoculation, 173 Hypha, 232 Hypochlorite of calcium, 204 Hypodermic inoculation of animals, 92) 931 94 204, Ice, bacteria of, 125, 133 Ice-cream poisoning, 134 Illumination for the microscope, 21 Imbedding, 37 Immune-body, 191 Immunity, 16, 170 acquired, 170 active, 170, 177 antitoxic, 175 bacteriolytic, 178 by injection of cultures, 172 duration of, 177 individual, 170 35° Immunity, natural, 170 passive, 170, 177 racial, 170 side-chain theory, 182 theories of, 178 unit, 284 Impression-preparation, 25 Incubator, 75 Indol, 118 test for, 118 Infected wounds, 219 Infection, bodily conditions favoring, 155 local conditions favoring, 156 of investigators with pathogenic bacteria, 104 of wounds, 156 mixed, 158 secondary, 158, 159, 238 terminal, 158 Infectious disease, definition, 147 diseases not followed by immu- nity, 171 Inflammation, 233, 239 diphtheritic (see also Pseu- domembranous inflammation, 246, 280, 282 Influenza bacillus, 275 Infusorial earth in filters, 57 Inoculation of animals, 92 in isolating bacteria, 84 of tube-cultures, 73 Inoculations, preventive, 172 for anthrax, 172, 274 for black-leg of cattle, 16, 172 for bubonic plague, 172, 265 for cholera, 321 for erysipelas of swine, 172 for fowl-cholera, 172 for hydrophobia, 173 for small-pox, 10, 171 for tuberculosis, 293 for typhoid fever, 310 Insects, destruction of, 203, 206 infections spread by, 153, 154, 309; 321, 328, 333, 336 Instruments, surgical preparation, 210, 213 Intermittent sterilization, 51 Intestine, bacteria of, 143, 144 Intravenous inoculation, 93 Invisible growth on potato, 303 microbes, 110, 150 Involution forms of bacteria, 110 Iodide of mercury, 199 INDEX. Iodine solution, 30 Todoform, 206 Tris diaphragm, 18 Itch, 14 JENNER, I0 Journals of bacteriology, 7 KANGAROO tendon, surgical prepara- tion of, 216 Kerosene, 206 Kirkbride forceps for slides, 27 Kitasato filter, 57 Klaischpreparat, 25 Klebs-Léffler bacillus (see also B. diphtheriz), 277 Knives, sharpening of, 39 Koch, 15 Koch’s gas-burner, 78 method for anaérobes, 82 plate-cultures, 15, 84, 93 rules, 147 steam sterilizer, 54 tests for germicides, 194 Lactic acid, 119, 134 Lactose, 62 Leeuwenhoek, 9 Leprosy bacillus, 287, 294 Leptothrix, 228, 229 buccalis, 141, 229 innominata, 229 maxima buccalis, 229 Leucin, 118 Leucocytosis, 179, 180 artificial, 180 Leucomaines, 163 Ligatures, surgical preparation, 197, 214, 216, 217 Light, influence on bacteria, 116 Lime as a germicide, 205 Liquefaction of gelatin, 117 Lister, 14 Lithium-carmine, 43 Litmus-agar, 65 -milk, 67 Lockjaw (see Tetanus) Léffler’s bacillus of diphtheria, 104, 152, 277 blood-serum, 68 methylene-blue, 29 stain for flagella, 46 INDEX. Lump-jaw, 299 Lungs, bacteria of the, 140 Lustgarten’s bacillus of syphilis, 149 Lymphoid tissues, relation of bac- terla to, 140, I51 Lysins, 165, 166 inactivated, 191 reactivated, 191 Lysol, 200 MaAcropHaGEs, 178 Madura disease, Madura foot, 300 Magnifying power of objectives, 21 Mails, transmission of specimens of bacteria in, 100 Malachite-green as a germicide, 200 Malaria, 154, 203, 206 parasite of, 331 Malaria] parasite, staining of, 44, 98 Malignant edema, bacillus, 122, 268 pustule, 274 Mallein, 164, 298 Malta-fever, micrococcus of, 253 Marmorek’s antistreptococcus serum, 247 serum-medium, 68, 247 Massachusetts steam sterilizer, 53 Mastzellen, 40 Mayer’s glycerin-albumen, 38 Measles, 149, 246, 283 Meat, tubercle bacilli in, 137 Mechanical filter, 127 Medium, culture- (see Culture-me- dium) Membranous croup, 283 rhinitis, 283 Meningitis, 246, 252, 255, 259 cerebro-spinal, 255 Mercuric chloride (see Mercury bi- chloride) iodide, 198 Mercurol, 199 Mercury bichloride, 195, 196, 199 stock solution, 199 Metachromatic granules of bacteria, 110 Metastatic abscesses, 241 Metchnikoff, theory of phagocytosis, 178 vibrio of, 324 Methyl alcohol lamp in formaldehyde disinfection, 202 Methylene-blue, 28, 29, 30 as a germicide, 200 351 Methylene-blue, Loffler’s, 29 Methyl-violet as a germicide, 200 Miasmatic disease, definition, 148 Microbe, use of the word, 3 Micrococcus agilis, 220 amylovorus, 147 definition, 4, 107 gonorrhoez, 255 lanceolatus, 235, 249 melitensis, 253 of sputum septicemia, 249 Pasteuri, 249 pneumoniz croupose, 249 pyogenes tenuis, 235, 253 tetragenus, 235, 248 ures, 220 Micromillimeter, 21 Micron, p, 21 Microphages, 178 Microscope, 21 Microscopic examination of teria, 21 Microtome, 38 Miliary tubercle, 291 tuberculosis, 292 Milk as a culture-medium, 67 bacteria of, 123, 153 number of bacteria in, 135, 136 of lime, 205 pasteurization, 55, 136 pathogenic bacteria in, 134 -poisoning, 135 samples of, 97 staining bacteria in, 138 sterilization in infant feeding, 136 tubercle bacilli in, 137, 138 Miller’s spirillum, 326 Milzbrand (see Anthrax) Mixed infection, 158 Moisture, effect on growth of bac- teria, 115 Mosquitoes as carriers of infectious disease, 154, 333, 336 destruction of, 203, 206 Motility of bacteria, 24, 113 Moulds, 103, 124, 229 cultivation, 69 Mouth, bacteria, 141, 226, 229, 251, 259 Movement, Brownian, 24 Mucor mucedo, 231 Mucous membranes, bacteria of, 140 14 Multiplication of bacteria, 111 Mumps, 149 bac- 352 Mustard as a deodorizer, 206 Mycelium, 232 Mycetoma, 300 NASAL cavity, bacteria of, 141, 259 Natural immunity, 170 Neisser’s gonococcus, 255 stain for diphtheria bacilli, 277 Neutral red.in culture-media, 65, 302, 311, 314 Neutralization of culture-media, 59, 60, 61 Nitrate of silver, 199 Nitrifying bacteria, 119, 122 Nitrogen fixation by bacteria, 123 liberation by bacteria, 119 Nitroso-indol reaction, 119 Noma, 229 Non-pathogenic bacteria, 220 definition, 110 Normal solutions, 61 Nose-piece, 18 Novy’s method for anaérobes, 82 Nucleins, 199 Number of bacteria in feces, 144 milk, 136 soil, 122 water, 128 of species of bacteria, 220 Nutrient agar-agar, 64 bouillon, 59 gelatin, 62 Nutrition of bacteria, 114 OBERMEIER’S spirillum, 327, 328 Objectives, 18 Ocular, 18 Odors developed by bacteria, 119 from water, 126 Oese, 22 Oidium lactis, 230 Oil, aniline, 29, 41 cedar-wodd, 20 culture-media containing, 264 -immersion objective, 19, 20 kerosene, 206 Oils, essential, as germicides, 206 Opsonin, 181 Osteomyelitis, 243, 309 Otomycosis, 232 Ovum, bacteria conveyed in, 150 Oxalic acid, 211 Oxygen, relation of bacteria to, 115 INDEX. Oysters, typhoid fever conveyed by, 139 Ozena bacillus, 259 Ozone in purifying water, 128 PARACOLON bacillus, 313 Paraffin imbedding, 37 Paraform or paraformaldehyde, 201 Parasite, definition, 105 Paratyphoid bacillus, 313 Parietti’s method for examination of water, 132 Park, Roswell, method for disinfect- ing hands, 211 Park, W. H., method for cultivating anaérobes, 82 Passive immunity, 177 Pasteur, 13, 16 Pasteur-Chamberland filter, 57, 128 Pasteurization, 55, 136 Pathogenic bacteria, definition, 110 Pear-blight, 6, 147 Penicillium glaucum, 230 Peptone, 59, 118 Dunham, 67 solution, concentrated, 324 Peptonizing ferments formed by bacteria, 117 Pericarditis, 239, 244, 246, 253 Periostitis, 309 Peritonitis, 239, 243, 245, 261. 263, 313 Perlsucht, 289 Permanganate of potassium, 206, 211 Peroxide of hydrogen, 205 Petri dishes, 86 Petroleum for destroying insects, .206 Pfeiffer’s capsule bacillus, 259 reaction for cholera spirillum (Pfeiffer’s phenomenon), 192, : 320 Phagocytosis, 176, 178, 233, 335 Phenol (see also Carbolic acid), 118 Phenolphthalein, 60 Phosphorescence of bacteria, 117, 224 Picric acid, 28 Piorkowski’s culture-medium, 303 Piroplasma, 154. Placenta, bacteria through, 150 Plague, bubonic, bacillus of, 263 Plants, diseases of, 6, 147 Plasmodium of malaria, 331 staining of, 43, 98 transmitted INDEX, Plasmolysis, 110 Plate-cultures, 84 Platinum wire, 22 rules for use, 22, 73 Pleuritis, 239, 244, 246, 252 Pleuro-pneumonia of cattle, 150 Plugs, cotton, for tubes, etc., 70, 80 Pneumococcus of Frankel (see also Micrococcus lanceolatus), 239, 249 Pneumonia, broncho-, 239, 246, 258, 261, 301, 308 croupous, 239, 251, 259 diagnosis, 251 Pneumonomycosis, 232 Poisoning by food, 134, 139 Porcelain filter, 57 Post-mortems, disinfection at, 95, 96, 208 Post-office rules for mailing speci- mens of bacteria, 100 Potassium permanganate, 206, 211 Potato as a culture-medium, 66 bacillus, 83, 102, 112, 135, 223 invisible growth on, 303 Precipitins, 167 for albumen, 167 for bacteria, 168 Predisposition to infection, 155 Products, bacterial, 117, 134, 139, 161 Propionic acid, 119 Protargol, 200 Protective inoculation, 172 for anthrax, 172, 274 for Asiatic cholera, 321 for black-leg of cattle, 172 for bubonic plague, 173, 265 for erysipelas of swine, 172 for fowl-cholera, 172 for hydrophobia or rabies, 173 for small-pox, 10, 171 for tuberculosis, 294 for typhoid fever, 310 Proteus mirabilis, 262 vulgaris, 262 Zenkeri, 262 Protoxoid, 189 Protoxonoid, 189 Protozoa, pathogenic, 16, 154, 329 Pseudo-diphtheria bacillus, 281 -gonococcus, 256 -membranous inflammations 246, 252, 280, 283, 315 -pneumococcus, 253 -tuberculosis, 294 3° 353 Ptomaine poisoning, £39 Ptomaines, 162 Puerperal fever, 11, 245, 283 Pure cultures, 15, 84, 91 Pus, blue, 261 cells, 234 formation, 234 green, 261 samples of, 97, 99 Putrefaction, 13, 120 Pyemia, 241 Pyocyanin, 164, 261 Pyogenic bacteria, 235, 237 Pyoktanin, 200 Pyosalpinx, 258 Pyrogallic acid for cultivating anaé- tobes, 79 Pyroxylin, 37 QUARANTINE, g RABIES, 149, 173 diagnosis of, 175 Pasteur treatment for, 174 virus fixe of, 174 Racial immunity, 170 predisposition to infection, 156 Rats, acid-proof bacilli of, 294 relation to bubonic plague, 265 Rauschbrand, 172 Ray-fungus of actinomycosis, 235, 298 Reactions of culture-media, 59, 61, 63, 115 Receptor, 183 first-order, 184 second order, 185 third order, 186, 187 Recovery from infectious disease, 160, 170, 177 Reichert’s gas-regulator, 77 Relapsing fever, spirillum, 328 Rheumatic fever, 149, 240 Rheumatism, 149, 240 Rhinoscleroma, bacillus, 259 Ricin, 164, 177 Ripening of cream, 137 ‘Robin, 164 Roll-tubes of Esmarch, 88 Rooms, disinfection, 201, 203, 204 Root-tubercle organisms, 123 Rosolic acid, 67 Rouget, 172 354 Rubber caps for culture-tubes, 74, 79 gloves, 212 stoppers for culture-tubes, 74, 79; 80 Rules for students, 86, 94, 95, 104 of Koch, 147 of post-office, 100 SABOURAUD’S culture-medium, 69 Saccharomyces cerevisiz, 230 Saccharose, 62 Salt-agar, 264 Sanarelli’s bacillus of yellow fever, 149 Sand filter, 127 Sapremia, 160 Saprophyte, definition, 109, 146 Sarcina, 108, 221 pulmonum, 221 ventriculi, 144, 221 Sarcoma, toxins of streptococcus for, 248 Scarlet fever, 149, 246, 247, 283 Schatz’s method for disinfecting hands, 211 Schizomycetes, definition, 3 Schultz’s method for neutralizing culture-media, 61 Schweinerothlauf, 172 Scrofula, 291 Sealing culture-tubes, 79 Secondary infection, 159, 238 Section-cutting, 39 Sections, staining bacteria in, 39 carmine, 43 Gram’s method, 41 hematoxylin, 43 tubercle bacilli, 42 Weigert method, 41 Sedgwick’s test for germicides, 196 -Tucker aérobioscope, 125 Self-purification of water, 126 Semen, transmission bacteria by, 150 Semmelweis, 11 Separator for milk, 136 Septicemia, 160 Serum (see Blood-serum) -test for typhoid fever, 166, 306 Shiga’s bacillus of dysentery, 138, 314 Side-chain theory of immunity, 182 Silk threads in testing germicides, 194 surgical preparation, 217 Silkworm gut, surgical preparation, 217 INDEX. Silver, germicidal power of, 217 nitrate, 199 wire in surgery, 217 Sinuses, cranial, bacteria of, 140 Size of bacteria, 4, 110 Skatol, 118 Skin, bacteria of, 140 disinfection, 141, 21T Sleeping sickness, 337 Slides, forceps for, 26 glass, 27 Small-pox, 171, 334 inoculation of, 10 vaccination for, 171 Smear-culture, 74 preparations, 25 Smegma bacilli, 143 Snake-venom, 164 Sodium hydroxide, 60, 80 Soft chancre, bacillus of, 258 Soil, bacteria of, 122, 153 Solutions, normal, 61 Species of bacteria, 107 Spirilla in the mouth, 141, 220, 226, 326 in water, 102, 131, 226, 227, 327 Spirillum, definition, 4, 107, 109 dentium, 226 of Asiatic cholera, 126, 131, 134, 144, 316, 317 of Deneke, 326 of Finkler and Prior, 325 of Metchnikoff, 325 of Miller, 326 of Obermeier, 327, 328 of Vincent, 228 plicatile, 227 relapsing fever, 327, 328 rubrum, 226 rugula, 226 sputigenum, 226 tyrogenum, 326 undula, 227 volutans, 227 Spirocheta, definition, 109 dentium, 226 Obermeieri, 327, 328 of syphilis, 328 pallida, 328 plicatile, 227 refringens, 328 Splenic fever (see Anthrax) puncture in typhoid fever, 308 Sponges, surgical preparation of, 217 Spontaneous generation, 3, 13 INDEX. Spores, 3, 14, 111 arthro-, 112 endogenous, 112 of moulds, 232 of the malarial parasite, 331, 333 resistance to heat, etc., 112 staining, 44 Sporotricha or sporothrix, 232 Sputum, collection, 33, 97 disinfection, 33, 207 staining, 32, 97, 287 Stab-culture, 72 Staining, 27 bacteria in tissues, 37, 40 blood, 43 capsules, 45 diphtheria bacillus, 277 flagella, 46 gonococcus, 256 Gram’s method, 30, 41 malarial parasite, 44, 98 sections, 39 spores, 44 tubercle bacillus, 32 in milk, 32, 138 in sputum, 33, 97 in tissue, 43 a growth of plague bacillus, 264 Staphylococcus cereus albus, 235 flavus, 235 definition, 107 epidermidis albus, 137, 141, 235, 249 pyogenes albus, 235, 244 aureus (see also Suppura- tion), 137, 235, 239, 240, 241, 243 citreus, 235 Steam sterilization, 210 Stegomyia, 154, 336 Sterilization, 51, 210 after autopsies, 94, 95, 208 by boiling, 51, 128, 210, 213 by filtration, 57,'128 by steam, 51, 210 by the autoclave, 55 by the naked flame, 49 fractional, 51 hot-air, 49 intermittent, 51 of blood-serum, 55, 69 of culture-media, 51, 59, 63, 68, 7t 355 Sterilization of cultures, 86, 104 of dressings, 217 of glassware, 50 of gloves, rubber, 212 of hands, 211 of instruments, 213 of ligatures, 215 of milk in infant feeding, 135 of test-tubes, 70 of water, 127 steam, 51, 210 Sterilizer, Arnold, 52 hot-air, 50 Koch, 54 Massachusetts, 53 steam, 52 Sternberg’s bulbs, 99 determination thermal point of bacteria, 114 tests for germicides, 194 Stewart’s forceps, 26 Stick-culture (see Stab-culture) Stitch-abscesses, 244 Stoddart’s culture-medium, 303 Stomach, bacteria of, 143 Stools, disinfection, 207 Storage of water, 127 Straus’s method for diagnosis of glanders, 297 Streptococcus brevis, 244 definition, 107 lanceolatus, 249 longus, 244 MUCOSUS, 253 of erysipelas, 235, 248 pyogenes (see also Suppuration), 164, 235, 239; 244 serum, 247 Streptothrix, 228, 301 actinomyces, 298 cuniculi, 229 Stropping knives, 39 Substance sensibilisairice, 191 Sugar-free bouillon, 63 Sugars in culture-media, 63, 65 Sulphur, use in disinfection, 203, 209 Sunlight, influence on bacteria, 116 Suppuration, 233 Surgical disinfection, 210, 218 infection, 156, 236 Surra, 336 - Swarming islands, 262 Swine erysipelas, 172 Symptomatic anthrax, 172 Syntoxoid, 189 death- 356 Syntoxonoid, 189 Syphilis, 149, 150 spirochete in, 328 Systematic study of species of bac- teria, 101 TEACHING bacteriology, suggestions for, 101, 102, 103, 104, 105 Teeth, bacteria of, 142 caries of, 142 Tendons, animal, as ligatures, 197, 214 Terminal infections, 158 Test-tubes, 70 inoculation of, 72 manner of holding, 73 plugs for, 80, 82 sealing of, 79 sterilization, 70 Tetanus antitoxin, 176, 271 bacillus, 84, 122, 269 toxin, 162, 163, 164, 177, 187, 270 Tetrad, definition, 108 Texas fever, 154 Thermal death-point of bacteria, de- termination, 114 Thermophilic bacteria, 114 Thermostat (see Gas-regulator) Thiothrix, 228 Thrush, 232 Thymol, 97 Tinea favosa, 232 trichophytina, 232 Tissues, fixation and hardening, 37 staining bacteria in, 36, 40 Titration of culture-media, 61 Toxemia, 159 Toxin, definition, 163 endo-, 176 Toxins, 118, 164, 176 extracellular, 162, 176 intracellular, 162, 176 necrosis produced by, 164 of diphtheria, 162, 164, 177, 187, 282 of tetanus, 162, 163, 164, 177, 187, 270 spectra of, 188, 189 Toxoid, 189 Toxon, 189 Toxonoid, 189 Toxophore, 183 Trichophyton, cultivation, 69 Trypanosome, 336 INDEX. Tsetse-fly disease, 154, 337 Tubercle bacillus, 150, 286 in butter, 137 in meat, 137 in milk, 138 gray, miliary, yellow, 291 structure, 290 staining, 32, 42, 286 in milk, 138 in sputum, 33, 34, 97) 289 in sections of tissues, 42 Tuberculin, 164, 293 R, 293 Tuberculosis, 290 acute miliary, 292 bovine, 137, 289, 292, 293 diagnosis, 33, 286, 292, 293 frequency, 137, 291 immunity, 294 of birds, 294 organs affected by, 292 pseudo-, 294 spread of, in the body, 291 Typhoid fever bacillus, 126, 131, 133, 134, 150, 30% contrasted with colon ba- cillus, 311 fever diagnosis, 303, 307 serum-test, 166, 306 Typhus fever, 149 Tyrosin, 118 Tyrotoxicon, 135 ULTRAMICROSCOPIC organisms, IIo, 150 Unit, immunity, 284 Urea, decomposition by bacteria, 118 Urethra, bacteria, 142 Urethritis, gonorrheal, 257 Urinary bladder, bacteria of (see also cystitis), 140 Urine, disinfection, 207 samples, 97 -serum-agar, 257 typhoid bacilli in, 308 Uterus, bacteria of, 140 VACCINATION, 10, 171 and tetanus, 270 Vaccinia, parasites in, 335 Vagina, bacteria of, 142 INDEX. Vaginitis, gonorrheal, 257 Van Ermengem’s method for staining flagella, 47 Vegetative forms of bacteria, 111 Venom of snakes, 164 Vibrio aquatilis, 327 Berolinensis, 326 definition, 109 Metchnikovii, 324 proteus, 325 tugula, 229 Schuylkiliensis, 327 Vibrion septique, 268 Villemin, 12 Vincent, bacillus of, 228 Vinegar, bacteria in, 5 Violet, gentian-, 28, 30 methyl-, 200 Virulence of bacteria, 115, 157 WarmTH, effect on growth of bacteria, 114 Water, bacillus coli communis in, 132 typhosus, 131 bacteria of, 126 conveyed by, 125, 153 filtration, 127 ground-, 125 infections carried by, 125 number of bacteria in, 128 pathogenic bacteria in, 126, 131 purification by ozone, 128 samples of, 97, 128 self-purification, 126 spirilla in, 102, 226, 327 sterilization of, 128 storage of, 127 Watery solutions of aniline dyes, 28 Weigert’s stain for fibrin and bac- teria, 41 357 Weir’s method for disinfecting hands, 212 Welch’s stain for capsules, 45 Whooping-cough, 149 Widal’s serum-test for typhoid fever, 306 Wire baskets, 70 platinum, 22 silver, 217 Wolffhiigel plate, 129 Wool-sorters’ disease, 124, 153, 274 Wounds, infected, 219 infection of, 156, 237 irrigation of, 219 Wright’s stain for blood, 43 method for anaérobes, 80 Wurtz’s culture-medium, 302 Wurzelbacillus, 224 XEROSIS bacillus, 281 X-rays, 116 Xylol, 37, 40, 41 YEASTS, 103, 124, 144, 229 Yellow fever, 149, 150, 154, 203, 206, 336 tubercle, 291 Yersin’s serum for plague, 265 Z1EBL’s carbol-fuchsin, 34 Zinc chloride, 207 sulphate, 207 Zodgloea, 111 Zymophore group, 186 Zymotic group, 186 iets Ritter: boc tiey 3 Peeecpces boven set NOL ST thet ST ede ot 1a Paty F eres ot be WP ta) Fab e be Se ts Te ws oe