a er ee 2S ee eS ty Ate OA ON mane 00, atm = Nae RE ea eva ines wep fee PP ts. PAP TR OE IS BT Beet 28 ee at ae fil OH - SEs eo Os RP ores Pe Ee: Aieeot » -, £7, 4 5 ne FR et. ie PR OR POD, = nm gre ao 4 —s Ra Oe hae ree a Ps : SA ee the ee : a LO Oe Lame te me tee aay 33 SRS, Ag mae ae ee ae | SEP CRE SES THE UNIVERSITY OF ILLINOIS LIBRARY O16.07 $i 54 rd 26 1934 etry tt sh tbtareialese gst Synnn y ‘ e235 eens Hh , sae tit aeay redoliilisieieh enget he eee BEBE) agehay ore Sen gegen one poeemtanee ° . bit} y ae CLINICAL DIAGNOSIS BY MEANS OF MICROSCOPIC AND CHEMICAL METHODS FOR STUDENTS, HOSPITAL PHYSICIANS, AND PRACTITIONERS swe). CHARLES E. SIMON, B.A., M.D. FORMERLY OF THE RESIDENT STAFF OF THE JOHNS HOPKINS HOSPITAL; PROFESSOR OF CLINICAL PATHOLOGY AT THE BALTIMORE MEDICAL COLLEGE 5; CLINICAL MICROSCOPIST TO THE UNION PROTESTANT INFIRMARY. SIXTH EDITION, THOROUGHLY REVISED ILLUSTRATED WITH 177 ENGRAVINGS AND 24 PLATES IN COLORS LEA BROTHERS & CO. PHILADELPHIA AND NEW YORK OOF, aa etost cf € . a c e« ta c CoS £ b so c 6-.t.e' 0 od . © .¢ < c < CSC Ce mA € as c € a" 8 ¢ er Coo o.« 2 jan f yee ae € y) € Chee ny a 8 . cc C8 c ec ¢ b. GG) Oe ° ove Cra ec te S' ri @ « c cs Entered according to Act of Congress, in the year 1907, by LEA BROTHERS & CO. in the Office of the Librarian of Congress. All rights reserved. MY WIFE FAITHFULLY AIDED IN ITS PREPARATION oe =a 7 * ox ‘THIS EDITION ALSO nah Is a eee AFFECTIONATELY DEDICATED > PREFACE TO SIXTH EDITION. IN preparing the sixth edition of the Clinical Diagnosis the author was confronted with an important problem. A great deal of new mate- rial had to be introduced, but the size of the volume, which had steadily grown within the ten years of its existence, could not be exceeded. It was accordingly necessary to go over the entire work carefully and to cut out everything that was not of clearly practical value, to con- dense, and to rewrite. ‘The amount of labor involved was consider- able, but the object has been, it is hoped, satisfactorily achieved. The chapter on the Blood has been further enlarged and brought thoroughly to date. Every page in the work has undergone a radical review. A new chapter on the Opsonins has been introduced, in which the subject-matter has been conservatively and, it is hoped, fairly pre- sented; full details are given regarding the technical portion of the subject, in which the writer’s experience as a pioneer worker may prove of value. ‘Two appendices have been added. ‘The first deals with the prepa- ration of culture media, and may prove of service to teachers who use the book not only asa text-book of clinical diagnosis, but also as a guide to the student’s work in bacteriology. ‘The second represents an out- line of a course in clinical laboratory methods, and is presented at the request of teachers in clinical microscopy in many of our medical schools, in which the subject is steadily growing in importance. ‘The “course” is based upon the work which the writer has conducted for post-graduates during the past ten years in his own laboratory, and is designed to be thoroughly practical and comprehensive. Numerous illustrations in black and white, as well as a number of colored plates, mostly from the brush of Mrs. Simon, have been added. ‘The author wishes to thank the medical profession for the continued favorable reception of the Diagnosis, the pioneer work in America, and trusts that the present edition also will serve its purpose as a trust- worthy guide to the medical student, general practitioner, and the laboratory research worker. CHARLES E. SIMON. 1302 Mapison AVE., BALTIMORE, 1907. ey7 2 Tae ae ech CONTENTS. CHALE eRe wl. THE BLOOD. General considerations General characteristics of the blood . color odor. specific oravity : determination according to ‘Hammerschlag ‘ determination according to Schmaltz and “‘Peiper indirect estimation of the hemoglobin reaction estimation of the alkalinity according to Lowy . estimation of the alkalinity according to Engel . estimation of the alkalinity according to Dare Chemical examination of the blood . Ro dae general chemistry of the blood . coagulation of the blood blood pigments hemoglobin and oxyhemoglobin hemoglobinemia , : carbon monoxide hemoglobin ; nitric oxide hemoglobin as hydrogen sulphide hemoglobin carbon dioxide hemoglobin . hematin tee oe hemin methemoglobin hematoidin : hematoporphyrin the proteids of the blood the carbohydrates sugar . estimation of the sugar in the blood. Williamson’s diabetic blood test . glycogen . 3 cellulose urea : uremia ammonia ; uric acid and xanthin bases fat and fatty acids lactic acid... biliary constituents acetone. cholin Microscopic examination of the blood the red corpuscles. variations in the size of the red corpuscles variations in the form of the red corpuscles . viii CONTENTS Microscopic examination of the blood—Continued. variations in the color of the red corpuscles and color index variations in the number of the red corpuscles . behavior toward aniline dyes (poly chront ayo ee granular degeneration ne te : Cabot’s ring bodies . Ehrlich’s hemoglobinemic Innenkérper nucleated red corpuscles normoblasts . Thais megaloblasts . the leukocy tes. : general differentiation of the various forms of leukocytes ee differentiation of the leukocytes according to their behavior toward aniline dyes HL 5 ae Se iat dene a ae the small mononuclear leukoey tes the large mononuclear leukocytes the polynuclear neutrophilic leukocytes the polynuclear eosinophilic leukocytes . the mast-cells ; the myelocytes . Tiirck’s irritation forms (phlogoey tes) iodophilia leukocytosis ; f polynuclear neutrophilic hyperleukocytosis ; polynuclear neutrophilic hypoleukocytosis neutrophilic karyomorphism . polynuclear eosinophilic hyperleukocy tosis poly nuclear eosinophilic hypoleukocytosis ymphocytosis OP. wend lymphopenia : variations in number of the large mononuclear leukocytes. variations in number of the mast-cells te aoe myelocytosis . the plaques. the dust particles of Miiller . General technique . slides and cov er-glasses the blood mount. . fixation The aniline dyes and principles of staining . Methods of staining . the eosinate of methy lene blue . Ehrlich’s triacid stain the Romanowsky methods method of Hastings method of Leishman method of Wright method of Giemsa . method of Goldhorn Demonstration of iodophilia .. Enumeration of the corpuscles of the blood method of Simon . enumeration of the leukocytes enumeration of the red cells differential leukocyte counting enumeration of the plaques The hematokrit ee Loe volume index . Estimation of hemoglobin Dare’s hemoglobinometer Fleischl’s hemoglobinometer . Gowers’ hemoglobinometer (Sahli’s ‘modification) Talquist’s method sigs < CONTENTS a LAGE Estimation of blood iron . Sy Ae, Se Se 153 Kryoscopic examination of Jusyitteaa! = nein a ie Rel man = JY Osmotic resistance of the red cells. . ee eee mR ep uueer LOT, Bacteriology and parasitology of the blo> ana tia ya Stet te Re 83158 typhoid fever... ; OS ane, Sie, eh ota iene LOO agglutination (es PEPER Os Oeste ric 2 160 paratyphoid fever ae : a tact Weigel Kis pneumonia. . hE Ce Se a A pat oe oe LOG pyogenic Bacterieniiane Mime yiatee ernest tate Fes yet 3s 81 G8 Bee OCU US CoD MCCTn aaa wan ati ore An a LO te ou L7 anthrax ey BL ew ee eee 172 acute miliary POET HIOA MMM ene aera ees BOE heh Oo EET elanders 174 influenza 174 Malta fever 174 bubonic plague 175 malaria. |, LIRR eg) ar Lik gay TESST SUN BTS SA ey he. |! es a a a a 9'/ relapsing fever Mig ih Hee ee typhus fever 190 Kala-azar . 190 syphilis. 191 spotted fever . 191 filariasis rel ek thy CAE Se Re ot 191 distomiasis (bilharziasis) TS CGN eile | Seeks OO chemistry of the saliva . aR Page rth eee eh aL microscopic examination of tiensali vate tenths soar oae ete 1 OO pathological alterations . Paha bie a toe tbe Sie ar ee oh ks, te Oe Bie incisenses OMtne mOute Wo 8 Ss a Pha ee eee 202 Bape CuomiOmunednNOUth ste rea - Pe tell a re 202 actinomycosis. it Se a Sed Cale Yj Pane At ie oo Parrumintanatitisie wee leet crts ne cee ep aye, 7202 MCAT eriveen Oia tic menente tll Mow cr tego i way Tho Gy Se 202 POROEPUC DISS LOMO duicn mnn enw Aen tat Vn tgy .) Bo eigs 2 )), »« e2O02 tc oe CoP Pnr a i Mr ee a TT Nl at ee, 202 Patter oe. ee ee ere er tee A ath eee, aes 208 Coating of the tongue Pe ee A os a eee os ey Stig ee 200 Coating of the tonsils . Peat hhh rae ernie pears De e740. pharyngomycosis leptothrica Ewe ie ee ee): adie we: 12209 tonsillitis LA pie eRe Py coal sg RCE eRe AN Vincent’s angina . MA MeN ee i ek drag COS a ieee Memeo ee i aig a.” Ly 205 eGo ee eee LP i 8) ae ee i 208 Peach ev Cte Ree ee ol eee ae ee Co 208 Oils /. "al 224 NE Sg ema 00 Oe Ie THE GASTRIC JUICE AND THE GASTRIC CONTENTS. SeMmeereWOU On Casbric iilicegem. .. AOS oes a. we a.) 2209 Test meals . ee ee ager hae ere oe LD the test breakfast of Ewald and Boas . ee ES Aa, ie eee 4 OE ee CONTENTS Tests meals—Continued. the test breakfast of Boas the test dinner of Riegel the double test meal of Salzer The stomach tube... contra-indications to the use of the tube introduction of the tube 4 General characteristics of the gastric juice : amount. Chemical examination of the gastric juice the acidity of the gastric Juice .. ; determination of the acidity of the eastric juice ; the amount of free hydrochloric acid ; euchlorhydria 2 ee hypochlorhydria anachlorhydria hyperchlorhydria test for free acids : test for free hy drochloric acid the dimethyl-amino- azo-benzol test the phloroglucin-vanillin test the resorcin test the tropeolin test. the combined hydrochloric acid. quantitative estimation of the hydrochloric acid . Topfer’s method Me 5) Pk ess deficit of hydrochloric acid Sahli’s method .. Martius and Liittke’s method . Leo’s method the ferments of the gastric juice and their. zymogens. pepsin and pepsinogen . ae ena tests for pepsin and pepsinogen quantitative estimation : chymosin and chymosinogen ; tests for chymosin and chymosinogen quantitative estimation ° analysis of the products of albuminous digestion tests for the products of carbohydrate digestion . lactic acid . : mode of formation and clinical significance ; tests for lactic acid . Kelling’s test Uffelmann’s test Strauss’ test Vournaso’s test Boas’ test quantitative estimation of lactic acid according to Boas’ method the fatty acids . 2 oe eee Pe urohematin 506 uroroseinogen —.. » ee hae aa, BLOW pathological pigments and chromogens 5,» bly Ay Be nee ee ie gL blood pigments ei aein 508 hematin S eet sg cote OUD urorubrohematin and urofuscohematin . . . . . 508 urohematoporphyrin..)-«.- <)° -*- 3h ee ues biliary pigments oie 510 Smith’s test 512 Huppert’s test. A eee ee al te 2 LE. Gmelin’s test (as modified by Rosenbach). BY, YY y que fete Gmelin’s test . ee ore «Mis OE ee ge ae em oe biliary acids 512 cholesterin pte a LN Sle, rR De eee oe pathological urobilin 7°... s “wh 5 ae) a melanin and pe ene er Va a ES ee en phenol . 6 3: LW “Eels, ae en be alkapton (homogentisinie acid) . Logo)! 6 is Jee ial cer en blue urines. Rage tee) bie 521 green urines 521 pigments referable to drugs 521 Ehrlich’s diazo reaction . 522 benzaldehyde reaction . 526 I CELODEG ee Ses a MENS 2288 527 tests for acetone 529 Legal’s test . 529 Lieben’s test 529 Frommer’s test 530 Dennigeé’s test 530 quantitative estimation. 531 Diacetic acid 532 Gerhardt’s test 532 Arnold’s test 533 Oxybutyric acid 533 estimation . 534 Crotonic acid 535 Lactic acid . 535 Oxyamy edalic acid. 536 Volatile fatty acids 537 Amino-acids 538 Fat 539 Ferments 540 Gases 541 Ptomains 542 isolation of diamins RET eters 543 Kryoscopic examination of the uring "ys a Son) ee ee Sediments ee Sie OE ye Aa ee ae ees Microscopic examination of the urine. =. . % -.) 2) ac eee ees non-organized sediments : oats oa Ss Sete eee sediments occurring in acid Urines >; |: 5 ie eet uri¢e acid. ae 549 amorphous urates . 550 calcium oxalate UEC ptr ees Oy AE SS monocalcium phosphate arene ret AF I leh hippuric acid Liege 552 calcium eae 553 cystin 553 leucin and tyrosin . 554 xanthin . 556 soaps of lime and MAGNESIA. 64. SS ee ea CONTENTS Microscopic examination of the urine—Continued. bilirubin and hematoidin fat ; sediments occurring i in alkaline urines basic phosphate of calcium and magnesium neutral calcium phosphate magnesium phosphate ammoniomagnesium phosphate calcium carbonate . ammonium urate indigo organized constituents of urinary sediments epithelial cells leukocytes red blood corpuscles tube casts examination true casts hyaline casts En granular casts waxy casts pseudocasts eylindroids : clinical significance of tube casts spermatozoa. parasites vegetable parasites animal parasites tumor particles. foreign bodies . Pik eRe Ven TRANSUDATES AND EXUDATES. Transudates general characteristics specific gravity : lemistry of transudates. microscopic examination. Exudates serous exudates technique bacteriological examination of inoscopy chemistry of PUM MeR ee S general characteristics of pus chemistry of pus. microscopic examination of pus leukocytes . giant corpuscles detritus . red blood corpuscles pathogenic vegetable parasites protozoa. Je ae , vermes crystals technique gonorrheal pus the gonococcus putrid exudates chylous and chyloid exudates XVII ‘PAGE eae CONTENTS Exudates—Continued. ) PAGE syphilitie- material ** 700." aa SS; cee Gia. i ee oe Spirocheete-pallida:<)° -5° eee ee CHAT Pi ie rlex: THE CEREBROSPINAL FLUID. Amount bo ee pt See alee Sg eras oe SO 2 oe Appearance POE ee Tre bh aie ee eee i a Specific gravity 97.) 0. See, SC ee Reaction... a er renee VM Mer cutee ty oe tie dae Chemical composition re, Tae emer en eR a ee ei B Microscopic‘examination .°...) vad. Wo) ce Bes ee Cytology po oe5) <4 0 9 Ee ey ee Bacteriology. «2 sa § a ae a ee ee a Toxicity. eR Ba re CHAPTER X. THE EXAMINATION OF CYSTIC CONTENTS. Cysts of the ovaries and their SP Ren ane: rane oe ae a A ce ye a! Rs (Feil?) Hydatidiecysts 2. 0e. oan eee re aR rt hy VER AR. Lie Hydronephrosis. °°. < “(ie (eg oat ee a ae Pancreatic.cysts .» >. SBS age fs ee EBS Be 5h ead ban Bay a 68 THE SEMEN. General characteristics: 56... Spe oe > oe ee et Chemistry of the semen . eT 2 Soe oo" os ae arose oes Microscopic examination of the semen). io hh ee ee Pathology of the semen . a? ry ee ie ie IEG oll er rr oe The recognition of semen in stains .....c OMe ees. CIVAIE TE ives 13 VAGINAL DISCHARGES. Bacteriology. i mr eenrrde he! Mn by ee PANY Vaginal blennorrhea “2 3.) v 5 se 2 pee ae Menstruation “24.80 a I EL ee ee The lochia .. rrr mr pe ee ke, es, Vulvitis and vaginitis re me Me RS ee! Membranous dysmenorrhea)... 1's, 2) 7 at Cancer eso ap Se OI Gonorrhea oS 8 8 Se a a ee Abortion 2220 26.8 a COHRACP TF Rallis THE SECRETION OF THE MAMMARY GLANDS. Lhe secretion of. milk in the'newly born". 2)? fei.) eee Colostrum . Pee A Ne a) ON The secretion of milk i in the adult female . 5 ae Go OG ee a Human ‘milk! och ee ee 2 a me The milk in.disease’ 4.5/0) sah te ee ee . , 634 nation of the fat . imation of lactose . hn nique : 5 ~ opsonic index . a reparation of culture media fe 2 Lectures _ Laboratory exercises . ales of the specific gravity OAS PR OX LV: THE OPSONINS. fd 25) 2a they pa BD A. B. ; oO iting of a course in clinical Spicroacopy 637 637 639 642 643 _ 649 654 654 656 a} > ’ >> 9 . o..(<4U 26.78 a ’ oo ‘ A CLINICAL DIAGNOSTS: reas el Behe i THE BLOOD. GENERAL CONSIDERATIONS. Ir blood is allowed to flow directly from an artery into a vessel surrounded by a freezing mixture, and containing one-seventh its volume of a saturated solution of sodium sulphate, or a 25 per cent. solution of magnesium sulphate (1 volume to 4 volumes of blood), it will be observed that after some time a sediment, pre- senting the color of arterial blood, has formed at the bottom, which is covered by a layer of clear, straw-colored fluid—the blood plasma. Upon microscopic examination the sediment will be seen to contain: (a) Numerous homogeneous, non-nucleated, circular, biconcave disks. ‘These measure on an average 7.5 4 in diameter, and are of a faint greenish-yellow color when viewed through the microscope, while en masse they present the color of arterial blood—the erythro- cytes or red corpuscles of the blood. (b) Roundish or irregularly shaped nucleated cells which are for the most part granular and far less numerous than the red corpus- cles, and devoid of coloring matter—the leukocytes, colorless or white corpuscles of the blood. (c) Minute colorless disks, measuring less than one-half the diam- eter of a red corpuscle—the so-called blood plaques, or blood plates of Bizzozero. GENERAL CHARACTERISTICS OF THE BLOOD. Color.—Chemical examination of the blood shows that its color is referable to the presence of an albuminous, iron- -containing substance —hemoglobin—in the bodies of the red corpuscles, which is character- ized by its great avidity for oxygen, and forms a compound therewith, known as oxy hemoglobin. The relatively larger amount of the latter encountered in ithe arteries, as compared with the veins, causes the difference in the appearance of arterial and venous iene the 2 i8 THE BLOOD former presenting a bright scarlet-red, the latter a dark-bluish color. A bright cherry-red ecloris noted in poisoning with carbon monoxide, while a brownish-red or chocolate color is observed in poisoning with potassium chlorate, anilin, hydrocyanic acid, and nitrobenzol. A milky appearance is frequently seen in well-marked leukemia. In chiorosis and hydremic conditions, as would be expected, the blood is pale and watery. Odor.——The peculiar odor of the blood, which varies in different animals, the halitus sanguinis of the ancients, is due to the presence of certain volatile fatty acids, and may be rendered more distinct by the addition of concentrated sulphuric acid. Specific Gravity.—The specific gravity of the blood in healthy adults varies between 1.058 and 1.062, being higher on an average in men, 1.059, than in women, 1.056, and children—boys 1.052, girls 1.050. Generally speaking, it is proportionate to the amount of hemoglobin and the volume of red corpuscles. It is diminished by fasting, the ingestion of solids and liquids, gentle exercise, preg- nancy, etc. It depends, moreover, upon the bloodvessel from which the specimen is taken, being higher in venous than in arterial blood. Under pathological conditions the specific gravity may vary be- tween 1.025 and 1.083. In nephritis, chlorosis, the anemias in general, and in cachectic conditions (carcinoma of the stomach, etc.) it may diminish to 1.031. In phthisis it is diminished in the third stage val .040 to 1.042), and in the first stage (1.049) in those patients in whom the onset has been very gradual. In the second stage normal figures are obtained (1.058 to 1.060), corresponding to the relatively high percentage of hemoglobin (90 to 95 per cent.) which is then noted, and which is referable no doubt to a concentration of the blood. An increased specific gravity is met with in febrile diseases (typhoid fever, 1.057 to 1.063), conditions associated with pronounced cyanosis (emphysema, fatty heart, uncompensated val- vular disease, 1.054 to 1.068), and obstructive jaundice, 1.062. ‘The highest values have been found in enterogenous cyanosis, 1.067 to 1.083. Method of Determining the Specific Gravity of the Blood. Hammerschlag’s Method.—A carefully dried cylinder, measuring about 10 cm. in height, is partly filled with a mixture of chloroform (sp. gr. 1.526) and benzol (sp. gr. 0.889), having a specific gravity of 1.050 to 1.060. Into this solution a drop of blood is allowed to fall directly from the finger, pressure being avoided, and care taken that the drop does not come in contact with the walls of the vessel. The drop should not be too large, as otherwise it will separate into droplets, giving rise to inaccurate results. Should the drop sink to the bottom, it is apparent that the specific gravity of the mixture is lower than that of the blood, necessitating the addition of chloroform. This should be added drop by drop while the mixture is thoroughly ee GENERAL CHARACTERISTICS OF THE BLOOD 19 stirred. If, on the other hand, the drop should tend toward the surface it is best to add an amount of benzol sufficient to cause the blood to sink to the bottom, and then to bring it to the proper degree of sus- pension by the subsequent addition of chloroform. As soon as the drop remains suspended the mixture is filtered, and its specific gravity ascertained by means of an accurate areometer registered to the fourth decimal. ‘The figure obtained is the specific gravity of the blood. ‘lhe chloroform-benzol mixture may be kept indefinitely. _ With practice, results sufficiently accurate for clinical purposes may thus be obtained with an expenditure of very little time. ‘The examination should in each case be made at the same hour, as the specific gravity undergoes diurnal variations. Instead of the chloroform-benzol mixture, one of chloroform and olive oil may be employed, as suggested by Van Spanje. It has the advantage of being less volatile than the other. ‘Three parts of chloroform and one of oil give a mixture with a specific gravity of 1.056. - $chmaltz and Peiper’s Method.—Where delicate scales are avail- able the method of Schmaltz and Peiper may be employed. A capillary tube, measuring about 12 cm. in length and 1.5 mm. in width, with its ends tapering to a diameter of 0.75 mm., is filled with blood and carefully weighed. ‘The weight of the blood, divided by the weight of an equivalent volume of distilled water, indicates the specific gravity. As the result of numerous investigations it may now be regarded as an established fact, that with the exception of nephritis, circulatory disturbances, leukemia, posthemorrhagic anemia, and that resulting from inanition, the specific gravity of the blood varies directly with ‘the amount of hemoglobin and the volume of the red corpuscles. ‘A simple method is thus given by means of which hemoglobin esti- ‘mations can be made in the absence of more expensive instruments. In the following table the specific gravities, as obtained with Ham- ‘merschlag’s method, and that of Schmaltz and Peiper are given, with the corresponding amounts of hemoglobin: Specific gravity Specific gravity according to Hemoglobin. according to Hemoglobin. Hammerschlag. Schmaltz and Peiper. 1.033-1.035 . . 25-30 per cent. £0380 Site? ae. 20 per cenit. 1.035-1.038 . . 30-35 - 1.035 i * aoe BU v 1.038-1.040 . . 35-40 os 1.038 Die Woke OD Y 1.040-1.045 . . 40-45 oa 1.041 none aU .* 1.045-1.048 . . 45-55 yi UN 4, fale ma alee ae: 15% a 1.048-1.050 . . 55-65 ee LAM Sao vista OU 1.050-1.053 . . 65-70 1.048 parses het. OO a 1.053-1.055.. . 70-75 uA 1.049 he eos OU ee 1.055-1.057 . . 75-85 ra 1.051 aes 4 8- OO « 1.057-1.060 . . 85-95 i 1.052 APU Per a atl) . BOSBD fo eA te 16 ‘a 1.056 Motes ee OO . TOR CRM eS tii. 90 . POO iio aa MOU on 20 THE BLOOD LITERATURE.—Schmaltz, Deutsch. Arch. f. klin. Med., vol. xlvii, p. 145; and Deutsch. med. Woch., 1891, No. 16. Stintzing u. Gumprecht, Deutsch. Arch. f. klin. Med., vol. lili, p. 265. Siegl, Prag. med. Woch., 1892, No. 20; and Wien. med. Woch., 1891, No. 33. Hammersthlag, ibid., 1890, p. 1018; and Zeit. f. klin. Med., 1892, vol. xxii, p. 475. Schmaltz, Deutsch. Arch. f. klin. Med., 1890, vol. xlvii, p. 145; and Deutsch. med. Woch., 1891, vol. xvii, p.555. Appelbaum, Berl. klin. Woch., 1901, vol. xxxix, p. 7. Reaction.—The reaction of the blood during life, owing to the presence of disodium phosphate and sodium carbonate, is alkaline. The degree of alkalinity in healthy adults, while fasting, corresponds to about 300 to 325 mgrms. of sodium hydrate for 100 c.c. of blood (Lowy). Variations amounting to 75 mgrms. plus or minus are, however, not uncommon and in part due to unavoidable errors of technique (50 mgrms.). Generally the alkalinity of the blood is lower in women and children than in men, and is influenced by the process of digestion, exercise, etc. At the beginning of digestion, when hydrochloric acid is being freely secreted, the alkalinity of the blood increases; while later on it diminishes. Higher values are usually found during pregnancy than in the non-pregnant state. A decrease is observed following violent muscular exercise and also after the prolonged use of acids, while an increase is brought about by the ingestion of alkalies. An increase in the alkalinity of the blood occurs after a cold bath, and it is interesting to note that this is apparently associated with an increase in the bactericidal power of the blood. Under pathological conditions the alkalinity may be diminished or increased, as is shown in the table below. Unfortunately we are not able to account for these fluctuations in a satisfactory manner and the data are thus of little value. A marked decrease in diabetes may be viewed as of serious prognostic omen and as indicating acid intoxication. During diabetic coma the reaction owing to the pres- ence of large amounts of beta-oxybutyric acid may actually be acid. The supposition that in gout a diminished alkalinity exists in the intervals between attacks, and that this increases beyond the normal during the attack, has been proved unfounded. Orlowsky has recently expressed the opinion that the variations in the alkalinity of the blood which have been noted in various diseases and sometimes in one and the same disease, by various investigators working with the older methods, are referable to the varying tonicity of the blood and its varying richness in red corpuscles. Working with blood plasma Orlowsky found a marked diminution of the alkalinity in advanced uremia, in cancerous cachexia, and in — severe cases of diabetes, while in other diseases normal values or - at most but slight and exceptional variations were observed. The following table gives some of the results which have been obtained with Lowy’s method: GENERAL CHARACTERISTICS OF THE BLOOD 91 Pe TPOODNETT val ry ce Oy eG 4 227-643 Carcinoma ventricull Wee ee es a oe ta © 2H6=O35 Uleus ventriculi .. ee ee a - BO2=460 Anadeny of the stomach | i eee ea kt Cy er ODA -SOO EM URME Tee eee eas eS et. | 848-879 DR TUALiG ioc PC ee we ee ee we 8 Q1D-279 Sums PUMONals.. 9). . ke we LL, a 450-468 Ce Sete) ke es ete ooh OP we 2239-3435 CT te RP Bel Oey ee DOR ADG DINMUGTONIG Th) Muar ee pees 208-344 ETO inN soe eee ee mee eS cs “8368-465 Erysipelas . AME eo Wey le a ee wee", ~ 498 eMEUICEMOMGD 9 Gg th RCE imi ete a 270-640 ee ee eee er oe ane Ss . 263-464 TC ee eke eee West ee et 44g Leukemia .. het eRe yan ete ee oe Co BBR RSA Pernicious anemia ee ee eee a ee | , AQ2O Diabetes mellitus Ca erg ea ee 362-447 Chronic interstitial nephritis (pee ficae ok wand SLOADD Chronic parenchymatous nephritis. . . . . . . 3812-490 eee NGO VOCs, mee ile eth keen Sh as cg RPK OtO The alkalinity may be measured according to one of the following methods: Lowy’s Method.—F ive c.c. of blood, obtained from one of the superficial veins of the arm (preferably the median cephalic), are allowed to flow into a small flask provided with a long and partially graduated neck, and containing 45 c.c. of a 0.25 per cent. solution of ammonium oxalate. Coagulation is thus prevented and the blood made lake-colored—. e., the hemoglobin is dissolved from the stroma of the red corpuscles. ‘The mixture is then titrated with a ys normal solution of tartaric acid, using lacmoid paper, soaked in a concentrated solution of magnesium sulphate, as an indicator. As a normal solution of tartaric acid contains 75 grams _ to We liter, a 5; normal solution will contain 3 grams, and 1 c.c. of the ¥ normal golution will corrrespond to 0.0016 gram of sodium nes Supposing that 10 ¢.c. of the 5; normal solution were necessary to neutralize 5 c.c. of blood, the alkalinity of these 5 c.c. in terms of sodium hydrate would correspond to 0.016 gram, and the alka- linity of 100 c.c. of blood to 0.016 20=0.320 gram—. e., to 320 mgrms. Engel’s 9Method.—'lhis is essentially a modification of Loéwy’s method, and is well adapted for clinical purposes, as the amount of blood required for a single examination can readily be obtained by ordinary puncture. The blood is measured and rendered lake-colored in a specially constructed pipette (Fig. 1). ‘To this end the blood is drawn to the 0.05 ¢.c. mark and diluted with neutral distilled water, so that the volume of the mixture reaches the 5 c.c. line. After slight agi- ' Regarding the standardization of normal solutions the reader is referred to special works on quantitative analysis, 22 THE BLOOD tation the solution is placed in a small beaker and titrated with a =; normal solution of tartaric acid, from a special burette which fo accompanies the pipette. This is so constructed that each cubic centimeter is divided into twenty parts. Before and after the addition of every drop of the titrating fluid the reaction of the mix- — Wim 7 ture is tested by placing a drop upon lacmoid paper. The end > reaction is reached when the yellow drop of the blood mixture shows . a distinct red line along the margin. ‘The result is expressed in terms of milligrams of sodium hydrate per 1 ¢.c. of blood. Normally about 10 c.c. of the acid solution are employed. ‘The tartaric acid solution contains 1 gram of Merck’s crystals (crystallized reagent) to the liter, — so that 1 c.c. corresponds to 0.533: mgrm. of sodium hydrate. Fic. 1.—Engel’s alkalimeter. Supposing that 0.6 c.c. of the acid solution was required to neu- tralize the 0.05 c.c. of blood, then 12 c.c. would be necessary for 1 c.c. of blood. As 1 c.c. of the acid solution represents 0.533 mgrm. of sodium hydrate, the alkalinity of 1 c.c. of blood would correspond to 120.533—~. e., to 6.396 mgrms. Dare’s Method.—'l’his method is based upon the fact that the characteristic spectrum of oxyhemoglobin disappears at the point of exact neutralization when the blood is titrated with a dilute solu- tion of tartaric acid. The examination is made with the aid of a special instrument, the hemo-alkalimeter, which is pictured in the accompanying illustration (Fig. 2). B is a glass stopper through which passes an automatic capillary blood pipette of 20 c.mm. capacity, the exposed end of which is ground to a tapering point. ‘The stopper fits into the tube : GENERAL CHARACTERISTICS OF THE BLOOD Ae A, which has a capacity of 3 ¢.c. and is graduated in cubic cen- timeters. ‘The upper end of the tube is blown into a bulb with a minute aperture at C. A 2 c.c. dropping tube provided with a short piece of rubber tubing accompanies the instrument. To neutralize the blood a zxyo normal solution of tartaric acid is used, which should contain an amount of alco- hol sufficient to prevent the growth of bacteria, but insufficient to precipitate the albumins of the blood. ‘The reagent may be prepared by dissolving 0.075 gram of tartaric acid (Merck’s crystals; guaranteed reagent) in a small amount of distilled water, adding 20 c.c. of alcohol (93-94 per cent.), and diluting to 200 c.c. with water. For the spectroscopic examination a Browning instrument (Fig. 3) will suffice. Fic, 2.—Dare’s hemo- : F alkalimeter. Fic. 3.—Browning’s spectroscope. (Zeiss.) Mernop.—A drop of blood is obtained from the finger-tip or the lobe of the ear in the usual manner. ‘The blood pipette is filled im situ by capillary attraction, holding the instrument horizontally to the drop of blood as it emerges from the wound. With an ordinary medicine dropper filled with distilled water the blood is washed into the bottom of the tube, connecting the dropper with the pipette by means of a short piece of rubber tubing. Blood and water should just reach the zero mark, and are intimately mixed by closing the aperture in the bulb with the finger and inverting the tube several times. ‘The reagent pipette is then filled with the tartaric acid solu- tion and the rubber tubing slipped over the outer end of the blood pipette; by compressing the rubber bulb the acid solution is forced through the pipette into the test-tube, the aperture in the glass bulb being closed before the pressure is relaxed. Having done this the tube is inverted several times while still attached to the reagent pipette, taking care that this is held vertically so that the acid solu- tion does not get into the rubber bulb. ‘The tube is clamped in front 24 THE BLOOD of the spectroscope and examined for the two bands of oxyhemoglobin. (See Fig. 6.) So long as these are visible more of the acid is added, inverting the tube after each addition; as the bands become fainter one drop at a time is allowed to enter. At first this 1s rather tedious, but after several examinations have been made it will be found unneces- sary to apply the spectroscope so frequently to determine the point of neutralization, as the eye rapidly learns to recognize this by the characteristic change of color of the blood mixture. ‘The observation is at an end when the oxyhemoglobin bands have just disap- peared. ‘The examination is made with artificial light, keeping the distance from the light constant. Dare suggests that for sake of convenience the results be expressed in terms of the number of cubic centimeters of the tartaric acid solution instead of in mgrms. of sodium hydrate, as has been cus- tomary. ‘lhe corresponding values are given in the table below, and have reference to 100 c.c. of blood. His normal values range between 266 and 292. Equivalent in terms C.c. of reagent: of mgrms. of NaOH per 100 c.c. of blood. 2.6. 345.0 2.4. 319.0 2.2. 292.0 2.0. 266.0 1S 7 239.0 Leb 212.0 1,4. 176.0 i Marae 169.0 194 Oe 130.0 OS: 96.0 OsO% 79.0 0.4. 53.0 22 26.6 Dare has ascertained with his method that there is a more or less constant relationship between the alkalinity of the blood and the color index, and he suggests that this may be the reason why the results obtained by different investigators differ so widely, as at different stages of the disease the color index may change. ‘The iiteaitionl & is quite convenient and merits the careful attention of all laboratory workers. LirpratTurE.—y. Jaksch, Zeit. f. klin. Med., 1887, vol. xiii, p. 350. A. Lowy. Arch. f. d. gesammte Phy siol., 1894, vol. lviii, Dp. 462. Loéwy u. Richter, Deutsch. med, Woch., 1895, vol. xx, p. "526. Peiper, Areh. f. path, Anat., 1889, vol. CXVI, p. 337. Rumpf, Centralbl. f. inn. Med., 1891, vol. xii, p. 447. ’ Kraus, Arch. f. exp. Path. u. Pharmakol., vol. xxvi. Engel, Berlin. klin, Woch., 1898, p. 308. Brandenburg, Zeit. f. klin. Med., vol. xxxvi, p. 267. Orlowsky, Wratch, 1902, vol. xxii, pp. 1190 and 1222. A. Dare, Phila, Med, JOU Jou 1903: and Jchns Hopkins Hospital Bull., July, 1903, CHEMICAL EXAMINATION OF THE BLOOD 9 or CHEMICAL EXAMINATION OF THE BLOOD. Chemical Composition of the Blood.—A general idea of the chemical composition of the blood may be formed from the accom- panying table of C. Schmidt, calculated for 1000 parts: Man. Woman. Oe a ag wa recs me he oS 618.00! 369.20 Water... Ae ee ye 40-70 272.60 Hemoglobin and elobulins ert 9A Pree ees 150/60) 120.10 Miferabanltga lbw Mee) Oe ae 3,10 3.09 REL ee est oe we Reh te en, 5° 0486,90 603.80 een wee eee oe Gale ee ee tc) ho. 39.00 552.00 PApTIN: #2, 2 er eRe > at Sr, 3.90 1.91 Albumins and extractiv Coc RU eee urs? oe ey 39.90 44.79 erates Loren eee Maee toe NR ees LS. orem 4.14 5.07 Blood plasma differs from blood serum in the presence of fibrinogen in the former and its absence in the latter. ‘he substance is used up during coagulation, fibrin and a small amount of fibrinoglobulin resulting. The albumins which are common to both plasma and serum are serum albumin and serum globulin. Of these the globulin is the larger fraction (3.84 as compared with 2.6 per cent., in horses’ blood). From the following table it will be seen that a marked difference exists in the nature i the mineral ingredients between serum and the red corpuscles, the latter being relatively rich in potassium salts and phosphorus, and poor in atin salts and chlorine. The fioures have reference to 1000 parts of blood Man. Woman. Red Red corpuscles. Serum. corpuscles. Serum. Pee ine fie” Son 1 BSG 0.1535 1412 0.200 er ee a ee Oe es O24) 1.661 0.645 1.916 CaO . : : ne MgO Fe.O; he ne te ee ee 78 bs es Cl ae 2. ieee the a SUS 13722 0.362 1.440 P.O. Se) i Sy Fe 0 15 0.071 0.643 2.202 It is noteworthy that the amount of sodium chloride in the serum, 6 to 7 pro mille, remains fairly constant no matter whether large amounts are ingested or none at all is given. ‘he term “‘isotonic”’ has been applied to a salt solution which is just strong enough to pre- vent the solvent action of the water upon the hemoglobin of the red corpuscles. In the case of the serum we meet with a condition of hyperisotonia—z. e.,an amount of salt in excess of that actually required in order to prevent the destruction of the red corpuscles. ' This figure is too high; in man it varies between 420 and 470 for 1000 parts of blood. 26 THE BLOOD Fat occurs in amounts varying from 1 to 7 pro mille in fasting animals, while following the ingestion of a meal rich in fats as much as 12.5 pro mille have been encountered. Soaps, cholesterin, and lecithin have likewise been found. Glucose is a normal constituent of the plasma, amounting to from 1 to 1.5 pro mille in man. While it is possible to increase this amount to a certain degree by the ingestion of large quantities of sugar, this appears in the urine, according to Claude Bernard, as soon as 3 pro mille have been exceeded. In addition to glucose, another reducing substance has been found in the blood, which differs from the former in not being fermentable. According to the re- searches of P. Mayer,’ this is in all probability a glucuronic acid compound. Whether jecorin also occurs in the blood is doubtful. Among the extractives which have been found there may be men- tioned urea, uric acid, kreatin, carbamic acid, sarcolactic acid, gly- cogen, hippuric acid, and under pathological conditions xanthin, hypoxanthin, paraxanthin, adenin, guanin, leucin, tyrosin, lactic acid, cellulose, 3-oxybutyric acid, acetone, and biliary constituents. It has been pointed out that the color of the blood is referable to the presence of hemoglobin in the red corpuscles, and that it varies from a bright scarlet-red in the arteries to a dark bluish-red in the veins, the exact shade depending upon the amount of oxygen present in combination with hemoglobin as oxyhemoglobin. Upon chemical examination two other gases may be demonstrated under physiological conditions, viz., carbon dioxide and nitrogen. Of these, the latter appears to play no part in the body economy, and the amount present merely corresponds to that which would be absorbed by an equal volume of distilled water, viz., 1.8 vol. per cent., calculated at 0° C. and 760 Hgmm. pressure. The amount of oxygen and carbon dioxide, on the other hand, undergoes considerable variation, depending upon the particular bloodvessel from which the specimen is taken—. e., whether this be an artery or a vein, and, furthermore, upon the velocity of the blood current, the temperature of the body, rest, exercise, ete. The relation existing between the amounts of these gases in arteries and veins may be seen from the following table: Arterial blood. Venous blood. Oxygen. . Meena eee Lat per cent. 6.8 percent. Carbon dioxide . . . 40.3 2 Bad WR Nitrogen.) (ols) eeseen ge eats Ss IB ae are Oxygen, as already pointed out, occurs principally in chemical combination with hemoglobin (oxyhemoglobin), only 0.26 per cent. being present in solution in the plasma. Zeit, f. physiol, Chem., vol, xxxii, p. 518. a CHEMICAL EXAMINATION OF THE BLOOD af Of the carbon dioxide which may be obtained from the blood, only one-tenth is held in solution. One-third is found in the red corpuscles, in the form of a loose compound with the alkalies of the corpuscles, and possibly also in combination with hemoglobin. ‘The remaining portion is held in chemical combination by the alkalies of the plasma and albuminous bodies. Coagulation.—_If blood is allowed to flow into a vessel and set aside, it will be observed at the expiration of a few minutes that the entire mass has become transformed into a semisolid, gelatinous material, which is spoken of as the blood clot or the placenta sanquinis. Still later it will be seen that a small amount of straw-colored fluid appears on top of the clot, which gradually increases in amount, while the clot itself undergoes shrinkage, until finally it floats, greatly diminished in size, in the surrounding fluid. The straw-colored fluid which has thus been obtained during the process of coagulation is spoken of as the blood serum. If a bit of the clot is examined microscopically, it will be seen to consist of a more or less dense network of fibers, the meshes of which are filled with blood corpuscles, which may be washed out, leaving the fibrous network, fibrin, behind. Chemically speaking, fibrin belongs to the class of the so-called coagulated albumins; it does not occur in the circulating blood, but is formed only during the process of coagulation. Under normal conditions blood clots in from two to six minutes after being shed, while in disease, notably in hemophilia, coagula- tion may be greatly retarded or does not occur at all, so that fatal hemorrhage may follow the infliction of trifling wounds. A ten- dency to hemorrhage is also observed in scurvy, purpura, In some infectious diseases, such as typhoid fever and yellow fever, in poison- ing with phosphorus,’ ete. Sicard? has pointed out that in purpura primary coagulation occurs as with normal blood, but that sub- sequent retraction of the clot and exudation of serum take place to only a very limited extent. Normal serum when added to fluids, such as hydrocele fluid, which are not spontaneously coagulable, in the proportion of 1 to 80, induces coagulation i in from four to six hours. The serum of purpuric patients, on the other hand, is either entirely devoid of this property or possesses it to only a very slight degree. The addition of a trace of calcium chloride, however, causes such serum to behave very much like normal serum. Sigard hence suggests that in certain cases of purpura the fibrin ferment or its pro-enzyme is not present in sufficient quantity to cause more than a primary coagulation. 1 Schmidt, Pfliiger’s Archiv, vol. xi, pp. 291 and 515. Bojanus, Inaug Diss., Dorpat, 1881. * Compt.-rend. Soc. biolog., vol. li, p. 579, 28 THE BLOOD o_ Wright’s Coagulometer.—Wright’s coagulometer may be con- veniently employed to determine the rapidity of coagulation. ‘The instrument is shown in the accompanying illustration (Fig 4). ‘The essential parts are a tin water can, a thermometer registered to about 50° C., and a so: of glass tubes measuring about 10 cm. in length with a lumen of 0.25 mm. and marked at 5 cm. When the instrument is to be used, * can is filled with water having a temperature about that of the body. ‘The tubes are covered at their distal ends with little rubber caps and placed in their respective positions in the water bath, where they remain until they have acquired a similar temperature. Fie, 4.—Wright’s coagulometer. ‘They are then successively filled about one-half by aspiration from a drop of blood obtained from the finger or the lobe of the ear and replaced (properly numbered) tips down into the water in their proper positions. Careful note is kept of the exact time when they are filled. When a series of six or eight tubes has been filled, tube No. 1 is withdrawn from the water, and is held over a piece | of white filter or blotting paper. The condition of the coagulation is then tested by blowing into the tube, the time of testing and the — result being noted on the record. THE BLOOD PIGMENTS 29 1. If the contents cannot be blown out, an entry is made on the record that coagulation is “complete.” 9 Tf the contents can be blown out, but if shreds of fibrin are found adhering to the inside of the tube or to the filter paper, coagula- tion is recorded as “incomplete.” 3. If, lastly, the contents can be blown out cleanly, and if no trace of fibrin is seen on the filter paper, a note is made that coagulation has not yet begun. In the first case the second tube is immediately taken in hand and is tested in the same manner as the first. If this is found clotted the next in series is tested, and so on, until a tube 1s found in which coagulation is still incomplete. In the second case, 7. ¢., in the case where coagulation is found to be still incomplete, a slightly longer interval—reckoning from the time at which the second tube was filled in—is allowed to elapse before the tube next in series is tested. Lastly, if it is found that coagulation has not yet begun, an inter- val of one minute or more is allowed to elapse before testing the tube next in series. When the condition of the blood in a coagulation tube has been once tested, the tube in question must be put aside. Even if it has not all been blown out of the tube, its rate of coagulation will have been disturbed by the movement. Under normal conditions the coagulation time with these tubes will be found to vary between three and five minutes. ‘The tempera- ture of the water in the can should be kept uniform during the exami- nation by adding hot water if necessary. The tubes are cleansed by removing the clots with a fine wire; they are then washed with water, with alcohol, and finally with ether. THE BLOOD PIGMENTS. Hemoglobin and Oxyhemoglobin.—Hemoglobin is a proteid which is composed of an albuminous radicle, globin, and a non-albu- minous pigment radicle, hemochromogen. Upon the presence of the latter depends the readiness with which hemoglobin forms compounds with certain gases, such as oxygen, carbon monoxide, carbon dioxide, nitric oxide, and cyanogen. Hemochromogen in combination with oxygen is known as hematin. Oxyhemoglobin thus differs from hemoglobin merely in the fact that the pigment radicle is present in combination with oxygen. By itself hemoglobin is largely found in the blood of asphyxia. Under ordinary conditions it is principally present as oxyhemoglobin ; in arterial blood this preponderates, while in venous blood a mixture of both is found. 30) THE BLOOD On spectroscopic examination hemoglobin in suitable dilution shows a single band of absorption between D and E, extending slightly beyond D to the left (Fig. 5). Oxyhemoglobin shows two bands of absorption between D and E. One band, a, which is not so wide as the second, B, but darker and more sharply defined, borders on D; the second, which is wider but less sharply defined, lies at E (Fig 6). ‘This spectrum can be readily transformed into that of hemoglobin by the addition of a re- ducing agent, such as ammoniacal solution of ferrous tartrate (Stokes’ fluid), ammonium sulphide, or cuprous salts. Under normal conditions the amount of hemoglobin is fairly con- stant, but varies somewhat in different countries with the habits of the people, the character of the diet, ete. In Germany, as the result of 61 estimations, Leichtenstern found 14.16 per cent. by weight as the average in healthy men, and 13.10 per cent. in women. Red Orange Yellow Green Cyan-blue PANO ia Baur D Eb 1 Fia. 5.—Spectrum of reduced hemoglobin. (v. Jaksch.) Red Orange Yellow Green Cyan-blue AD eG: D Eb F Fic. 6.—Spectrum of oxyhemoglobin. (v. Jaksch.) Clinically we express the amount of hemoglobin by relative figures as compared with the average normal percentage by weight; on this basis the scale of the various hemoglobinometers is constructed. On these instruments the figure 100 represents the average normal value; this, however, varies somewhat with the various forms of hemo- globinometers according to the average percentage by weight which has been taken as a standard in establishing the 100 mark. With the Gowers instrument Strauss and Rohnstein obtained figures varying between 85 and 125 as normal values; this would furnish an average of 105. Schaumann and v. Willebrandt give 88 as the average normal. With the v. Fleischl instrument I rarely find higher values than 90 per cent. in inhabitants of large cities, but with the Dare apparatus the average results more nearly approach the 100 mark. In children the average values are somewhat lower than in the THE BLOOD PIGMENTS 31 adult. Stierlin gives 79.7 per cent. for boys and 82.1 for girls. Borchmann’s values are even lower, viz., 55 and 80; Gundobin gives 70 and 95. The ingestion of large amounts of water does not cause a dilution of the blood and hence a diminution of the amount of hemoglobin; but relatively higher values are found upon the withdrawal of liquids, owing to a concentration of the blood as a whole. Fat persons show smaller values than correspond to their age. Pathological Variations.—Abnormally high values, hyperchromemia, viz., 120 to 150 per cent., occur in cases of chronic enterogenous cyanosis, and may also be observed in congenital heart disease. The hyperchromemia in these cases is associated with polyglobulism. A pathological decrease is spoken of as oligochromemia, and is observed in all forms of anemia from whatever cause. The lowest values are found in chlorosis, in which the oligochrome- mia far exceeds the oligocythemia, viz., the diminution in the number of the red cells. In an analysis of 94 cases I found an average of 42.5 per cent.; the lowest value was 17.5 (Fleischl). There are instances on record in which the reading was still lower. Very low figures are seen in splenic anemia, and it is rare, excepting in chlorosis, to find such a low grade of chromemia associated with a blood count which is normal or may indeed be above normal. ‘The average of 13 estimations given by Osler was 47 per cent. In pernicious anemia the oligocythemia exceeds the oligochro- memia. ‘The loss of hemoglobin is, however, also quite marked and may be as great as in the most extreme cases of chlorosis. In the series of 23 cases collected by Strauss and Rohnstein the average value was 25 per cent. (Gowers); in 9 cases it was lower than 20 per cent. A. Meyer reports a bothriocephalus case with only 10 per cent. In the early stages of leukemia the loss of hemoglobin is often not especially marked; later the anemia may become quite intense, but the oligochromemia is not necessarily of high grade even in well- developed cases. Ehrlich cites cases in which the Gowers instrument gave readings of from 60 to 70 per cent. On the other hand, there are eases in which the oligochromemia js an early feature of the disease, and in one instance of this kind I obtained a reading of only 27 per cent. Cases of this order have been described as leukanemia. ‘The blood picture is essentially a composite of leukemia and pernicious anemia. While in the course of typhoid fever the amount of hemoglobin is always reduced (Osler), and usually to a greater extent than the number of the red corpuscles, the most severe grades of anemia may be encountered during convalescence, when the amount of hemoglobin may fall to 20 per cent. In the early stages of carcinoma of the stomach the cachexia is not well pronounced. Schiile states that in his analysis of 198 cases it 39 THE BLOOD occurred in only 30 per cent. Later the loss of hemoglobin is quite marked; the values may indeed approach those seen in chlorosis and pernicious anemia. An intense grade of anemia is seen in generalized septicemia, and, as Ewing remarks, no form of the acute disease appears to act more violently than does puerperal or uterine sepsis. A diminution in the amount of hemoglobin to 20 per cent. is here not uncommon. In the chronic cases also a high grade of oligochromemia is. a constant feature. In a case of lumbar abscess of six months’ duration I found 21 per cent. of hemoglobin, with 1,025,000 red cells. ‘The hemo- globin in all these cases diminishes more rapidly than the number of the red cells. In pulmonary tuberculosis a diminution in the amount of hemo- globin is seen essentially in the third stage of the disease (40 to 45 per cent.), while previously fairly normal values are obtained (90 to 95 per cent.). It is to be noted, however, that a certain grade of anemia (69 per cent.) is quite commonly observed, even in the first stage, in those cases in which the disease has been of very gradual onset, viz., in patients who often have suffered from tuberculous affections (scrof- ula) since childhood. In the third stage the anemia is well marked (40 to 50 per cent.). A notable diminution in the amount of hemoglobin is observed in chronic nephritis, chronic enteritis, in chronic lead and mercurial poisoning, in syphilis, ete. In syphilis the anemia develops at a time when the entire organism has been thoroughly infected. ‘lhe lowest hemoglobin values are reached just before or coincidently with the appearance of the rash. In the secondary stage the degree of oligochromemia, ceteris paribus, may be regarded as a fair index of the severity of the infection. In untreated cases the hemoglobin remains low for scveral days or even for weeks. A gradual rise then occurs which is associated with beginning involution of the exanthem.* In uncomplicated cases normal values may subsequently be reached even without treatment; a fall again occurs with relapses. Similar changes are observed in the tertiary stage. specially interesting are the observations of Justus on the blood changes which occur in the course of mercurial treat- ment; Justus ascertained that a rapid and material diminution of the hemoglobin (10 to 20 per cent.) occurs when a large (medicinal) amount of mercury is introduced at one time into the body of the infected individual. ‘This decrease is only observed in the blood of patients with florid syphilis; it is specific and does not occur in healthy nor in otherwise diseased individuals. ‘The reaction is demonstrable in every form of syphilitic infection (secondary, tertiary, and heredi- tary) as soon as the more distant lymph glands begin to swell. It disappears, or is at least no longer demonstrable, with beginning involution of the symptoms. ———° . ae ee —— as ee ee See ee Pe OF OL BE DEE Th Ctr es kn. Ce ee - % > ‘ f ¥ THE BLOOD PIGMENTS 33 The practical value of this syphilitic blood test has not yet been definitely established. While some observers have expressed them- selves against its value, it must be recognized that in discussing his adversaries’ criticisms Justus seems to have maintained the upper hand. During anesthesia by ether the amount of hemoglobin is always absolutely reduced. In some instances there is an apparent increase, but this is never proportionate to the rise in the number of the red cells which is simultaneously observed (Da Costa, Kalteyer). Owing: to the hemocytolysis which thus undoubtedly takes place a very low percentage of hemoglobin should be regarded as a counterindication to general anesthesia. A lower value than 50 per cent. 1s now re- garded by many as a dangerous figure. For the estimation of hemoglobin see p. 147. LITERATURE,—Strauss u. Rohnstein, Die Blutzusammensetzung b. d. verschied- enen Anwmien, Hirschwald, Berlin, 1901. Appelbaum, Berl. klin. Woch., 1901, vol. xxxix, p. 7. Quincke, “Zur Pathologie d. Blutes,” Deutsch. Arch. f. klin. Med., vols. xxv and xxvii. Leichtenstern, Unters. tiber d. Hemoglobingehalt d. Blutes im gesunden u. kranken Zustande, Leipzig, 1878. W. Osler, “On Splenic Anemia,” Am. Jour. Med. Sci., 1902, vol. exxiv, p. 763. Justus, Virchow’s Archiv, vol. exl, p. 1; and Deutsch. Arch. f. klin. Med., 1902, vol. lxxv, p. 1. Hemoglobinemia.—The term hemoglobinemia has been applied to a condition in which the hemoglobin is dissolved out from the red corpuscles, and, appearing in the plasma as such, leads at first to a very decided choluria and in extreme cases to hemoglobinuria. Various poisons, such as potassium chlorate, carbolic acid, pyro- gallic acid, naphtol, arsenic, antimony, hydrochloric acid, sulphuric acid, antifebrin, antipyrin, phenacetin, sulphonal, tincture of iodine, when given hypodermically, or even internally in sufficiently large doses, will call forth a hemoglobinemia which is followed by hemo- globinuria. Fresh morels also contain a poison which is capable of producing an intense hemoglobinuria, and which may be extracted with hot water. In acute and chronic infectious diseases of a severe type, such as scarlatina, typhoid fever, intermittent fever, icterus gravis, syphilis, as also in diseases depending upon a hemorrhagic diathesis, such as variola hemorrhagica, scurvy, as also following insolation, extensive burns, and frostbite, hemoglobinemia, leading to hemoglobinuria, 1s not infrequently observed. ‘The same has been noted in splenic anemia and in Raynaud’s disease. In syphilis a moderate grade of hemoglobinemia can be demonstrated by spectroscopic examination of the serum within two or three minutes following an intravenous injection of mercuric chloride in medicinal doses. (See also Justus’ test.) An epidemic hemoglobinuria of the newly born and a paroxysmal 3 24 THE BLOOD or intermittent hemoglobinuria, both of unknown origin, have like- wise been described. Hemoglobinemia also follows the infusion of blood of animals of one species into the circulation of animals of a different species. In some cases, and particularly in those following poisoning with chlorates, ete., the hemoglobinemia ultimately leads to a well-pro- nounced methemoglobinemia (see below). A hemoglobinemia, aside from the urinary examination, may be readily recognized by a spectroscopic examination of the serum, when the two bands of absorption of oxyhemoglobin will be observed. A very simple method which may be employed for the same purpose is the following: One-half to 1 ¢.c. of blood is collected in a small glass tube, drawn out and sealed at one end. ‘This amount can be readily obtained by puncturing the ear and milking out the blood, which is transferred to the tube by means of a little pipette. After the blood has clotted, the clot is separated from the walls by means of a wire or a glass rod and the corpuscles packed down by centri- fugation. With normal serum the supernatant fluid presents a straw-yellow color, while in hemoglobinemia it is colored a more or less intense red. If the supernatant fluid is withdrawn, diluted with a little water, and heated to 70 to 80° C., the coagulum in the presence of hemoglobin will present a brownish color. _ Lirerature.—Ponfick, Verhandl. d. Cong. f. inn. Med., 1883, vol. ui, p. 205. Stadelmann, Arch. f. exp. Path. u. Pharmakol., 1882, vol. xv, p. 337, and 1884, vol. xvi, pp. 118 and 221. Afanassiew, Zeit. f. klin. Med., 1883, vol. vi, p. 281. v. Jaksch, Verhandl. d. Cong. f. inn. Med., 1891, vol. x, p. 353. Carbon Monoxide Hemoglobin.—In cases of coal-gas poisoning the blood, both of arteries and veins, presents a bright cherry-red ~ color, owing to the presence of carbon monoxide hemoglobin. Such blood, when properly diluted, like oxyhemoglobin, shows two bands of absorption between D and FE (Fig. 7), which are nearer the Red Orange * Yellow Green Cyan-blue ab ttm Gap 6: D Eb F Fic. 7.—Spectrum of carbon monoxide hemoglobin. (v. Jaksch.) violet end of the spectrum, however, and may readily be distinguished from those referable to oxyhemoglobin by the addition of a reducing agent. ‘lhis will not affect the spectrum of carbon monoxide hemo- globin, while that of oxyhemoglobin is transformed into the spectrum of reduced hemoglobin. THE BLOOD PIGMENTS Bd For medico-legal purposes a number of additional tests have been devised, among which that suggested by Hoppe-Seyler is one of the simplest and at the same time most reliable. ‘The blood is treated with double its volume of a solution of sodium hydrate (sp. gr. 1.3). Normal blood is thus changed into a dirty-brownish mass, which exhibits a trace of green when spread upon a porcelain plate, while carbon monoxide blood yields a beautiful red under the same con- ditions. Nitric Oxide Hemoglobin.—The blood in cases of poisoning with nitric oxide, owing to the presence of nitric oxide hemoglobin, yields a spectrum which is similar to that of carbon monoxide hemo- globin; the bands, however, are less sharply defined and paler than those of the latter, and, like these, do not disappear on the addition of a reducing substance. Sulphohemoglobin (Methemoglobin Sulphide).—In cases of poisoning with hydrogen sulphide no definite changes can be dis- covered in the blood upon spectroscopic examination, although Hoppe-Seyler has shown that hemoglobin may enter into com- bination with this gas. It is stated, however, that in such cases the blood becomes dark and of a dull-greenish tint, and that the distinc- tion between arterial and venous blood is lost. A remarkable instance of sulphohemoglobinemia has been de- scribed by v. d. Berg,’ in a case of autotoxic enterogenous cyanosis. In this case an organism producing hydrogen sulphide was isolated from the stools. When grown in a solution of normal oxyhemoglobin sulphohemoglobin resulted. Carbon Dioxide Hemoglobin.—With carbon dioxide, as men- tioned above, hemoglobin is also thought to enter into combination, the spectrum being similar to that of reduced hemoglobin. ‘The latter, in fact, is formed artificially when carbon dioxide is passed through a solution of oxyhemoglobin. If this process is carried farther, the hemoglobin is decomposed and globin is thrown down; an absorption band is then obtained which is similar to that result- ing when hemoglobin is decomposed with acids (see below), and is no doubt referable to the presence of free hemochromogen. Of the blood changes occurring in cases of poisoning with hydro- cyanic acid and acetylene but little is known, and the reader is referred to works on toxicology for their consideration. Hematin.—If oxyhemoglobin in aqueous solution is heated to a temperature of from 60° to 70° C., it is decomposed into globin and hematin. ‘The same result is reached by treating the aqueous solu- tion with acids, alkalies, or the salts of various heavy metals. Hematin is an amorphous, blackish-brown, or bluish-black sub- stance which is frequently encountered in old transudates, in the 1 Arch. f. klin. Med., 1905, vol. lxxxiii, p. 86. CY oe THE BLOOD stools after hemorrhages, and after meals consisting largely of red meats. It is said to occur in the urine in cases of poisoning with arsenic, and in the blood of animals poisoned with nitrobenzol its presence can likewise be demonstrated with the spectroscope. Red Orange Yellow Green Cyan-blue ——_-”- SS @*89M?—_—_—_______—™ —_—[[—["[[——“. (As Oa Dae C D ib F ' 40 50 60 70 80 90 100 110 Fic. 8.—Spectrum of hematin in alkaline solution. (v. Jaksch.) In acid solution it shows a well-defined spectral band between C and D (Fig. 10). Between D and F a second band is seen, which is much wider but less sharply defined than the first, and may be resolved into two bands by dilution, one between b and F, near FP, and another between D and FE, near /; a faint fourth band may also be seen between D and E, near D. As a rule only the two bands between D and F are visible. Red Orange Yellow 4 on Green Cyan-blue Aca WeBae. G D Eb F Fie. 9.—Spectrum of reduced hematin. (v. Jaksch.) In alkaline solutions it shows but one broad band, the greater ey of which lies between C and D, extending slightly beyond D (Fig. 7). If an alkaline solution of hematin is treated with a reducing sub- stance, reduced hematin (hemochromogen) results, which gives rise to two absorption bands between D and F (Fig. 9). Hemin.—Hematin readily combines with one molecule of hydro- chloric acid to form hemin. ‘This substance crystallizes in light- brown or dark-brown rhombic plates or columns, which are quite characteristic (Plate 1). They bear the name of their discoverer, Teichmann. ‘The size of these crystals varies with the manner in which they are produced, the largest specimens being met with when the glacial acetic acid (see below) is allowed to evaporate as slowly as possible. Specimens measuring from 15/ to 18 in length may then be seen. Smaller crystals will be present at the same time, occurring either singly or in the form of stars, rosettes, and crosses. As these crystals may be obtained from mere traces of blood, their PLATE I. stals. Hemin Cry ; a ‘ a Ms THE BLOOD PIGMENTS 37 formation must be regarded as conclusive evidence in medico-legal examinations. Lewin and Rosenstein have pointed out, however, that under certain conditions a negative result may be reached, even if the coloring matter is derived from the blood. ‘This is the case especially when the hemoglobin has been transformed into hemo- chromogen or hematoporphyrin, or when substances have been mixed with the blood which are either capable of altering its general composition or which, through their mere presence, interfere with the reaction. Such substances are certain salts of iron (rust), lead, mer- eury, and silver; further, lime, animal charcoal, and sand, when inti- mately mixed with the blood. In medico-legal cases a spectroscopic examination should hence be made whenever the hemin reaction is not obtained. Mernop.—A small drop of normal salt solution is carefully evaporated on a slide, when a few particles of the suspected material, powdered or teased as finely as possible, are placed on the delicate layer of crystallized salt. Glacial acetic acid is now added drop by drop and the specimen carefully heated (three-quarters to one minute) until bubbles begin to form. ‘While evaporation is being continued glacial acetic acid is further added until a light-brown tint appears. As soon as this point is reached, the last traces of the acid are allowed to evaporate, the specimen being held at a greater distance from the flame. A drop of glycerin is then added and the preparation covered with a cover-glass. ‘lhe examination is made with a one-fifth or a one-sixth objective. Attention is especially directed to brownish streaks or specks, which, in the presence of blood, can usually be made out with the naked eye. Methemoglobin.—Methemoglobin is a pigment closely related to oxyhemoglobin, and is frequently encountered in hemorrhagic transu- dates, cystic fluids, and in the urine in cases of hematuria and hemo- globinuria. In the circulating blood methemoglobin is found after Red Orange Yellow Green Cyan-dlus Ae BC D Eb F 40 50 60 70 80 90 100 110 Fig. 10.—Spectrum of methemoglobin in acid and neutral solutions. (v. Jaksch.) the ingestion of large quantities of potassium chlorate, notably in children, as also after the inhalation of nitrate of amyl, the use of kairin, ionilin: hydrochinon, pyrocatechin, iodine, bromine, turpen- tine, phen: perosmic acid, permanganate of potassium, and enutanen (see Hemoglobinemia). Most remarkable is the occurrence of met- hemoglobinemia in cases of so-called autotoxic enterogenous cyanosis, had 38 | THE BLOOD as reported by Stokvis and v. d. Berg. In one case the latter found sulphohemoglobin in the place of methemoglobin. ‘The spectrum of an aqueous or slightly acidified solution of met-— hemoglobin (Fig. 10) closely resembles that of an acid solution of hematin, but differs from this in the ease with which it is transformed into that of hemoglobin when an alkali and a reducing substance are added. ‘The spectrum of hematin under the same conditions is trans-_ formed into that of an alkaline solution of hemochromogen. In alkaline solutions, on the other hand, two bands of absorption are observed, which are similar to those of oxyhemoglobin, but differ from these in the fact that the band nearer LE, b, is more pronounced than the one at D,a. A third, but very faint, band may further be observed between C and D, near D. Hematoidin.—Small amorphous particles of an orange or ruby- red color, or crystals belonging to the rhombic system, occurring either singly or in groups, are frequently met with in the sputum, the urine, and the feces, as well as in old extravasations of blood. They were discovered by Virchow, who applied the term hematoidin to this particular pigment, the hemic origin of which is undoubted. It is supposedly identical with bilirubin. Hematoporphyrin.—Hematoporphyrin is likewise a derivative of hematin, and, according to Nencki and Sieber, isomeric with biliru- bin. In dilute solution with sodium carbonate it shows four bands — Red Orange . Yellow Green Cyan-blue Arp Beg D Eb F 40 50 60 70 80 90 100 110 | Ee il Fic. 11.—Spectrum of hematoporphyrin in alkaline solution. of absorption, one between C' and D; a second one, broader than the first, about D, especially marked between D and FE; a third one, not so broad and less sharply defined, between D and LE, and a fourth one, broad and dark, between b and F (Fig. 11). The clinical significance of this body, which also appears in the urine, as well as the causes which give rise to its formation, are — unknown (see Hematoporphyrinuria). It has been found postmortem in the blood, in a case of sulphonal poisoning, by Taylor and Sailer.’ 1A. KE. Taylor and J. Sailer, Contrib. from the William Pepper Laboratory, Phila., 1900, p. 120. i at i THE PROTEINS OF THE BLOOD 89 a THE PROTEINS OF THE BLOOD. In considering the proteins of the blood from a clinical point of view, it is necessary to distinguish between an increase and a dimi- nution in their normal amount, constituting the conditions of hyper- albuminosis and hypalbuminosis, respectively. As may be expected, the former is met with whenever water is more rapidly withdrawn from the system than it can be supplied, and is hence observed in cases of cholera, acute diarrhea, following the use of purgatives, etc. This increase in the amount of proteins is only a relative increase, however. ‘The occurrence of an absolute increase has not been satisfactorily demonstrated. An absolute hypalbuminosis, on the other hand, is observed following a direct loss of proteins from the blood, as in hemorrhage, dysentery, albuminuria of high degree, the formation of large collections of pus, ete. ‘This is generally associated with a relative increase in the amount of water—1. ¢., a hydremia—which is particularly noticeable after hemorrhages, and referable to a diminished secretion and excretion of water, as well as to a direct absorption from the tissues. Hypalbuminosis has also been observed in pernicious anemia, and is dependent partly upon a diminution in the amount of the albumins of the serum and partly upon a decrease in the weight of the corpuscular solids. ‘lhe amount of serum-albumin is about normal, while the globulins are much diminished.* The term hyperinosis has been applied to a condition in which the amount of fibrin (normally 0.349 to 0.425 per cent.) is increased. This is said to occur in various inflammatory diseases, such as pneu- monia, pleurisy, scarlatina, acute articular rheumatism, and ery- sipelas, while a diminished amount of fibrin, hypinosis, or normal values are seen in malaria, nephritis, pyemia, pernicious anemia, typhoid fever, and leukemia (both lymphoid and myeloid). In order to determine the amount of fibrin, 30 to 40 c.c. of blood, obtained by means of cupping glasses or venesection, are placed in a previously weighed beaker, fitted with an India-rubber cap, through the centre of which passes a piece of whalebone, firmly fixed. ‘Ihe blood is defibrinated by beating with the whalebone, when the beaker with its contents is weighed, the difference indicating the weight of the blood. ‘The beaker is then filled with water and the mixture again beaten. The fibrin is allowed to settle and after being washed with normal salt solution collected on a filter of known weight. It is further washed with normal salt solution until free from color- ing matter, then boiled in alcohol to dissolve out fat, cholesterin, and lecithin, dried at 110° to 120° C., and on cooling weighed over sulphuric acid. 1 Erben, Zeit. f. klin. Med., 1900, vol. xl, p. 266. 40 THE BLOOD Fairly satisfactory results may also be obtained by simply making — wet mounts (which see), ringing with vaselin and setting aside for several hours, when they are examined microscopically. In cases of pneumonia and acute articular rheumatism marked fibrin formation — will be observed, starting from clumps of blood platelets. The presence of albumoses and peptone bodies in the blood of leukemic (myeloid) patients has been repeatedly observed after the blood has stood for some time, or after the death of the patient (v. Jaksch.t Matthes,? Erben,*? Schumm*). ‘Their formation is due . to the liberation of a proteolytic ferment, derived from the polynuclear neutrophiles. Schumm also found leucin and tyrosin. In normal human blood Schumm found no albumoses after death. In intersti- tial nephritis a fair amount could be demonstrated. Albumoses have also been found in a case of abscess of the brain, associated with albumosuria. Freund? claims that they are met with in sarcoma, while they are absent in carcinoma (not confirmed). Following the injection of nuclei and spermin albumosemia appears to occur quite constantly both during the stage of hypo- as well as hyperleukocytosis. After injections of pilocarpin albumo- suria is observed only in association with hyperleukocytosis. In order to test for albumoses, the coagulable albumins should first be removed, when a positive biuret reaction in the filtrate will indicate their presence (see also Salkowski’s test). Carbohydrates. Sugar.—Sugar, as indicated above, is a normal con- stituent of the blood, its quantity varying between 1 and 1.5 pro mille. Under pathological conditions this amount may be exceeded and notably so in diabetes, in which Hoppe-Seyler found as much as 9 pro mille in a given case. In addition to sugar, a non-fermentable reducing substance has been encountered in the blood, which, according to Mayer, appears to be a compound glucuronate.° ‘The presence of jecorin in the — blood still remains to be proved. Large quantities of a reducing substance, the greater portion of — which consisted of sugar, have been met with by Trinkler in carci- noma; it was observed at the same time that carcinoma of inter- nal organs was associated with far greater amounts of sugar than cancerous disease of the skin and the mucous membranes. It is also interesting to note in this connection that an increase in the degree of the cachexia was not accompanied by an increase in the percentage of sugar. 1 Zeit. f. physiol. Chem., vol. xvi, p. 243. 2 Berlin. klin. Woch., 1894, Nos. 23 and 24. * Zeit. 1. Heéilk,,41903)2vol. Sexy. * Hofmeister’s Beit., vol. v, p. 442. ® Freund u, Obermayer, Zeit. f. physiol. Chem., vol. xv, p. 310. OO LDIGRARVOl > Xx peo, THE PROTEINS OF THE BLOOD 41 The results reached by ‘Trinkler' apparently also bear out the correctness of the conclusions formed by Freund, who claimed that a differential diagnosis between carcinoma and sarcoma, in which latter condition no increase in the amount of sugar was noted, can always be effected upon the basis of an examination of the blood in this direction. Further examinations in this direction are lacking. In the following table the percentages found in the different dis- eases investigated are given, from which it is apparent that, next to carcinoma, the largest quantities of sugar are met with in the infec- tious diseases and the lowest figures in diseases of the kidneys: Average. Minimum. Maximum. Per cent. Per cent. Per cent, RCO ese eee». * 0.1819 0.1023 0.3030 mena iever <')) 4. -- ..- .. -0.0950 0.0875 0.1022 Smo ne) =, ee SS O,0943 0.0813 0.1092 eer ese he 8 2 8 ye 0.0838 0.0796 0.0915 ener ts. | 0737 0.0664 0.0897 Serer Pee ue Ss oe 0.0701 0:0450 0.0917 Maaercuiosigd © .~ . 2. ~ . 0.0653 0.0450 0.0817 thee woe sg 6S 0.0553 0.0449 0.0748 Nephritisand uremia . . . . 0.0489 0.0321 0.0559 In order to demonstrate sugar in the blood, 15 to 30 grams, ob- tained by venesection or cupping glasses, are placed in an evapo- rating dish and treated with an equal weight of finely powdered sodium sulphate and a few drops of acetic acid. ‘The mixture is brought to the boiling point and passed through a muslin filter as soon as the coagulum has become black and spongy, water having previously been added to the original volume. ‘The filtrate is passed through Swedish paper. In the final filtrate the sugar is then esti- mated as described elsewhere (see Urine). Cavazzani has drawn attention to another method of freeing the blood from proteids, which is said to be entirely satisfactory. ‘To this end, 20 to 30 c.c. of blood are added to 200 c.c. of distilled water in a porcelain dish and treated with 5 or 6 drops of a solution consisting of 10 parts of acetic acid (sp. gr. 1.040) and 1 part of lactic acid. ‘The mixture is boiled for eight to ten minutes, filtered, and the coagulum washed repeatedly with hot water and finally pressed out in a piece of muslin. ‘The resulting filtrates, which are prac- tically colorless, are then concentrated to a small volume, and any traces of albumin, which may still separate out, filtered off. If an excess of the acid solution has been added, it may happen that the mixture does not clear up on boiling. It is then only necessary to add a few crystals of sodium carbonate, when coagulation will occur at once. On the other hand, it may at times be necessary to add a few more drops of the acetic acid solution. 1 Centralbl. f. d. med. Wiss., 1890, p. 498. Freund u. Obermayer, loc. cit. 42 THE BLOOD Williamson’s Diabetic Blood Test.—This test is of much interest, and may possibly serve to differentiate the ordinary forms of diabetes — from that in which the blood sugar is not increased. It is based upon the observation that a warm alkaline solution of methylene blue is decolorized by grape sugar. A positive result may at times be obtained when the sugar has temporarily disappeared from the urine. * MetnHop.—Twenty cbmm. of blood, obtained from the finger or the ear, are carefully measured off with the aid of the capillary pipette, ~ which accompanies Gower’s hemocytometer, and mixed in a test- tube of small caliber with 40 cbmm. of distilled water. ‘To this mixture 1 c.c. of an aqueous solution of methylene blue (1 to 6000) and 40 cbmm. of a 6 per cent. aqueous solution of potassium hydrate are added. A control tube is similarly charged with non-diabetic blood. ‘The two specimens are placed in boiling water and allowed to remain for three to four minutes, without shaking. At the end of this time it will be seen that the diabetic blood has decolorized the methylene-blue solution, which has turned a dirty yellowish green or yellow, while the non-diabetic specimen has retained its — original color. The quantity of blood used should not exceed the amount indi- cated, as a decolorization of the methylene blue also results with non-diabetic blood if large amounts, such as 60 cbmm., are em- ployed. The reaction is supposedly due to an increase of glucose in the blood, and was obtained in all of forty-three cases of diabetes which were examined. It is said to be obtainable for a considerable time after death. Adler’ found the reaction in all of nine cases of dia- betes, while in one hundred and twenty-one non-diabetic cases nega- tive results were reached. Very curiously, it was absent in non- diabetic glycosurias. Adler believes the reaction to be referable to a diminished alkalinity of the blood. Glycogen.—'‘I‘here appears to be no doubt that glycogen normally occurs in the blood of various animals. Huppert* succeeded in demonstrating its presence in all animals examined, the amount vary- ing between 0.114 and 1.560 grams for 100 parts of blood (see Todophilia). Cellulose.—Cellulose has been found in the blood of tuberculous — patients. Urea.—Urea occurs normally in the blood in traces—O0.016 to 0.020 per cent. Larger amounts are encountered whenever, as — in nephritis, various diseases of the urinary organs, cholera Asiatica, cholera infantum, eclampsia, etc., its elimination is impeded, or when- 1 R. T. Williamson, Centralbl. f. inn. Med., vol. xvii, No. 33. Zeit. f.. Heilk., 1900, vol. xxi, No. 11. * Zeit. f. physiol. Chem., 1893, vol. xviii, p. 144. THE PROTEINS OF THE BLOOD 43 ever, as in fever, owing to increased albuminous decomposition, urea is formed in abnormally large quantities. It is interesting to note that a smaller amount of urea is found in fatal cases of eclampsia than in those ending in recovery, which has been explained by the assumption that in this condition the functional activity, not only of the kidneys, but also of the liver, is lost. The methods which are available for the detection of urea in the blood are still too complicated for clinical purposes, and the value of the information derived so small as hardly to warrant the labor involved. Hoppe-Seyler’s method should be employed whenever an examination in this direction is deemed advisable. Uremia.—lormerly, it was thought that the complex of symp- toms generally spoken of as uremia was referable to the retention in the blood of urea or ammonium carbonate. ‘This view has since been disproved, although it must be admitted that in uremia an increased amount of urea is frequently noted. Other views, accord- ing to which uremia is referable to an accumulation of potassium salts, of extractives, and especially of kreatinin, or of ptomains in the blood, must be regarded as being sub judice. ‘There is no reason, however, to ascribe the uremic condition to the retention in the blood of one particular constituent of the urine, and it is not improbable that a retention of all may be responsible for the symptoms observed. LirERATURE.—Feltz and Ritter, De l’uremie exper., Paris, 1881. Astaschewsky, St. Petersburg med. Woch., 1881, No. 27. | Bouchard, Le¢ons sur l’autointoxica- tion, Paris, 1887. Rovighi, Rivista clinica, 1886. Ammonia.—Normal venous blood, according to the researches of Winterberg, contains about 1 mgrm. of ammonia for each 100 c.c. In febrile conditions variable results are obtained, but it appears certain that a definite relation between the height of the fever and the amount of ammonia does not exist. In chronic hepatic diseases, and notably in cirrhosis, it is not increased. Acute yellow atrophy also is not necessarily associated with an increase. Very significant is the observation that in uremia following extirpation of the kid- neys no increase is observed. An ammoniemia in the sense of v. Jaksch can hence scarcely be said to exist. Lrrerature.—Nencki, Pawlow, and Zaleski, Arch. f. exp. Path. u. Pharmakol., 1896, vol. xxxvii, p. 26. Winterberg, Wien. klin. Woch., 1897, p. 330. Uric Acid and the Xanthin Bases. Uric Acid.—Formerly, the presence of appreciable amounts of uric acid in the blood was regarded as pathognomonic of gout. But we now know that a lithemic con- 1 See Hoppe-Seyler, Handbuch der physiologisch und pathologisch-chemischen ‘Analyse. At THE BLOOD dition may occur also in other diseases. ‘T'races of uric acid are indeed encountered under normal conditions. A definite lithemia has been observed in a variety of disorders, such as pneumonia, acute and chronic nephritis, leukemia, conditions associated with an insufficient aération of the blood, as in the various diseases of the heart, in pleurisy with exudation, emphysema when accompanied by cyanosis, the severer forms of anemia, etc. v. Jaksch_ claims to have found uric acid in the blood in 88.88 per cent. of his_ cases of nephritis. Fever in itself does not appear to lead to an increased production of uric acid, as negative results were obtained in nine cases of typhoid fever out of eleven, in five cases of acute” articular rheumatism out of six, ete. j ‘The assumption that acute attacks of gout are referable to increased alkalinity of the blood, and a consequent increase in the amount of circulating uric acid, has been disproved. ; In order to estimate the amount of uric acid in the blood, the fol-— lowing method may be employed: 100 c.c. of blood, obtained by means_ of venesection or of cupping glasses, are at once diluted with three or four times their volume of water and heated on a water bath. As_ soon as coagulation sets in, a few drops of a 0.3 to 0.5 per cent. solution of acetic acid are added until a feebly acid reaction is obtained. — After having been kept upon the boiling water bath for from fifteen to twenty minutes longer, until the albumin has separated out and settled in brownish flakes, the mixture is filtered while hot, and_ the precipitate washed repeatedly with hot water. Filtrate and washings, which usually present a slightly yellow or brownish color, are again brought to the boiling point after the addition of 0.3 to~ 0.5 per cent. of acetic acid, decanted, filtered, and after the addition — of a small amount of disodic phosphate further treated according — to Folin’s method (see Urine). LITERATURE.—Picard, Virchow’s Archiv. vol. ii, p. 189. Garrod, Med.-Chir.— Trans., 1854, p. 49. Salomon, Zeit. f. physiol. Chem., vol. 11, p. 65; and Charité — Annalen, 1880, vol. v, p. 1372. Klemperer, Deutsch. med. Woch., 1895, No. 40. Weintraud, ibid., V. B. p. 185. } ; Xanthin Bases.—Xanthin bases do not occur in normal blood or are present only in exceedingly small amounts. Under pathological ” conditions they may be encountered in recognizable quantities, ; so in leukemia, typhoid fever, lymphatic tuberculosis, emphysema, | phthisis pulmonalis, pleurisy, and chronic nephritis. ‘To demonstrate the xanthin bases in the blood the albumins are first removed as just described (see Uric Acid) and the filtrate then examined according to Salkowski’s method (see Urine). | LITERATURE.—A. Kossel, Zeit. f. physiol. Chem., 1882, vol. vii, p. 22. Scherer, Verhandl. d. physik. med. Ges. z. Wurzburg, 1852, vol. ii, p. 325. ; THE PROTEINS OF THE BLOOD 45 _ Fat and Fatty Acids.—Engelhardt has pointed out that the amount of fat which is contained in normal human blood may be subject to considerable variations, and gives 0.194 per cent. as the average. The lowest figure which he obtained was 0.101 and the highest 0.273 per cent. ‘These figures differ very materially from those of older observers, who have found from 0.73 to 1.4 per cent., but it is quite likely that Engelhardt’s method is responsible for these _ differences, and is probably more reliable (see below). Unfortunately only a few analyses of pathological material have been made with this method, and these have reference only to the blood of cachectic Po es 949 O%P% «@ 22.0 0 o% © ot % e *e%o e549 at Pa Oar ae ea ced ere Ss te *¢ ry "eo ° @ Yeoo,e.° ge afer’ .. e Fy 9, < » %¢: e oe e® Fie, 12.—Pronounced lipemia. Specimen treated with osmic acid. Lower half shows extra- cellular fat globules, upper half having been cleared by oil of turpentine. (Gumprecht.) individuals. An increase in the amount of fat has here not been demonstrated, the results varying between 0.112 and 0.284 per cent., with 0.174 as an average. ‘The cachexias in question were of tuber- culous and carcinomatous origin. With the older methods an increase in the amount of fat, aside from that observed after the ingestion of large amounts of fatty food, has been met with in cases of obesity, chronic alcoholism, in phosphorus poisoning, in injuries affecting the long bones and the spinal cord, in various hepatic diseases, chronic nephritis, tuberculosis, malaria, cholera, during starvation, ‘pregnancy, in nursing infants, etc. ‘The greatest increase, however, is observed in certain cases of severe diabetes, in which amounts varying between 1.276 and 18.12 per cent. have been encountered, and in which the fat may be visible with the naked eye (see below). In such cases fat emboli may be found postmortem, plugging the vessels of various 46 THE BLOOD organs, and notably the brain, the lungs, and the kidneys. ‘This increase in the amount of fat constitutes the condition spoken of as lipemia. The term lipacidemia has been applied to the occurrence of fatty acids in the blood. ‘This has been noted in various febrile diseases, leukemia, and especially in grave cases of diabetes, where beta- oxybutyric acid may be found in large amounts, and is no doubt directly concerned in the production of coma. ‘To demonstrate the presence of fat in the blood, it is best to pre- pare cover-glass specimens, and to mount these in a drop of a 5 per cent. solution of osmic acid. The fat droplets are thus colored black, and appear about as large as the finest fat granules which are found in milk or butter. ‘They may also be stained with Sudan III, or Biebrich scarlet, and are thus colored red. In every case the necessary instruments and glasses should be carefully cleansed with - ether, so as to avoid the accidental introduction of fat. As a quantitative estimation of the fat is not always possible, Landy recommends the following simple procedure to demonstrate the presence of an excess of fat: A small drop of blood is received upon a cover-glass, which is then adjusted over the depression of a cupped slide and ringed with vaselin. On standing, the serum separates out concentrically or excentrically from the small blood clot, and normally or in the presence of no excess of fat appears perfectly clear. If, however, much fat is present, it becomes cloudy after several minutes or hours, and then appears bluish-white, gray- — ish-white, or even milky-white. To ascertain positively that the turbidity is due to fat, a microscopic examination of the hanging drop is made within a few hours following the preparation of the specimen, so as to exclude fibrin as the possible cause of such tur- bidity. | Quantitative Estimation—The apparatus which is best used is a modification of that of Nerking, as suggested by Engelhardt.’ As seen from Fig. 13, it consists of the ether flask A, which is placed on a permanent water bath, such as that of Miinke. a represents the escape tube for the ether vapor; at b there is a closure by means — of mercury, the upper escape tube ¢ dipping into the mercury over the mouth of 6. B is the cooler for the ether vapor; C, the water condenser. ‘The cooled ether falls through the cooler into d. ‘This ends below with a funnel-shaped mouth, close to the bottom of the extraction flask E, with five apertures, and has a small open side tube, f, which counteracts any negative pressure that may occur above the liquid in the extraction flask. ‘The fluid to be extracted extends to within 1 to 2 cm. from the aperture of the off-flow tube 7. When the ether layer extends to the level with & the tube 7 acts as a * The apparatus may be procured from Arno Haak, Jena. Price, 12 marks. THE PROTEINS OF THE BLOOD 47 siphon and draws off the fatty ether into A again by way of the tube L, which is likewise provided with a mercury stop. The blood, about 10 c.c., is received in a graduate and weighed. It is washed into the extraction flask with about ten times its volume of 2 per cent. hydrochloric acid and boiled for three hours (with inverted condenser). On cooling, the material is extracted in the appa- ratus described for about forty-eight hours. At the expiration of this time the fatty ether in A is poured into a separating funnel together with the ethereal washings, which are used to remove all the material from the flask, the idea being to get rid of any water or bits of the bloody material that may by chance have been siphoned into A. The ether is then evaporated in an open glass dish. ‘lhe residue is dis- solved in absolute ether and filtered through a double folded filter (so as to absorb any traces of water remain- ing) into a beaker, when the ether is allowed to evaporate. ‘The residue is placed in a drying oven at 40° C. for one hour, and after remaining in the vacuum over sulphuric acid for twelve hours it is weighed. With this method lecithins, choles- terins, and fatty acids are obtained conjointly with the fat, which Engel- hardt does not regard as objectionable, as they are present only in traces and may be regarded as_ physiologically equivalent to neutral fat. Estimation of Fatty Acids.—This is carried out along the same lines as des- cribed in the Urine (Lipaciduria), after removal of the coagulable albumins. At 1 Fea ——=- | least 20 to 30 c.c. should be available. fy. 13—Fat-extraction app aratus. Cholesterin.—T'races of cholesterin are normally met with in the blood. Larger amounts have been observed in diabetes (0.478 per cent.) in association with marked lipemia.* Hale White’ reports a case in which microscopic examination showed a granular precipitate, which did not stain with osmic acid. Chemi- 1 Virchow’s Archiv, vol. clxxii, Heft 1 and 2. ? Lancet, October 10, 1903. 48 THE BLOOD ! cal examination led to the conclusion that the substance was‘an ester | of cholesterin with one or more of the higher fatty acids. \. 2 a LITERATURE.—M. Bonninger, “ On the Methods for the Estimation of Fat in the Blood, and the Amount of Fat in Human Blood,” Zeit. f. klin. Med., vol. xlii, partsi and ii. TT. B. Futcher, “ Lipemia in Diabetes Mellitus,” Jour. Am. Med, Assoc., 1899, p. 1006. S. Watjoff, “ Ueber d. Fettgehalt d. Blutes b. Nierenkrank- heiten,’’ Deutsch. med. Woch., 1897, p. 559. v. Jaksch, “ Lipacideemie,”’ Zeit. f. klin. Med., vol. xi. W. Ebstein, “ Beitrag z. Lehre v. d. Lipemie u. d. Fettembolie,” etc., Virchow’s Archiv, 1899, vol. clv, p. 571. M. Engelhardt, Deutsch. Arch. f. klin. Med., 1901, vol Ixx, p. 182. Zandy, ibid., vol. lxx, p. 301. Lactic Acid.—There appears to be some doubt whether or not lactic acid normally occurs in the blood of man during life.’ In the blood of dogs, Gaglio, could always demonstrate the presence of the acid during the process of digestion, after feeding with meat. ‘The amount varied between 0.3 and 0.5 pro mille. During starvation smaller amounts were found, but it never disappeared altogether. In one instance Gaglio obtained 0.17. pro mille after fasting for forty-eight hours. Similar results were obtained by Ivisawa, who noted that the amount of lactic acid in the blood stood in direct — relation to the degree of anemia which was produced. In the human being Irisawa found lactic acid fairly constantly after death, the amount, determined as zinc lactate, varying between — 0.233 and 6.575 pro mille. ‘These extensive variations he was unable to explain by the character of the disease causing the fatal termina- tion, and it is possible that the cause lies in the fact that in some cases the blood was obtained shortly after death, while in others many hours had elapsed, as Irisawa himself suggests. The following method may be employed: 100 to 300 c.c. of blood are extracted with three times its volume of alcohol, filtered, and the filtrate evaporated to a syrupy consistence. ‘his is then made — strongly alkaline with barium hydrate and shaken with large quan- tities of ether, in order to remove the fats which are present. ‘The residue is acidified with phosphoric acid and again shaken with ether for twenty minutes at a time, until the process has been repeated five or six times, the lactic acid passing over into the ether. ‘The ether is distilled off from the extract, the residue taken up with— water, and the solution carefully evaporated in order to drive off any ether still remaining, as well as the fatty acids. Carbonate of zine — is now added and the solution heated to 100° C. and filtered. ‘The - filtrate is evaporated on a water bath until crystallization begins, when it is allowed to cool and treated with a few drops of absolute | alcohol, in order to effect a complete separation of the lactate of zinc. The solution is allowed to stand exposed to the air until a a constant weight is obtained. | LITERATURE.—G, Gaglio, “ Die Milchsiiure d. Blutes,’’ Du Bois Archiv., ian p. 400. ‘T. Irisawa, “ Ueber d. Milchsiiure im Blut und Harn,” Zeit. f. physiol. Chem., 1892, vol. xvii, p. 349. “ THE PROTEINS OF THE BLOOD 49 Homogentisinic Acid.—Homogentisinic acid has been demon- _ strated in the blood serum of an alkaptonuric, by Abderhalden and - Falta.’ Biliary Constituents and Urobilin.—Bile pigment does not occur in the blood under normal conditions, but may be demonstrated _ whenever it is present in the urine (obstructive jaundice, hepatic cirrhosis, acute yellow atrophy, phosphorus poisoning, ete.). It _ appears, moreover, that bilirubin 1s present in the blood in nearly every case where urobilin is found in the urine. In pernicious anemia bilirubinemia is thus quite constantly associated with uro- _bilinuria. ‘At the same time urobilin can usually be demonstrated in the blood. In chlorosis bile pigment does not occur in the blood. ‘The demonstration of bilirubinemia constitutes the most delicate test for the entrance of bile into the blood and lymph; it is a much more delicate indication than the occurrence of bilirubinuria. Bilirubin can be demonstrated in the blood most readily in the fol- lowing manner: A short piece of glass tubing is drawn out so as to form a tapering lower end, which is then sealed. By means of a pi- pette 10 to 15 drops of blood, obtained by free puncture of the finger or ear, are transferred to the first tube and the serum separated from the corpuscles by centrifugation. ‘The coagulum which forms is sepa- rated from the walls and packed down into the lower portion of the tube. ‘The supernatant fluid is normally clear or but faintly turbid, and of a straw color: in the presence of bilirubin it is colored a bright yellow, which on exposure to the air gradually turns to a gteenish tint. For more exact information the method of Syllaba may be used: 10 to 15 c.c. of blood are placed in a cool place for sedimentation. ‘The serum which separates out is removed with a pipette and 5 c.c. diluted with double the amount of water and coagulated by boiling after the addition of a pinch of sodium sulphate and acidifying with dilute acetic acid. Any bilirubin that may be present is carried down in the coagulating albumin while urobilin remains in solution. The fluid is then filtered and the filtrate tested by boiling to make sure that the coagulation is complete. If no urobilin is present the filtrate is clear, colorless and spectro- scopically free from absorption; if, however, urobilin is present in the serum, as is usually the case in pernicious anemia, then the filtrate presents a reddish color and shows a narrow band of absorption between band F. The collected precipitate in the absence of bilirubin (in normal serum and the serum of chlorosis) is white, but in.the presence of bilirubin (in the serum of pernicious anemia) of a slight yellowish color. ‘The precipitate is washed with hot water, boiled with acidu- ! Zeit. f. phys. Chem., vol xxxix, p. 143, 50 THE BLOOD lated alcohol (sulphuric acid) and the mixture filtered. In the — presence of bilirubin the alcohol is colored a fine green and_ the coagulum presents the same color; in the absence of bilirubin the © aleohol remains colorless. In order to test for biliary acids, the blood is first treated with alcohol, in order to remove the proteids. ‘The biliary acids which are present in the filtrate are next transformed into their lead salts by means of lead acetate and ammonia and thus precipitated. After washing with water the precipitate is boiled with alcohol and filtered. ‘The lead salts are decomposed by means of sodium carbo- nate, the solution is again filtered, the filtrate evaporated to dryness, and the residue extracted with absolute alcohol. ‘The alcohol is dis- tilled off, when the biliary salts of sodium will crystallize out or remain behind as an amorphous mass, which may be tested directly according to Pettenkoffer’s method. ‘l’o this end, some of the residue is dissolved in water and treated with two-thirds of its volume of concentrated sulphuric acid, care being taken that the temperature — does not rise beyond 60° C. ‘To this mixture a few drops of a 20 per cent. solution of cane sugar are added, when in the presence of biliary acids a beautiful violet color is obtained, which is referable to the action of furfurol, formed from the cane sugar and the acid, upon the biliary acids. Acetone.—Acetone has been found in the blood in considerable amounts under various pathological conditions, and especially in diabetes and fevers. | In order to demonstrate its presence, Dennige’s test may be employed: 5 c.c. of blood are treated with about 30 c.c. of Dennigé’s reagent and allowed to stand until the dark-brown precipitate has settled to the bottom. ‘The supernatant fluid is filtered off and_ treated with a little more of the reagent, so as to ensure complete precipitation. It is then acidified with sulphuric acid and heated as— described. ‘lhe formation of a white precipitate, which is soluble in an excess of hydrochloric acid, is referable to acetone or diacetie acid. (See Urine.) LITERATURE.—v. Jaksch, Acetonurie u. Diaceturie, Berlin, 1885. Reale, Schmidt’s Jahrbuch., 1892, p. 106 (Extract). Choiin.—Cholin has been demonstrated by Moth and Halli- burton in the blood in diseases of the nervous system which are associated with a destruction of nerve tissue; notably in general paresis, tabes, combined sclerosis, disseminated sclerosis, alcoholic — polyneuritis, beriberi, and following the division of both sciatic. nerves in cats, Meriop.—Five c.c. of blood are treated with from six to eight times that amount of absolute alcohol and filtered. The filtrate is dried at 40° C., and the dry residue extracted three times with absolute | itl | MICROSCOPIC EXAMINATION OF THE BLOOD 51 alcohol, filtered, and the solution evaporated. The alcoholic solution of the residue is precipitated with a 10 per cent. alcoholic solution of platinum chloride and the precipitate decanted from the absolute alcohol. ‘The precipitate is finally dissolved in 15 per cent. alcohol, the solution filtered and evaporated in a watch crystal at 40° es With a low power the octahedral crystals of cholin-platinochloride ean then be seen. ~ Normal human blood (in the amount mentioned) rarely gives rise to such crystals, so that the result is practice ally negative. Sine qua non for the success of the method is that the ean is absolute; 99 per cent. will not suffice. [See also Donath’s method (Cerebro- spinal fluid). MICROSCOPIC EXAMINATION OF THE BLOOD. The Red Corpuscles. Variations in Size and Form.—The normal red blood corpuscles are greenish-yellow, circular bodies, which in postembryonic life are non-nucleated. While it has been generally accepted that the red cells are biconcave several writers have recently nsisted that they are bell-shaped (Weidenreich, F. 'T’. Lewis). ‘They ire possibly composed of fluid contents within a membrane of some ‘atty substance. ‘Their diameter varies between 6 and 9 y, with an werage of 7.5 4. ‘The presence of larger or smaller cells is abnormal. Smaller cells are termed microcytes and measure from 3.5 to 6 4; larger cells are known as macrocytes or megalocytes, and usually have a diam- oter of from 9.5 to 12 4; still larger specimens are spoken of as giant corpuscles (Hayem);' they may attain a diameter of 16 4. ‘The terms macrocytosis or microcythemia and macrocytosis or macrocythemia are used to designate a predominance of the corresponding variety. | As regards the origin of the macrocytes, there is evidence to show that they may result from the common normocytes in the circulating vlood through imbibition of water, so that their occurrence from his point of view could be regarded as a degenerative phenom- ¢non. But, onthe other hand, the presence of macrocytes may be nterpreted as evidence of a regenerative process, bearing in mind that in the bone-marrow the size of the erythroblasts is larger than that of the common normocytes; the macrocytes would thus repre- sent young normocytes which have prematurely found their way nto the circulation. ‘The microcytes probably result from the normo- sytes in the circulating blood through loss of water r; whether their presence may at any time be regarded as the expression of a regen- -rative process seems questionable. Not infrequently microcytes ire formed artificially during the preparation of the specimen. 1 Le Sang, Paris, 1891. ) 0) — 52 THE BLOOD Microcytosis is, on the whole, of comparatively little clinica interest, and may be observed in any severe anemia. Macrocytosis is more important. ‘To a certain extent it is seen in severe forms of anemia of whatever origin, but it is noteworthy that the presence of macrocytes in large numbers is essentially observed in pernicious’ anemia. During the active period of the disease the macrocytes— may here represent 70 per cent. of all red cells (Lazarus). ‘The condition, however, is not constant. } Going hand in hand with pathological variations in the size of the. red corpuscles—anisoc ytosis—there are variations in form which may affect not only the microcytes and macrocytes, but also the corpuscles of normal size. Cells may thus be seen which resemble a flask, a kidney, a biscuit, a boat, a balloon, a dumb-bell, or an anvil, while others are altogether irregular in appearance. Os # > wer Pe noe - Pe g” ed bs ese ae ees tt "he S,, ‘ > eo ii. 12 ‘ ca ; Eee Le £m, & “> P 2 . = | ‘ | Ms, iy (en hy, Ps ae & eee >» & . i Loe . oo i Z Fig % - v fr a ed Fig. 14.—-Poikilocytosis., 5 ‘ Especially interesting is the fact that such abnormally formed cells, — which are generally spoken of as povkilocytes (Fig. 14), may manifest a certain degree of motility, so that they have at times been mistaken for microparasites. ‘This is seen especially in marked cases of per- nicious anemia, and is most noticeable in the smaller forms. In pernicious anemia povkilocytosis is most pronounced, and at one time it was thought that the condition was characteristic of the disease. — It has been shown, however, that it occurs in other anemias as well, though its occurrence is probably always evidence of a specially severe form. In chlorosis it is usually only seen in the most severe | cases, and particularly in those manifesting a tendency to throm-— bosis and embolism. In this connection a special deviation from the normal form of the red corpuscles also requires consideration, viz., the prevalence of | PLATE II. The Elements of Normal Blood. , red cells in rouleaux; b, crenated red cells; c, finely granular (neutrophilic) leukocytes; d, rsely granular (eosinophilic) leukocytes; e, small, and f, large mononuclear leukocytes; alaques. Le J * om ‘ - ace oo i. ed a aS, 4 a ae a - : _s. A> A) : . = . } 4 1). ae ys . [ J ' ~ - . 4 : ‘ e - 7) , \- MICROSCOPIC EXAMINATION OF THE BLOOD 53 oval cells. ‘hese are notably observed in pernicious anemia and seem to be of distinct diagnostic importance. ‘hey are found not only during the active periods of the disease, but frequently also in the interval between exacerbations. Poikilocytosis is a degenerative phenomenon, and it is essential not to confound true poikilocytes with certain abnormal forms, _ which may be seen in any normal preparation and which are the result _of mechanical injury, mutual compression, etc., and can readily be distinguished with practice. ___In wet preparations red cells will be seen near the margin of the _drop where evaporation is actively going on, which present little knobs or spicules on their surface and along the periphery. Such cells are spoken of as crenated cells. ‘he phenomenon in itself is normal, but it is noteworthy that crenation may at times be observed in the centre of a carefully prepared specimen after a few seconds already, while as a rule from fifteen to thirty minutes elapse before _ the process begins to attack cells in this location. ‘The significance of this early crenation is not known. ‘This is also true of delayed money-roll formation, which is observed in various hepatic diseases, in pneumonia, nephritis, etc., whereas normally the red corpuscles tend to agglutinate in this form immediately unless special pains have been taken to secure the separation of the individual cells. (See Plate II.) Variations in the Color of the Red Corpuscles.—'[‘he degree of color- _ ing of the red corpuscles depends upon the amount of hemoglobin. _ The centres of the cells in well-mounted specimens are always paler than the periphery, and any deficiency in the amount of coloring matter is here at once apparent. With a moderate grade of anemia the cell as a whole looks paler, and the pale central area is increased in size. With a further increase in the loss of coloring matter the central area is absolutely colorless and encroaches upon the peripheral colored zone more and more until finally the so-called pessary forms result, in which only a narrow rim of hemoglobin remains. ‘These changes can be made out in wet preparations, but are especially well seen in stained specimens. - ‘The central pale area is, however, visible only in well-preserved cells and not in flattened out cells, which are stained uniformly throughout and which may also be seen in any specimen. The color of the normal red cells in wet specimens is a pale greenish- yellow. In malaria curiously discolored corpuscles are seen, which present a bronzed appearance; their presence should always excite Suspicion. ‘The meaning of the discoloration is not known, but in all probability it is evidence of a degenerative process. The Color Index.—The term color index is used to designate the _ Telative amount of hemoglobin which is contained in each corpuscle. It is determined by dividing the percentage of blood coloring matter - 54 THE BLOOD by the percentage of red cells as compared with the recognized normal, viz., 5,000,000. Exampte.—The percentage of hemoglobin is 50, the red count — per cbmm. is 2,000,000, viz., 40 per cent. of the recognized normal, 5,000,000. ‘The color index is then 50 divided by 40—~. e., 1.25. Under normal conditions the color index is about 1, but may vary from 0.95 to 1.17; it is slightly higher in men than in women. In the secondary anemias, in which the decrease in the amount of hemoglobin is proportionate to the diminution of the red corpuscles, the color index is approximately normal. But in the majority of cases the diminution of the hemoglobin somewhat exceeds that of — the red cells, so that lower values are commonly met with. In pernicious anemia, on the other hand, where the corpuscular decrease usually exceeds the diminution of the hemoglobin, a high color index — is the rule. ‘There may be periods in the course of the disease, how- ever, in which a normal index and even subnormal values are found. In the chronic cases lower figures are more commonly obtained than — in the acute cases. In the series of 22 cases collected by Strauss and — Rohnstein the value of the color index varied between 0.5 and 1.95. — In 8 eases of the series variations from 1.13 to 1.95 were observed, and in 6 lower values than 1 were noted, viz., 0.5 to 0.9. Cases in which the color index falls as low as 0.5 are rare in pernicious anemia. In the one instance in the series in which this was found, the hemo- — globin was only 10 per cent., while the red cells numbered 1,048,000; there was a high grade of poikilocytosis and all transitions between — the smallest microcytes and the largest types of macrocytes. In well-established hookworm anemia in contradistinction to the cryptogenetic type of pernicious anemia the color index is low (Ashford). In the secondary anemia of carcinomatosis the color index rarely exceeds 1. In Strauss and Rohnstein’s series! of 35 cases the highest value was 1.1 (in one case only); in the rest it varied between 0.53 and 0.96. In chlorosis, in which the degree of oligochromemia exceeds the corpuscular loss the color index is markedly lowered; in especially severe cases it may fall to 0.3 and even lower. But it is not ad- missible to make the diagnosis of chlorosis on this basis only, as it is fairly common to meet with a markedly lowered color index in some | secondary anemias also, and especially in the form which is referable to carcinoma, as has just been mentioned. In splenic anemia like-. wise the degree of oligochromemia may far exceed the degree of oligocythemia. Variations in Number.—'’he number of red corpuscles in the blood of healthy adults is fairly constant. In man 5,000,000 may ‘ Die Blutzusammensetzung b. d. verschiedenen Anemien. Berlin, 1891. MICROSCOPIC EXAMINATION OF THE BLOOD 55 be considered a fair average, and in women 4,500,000. Higher -yalues are not uncommon, but rarely exceed 6,000,000 in perfectly - normal individuals. The largest number is found on the first day after birth, average 6,985,428. It diminishes until the third day. Following a temporary rise it drops farther and becomes fairly constant between the sixth and the tenth day.’ In 20 healthy infants Karnizki’ obtained the following values: Age. nana Tee | 6.239,725 See se) S68 8,703,000 to 5,848,000 8-12 “ eee eee. 0,551,000 to 5,590,521 Then the number increases, especially after the sixth year, and remains on an average higher during childhood than in babyhood. A somewhat higher average is found among people living at a _ considerable elevation above the sea level, and it is interesting to note that an increase in the number occurs whenever a change in the habitation is made from a lower to a higher level. ‘This increase is frequently quite marked, as is apparent from the following table, which is taken from Ehrlich: Altitude Increase of 561 meters em Cee Rea er ae > tS mee 800,000 700 tet cl ee oy ae Ry oy 1,000,000 1800 em sem! ete) mee sx) 2,000,000 4392 a aera ee ee 3.000 000 A corresponding diminution occurs when a change is made from a higher to a lower level. In this connection Gaule’s* observations are of interest. On - the occasion of a balloon ascension to a height of from 4200 to 4700 meters he counted 7,040,000, 8,800,000, and 7 ,480,000, respectively, in the three participants of the journey. ‘T he hemoglobin was at the same time diminished, and he accordingly concluded that the increase during the ascent was due to an increased production of red cells; the probable nature of this conclusion was strengthened by the fact that numerous normoblasts were found in the blood, many under- going division. Jolly and Bensaude’ and others on similar expeditions were unable, however, to demonstrate the presence of nucleated red cells or to note the occurrence of an increased number of red cells. According to Weinzirl," the increased counts due to high altitude are * Scipiades, Arch. f. Gyn., vol. ixx, p. 630. 2 Arch. f, Mivirlerheilic: 1903, vol. xxxvi. * “Die Animie,” Nothnagel’s ’specielle Path. u. Therap., vol. viii, part. i. ; Compt. -rend., vol. exxxiii, p. 903. * Compt.-rend. ’ Soe biol., vol, liii, p. 1084. Saint Martin, Soe de biol., July 23, 1904. é Amer. Jour. Med. Sci., 1903, vol. exxvi, p 299. 56 THE BLOOD temporary and in part at least referable to cold. He showed that in rabbits a certain increase in the number of red cells occurs when they are removed from warm to cold quarters, and that their subsequent removal to a higher altitude does not lead to a further increase. Clinically we distinguish between relative polycythemia in which | the condition is due to a diminution in the quantity of plasma, and true polycythemia in which there is an actual increase in the number of the red corpuscles. Relative polycythemia is much the more common. In some instances it is due to loss of liquid by sweating, | diarrhea, or increased diuresis. In another group of cases there is loss of liquid by secretion or transudation, as in obstruction of the pylorus with dilatation of the stomach, and in the constant loss of liquid from the blood in recurring ascites. In, some of these cases the polycythemia is of high grade, and may persist for years. In advanced cases of nephritis, phthisis, malignant disease, etc., there is also a certain grade of relative polycythemia, due to loss of water from the body at large. ‘The polycythemia which is noted in poisoning by phosphorus and carbon monoxide (in one case of coal-gas poisoning a count of 11,200,000 is reported), various ‘coal-tar products, during and immediately after the administration of ether, following cold baths and severe muscular exercise, also belongs to this order and is no doubt referable to vasomotor disturbances. Of similar origin probably is the polycythemia which is noted in disease of the adrenal glands, where counts of from 6,000,000 to 7,000,000 have been repeatedly noted; and the same is probably true of diabetes, in which poleythemia may be observed both while fasting and while much fluid is being ingested. ‘True polycythemia is met with in diseases in which there is difh- culty in proper aération of the blood, as in heart disease, and in a peculiar type of chronic cyanosis which has been described by Osler’ as a. new clinical entity, the so-called autotoxic enterogenous cyanosis. In acquired heart disease with continued inadequacy of the circulation of slight degree a moderate grade of polycythemia is very common; in the congenital form the figures often reach 8,000,000 to 9,000,000. ‘The highest values are seen in Osler’s disease, however. In the first nine cases which have been reported the highest count was 12,000,000; in eight it was above 9,000,000, and in the ninth it was 8,250,000. ‘The usual range of hemoglobin at the same | time was from 120 to 150; the specific gravity varied between 1.067 and 1.083, and the leukocyte count between 4000 and 20,000; as a rule it was below 10,000. Vaquez? notes that whereas in congenital - heart disease and the coincident polyglobulism the diameter of the | ‘Stengel, Proc. Path. Soc. Phila., 1899. Oertel, Deutsch. Arch., vol. i, p. 293. * “Chronic Cyanosis with Polycythemia,’”’ Amer. Jour. Med. Sci., 1903, vol exxvi, p. 187. * Soc. biol., 7 Mai, 1892 MICROSCOPIC EXAMINATION OF THE BLOOD 57 red cells’ is increased from 7.5 to 8.5, this is not observed in the idio- athic form of cyanosis. While there can thus be no doubt that a true polycythemia does occur, it has been conclusively demonstrated that such a condition does not exist in what is generally termed plethora, and that the _yarious symptoms of plethora formerly attributed to a general increase in the amount of blood are referable to vasomotor disturbances. Oligocythemia, viz., a diminished number of red cells, is much more common than polycythemia. It may be temporary or perma- nent, and is seen in all forms of anemia of whatever origin. It is most marked in pernicious anemia. ‘The exact figure will here, of course, depend upon the stage of the disease and the individual case. _ A decrease to one-half of the normal number may be seen in com- paratively mild cases; a million red cells is a common count. ‘The number may fall to 500,000 and even lower. In one case reported by Quincke* a count of 143,000 was observed, and it is interesting to note that seventy-four days later the same patient had 1,254,000 ercbmm. Osler’ reports a case in which shortly before death the red cells fell below 100,000. ‘This is the lowest count that has been recorded. In the stage of amelioration they may rise to 4,000,000 and even higher. In the series collected by Strauss and Rohnstein® 1,240,000 was the average at the time when the patient first came under observation, and in Cabot’s series of one hundred and ten cases the average number is almost identical—1,200,000. In chlorosis, contrary to what is found in pernicious anemia, the red cells are usually not much diminished. In Cabot’s’ series of seventy-seven cases the average count was 4,050,000. At times, however, cases are met with in which the diminution of the red cells _ almost keeps step with the diminution in the amount of hemoglobin. Von Limbeck cites three cases with 1,750,000, 1,850,000, and 1,930,000, respectively; and Hayem mentions an instance in which only 937,360 cells were counted. Such cases are exceptional. As in chlorosis so also in splenic anemia, the corpuscular anemia is of very moderate grade, even though the diminution in the amount of hemoglobin may be considerable. Of the forty-one cases collected by Osler, the average was 3,425,000; the lowest count was 2,187,000 and the highest 5,200,000. A similar condition is found in Kala-azar, where the number of red cells is commonly reduced to 2,000,000 to 3,000,000. In leukemia the red cells are usually not diminished to a very great extent; and the oligocythemia is generally more marked in the lymphatic than in the myelogenous variety; the average figures 1 Centralbi. f. d. med. Wiss., 1877, No. 47; and Deutsch. Arch., 1877, vol xx. * Johns Hopkins Hosp. Bull., 1902, vol. xiii, p. 251. . Loe. eG. * Clinical Examination of the Blood, Wm. Wood & Co. bs 58 THE BLOOD in Cabot’s series are 2,730,000 and 3,120,000, respectively. Counts of 1,000,000 or thereabout may, however, be met with. In pseudoleukemia the red cells may be only moderately dimin- ished, viz., between 3,000,000 and 4,000,000, but in some cases the corpuscular destruction is quite active, and in the last stages of the disease values may be found which are not much above 1,000,000_ or 1,500,000. | The count which is obtained in post-hemorrhagic cases will depend very largely upon the amount of blood lost and the time at which | the examination is made. ‘The lowest counts, according to Lyon,’ Hiihnerfauth,’ and Siegel-Maydl,’ are found between the second and the eleventh day. In Rieder’s* case the figures varied between 1,300,000 and 3,335,000; in those of Strauss and Rohnstein,® be- tween 1,119,000 and 4,420,000. A sudden reduction in the number to 1,000,000 or less is usually followed by a fatal result. ) In the anemias of infancy and early childhood the oligocythemia is often very pronounced. In the infantile pseudoleukemia of v. Jaksch especially low values may be found associated with an increase of the leukocytes of such extent that the ratio between the two may be suggestive of true leukemia; there is, however, no myelemia, but an increase of the normal types. In infantile leu- kemia of the lymphatic variety McCrae found 2,350,000 as the highest count. An extreme and rapidly progressive anemia is frequently noted in acute streptococcus infections. Grawitz® states that according to Rocher’s investigations it is probable that the diminution of the red cells in septicemia is greater than in any other infectious disease and appears in a shorter time. Cases may indeed be encountered in which the question of pernicious anemia may enter into the diag- nosis, as occurred in two cases of gonorrheal endocarditis which were observed by Osler. An extreme grade of corpuscular destruction is also noted in malaria. In acute cases the loss of red cells during the first twenty- four hours may reach 1,000,000, and in two days even 2,000,000. In neglected chronic cases the count usually varies between 3,000,000 and 4,000,000; the oligocythemia may, however, be far more ex- tensive, and Ewing’ cites a case observed by Kelsch, with only 583,000 red cells per cbmm. The anemia observed after typhoid fever is as a rule not very severe, but exceptional cases occur in which the loss of red corpus- 1 Virchow’s Archiv, 1881, vol. xciv. AP DIDS Val xs Vas ’ Wien. med. Jahrbuch., 1884. * Beit. z. Kenntniss d. Leukocytose, Leipzig, 1892. PLLOEACIG, * Klin, pathol. d. Blutes, Enslin, Rerlin, 1902. 7 On the Blood, Lea Bros. and Co., 1901. MICROSCOPIC EXAMINATION OF THE BLOOD 59 cles is considerable. Osler cites an instance in which the number fell to 1,300,000. The post-rheumatic anemia is usually not so pronounced. In acute endocarditis Stengel’ has noted a rapid decrease of the red corpuscles, often to 50 and even 40 per cent. In pulmonary tuberculosis the number of the red corpuscles runs a course parallel to that of the hemoglobin. Oligocythemia is really only seen during the third stage (2,000,000 to 2,500,000); while during the second stage, owing to an actual concentration of the blood (Grawitz), normal figures are the rule. In the first stage a diminution of their number (3,800,000) is only seen in patients who have repeat- edly suffered from tuberculous affections (scrofula) since childhood, and in whom the onset of the pulmonary disease has been gradual, while, on the other hand, normal values are found in individuals who appear to be in perfect physical health, who are well nourished, with well-shaped chests, and without hereditary predisposition (Appel- baum).’ In acute gastritis, and usually in chronic gastritis also, the number of the red corpuscles is not diminished, while in carcinoma a marked oligocythemia occurs at some time in the course of the disease. In the earlier stages, however, this is often but little marked, and at times an apparent increase of the red cells is noted (relative poly- cythemia). Later a diminution is probably always found. In the severer forms of chronic gastritis a diminution is also fairly constant, but rarely so marked as in carcinoma, if we except those cases of gastric anadeny which present the clinical picture of a pernicious anemia. In the differential diagnosis between carcinoma of the stomach and pernicious anemia a count below 1,000,000 points to the latter disease. In ulcer of the stomach anemia of the chlorotic type “is very common. In Cabot’s series of 51 cases, 42 (80 per cent.) had a hemoglobin value of less than 50 per cent., and in the Hopkins series, reported by Futcher, the average value was 58 per cent., with a red count of 4,071,000. When hemorrhages have recently occurred the blood count may of course be very low. In the majority of cases of rickets there is no material diminution in the number of the red cells, while the hemoglobin may be much reduced, but in the severer forms with visceral complications there may be oligocythemia of extreme grade. vy. Jaksch cites a case in which the red count fell from 1,600,000 to 750,000 within three months, and Luzet noted a drop to 500,000 within three weeks (Ewing). In congenital syphilis the oligocythemia is usually marked, except- ing in very mild cases, and in the severer infections the blood picture may simulate that of pernicious anemia. © 0Gs Gits,, De 3nd. 2 Berl. klin. med. Woch., 1901, vol. xxxix, p. 7. 60 THE BLOOD Behavior toward Aniline Dyes. Polychromatophilia (Polychro- masia).—'[he normal living red cell possesses no affinity for dyes; it is achromatophilic. ‘The normal fixed cell of the circulating blood, — on the other hand, has a marked affinity for acid dyes, such as eosin, ~ orange-G, acid fuchsin, etc.; it is accordingly said to be oxyphilic, and as it takes up only one color from a mixture of different dyes it is termed monochromatophilic. Under various pathological condi- — tions which are associated with a marked grade of anemia cells are — met with which are polychromatophilic. Such cells manifest an affinity not only for acid dyes, but simultaneously also for basie dyes, so that with a mixture of hematoxylin and eosin, or eosin and — methylene blue, the red cells are not stained in the usual tint of the — hemoglobin, but present a mixed color in which the tint of the basic | dye is more or less apparent (Plate IIT). As regards the significance of the polychromasia, Ehrlich main- | tained that: the condition was evidence of a degenerative process— of a coagulation necrosis of the discoplasm as a consequence of which — this takes up albumins from the blood plasma, while it loses the — power of holding its hemoglobin. ‘The oxyphilia hence diminishes, ~ while owing to the absorption of albumins a more or less well-marked — basophilia develops. As a matter of fact polychromatophilia is often — seen in cells which are manifestly degenerating, and in myelogenous — leukemia especially one frequently meets with nucleated red cor- — puscles which are markedly polychromatic and in which the proto- — plasm is evidently undergoing destruction, often appearing merely — as a little hood attached to one side of the nucleus (see Plate III). © Ehrlich accordingly speaks of an anemic or polychromatophilic — degeneration of the blood. But, on the other hand, there is evidence to show that polychromasia may be the expression of a regenerative process, and we find as a matter of fact that the erythroblasts of the normal bone-marrow are for the most part polychromatophilic, and the more markedly so the younger they are. Megaloblasts are proba- — bly always polychromatophilic (Plate III). Welker has shown that basophilic red cells are normally found in pigeons, mice, guinea- pigs, cats, and dogs, while they are absent in the horse and the ox. I have also found them in the blood of birds, reptiles, amphibia, and — fishes. In those animals, moreover, in which the red cells of the © circulating blood are normally nucleated a certain grade of poly-— chromasia, according to my experience, appears to be the rule in all the younger cells; the pure hemoglobin tint is only found in the mature forms. Of late, Ehrlich has admitted the existence of a physiological polychromasia, but he still maintains that it may also occur as the expression of a degenerative process. = * r* ia” a, a group of red cells undergoing granular degeneration; b, red cells showing Cabot’s ring eS; c¢c,normoblasts with nuclei undergoing karyolysis; the bodies of the cells show granular wneration ; d, normoblast with pyknotic nucleus; /, red cell, suggesting loss of nucleus by extrusion; cell undergoing mitosis; h, megaloblasts with polychromasia of protoplasm; 7, gigantoblast ; pung normoblasts, showing spoke-shape arrangement of the chromatin; /, a group of plaques. il m MICROSCOPIC EXAMINATION OF THE BLOOD 61 Lirerature.—Ehrlich, Charité Annalen, vol. x, p. 136. Engel, Deutsch. med. Woch., 1899, p. 209. Gabritschewsky, Arch. f. exp. Path., vol. xxviii, p. 83; Zeit. f. klin. Med., vol. xxvii, p. 492. Askanazy, ibid., vol. xxi, p. 415. Maragliano and Castellino, ibid., vol. xxi, p. 415. Diabetic Chromatophilia—Bremer has pointed out that a distinct difference exists in the affinity of diabetic blood for certain aniline dyes, as compared with non-diabetic blood. For, whereas non- diabetic blood is readily stained with Congo-red, methyl blue, eosin, etc., diabetic blood is distinctly refractory, while such dyes as Biebrich scarlet, which readily stain the diabetic blood, do-not color non- diabetic blood. Regarding the nature of the substance in diabetic blood which is responsible for this peculiar behavior, little is known, but it appears certain that the reaction is not dependent upon the presence of glu- cose nor upon the degree of alkalinity of the blood, as suggested by Lépine and Lyonnet. Bremer’s claim that the reaction is pathog- nomonic of diabetes and glucosuria and may even yield positive results in the pre-diabetic stage of the disease, and when the sugar has temporarily disappeared from the urine, has been confirmed in all essential points, both in this country and abroad. A few inter- esting exceptions, however, have been noted. In animals, for example, in which glucosuria has been artificially produced by means of phlorhizin, the reaction does not occur, whereas in phloro- glucin-diabetes positive results are obtained. In Bremer’s entire series of diabetic cases a negative result was obtained but once, and in this instance he believes that the diabetes was of the renal type, and analogous to the phlorhizin-diabetes of animals. He suggests that it may thus be possible to differentiate this form from the hema- togenic variety, using the latter term in its widest sense. Lépine and Lyonnet report a positive result in one case of leukemia, but Bremer believes this to have been due to faulty technique. Hartwig finds that Bremer’s reaction is constant in diabetes, but that it may also occur at times in other conditions. The description of Bremer’s diabetic blood test is omitted at this place, as it has not proved practical for routine work. For a consideration of the technique see the Literature below. LireraturEe.—L. Bremer, “ An Improved Method of Diagnosticating Diabetes from a Drop of Blood,” N.Y. Med. Jour., 1896; Centralbl. f. inn. Med., 1897, p. 521. Le Goff, React. chrom. du sang diabet., Paris, 1897. Lépine and Lyonnet, Lyon méd., vol. Ixxxii, p. 187. Hartwig, Deutsch. Arch. f. klin. Med., vol. lxii, p. 287. Granular Degeneration of the Red Cells.—Under pathological conditions red cells may be met with which contain basophilic gran- ules. ‘These are readily stained with methylene blue, methylene azure, thionin, ete. Methyl green, however, which is a specific unclear dye, does not stain the granules. ‘Their size, form, and number 62 THE BLOOD are variable. While the majority are round, others are rod-shaped — or biscuit-shaped. ‘The largest granules are found in pernicious : anemia and in cases of lead poisoning with intestinal manifes- tations. ‘They are then quite readily seen and attract attention at once (Plate III). In most other diseases in which they occur they — are much smaller, and on superficial examination they may indeed be overlooked; some cells at first sight merely look a little off-color, — and it is seen only on very careful examination that the apparent polychromasia is in reality due to the presence of large numbers of minute dots. Very often, in especially anemic cells, the granules — are arranged in the peripheral portion of the cell. ‘Their number is exceedingly variable; generally speaking, it depends upon their size; when they are especially large they are relatively less numerous. The granules may occur in cells of normal size or color, in poiki- locytes, and in nucleated red cells, both of the normoblastic and the megaloblastic type, especially the former. Not infrequently they are seen in cells which are markedly polychromatic, but, like Grawitz, I do not believe that granular degeneration represents a phase of — polychromasia. : In disease they are most constant and numerous in pernicious anemia, in lead poisoning, and in malaria; they are less constant and less numerous in the leukemias, in pseudoleukemia, in the cachexias referable to septic infection, syphilis, carcinomatosis, and in the final stages of tuberculosis. In chlorosis and in the anemia of chronic nephritis they are absent; in two cases of v. Jaksch’s anemia, in which nucleated red cells were quite numerous, I ob- tained negative results. In pernicious anemia granule cells are frequently found in the interval and at a time when the blood picture is otherwise practically normal. I have seen them most numerous in a case in which blood crises occurred from time to time (see page 60); almost every nor- moblast contained granules; non-nucleated granule, cells were how- ever, at the same time present in large numbers Late in the disease, or in aplastic pernicious anemia, granule cells in my experience may be absent. In lead poisoning granule cells are practically found without — exception, and may be encountered at a time when no clinical symp- _ toms are manifest. ‘The amount of lead which is necessary to call — forth their appearance is quite small, and it is a common experience — to meet with a small number after the administration of lead in medicinal doses. I have found them after the ingestion of only 0.5— gram given in divided doses in the course of forty-eight hours. In | cases of lead poisoning they persist for a long time after exposure has ceased. In one case of double wrist- and ankle-drop I could — still demonstrate granule cells after five months. In malaria granule cells are also common. Plehn found them q , MICROSCOPIC EXAMINATION OF THE BLOOD 63 in Europeans after a short sojourn in the tropics, and looked upon the granules as spores of the malarial parasite. In septic cases and in the cachexia of carcinomatosis they are not - numerous; in a case of cancer of the stomach with only 27 per cent. of hemoglobin, which I recently observed, I found no granule cells. In the early stages of phthisis granular degeneration is not seen, _ but it may occur later, when a general septicemia has supervened. Takasu’ notes the occurrence of granule cells in infants affected with beriberi, especially in the acute severe cases. ‘The same appar- ently occurs in the adult. __ As regards the significance of the granules, Engel, Ehrlich, and others have suggested that they are most likely products of karyor- -rhexis. Others maintain, and I think rightly so, that they are not of nuclear origin. ‘They may be found at a time when not a single nucleated red cell is demonstrable in the blood and nucleated red cells _may be seen in which no sign of karyorrhexis is manifest, while the _body of the cell is studded with granules. ‘They may be found in nucleated cells which are undergoing karyokinetic division. Unlike the nuclei of the erythroblasts, the granules have no affinity for methyl green, which is a specific nuclear dye. ‘This can be shown very well by staining with methyl-green-pyronin, when granular products derived from nuclei are stained green, while the stippling in the same cell appears red. A few observers claim to have stained the granules with methyl green; this merely shows that their dyes _ were contaminated with methylene blue. According to Grawitz and others granule cells are not commonly found in the bone-marrow even when they are numerous in the cir- culating blood; when they do occur, they are not more numerous than in the peripheral vessels, Grawitz hence regards their presence as an indication of a degenerative change in the hemoglobin, and speaks of the phenomenon as “granular degeneration.” Others regard the bone-marrow as their place of formation. Niageli’ thus comes to the conclusion that they are formed in the bone-marrow, because they only appear in artificial lead intoxication, when this is continuously established, and disappear when larger doses are given. Preceding the death of the animal they are not found: Opposed to the peripheral formation of the granules and Grawitz’s degeneration hypothesis is the occurrence of granule cells in the blood of embryos. According to Pappenheim stippling is not found in erythroblasts i in the bone-marrow under normal conditions, but only when there is excessive regeneration, as in the embryo, in pernicious hemolytic anemia, in myelophthisic neoplastic anemia, in myelogenous pseudo- leukemia and lymphadenoid leukemia and lymphosarcomatosis of the bone-marrow. Schmauch has observed similar appearances in the ? Folia hemat., vol. i, p. 501. * Minch. med. Woch., 1904. 64. THE BLOOD blood of healthy cats, and Engel has’ described the occurrence of granule cells in the blood of early cat embryos. I have found granule cells in the blood of various animals and occasionally one meets with an isolated cell in apparently normal individuals. Whether or not the granule cells which Vaughan* has demonstrated in normal wet specimens with Unna’s polychrome methylene blue are identical with the variety described above is not certain. ‘Their number varied quite constantly between 1.8 and 5 per cent. ‘The examinations were conducted with wet blood, a drop of the staining fluid being placed upon the site of the puncture. At first the granules are red, but after some time they change through a purple to a pro- nounced bluish. Positive results were also obtained under various pathological conditions, especially in pernicious anemia, where their number was about ten times as great as in normal blood. In new- born infants they average 4.7 per cent. Vaughan regards the gran- ules as nuclear remains and states that he rarely found stippling and nuclei in the same cell. In my own experience normoblasts in pernicious anemia are very frequently granular (see Plate III). Analogous results have been obtained by Cadwalader.’ Not to be confounded with “granular degeneration’’ is the stippling of Schiiffner,* Ruge,* and Goldhorn, which is seen in many red cells” infected with tertian parasites. ‘This is brought out with methylene azure and may also hide the parasite from view. LITERATURE.—E. Grawitz, “ Ueber kérnigeDegeneration d. rothen Blutzellen,” Deutsch. med. Woch., 1899, No. 36, p. 585; “ Klinische Bedeutung u. experiment, Erzeugung kérniger Degenerationen,” etc., Berlin. klin. Woch., 1900, p. 181; “Granular Degeneration of the Erythrocytes,” ete., Amer. Jour. Med. Sci., 1900, vol. exx, p. 277. Bloch, Deutsch. med. Woch., 1899, V. B. p. 279. Litten, ibid., No. 44. Behrendt, ibid., No. 44. White and Pepper, “Granular Degeneration of the Erythrocyte,’ Amer. Jour. Med. Sci., 1901, vol. exxii, p. 266. C. E. Simon, International Clinies, 1902, vol. i, p. 69. Stengel, White, and Pepper, Amer. Jour, ~ Med. Sci., 1902, vol. exxiii, p. 873. Cabot’s Ring Bodies.—Cabot has drawn attention to the occa- sional occurrence in red cells of curious ring bodies which are usually stained red with Wright’s modification of Leishman’s stain, but which may also take on a blue color. He found such rings in per- nicious anemia, in lead poisoning, and in lymphatic leukemia. I have been able to demonstrate the same structures with the eosinate of methylene blue, and could verify Cabot’s observation that they _ occur in granule cells, but may also be found in apparently normal red corpuscles (Plate III). No doubt they bear some relation to the nucleoids. (See Fig. 15.) LITERATURE.—Cabot, Jour. Med. Research, 1903, vol. ix. 1 Jour. med. Res., December, 1903. 2 Amer. Jour., February, 1905, p. 213. 3 Deutsch. Arch. f. klin. Med., 1899, vol. lxiv, p. 428. * Zeit. f. Hyg. und Infectkrht., 1900, vol. xxxiii, p. 178. | | : | } MICROSCOPIC EXAMINATION OF THE BLOOD 65 Ehrlich’s Hemoeglobinemic Innenkorper. —'‘l‘hese structures may be encountered in red cells in conditions associated with extensive hemocytolysis the result of specific blood poisons. ‘The individual, body is round and characterized by its affinity for acid dyes. Fia, 15.—Cabot’s ring bodies. Nucleated Red Corpuscles. Erythroblasts.—Nucleated red cor- puscles are not found in the circulating blood of normal individuals, excepting at birth and during the first days of life, when it is not unusual to meet with an occasional cell of this type. In the bone- marrow, however, they are always found. It is here possible to dis- _ tinguish two types, viz., the normoblast and the megaloblast. The latter is ontogenetically the older and gives rise to the normoblast through a process of homoplastic differentiation by cell division; it thus bears the same relation to the normoblast which exists between the large lymphocyte and the small lymphocyte, and the amblychro- matic myelocyte and the trachychromatic myelocyte (which see). The megaloblast itself results from the large lymphocyte through direct heteroplastic transformation and ages into the macrocyte, while the normoblast similarly develops into the normocyte. (See schema on p. 72.) While at a certain period of embryonic life megaloblastic blood corpuscle formation plays a prominent role, megaloblasts are found only in small numbers in the bone-marrow of the normal adult. Normoblasts, on the other hand, are numerous and control the usual red corpuscle production exclusively. The Normoblasts—The normoblasts (see Plate III), like the normal red cells of the circulating blood, have a diameter which 5 i 66 THE BLOOD varies from 6 to 9 y. ‘The nucleus in the youngest cells occupies a central position, and is larger and relatively poorer in chromatin ‘than in the older cells, where it is frequently located eccentrically. The size varies between 2 and 4 y. ‘The appearance of the normo- blast in the peripheral circulation is variable (Plate III). In most cases young cells are seen with a radiary arrangement of the chro- matin and polychromatophilic protoplasm. At other times older cells with densely staining pyknotic nuclei and oxyphilic protoplasm are encountered and again we may meet with cells in which manifest karyolysis is going on, as evidenced by budding of the nucleus and diminished chromatophilia. Fragmentation of the nucleus (karyor- rhexis) may also be seen, as also free nuclei as such. Mitoses are not uncommon in pernicious anemia and leukemia.* In the majority of cases in which normoblasts are found in the blood these are well preserved, but in myeloid leukemia more — especially it is common to meet with cells in which the protoplasm surrounding the nucleus is much diminished in amount and presents a ragged outline. ‘These cells are manifestly degenerating, and in many specimens the protoplasm will be seen reduced to a little hood which is attached to one side of the nucleus (Plate III). Such cells in my experience are always polychromatophilic and are apt to be mistaken by the beginner for lymphocytes. The occurrence of normoblasts in the circulating blood is always evidence of stimulation of the bone-marrow, which may occur either indirectly, as the result of an “anemic” condition of the blood (second- ary myelopathy), or directly, as in disease of the bone-marrow per se (primary myelopathy). We may accordingly meet with normo- blasts in almost any form of anemia, be this the result of traumatism (posthemorrhagic), of inanition, or of organic disease. In the acute forms of anemia they are apt to be most numerous, but even in the more chronic cases and in cachectic conditions specimens of blood may be obtained in which one or more normoblasts are seen in every field. In the secondary anemias, however, they are less common. In active cases of pernicious anemia and the different leukemias normoblasts are quite constantly met with in fairly large numbers. Their continued absence in pernicious anemia is usually evidence of an aplastic condition of the bone-marrow and a bad omen. At times there occur sudden invasions of the circulating blood by red cells, many of which are nucleated; this phenomenon y. Noorden terms a blood crisis, and it is noteworthy that the invasion of the red cells may be preceded and accompanied by a very ex- tensive increase of the leukocytes. Ehrlich cites a case of hemor- | rhagic anemia, reported by v. Noorden, in which at the time of such a blood crisis the normoblasts were so numerous, while hyperleuko- *G. Dock,“ Mitosis in Circulating Blood,” Trans. Assoc. Amer. Phys., 1902, p. 484. al MICROSCOPIC EXAMINATION OF THE BLOOD 67 cytosis of a high grade existed at the same time, that the blood con- dition strongly suggested the existence of a myeloid leukemia. ‘The increase of the red cells in this case amounted to almost double their original number. To estimate the extent of a blood crisis, the following examina- tions are necessary: (a) A determination of the absolute number of red corpuscles. (b) A determination of the ratio between the white and red cells. (c) Adetermination of the ratio between the nucleated red and white cells. EXAMPLE.—Supposing that in a given case 3,500,000 red corpus- cles are found in the cbmm., while the ratio of the white to the red corpuscles is 1 to 100, and that of the nucleated red to the white 1 to 100; 3500 nucleated red corpuscles must hence be present in each — ebmm. of blood—i. e., | for each 1000 of normal red corpuscles. The Megaloblasts.—These are usually from two to three times as large as the normoblasts, and may attain even more extensive propor- tions (Ehrlich’s gigantoblasts). (See Plate III.) But some specimens are only a very little if at all larger than the common red cells; these probably represent young daughter cells. ‘The megaloblasts are rovided with a relatively large centrally located nucleus, which is wide-meshed and which with the triacid stain is not colored nearly so deeply as the normoblastic nucleus. In some specimens, indeed, the affinity for methyl green is so little marked that at first sight a nucleus can hardly be distinguished. With those staining mix- tures, on the other hand, which contain methylene blue as base, it _ can always be fairly well made out. But owing to the fact that these cells are almost invariably polychromatophilic, the nucleus may at first be overlooked, as the ploychromatic protoplasm appears in the meshes of the nucleus and sometimes differs but little in color from the chromatin. ‘The inexperienced not infrequently mistake such cells for large mononuclear leukocytes that are somewhat off-color; the character of the nucleus, however, viz., its wide meshwork, should prevent this mistake. Mitoses in megaloblasts are at times seen. | As already mentioned the megaloblast is essentially a cell of embryonic life. After birth, under normal conditions a few megalo- blasts may be found in the blood of very young infants, and it is noteworthy that in the severe types of secondary anemia megalo- blasts are far more apt to occur in children than in adults. But even then they are rare. In the bone-marrow of the adult they are | present in very small numbers. According to Ehrlich, the presence of megaloblasts in the blood is evidence of a reversion of blood formation to the embryonic type and of grave prognostic import. He regarded their presence as indicative of essential pernicious anemia; and, as a matter of fact, they are here quite constantly 68 THE BLOOD met with and represent one of the most important features of the disease. ‘They are rarely numerous, however, and there are cases in which they are absent’ (aplastic anemia). The modern tendency is to regard the appearance of megaloblasts in the blood as evidence of an anemia of unusual severity, viz., as a degenerative-regenerative symptom, and not as an indication of any one disease. While they are undoubtedly most constant in per- nicious anemia, they may also be met with in other forms. ‘They have been found in leukemia, in the pseudoleukemia of infants, in lead poisoning, and, even in chlorosis, and as I have pointed out already, in some of the severe types of secondary anemia occurring in young children. In cancer of the stomach, according to Osler and McC Tae, they are rarely if ever found. Askanazy’ has. reported an interesting case of bothriocephalus infection in which the megaloblastic type of blood regeneration disappeared after expulsion of the parasites— sixty-seven in number—and was replaced by the normoblastic type, the case ending in recovery. The appearance of megaloblasts in extra-uterine life merely in- dicates an incomplete maturation of young elements, their consump- tion and consequent increased production. ‘The following sketch, taken from Pappenheim, gives an idea of the relation of normoblasts and megaloblasts to the different types of anemia: Under normal conditions Pappenheim’s large lymphocyte (see schema, p. 72) gives rise to the young megaloblast, which in turn dif- ferentiates itself at once into young normoblasts. ‘The young normo- blast ages to the pyknotic normoblast and loses its basophilic nuclein — as a result of chemical karyolysis. In this manner an apparentiy non-nucleated erythrocyte results, which loses its nucleoid later, in the blood, as blood platelet in consequence of variations in the tonicity of the plasma. In severe toxogenic anemias, on the other hand, there is an arrest of development upon an embryonic basis. A certain propor- tion of young megaloblasts multiplies homoplastically; another portion matures to reid megaloblasts, while a third fraction only becomes differentiated to young normoblasts. Of these in turn one portion matures to the old fount which dislodge their nuclei in the anemic serum in toto, while another portion loses the nucleus during the process of hastened maturation by karyorrhexis. As a consequence & many of the anemic normocytes contain no nucleoids, and the blood _ as a consequence contains only small numbers of blood platelets. Pyknotic normoblasts, as also young megaloblasts (of the type of - the large lymphocyte), may thus be encountered in all forms of severe’ anemia of whatever origin. In the kryptogenetic type of pernicious — anemia and bothriocephalus anemia, however, old megaloblasts (of the — ‘ Pane, “ Sull’ anemia progressiva mortale senza corpuscoli rossi, nucleati nel sangue,’’ Riform. med., 1900, No. 263. * Zeit. f. klin. Med., "1895, vol. xxvii, i MICROSCOPIC EXAMINATION OF THE BLOOD 69 type of the large mononuclear leukocyte) are further seen, as also young normoblasts (of the type of the small lymphocyte) under- going karyorrhexis. Generally speaking the number of erythroblasts is no indication of the severity of the case, but merely indicates the extent to which the bone-marrow responds to the blood destruction. ‘The appearance of megaloblasts is hence not necessarily an absolutely unfavorable symptom, but simply the expression of an unusually high activity of the erythropoietic tissue. In cases of traumatic anemia unusually small nucleated red cells have at times been observed. ‘hese are termed microblasts. ‘They have attracted but little attention and are quite rare. I have seen such cells, measuring not more than 3 to 3.5 4, In a case of pernicious anemia at the time of the blood crisis, when large numbers of normo- blasts were also present. The Leukocytes. General Characteristics.—The leukocytes, or white corpuscles of the blood, as seen in the wet preparation (Plate II), are roundish or irregularly shaped cells, which very in size, but for the most part are larger than the red corpuscles. ‘They are all nucleated, and, as the term indicates, devoid of coloring matter. In a general way they may be divided into two distinct classes, viz., those which are granular and those which are not granular. The granular cells (granulocytes) are by far the most numerous, and are characterized by the fact that they are capable of active loco- motion. Even without a warm stage it is almost always possible to observe this in the ordinary wet preparation. ‘lhe moving cells at once attract attention by their irregular outline. On careful exami- nation with a high power it will be noted that the cell advances in a definite manner, which is quite analogous to what is seen in the ameba. The protoplasmic portion manifestly consists of two parts, viz., a non- granular hyaline ectosare and a granular endosare. As the leukocyte progresses the hyaline ectosare advances with a flowing motion, form- ing a distinct layer in front of the granular endosarc, which itself then merges into the non-granular portion. The moving leukocyte is roughly pear-shaped, with the base in advance, while the rear end tapers markedly and frequently seems to drag behind it a small, roundish mass which, like the main body of the cell, is also granular. These granular leukocytes are true phagocytes and take up foreign matter into their interior like amebas. According to Metchnikoff, the phagocytic function is the most important function of the leukocytes, and the outcome of a bacterial invasion, figuratively speaking, will depend upon the superiority of the org: anisms engaged in wi aifare. The nucleus of the eranular leukocytes is either polymorphous— 7. e., it is composed of different lobes which are joined together—or 7a THE BLOOD it may be multiple. Such cells are hence spoken of as polymorpho- nuclear and polynuclear leukocytes, respectively. ‘he polymorphous cells represent an earlier stage in the development of the polynuclear cell. While the granules in the majority of the leukocytes are fine (Plate II), on careful search some cells will be found in which they are coarse and highly refractive. ‘This coarsely granular variety is very characteristic in appearance and at once attracts attention. ‘The cells are far less numerous, however, and, as a matter of fact, represent — only from 1 to 4 per cent. of the total number of the leukocytes, while ~ the finely granular variety represents from 60 to 70 per cent. The non-granular leukocytes, in contradistinction to the granular variety, are mononuclear, with very little tendency to polymorphism. They are quite hyaline in appearance, and are readily overlooked by the beginner unless a somewhat subdued light is used in the exami- nation. ‘I'wo varieties may be recognized: one about the size of a red corpuscle, the other somewhat larger. ‘The nucleus in both varieties occupies a considerable portion of the cell and is surrounded by a | : layer of protoplasm which is practically hyaline. Every cell, it is true, contains a few granules collected at a certain point along the periphery, where the protoplasm is more extensively developed than elsewhere; but these granules, in contradistinction to those which we see in the polynuclear varieties, probably represent nodal points in the cytoreticulum, and not a specific secretory product, as which Ehrlich ~ and his school view the granules of the polynuclear variety. In the small mononuclear form one or sometimes two small, brownish granules can usually be discerned somewhere in the peripheral layer of the protoplasm. Of the significance of this granule, so far as I am aware, nothing is known, nor has its presence been previously described (Plate I). The non-granular mononuclaer leukocytes, in contradistinction to the polynuclear granular variety, were formerly regarded as non- — motile. Jolly, Wolff, and others have shown, however, that they also _ are capable of changing their form even though progressive loco- motion may not occur. ‘lhe change in form can readily be demon- strated even without a warm stage, and it will be observed that the nucleus takes an active part in these changes. Classification.—While it is possible to distinguish the different varieties of leukocytes in the wet and unstained preparation, a more complete picture of the structure of the individual forms may be obtained from a study of stained preparations. ‘The study of such preparations, moreover, forms the most satisfactory basis for the classification of the different forms. We distinguish the following — varieties: 1. The Lymphocytes (Small Mononuclaer Leukocytes, or Micro- lymphocytes) (Plate IV).—The lymphocytes which occur normally i PLATE IV. Lymphocytes. The cell a shows nucleus after division, each with a nucleolus ; 6, a plasma cell (irritation form, phlogoecyte). ¥ se 7a ' {Sty a el el " . 4 Pte eon * = y LISRAKY @F THE AVERSITY of ILLINOIS eho MICROSCOPIC EXAMINATION OF THE BLOOD 71 in the blood are for the most part a little smaller than the red corpuscles or of equal size. ‘lhe nucleus is single and surrounded by a narrow rim of protoplasm which is generally described as non-granular; but, as I have pointed out, a few granules can almost always be made out in the wet preparation at a certain point along the periphery, where the protoplasm is a little more extensively developed. ‘These granules, however, probably represent nodal points of the cytoreticu- lum, and are not to be regarded as in any way analogous to the granules which are met with in the polynuclear leukocytes. Nucleus and pro- toplasm are both basophilic, and, generally speaking, the protoplasm is so more markedly than the nuclus. ‘This is best seen in specimens that have been stained with a methylene-blue mixture, where the lym- phocytes for the most part present a comparatively feebly staining nucleus which is surrounded by a rim of dark blue. Other cells belonging to the same group, however, will also be seen in which this is not marked, but in which the staining affinities of both nucleus and protoplasm appear about the same or in which the protoplasm may even be lighter in color. ‘These cells are generally a little larger than the first variety, with a somewhat broader zone of protoplasm and an eccentric position of the nucleus. ‘They represent a later stage in the development of the deeply staining cell, and are sometimes termed medium-sized lymphocytes. A still larger form may also be met with, but is rarely seen under normal conditions. ‘The staining properties of these large lymphocytes (macrolymphocytes) are essentially the same as those of the smaller varieties. ‘lhe position of the nucleus may be either concentric or éccentric, as in the smaller forms, and a nucleolus is frequently demonstrable. ‘This large type is notably seen in acute lymphatic leukemia, where it is usually the predominating cell. In smaller numbers it 1s also found under other pathological conditions which are associated with a hyperplasia of the lymphadenoid tissue. According to Pappenheim, the large lymphocyte represents the ancestral cell (Ur or Stammzelle), from which all other leukocytes, as well as the red cells, are indirectly derived as the result of heteroplastic differentiation. (See schema, p. 72.) ‘The large lymphocytes are identical with Benda’s lymphogonia, ‘l'roje’s lymphoid marrow cells, Niigeli’s myeloblasts and the undifferentiated lymphoid cell of Michaelis, Wolff, and 'Tiirck. With certain dyes, like methylene blue, the protoplasm of the lymphocytes does not appear perfectly homogeneous, but presents a peculiar granular appearance. ‘This is referable to nodal points of the cytoreticulum and does not represent a true granulation. With methyl green, and hence with Ehrlich’s triacid stain, the protoplasm is perfectly homogeneous and appears as a pale rim about the some- what more deeply staining nucleus. While it is thus impossible with the usual dyes to demonstrate the existence of a true granula- 79 THE BLOOD tion in the lymphocytes, Michaelis' has called attention to the fact that with eosin-methylene-azure solutions (p. 132) distinct granules can be seen (azurophilic granules). ‘Their significance, however, has not been established. Very curiously these granules could not be demonstrated in the tymphocytes obtained from the lymph glands directly, and it appears that they are present in only a certain percent- age of those occurring in the blood. ‘The number of granuies in a cell is variable; in some ‘only two or three are seen, while in others the pr otoplasm i is literally studded with them. Their size varies between that of the common neutrophilic and that of the eosinophilic varieties (Plate V). | INTERRELATION OF LEUCOCYTES AND ERYTHROCYTES ERYTHROBLASTS LYMPHOCYTES GRANULOCYTES Large lymphocyte —>Large mononuclear —>Transition form glee a ~*~ 7 Megalocyte <—Old megaloblast <—Young megaloblast Young amblychromatie —>Older form — Mature form myelocyte @,7Y,€ a, 756 ye Normocyte <—Old normoblast <— Young normoblast Trachychromatie —> Polymphorpho-—>Polynuclear myelocyte @,Y,€ nuclear leucocyte leucocyte a.Y-€ a,y,€ Young small lymphocyte —> Older form —>Rieder’s form ——> Direct cytogenetic development ee ee ee differentiation by cell division ——> —> Direct heteroplastic transformation In wet specimens, as I have pointed out, one or two reddish- brown granules are quite commonly seen in most of the lymphocytes. In stained preparations these cannot be demonstrated. The outline of the cell in the smaller forms is usually fairly smooth, but in the larger varieties it is often shaggy, and at times specimens are seen with a number of distinct knobs. The nucleus, in the smaller forms especially, is concentrically located, while in the larger varieties, in which the protoplasm is more extensively developed, it commonly occupies an eccentric position. In the stained specimens, especially in the larger cells, it — is sometmies surrounded by a faint areola, which is probably owing to artificial retraction. ‘The nucleus is more commonly oval or bean- : 4 shaped than round; deep invaginations are not often seen and frag- mentation of the nucleus is rare. Such cells present an appearance which is altogether different from that of the true polynuclear elements. | Lymphocytes undergoing mitosis are sometimes seen in the blood ~ i of lymphatic leukemia. Characteristic figures, however, are com- paratively rare, and it is more common to meet with cells in which 1 Michaelis and Wolff, Virchow’s Archiv, 1902, vol. elxvii, p. 151. | Leukocytes. | @, microlymphocytes: a!, same, showing azurophilic granules; b, large mononuclear leukoeytes; neutrophilic polymorphonuclear elements, d, adult eosinophile; e, neutrophilic myelocytes; eosinophilic myelocyte; g, mast-cell; 4, karyokinetic normoblast. Stained with Wright’s stain. 1: f La ‘ <7 MICROSCOPIC EXAMINATION OF THE BLOOD 73 division of the nucleus has already occurred (Plate IV). In hema- toxylin-eosin specimens it is usually possible to demonstrate a nu- cleolus, but in eosin-methylene-blue preparations my experience has been that they are not usually seen in the lymphocytes of the normal blood, and seem to be comparatively infrequent also in the blood of lymphatic leukemia. Occasionally, however, specimens are ‘met with in which they are distinct, and at the same time multiple; in such cases active cell division seems to take place in the circulating blood. In adults the number of the lymphocytes normally varies between 20 and 30 per cent. Higher values are found in young children, especially during the first year of life, when the ly mphocytes con- stitute from 50 to 60 per cent. of the total number. At birth, how- ever, they are less numerous than in adult life, viz., only about 15 to 16 per cent. Later they increase and by the tw elfth day it is usual to have from 40 to 50 per cent. After the fifth year adult values are normally the rule. In disease the number of the lymphocytes may be increased or diminished, conditions which are spoken of respectively as lympho- cytosis and lymphopenia. _ While it was formerly supposed that the lymphocytes originate only in the lymph glands proper, there is evidence that they may be formed wherever there is lymphoid tissue, and hence also in the ‘Spleen and in the bone-marrow. ‘They are probably derived from the large lymphocytes of the germinal centres indirectly through a process of differentiating karyokinesis, and represent ‘fully differ- entiated cells which are incapable of further development. 2. The Large Mononuclear Leukocytes (Splenocytes).—'[hese are mostly two or three times as large as the red corpuscles and pro- vided with a large single nucleus, which is surrounded by a relatively wide zone of non-granular protoplasm (Plate V). The nucleus in some cells is oval or elliptical, while in others it is more or less invagi- nated (Ehrlich’s transition forms). In the wet preparation the large mononuclear leukocytes are exceedingly hyaline, so that they are readily overlooked by the beginner. Both nucleus and protoplasm are basophilic, but much less markedly so than in the lymphocytes, and it is noteworthy that the protoplasm usually possesses a less marked affinity for the basic dye than the nucleus. Cells are also met with, however, in which the affinity for the dye is about the same in both. If by chance this occurs in specimens which are somewhat smaller than usual, a cer tain amount of difficulty arises in differentiating such small “large” mono- nuclear leukocytes from the older ly mphocy tes. A hard-and-fast line of distinction cannot here be drawn, and in every differential leukocyte count the personal equation will of necessity enter into consideration. ‘The salient characteristics of the two ty pes should, : 4 74 THE BLOOD however, be borne in mind; in the lymphocytes the protoplasm is but feebly developed in relation to the size of the nucleus, while in the large mononuclear leukocyte the reverse is true. ‘lhe proto- plasm in the latter, moreover, is apparently much more delicate in structure, and is readily wrinkled by contact with adjacent cells; not infrequently cells of this type are found which have manifestly been torn or otherwise injured during the preparation of the speci- men; the lymphocytes, on the other hand, are usually well-preserved and clear-cut, sharply defined cells. In preparations that have been stained with Ehrlich’s triacid both nucleus and protoplasm are very faintly colored and the latter appears perfectly homogeneous; but in specimens which have been stained with mixtures containing methylene blue as the basic component, the protoplasm presents a somewhat granular appearance, which, as in the lymphocytes, is referable to the existence of a cytoreticulum, A certain proportion of the large mononuclear leukocytes (including the transition forms), as in the case of the lymphocytes, also contain azurophilic granules. Inclusive of the transition forms the large mononuclear leukocytes normally represent from 1 to 6 per cent. of the total number. ‘They are relatively more numerous in young children, in whom the highest values are found between the sixth and ninth day after birth. Many of the cells at this time are of the type of the transition form; they may number 18 per cent.; but even in older children one commonly finds a larger proportion of these cells than in adults. According to Pappenheim the large mononuclear leukocytes develop directly, cytogenetically, from the “‘large’ lymphocytes, and then age into the “transition forms” which represent the final stage in the development of this type. ‘The former view, according to which the large mononuclear leukocyte develops directly cytogenetically from the small lymphocyte and later ages into o the polynuclear neutrophile, has been abandoned. For the most part the large mononuclear leukocytes develop in the spleen (hence the term splenocytes). 3. The Neutrophilic Polynuclear Leukocytes (Plate VI).—These cells are a little smaller than the large mononuclear leukocytes and represent the finely granular variety already mentioned. ‘hey are active phagocytes and as such capable of progressive locomotion, — The nucleus in the younger cells is polymorphous, while the older cells are actually polynuclear, the number of lobes varying from two to six. In stained specimens the nucleus shows a coarsely reticular structure with nodal thickenings and is very markedly basophilic. The protoplasm, on the other hand, is very feebly oxyphilic. Embedded in the protoplasm are numerous fine granules—the e-eranulation of Ehrlich—which are characterized by their affinity for neutral dyes. Hence the term polynuclear neutrophilic leukocytes. el OF Id eae Granulocytes. a, polynuclear neutrophilic leukocytes; 6, polynuclear eosinophilic leukocytes; ¢, mast-cells; oung eosinophilic myelocytes; e, neutrophilic myelocytes; 7, the nucleus here has just undergone ‘ision; the clear space is a vacuole. MICROSCOPIC EXAMINATION OF THE BLOOD 75 These granules are ordinarily very abundant; but in disease they may diminish in number until very few are left, and in some cases they may indeed be absent. Ewing" has called especial attention to the decrease in the number of the granules in the acute leukocytosis. I have observed total absence of granules in a case of trichinosis at a time when marked eosinophilia existed. Kast mentions an instance of general carcinomatosis with a leukocytosis of 120,000 in which 1.68 per cent. of the cells contained no granules. Hirschfeld de- scribes the same occurrence in connection with growths involving the bone-marrow, and others have noted it in myeloid leukemia, where toward the end, in chronic cases, it is a fairly common phenomenon. _ Associated with the diminution in the number of the granules there are frequently also degenerative changes affecting the nuclei. ‘These may be of the type of karyolysis with swelling and loss of chromatin, or of karyorrhexis with hyperchromatosis and fragmentation of the nucleus. ‘lhe former is the more usual in the acute leukocytoses, while the latter is seen especially in leukemia. In cases of the myeloid yariety it is quite common to note complete fragmentation of the nucleus into six to ten segments. ‘l'his phenomenon was first observed by Ehrlich in a case of hemorrhagic smallpox, and is of common occur- rence in fresh exudates. Cell degeneration associated with loss of chromatin and swelling, while it no doubt occurs to a greater degree in disease, may also be observed under normal conditions. In every dried and stained specimen a certain number of such cells will be found in which the nucleus appears as a much swollen and but faintly stain- ing shadow, the Kernschatten of the Germans, sometimes surrounded by some of the granules, which appear scattered as though the cell had been burst asunder by force; at other times the Kernschatten alone remains and nothing is seen of the body of the cell. I have stated that the loss of granules on the part of these cells may go on to a point where they are absent altogether. It may happen, however, that the granules are only apparently absent, and merely do not react as usual with ordinary dyes. A proper explana- tion of this peculiar behavior cannot be given, but every worker in blood is no doubt familiar with the phenomenon. Sometimes a change in the mode of fixation will cause the granulation to appear; at other times it may be demonstrated by the aid of some other dye. Vacuolization of the polynuclear leukocytes is very much less common than in the case of the mononuclear elements. While the neutrophilic leukocytes as a general rule are large cells, unusually small specimens are seen in the blood of myeloid leukemia. These dwarf forms must not be mistaken for the small cells which one may find in any specimen of blood where it is thick and where the pro- cess of drying has occurred slowly. In cells of this latter order the ' Clinical Pathology of the Blood, Lea Bros., Ist ed., p. 118 "6 THE BLOOD staining of the granules is also frequently deficient or they may not show at all. Neusser' some years ago called attention to the fact that with a certain modification of Ehrlich’s triacid stain it is possible to demon- strate the presence of basophilic granules about the nucleus of some of the polynuclear leukocytes, as well as the mononuclear elements. He, as well as Kolisch,’ regarded the presence of these perinuclear granules as characteristic of the so-called uric acid diathesis. As tubercular disease, moreover, is usually not seen in such cases, Neus- ser thought the presence of these granules in cases of phthisis to be a favorable symptom. Futcher,’ on the other hand, was unable to confirm these observations, and my own investigations® are likewise opposed to Neusser’s conclusions. I was able to demonstrate the gran- ules both in health and disease in almost every case, and was at one time even led to think that their absence was of more significance than their presence. A relation between their presence and the elimina- tion of uric acid or xanthin bases certainly does not exist. Within recent years the subject has received no further attention, especially since Ehrlich expressed the belief that the granules are artefacts, He states that they are only exceptionally seen when solutions of chemically pure crystalline dyes are used, from the Actiengesellschait fiir Anilinfarbstoffe in Berlin. The polynuclear neutrophilic leukocytes are derieed from corre- sponding mononuclaer forms—the neutrophilic myelocytes—which are normally found only in the bone-marrow. ‘They result from these directly and represent their adult form. | Arneth? divides the polynuclear neutrophiles into five classes accord- ing to the number of nuclear lobes. Under normal conditions the percentage numbers of the different varieties remain fairly constant for one and the same individual, but vary somewhat in different people. The first class is represented by mononuclear forms and is subdivided into (a) mononuclear forms, corresponding to and identical with Ehr- lich’s m2 yelocytes (see below): (b) forms with but slightly indented nuclei, the invagination not extending to a greater depth than the middle of the nucleus (the metamyelocytes); (c) cells in which the invagination extends farther than in form (6), but in which no separa- tion into isolated loops or lobes has as yet occurred—the true poly- morphonuclear variety. "The two first varieties are essentially only seen under abnormal conditions, although an occasional metamyelo- cyte may at times be encountered in health, Cells of type (c) are present to the extent of 4 to 9 per cent. ‘The second class com- prises cells with two distinct nuclear fragments, which may appear 1 Wien. klin. Woch., 1894, p. 71. 2 \Ibide, 1895, .p, “797 * Johns Hopkins Hosp. Bull., May, 1897. * Amer. Jour. Med. Sci., vol. exvil, p. 139. ® Die neutrophilen weissen Blutkérperchen. G x. Fischer, Jena, 1904. e MICROSCOPIC EXAMINATION OF THE BLOOD 77 either as two loops or two lobes. ‘They constitute from 21 to 47 per cent.; the number, as already stated, varies somewhat with the individual, but is quite constant for one and the same person. In | this class the cells with two loops normally always exceed those ‘with one loop and one lobe, while true bilobes are rare. The third class presents three nuclear divisions and can be subdivided into four groups in accordance with the number of loops or lobes (see ‘p. 81). Cells with two lobes and one loop approximate those with ‘two loops and one lobe, while cells with three loops or three lobes respectively are in the minority. Conjointly the groups of the third ‘class represent 33 to 48 per cent. Their number thus about equals ‘that of group two, but has a tendency to be somewhat in advance. The fourth class is provided with four nuclear divisions with five ‘subgroups and numbers 9 to 23 per cent. he fifth class finally ‘comprises cells with five or more nuclear subdivisions and may be ‘subdivided according to the same principle. Only 2 to 4 per cent. of the neutrophiles normally belong to this order. The various ‘classes as just described represent different stages in the develop- ment of the neutrophilic cells, the myelocytes on the one hand being ‘the youngest, and the polynuclear leukocytes with many lobes the oldest. The polynuclear neutrophiles are the most common leukocytes of the blood and normally constitute from 60 to 70 per cent. of the total number. In young children they are relatively less numerous excepting during the first twenty-four “hours of life, when they may number 73 per cent. But they rapidly diminish, so that values of from 20 to 40 per cent. may be regarded as normal during the first year. Low values continue practically to the twelfth year, though the numbers gradually rise. From the twelfth to the fourteenth year ‘60 per cent. may be regarded as an average; after that age the values given for the adult hold good. 4. The Polynuclear Oxyphilic or Eosinophilic Leukocytes (Plate V1). —In size and general appearance these cells resemble the polynuclear neutrophiles, and, like these, are capable of progressive locomotion. ‘The granules—the g-granulation of Ehrlich—however, are much larger and highly refractive, and possess a marked affinity for acid dyes, such as acid fuchsin and eosin. Hence the term oayphilic or eosinophilic leukocytes. With neutral dyes or basic dyes they will not stain. The appearance of the individual granules varies somewhat in stained preparations. Some are round, others oval; ‘some appear to stain throughout, others make the impression of little vesicles with a limiting ‘membrane, which alone takes the dye, while the interior remains unstained. ‘This bleb-like appearance of the granule is one of the most marked characteristics. Barker' : ‘ Johns Hopkins Hosp. Buli., 1894, p. 93. 78 THE BLOOD has shown that the granules contain iron. ‘They are insoluble in ether and cannot be stained with osmic acid. ‘They are therefore not composed of fat. The protoplasm of the eosinophilic leukocytes is usually almost altogether hidden from view, owing to the dense packing of the granules; it is slightly basophilic. ‘The nucleus is mostly bilobed, sometimes trilobed, and in stained specimens it is quite common to find the individual lobes unconnected by threads of chromatin; often the two lobes are situated at opposite poles. As a rule the nucleus is less markedly basophilic than that of the neutrophilic variety. A nucleolus is not seen. The same degenerative changes which have been described in con- nection with the polynuclear neutrophiles may also be observed in the eosinophiles, and here, as there, one can at times note a material diminution in the number of the granules. I have never observed their entire absence, however, and it is noteworthy that in those cases of chronic leukemia in which the neutrophilic granulation may disappear the eosinophilic variety remains. While the common eosinophile is a large cell, unusually small eosino- philes are frequently seen in the blood of myeloid leukemia. ‘These should not be confounded with the small forms which may be seen in the thicker portions of almost any normal specimen, and which latter owe their small size to a gradual contraction during the process of drying. Under normal conditions the percentage of the eosinophiles varies between 1 and 4. While repeated attempts have been made to connect the eosino- philic leukocytes of the blood cytogenetically with the neutrophilic variety, there is no satisfactory evidence to support this view. On the contrary, there are strong reasons for believing with Ehrlich that, analogous to the neutrophilic variety, the polynuclear eosino- philes are normally formed in the bone-marrow, and here only from mononuclear eosinophilic cells—the eosinophilic myelocytes. 5. The Mast-cells (Polynuclear Basophilic Leukocytes) (Plate V1). —The mast-cells which are normally found in the blood are approxi- mately of the same size as the polynuclear neutrophiles and eosino- philes. In myeloid leukemia, however, in which they are espe-— cially numerous, the size is more variable; on the one hand, they may — measure only 5.5 in diameter, while on the other they may attain a dimension of 22 4. ‘lhe nucleus is polymorphous; but the ten-— dency to form individual lobes is far less marked than in the corre- — sponding eosinophilic and neutrophilic elements. Quite commonly — it is leaf-like and flat in appearance. Its affinity for basic dyes 1s — quite feeble, so that it is often difficult in stained preparations to — make out the boundary line between nucleus and protoplasm. It is _ almost always excentrically located and usually has a fairly uniform — a MICROSCOPIC EXAMINATION OF THE BLOOD 79 diameter of 4 4. In the smaller specimens the nucleus occupies almost the entire cell. _ Embedded in the protoplasm lie granules of variable size—the 7 granulation of Ehrlich—some of which are fully as large as or even larger than the eosinophilic granules, while others are much finer. They are characterized by their affinity for basic dyes and the fact that with certain ones they stain metachromatically, viz., in a color which is different from that of the dye itself, which latter must be simple and not compound. ‘Tissue elements which will stain in this -manner are spoken of as chromotropic elements. Only a limited number of dyes have metachromatic properties. ‘lhe most notable ones are the violet basic dyes hexamethyl violet, cresyl violet, thionin, neutral violet, and amethyst violet; further, the blue dyes methylene _azure, cresyl blue, and toluidin blue, and the red basic dyes pyronin, -acridin red, neutral red, and saffranin. With the latter group the mast- cell granules are colored yellow, with most of the violet dyes red, and with cresyl-violet R (extra) almost a pure brown. Methyl green does not stain the mast-cell granules unless it is contaminated with methy! violet, and for this reason the granules remain colorless in ‘specimens stained with Ehrlich’s triacid stain. In specimens fixed by heat and stained with aqueous alum hematoxylin solution the a-granules are also not demonstrable. ‘They have been dissolved; ‘but there remains a well-defined spongioplasm, upon which the gran- ules were deposited. _ The mast-cell granules are absolutely basophilic, viz., they can only be stained with basic dyes, and retain the basic dye on subse- quent differentiation in acid media. ‘lhey are capable, moreover, of taking up the basic dye from its acidified solutions, as in the case of Ehrlich’s dahlia-acetic acid mixture. The granules of the common mast-cells of normal blood are re- sistant to water, while in myeloid leukemia cells are met with the granules of which dissolve with great readiness. ‘heir chemical jature is still a matter of dispute, but there is a tendency to associ- ate the mast-cell with the formation of mucin. ‘This presupposes che identity of the blood mast-cell with the common mast-cell of connective tissue. In the past this has been tacitly assumed, but Pappenheim more especially has called attention to the fact that he hematogenic mast-cell differs from the histogenic form, and chat the two probably represent different species. Pappenheim nelines to the view, however, that the granulation of the hema- ogenous mast-cells is not a true morphological granulation, but nerely chemically altered lymphocytic spongioplasm, or a transport substance which has been taken up and metabolized. _ The number of mast-cells varies between 0.2 and 1.0 per cent. dwing states that he constantly failed to find mast-cells in the better lass of healthy subjects, while in hospital and dispensary cases with } 80 THE BLOOD | minor ailments they appeared to be more numerous. My own obser vations do not bear this out; in my experience they are invariably pres ent in health irrespective of the general nutrition of the individual. The origin of the mast-cells of the blood has not been definitely ascertained. Ehrlich supposed that they originated from the con nective-tissue cells as the result of hypernutrition, while Harri suggests that they may be derived from the large mononucleai leukocytes. According to Pappenheim, the mast-cell originates ir the bone-marrow from a granular mononuclear ‘type which corre} sponds to the eosinophilic and neutrophilic myelocytes. 6. The Myelocytes.— he myelocytes are mononuclear granulai cells, which are normally not found in the circulation, but are en. countered only in the bone-marrow. Generally speaking, they represent the juvenile forms of the poly: nuclear leukocytes of the blood, and we accordingly distinguish three varieties, viz., the neutrophilic, eosinophilic, and basophilic myelo-| cytes. ‘The two last-named varieties, according to our present ideas age directly into the corresponding polynuclear forms—2. e., they become the common eosinophiles and the mast-cell. In the case oj the neutrophilic variety it appears that two types exist, a smaller and a larger form, which Pappenheim’ designates respectively as the trachychromatic and the amblychromatic ‘form. ‘These are onto- genetic ally derived, the first from the last, but only the trachychro- matic variety ages into the common polynuclear neutrophile of the circulating blood. The nucleus of the amblychromatic form as it matures likewise becomes polymorphous, but normally the cell remains an inhabitant of the bone-marrow even then. | As regards the origin of the myelocytes, I incline toward Pappell heim’s view, according to which all three varieties result from the| large lymphocytes through a process of heteroplastic differentiation. (a) ‘THe NeEvutTRopHILIC Myrtocytes.—These, as I have stated, are of two kinds. ‘The one type, the amblychromatic myelocyte ot Pappenheim, is a large cell provided with a relatively large, cen- trally located, round nucleus which stains but feebly with basic dyes. This is surrounded by a comparatively narrow zone of basophilic protoplasm which contains very fine neutrophilic granules. As the cell matures the nucleus becomes more or less invaginated and ultimately distinctly polymorphous. ‘The protoplasm at the same time becomes relatively more abundant. Pappenheim speaks of this type as the heteroplastic promyelocyte. Such cells differ mark- edly in size from the common polynuclear elements which result from the second type of myelocyte. The second type, viz., the trachychromatic myelocyte, is a smaller cell, which is essentially characterized by the fact that its nucleus — oe 1 Virchow's Archiv, vols, clix and clx, | MICROSCOPIC EXAMINATION OF THE BLOOD 81 ‘stains quite intensely with basic dyes. The protoplasm is faintly oxyphilic and the granulation rather coarser than in the amblychro- matic variety. As this cell matures the protoplasm becomes rela- tively more abundant and the nucleus distinctly polymorphous; it ‘then constitutes the common neutrophile of the circulating blood. ‘Between these two extremes there are transition forms, in which the nucleus is still single, but already shows a marked tendency toward polymorphism. ‘These cells do not occur in normal blood. They have been described especially by Arneth. Pappenheim terms them metamyelocytes or proleukocytes (see Fig. 16). la Ib II III ri’) 100 BGq CE ® 66, ee EOn6o @ S|, 0© 989g) at S00," Fic. 16.—Karyolobism and polynucleosis of neutrophilic leukocytes. Neutrophilic myelocytes undergoing mitosis are sometimes seen n the circulating blood in cases of myeloid leukemia; on the whole, 1owever, they are rare, and it is more common to meet with cells n which the division of the nucleus has already taken place (Plate VI). Miiller and Jolly have shown that the neutrophilic myelocytes of he circulating blood are capable of active locomotion. (b) Hr Eostnopumic Myetocytes.—lIn the more mature forms he color of the eosinophilic granulation on staining with eosin- nethylene-blue mixtures is a pure eosin red, ‘The younger forms, 82 THE BLOOD however, present a purplish-violet color, and some granules may | indeed be a pure blue (Plate VI). This appearance is owing to the fact that the young eosinophilic granule is physically cyanophilie | and chemically amphophilic, whereas the mature granule is physically | erythrophilic, but chemically absolutely oxyphilic. This is well | shown by staiming such young cells with a mixture in which the | basic dye is of a light color and the acid component dark, such as. vesuvin on the one hand and water blue on the other. ‘The mature | eosinophilic granules will then take on the blue color of the water blue, while the young granules which stained blue with the eosin- | methylene-blue mixture, and which we might accordingly have — regarded as basophilic, are now likewise colored by the acid blue | instead of the basic vesuvin, thus showing that they are in reality | not basophilic, but amphophilic-cyanophilic t The protoplasm of the eosinophilic myelocytes is basophilic. The size of the cells is quite variable; some are considerably larger | than the corresponding polynuclear form, while others are much | smaller. ‘The cyanophilic cells are, generally speaking, the largest. _ According to the observations of Miiller and Jolly the eosino- philic myelocytes are capable of progressive locomotion. | (c) ‘Tur Basopuitic Myexocyres, like the eosinophilic and neu- | trophilic varieties, may be of variable size and are provided with a | large centrically located nucleus, which is often distinguished only with difficulty from the surrounding protoplasm. | 7. Irritation Form (STIMULATION Forms, or PHLoGocyTEs).—_ ‘These are mononuclear non-granular cells, the protoplasm of which | is stained a rich brown by the triacid mixture. ‘The nucleus is round, | eccentrically located, and colored a bluish green. Oftentimes it — shows a distinct wheel-spoke structure. According to Tiirk, who | first described these cells, they are met with under the same conditions | as the myelocytes. Pappenheim regards them as plasma cells and as __ largely derived from histogenic lymphocytes as the result of a retro-_ gressive degeneration, and characterized by hypertrophy of the | cytoreticulum, increase of chromatin and chromatokinesis of the — nucleus with coincident appearance of a markedly chromatophilic sub- _ stance of exogenic origin. As intermediary cells Pappenheim regards __ lymphocytes without chromophilic protoplasm, but with radiary nuclei. It is thus essentially a pathological product. The cells have a spongioplastic cytoreticulum and vacuoles. ‘They may attain | a size of 30 4. ‘These cells, in my experience, are most frequently _ met with in the blood of children, where their number may attain 5 per cent. of all leukocytes. Wrench and Bryant' found this propor-— tion in a girl of 10, in which, possibly as the result of gas poisoning, a severe anemia had developed. 1 Guy’s Hosp. Repts., 1905, vol. lix, p. 333. ) MICROSCOPIC EXAMINATION OF THE BLOOD 83 According to Pappenheim the occurrence of plasma cells in the ‘blood is indicative of a chronic inflammatory process, either of the ‘connective tissue or of the hemopoietic apparatus (tuberculosis, Hodgkin’s disease, myeloma, etc.). I have found them relatively ‘numerous in inflammatory conditions of the abdominal viscera (peri- tonitis, appendicitis, typhoid fever), and occasionally in measles. The term irritation or stimulation forms indicates that the cells are found in connection with infectious, toxic, viz., inflammatory “irritation.” Iodophilia.—On staining blood smears of normal individuals with iodine (see p. 137) the protoplasm of the leukocytes is colored a bright yellow, while the nucleus is somewhat refractory and takes on a lighter tint. Under certain pathological conditions this staining quality is modified; cells are then seen in which reddish-brown granules appear in the protoplasm or it may occur that this presents a diffuse brownish color throughout. ‘This intracellular reaction affects the polynuclear neutrophiles almost exclusively; the mono- nuclear elements may, however, also react, in which case one com- monly sees large, pale-brown granules arranged about the nucleus in a single row. In eosinophiles the reaction does not occur. ‘he extent to which the leukocytes are involved is quite variable; in some cases a few cells only are affected, while in others one is scar cely able to find a normal cell in an entire preparation. An extracellular reaction also occurs, but is of little clinical in- terest, as it is not infrequent even in health: it occurs in small, round- ish or oval masses, which are possibly true plaques, but which may also be small bits of protoplasm derived from leukocytes. As to the nature of the substance which reacts with the iodine in the manner indicated, there is no uniformity of opinion. Ehrlich cegards it as glycogen, and assumes that this 1s present normally in every cell in the form of a colorless compound, from which the free zlycogen is under certain conditions split off, and can then be demon- strated as such. Czerny, on the other hand, looks upon the iodo- yhilic substance as an antecedent of amyloid, while Goldberger id Weiss view it as peptone. Kaminer has shown that normal -one-marrow does not contain iodophilic leukocytes, but that they may here be found when they are present also in the blood. He concludes that the reaction is a degenerative phenomenon and not an evidence of regeneration. _ The occurrence of the reaction in disease has been studied especially oy Gabritschewsky, Czerny, Livierato, Kaminer, Cabot, and Locke. from these investigations it appears that septic conditions of all -sinds may furnish a positive reaction, but that active suppuration nay also occur without iodophilia (Reich, Kiittner). Locke’s list of diseases of this order includes general septicemia, abscesses (ex- epting in the earliest stages), appendicitis accompanied by abscess } a 84 THE BLOOD formation, general peritonitis, empyema, pneumonia, pyonephrosis, salpingitis with severe inflammation or abscess formation, tonsillitis, gonorrheal arthritis (in contra-indication to other forms), and acute | intestinal obstruction where the bowel has become gangrenous, Locke concludes that no septic condition of any severity can exist without a positive reaction. In puerperal sepsis also it is said to be. constant (Kaminer). In pneumonia with frank resolution it dis- | appears in from twenty-four to forty-eight hours following crisis, | In typhoid fever a positive reaction is not commonly obtained before the end of the second week, and it may indeed remain absent through- | out the course of the disease. In the differential diagnosis between a serous and a purulent pleuritic effusion the absence of the reaction points to the former condition. Cerebral abscess may show the re- action, while in brain tumor it is absent (Gulland). In diphtheria | it is only seen when there is much inflammation; it is never intense (Gulland). In contradistinction to chlorosis, pseudoleukemia, and the common forms of secondary anemia of moderate intensity, iodophilic leuko- cytes are found only in the severer forms of anemia, such as perni- clous anemia, leukemia (notably in acute cases), and the severe forms of secondary anemia. , 3 In animals the reaction can be produced artificially by infection with the streptococcus, the staphylococcus, the Bacillus pyocyaneus, Loffler’s bacillus, the anthrax bacillus, that of Friedlander, the Bacillus coli communis, or the typhoid bacillus; as also by means of ricin, abrin, and the diphtheria toxin. Following the injection of oil of tur-| pentine, croton oil, mustard. oil, and silver nitrate, the reaction may occur even though bacterial infection has been avoided. In man it is also said to occur following narcosis. LireRATuRE.—Ehrlich, Zeit. f. klin. Med., 1882, vol. vi. Gabritschewsky, Arch, f. exp. Path. u. Pharmak., 1891, vol. xxviii. Czerny, ibid., 1893, vol. xxxi. Goldberger u. Weiss, Wien. klin. Woch., 1897. Hofbauer, Centralbl. f.: inn. Med., 1899. Livierato, Deutsch. Arch. f. klin. Med., 1894, vol. iii. Kam-) iner, Berl. klin. Woch., 1899, p. 119; and Deutsch. med. Woch., 1899, p. 206. | Cabot and Locke, Jour. Med. Research, 1902, vol. vii. Locke, Boston Med. and: Surg. Jour., 1902, p. 289. Reich, Beit. klin. Chir., xlii, 2. Kiuttner, Arch. f.| klin. Chir., Ixxiii, 2; and Centralbl. f. Chir., 1904, No. 27, Beil., pp. 3-5. : Leukocytosis.—While the number of red corpuscles is normally fairly constant, that of the leukocytes is subject to not inconsiderable’ variation. It is influenced by the age and sex of the individual, the process of digestion, menstruation, pregnancy, the bloodvessel from. which the specimen is taken, ete. Generally speaking the number of the leukocytes varies between 3000 and 10,000, the exact num- ber, ceteris paribus, depending upon the state of nutrition of the individual. In ill-nourished persons low values are the rule, while maximum numbers are generally associated with a state of excep- i | MICROSCOPIC EXAMINATION OF THE BLOOD 85 ional vigor and good nutrition. ‘These extreme figures, however, are uncommon, and as a general rule a count of 10,000 may be re- : zarded as abnormal; 5000 to 6000 are the most common values which one finds if the examination is made with the individual in a “fasting condition. During the process of digestion the figures are higher (see below). _ An increase in the number of leukocytes is met with under the most diverse conditions, both in health and disease. When transi- tory, it is designated as leukoce ytosis. But it would be better to restrict this term to indicate the number of the leukocytes in a general way, and to speak of an increase as hyperleukocytosis, and of a decrease as hypoleukocytosis. _ It will be convenient to consider the subject of leykocytosis under the following headings: la. Polynuclear neutrophilic hyperleukocytosis. 1b. Polynuclear neutrophilic hypoleukocytosis. 2a. Polynuclear eosinophilic hyperleukocy tosis. 2b. Polynuclear eosinophilic hypoleukocy tosis. 3a. Mast-cell hyperleukocytosis. 3b. Mast-cell hypoleukocytosis. 4a. Large mononuclear hyperleukocytosis. 4b. Large mononuclear hypoleukocytosis. 5a. Lymphocytosis. 5b. Lymphopenia. The term myelemia, or myelocytosis, may be used to designate the appearance of myelocytes in the circulating blood, and in confor mity with the three recognized forms we may speak of a neutrophilic, an 2osinophilic, and a ‘basophilic or mast-cell myelocytosis. Until quite recently the general tendency in clinical laboratories has seen to lay especial stress upon the absolute leukocyte count and to leave the relative values of the different forms out of sight. This should not de, and I cannot insist too strong gly wpon the «mportance of the relative ‘ount, which in many respects is far greater than a knowledge of the otal number. For this reason also I have chosen the consideration of he subject of hyperleukocytosis on the basis of the classification just yutlined. Polynuclear Neutrophilic Hyperleukocytosis.— This is the most common form of hyperleukocytosis, and, as the term indicates, mincipally affects the polynuclear neutrophiles. Exceptionally it may be associated with a polynuclear eosinophilia, as well as with a -ymphocytosis; but as a general rule both eosinophiles and lympho- *ytes are diminished. ‘This diminution is often not only relative, out absolute as well. In very marked cases of hyperleukocy tosis of his type it is not uncommon to meet with a few myelocytes which ire then also of the neutrophilic variety; this is especially the case jn children in whom the bone-marrow reacts more readily to stimu- 86 THE BLOOD lation. Eosinophilic myelocytes, on the other hand, are but rarely seen. Clinically we must distinguish between an increase of the poly- hyperleukocytosis which is observed in disease. We may accord- ingly speak of a physiological and a pathological form. Physiological Hyperleukocytosis.—As physiological increase in number of the leukocytes is notably observed at birth, during th process of digestion, in pregnancy, in association with severe mus- cular exercise, following the use of cold baths, ete. pet ct io, the Newborn. ein: to the expen trophiles. The number then falls and at the same time oe a 0- cytes increase. ‘The curves of the two varieties cross between the sixth and the ninth day, and by the twelfth the lymphocytes are in excess. From the end of the first month to the fourteenth year ie latter. The eosinophiles are relatively at least didinished 1e total increase rarely exceeds 3500 in normal adults, while in young children it may be ‘much more marked. Schiff‘ cites an instance in which 19,500 leukocytes were counted one hour after birth, 27, 625 after the first meal, and 36,000 after the fourth meal. It is especially pronounced after a preliminary period of fasting and following a me: al rich in proteids. ‘The maximum increase is usually observed between the third and fourth hour. ; In cases in which a hyperleukocytosis exists from other causes, as in pregnancy, in inflammatory diseases, etc., digestive hyperleuko- cytosis does not occur. Lobenstine’ in analyzing 20 cases of pregnancy in this direction found digestive leukocytosis in 13, no change in 1 and an actual decrease in 6. Apparently, however, he only made his examinations following the ordinary midday meal. In a few isolate ed instances it has also ee found absent in apparently normal individ- uals without assignable cause. Under pathological conditions its absence is not uncommon, even though hyperleukocytosis referable to other factors may not exist. ‘This is notably the case in carcinoma 1 Zeit. f. Heilk., vol. xi, p. 30, and 1890, p. 1. 2 Amer. Jour. Med. Sci. . August, 1904. MICROSCOPIC EXAMINATION OF THE BLOOD 87 | of the stomach, and it was once thought that the absence of digestive _ hyperleukocytosis in doubtful cases could be interpreted as evidence A in favor of its existence." Generally speaking, this is true even now, » and we may say that in about 90 per cent. of all cases of carcinoma of | the stomach digestive hyperleukocytosis does not occur. ‘The symp- - tom, however, is not pathognomonic, as a number of instances of | carcinoma have been reported in which there was a distinct increase, | and as digestive leukocytosis may also be absent in other conditions. _ In anemic individuals, from whatever cause, especially large amounts _ of proteids are sometimes necessary to elicit an increase of the leuko- ' eytes (Miiller*) and in some cases a subnormal number may even be - encountered (Rieder*). To study digestive hyperleukocytosis, it is well to proceed as follows: _ (a) The first blood count should be made after the patient has ' fasted for about seventeen hours. ' (b) After this period he receives a test meal consisting of from | 200 to 1000 c.c. of milk and one or two eggs, the amount varying ' with the condition of the patient. (c) Further blood counts are made one, two, three, and four hours ' later. (d) ‘The existence of a digestive hyperleukocytosis should only be | regarded as proved if an increase of at least 1500 cells occurs, pro- _ viding that maximal amounts of food have been taken. If smaller ' amounts have been given, an increase of 1000 cells is sufficient to : establish its existence, provided that the same result is observed on _ repeated examination. Leukocytosis of Pregnancy and Parturition—The hyperleukocy- _ tosis which is observed in pregnancy is particularly marked during ' the last five months, and appears to occur quite constantly in primi- | pare, while in multiparee exceptions are common. In an analysis ' of 55 cases Hubbard and White* obtained positive results in 44— _ 1. e., in 80 per cent.—most marked and constant in young primipare. : Rieder in an analysis of 31 cases noted a hyperleukocy tosis in 20, ' all the negative cases being multipare. In a series of 17 multipare ' an increased number of leukocytes was noted in only 7. In Rieder’s ’ series the number of leukocytes varied between 10,000 and 16,000, with an average of 13,000. ‘This represents the usual increase, but _ at times much larger numbers may be observed; Cabot thus reports | 3 cases with a leukocytosis of from 25,000 to 37,000 _ Lobenstine gives the following figures as the result of an analysis _ of 50 cases in the ninth month: _ * Schneyer, “ Das Verhalten d. Verdauungsleukocytose b. uleus rotundum u. | carcinoma ventriculi,” Zeit. f. klin. Med., vol. xxvii, p. 249. ? Zeit. f. Heilk., 1890, p 213. rs ; Beit. z. Kenntniss d. Leukocytose, 1892. * Jour. of Exper. Med., 1898, p. 639. | ‘a 88 THE BLOOD Average. count*:-"2 >! 2a FSS ge oe ee Highest counties Pew ad ts) a es Ae ee ee ce Lowest cOouUntS 2 suk eo 0 ptae ae 5,400 AVerage iniprimipares wae) cis» pg a ee 9,346 Average in multipare .. Nk Cage SION aes ae Le Absence of leukocytes in 7 cases. During actual labor there is an increase of the leukocytes over and _ above the numbers previously observed in pregnancy; 30,000 cells | may then be noted. Lobenstine’s figures in his series of 50 normal cases on the third day of the puerperal period are the following: Average COUDb Es U0) f= het OR PE 9 en ea Highest-count so “asd 390 ee? So aie St A ee ae Lowest contite go> >

percentage of lymphocytes, at least equalling if not exceeding that of! the polynuclear neutrophiles, is a valuable aid in the diagnosis of whooping-cough before the characteristic symptoms of the disease. have appeared. Exceptions, however, occur, in which the lympho- cytosis does not reach the usual high figures. | In rickets a well-marked lymphocytosis is the rule, which is both relative and absolute; the same holds good for congenital syphilis and for the secondary stage of the acquired clisease. In bronchopnewmonia there is at times a well-marked lympho- cytosis instead of a polynuclear hyperleukocytosis. Cabot cites an) instance with a total leukocyte count of 94,600 and 66 per cent. P| lymphocytes. In measles there is at first an increase of the polynuclear neutro- philic elements; later the lymphocytes increase in inverse proportion to the neutrophiles, the total number being largely dependent upon the degree of glandular involvement. In typhoid fever a relative lymphocytosis begins about the end of the first week and reaches its highest point in the stage of defer- vescence. Ewing states that he has found a uniform relation in, this disease between the lymphocytosis in the blood and the grade of lymphatic hyperplasia found at autopsy. He records an instance in which the examination of the blood led to a strong suspicion of lym- | phatic leukemia, and in which at autopsy the mesenteric glands were of unusually large size, and the edges of the partly necrotic intestinal ulcers rose 1.5 cm. above the mucosa. In smallpox there is a general tendency to an increase of the | nuclear elements. ‘The same is seen in varicella. | In tuberculosis, when well advanced, the lymphocytes are usually” diminished, and the more so the more prominently the patients have’ become septic. Early in the disease and in convalescent cases there is often a distinct tendency to lymphocytosis. In this respect my observations agree very well with those of Holmes.’ In a series of 202. 1 Clin. Med. Ital., 1899, No. 1. 2 Amer. Med., 1902. * Jour. Amer. Med, Assoc,, Jan, 28, 1905, MICROSCOPIC EXAMINATION OF THE BLOOD 111 dis he found, associated with a lymphocyte count of 10 or lower, only ) cases w Pash, could be viewed as recoveries or convalescents; with 10 to 20 per cent., 14 cases, and with more than 20 per tenth 39 cases. | In uncomplicated influenza lymphocytosis is the rule during the active period of the disease, while in convalescence the neutrophiles nay show maximum Raratal values. _ Inepilepsy there may be distinct hyperleukocytosis, referable to an nerease of the small mononuclear elements. Boston and Pearce ‘ound the neutrophiles down to 29 per cent. In pellagra mononucleosis apparently occurs with characteristic vegularity, which may be of service in the diagnosis of the disease ‘rom other erythemas.* In leprosy both lymphocytes and large mononuclear elements, but especially the former, are increased. | A relative as well as absolute lymphocytosis occurs in the helmin- hhiases in which the eosinophiles are markedly increased. It is espe- cially pronounced in trichinosis. In general paresis, during the first stage, there is a tendency to ayperleukocytosis of the neutrophiles (70 to 80 per cent.); but in the hird stage the latter fall as low as 40 per cent. while the lymphocytes ire increased.” A well-marked lymphocytosis is seen in Kala-azar. , According to Sahli a decrease of the total leukocytes, associated with a relative increase of the lymphocytes, may be observed in hemo- »hilia. In uncomplicated cases of pseudoleukemia an absolute increase of he leukocytes does not occur; but there is usually a relative increase of the lymphocytes of such extent that the normal ratio to the poly- auclears 1 to 3 rises to 2 to 3to 1. This relative lymphocytosis Ehr- ich and Pinkus regard as characteristic of true pseudoleukemia, n the differential diagnosis from sarcomatosis and other lympho- natous growths.* _ Grawitz,* on the other hand, maintains that from the leukocyte ‘ount no diagnostic conclusions can be drawn, and cites cases in vhich the ratio was either normal or in which the lymphocytes were icaaely diminished. When the pseudoleukemic process involves the bone-marrow the slood findings may be very variable. As a result of stimulation of the ayeloid tissue myelocytosis may occur; in other cases the blood pic- ure closely resembles that of lymphatic leukemia (leukanemia, ' Grigorescu and Galasescu. Spitalul, 1903. ? Bruce, Scott. Med. and Surg. Jour., June, 1903. * Pinkus, Die Leukemie, Nothnagel’s Encykl. * Klinische Pathol d. Blutes, 2d ed, iz 18 THE BLOOD pseudopernicious anemia). In all such cases anemia is at the same time a prominent symptom owing to the replacement of the ery- throblastic by lymphadenoid tissue. The highest grade of lymphocytosis is met with in the so-called ° lymphatic form of leukemia. As in the myeloid variety, the total number of leukocytes is here also very much increased, though not to the same extent. ‘The highest count in Cabot’s series was 220,000 and the lowest 40,000, so that we may regard 130,000 as an average. ‘Ihe lymphocytes usually number more than 90 per cent. In the chronic cases the small lymphocyte prevails, while in the acute cases the large lymphocyte controls the blood picture. When septic complications develop, the total number of the leukocytes, as in the myeloid form of leukemia, likewise undergoes a considerable - diminution, but the lymphocytes still remain relatively increased. In one of Cabot’s cases, in which as the result of septicemia the total number of leukocytes fell to 471 per cubic millimeter, the percentage of lymphocytes still was 94.7. In the majority of cases. of chloroma there is a moderate leukocyto- sis with lymphocytosis of the small or large cell variety, but in others _ myelocytes enter more or less prominently into the blood picture. An experimental. lymphocytosis has been observed following the — injection of tuberculin and of extract of carcinomatous tissue (Gra- witz). Waldstein claims to have produced a marked increase of the lymphocytes by hypodermic injections of pilocarpine, but, according — to Ewing, this increase is only relative and brought about by a diminution of the polynuclear cells. Wilkinson speaks of a lymph-_ ocytosis following injections of quinine hydrochlorate and Perry | has noted the same after the administration of thyroid extract.’ | Lymphopenia.—Lymphopenia is notably observed in the acute infections which are associated with an increase of the polynuclear neutrophiles, and is almost always relative. ‘The condition per se / has received but little attention, and is relatively unimportant from the clinical standpoint. Variations in the Number of Large Mononuclear Leukocytes.— Variations in the number of the large mononuclear leukocytes are as a rule not sufficiently marked to cause either a distinct increase or decrease of the total number of the leukocytes. One notable exception to this rule, however, exists in the cases of the acute type of lymphatic leukemia, in which the predominant cell is the large lymphocyte, viz., the juvenile form of the common large mononuclea! leukocyte, in the sense of Pappenheim. At the same time it mus’ be noted that some cases of chronic lymphatic leukemia also occu in which the large mononuclear leukocyte and Ehrlich’s transitior form represent a large percentage of the leukocytes. ‘These relations * Cited by Da Costa. MICROSCOPIC EXAMINATION OF THE BLOOD 113 however, are not constant. A decrease is notably seen in myelocytic leukemia. In the so-called pseudoleukemia infantum of v. Jaksch a marked increase of the mononuclear elements is observed in a certain per- centage of cases, but in the larger number the general increase of the leukocytes is pert bie to an increase of the polynuclear cells. | A relative as well as an absolute increase of moderate grade is observed in many of the diseases in which the lymphocytes are increased, as in rickets, syphilis, measles, scarlatina, smallpox, and according to my experience also in typhoid fever, etc. I have ob- served a marked increase in a case of Addison’s “Rents a few days before death, and found notable numbers in debilitated individuals, in association with sloughing epithelioma, etc. In a fatal case of epidemic cerebrospinal meningitis with a high leukocytosis and polynucleosis which I recently saw there was both -a relative and absolute increase of the mononuclear leukocytes. Some of these as well as some of the neutrophiles contained menin- gococci. In mycosis fungoides an increase of the large mononuclear elements has been noted. by Hodara* and Pappenheim.’ A distinct increase is further observed in chronic malaria. In this connection Krauss* remarks that it is not so much the absolute increase of these cells which is diagnostic of malarial infection as the relative increase over the small mononuclears. In cases of malarial infection without much fever and without quinine the polynuclears are at the same time markedly diminished, while during the rise of a malarial fever, or as a result of quinine therapy, the polynuclear neutrophiles may reach 80 per cent.; but even then the large mononu- clears exceed the small mononuclears. In Kala-azar, as in malaria, there is a distinct tendency to an in- crease of the large mononuclears. In a series of 10 cases reported by Donovan, the average was 21.58 with variations from 6 to 48 per cent. _ Variations in the Number of the Mast-cells—A small number of mast-cells is found in the blood under normal conditions. The presence of more than 1.5 per cent. is probably always pathological. A remarkable increase is noted in the myeloid type of leukemia and is one of the most constant features of the disease; more constant, in fact, than the increase of the eosinophiles. The REECE is not necessarily above normal, but not infrequently values of from 5 to 10 per cent. are found. It is noteworthy that this increase of the mast- sells may be demonstrable at a time when the disease is apparently quiescent. In one instance of this kind the total number of the leuko- ‘ Monatsheft f. prakt. Dermat., 1904. ? Folia hemat., vol. i, p. 487 > Jour. Amer. Med. Assoc., October 22, 1904. 114 | THE BLOOD cytes had been 350,000; three months later I counted but 2080, of which 10.9 per cent. were mast-cells, and later they rose to 15 per cent. The only other condition in which I have found such high values occurred in a patient, following fracture of the ankle and consequent cellulitis. In this case they rose to 17 per cent. and the blood in general presented a typical leukemic picture. A few days later normal values were found. | A more moderate increase is noted in many other diseases. Gen- erally speaking, my experience has been that they are more numerous. in conditions in which the eosinophiles also are increased, and are. generally diminished when the eosinophiles are below normal. ‘This! rule, however, is not absolute. I have found values above the normal) in various skin diseases, in gonorrhea, in certain cases of malignant disease, associated with eosinophilia. In one case of renal carcinoma, a few weeks after the removal of the growth. I counted more than) 2 per cent. of mast-cells, with but 1.9 per cent. of eosinophiles. Canon reports an increase of mast-cells in chlorosis; Sherrington, in cases of Asiatic cholera, dying in the reactive stage; ‘Taylor, in two cases of septic bone disease; Da Costa states that an increase has also been observed in some cases of splenic anemia. I have found the number diminished or entire absence of mast- cells in some cases of malignant endocarditis, appendicitis, empyema, | influenza, tonsillitis, intestinal obstruction, lumbar abscess, periproc- titic abscess, pernicious anemia, hematoma of the abdominal walls, diabetes, carcinoma of the cervix (septic), “black” jaundice, pneu- monia (unresolved). In malaria the number is usually normal. Mye:ocytosis.—At birth and during the first weeks of life it is usual to meet with a small percentage of neutrophilic myelocytes in the circulating blood under perfectly normal conditions, while in adults their presence is always abnormal. In children the tendency to mye- locytosis is always more pronounced than in adults. Zelinski- and Cybulski," who have studied this question more particularly, have found myelocytes in a great many diseases. In the pseudoleukemia) of v. Jaksch the number varied between 1.5 and 17.4 per cent., in congenital debility from 3.5 to 12.5 percent. In congenital syphilis they found myelocytes quite commonly, the number—usually moderate —depending upon the intensity and duration of the morbid process. They disappear upon institution of mercurial treatment. In rickets the number of myelocytes 1s dependent upon the intensity of the morbid process. In the lighter cases they are scanty or absent. Scrophulosis is not associated with myelocytosis. ‘Tuberculosis and catarrhal processes involving the digestive apparatus, of long duration, cause the appearance of myelocytes in fairly large numbers. Very curiously acute dyspeptic processes were also found associated with 1 Jahrb. f. Kinderheilk., 1904, vol. lx, p. 884 ) MICROSCOPIC EXAMINATION OF THE BLOOD 115 anyelocytosis in very young children. In a case of congenital atresia of the anus they found 20 per cent. of myelocytes. - Tiirk has shown that they are quite common in the acute infectious diseases of childhood, and in diphtheria Engel ascertained that chey are especially numerous in the severe cases (3.6 to 16.4 per eent.). In mild infections they are not usually seen, and when yresent they are found in only very small numbers. In pneumonia hey are absent or very few in number at the beginning of the dis- ease, while at the time of the crisis or immediately thereafter they yecome more numerous and in some cases represent 12 per cent. of ill neutrophilic cells; such high percentages, however, are rather amcommon and are more apt to be encountered in children than in idults. In acute septic conditions a small number of myelocytes nay also be observed; larger numbers are found in the more chronic vases, which are Peociied with marked anemia. In a case of umbar abscess which had been discharging for six months I found "8 per cent. In a case of “black” jaundice I found 2.2 per cent. Neusser has soted their presence in asphyxia and acute mania; Ewing states that hey have been found in considerable numbers in rickets, osteomyeli- is, and osteomalacia. Da Costa speaks of their occurrence in poison- ug by carbon monoxide, in hepatic cirrhosis, acute gout, malignant peers, and exophthalmic goitre. _ The occurrence of myelocytes under these conditions is to be egarded merely as a quantitatively or gradually increased polynu- leosis of the corresponding granular cells, the result of an increased jestruction of the adult cells and consequent increased production amisohypercytosis). ‘This is in contradistinction to the myelocytosis -ssociated with myeloid leukemia where there is a primary increased ormation referable to myeloid hyperplasia; this form is*essentially a vassive myelocytosis, while the former is active. ' In anemic conditions of whatever origin it is common to meet vith a moderate number of neutrophilic myelocytes. In pernicious ‘nemia they are quite constant in the active stage of the disease; ‘s arule the values do not exceed 0.5 to 1 per cent., but at times they pay reach 7 per cent. _ Pappenheim makes a distinction between primary hemophthisic vernicious anemia of the Biermer type and the form referable to “othriocephalus infection, on the one hand, in which myelocytes in is experience do not occur, and primary myelophthisic spleno- hedullary anemia, myelomatosis, and myelogenous pseudoleukemia tumor anemia, pseudopernicious anemia) on the other, in which Pe may be present. | _ According to Kurpjuweit? the occurrence of myelocytes in large | 1 Deutsch. Arch., vol. lxxvii. 7 116 THE BLOOD numbers (4 to 17 per cent.) in connection with the symptom complex | of a severe anemia is to be viewed as almost pathognomonic of malig- nant growth with bone-marrow metastases, even when a prima tumor cannot be found. In the secondary anemia associated with syphilis and malignant disease, as also in the pseudoleukemia of v. Jaksch, variable figures are | found (1.5 to 17 per,cent.). In a young child in which a notable | anemia had developed as the result of amebic dysentery, Amberg | counted 9 per cent. In the estivo-autumnal type of malaria they | are quite common. ; The neutrophilic myelocytes which are met with under these various conditions are almost without exception of the small trachy-| chromatic variety. ‘The amblychromatic variety is practically only | encountered in the myeloid type of leukemia, which is really the one disease in which large numbers of myelocytes of all kinds find) their way into the blood. Upon their presence in numbers exceeding those found in all other diseases the diagnosis is largely dependent. The blood state is that of a true myelemia. ‘The number of neutro- philic myelocytes in myeloid leukemia is often most remarkable, and a count of from 50,000 to 100,000 per cubic millimeter is by no means_ exceptional. ‘The average percentage of 18 cases reported by Cabot was 37.7, corresponding to a total number of 162,000 leukocytes. Coincidently with the neutrophilic myelocytes eosinophilic myelocytes also appear in the blood and may constitute the majority of the eosino-. philic cells seen in this type of the disease; their percentage, how- ever, is rarely large. ‘The total number of the polynuclear eosino- philes is at the same time increased, although the relative percentage may be normal or even slightly below normal. ‘The polynuclear neutrophilic cells and the lymphocytes, while absolutely increased, are relatively much diminished. Of the latter, only 7.6 per cent. are found on an average, and of the former 49.2 per cent., as com- pared with 20 to 30 and 60 to 70 per cent., respectively, under nor- mal conditions. ‘lhe mast-cells, as I have pointed out, are inya- riably present in increased numbers in the myeloid type of the disease. While the majority of the neutrophilic and eosinophilic cells present a normal habitus, it is common in myeloid leukemia to meet with dwarfed forms. Occasionally also leukocytes are ob- served which are undergoing mitosis. Of special interest is the fact that in certain chronic cases of the disease the neutrophilic cells apparently lose the power of forming neutrophilic material. Non-granular polynuclear cells and myelocytes then appear in the blood and may give rise to much confusion. In one case of this kine reported by Ehrlich the great majority of the mononuclear elements which constituted 70 per cent. of the total number, were entinel free from neutrophilic granules. The total number of the leukocytes in myeloid leukemia in the MICROSCOPIC EXAMINATION OF THE BLOOD ak active stage of the disease is much increased. In Cabot’s series vof 30 cases the average was 438,000. If at the same time, as not ‘infrequently occurs, there is a coincident anemia with marked diminu- tion of the red cells the ratio between the whites and reds may fall to 1 to 2 or even | to 1; there are cases on record, indeed, in which ‘the leukocytes outnumbered the red cells. Formerly much. stress ‘was laid upon this ratio in the diagnosis of the disease; leukemia “was regarded as a hyperleukocytosis in which the ratio exceeded .a definite proportion that was generally placed at 1 to 50. As a matter of fact, there is probably no other disease in which so great an increase of the leukocytes is observed, and even at the present day the diagnosis ‘is usually justifiable when an increase of such proportions is noted. But, as I have pointed out, myeloid leukemia is essentially a myelemia | and not a hyperleukocytosis. ‘here are cases, moreover, exceptional _/to be sure, in which the increase of the leukocytes i is not so extreme. I have ese one case in which the total number was only 2080 and the ratio of the whites to the reds 1 to 1015. The diagnosis of the disease should hence be based primarily upon qualitative changes in the morphology of the blood and only secondarily upon an increase of the leukocytes as a whole. When septic complications supervene in the course of the disease the blood condition may undergo marked changes. ‘I’hus, in propor- (tion to the degree of infection the myelemic picture oradually dis- ‘appears and is replaced by that seen in simple septic conditions. ‘The polynuclear neutrophiles may then increase to 90 per cent., and even more, while the eosinophiles diminish and may almost ‘disappear. | A-ray treatment in a certain number of cases may cause a marked ‘change in the blood picture. ‘The total number may fall rapidly jand there is a general tendency to normal conditions; a complete disappearance of myelocytes 1 is, however, very rare. The mast-cells -very curiously remain above normal, In the purely lymphatic form of leukemia neutrophilic myelocytes ‘are scanty; there are cases of mixed leukemia, however, in which at some stage of the disease the blood picture is essentially of the Hymphatic type, while at another period there is a marked myelo- cytosis.? _ Ina case of compound fracture with consequent cellulitis I found a blood picture which was typical of myelocytic leukemia, with large ‘numbers of myelocytes (15 per cent.). After a few days there was a /return to normal. Hastings tells me that he has found myelocytes In 3 to 7 per cent. of cases of fracture. ___* For a detailed consideration of the blood changes in leukemia see especially : Pinkus, “ Die Leukaemie,”’ Nothnagel’s Encyel. Ewing, Clinical Pathology of the Blood, Lea Brothers. Cabot, Clinical Exam. of the Blood, Wm. Wood & Co. |Pappenheim, Zeit. f. klin. Med. Haematologische Streitfragen, 1903. } | | 1138 THE BLOOD In pseudoleukemia myelocytes are essentially seen in cases where the pathological process has affected the bone-marrow (myeloid pseudoleu- kemia) and where, as a result of lymphadenoid substitution of the | myeloid tissue, an irritative myelocytosis develops. | E osinophilic myelocytes, aside from their occurrence in myeloid | leukemia, are comparatively rare. ‘They have been found in the | pseudoleukemia of infants; Mendel’ speaks of their occurrence in_ a case of myxedema; ‘Tiirk” reports that they are occasionally seen | in some of the infectious diseases, and Bignami claims to have seen | them in pernicious malaria. In one case of posthemorrhagic | anemia referable to a ruptured tubal pregnancy I found 1 per cent. | of eosinophilic myelocytes, and in a case of myelogenous leukemia, | in which the eosinophiles were absolutely much diminished, the only | eosinophile that I could find in many slides was a myelocyte. Ina case of trichinosis they were also occasionally seen at a time when the eosinophiles were much increased. The Plaques. In addition to the leukocytes and red corpuscles large numbers of | small, roundish elements are encountered in the blood which measure | about 3 4 in diameter and are free from coloring matter (Plate Il, Fig. 1). They are frequently seen collected into groups resembling bunches of grapes. ‘These are the blood plates or plaques of Bizzozero. - According to Hayem, they represent red corpuscles in an early stage of development, and are themselves derived from leukocytes within the | lymph channels. He terms them hematoblasts. ‘This view is not. shared by modern hemotologists. Lilienfeld, Hauser, Howell, and others regard the plaques as disintegration products of leukocytes, while still others look upon them as precipitated globulins derived | in part from the morphological elements of the blood and in part. originating directly in the plasma. More generally accepted is the view expressed by Engel, Bremer, Maximow, Pappenheim, and others, according to which the plaques are derived from the red cells by extrusion. ‘They are originally contained in the interior of the cells | as so-called nucleoids, and represent the remains of the original nucleus, which has lost its individuality as the result of chromatolysis. | As a matter of fact, it is possible by suitable staining to demonstrate | the plaques not only within the red cells, but also their extrusion | from the cells, so that the erythroglobular origin of some of these’ formations at least can scarcely be doubted. Jost, moreover, has shown that in the blood of sheep and calf embryos they appear at a 1 Berl. klin. Woch., 1896. No. 45. ? Klin. Untersuch. d. Blutes, ete., Wien u. Leipzig, 1898. MICROSCOPIC EXAMINATION OF THE BLOOD 119 time when leukocytes are not as yet demonstrable. But, on the other hand, there is a possibility that what we generally designate as plaques does not represent a unity, and that some of the elements which resem- ble the true blood platelets may be of different origin. ‘lo a certain ‘extent such ill-defined little bodies are without doubt derived from leukocytes by a process of plasmorhexis—i. e., by the liberation of small bits of protoplasm. ‘This may be observed under the microscope directly. | _ Deetjen has shown that the true plaques are capable of executing ameboid movements when the blood is placed on a slide which has been covered with a thin film of agar containing a certain amount of sodium chloride, sodium metaphosphate, and dipotassium phos- phate. He also believes to have demonstrated a nucleus in the in- dividual plaque, and concludes that the bodies in question do not represent artefacts or products of degeneration, but are true cellular elements. According to Osler, the number of plaques varies normally between 200,000 and 500,000 per cubic millimeter. Brodie and Russell claim that this number is too small, and that with their improved method of counting an average of 635,300 is obtained. ‘lhe normal ratio between the plaques and the red corpuscles would thus be 1 to 7.8, taking 5,000,000 as the average normal for the red cells. More recently Helber found variations between 192,000 and 264,000. _ Under pathological conditions the plaques may be increased or diminished. In pernicious anemia their number is very low; Van Embden found 64,000 and 32,000 in two cases. At times they are apparently absent, but in some cases increased numbers have been observed. - According to Pappenheim the plaques are diminished in pernicious anemia owing to over-rapid maturation of the red cells. As a result the nuclei of the erythroblasts either do not become pyknotic and un- dergo chemical chromatolysis with consequent formation of oxyphilic, \vlZ., azurophilic nucleoids, but are destroyed already in an early stage by karyorrhexis; or, if they do become pyknotic, they are expelled from the cells plasmolytically in the anisotonic (anemic blood serum). A nucleoid thus does not remain which could later escape as a plaque. In leukemia the plaques are often greatly increased. A large Increase is at times observed in posthemorrhagic anemia and in chlorosis, but the results are not constant. In the secondary anemias referable to carcinoma, sepsis, tuberculosis, etc., the findings are variable; sometimes an increase is observed, at others a decrease, and then again normal values; the results, moreover, are inconstant in one and the same case. In the acute infectious diseases their number is the smaller the more severe the course of the disease. In pneu- monia they are often diminished during the fever, but increased after the crisis. Similar results have been obtained in typhoid fever, while in ¥ 120 THE BLOOD erysipelas they are found increased from the start. Enormous numbers | of plaques may be seen in the course of trichinous infection. Schleip | looks upon their appearance in large numbers as evidence of approach- ing convalescence. In my own case, however, they seemed to be | most numerous at a time when the clinical symptoms were most active. (For the enumeration of the plaques see p. 144.) LITERATURE.—Bizzozero, Virchow’s Archiv, vol. xc. Hayem, Le sang, Paris, 1889. Howell, Jour. of Morph., 1891, vol. iv, p. 57. Maximow, Arch. f. Anat., 1899, vol. i, p. 33. Jost, Arch. f. mik. Anat., 1903, vol. lxi, p. 667. Determann, Deutsch. Arch. f, klin. Med., vol. lxi, p. 365. Deetjen, Virchow’s Archiv, 1901, | vol. elxiv, p. 239. Brodie and Russell, Jour. Physiol., 1897, Nos.4 and 5. Heller, Deutsch. Arch., vol. xxxi, Heft 3 u. 4. The Dust Particles or Hemokonia of Muller. These may be seen in any fresh specimen of blood mounted in the usual manner. ‘hey are small, generally round, sometimes dumb- bell-shaped, colorless, highly refractive granules, which manifest very active molecular movements. ‘They occur in the plasma of the blood and are apparently not connected with the process of coagula- tion. Miiller found them abnormally numerous in a case of Addi son’s disease, while they were diminished during starvation and im various cachectic conditions. Stokes and Wegefarth regard these granules as identical with the neutrophilic and eosinophilic granules of the leukocytes. ‘They suppose that the bactericidal power of the | leukocytes and of the serum of man and many animals is due to their presence. As a matter of fact, the origin of the hemokonia from the granular leukocytes can frequently be directly observed. I have quite constantly found the hemokonia increased at the height of digestion, and have then repeatedly observed their extru- sion from both neutrophilic and eosinophilic cells. LireRATURE.—H., F. Miller, ‘‘ Ueber einen bisher nicht beachteten Formbe- | standtheil d. Blutes,”’ Centralbl. f. allg. Path. u. path. Anat., 1896, p. 929 W. R. Stokes and A. Wegefarth, “The Presence in the Blood of Free Granules, ete., and their Possible Relation to Immunity,’ Johns Hopkins Hosp. Bull., ‘1897, p. 246. E. B. Sangree, “Leukocytic Granules,” etc., Phila. Med. Jour., 1898, p. 472. General Technique. Slides and Cover-glasses.—T’o obtain satisfactory results, it is essential to have glassware of the best quality. ‘The cover-glasses should not measure more than 0.08 to 0.10 mm. in thickness and must — be cleansed with care. ‘The same holds good for the slides, which — should have a level surface; many of those furnished by dealers _ are unsatisfactory for work with i immersion lenses. ee ee ———- MICROSCOPIC EXAMINATION OF THE BLOOD 4 HPA _ Both covers and slides should be placed in concentrated sulphuric acid or in glacial acetic acid for several hours. ‘They are thoroughly ‘washed in running water and distilled water and then placed in alcohol and finally in ether, where they remain for several hours. ‘During this process care must be had that they are well separated from each other. Subsequently they are kept in jars with absolute alcohol, and are dried just before use, or they may be dried at once with fine linen or Japanese lens paper and stored in dust-proof -receptacles. When once cleansed, the cover-glasses should be ‘handled only with forceps. To cleanse slides that have been used, the covers must first be removed by immersion for several days in xylol or turpentine. They are then placed in hydrochloric acid to which about a tea- spoonful of potassium chlorate has been added for every 30 c.c. The mixture is kept on the boiling water bath to the point of decolorization. ‘The slides are next rinsed in hot water, heated for ‘a half-hour in a thin mush of equal parts of washing soda, sawdust, and talcum, prepared with the aid of water and stirring frequently, then washed off with hot water acidified with hydrochloric acid, and ‘finally with pure hot water, alcohol, and ether. The Blood Mount.—We distinguish between wet mounts and dry mounts. Wet specimens can only be utilized successfully if the patient is near at hand to the laboratory, as in office work and in the hospital; where several hours must elapse before the preparation can ‘be examined, it will usually be best to resort to the dry specimen. Wet preparations, however, are very convenient and yield a large ‘amount of information without delay, and a rapid survey will indi- cate whether or not it will be necessary or advisable to resort to a more detailed examination. ‘The grade of an anemia: the degree, icharacter, and extent of a hyperleukocytosis; the presence of malarial organisms, can all be told from the wet preparation. With the dry _and stained specimen, on the other hand, all these points are brought out more distinctly, and other information is further afforded which ;cannot be obtained from the wet specimen alone. _ To prepare a blood specimen, the tip of a finger, or in children especially the lobe of the ear, is first cleansed with ether and then punctured with a suitable instrument, such as a fine lancet or a ‘Stout needle. ‘he puncture should. be sufficiently deep that the blood will flow from the wound without undue pressure. To prepare a wet specimen, a clean cover-glass is taken up with a /par of forceps, with flat blades and a light spring, touched to the ‘drop without coming in contact with the skin, and immediately transferred to a clean slide. If suitable glassware is used that is perfectly clean, the drop will immediately spread out between cover- ‘glass and slide, and on examining with a low power, which should always precede examination with a high power, it will be noted that 122 THE BLOOD in the central portion of the specimen especially the red cells will be well separated from one another and will not have run into rouleaux. This will only occur if the glassware is imperfect, if it is not perfectly clean, or if the drop has been too large. ‘To gauge the proper size of the drop requires a little practice. Along the margin of the speci- men, where a certain amount of evaporation is going on, it is usual to find rouleaux and crenated red corpuscles, even though the rest of the specimen is perfect, and in the course of time postmortem changes will also become noticeable throughout the preparation. If the specimen is ringed with a little paraffin, however, a satisfactory examination is still possible after a number of hours, and even without being ringed such preparations can be kept for at least one hour. To prepare dry specimens, which are subsequently to be stained, the blood is spread between cover-glasses or on slides. Personally I have almost abandoned the use of cover-glasses, and much prefer slides for routine work. But little practice is Fic. 17.—The preparation of blood smears on slides. required to obtain very satisfactory results, and it is possible to control the quality of the individual smears with a degree of precision which — is but rarely attained even by the most experienced workers with cover-glasses. ‘The spreads, moreover, are much larger; so that there will always be a sufficient number of leukocytes available even under normal conditions to permit a count of at least a thousand cells. At the same time it is possible to spread portions of the drop — so thin that the individual cells are well separated the one from the | other, while other portions can be made a little thicker. The slides are cleansed in the same thorough manner as in the case of the cover- glasses. A fair-sized drop of blood is then mounted near the end of — MICROSCOPIC EXAMINATION OF THE BLOOD 128 one slide and spread with an even sweep with the edge of a second slide; this should be done with a light hand, and holding the first slide in the left hand between the thumb and the second and third fingers. ‘I‘he second slide should also be held in this manner, but at an angle of 45 degrees to the first, as shown in the accompanying illustration (Fig. 17). Before commencing the sweeping movement I let the blood spread along the edge of the second slide by capillary attraction; then I move across, gradually raising the second slide to a vertical position. Pressure must be carefully avoided. _ If covers are to be used, one cover-glass is locked in a pair of forceps such as those devised by Ehrlich and pictured in the accom- panying illustration (Fig. 18). A second cover is taken up with a pair of forceps without a lock, but with flat blades and a light spring; this is held to the drop of blood just as it emerges from the puncture, and is then immediately laid upon the first cover. If the glasses are of satisfactory quality and clean, the blood will at once spread in a capillary layer; the top cover is then drawn from the lower cover by grasping the edge firmly with the fingers and making even traction in a plane parallel to the other. Here also a certain amount of experience is necessary in gauging the size of the drop | A } a Fie. i8.—-Ehrlich’s cover-glass forceps. in reference to the size of the covers. In no case should it be so arge that the top cover floats upon the blood. If the drop is rather small, the two covers should overlap only to such an extent as to furnish a space which is just filled by the blood. If the drop is arger, they should overlap over a larger surface. After being allowed to dry in the air the specimens are placed between layers of filter paper and may then be stained at leisure. [ff several days must elapse before the examination, it is well to place them, wrapped in filter paper, in closed jars. Should it be desired to preserve the specimens for a long time—‘. e., for months or years—it is best to coat the films with a thin layer of paraffin, which later is dissolved by immersion in toluol. In this manner especially valuable and rare specimens may be kept almost indefi- aitely. Unless this precaution is taken, the staining qualities of all the morphological elements of the blood will undergo changes which render the specimens unfit for color analysis. | Fixation.—The selection of the method of fixation depends very jargely upon the stain which is to be employed. If strongly alcoholic ’ | solutions are used no previous fixation is necessary, but with aqueous | solutions fixation must precede the staining. ‘l’o this end several | methods may be employed. The best results are obtained by heat. | For this purpose a copper plate may be used measuring about 10 em. | in width by 40 cm. in length and 3 to 5 mm. in thickness; this is heated | by a Bunsen burner or a small coal-oil stove. After the plate has a_ fairly constant temperature, the desired degree is ascertained by a series of drops of water, toluol (boiling point, 110° to 112° C.), or xylol (137° to 140° C.), ete., noting the line at which ebullition occurs, | If the distance of the plate from the flame and the size of the flame, etc., are constant, the apparatus requires practically no attention | and serves its purpose very well. As a rule a brief fixation only is | necessary—1. €., exposure to a temperature of from 100° to 126° C. | for one-half to two minutes, while in special cases Ehrlich recom- | mends a more prolonged exposure or a higher temperature. Very) good results are obtained for most purposes by heating the blood | films to a temperature of 140° C. for thirty to forty-five seconds, | as suggested by Rubinstein. This point is conveniently ascertained on the copper plate by noting the line at which the so-called Leiden-| frost phenomenon begins to occur, viz., the point at which a drop of) water assumes the spherical form and rolls about on the plate. In the place of the copper plate an ordinary drying oven provided with a thermostat and thermometer or a_ so-called Victor Mayer | Siedekessel may also be employed. ‘The latter is a small copper kettle covered with a thin plate, which is perforated for the recep-. tion of the boiling tube. If a small quantity of toluol is boiled in this kettle for a few minutes, the copper plate will acquire a tem-: perature of from 107° to 110° C., and retains this sufficiently long for ordinary purposes (Ehrlich). Absolute alcohol or a mixture of equal parts of absolute alcohol | and ether (Nikiforoff) have also been recommended as fixing agents | for blood films, but are not very satisfactory for the study of the neutrophilic granulation. With Ehrlich’s triacid stain especially it” will frequently be noted that the granules are stained imperfectly or not at all. For the study of nuclear structures, however, both are quite satisfactory. In the case of absolute alcohol alone immersion of the blood films for a few minutes is sufficient; with alcohol and ether fixation for one-half to two hours is necessary. Formalin is useful as a fixing agent and may be used in con- nection with practically all the common blood stains. 1000c.c: Semerecont: SOsin BOlUfION ys. =. Te 100 c.e. Pervenrome-blué solution --. 4. 6. Ce 200 c.c. 1 per cent. methylene-blue solution . . . . . . 70 cle: ) | The mixture is stirred. A green, metallic-looking scum appears on the surface and a fine precipitate separates out. ‘To bring this about it may be necessary to add a little more of the 1 per cent. nethylene-blue solution, viz., 80 instead of 70 c.c. The mixture may be filtered at once or after standing for twenty ‘o thirty minutes. ‘The residue is allowed to dry in the air or in the Irying oven at a temperature not above 60° C. It is finally pulverized und can be stored in this form. ‘The amounts of the dyes indicated ibove furnish from 0.7 to 1 gram of the ultimate product. ' For staining purposes a 0.25 per cent. solution in absolute methyl weohol is used, which is prepared by rubbing up the dye with the ilcohol in a mortar. If successful the solution has a purple plum color. _ Care should be had that the alcohol is neutral. Some lots of methyl] uecohol show an acidity of 1 to 2 cc. of 7% alkali for 100 c.c. such specimens must be neutralized by the addition of 0.05 to 0.1 gram of dry sodium carbonate for 100° c.c. _ Previous fixation of the blood specimens is not necessary, as the ucohol fixes while the staining is going on. ‘The films are covered _ vith the solution and left for one minute, after which they are differ- mtiated by the addition of water until a greenish, metallic-looking ‘cum appears on the surface (15 drops to a slide). ‘This is continued or five minutes, when the preparations are rinsed for two to three econds in water and immediately dried by blotting. ‘This procedure vill answer for all ordinary purposes, and for bringing out the young orms of the malarial parasite, but for the maturer forms it is better 0 stain for two minutes and to differentiate for ten. _ The negative surface of the specimen should be carefully inspected nd washed if necessary, to remove any dried stain that may be present ind which appears as a thick, greenish coating. _ In a properly stained specimen the red-cells appear red; in over- : : ! | | _ 134 THE BLOOD stained or old specimens light gray or light blue. Polychromatophilia and granular degeneration are well shown. ‘The neutrophilic gran- ules are bright red, the eosinophilic granules eosin colored, and the mast-cell granules dark red. ‘The nuclei of the lymphocytes, large mononuclear leukocytes, and myelocytes are magenta red; those of the polynuclear leukocytes a bluish violet. In some of the lympho- cytes and large mononuclear leukocytes Michaelis’ granules will be seen. ‘The blood plates are pale blue with red nuclei. ‘The nuclei of the red blood corpuscles are red. ‘The malarial organisms present a blue body with one or more intensely red nuclear structures, varying in size from that of a tiny dot in the youngest forms to a structure which | in the microgametocytes fills the entire body of the parasite in the form of a fine reticulum. In the segmenting bodies each segment contains a red nucleus, while the body is blue. | LerIsHMAN’S Metuop.’—Leishman also makes use of the isolated | eosinate of methylene blue mixed with eosinate of methylene azure. : He proceeds as follows: ‘wo solutions are prepared: one a 1 pro. mille solution of eosin (Griibler’s extra B.A.) in distilled water; the other a 1 per cent. solution of medicinal methylene blue (Gribler), also in distilled water, and alkalinized with sodium carbonate to the | extent of 0.5 per cent. ‘This last solution is heated to 65° C. for twelve hours, and is then allowed to stand at the temperature of the room for ten days before using. Equal volumes of the two solutions are mixed in a large open basin and allowed to stand for from six to twelve hours, the mixture being stirred from time to time with a glass | rod. ‘The resulting precipitate is collected on a filter, thoroughly . washed with distilled water, dried, and powdered. A 0.15 per cent. solution of the dye in pure methyl alcohol serves as stain and does not | deteriorate on keeping. Special fixation is not required. The blood film is covered with the solution and stained for about one- half minute. Double the amount of distilled water is then added and allowed to mix with the alcoholic solution. After five to ten minutes: the stain is washed off with distilled water, a few drops of water being: allowed to rest on the film for a minute. ‘The specimen is next dried (without heat) and can be examined as usual. ‘The soaking in water for a minute after the staining is important, as it intensifies the Roman- owsky stain; it changes the tint of the red corpuscles from a greenish-. blue to a transparent pink or greenish color, while the nuclei of the leukocytes are usually a ruby red. ‘The nuclei of nucleated red cells are almost black and the extranuclear portion gray. ‘The blood plates: are a deep ruby red with shaggy margins, frequently showing a pale-. blue peripheral zone surrounding the red ¢entre. The body of the malarial parasite stains blue and ‘its chromatin a ruby red. In the case of the tertian parasite Schiiffner’s dots are well marked in the containing red corpuscles. | 1 Brit. Med. Jour., September 21, 1901. MICROSCOPIC EXAMINATION OF THE BLOOD 135 _ Wricut’s MopiricaTion oF LerIsHMAn’s Mernop.’—Wright has simplified Leishman’s method in several important particulars, which render it even more convenient for routine work; he has ascer- tained, moreover, that any one of the Griibler methylene blues can be employed for the purpose of obtaining a sufficient quantity of methylene azure. _ ‘The staining fluid is prepared as follows: 1 per cent. of methylene blue is added to a 1 per cent. aqueous solution of sodium bicarbonate, when the mixture is steamed in an Arnold steam sterilizer for one hour. On cooling, the solution is poured directly into a large dish or flask and treated, while stirring or shaking, with a sufficient quantity of a 1 pro mille solution of eosin (yellow shade) until the mixture has assumed a purple color and a scum with a metallic lustre forms on the surface. ‘This will require about 500 c.c. of the eosin solution for 100 c.c. of the methylene-blue solution. ‘The resultant precipitate, which contains both eosinate of methylene blue and eosinate of methylene azure, is collected on a filter, and without further washing allowed to dry. When thoroughly dry, a 0.3 per cent. solution in pure methyl alcohol is prepared (this is practically a saturated solu- tion). ‘The solution is filtered and to the filtrate 25 per cent. methyl alcohol further added so as to dilute the stain somewhat and to lessen the tendency of the dye to become precipitated during the process of staining. | The air-dry blood films are covered with the stain for one minute; water is then added drop by drop until the staining fluid becomes isemitranslucent and a reddish tint becomes visible at the margins, while a scum with a metallic lustre forms on the surface. ‘lhe amount of water required will vary with the amount of staining fluid on the ‘preparation, but in general it may be said that 8 or 10 drops will suffice if a seven-eighths inch square cover-glass is used. ‘The staining fluid, thus diluted, is allowed to remain on the preparation for two or ‘three minutes, during which time the real staining of the specimen takes place. It is then washed off, when the blood film will be seen to have a blue or purple color. The next step is to develop the differential staining of the various elements in the preparation. This is done by washing the prepara- ion in water, preferably distilled water, until the better-spread por- tions of the film appear yellowish or reddish in color. If desired, ‘the process of differentiation may be readily observed by placing the cover-glass, film side uppermost, on a slide, covering it with water, and examining it with the microscope under a low magnifying power. The red blood corpuscles, which, as before stated, at first have a blue ‘color, will become greenish, then yellowish, and finally orange or pinkish in color, depending upon the depth of the original staining, ' Jour. Med. Research, 1902, vol. vii. 136 THE BLOOD which varies with the length of time that the diluted staining fluid has been allowed to act, and with the degree of its dilution. The differentiation by washing in water seems to be essentially a a process of decolorization by which some of the blue constituent of the dye is removed, for the water that drains off from the preparation has a blue color. ‘This differentiation or decolorization proceeds slowly, and may require from one to three minutes, depending upon the intensity of the staining and upon the tint sought to be obtained in the red corpuscles. It is apparent from the above that with a little experience with the method the color of the red corpuscles may be made either orange or pink. When the desired color is obtained in the red corpuscles the preparation is quickly dried between layers of filter paper and mounted in balsam. It is important to arrest the decolorization by drying the preparation as soon as the desired tint in the red corpsucles is obtained, for it may be carried too far. Dried blood films may be kept for weeks without impairment of their staining properties. Films months old will probably not give good results. In a suitably stained specimen the red cells are either orange or pink; polychromatophilia and granular degeneration are well shown (the granules blue); the neutrophilic granules are a reddish lilac; the eosinophilic granules eosin colored; the mast-cell granules a dark blue, a dark purple, or even black. ‘The lymphocytes have dark purplish-blue nuclei with robin’s egg blue protoplasm, in which the granules described by Michaelis appear dark blue or purplish. ‘The large mononuclear leukocytes present a blue or dark lilac colored nucleus, and in some Michaelis’ granules can also be made out. The blood plates are stained a deep blue or purplish and the malarial — organisms are colored as with Leishman’s method. Giemsa’s Method.'—Giemsa’s stain has the following composition: Azure IT (azure plus preranpe blue Ad) sits Gee ae rane 3.0 Kosin (B. A.) on} Se sank Sale aaa? ean ee 0.8 Glycerin (Merck, C. P.) MS iar ee Peay et Ah Methyl alcohol (Kahlbaum Thy. 5h rece od De er ee ae eee a It is prepared by grinding up the dyes in the absolute alcohol and | then adding the glycerin. ‘The blood films are fixed for a minute in absolute methyl alcohol and then stained for five minutes in a _ mixture of 14 drops of the dye to 10 c.c. of distilled water, which is always freshly prepared; a trace of sodium carbonate may be added to the water to intensify the basic colors. After washing in water the films are blotted and are then ready for examination. The various elements are stained as with the methods already — described. 1 Centralbl. f. Bakter. Abt. I, vol. xxxvii, 2, p. 308 ENUMERATION OF THE CORPUSCLES OF THE BLOOD 137 - Goldhorn’s Method.'—The blood smears are fixed with pure methyl alcohol for fifteen seconds, washed in running water, stained for ‘thirty seconds in a 1 per cent. aqueous solution of eosin, washed, stained for one minute in Goldhorn’s polychrome methylene blue, again washed and dried in the air. | The polychrome methylene blue is prepared as follows: 2 grams of ‘methylene blue and 4 grams of lithium carbonate are dissolved in 300 ‘ec. of warm water. ‘The solution is heated in a porcelain dish on a. boiling water bath for 15 minutes, then poured into a glass-stoppered ‘bottle and set aside for several days. Finally it is rendered only slightly alkaline by the careful addition of 4 to 5 per cent. acetic acid solution (test with litmus paper). ‘The method gives excellent results. DEMONSTRATION OF IODOPHILIA, Cover-glass specimens are prepared as usual; after drying in the air they are placed in a small jar containing a few crystals of iodine. After several minutes the films assume a dark-brown color, when they -are mounted in a drop of a saturated solution of levulose and examined with an-oil-immersion lens. The red corpuscles are stained light yellow, while the leukocytes are almost colorless. All glycogen granules, whether contained in leukocytes or free in the blood, are stained a distinct mahogany. - This method furnishes better. results than the older method of staining with a solution composed of 1 gram of iodine and 3 grams ‘of potassium iodide in 100 grams of a concentrated solution of muci- lage (1 part of Lugol’s solution to 100 parts of a thick mucilage.” . ENUMERATION OF THE CORPUSCLES OF THE BLOOD. Method of Thoma (Author’s Modification) .’—The instrument con- sists of two diluting pipettes and a counting chamber (Fig. 19). ‘I'he latter is ruled into 100 large squares (A, A, A), each occupying an area of s5 sq. mm. (Fig. 20). They are separated from one another by double guiding lines (a b, a b) with an intervening distance of 3'y mm. Where the horizontal and vertical lines intersect small squares (a, a, @) result, 100 in number, which accordingly have an area of 7)> Sq. mm. each. The large squares are thus bounded by rectangles (b, b, b), measuring 3); mm. in width by 54; mm. in length, representing an area of shy sq. mm. As the little platform (/) carrying the ruling is exactly jy mm. * The New York Univ. Bull. of Med. Sci., 1901, vol.i, No. 2. ® Ehriich-Lazarus, Die Anaemie, loc. cit. | * The counting chamber can be procured from Ernst Leitz & Co., New York. 138 THE BLOOD lower than the outside glass plate (e), each large square represents 1 efi ic yal the base of a cube the contents of which are 5; TO = 350 cb. mm.; each small square similarly corresponds to <)> X qo = topo Cb- mm., | and each rectangle to z45 X zo = rd0 Cb. mm. | 1. Enumeration of the Leukocytes.—A drop of blood is procured by | freely puncturing the finger or the lobe of the ear, after cleaning and | drying the skin, wiping away the first drop or two, and avoiding un- | due pressure. It is drawn into the 1 to 10 diluting pipette to the mark egstge C Frc. 19.—Thoma-Zeiss blood-counting apparatus. A and E£, red and white diluting pipette, respectively; B, counting chamber, seen from above; C, profile of counting chamber. “4 1, and after carefully wiping the end is immediately mixed with a 1 per cent. solution of glacial acetie acid, containing a small amount of an aqueous - gentian violet solution (1 c.c. for 100 c.c. of the dilute acid), by drawing up the mixture to the 11 mark. The rubber tube of the pipette is detached, both ends of the pipette closed with the thumb and middle finger, and blood and diluent well agitated. A couple of drops are then blown out, so as to clear the capillary tube of the diluting fluid which has not entered the bulb of the pipette. A drop of the diluted blood is now placed upon the platform of the counting slide, and one of the cover-glasses which accompany the instrument adjusted in such a way as to exclude bubbles of air. ‘The size of the drop should be such that, when the cover-glass is in place, it does not run over into the moat (g) surrounding the circular platform, nor even project over the sides. ‘Tiirk advises that a tiny droplet of the’ pure diluting fluid be placed upon the plate D, before the diluted blood is placed upon the counting platform. If cover and slide have been previously scrupulously cleansed and slight pressure is now made upon the cover where it overlies the plate D, Newton’s anting Chamber. iled in red, is used in counting the red ugh large square 1/250 cb. mm. ytes; the central block of 16 asure 1/4000 cb. mm., and oj PLATE VII. a a cabbelened ag aa a Bl Ba Pale bi eth oes —} ee eee else [2 asa soa a a eee ene ee ee ee eee a : ee See | 4 PP} ieee a Cees oe eee Se | | SS Ee ee ed ee ee a ad ie i ape ll ce se eel bel I A St Sa aa ncieal ued el ae a ee (TE | | | SE a er geae eae i eek i a I a a OG [eerie Sted ed ahead ecm ae a a ee ee eceen | . 2 a a ees ee aa a a ae a py} |__| | a bi eS eS ~ SS e ee ; ol Le eH ‘gee aa A ae sae ea ie ae i || le ge Turk’s Counting Chamber. There are in all 144 large squares, for counting the leukocytes; the central block of 16) ruled in red, is used in counting the red cells. The cubic contents of each small square measure 1/4000 cb. mm., and of eagh large square 1/250 cb. mm. BNUMERATION OF THE CORPUSCLES OF THE BLOOD {39 olored rings will become visible—a sign that a successful mount has wen made. ‘Lhe slide is set aside for a few minutes, so that the rpuscles settle down, when it is examined with a high power (Leitz Jeular 2 and Obj. 6).* It will be noted that the red corpuscles are visible and that the leukocytes are colored blue. For counting , mechanical stage is very convenient. Starting with the top row f large squares at the left corner, the total number of leukocytes the 100 large squares is now carefully counted. his number Fig. 20.—Simon’s counting chamber, ivided by 100 gives the average number of leukocytes for one large quare. As the cubic contents of each large square are 5}; cb. mm., Js only necessary to multiply the number of leukocytes in one square y 250 in order to find the number for 1 cb. mm. of diluted blood, and his by the degree of dilution (in the above instance by 10) to find he number for 1 cb. mm. of diluted blood, and 1000 * 10 the number 11 ch. mm. of non-diluted blood. 7 To focus through the thick cover the objective should have a long working istance, 140 THE BLOOD Exampie.—Total number of leukocytes counted in the 100 large squares = 400; hence 494, viz., 4 = number of leukocytes in a single square, 1. é., in-zty cb. mm. of diluted blood; hence 250 x 4 = 1000 the number of leukocytes in 1 cb. mm. of non-diluted blood, anc 1000 10 the number in cb. mm. of non-diluted blood. When counting the cells note should only be taken of such tha lie within the squares or upon the upper and left boundary lines cells upon the right and lower lines should be omitted. In the above instance a dilution of 1 to 10 has been advocated. ‘T’his may be used as a matter of routine. If a marked grade of leukocy- tosis is anticipated a dilution of 1 to 20 will be found more convenient Fie. 21.—Tiirk. Fic. 22._-Thoma; centre part, Fic. 23.—Zappert-Ewing. Fic, 24.—-Thoma, Blood-counting chambers. Tf desired even higher dilutions may be used, in which case the red pipette permitting of a dilution of 1 to 100 or more is employed. 2. Enumeration of the Red Cells —'l‘he blood is diluted 100 times by filling the red pipette with blood to the mark 1 and with the diluent to 101. For. diluting the blood in the enumeration of the red cor- puscles Toison’s solution is most convenient: Socditim chloridey fay h er. oat eee ee eee ees 1.0 Sodium ‘sulphate® 455. 292550 oe) eines oy ee eee ee 8.0 Netltral vlycerin fai inc 3c, be 3) 22 ee eee 30.0 Distilledswater? x4. Re TSS 2) ee eee eee Methyl violet (5 B.) . 0.025 ENUMERATION OF THE CORPUSCLES OF THE BLOOD 14] _ To prevent the development of molds the solution should further sontain about 1 pro mille of thymol. _ After mixing the diluent and blood thoroughly and blowing out he pure diluting fluid in the capillary tube a drop is mounted as lescribed. All the red corpuscles are then counted—in the 100 mall squares, if no marked degree of anemia exists, or in 40 or more ectangles if the corpuscles are distinctly diminished. ‘The calculation 3 then made as follows, bearing in mind the cubic contents, corre- ponding to the small square and the rectangle, viz., ¢o'99 and poly b. mm., respectively: ExampeE 1.—Number of red cells in 100 small squares = 1000; in 1 herefore 10, viz.,in z4ya cb. mm.; in | cb. mm. of diluted blood 4000 x 0 = 40,000 and in 1 cb. mm. of non-diluted blood 40,000 * 100 = 000,000. EXAMPLE 2.---Number of red cells in 40 rectangles = 800; in 1] eactangle therefore °° = 20,1. ¢., in zoo cb. mm.; in 1 cb. mm. of liluted blood hence 20 * 1000 = 20,000, and in 1 cb. mm. of non- iluted blood 20,000 x 100 = 2,000,000. If for any reason a larger area is to be counted for red cells, this an, of course, be readily done by going over a larger number of rect- ngles, or by combining small squares and rectangles, due allowance veing made for the cubic contents of the ground covered. Other counting chambers are also in existence. ‘The form of the uling of various models is shown in the accompanying figure (Figs. ‘1 to 24). ‘They are used in the same manner as that of the author. “he calculation in each case depends upon the number of squares ounted, and its corresponding cubic contents and the degree of lilution. _. Second Mernop.—lIf a counting chamber with one of the more nodern rulings is not available, but if a mechanical stage is at hand, he leukocytes can also be counted with the old Thoma counter n the following manner: A drop of the diluted blood is mounted as isual. With the mechanical stage a field corresponding to the osition of 1 in the accompanying diagram (Fig. 25) is then selected is the starting point. ‘The presence or absence of leukocytes is noted ind’ the field changed, so that an adjoining circle is brought into ‘ew, and so on. In this manner at least 100 circles are gone over, ising a corpuscle to the side or above or below as a guide to the next ield. ‘The total number of leukocytes is noted and the average or one circle calculated. If the cubic contents corresponding to ach circle are known, the calculation of the number of leukocytes nicb. mm. of blood becomes a simple matter. ‘The determination f the cubic contents corresponding to a circle is made as follows: Noting the number of the eyepiece and the objective, the diameter of he field of vision is measured with a stage micrometer, or with the iid of the rulings of an ordinary Thoma-Zeiss counter, bearing in ‘ ; + 142 THE BLOOD mind in the latter case that the distance between two vertical line is yy mm. ‘The area of the circle, according to geometrical law, wil then be equivalent to zp’, in which z is a constant factor—. e., 3.1416 and p the radius, from which the corresponding cubic contents ar calculated by multiplying the result by 0.1—. e., the depth of th counting chamber. ‘The resultant value, which should be ascertaine for every instrument separately, will, of course, be constant for the sys tem of lenses and the counting chamber used. With a Bausch @ Lomb ¢ (long-working distance), the 1-inch eyepiece, and 160 mm tube length, the cubic contents of the field are 0.009 cb. mm. Example.—The blood was diluted 100 times. In 100 fields 5| leukocytes were noted—+. e., 0.5 for 1 field, or for 0.009 cb. mm. in 1 cb. mm. of diluted blood there would hence be 0.5 divided b: | Fic. 25.—Schema of circles for counting leukocytes: a, moat surrounding central platform, b, of counter; 1, starting point. 0.009 = 55.5, and for 1 cb. mm. of undiluted blood 55.5 x 100 = 5550 leukocytes. | Cleaning of the Apparatus.—After use the apparatus must be care fully cleansed. ‘The pipette is washed out with the diluting fluid then with water, next with absolute alcohol, and finally with ether The washing will be facilitated by slipping the rubber tube over thi long arm of the pipette and blowing the contents of the bulb out 0 the short arm. In laboratories which are equipped with a suctiol pump this may be conveniently employed; the entire process thei occupies only two or three minutes. The counting chamber is washed with water only; alcohol and ethe dissolve the substance with which the platform is cemented to the slide . ENUMERATION OF THE CORPUSCLES OF THE BLOOD 143 | Differential Enumeration of the Leukocytes.—The differential enu- meration of the leukocytes is usually made in dried and stained speci- mens. A mechanical stage is a great convenience, but not a necessity. The idea is to go over a large number of cells, for ordinary purposes not less than 500 to 600, to classify these, and finally to calculate the percentages. ‘he cells are charted as shown below: S. M. (small mononuclear leukocytes): AY FW RL AW TH THK TA TK TK = 45 L. M. and T. F. (large mononuclear leukocytes and transition forms): FY TL YY = 15 } P. (polynuclear neutrophiles): WZ TRL HL ML ML RL TK TA THOT oT THT TA KK WL TA TOOTH oT OK EK Mh WA TK TAL MM TK = 155 E. (eosinophiles) : }AY — 5 M. (mast-cells): // =— 2 222 Result : Total number of cells counted, 222, of which: 45x 100 small monos., GODS sine 20.2 per cent. 15X 100 large monos., Ly hee 6.7 155X100 polys., 999. ~= 69.8 # 5x 100 ‘a eOsiNs., 992 = 2 100 e mast., 999. = U9 While making a differential count it is always well to keep note of the time, as it is often possible in this way to form a fair idea. of the actual number of the leukocytes without an absolute count. ‘This, of course, requires a certain amount of experience in the preparation of the smears, which should be uniformly of nearly the same thick- ness. After one has then learned by control how many leukocytes in a blood smear, observed within a certain length of time, may be considered as normal, it is not difficult to judge the grade of a hyper- leukocytosis by the increase in number noted within the same length of time. Everyone must here work out his personal equation. A ‘general idea of the degree of increase can, of course, be formed by examining the specimen with a low power—a Bausch & Lomb 4, for example—but in the manner indicated one gets a numerical expression which is at times quite helpful. ‘The count itself can be readily made with a 3; it requires a little practice, but proves a great saving of time. , | | | | 144 THE BLOOD Enumeration of the Plaques.—For this purpose the method of Broide and Russel has been advocated. ‘The method is an indireet | one. First, the red corpuscles are counted in the usual manner. A drop of the staining fluid, composed of equal parts of a 2 per cent. solution of common salt and a saturated solution of dahlia in glycerin, is then placed upon the finger, when this is punctured through the | drop and the blood allowed to mix with the reagent. In this mixture the ratio between the plaques and the red corpuscles is ascertained, | and the total number of plaques contained in 1 cb. mm. of blood | determined by calculation. ‘The plaques are stained the color of | dahlia and can readily be counted. Rapid work is essential, as the | staining fluid soon attacks the red corpuscles. Other writers determine the ratio of plaques to red cells in smears and then calculate their number after an absolute red-cell count. Jenner’s stain or any one of the methylene-azure mixtures (Hastings’, — Giemsa, Wright) will answer the purpose. The Hematocrit.—The use of the hematocrit for counting the red blood corpuscles has been repeatedly advocated, but has not met with favor. ‘The method is inapplicable whenever there is any material variation in the size and form of the red corpuscles and when- ever the number of the leukocytes is greatly increased. ‘This means that the method cannot be employed in the majority of cases in which we are especially interested in the blood count. If, however, it is desired to ascertain the volume of the red corpuscles in relation to | the amount of plasma, the instrument will furnish satisfactory results. | A centrifuge run by electricity is practically a necessity; in this way alone is it possible to maintain the proper rate and uniformity of speed. | Hand centrifuges are, in my experience, totally inadequate, and with instruments driven by water power it is impossible to attain a sufficient — rate of speed for this purpose. An apparatus like the one pictured in the accompanying illustration (Fig. 26) answers the purpose best. | It is connected with the street current or with a small battery, a_ rheostat being interposed to control the current and the rate of speed. | At the same time a speed indicator can be attached which strikes a bell for every 100 revolutions. For the hematocrit a speed of 8000 to - 10,000 revolutions per minute is required. | The hematocrit which is almost exclusively used in the United | States is that of Daland (Figs. 27, 28, 29). It consists of a metallic | frame which carries two glass tubes measuring 50 mm. in length | and 0.5 mm. in diameter. Each tube bears a scale ranging from | 0 to 100, the individual divisions of which are rendered more easily visible by a magnifying lens front. In the frame the outer end | of each tube fits into a small depression, the bottom of which is cov- ered with thin rubber; the inner ends are held in position by springs. The instrument is screwed to a firm table and is oiled daily when in use. — If the patient is directly available, undiluted blood is used, ‘The J nq EN ~ TR 4 4 TIO N Oo a) F TH yl & 4 YOR PU USCLES DF r y BIL LOOL ] 45 by ing er is ‘reel wasl y ied ond I net vitl OI ( 30a + The the A smal ae watel the rubh pee ‘bevel endo si Sata a en u e 10 n th er tul as be d « IES; 1s as Be sn, en f the whi rae naa i foci Baio been previous! tube ee h is cE caine and osite sid rtment rected ia epi quickh »mpletel over ae e and ce the fr nd the fee eee ati filled fe e “am. as 1th 2 vy ins e. s tt a witl trument Its mate snetiatel e fe roti 1S di elir ated r apl ately fi , a r pl ced spe a - ed O of frc n 1 = ame a = = = —S Fic. jae. mer rh Im to eo pro sen crit stat, ved i e n rivattachment lectric h evol ent speed ema re laaprceed ae a t at wl Bare He ene t The ube s j, Fie il ] > oF — Tal and’s d’s he matc serit ae. 10,000 he a4 is s aeeieds ase a aah PU tly re ions per mi fc: 4 minute ford in Bui ee mal, nat oe the Oe Be uals ale when i . peer nine a. that eae es at a Stee s age of compels i t itera pane ate 1e figure i a with yichro- 146 THE BLOOD mate, as proposed by Daland. As Ewing suggests, this can be) done with the pipette which accompanies the ‘Thoma-Zeiss blood counter. In the case of the red pipette the capillary tube is filled with blood to the mark 1, then a small air bubble is drawn in, fol- lowed by another tube length of blood. Three or four volumes of blood are obtained in this way and diluted at once with an equal. quantity of the bichromate solution. In the case of the white pipette a single tube length of blood and the diluent 1s sufficient. Blood and diluent are thoroughly mixed, care being had not to} include any air bubbles. In this form the blood is carried to the. laboratory, where both tubes are filled by allowing the drops to flow. in from the point of the pipette. ‘To obtain the percentage volume: the resultant figure is in this case, of course, multiplied by 4. | In the case of normal blood it has been ascertained that 1 per cent. by volume, as read off from the scale, corresponds to almost: 100,000 red corpuscles per cb. mm.; to obtain the total number of aq Fig. 28.—--Daland’s hematocrit. ge : \M\ 3 i | i | i] ) | i Ul} WL Kin, | rf Af a WY po! : pH $+ titttity ety tosis tte te tee AP MO CRB EA TT A | So a 25 9 ee 5 + rr ¢ | eee (yee re eee fee Ft? ts ha 20 ee J 1 Fic. 29.,—Daland’s hematocrit tube. } red cells per cb. mm., it is hence only necessary to add five ciphers to the percentage indicated on the scale. Example—Undiluted blood was used; the reading on the scale was 45. The volume per cent. of the red corpuscles would hence be 90, and the number of red cells per cb. mm. 4,500,000. | But, as I have pointed out, this calculation presupposes that the size and form of the red cells are practically normal, and that the leukocytes are not materially increased. | With normal blood the leukocytes appear only as a narrow, indis- tinct, milky band at the central end of the column of red cells, whieh: with a material increase of the leukocytes becomes more marked and reaches its greatest extent in well-marked cases of leukemia. | Aspelin has recently suggested that with a suitable modification of the Daland apparatus quite accurate leukocyte counts can be ob- tained by centrifugation; but bearing in mind the variations in the size of the different leukocytes and the varying degree in which the ‘ >, ESTIMATION OF HEMOGLOBIN 147 lifferent forms take part in the production of the different types of ayperleukocytosis, it is evident at once that still less is to be antici- sated from the centrifugal method in this direction than in.the case of the red cells. + Volume Index.—The term volume index has been introduced by “apps to designate the relation existing between the volume of red ‘ells determined by centrifugation (see above) and their number. if both are normal the ratio ore Ci bercen = (0.99 average of 10 hormal individuals). In 29 cases of pernicious anemia. the volume ndex was high during the active stage of the disease, ranging from 05 to 2.0. During periods of improvement it steadily fell, while n periods of decline it steadily rose. In chronic secondary anemia vf moderate intensity normal values are the rule; in a few they are ow. In acute secondary anemia (sepsis, hemorrhage) the index may ve low (0.72); so also in chlorosis of the severer type. In a few cases f chronic severe secondary anemia (as in uncinariasis) Capps found he volume index high. Analogous results have been obtained by Vroth. LirerRATURE.—Hedin, Arch. f. ges. Phys., vol. xl, p. 360. Giirtner, Wien. klin. Voch., 1892, No. 2. Daland, Fort. d. Med., 1891, No. 21. Aspelin, Zeit. f. klin. led., 1903, vol. xlix, p. 393. J. A. Capps, Journ. Med. Research, December, 103. P. Wroth, Johns Hopkins Hospital Bull. February, 1907. ESTIMATION OF HEMOGLOBIN. 'Hemoglobinometers.—While it is usually possible to form a urly clear idea of the degree of anemia by direct inspection of the atient, the appearance of the mucous surfaces, etc., it is often de- rable to obtain more definite information, and, above all, a numer- val expression of the extent of the anemia. ‘This is especially im- ortant in the diagnosis of certain forms of anemia, in which the color index” plays an important part—1t. e., the ratio between the ercentage of hemoglobin and the percentage of the red corpuscles, s compared with the normal. ‘To this end, special instruments have een devised, which are termed hemoglobinometers or hemometers. ‘f the various forms which are now in the market, the hemoglobin- meter of Dare is probably the best, and is rapidly replacing the old istrument of y. Fleischl, which for many years was the standard. - Is more exact and more convenient. Miescher’s modification of te Fleisch] instrument is possibly still more accurate, but too costly general adoption. ‘The little instrument of Gowers, in the modifica- on of Sahli, when obtained from a reliable source will also furnish od results. Unfortunately many of those which have been placed 1 sale are worthless. Oliver’s instrument has some advantages over je Fleischl, but none over the Dare. The Talquist method is : ’ | warmly recommended by Cabot, and may be used to advantage in routine work by the general practitioner; for exact work it is insu ficient. | Dare’s Hemoglobinometer.—' he essential parts of Dare’s hemo- globinometer (Fig. 30) are an automatic pipette for collecting the blood (Fig. 31) and a graduated color scale (Fig. 32) to measure the corresponding percentage of hemoglobin. ‘This latter reads. from 10 to 120, the 100 mark corr esponding to the color of a solution of 13.77 grams of hemoglobin: in 100 cc. of serum. The various shades of color cor- responding to the scale are ob- tained by rotation of a pris- matic glass semicircle tinted with golden purple of Cassius, z (Fig. "32 , E), which is secured to a thin white glass disk (1). The numerical scale is placed on the edge of a correspond- =i calla el ing semicircle (47) of thick ated white glass (/). This pari of the apparatus is enclosed in¢ dust-proof hard-rubber case and is rotated from the outside by the aid of a rubber-covered roller which runs on the edge of the disk and is turned by a milled wheel at R (Fig. 30). In the rubber case is a little circular window through which the color of the prism is viewed by means of a small telescoping camera tube (Fig. 33, N)) 148 THE BLOOD Fic. 30.—Dare’s hemoglobinometer. Fic. 31.—Automatic pipette. Fic. 32.—Graduated color scale. ' provided with a magnifying lens of low power. The color aperture represents a surface about equal to 3 per cent. of the color scale Looking through the tube a corresponding window will be seen sid¢ by side with the one through which the color scale is visible. In froni of this the blood pipette is secured. ‘The essential part of this is ar oblong plate of white glass (Fig. 31, A), into the end of which ¢ depressed surface of measured depth i is ground, the floor being ex actly parallel to the plane surface of the glass. ‘This depressior forms a capillary chamber (D) when the transparent glass plate (6B. i ESTIMATION OF HEMOGLOBIN 149 ‘is firmly clamped upon it by the pipette clamp C; it is filled by capillary attraction when either of the three free edges is touched to the blood drop. ‘The pipetteyis le d in position on the stage of the instrument by guides which run in grooves on the lower part of the clamp. ‘The plate of white glass is toward the light. , The camera tube screws into a movable shutter (Fig. 30); when | this is swung outward the two aper- tures become visible through which the blood and the colored scale are viewed. In front of the pipette a candle is clamped in such a position that both the blood and the color scale are equally illuminated. Mernop or Usre.— As the compari- son of the color of the blood with that of the color scale should be made as Fic. 33. Horizontal section of Dare’s hemoglobinometer (on a level with centre of comparison ‘apertures): J, candle; K, white | glass disk of color prism; L, color prism; M, aperture through which color of blood film is viewed; M’, aperture through which the illum- inated color prism is viewed; JN, camera tube; O, transparent glass of pipette; P,.white glass of ; : pipette. Fic. 34.— Filling the automatic blood pipette. soon after filling the pipette as possible, the apparatus is prepared for use beforehand by screwing the camera tube into place and ad- justing the candle; this should be at such a level that the blue flame of the candle is below the color aperture, care being taken to have the wick of proper length (half-inch) and not charred at the tip. Curved or eccentric wicks should be turned so that the intensity of light in a vertical position is midway between the two color aper- tures. _ The glass plates of the pipette having been thoroughly polished and vefastened in the clamp, the finger or ear is freely punctured as usual and the capillary space of the pipette filled with the blood, by hold- ing one of the three edges horizontally to the drop (Fig. 34). Any blood adhering to the flat surfaces of the glass plates is carefully wiped away and the pipette placed in position. ‘The candle is lighted, the shutter thrown out, the camera tube focused, and the color of the blood (on the left) compared with the color scale (on the right). ‘The two are matched by rotating the color disk by means 150 THE BLOOD of the milled wheel, which should be done in an abrupt manner, and frequently resting the eye. ‘Io this end the shutter is dropped and thrown out again as the case may be. ‘The examination need not be conducted in a darkened room, but it is important to turn the instrument toward a dark background, so as to eliminate all direet or reflected light. ‘he reading is indicated by the bevelled edge of the rectangular opening on the side of the case; the figure immedi- ately beneath this represents the percentage of hemoglobin. Im- mediately after use, the two glass plates of the pipette are cleansed with water and a little acid alcohol, dried, and again replaced. Fur-' ther details in regard to technique accompany the instrument. | My personal experience with the instrument has been quite satis- factory. LITERATURE.—A. Dare, Phila. Med. Jour., Sept. 22, 1900. Fleischl’s Hemoglobinometer.—'I‘he principle underlying the vy. Fleisch! method is essentially the same as that of the Dare method; the color of the blood is compared with the color of a glass wedge stained with the golden purple of Cassius or a similar pigment, a scale indicating the corresponding amount of hemoglobin. With the Fleischl instrument, however, diluted blood is used, which is one} of the disadvantages of the method. | The instrument (Fig. 35) consists of the glass wedge a, to which a scale, b, is attached, ranging from 0 to 120, 0 being placed at the thinnest, 120 at the thickest portion of the wedge. By means of a rack and pinion this may be made to slide from side to side beneath a platform corresponding to the stage of a microscope. In the centre of the platform there is a circular opening into which artifi- cial light (daylight is not permissible) is projected from a circular plate of plaster of Paris mounted beneath, in the position of the mirror of the microscope. Into the circular opening a metallic tube, 1.5 em. in height, is fixed, which is closed at the bottom with a) plate of glass and divided into two equal compartments by a metal partition. One compartment receives the light through the glass wedge—the red chamber; the other, directly from the plaster-of- Paris reflector—the white chamber. | Capillary pipettes of known capacity accompany the instrument, This capacity is somewhat variable and is indicated on the handle of each, which number must correspond with that marked on the tof screw head of the instrument. Generally speaking, the capacity ol each pipette is such that with the blood of a perfectly normal ind vidual the mixture of blood and water in the white chamber wil correspond in color to that of the colored wedge at the mark 10( (a 13.77 per cent. solution of hemoglobin). The pipette is filled by capillary attraction from a drop of bloo¢ ESTIMATION OF HEMOGLOBIN 151 btained in the usual manner. If on trial it is found that the blood joes not immediately run up in the tube, this is repeatedly washed ut with water and then dried. If this is always done after the xamination, the pipette will be in working order on the next oc- asion. While filling the pipette care should be had that it is not mmersed in the blood, but only brought in contact with it. ‘The wo compartments of the cell having been previously partly filled with vater, the charged pipette is at once placed in the white chamber nd rapidly moved to and fro until the blood is well mixed with he water. Any trace remaining in the pipette is carefully washed ut with water by the aid of a medicine dropper. ‘lhe contents of ’ Fic. 35.—v. Fleischl’s hemometer., 1e chamber are stirred with the handle of the pipette when both ompartments are filled with water, using the same dropper, so that qere is a convex meniscus over each. ‘The color of the blood is ten matched on the wedge, which should be moved by quick turns f the adjustment screw rather than in a gradual way, as the eye will therwise be less apt to appreciate fine shades of difference. Day- ght, as I have said, is not permissible; a candle or gas flame of ioderate intensity placed about a foot and a half distant is best. ‘he eye should be perpendicularly above the cell, and it is well to tew the colors through a paper tube which is placed over the two ompartments. ‘The number facing the notch in the little well | ; 152 THE BLOOD immediately behind the cell indicates the percentage of hemoglobin, The readings corresponding to the middle portion of the wedge are: apt to be more nearly correct than the lower values. For this reason it is well, when a preliminary examination has shown a low figure, to repeat the test, using two or three pipettefuls of blood instead of one, the result, of course, being divided by 2 or 3, as the| case may be. On the whole, the Fleisch! method furnishes results which are somewhat lower than those obtained with the Dare; this is true especially of the older models, with which a percentage of 100 was only rarely observed.. The instruments of more recent construction, however, are much better. Personally I regret to see the Fleischl apparatus supplanted by newer instruments; it was con- venient and neat. It has its defects, to be sure, and it is unfor-) tunate that the Miescher modification, in which these have been elimi- nated, and which unquestionably gives the most accurate results, is still so costly that its general use is out of the question. Gowers’ Hemoglobinometer (Sahli’s Modification)—'lhe apparatus (Fig. 36) consists of two glass tubes (A and B) which are of the same diameter. One of these (A) is closed and contains a solution of hematin hydrochlorate in a concentration corresponding to a 1 per cent. solution of normal blood. ‘The other tube is provided with an ascending scale of 140 divisions, each degree corresponding to 20 cb. mm. A capillary pipette marked at 20 cb. mm., a guarded lancet, a dropping bottle, and a small stand accompany the instrument. The finger is punctured as usual and the pipette filled to the 20) cb. mm. mark; the blood is immediately discharged into the graduated | tube and mixed with one-tenth normal hydrochloric acid (saturated | with chloroform as a preservative) which has been previously filled’ in to the mark 10. When the color of the mixture has become a clear . dark brown, water is added drop by drop, shaking after every addition, until the color matches that of the standard solution. ‘The division on the scale ultimately reached indicates the percentage of hemoglobin. The examination can be conducted with natural and _ artificial light. | ‘The method, as I have indicated above, is satisfactory if the instru-, ment has been obtained from a reliable source. Its low cost makes) it especially serviceable in large clinics and for purposes of teaching, in the clinical laboratory. But in every case it is advisable to com- pare its scale with a standard instrument | Talquist’s Method.—'The color of the blood, in this case undiluted, is compared with a series of lithographed standard tints, which repre- sent a scale ranging by tens aon 10 to 100. ‘The technique i is very simple: drops of blood are received on pieces of white filter paper of suitable thickness which accompany the color scale, and are compared: with the tints on the plate, using ordinary daylight. Accuracy is, of course, not to be expected from so crude a methodly| ESTIMATION OF HEMOGLOBIN 153 ‘so that its use is of necessity limited. It will suffice in a very general way to control the result of treatment, but it is inapplicable in the ‘determination of the color index. ’ Estimation of Blood Iron with Jolles’ Ferrometer.—'l'he estimation of the hemoglobin from the amount of blood iron, as originally sug- ‘gested by Jolles, is unfortunately not possible, as it has been shown ‘that constant relations between the two bodies do not exist. All the ‘iron of the blood is not present in this form, nor does it all occur in the form of colored compounds. Jolles’ method B A of estimating the total amount of blood iron ‘deserves consideration, however, as it Is a practical method and discloses facts which ‘are of clinical interest. It is desirable that it ‘should be introduced in the clinical laboratory ‘as a routine method. The principle is the following: A small ‘amount of blood is incinerated, and the re- ‘maining red oxide of iron brought into solu- tion with a little monacid potassium sulphate. In this solution the iron is then estimated Ppeminetrically with an instrument which is constructed upon the principle of Fleischl’s hemometer and which is termed the ferrorm- eter. It is made by Reichert in Vienna and can be readily transformed into the hemom- ‘eter proper. Full directions accompany the ‘apparatus. ‘The results are expressed in rel- ative terms, the number 100 on the scale ‘corresponding to 0.0425 per cent. by weight of iron. Some of the results which have been ‘obtained with the clinical ferrometer are given Hl ue below, together with the corresponding ire ——=—= Fic. 36.—Sahli’s hemo- indicating the amount of hemoglobin: Polabmormetar Ferrometer Hemometer number. number, Bettini ee wo eS ee | e &* 108.0 100 Dee eee ON, Me fe el) Oe Oe, 92.6 105 Sone ae ee ee 95.5 100 eS Te eae, al i 110.0 105 Denese asa 2) go eS 83.8 92 rereeE ee, ett. 3g SO nd 9 82.1-68.2 30-65 BreeSOUNE oe i ae es 8B, 2-74.7 15-40 ee nT bis 55.0 SO Leukemia Pr oF nt age Le Sse 40.7 D2 Leukemia ee te en eee ee) P: 38.6 35 Pseudoleukemia bh Og et Chet 75-80 SOG a a (h-57 30 Severe diabetes .. Eee | re ae al OF 91.4 35-40 Parenchymatous ne phritis ee BO ee 1.7 50 154 THE BLOOD These figures at once illustrate the lack of relationship which exists between the amount of hemoglobin and that of the blood iron as a whole. In a series of cases Jolles also examined into the presence of iron. in the serum, by centrifugating a given volume of blood mixed with | an 0.8 per cent. salt solution, ed found that in health the serum contains no iron. In 3 cases of chlorosis, in 1 case of leukemia, in 1 of neoplasm, and | of interstitial nephritis, negative results were like- wise reached. In 2 cases of severe diabetes, on the other hand, notable quantities were found. Deganello' has studied the relation between the amount of blood sell Hb and found that this ratio remains normal, until the Hb has reached iron and hemoglobin ( in different forms of secondary anemia, | | | | a certain minimum—46 to 58 per cent.; from this point off the value | se surpasses the normal the more the deeper the Hb value falls. Mere mechanical loss of Hb does not materially alter this value, however, even in cases of marked oligochromemia. When toxic | influences are at play marked discrepancies will result. Mitulescu’ comes to quite analogous conclusions. He thinks that | the hemoglobin estimation only is required as a rule, from which the | iron value ans be calculated according to Hoppe-Seyler’s formula: Hee . If hemolytic processes are suspected, or if albumin- | Cin uria exists, both methods are to be employed. LITERATURE.—A. Jolles, “ Ferrometer,’’? Deutsch. med. Woch., 1897, No. 10; | ibid., 1898, No. 7. Hladik, “ Untersuchungen uber d. Eisenghalt d. Blutes> gesunder Menschen,’’ Wien. klin. Woch., 1898, No. 4. 8. Jellineck, “Ueber | Farbekraft und Eisengehalt d. Blutes,”’ ibid., Nos. 33, 34. -volsix xia, pir i2l: Agglutination Test (Pfeiffer- Widal Reaction).—Owing to the develop- | ment of specific agglutinins in the blood serum of typhoid patients, as a_ consequence of infection, such serum possesses the property of causing | arrest of motility and agglutination of the corresponding bacilli. ‘This | observation, originally made by Pfeiffer, was utilized for diagnostic purposes by Widal, in 1896. ‘The method which bears his name has | been generally adopted in the clinical laboratory, and must be regarded | as a most valuable aid in the diagnosis of typhoid fever. ‘The reaction — occurs in over 95 per cent. of undoubted cases, and may appear as early as the first day of the disease, meaning thereby the first day that the patient spends in bed or the fifth day of general malaise. Such instances, however, are uncommon, and, as a general rule, a positive result is obtained only after the fifth or sixth day in bed. | In a small number of positive cases, on the other hand, the patient may pass through the entire course of the disease, and present typ- ical clumping only during convalescence or a subsequent relapse. In every case, therefore, in which no reaction is obtained upon first trial, the test should be repeated at regular intervals throughout the disease. 1 At first the bacilli are but little active, but on further cultivation and rein- oculation their motility increases. | BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD 16] -ntermittence of the reaction, moreover, is very common, and empha- izes still further the necessity of frequent examinations in apparently iegative cases. _ While in some instances the reaction disappears very soon after he temperature reaches normal, and even earlier, it generally con- inues into convalescence, and may in some cases be observed months vnd years after the attack. Cases have been recorded in which a yositive reaction could be obtained thirty-seven years after infection. - Ina series of 71 cases who had had typhoid fever in the past Krause’ ound the reaction in 36—in 1 instance twelve years after the illness. )£ 26 cases examined within a year 16 gave a positive result, of 21 rom the second to the fifth year 12, of 19 from the fifth to the tenth ear 7, and of 5 from the tenth to the twentieth year 1. In 3 in- tances no reaction could be obtained within a month of the disease. - Such observations, of course, entail the usefulness of the test to a ertain extent. For this reason the demonstration of a negative eaction early in a case which is followed by a positive reaction is a particularly valuable symptom, the early negative result eliminating he possibility that the subsequent positive finding could be referable 9 an antecedent typhoid. | The question whether or not Widal’s reaction is a specific reac- ion of the typhoid organism can be answered in the affirmative, otwithstanding the fact that at times cases of apparently true typhoid ever are seen in which no clumping is obtained, and that the reaction as been observed in cases which were apparently non-typhoid. ouch exceptions are due in part to faulty technique, viz., to too low a _egree of dilution of the serum, the use of old or impure cultures, too ong a time limit of observation, single negative tests, etc. On the ther hand, there can be no doubt that typhoid bacilli are at times resent in the body without giving rise to symptoms of typhoid fever. na case of cholelithiasis, reported by Cushing, typhoid bacilli were ound in the gall-bladder, and distinct clumping was observed with dilution of 1 to 30, although no history of typhoid fever could be btained. Another interesting apparent exception to the rule that the Vidal reaction is only obtained in cases of typhoid infection is re- orted by Griinbaum,’ who notes that he obtained a positive reaction 1 cases of febrile jaundice. His observations have since been amply onfirmed. ‘The biliary components pro se have manifestly nothing » do with the production of the reaction, however, as is shown by 1e observation of Kiimmerer* who obtained agglutination in only 3 tundice cases out of 50 (from the most diverse hepatic diseases). In le positive cases no doubt infection had occurred by some organism ) ' Zentralbl. f. Bact., 1904, vol. xxxvi, No. 1. ? Cited by Durham, Brit. Med. Jour., 1898, vol. ii, p. 600. * Berlin. klin, Woch,. No, 25, 1904. |e 162 THE BLOOD of the paratyphoid group, standing nearer to the typhoid than the colon bacillus. There can further be no doubt that individuals exist who are naturally immune against typhoid fever, and that some of the positive | results which have been obtained in healthy individuals who haye never had typhoid fever may be explained in this manner. So far as the non-occurrence of the reaction in cases of apparently | true typhoid fever is concerned we now know that infections occur with organisms which are closely related to the typhoid bacillus, (the paratyphoid group) and which clinically resemble typhoid fever, but which give no reaction with the typhoid bacillus, unless in | low dilutions. (See Paratyphoid Fever.) Widal’s test is a most valuable aid in the diagnosis of typhoid fever, but cannot be relied upon to the exclusion of other symptoms. Technique.-—The method is based upon the fact that typhoid serum will cause arrest of motility and agglutination of the specific bacilli even when diluted, whereas clumping of the same organism is obtained only with sera from other diseases and healthy individuals when these are used in a more concentrated form. ‘The time limit. at which clumping occurs is likewise an important factor, as non- typhoid sera are at times met with in which, notwithstanding a) certain degree of dilution, agglutination occurs, providing that the) specimen is kept for a long time. Both factors—viz., the degree of dilution necessary to eliminate the agglutinating power of non-typhoid sera, as also the time limit of observation—have been arbitrarily de- termined. Widal originally advised a dilution of 1 to 10, and Griiber a time limit of one-half hour. It was soon ascertained, however, that this dilution was too low, and most observers have fayored a dilution of 1 to 40 or 1 to 50. At the present time there is a ten- dency to further increase this even as far as 1 to 200 with a time limit of one-half hour. With the original method only a full virulent, fresh bouillon culture of the typhoid bacillus, viz., one not older than sixteen to twenty- four hours, is employed. ‘The further technique is simple: 1 volume of blood serum is diluted with the requisite amount of normal salt solution to 20, 25, 50, or 100 volumes, as the case may be. Of this mixture one droplet is mounted on a cover-glass, mixed with a droplet of the typhoid culture (dilutions of 40, 50, 100, or 200 thus resulting), and inverted over a cupped slide, with a little vaselin along the edges. ‘The examination is conducted with a medium power (Leitz, 6 or 7; Bausch and Lomb, ¢). .| If the case in question is one of typhoid fever, it will be observed that after a variable length of time the individual bacilli, which at first actively dart about the field of vision, become quiescent and tend to gather in distinct clumps, while the interspaces become entirely free from bacilli or very nearly so. After one-half hour, or one = Rg BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD 163 or two hours, according to the degree of dilution, all motion has ceased. When the time limit has expired and loss of motility and agglutination have not occurred the result is negative. In such an event further examinations should be made on the following days. In every case it is well to make a control test with the simple bouillon culture, so as to ensure the absence of preformed clumps and _ the virulence of the organism; of the latter, the degree of motility is the oest index. In order to secure the necessary degree of dilution, various methods have been suggested. The simplest, and the one generally em- aloyed in municipal bacteriological aboratories, is to receive a large drop of blood upon a slide or slip of glazed aper, and allow it to dry. A drop or ‘wo of distilled water is then placed on ‘he blood and allowed to remain for sey- eral minutes, when it is further diluted md examined as described. The wrincipal advantage of this method is ts simplicity and the fact that the dried dlood retains its agglutinating proper- jes for weeks and months. ‘The esults, however, are less reliable than vith the use of liquid blood. This an be readily collected in little glass apsules, such as Wright has recom- aended for opsonic work (Fig. 40, 5). vhe finger or ear is pricked as usual ind the blood allowed to enter the ent capillary arm of the capsule by qerely being held in contact. When ‘nough has been collected, the far end f the capsule is warmed and_ the ‘raight end sealed, when the blood will tount into the body of the capsule. “he bent arm is then also sealed. In dis manner the blood can be kept for Rees trite) faong time. At the laboratory it is ™% *°— Wrisht’s pipette; b, Wright's ung into the centrifuge, if the serum as not already separated out, briefly centrifugated, and the capsule it with a file. The serum is then diluted with the aid of a Thoma- elss pipette or a common capillary pipette such as anyone can onstruct and is pictured in Fig. 40. ‘These pipettes are destroyed fter use. | Avery material advance in the practical application of the agglutina- i _ | me 164 THE BLOOD tion test was made by the discovery that it is not necessary to work, with living cultures of the typhoid bacillus, but that dead bacilli will answer just as well, providing they are killed off when in| a virulent condition. To this end formalized cultures are espe- cially convenient. ‘To prepare this a twenty-four to forty-eight hours’ bouillon culture of an actively agglutinable strain is treated with formalin to the extent of 1 per cent. of the solution, and set aside fora week. ‘The bacteria are allowed to settle, when the supernatant! fluid is poured off and replaced by formalized normal salt solution, In this form the material will keep for months. Before use it should be well agitated and examined to see that no artificial clumps are present. With the formalized culture the microscopic examination can then be made, or one can proceed macroscopically. If the micro- scopic test is used the examination is made after two to twenty hours. | The so-called Ficker Diagnosticum is a suspension of typhoid bacilli which have been killed off by a special process, which has) not been made public. The outfit is sold by Merck and is used in the macroscopic application of the test. It consists of a series of small corked tubes, a graduated dropping tube, a bottle of the diag- nosticum and one of normal salt solution, a small cupping glass and lancet. Cupping glass, rubber stopper, and lancet must first be steril- ized by boiling in water. ‘The blood is obtained from the back of the patient by making three or four deep punctures’ and applying the cupping glass in the usual manner, viz., after placing a few drops of alcohol in the bottom and igniting it and rapidly placing the bottle to the skin before the flame is extinguished. ‘The skin of the back is first cleansed with soap and water, alcohol, and ether. About 1 ec. of blood is thus drawn, the bottle closed with the rubber cork and set aside in a cool place until the serum has separated. ‘The test-tube and pipette are sterilized by means of alcohol and ether and the corks by boiling in water; 0.1 c.c. of the clear serum is now placed in one of the test-tubes, and after washing the pipette with water, alcohol, and ether, diluted with 0.9 c.c. of normal salt solution. A dilution of 1 to 10 thus results. Che mixture is well shaken, and 0.1 ¢.c. placed in a second tube and 0.2 c.c. in a third. With the carefully washed pipette 0.9 c.c. of the diagnosticum is added to test-tube No. 2 and 0.8 c.c. to No. 3) Dilutions of 1 to 100 and Ito 50 thus result. A further tube (No. 4) receives 1 c.c. of the diagnosticum alone. All tubes are closed, well agitated, and set aside in the dark at room temperature. ‘They aré inspected after ten to twelve hours, when as a rule a positive reactior’ can be detected. Sometimes it is necessary to wait for twenty hours; if after that the result is negative it is so reported. If the reaction is positive the bacilli in tubes 2 and 3 will have fallen to the 1 | find it more convenient to collect the necessary amount of blood from thi ear; from 1 to 5 ¢.c. can be obtained by ordinary puncture without difficulty. | _— x _ BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD 165 vottom, leaving the supernatant fluid clear, while the control tube 4 remains turbid. All tubes should be viewed against a dark back- ground. ~ The results which are obtained with Ficker’s diagnosticum are very satisfactory. ‘The method has been amply investigated and uniformly endorsed. The formalized cultures described above can be utilized just as well as the diagnosticum and in the same manner or any other modifi- cation that may suggest itself to the individual worker’. Capillary pipettes such as the one pictured in Fig. 40, a, can be used in the | place of the special, calibrated instrument mentioned. _ Paratyphoid Fever.—In cases of so-called paratyphoid fever organisms may be met with in the blood which apparently occupy a oosition intermediate between the typhoid bacillus and the organisms velonging to the colon group. Collectively they are spoken of as oaratyphoid bacilli, though the question whether or not they represent well-defined species has not been definitely settled. Cases of paratyphoid fever clinically resemble true typhoid, but are on the whole milder in their course. As a rule the serum does not react with the typhoid bacillus, while the organism which appears “o be pathogenic in the individual case is agglutinated in a typical manner. Unfortunately, however, the serum of one case will not always react with the organism of a second case; so that the serum reaction will not always make it possible to distinguish the inter- mediates as a group from typhoid on the one hand, and the bacillus coli on the other.” Moreover, it has been shown that the serum of true typhoid may agglutinate the paratyphoid bacillus in higher dilutions even than the typhoid bacillus, although this is probably not usual (Griinberg and Rolly). _ The paratyphoid group includes Widal’s paracolon bacillus, Gwyn’s bacillus, Cushing’s bacillus 0, Hewlett’s bacillus b, Noonan’s vacillus, Schottmiiller’s bacilli, etc. It is subdivided into two groups, A and B, of which B causes at first an acid reaction in litmus milk, which in about ten days changes to a permanently alkaline reaction, while group A causes permanent acidity. Unlike the typhoid oacillus the paratyphoid ferments dextrose with the formation of zas. ‘The intermediates do not form gas in lactose nor in saccharose media. On potato the growth is slight and there is no discoloration. They do not ferment milk or produce indol. The examination of the blood is conducted as in typhoid fever, out it is not always necessary to dilute it to the same degree. Some- ‘imes successful cultivation follows the spreading of a few c.c. of blood over the surface of agar tubes or plates. ia Riidiger, Jour. Infect. Dis., 1904, vol. i, p. 236. _* This only holds good for members of the paracolon group, while those of © paratyphoid groups interact without exception. ; : J 166 | THE BLOOD LirERATURE.—Gwyn, Johns Hopkins Hosp. Bull., 1898, vol. ix, p. 54. Cushing, ibid., 1900, vol. xi, p. 156. Schottmuller, Zeit. f. Hyg., 1901, vol. xxxviii. W.B, Johnston, Am. Jour. Med. Sci., 1902, vol. exxiii, p. 187 (analysis of all cases reported up to that time). A. W. Hewlett, ibid., p. 200. Coleman- Buxton, ibid., 1903, vol. CXAMl, p.979. sae also Ascoli, Zeit. f. klin. Med., 1903, vol. xlviii, p. 419, Pneumonia.—According to Rosenow the pneumococcus can be recovered in practically all cases of pneumonia, providing that large quantities of blood are used. He does not think that their presence indicates an especially unfavorable prognosis. He obtained positive: results in 160 of 175 cases with a mortality of 40 per cent. Positive results were obtained as early as twelve hours after the initial chill and as late as forty-eight hours after the crisis, although | negative results are the rule after crisis. Pneumococci were also obtained in the blood smears in 47 out of 80 cases. The results of other modern investigators are similar. | Prochaska, working under Ejichhorst’s direction, found pneu- mococci in the blood in each of 10 cases examined, and in a sub-| sequent series of 40 cases, of which 7 were fatal, he obtained the pneumococcus in 38. Frankel states that according to his experience, which is based | upon an examination of more than 150 cases, one may infer that. death will occur either with the symptoms of sepsis or that metas-. tasis will take place in the internal organs whenever a larger number of colonies develop on spreading 1 c.c. of blood upon a plate of agar. If, however, the number is so small that it is necessary to take lar ger amounts of blood to demonstrate their presence and to. oTow them in bouillon instead of on agar, so as to eliminate the bac- tericidal power of the blood altogether, then Frinkel- believes their: presence is of no significance, and does not warrant a fatal progno- sis. In the latter case he has found that the bacteria are frequently avirulent. | ‘The examination, which should be repeated every day, if necessary, | is conducted as follows: After disinfection of the arm in the usual) manner 10 c.c. of blood are aspirated and agar tubes—liquefied at 40° C.—inoculated, each with 1 or more c.c. of the blood. Plates are then prepared and kept at a temperature of from 35° to 37° C. ‘The colonies appear as small, round, grayish, jelly-like drops, which are quite characteristic. Rosenow finds that the best results are obtained with blood agar. Upon this the pneumococci, especially when very virulent, produce a hemolytic zone which is greenish in color. ‘This phenomenon, according to Schottmuiiller, may serve to distinguish the pneumococcus from streptococci, which cause hemolysis without pigment production. Instead of agar, bouillon may also be employed, and it is quite: likely, as Prochaska suggests, that in this manner positive results| may be more frequently obtained. Cole recommends the use of sterile litmus milk, of which portions of 150 ¢.c. each are employed. BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD 167 n Erlenmeyer flasks. Early acidification and coagulation occur, ind it is thus possible to determine more readily and quickly whether yrowth has taken place. Smears are then made and examined for sapsules (see below). ‘The identity is established by the characteristic shape and staining reactions of the organism, including the staining pe the capsules, by the typical growth in milk and agar, and by the absence of growth, or very slight growth, in gelatin at ordinary room temperature. Especially characteristic, further, is the fermentation of inulin by the pneumococcus. ‘lo this end serum water containing inulin is used as recommended by Hiss. The individual organism (Fig. 41) is capsulated, and usually oecurs in pairs, arranged end to end or in short chains. At times, however, the chains are quite long, and then it may be difficult Fie. 41.—Pneumococcus, showing capsule, to distinguish it from streptococci. It is easily stained with the com- mon aniline dyes. In order to differentiate the capsule, the method suggested by Epstein (see Sputum) should be employed. It should be remembered that capsules are only demonstrable in specimens obtained from milk or blood-serum cultures, while they are not shown in growths obtained from agar or bouillon. _ Agglutination According to Rosenow pneumococcic serum in- variably agglutinates the pneumococcus with a maximum dilution of 1 to 40 or 1 to 50. With rabbit-immune serum and using his serum- water medium for the growth of the organism, Hiss obtained aggluti- nation with dilutions up to 800 and over. | Lireraturrn.—Goldscheider, Deutsch. med. Woch., 1892, No. 14. Sittmann, ‘Deutsch. Arch, f. klin. Med., 1894, vol. lili, p. 323. Kiuhnau, Zeit. f. Hyg., 1897, jvol. xxy. Kohn, Deutsch. med. Woch., 1897, p.136. James and Tuttle, N.Y. Presbyterian Hosp. Rep., vol. iii, p. 44. Sello, Zeit. f. klin. Med., 1898, vol. xxxvi. White, Jour. of Exper. Med., 1899, vol. ii. Silvestrini and Sertoli, Riforma Med., 1899, No. 116. Abstr. in Centralbl. f. inn. Med., 1899, vol. xxi. ss) R. Cole, Johns Hopkins Hosp. Bull., 1902, vol. xiii, p. 236. Prochaska, Centralbl, f. gen. Med. 1900, No. 46. Prochaska, Deutsch. Arch. f. klin. Med., vol. lxx, p, 559. Frinkel, Deutsch. med. Woch., 1901, V. B., p. 212. Rosenow, Jour, Amer. Med. Assoc., 1905, No. 2, p. 851; Jour. Infect. Dis., March, 1904. 168 THE BLOOD Pyogenic Bacteriemia. Technique.—'l‘he general technique is the | same as that described before, but large amounts of blood are advised, viz., 20 to 25 c.c. ‘The media which are commonly employed are the ordinary laboratory media; in addition Libman has suggested the use of serum-glucose agar and serum-glucose bouillon. He has | pointed out that on these media the growth of most bacteria is more marked and more rapid than on ordinary serum agar. ‘This is true especially of the streptococcus, the pneumococcus, the gonococcus, and the meningococcus. With the solid media plates are employed almost altogether to the exclusion of media in tubes; 2 to 3 c.c. of | blood are used for 15 to 20 c.c. of the solid media. The number of organisms which may be found in the blood in septic conditions is exceedingly variable. On the one hand, but one | plate or flask out of several may show any growth, and then only after several days; while, on the other hand, the number of organ- isms may be quite large. Cole has reported a case of streptococcus septicemia in which the number of organisms amounted to 3642 per cubic centimeter of blood six days before death, and then rose to 10,716 per cubic centimeter two days before death. I have seen a case of meningococcus septicemia in which the organisms numbered 7,380,000 per cubic centimeter just before death. The time before death at which organisms may be found in the blood is also quite variable; sometimes they may be demonstrable | a month before, in other cases only a day or two before the fatal issue. | (a) Staphylococcus Bacteriemia.—Staphylococcus bacteriemia is more ~ common than was formerly supposed. ‘The variety usually seen is | the Staphylococcus aureus. ‘The albus is rare. Libman states that _ the latter plays an insignificant role in systemic infections; that in several years he has not met with a single instance in which he could ascribe a systemic infection to the Staphylococcus albus. He draws attention to the fact that the pigment production in the aureus may be delayed and that some of the positive albus cases recorded in the | literature may in reality have been aureus cases of this kind. He accordingly recommends that an apparent albus be observed for five days and grown upon potato and serum agar before the diagnosis Is made (glucose interferes with pigment production). Staphylococcus citreus also is very rare. Of the 28 positive findings in Libman’s large series of blood cultures many were instances of osteomyelitis, some were secondary to furun- cles or cellulitis, others were cryptogenetic, and 2 referable to post- partum infection (rare). All these were aureus cases. ‘The only positive albus cases were obtained within forty-eight hours before i BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD 169 death; Libman looks upon these as agonal invasions. ‘The Staphy- Jococcus citreus was isolated once in a case of osteomyelitis. F. Meyer and Michaelis and others report having found pus organisms in a large percentage of cases of advanced phthisis. ‘This is denied by ioermanit excepting as agonal infections. The Staphylococcus pyogenes aureus occurs in the form of spheri- cal bodies, averaging about 0.8 4 in diameter, which readily stain with the basic aniline dyes, as also with Gram’s method. ‘They usually occur in clumps, but may also be seen in pairs and in short chains. ‘The organism grows on all culture media, and in the pres- ence of oxygen gives rise to the formation of an orange-yellow pig- ment. Gelatin is rapidly liquefied; it coagulates milk with acid reaction and clouds bouillon. ‘The Staphylococcus pyogenes albus and citreus differ from the aureus by the absence of pigment in the inst and by the formation of a lemon-yellow pigment in the second. } Fig. 42.—Streptococcus pyogenes. > 800. (Frinkel.) (b) Streptococcus Bacteriemia.—In the large series of blood cultures eported by Libman streptococci were isolated in 58 cases. Strepto- -occus bacteriemia is thus more common than staphylococcus bacteri- mia. Some were instances of terminal infections, or infections aris- ng from the tonsils, the ears, and mastoid processes, or the genito- irmary tract (abortion and postpartum infections), while in “others nfections were referable to wounds, and still others were cryptogenetic. some cases were characterized by joint or bone lesions. Endocar- litis was frequent. One was a case of erythema nodosum. In several ses of mild acute endocarditis, following what clinically appeared 9 be typical articular rheumatism, Libman found atteniiated strepto- occi. ‘They could be demonstrated during extended periods of time. Streptococci have also been found in the blood in advanced cases of Athisis (probably as agonal invasions). ' Hektoen has pointed out that in scarlatina streptococci may be ound j in the blood during life in at least 18 per cent. of all cases. | 170 THE BLOOD I append his conclusions: Streptococci may occasionally be found | in the blood of scarlet-fever cases that run a short, mild, and uncompli- eated clinical course. They occur with relatively greater frequency | in the more severe and protracted cases of the disease, in which there | may also develop local complications and clinical signs of general | infection, such as joint inflammations; but even in the grave cases of this kind spontaneous recovery may take place. In fatal cases | streptococci may not be demonstrable. The theory that scarlet fever | is a streptococcus disease thus does not seem to receive direct support | from these investigations. | In diphtheria, measles, and smallpox infection with streptococci is also not uncommon. Other organisms may, however, also be met with, such as the various staphylococci, and quite commonly also, | according to Jehle, the bacillus of influenza. ; The Streptococcus pyogenes (Fig. 42) occurs in chains of spherical | cocci which usually vary from four to twenty in number. ‘The size of the individual organism is somewhat greater than that of the staphylococcus, but may vary even in one and the same chain. It is readily stained with the basic aniline dyes and also with Gram’s | method. It grows on all culture media at the temperature of the room, forming small, gray, granular colonies on agar and _ gelatin. Unlike the pneumococcus it does not ferment inulin media. As a rule, it does not liquefy gelatin, and it may or may not coagulate milk and cloud bouillon. Several varieties are recognized, viZ., | Streptococcus brevis, which forms short chains; Streptococcus longus, which occurs in long chains; streptococci, which render bouillon cloudy, and those which do not; streptococci, which form flocculent, | sandy, scaly, or viscous sediments. | The Streptococcus conglomeratus grows, without clouding bouillon, | in the form of dense, separate particles, scales, or thin mebmranes at the bottom and sides of the tube, and on shaking the sediment, it breaks up into little specks, without producing uniform, diffuse cloudiness. ‘The chains are long and interwoven in conglomerate masses (Welch). (c) Non-pneumonic Pneumococcus Bacteriemia.—In Libman’s series, apart from the pneumonia cases, pneumococci were found only. four times. ‘I‘wice there was an acute endocarditis of unknown source, once there was an infection between two toes, and once there was a suppurating ethmoiditis and frontal sinusitis, with abscess. | Other observers have found the organism in cases of billary abscess at the time of the chill, in suppurative odphoritis, in peritonitis, etc. It is interesting to note in this connection that Libman obtained only negative results in 25 cases of peritonitis, and also in a series of 25 cases of appendicitis, some of which were very severe. (d) Bacterium Proteus Bacteriemia.—Libman reports a case of uremia in which the proteus was found one day before death, together with streptococci. : i] kel BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD 17] - (e) Colon Bacillus Bacteriemia.—'l‘he colon bacillus also is rarely ound in the blood. Libman mentions a case in which it was emonstrated where the operation of internal urethrotomy had seen performed. An interesting case is also reported by Rochester see literature below). Paracolon Bacteriemia.—Aside from those cases in which para- ten bacilli have been found in so-called paratyphoid fever, para- olon bacteriemia is very rare. Libman and Berg report one case yhich clinically resembled cholecystitis. (g) Bacillus Pyocyaneus Bacteriemia.— I'he Bacillus pyocyaneus 5 rarely found in the blood. Libman and Brill report a case in which » occurred secondarily to a Staphylococcus aureus bacteriemia. (h) Gonococcus Bacteriemia.—Cases of gonorrheal septicemia in rhich the gonococcus was isolated from the blood of the patients uring life have been reported by ‘Thayer-Blumer, ‘Thayer-Lazear, tyelogoway, Wilson, Harris-Johnston, and others. In all these cases onorrheal endocarditis existed. In other infections of the same ature positive results were obtained by Ahmann, Colombini, Panichi, nd Unger, in association with polyarthritis, epididymitis, myositis, sndoyaginitis, inguinal bubo, and parotitis. In the endocarditis uses cultures were obtained after an illness lasting for from five ‘eeks to seven months, at times as early as the ninth to the eleventh ay preceding death, and on an average five days before death. To cultivate the gonococcus from the blood during life, it is neither ecessary to use a large amount of blood nor to dilute it greatly, or to employ any specially prepared medium. From 2 to 5 c.c. are ifficient. According to Harris and Johnston, it is more advan- eous to mix the blood with melted agar and to plate the same ian to use fluid media where the oxygen supply is more restricted. (For a description of the organism, see Gonorrheal Pus.) LireratureE.—N. M. Harris and W. B. Johnston, “ Gonorrhceal Endocarditis ith Cultivation of the Specific Organism from the Blood during Life,’ Johns | an Hosp. Bull., 1902, vol. xii, p. 236 (literature). Thayer and Blumer, 1896, vol. vi, p. "59. Thayer anc Lazear, Jour. Exper. Med., vol. iv, p. 81. _(}) Micrococcus Zymogenes Bacteriemia.—'his organism is appar- atly closely related to the Pneumococcus and the Streptococcus yogenes. It has been isolated from the blood in one instance y MacCallum and Hastings. (k) Meningococcus Bacteriemia.—In several instances of meningo- secus Meningitis the corresponding organism has been isolated from ie blood. In one case I found 7,380,000 diplococei_ per cubic entimeter. ‘The organisms could be demonstrated in large num- ers directly in the blood smear. Almost all were enclosed in poly nu- ear neutrophiles and in large mononuclear elements. Endocarditis.—In acute fendiocarditic or in acute exacerbations f chronic cases bacteriemia i is fairly common. Lenhartz obtained | ’ | 172 THE BLOOD positive results intra vitam in 16 cases out of 28, and Libman states that in cases of acute ulcerative endocarditis he has always found organisms in the blood. ‘The organisms which have been encountered are the Staphylococcus aureus, streptococci, pneumococci and the gonococcus. Of these the streptococcus cases are the most common, while the staphylococcus comes next in order. Pneumococcus and gonococcus endocarditis is relatively uncommon. Libman remarks that there is often a marked disproportion between the number of bacteria in the blood and the extent of the lesion. ‘here may be an almost countless number in the blood and only very small deposits on the valves, or there may be large vegetations with hardly any bacteria in the blood. As a rule they are present in fair numbers. In a series of 10 cases of mild acute endocarditis following what clinically appeared to be typical articular rheumatism Libman could demonstrate attenuated streptococci and diplococci during extended periods of time. Prognosis in Pyogenic Bacteriemia.—The prognosis in the pyogenic bacteriemias, aside from other considerations, is upon the whole unfavorable; recoveries, however, are possible. Each individual case must be judged separately. In Libman’s series of 50 cases of streptococcemia there were 6 recoveries (11 per cent.); of 28 cases of staphylococcemia 8 recovered (nearly 29 per cent.); of his 4 pneumococcus cases 1 recovered. Leaving out. the few pheumococcus cases there would be 86 cases with 16 per cent. recoveries. In Bertelsmann’s series of 48 cases of surgical bacteriemia 21 recovered, viz., 43 per cent. ; of these there were 28 streptococcus cases with 19 recoveries and 13 staphylococcus cases with 4 recoveries. In Lenhartz’s series of 77 medical cases (including several post- partum infections), there were 17 recoveries; of these there were 47 streptococcus cases with 6 recoveries and 13 staphylococcus cases with 1 recovery. LireraturE.—I, W. White, “Cultures from the Blood in Septicemia, Pneu- monia, Meningitis, and Chronic Diseases,’’ Jour. Exper. Med., vol. iv, p. 425. Petruschky, Zeit. f. Hyg., vol. xvii, p. 59. Sittmann, Deutsch. Arch. f. khn, Med., vol. liii, p. 323. Canon, Deutsch. Zeit. f. Chir., vol. xxxiii, p. 571; and Mitth. aus d. Grenzgeb. d. Med. u. Chir., 1902, vol. x, p. 41. Lenhartz, Miinch. med. Woch., 190), Nos. 28 and 29. Libman, Proc. N. Y. Path. Soc., 1903, vol. ii, pp. 5 and 57; “On Certain Features of the Growth of Bacteria,” ete., Jour Med. Research, 1901, vol. vi. Cole, Johns Hopkins Hosp. Bull., 1902, vol. xiii, p. 252. Wm. Welch, “Morbid Conditions Caused by the Bacillus Aérogenes Capsulatus,” ibid., 1899, vol. x, p. 134. Gwyn, ibid., 1900, vol. xi, p. 185 | (first. case); Cole, ibid., 1902, vol. xiii, p. 234 (second case). Hektoen, Jour. Amer. Med. Assoc., 1903, vol. xl, No. 11. Jehle, Zeit. f. Heilk., 1901, vol. xxii, p.190. Ewing, Trans. Amer. Assoc. Phys., 1902, vol. xvii, p. 208. D. Rochester, Jour. Amer. Med. Assoc., March 2, 1907. C. E. Simon, Meningococcus Septiczemia, Johns Hopkins Hosp. Bull., 1907. Anthrax.—The bacillus of anthrax, as first pointed out by — Pollender, Brouell, and Davaine, is frequently met with in the . BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD. 173 blood in the corresponding disease. As a rule the number is small. Smears are stained for five to ten minutes in a mixture of 30 c.c. of a concentrated alcoholic solution of methylene blue and 100 c.c. of a 1 to 10,000 solution of potassium hydrate; they are then washed for five to ten seconds in 0.5 per cent. acetic acid, washed with alcohol and dried. ‘hus stained, the bacilli appear as rods measuring from 5 to 12 in length by 1 + in breadth, and usually present a segmented appearance, the extremities being slightly thickened. Under suit- able conditions spore formation takes place. When present in large numbers it is not necessary to stain the blood, as the organism can then be seen without difficulty in the wet specimen. In doubtful cases, in which a microscopic examination of the blood yields negative results, a few cubic centimeters may be injected into a mouse or a guinea-pig, in the blood of which the bacilli will soon be found in enormous numbers if the disease is anthrax. McFadyean has described a color reaction of anthrax blood which seems to be pathogno- monic of the disease. Smears are prepared as usual and, when air-dry, fixed by heat— mntil the slide has become a Fre. 43.—Anthrax bacillus. Bi 200 diameters little too hot to be held against ; the skin. On cooling the specimens are stained for a few seconds with a 1 per cent. aqueous solution of methylene blue (medicinal of Merck), or with one of Griibler’s methylene blues, modified by boiling with 4 per cent. of sodium bicarbonate. After washing with distilled water they are dried with filter paper, subsequently by heat, and mounted in balsam. Anthrax blood then shows a distinct reddish or purplish tone, especially when held against the light, while other blood appears pure blue or greenish blue. Microscopic examination of the amorphous intercellular material shows the same result. According to Heim, who has described the same reaction inde- pendently of McFadyean, the color change is due to mucin derived from the capsules of the bacteria. LireraturE.—Pollender, Casper’s Vierteljahrsch. f. gerichtl. u. 6ffentl. Med., 1855, vol. viii, p. 103. Brauell, Virchow’s Archiv, vol. xi, p. 132, and vol. xiv, . 32. Davaine, Compt.-rend. de l’Acad. d. Sci., vol. lvii, p. 220. Blumer and oung, Johns Hopkins Hosp. Bull., 1885, p. 127. _ McFaydean, Jour, Comp. Pathol. and Therap., March and December, 1903. Heim, Miinch. med. Woch., 1904, No. 10, P7aps THE BLOOD Tuberculosis.—In acute tuberculosis tubercle bacilli have repeatedly been observed in the blood; but the search for them is most tedious and often in vain. Nevertheless a careful examination of the blood is indicated in doubtful cases; but only a positive result is of value. According to Liebmann, the tubercle bacilli are most numerous in the blood about twenty-four hours after the injection of tuberculin, Working in this manner he claims to have obtained positive results in 56 cases of 141. As a rule it is best to resort to the animal experiment. For methods of staining and a description of the tubercle bacillus, the reader is referred to the chapter on Sputum. [.irERATURE.—Liebmann, Berlin. klin. Woch., 1891, p. 393. Krénig, Deutsch, med, Woch., 1894, vol. v, p. 42. Leprosy.—In leprosy the corresponding bacilli have been found in the blood by Mitsuda.'| Their demonstration, however, is difficult. Glanders.—In glanders the specific bacillus is constantly present in the blood, and may be demonstrated by staining dried preparations for five minutes with a concentrated alcoholic solution of methylene blue mixed with an equal volume of a 1 to 10,000 solution of potassium hydrate just before using. From this mixture the specimen is passed for a second or two into a 1 per cent. solution of acetic acid which has been tinged a faint yellow by the addition of a little tropeolin 00 solu- tion; it is then decolorized by washing in water containing 2 drops. of concentrated sulphuric acid and 1 drop of a 5 per cent. solution of | oxalic acid for each 10 c.c. | In specimens thus stained the bacilli | appear as short rods measuring from 2 to 3 in length by 0.37 to 0. | in breadth, often containing a spore at one end. LirERATURE.—Duval, Arch. de méd expér., 1896, p. 361. Influenza.—The influenza bacillus has been found in the blood occasionally, but is more readily demonstrated in the sputum. Jehle found it in 22 cases of scarlatina out of 48 that ended fatally, in’ measles 15 times out of 23, and in 5 cases of varicalla out of 9. | In Hektoen’s series, on the other hand, the organism was not found; but it is noted that during the year influenza was not especially | prevalent in Chicago. (For a description of the organism see the Sputum.) LITERATURE.—Canon, Virchow’s Archiv, vol. exxxi, p. 401. Klein, Baumgar-_ ten’s Jahresb., 1893, p. 206. Kuhnau, Zeit. f. Hyg., vol. xxv, p. 492. Malta Fever.—In Mediterranean or Malta fever the specific organism, the Micrococcus melitensis (Bruce), has been isolated from the blood during life. It is said to be present in the peripheral - blood in all cases during the early stages and in severe febrile relapses. 1 Folia hematol., vol, i, p. 502. ee BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD 175 In the afebrile intervals and the subsequent cachexial stage it is not demonstrable. In no case are the organisms abundant, and for this reason the bacteriological findings are rather uncertain. Diagnosis is facilitated by the fact that a well-pronounced agglutina- tion is obtained with the patient’s serum. A positive reaction with a dilution of more than 1 to 20, according to Birt and Lamb, may be regarded as proof positive of the existence of the disease. As a rule agglutination can still be obtained with a dilution of from 1 to 600 to 1 to 700. _ It begins about the fifth day of the disease, and gradually diminishes in intensity during convalescence, but may persist for a year and a half and even longer. The organism in question is a coccus, measuring 0.3 4 in diam- eter, and occurs singly, in pairs, and sometimes in fours. Longer chains are not seen. It is motile. It is stained by the usual dyes and grows on nutrient agar and in broth. ‘The colonies are usually not visible until the third day. At first their color is that of a trans- parent amber, while later they are opaque. Liquefaction does not occur. LITERATURE.—C, Birt and G. Lamb, “ Mediterranean fever,’ Lancet, Sept. 9, 1899. Wright and Smith, Brit. Med. Jour., April 10, 1907. Musser and Sailer, Phila. Med. Jour., 1898, p. 1408, and 1899, p. 89. R. P. Strong and W. E. Musgrave. “The Occurrence of Malta Fever in Manila,’’ Phila. Med. Jour., 1900, p. 996. J.J. Curry, “Malta Fever,” Jour. Med. Research, July, 1901. Bubonic Plague.—In advanced cases of bubonic septicemia the specific organism may be found in the blood in small numbers. Toward the end of rapidly fatal cases they become more numerous, and may then be demonstrable directly with the microscope. Accord- ing to Bell’ the bacilli can be found in all cases and at all stages of the disease by using Ross’ dehemoglobinizing method (p. 177). The organism in question, the Bacillus pestis (Kitasato, Yersin), is a short, thick coccobacillus, with rounded ends, measur- ing 1.5 » to 1.75 » in length by 0.5 to 0.7 # in breadth. Examined in the hanging drop it is devoid of automobility. The polar regions are readily stained, while the interpolar area remains colorless. In many organisms a capsule can be made out by appropriate methods, but it is apparently not a constant feature. Oftentimes the form of the organism deviates from the normal. It may thus resemble a coccus on the one hand, while on the other it appears more elon- gated, and again it is common to meet with distorted and swollen, vacuolated forms, which are interpreted as involution or degeneration forms. ‘These latter are especially numerous in older cases and old cultures. ‘The organism is decolorized by Gram (Fig. 44). The blood smears are fixed by immersion in absolute alcohol for twenty-five minutes; or they are covered with absolute alcohol for about one-half minute, when the alcohol is burned off, For 1 Brit. Med. Jour. March 5, 1904. 176 THE BLOOD staining purposes, borax methylene blue (a solution of 2 per cent. methylene blue in 5 per cent. borax-water) or L6ffler’s alkaline methylene blue may be conveniently employed. In the first in- stance we stain for one-half minute, in the second for two to three minutes. ‘The polar staining is in this manner quite satisfactory. On gelatin and agar containing 2.5 to 3.5 per cent. of salt and in bouillon a fairly characteristic growth results. In the case of the agar involution forms are obtained, among which long, slender bacilli, which are segmented and present a vacuolated appearance, are especially noteworthy. In this state they stain quite badly and have lost a certain degree of their virulence. In bouillon the or- ganism often forms long chains of well-rounded bodies which are = te Ps : bd e ee © im, eos 2% a. eae 4 ht Qo "ee np A Pah * nba hy trace * “, 2%, ; ¥ S + a ' . * o* owe an 4 re wae .# *% ve tae. Fae, sys + Sed a ea: awe S'fe54 Vo Sie, 8 toe Fee y 3 RIE ore” S a A. ' oe “ts cs 7 “ays ~ a , “ >” a al Fig. 44.—-Plague bacilli from agar culture. >< 1100 diameters. (Park.) quite similar to a coccus. During its growth in bouillon it forms flakes or flocculi, which rapidly sink to the bottom of the tube, leaving the liquid clear above. Stalactite or stalagmite formations may also be seen, starting from the walls of the tubes or from sus- | pended droplets of oil or butter. Colonies on gelatin about thirty-_ six hours old are warty, strongly refractive formations, which often — present a delicate, irregularly indented margin. Even after twenty- | four hours one can obtain smears in which 50 to 100 bacilli are — grouped in little colonies of irregular form, while examination of the — plates with a magnifying power of 60 diameters reveals scarcely any growth. The organism does not liquefy gelatin. ‘The optimum temperature for growths is between 25° and 30° C. LrreraTuRE.—For Kitasato’s report see Annual Rep. of the U. S. Marine- — Hospital Service for 1894; W. Wyman, Bubonic Plague; U. S. Treasury Dept., | 1900. Kossel u. Overbeck, Arb. aus. d. Kais. Gesundheitsamt., 1901, vol. xviii. ANIMAL PARASITES 177 ANIMAL PARASITES. _ Malaria.—Malarial fever is referable to infection with a specific protozoan parasite belonging to the class of hematozoa, representa- tives of which are found in the blood of various animals, such as the rat, frog, turtle, carp, various birds, etc. ‘Three varieties are known to occur in the blood of man, viz., the parasite of tertian, quartan, and estivo-autumnal fever. ‘lhe life history of these organisms is now well understood, and it is known that in addition to the intra- corporeal cycle of development which takes place in the human body there is yet another, an extracorporeal cycle, which occurs in certain ‘mosquitoes of the genus Anopheles. Infection occurs through the ‘bites of such mosquitoes, which themselves have been infected by sucking the blood of malarial patients. ‘This has been abundantly demonstrated by Ross, Manson, Grassi, and others, and may be regarded as an established fact. Method of Examination.—When the patient is directly available at the laboratory, or if a few hours only need elapse before the examina- tion is made, wet mounts may be used, which are best ringed with a little vaselin or paraffin, if they cannot be examined at once. Other- wise dry mounts are prepared and stained with the eosinate of methy- lene blue, or one of the Romanowsky dyes, such as Hastings’, Wright’s, Giemsa’s, etc. (See Plate X.) With the Romanowsky mixtures, which all contain methylene azure, the chromatin (nuclear) granules are shown. It is best to procure specimens shortly before an attack, as adult ‘forms are then obtained; immediately after an attack is not the proper itime to hunt for parasites. In cases in which but few organisms are expected Ross has suggested ‘the advisability of spreading thick blood specimens and extracting the hemoglobin before staining. ‘The search for the youngest forms of the estivo-autumnal parasite especially is much facilitated in this manner. Ruge endorses this method in the following modification, but points out that the specimens are by no means beautiful. A large drop of blood (about 20 cb. mm.) is spread over a surface measur- ing about 18 square millimeters. ‘The air-dried preparation is then placed for a few minutes in a 5 per cent. solution of formalin,’ to which 0.5 to 1 per cent. of acetic acid has been added. In this manner the hemoglobin is all extracted, while at the same time the blood film is fixed; so that it can now be washed without fear of ruining the ‘preparation. ‘This is then stained either according to one of the ‘modifications of the Romanowsky method or with the eosinate ‘This solution would contain 2 per cent. of formaldehyde gas, as the commercial formalin is about a 40 per cent. solution. 12 178 THE BLOOD of methylene blue. Ruge further advises that specimens stained according to the Romanowsky method be subsequently stained with Manson’s solution,’ in order to render the smallest and medium- sized ring forms more readily visible, as their affinity for the dye is somewhat impaired by the fixation in formalin. My own experience with this method has been very satisfactory. Plasencia suggests the following method: Fixation in 0.5 per cent. formalin and absolute alcohol (equal parts); rapid drying in the air and washing in distilled water. ‘The specimens are then stained with a mixture composed of 80 c.c. of a saturated aqueous solution of toluidin blue and 60 c.c. of a 1 per cent. aqueous solution of eosin. After washing in water they are dried and examined as usual. Pla- senica regards this stain as better than Manson’s. The Parasite.—'he following forms of the parasite may be found in the blood: 1. Hyatrye NOoN-PIGMENTED INTRACELLULAR BopiEs.—These apparently represent the earliest stage in the development of the parasite, and are found in all forms of malarial fever; they are espe- cially abundant during the latter part of the paroxysm or immedi- ately thereafter. At first sight they may be mistaken for vacuoles, but upon closer examination it will be found that they exhibit dis- tinct movements of an ameboid character, and may thus _ easily be recognized with a little experience. The rapidity with which these changes in form occur in the tertian type of ague is most astonishing, and sketches of any one phase can often, indeed, be made only from memory; in quartan fever the movements are much slower and far less extensive. In the irregular fever of the estivo-autumnal form ameboid move-> ments may likewise be observed, but more commonly the para- site assumes a ring-like appearance, and does not throw out distinet pseudopodia. If these forms are carefully observed, however, it will. be found that they are not absolutely quiescent, but alternately ex- pand and contract. In tertian fever the organism (Plate VIII) is pale and indistinct, while in quartan fever it is sharply outlined and somewhat refractive — (Plate IX, Fig. 2). In the estivo-autumnal form the organism is_ usually much smaller than in the tertian type, and the ring-like bodies — frequently present at some point in their interior a distinctly shaded aspect which closely resembles the darker portion in the centre of a_ normal corpuscle (Plate [X, Fig. 1). It is thus possible, even at this stage in the development of the parasite, to distinguish between fever of the tertian, quartan, and estivo-autumnal type. | ' This is an aqueous solution of borax (5 per cent.) and methylene blue (2 per cent.). The blood films are stained with this solution .for about thirty seconds; they are then washed in water, dried with filter paper, and afterward by gently | warming them over the flame. . . gee Cot 8 Oe aT Ho The Parasite of Tertian Fever. 1,normal red corpuscle; 2 to 4, non-pigmented stage of the organism, showing ameboid move- ts; 5 to 7, progressive pigmentation and growth; § to 11, process of segmentation; 12, young 8; 13, large extracellular organism; 14, mode of formation of extra-cellular body; 15, small mented extra-cellular organism; 16, flagellated body and free flagella. Unstained specimen. sonal observation.) LIBRARY @F THE UNIVERSITY of HLLINOIS, ANIMAL PARASITES 179 2. PIGMENTED INTRACELLULAR ORGANISMS.—These represent a later stage in the development of the parasite, and, like the non- pigmented intracellular bodies, are met with in all types of malarial fever. ‘Their appearance, however, differs considerably in the vari- ous forms. In tertian fever minute granules of a reddish-brown color appear in the bodies of the organism soon after the paroxysm. ‘These gradually increase in number, while the invaded corpuscles proportionately become paler and paler, until finally only an indis- ‘tinct, shell-like outline can be discerned. In fresh specimens the granules, which often assume the form of little rods, resembling bacteria, exhibit most active molecular movements, attracting atten- tion at once. ‘The body of the parasite, which during its develop- ment has increased gradually in size, is probably hyaline, and may still be seen to undergo ameboid movements. ‘These are not nearly ‘so active, however, as in the non-pigmented stage. The move- ‘ments, moreover, cannot be followed so readily, owing to the pres- ence of the granules. At first sight these appear to be scattered ‘in small collections throughout the red corpuscles, and the impression may be gained that several organisms are present at the same time. Upon closer investigation, however, it will be seen that this is only apparently the case, and that the granules are confined to the bulb- ous extremities of the pseudopodia of a single parasite. Before ‘the end of forty-eight hours the organism has filled out the entire ted corpuscle, which at the same time has attained a larger size than normal. The ameboid movements become less and less marked, and the pigment granules, which may still be quite active, tend to collect about the periphery (Plate VIII). _ In quartan fever pigmented intracellular bodies likewise appear soon after the paroxysm. ‘The individual granules, however, are somewhat larger, of more irregular size, and darker in color than those seen in the tertian type (Plate IX, Fig. 2). Instead of ex- hibiting active molecular movements, moreover, they are almost entirely quiescent, and usually are grouped along the periphery of the organism. While ameboid movements can at first be observed, these become less and less marked, until finally, at the end of from sixty-four to seventy-two hours, they cease. he organism then presents a round or ovoid form, but does not fill the red corpuscle entirely. It is curious to note that in this form of ague the red corpuscles do not become decolorized, but rather darker than normally and at times specimens may be seen which present a distinctly green- ‘sh or brassy appearance. When the parasite has become fully developed the corpuscle is smaller than normally, and, on staining, t may be seen that the organism still is surrounded by a narrow zone of corpuscular protoplasm even when this is not apparent in Anstained preparations. | The pigmented intracellular bodies which may be found in estivo- . 180 THE BLOOD autumnal fever (Plate IX, Fig. 1) can readily be distinguished from those observed in tertian and quartan ague. As in those types, pigment granules also appear after the paroxysm; they are never numerous, however, and often only one or two minute dark granules can be detected near the periphery. ‘The organism, even in the later stages of its development, scarcely ever occupies much more than one-third of the corpuscles. Usually the granules exhibit scarcely any movements. As in the quartan type of ague, decolor- ization of the red corpuscles does not occur, and here, as there, a greenish, brassy appearance often is observed. At the beginning and during the paroxysm forms are at times seen in which the few pigment g granules that may be present have gathered in the centre of the parasite and formed a solid clump. From the facts that these are observed only during the paroxysm, and that central blocks of pigment are found only during the stage of segmentation (see below) in tertian and quartan ague, ‘l'hayer and others conclude that these bodies are presegmenting forms of the parasite. This belief is strengthened by the observation that pigment-bearing leukocytes are then also seen, which in the other types of fever likewise are found only at this time. 3. SEGMENTING Boptes.—In cases of tertian and quartan fever the process of segmentation may be observed directly under the micro- scope, if specimens of blood are obtained just prior to or during the chill. In tertian fever organisms will then be seen in which the de- | struction of the red corpuscles has advanced to a stage in which it is | only possible to make out a pale contour of the original host. ‘The parasite itself has gradually assumed a granular appearance, and the pigment granules, SNe until then have exhibited pronounced mo- lecular movements, now become quiescent, larger and rounder, and show a distinct tendency to collect in the centre of the body. Here they form a roundish mass in which the individual components can scarcely be made out. While this change in the position of the pig-_ ment is taking place, beginning segmentation of the surrounding granular protoplasm will be observed. ‘This at first is most marked at the periphery, from which delicate striae will gradually be seen to extend toward the central mass, dividing up the protoplasm into a number of oval bodies which closely resemble the petals of a flower (Plate VIII). Still later these bodies, which in reality are the sporules (merozoites) of the parasite, will be found scattered in an irregular manner throughout the interior of the organism. ‘The apparent envelope then disappears, and the sporules, which in tertian fever usually number from fifteen to twenty, lie free in the blood. Quite frequently, also, a sudden expulsion of the little bodies is observed and the impression gained as though the envelope had been burst asunder. Upon closer inspection, even at the petal stage, it will be seen that almost every sporule presents a tiny dot in its interior, 5 PLATE IX. PIG 1, A 22 L Schmidt fecit. The Parasite of Estivo-autumnal Fever. 1, normal red corpuscle; 2 to 10, gradual growth of the organism; 11 and 12, segmenting bodies ; , young forms ; 14 to 22, crescents, ovoids and spherical bodies, with and without bib; 23, flagellated dy. Unstained specimen. (Personal observation.) Cay PLCs o > LSchnudt fecut The Parasite of Quartan Fever. - f 1, normal red corpuscle; 2 to 6, gradual growth of the organism; 7, pigmented extracellular ly; §, segmenting body; 9, young forms; 10, vacuolated extracellular body ; 11, flagellated form. specimen, (Personal observation.) ANIMAL PARASITES 181 which may at first sight be mistaken for a pigment granule, but which in all probability is a nucleus. After the expulsion of the sporules these are frequently seen to move about in an active manner, _ but sooner or later they come to rest. While the progress of segmentation is very frequently observed to proceed in the manner described, this is not invariably the case. It may thus happen that segmentation occurs before the pigment granules have had time to gather at the centre, or that the parasitic rotoplasm breaks up into sporules directly without the intervention of the petal stage. In every case, however, the formation of sporules is associated directly with the occurrence of a paroxysm and repre- _ sents the asexual type of reproduction of the parasite (schizogony). giving rise to a new generation. As the process of se as the parasite approaches maturity become arran The sporules, unless destroyed by leukocytes, in turn invade new corpuscles, cause their destruction, and become segmented, thus omentation coincides in time with the occurrence of the chill, it is apparent that the interval elapsing between two consecutive chills—7. e., the type of the ague—depends upon the rapidity with which the organisms arrive at maturity. In quartan ague segmentation differs somewhat from that observed in the tertian form. It will here be observed that the pigment granules, which have gathered along the periphery of the organism, ged in a stellate manner, and apparently reach the centre through definite protoplasmic channels. Here they form a dense clump, and while the protoplasm assumes a finely granular appearance, segmentation proper begins -and proceeds as in the tertian form. ‘The number of segments, however, is smaller, varying between six and twelve. ‘The entire segmenting body, moreover, is smaller than in the tertian form, and the segments are arranged in a more symmetrical manner. Here, indeed, the most perfect rosettes are observed (Plate IX, Fig. 2). In estivo-autumnal fever segmenting bodies are only exception- ally seen in the peripheral blood, and it appears that the process of reproduction occurs principally in the spleen. ‘The segments, as a rule, number from ten to twenty. ‘The segmenting body itself, however, is much smaller than in either the tertian or quartan form, _and it is not possible to distinguish any remains of the original host. 4. CRESCENTS, Ovorps, AND SpHEROIDS (Plate IX, Fig. 1).— These are observed only in cases of estivo-autumnal fever when this has persisted for at least a week. At first sight they apparently bear no relation to the other forms which have been described, but it is known that they are derived directly from the pigmented intra- cellular forms. Specimens may thus be met with in which cres- -centic bodies are found in the interior of red corpuscles that have {lost but little of their original color. Such observations, however, are not common. ‘The typical crescents which are usually seen are / ! | 189 THE BLOOD highly refractive bodies, somewhat larger than a red corpuscle, measuring from 7 4 to 9 in length by 2 / in breadth. ‘Their ex- tremities are usually rounded off and joined by a delicate, curved line bridging over their concave border. ‘This is supposed to represent the remains of the original host. At other times this hood-like appendage is found along the convex border. ‘The little pigment granules and rods, which are always found in the interior of the crescents, are generally collected about the centre of the body, but they are occasionally also seen in one of the horns. While usually quiescent, a migration of some of the granules toward one extremity and back to the central mass may be observed. ‘The ovoid and J g Culex. Anopheles, Anopheles. Culex. Fie. 45.—(From Doflein.) spherical bodies, which are usually smaller than the crescents, exhibit | the same general features and often are provided likewise with a _ little hood. It is now known that the spherical bodies develop from the ovoids, and these again from the crescents. 5. EXTRACELLULAR PIGMENTED BovIEs oR GAMETES.—In tertian — and quartan ague some of the pigmented intracellular bodies, instead — of undergoing segmentation when they have arrived at maturity, — leave their hosts and appear as such in the blood. Some of them PLATE X. Malarial Organisms. ng estivo-autumnal ring bodies; b, young tertian form; c, tertian parasites in various levelopment; d, segmenting organism; e, estivo-autumnal crescents; /, large mononuclear te carrying pigment from ingested malarial organisms. Stained with Wright's stain. == _ ANIMAL PARASITES 188 at the same time increase considerably in size, and in the tertian form may become as large as a polynuclear leukocyte (Plate VIII). _ The pigment granules, moreover, exhibit an activity in their move- ments which is most astonishing and never observed under other conditions. ‘The outline of the parasite is then usually irregular and quite indistinct. Upon careful observation it will be seen that in some of these bodies the movements of the granules after a while become less and less marked, and finally cease, ~while the body of the parasite itself becomes still more irregular in outline. This appearance is undoubtedly referable to the death of the organism. In others a gradual fragmentation is observed, small particles of the pigmented mother-substance being cut off from the parent form. It is thus quite common to see the original parasite break up into four or five smaller bodies, in which the movements of the pigment granules persist for some time. Sooner or later, however, even these cease, the outlines of the bodies become more and more _ indis- tinct, and death occurs. In still others the formation of vacu- oles may be observed, the pigment granules at the same time becoming quiescent. ‘This process is likewise regarded as one of degeneration. Most interesting, however, is the fact that /lagel- lation may occur in some of these extracellular forms. ‘This may sometimes be hastened in the wet specimen by gently breathing upon the slide so as to form a thin film of moisture It will then be observed that the pigment granules which exhibit a most sur- prising activity tend to collect near the centre of the organism, while at the same time curious undulating movements may be made out along its contours. Suddenly one or more (one to six) slender fila- ments will be seen to protrude from as many points on the periphery, presenting minute enlargements here and there in their course (Plate IX) (polymites). ‘The length of these filaments, or flagella, as they are termed, varies considerably. As a rule, it does not exceed the diameter of from five to eight red corpuscles, but longer speci- mens are at times observed. With these flagella the organism makes most active whipping movements, scattering the red corpuscles. to the right and left. Attention is, indeed, usually first drawn to the presence of these bodies by the disturbance which they cause in the field of vision. Occasionally one of the flagella may be seen to become detached from the body of the parasite and to move rapidly about among the corpuscles in a snake-like manner. In microscopic speci- mens ‘they gradually come to a rest and often curl into a spiral. Beyond the fact that the flagellate organisms in tertian fever are larger than in the quartan form, no special points of difference exist (Plate IX, Fig. 2). In estivo-autumnal fever similar changes may be observed. ‘The flagellate forms are here direct derivatives of the crescents, which have changed to ovoids and these to spheroids. ‘The flaggellates, as in 184 THE BLOOD quartan fever, are smaller than those observed in the tertian form (Plate IX, Fig. 1). The significance of the flagellate organisms is now well under- stood. ‘They represent the male element in the sexual reproduction of the malarial parasite (microgametocytes) and the beginning of a cycle of development, which takes place outside of the human body, in the bodies of mosquitoes of the species Anopheles. ‘The beginning of this cycle was first observed by MacCallum in the blood of infected crows. He here discovered that when one of the flagella (micro- gametes) broke loose it almost always sought out another full- grown form of the parasite which had not undergone segmentation, and penetrated this, just as the spermatozoon penetrates the ovum. Schema of double cycle. L. B. Goldhorn, fee. ® 190k. 6)? 4( we ye Ue rest-body in Q / wall of stomach. a SRA cae Obkinet which penetrates ; lining of stomach-wall. ei h \ Mosquito ZN Salivary eesy gland. < q or am hy mosquu'd or Ny \ oN Flagellation / Ak ——— in stomach SS a of mosquito. Inner circle-asexual reproduction ; moist- Infection of man through gastro-intestinal and chamber observation shows no flagellation. respiratory tract, the infected mosquito dying | Outer circle-formation of (sexual: gametes ; in water, drying in air or sucking plant-juices, moist-chamber observation shows flagellation. infecting fresh vegetables (theoretical) Fia. 46.—Illustrating cycle of development. (Park.) Subsequently he observed the same process in the blood of the human being, which has since been confirmed by others. ‘The female cells are somewhat larger than the male cells and termed macroga- metes. ‘The further development (sporulation) of the fertilized forms (odkinetes), however, does not take place in the human blood, but in the mosquitoes. ‘The fertilized organism penetrates the stomach wall of the insect and here gives rise to the formation of little cysts (odcysts) in which after about seven days numerous irregular, rounded, ray-like strie appear. After a time the capsules of the cysts burst and the delicate, thread-like bodies (the sporozoites) are set free in the | body cavity of the mosquito, and shortly after appear in the salivary glands. ‘These bodies represent the young parasites, which result from the sexual reproduction of the adult organism. If at this stage ANIMAL PARASITES 185 of their development the infected mosquito is allowed to bite a human being malarial infection results, with the appearance in the blood of the hyaline forms already described. From the above description it will be seen that three forms of the malarial parasite may be found in the blood, viz., the parasite of tertian, quartan, and estivo-autumnal fever, and it has been shown that these forms may readily be distinguished from each other. In tertian and quartan fever several groups of the same organism may be present at one time, and as the process of : segmentation coincides with the occurrence of a paroxysm it will readily be seen that the Fic. 47.—Odkinetes of pernicious parasites in the stomach of Anopheles maculipennis thirty-two hours after having been sucked in. (Grassi.) Fic. 48.—Transverse section of the stomach of an anopheles, with cysts ot pernicious parasites. (Grassi.) number of paroxysms within a given time depends upon the number of groups which may be present in the blood. If a double infection with the tertian parasite has occurred, one group of organisms may thus have just reached the segmenting stage, while the second group has attained only a twenty-four hours’ growth, the result being that maturity is reached by the two groups on successive days. Quotidian fever is then the result. Should still other groups be present, the clinical picture will accordingly become more complicated. In quartan ague, similarly, double quartan fever will occur if two groups are present, and triple quartan fever if three groups are present at one time. Mixed infections, further, are also possible. PigMENTED LruKocytes.—In conclusion, it may not be out of place to refer to the presence of pigment-bearing leukocytes in the 186 THE BLOOD blood of malarial patients. (See Plate X.) These are quite constantly met with during the paroxysm, and it is indeed often possible to Q Fic. 49.—Four stages of sporulation of malarial parasites from Anopheles maculipennis, strongly magnitied: a-c, the pernicious parasite; a, four to four and a half days after ingestion; b and ¢, five to six days after ingestion; d, tertian parasite, eight days after ingestion. (Grassi.) Fic. 50.—Section through a tubule of the salivary gland of an anopheles, with sporozoites of th pernicious parasites; above an isolated sporozuite with higher magnification. (Grassi.) ; observe the process of phagocytosis directly under the microscope. The forms which are taken up are the small, fragmented, extra- cellular forms, the flagellate bodies, segmenting bodies, and free | ANIMAL PARASITES 187 pigment clumps. In every case where pigment-bearing leukocytes are observed, malarial fever should be suspected and a careful examination made, as a melanemia only occurs in this disease, in relapsing fever, and in connection with the rare melanotic tumors, in which not only leukocytes containing melanin may occur in large numbers, but also masses of pigment floating free in the blood. LirrratTuRE.—A, Laveran, Nature parasitaire des accidents de ’impaludisme, Description d’un nouveau parasite, Paris, 1881. P. Manson, Tropical Diseases, Cassell & Co., London, 1900, p. 1. For a full account of the literature, see the monograph by W.S. Thayer and J.;Hewetson,“ The Malarial Fevers of Baltimore,” Johns Hopkins Hosp. Rep., vol. v. On recent advances in our knowledge con- cerning the etiology of malarial fever, see W. 8. Thayer, Phila. Med. Jour., 1900, 4 1046, where a full account of the literature is given. T. B. Futcher, “A Critical ummary of Recent Literature concerning the Mosquito as an Agent in the Transmission of Malaria,’’ Amer. Jour. Med. Sci., 1899, p. 318. W.S. MacCallum, “On the Hematozoon Infection of Birds,’ Jour. Exper. Med., vol. ili, p. 117. EK. L. Opie, “On the Hematozoon of Birds,” ibid., p. 79. IF. Grohe, “ Zur Gesch. d. Melanaemie ” Virchow’s Archiv, 1861, vol. xx, 306. Fic. 51.—Trypanosoma gambiense (sleeping sickness) in blood of a rat. Two types are shown; the broad, pale form (female?) is dividing. Magnification 1500 times. MacNeal’s stain. (From Novy.) Trypanosomiasis.—The first authentic report concerning the occur- rence of trypanosomiasis in man was made by Dutton in 1902, while in animals their occasional presence had long been recognized (frogs, tats, dogs, groundhogs, etc.). In tropical regions certain species are pathogenic for certain domestic animals. ‘The tse-tse fly disease or -Nagana of Africa, the Surra disease of Asia, and the mal de caderas of South America are all referable to infection with trypanosomes (observed in the horse, the African buffalo, the ox, the donkey, thule, antelope, camels, and elephants). Especially interesting is the observation of Castellani and Bruce of the association of trypanoso- 2 188 THE BLOOD miasis with a certain symptom-complex, of which the so-called sleep-— ing sickness is one of the possible manifestations. Bruce could demonstrate the organism in the blood of 12 out of 13 cases, and in the cerebrospinal fluid in all of 38 cases. ‘The findings of these earlier observers have since been abundantly confirmed, and it is now generally conceded that the disease in question is referable to infection with trypanosomes. The Trypanosoma gambiense (Dutton) is from 8 to 25 y long, and from 2 to 2.8 y» broad. It is provided with an undulating mem- brane and a flagellum, which starts from a centrosome or micronucleus lying in the posterior end of the animal, and projects somewhat Fic. 52.—Trypanosoma gambiense from same preparation as preceding, showing the usual form; some cells in process of division. Magnification 1500 times. (From Novy.) beyond the anterior end. (See Figs. 51 and 52.) ‘There is an oval nucleus which is centrally located and is made up of chromatin granules. In the wet preparation the organism exhibits slow spiral move- ments. It is found free in the blood plasma, but may also be seen in the interior of leukocytes, which latter manifestly destroy the organisms exactly as the malarial parasites. In.dry specimens the trypanosomes can be readily stained with any basic dyes; with the Romanowsky stain or one of its modifications it is stained like the malarial organism. Levaditi' recommends the following method as especially valuable: Fixation in absolute alcohol and ether for five minutes; primary staining for two minutes with a saturated solution» of Bismarck brown, followed by washing and counterstaining with 1 Soe. de biol., November 23, 1903. ANIMAL PARASITES 189 Unna’s polychrome blue (diluted one-half with water) for two minutes. The specimens are rinsed in water, dried over a flame, and examined as usual. The number of organisms in a blood preparation is rarely large; as a rule, not more fen 3 to 8 are found to a cover-slip. ‘During apyrexia they are not seen. Infection in man probably occurs through a biting fly—the Glossina palpalis, which supposedly transmits the disease in a purely mechani- cal way. Novy and McNeal succeeded in cultivating the trypanosoma of Bruce in the water of condensation from a medium of agar mixed with defibrinated rabbit’s blood (1 to 1) at 25° C., and the rat trypano- some Tee lewisi) in a similar medium containing 1 part of blood for 2, 5, or even 10 parts of agar. LireraATURE.—Dutton, Thompson-Yates Laboratory Rep., 1902, vol. iv, part ii, p. 455; and Brit. Med. ’Jour., 1903, vol. i, p. 304. Castellani and Bruce, ibid., 1218 and 1431; Jour. Trop. Med., 1903, p. 167. Novy and McNeal, Journ. Amer. Med. Assoc., November 21, 1903. Novy, ibid., Jan. 5, 1907. Relapsing Fever.—Relapsing fever is characterized by the presence in the blood, and here only, of spirochetes which bear the name of their discoverer, Obermeier. In order to search for the organisms no special precautions are necessary. After having cleansed the finger a drop of blood is mounted on a very thin cover- glass. ‘This is inverted directly upon a slide, when the specimen is ready for examination; an oil-immersion lens is not required. Attention is drawn to the presence of the organisms by disturbances which are noticeable among the red corpuscles, and upon careful examination it will be seen that these are caused by the wriggling movements of the spirochetes. ‘Uhe Spirocheetee Obermeieri are long, slender filaments, measuring from 36 y» to 40 y in length by 0.3 # to 0.5 v in breadth, and present from eight to twelve in- curvations of equal size with tapering extremities. ‘These two last characteristics serve to distinguish this species from that described by Ehrenberg, in which the radius of the incurvations is not the same in all, and in which the extremities do not taper (Fig. 53). The number of spirilla which may be found in a drop of blood varies, being greater during the access of the fever, when twenty, or even more, may be observed in the field of the microscope. ‘They occur singly or in bunches of from four to twenty. In the quiescent stage they are arranged sometimes in the form of rings or of the figure 8. After the crisis they seem to disappear entirely, and their pres- ence during an afebrile period may therefore be regarded as indi- cating a pseudocrisis. During the afebrile periods small, bright, round bodies have been described in the blood, which according to some are spores, but according to others represent merely debris of the spirilla. 190 THE BLOOD Culture experiments have not been very satisfactory, although Koch observed an increase in their number at a temperature of from 102 sto alte Koch has shown that in African relapsing fever, which is likewise due to a spirocheta, infection occurs through the bite of a certain tick, Ornithodorus moubata, which acts as intermediary host in the development of the organism. Fig, 53.--Spirochetze Obermeieri; blood smear. » 1000 diam. (From Itzerott and Niemann.) ‘The tick fever of the Congo Free State is apparently identical with — the African recurrens described by Koch. Infection likewise occurs through the bite of infected ticks, Ornithodorus moubata. Hodlmoser has shown that the blood of recurrens is _ spirilla agelutinating. But as the culture of the organisms is practically not possible, the blood of a second case must be available for the test. LireRATURE.—Heidenreich. Untersuch. tiber d. Parasit. d. Ruckfallstyphus, Berlin, 1877. Moczutkowsky, Deutsch. Arch. f. klin. Med., vol. xxiv, p. 80, and vol. xxx, p. 165. Blisener, Inaug. Diss., Berlin, 1873. Engel, Berlin. klin. Woch., 1873, p. 409. J. E. Dutton and J. L. Todd, Brit. Med. Jour., November 11, 1905. 76 Versammlung d. Nat. u. Azt., Breslau, 1904. Typhus Fever.—According to Gottschalk’ a protozoon, closely related to Pyroplasma bigonicum, which he terms Apiosoma, can be demonstrated in the blood of typhus fever. He claims to have found sporulation cysts and flagellated forms. Infection according to Gottschalk may occur through bedbugs. Tropical Splenomegaly (Kala-azar). —Through the researches of Donovan, Leishman, and Ross especially it has been established that in tropical splenomegaly (cachexial fever, Kala-azar) parasites may be demonstrated in the blood which are probably etiologically connected with the pathological condition. ‘The organism in question has been termed the Leishmania Donovani (Leishman-Donovan body, Cunningham-Leishman-Donovan body). It represents a stage 1 Deutsch. med. Woch., 1908, No. 19. PLATE X! «<" a ba ~ 4 it Leishmania-Donovani. @, nuelei of leukocytes undergoing dissolution, (Stained with Leishman’s stain.) — a ANIMAL PARASITES 191 in the development of a trypanosome, as was suggested by Rogers and as has since been shown by cultural experiments by Leishman and ~ Statham. In the peripheral blood the organisms are rarely found and only when the temperature is high. Splenic puncture gives the best results. Donovan suggests that it is well to keep the patient flat on the back for twenty-four hours after the operation and to give a dose of calcium chloride immediately after and twice again at intervals of three hours (to prevent hemorrhage). ‘lhe parasites are princi- _- pally met with in large mononuclear cells. ‘The typical forms are oval or circular with a well-marked contour (Plate XI). There is a | deeply staining nucleus lying against the capsule and a deeply stain- ing, rod-like centrosome. ‘They may occur singly or in pairs or in _ zoogloea masses. ‘hey are readily stained with any: one of the methylene-azure mixtures (Hastings, Giemsa, Leishman, etc.). LITERATURE.—R. Ross, Brit. Med. Jour., July 9, 1904. L. Rogers, Lancet, ’ July 23, 1904. Leishman and others, Discussion, Brit. Med., Journ, September 17, 1904. Leishman and Statham, Jour. Royal Army Med. Corps, March, 1905. Syphilis.—The Spirocheta pallida (Treponema pallidum) has been demonstrated in the blood during life. Under ordinary circumstances, however, its search is here not likely to be attended by success. For diagnostic purposes it should be looked for in scrapings from chancres, papules, condylomas, in the aspirated juice of enlarged lymph glands, etc. (For a description of the organism see Examination of Syphilitic Material. ) Spotted Fever.—lIn the so-called spotted fever, which occurs in Montana, Nevada, Oregon, etc., an intracorpuscular ameboid, non- pigmented organism has been described by Wilson and Chowning, as also by Anderson, which they regard as the cause of the disease. They term this the Pyroplasma hominis. Infection supposedly _ takes place through ticks belonging to the species Dermacentor reticulatus. I have studied the blood in several cases which were placed at my disposal by Drs. McCalla, Maxey, and Pease, but was unable to find such structures. Craig and Stiles express themselves in a similar manner. LirerRATURE.—Wilson and Chowning, Jour. Amer. Med., Assoc., 1902, vol Xxxix, p. 131. J. F. Anderson, Amer. Med., 1903, vol. vi, p.506. Craig, Amer. Med., December 10, 1904. _ Filariasis.—According to Manson, the embryos of at least four, __ and possibly five and even more distinct species of nematodes may be _ found in the blood of man. ‘These various blood worms Manson designates as the Filaria nocturna, Filaria diurna, Filaria perstans, | Filaria demarquaii, Filaria ozzardi (a doubtful species), and a sixth, | which may or may not be connected with one of ‘the two last, the | ie 192 THE BLOOD Filaria magelhesi. ‘lwo of these at least are of pathological import, viz., the Filaria nocturna and the Filaria perstans. Filaria Nocturna (Manson): syn., Filaria sanguinis hominis (Lewis). his filaria is the embryo form of the Filaria Bancrofti (Cobbold), which inhabits the lymphatics and is unquestionably the cause of endemic chyluria, of various forms of lymphatic varix, of tropical elephantiasis arabum, and possibly also of other obscure tropical diseases. The organism in question is widely distributed. It is indigenous in almost all tropical and subtropical countries as far north as Spain in Europe and Charleston in the United States, and as far south as Brisbane in Australia. It is very common in Cochin and in some of the South Sea Islands, where one-third and one-half of the population, respectively, appear to be infected. In the following description of both parent and embryo form | quote largely from Manson’s account of the parasite in his admirable manual of tropical diseases. Fie. 54.—Filaria sanguinis. The parent filarias are hair-like, transparent worms measuring from 7.5 to 10 em. in length. ‘The sexes live together, often inextricably coiled about each other. Sometimes they are enclosed, coiled several in a bunch, and tightly packed in little cyst-like dilatations of the distal lymphatics; sometimes they lie more loosely in lymphatic varices; sometimes they inhabit the large lymphatic trunks between the glands, the glands themselves, and probably not infrequently the thoracic duct. ‘The female is the larger; there are two uterine tubes — which occupy the greater part of the body, and which are filled with ova in various stages of development. ‘The vagina opens near the mouth; the anus just in advance of the tip of the tail. The — cuticle is smooth and without markings. In both sexes the mouth end tapers slightly; it is clubbed and simple. The male is charac- terized by its marked disposition to curve. ‘The cloaca gives exit to — two slender, unequal spicules. . | ANIMAL PARASITES 193 In the wet preparations the /ilaria nocturna appears as a trans- parent, colorless little worm, which wriggles about most actively, constantly agitating and displacing the corpuscles in its vicinity. It will be noticed, however, that the animal does not propel itself through the drop of blood, but remains stationary. At first the movements are so active that it is impossible to make out any ana- -tomical details; after a number of hours, however, the movements become more sluggish, and it is then possible to study the worm with more ease. It measures about 0.31 mm. in length by 0.007 to 0.008 mm. in width. With the higher power it will be seen that the entire worm is enclosed in a delicate envelope, in which it moves _ backward and forward, the sheath being much larger than the worm (Fig. 54). It is owing to the presence of this sheath that active loco- motion on the part of the worm is not possible. About the posterior _part of the middle third of the parasite there is an irregular aggrega- tion of granular matter, which represents a viscus of some sort. With a high power one can further make out a delicate transverse striation ‘in the musculocutaneous layer throughout the entire length of the animal. In stained specimens two V-shaped light-spots can be smade out: one at a point about one-fifth of the entire length of the organism, backward from the head end; the other, very much smaller, a short distance from the tail. ‘The first Manson designates the “VY” spot, the second the tail spot. In stained specimens these two spots are readily made out, as they do not take the color. When ‘the movements of the animal have almost ceased, one can see on -eareful focussing that the head is constantly being covered and uncovered by a six-lipped or hooked’ and very delicate prepuce; ‘and, moreover, one can sometimes see a short fang of extreme itenuity suddenly shot out from the uncovered extreme cephalic end, jand as suddenly retracted. _ ‘TrecHnique.—The examination should be made late in the even- ‘ing, after the patient has rested for a number of hours. Drops of blood are then mounted, wet, on slides and ringed with vaselin to \prevent the specimen from drying. In such preparations the filarias keep alive for a week or longer. ‘They should be searched for with a low power—an inch objective is very convenient for the purpose. Attention is directed to their presence by the commotion which they cause among the neighboring blood corpuscles. _ To prepare permanent mounts blood smears are best made on slides, which are then stained with eosinate of methylene blue in the dsual manner. Working with the blood of infected animals, I have thus obtained very good results. The V and tail spots are very well orought out. To show anatomical details, however, staining with osin and hematoxylin, after fixing the smears with alcohol, gives the est results; in this manner the sheath is very well shown, as also ‘he structure of the musculocutaneous layer. | 13 194 THE BLOOD The number of worms which may be found in a specimen is very variable. During the daytime they are rarely seen, and, if at all, only one or two specimens at most are found. As evening ap- proaches, however, commencing about 5 or 6 o’clock, the filarias enter the peripheral circulation in increasing numbers. At mid- night the maximum number is about reached, with from 300 to 600 to the drop of blood. Later they gradually decrease, and by 8 or 9 a.m. they have again disappeared. ‘This periodicity, however, may be reversed if the patient is made to sleep during the daytime and remains awake at nights. During their absence from the peripheral circulation they may be found in the larger arteries and in the lungs. In non-active cases the number of filarias even at night is quite small. In one instance of this kind I found only the sheath of a single worm while examining perhaps fifty specimens. Infection occurs through the females of mosquitoes belonging to both the culex and anopheles family which have fed on the blood of filaria-infected individuals. The history of the parasite while in the body of the mosquito is in brief the following: After their arrival in the stomach the young worms shed the sheath and invade the thoracic muscles, where they increase in size (to 1.5 mm.), de- velop a mouth, an alimentary canal, and a trilobed tail. ‘They then find their way into the abdomen, where, in suitably prepared sections, they may occasionally be seen in the tissues about the stomach, and even among the eggs in the posterior part of the abdomen. ‘The majority now find their way to the base of the pro- boscis and under appropriate conditions out through the proboscis by a channel which they make for themselves. After introduction into the human body the organism finds its way into the lymphatics, where it attains sexual maturity; fecundation takes place and the new generation of filarias enter the blood current by way of the thoracic duct and the left subclavian vein. ‘The development of the embryc form in the mosquito occupies from sixteen to twenty days. Whether or not infection can occur in any other way is no known. We could conceive that some of the worms are eliminatec with the eggs of the mosquitoes, and that infection could then take place through contaminated drinking water. | Filaria Perstans.—This species is of interest, as it was though to be concerned in the causation of the so-called sleeping sickness 0 west tropical Africa. It has likewise been found in the Buck Indian) of British Guiana, among whom the same sickness also occurs." Th organism observes no periodicity, but is present in the blood bot! during the daytime and at night. | 7 1 More recent observations tend to throw doubt on this relationship and rathe suggest a connection between a species of trypanosoma and sleeping sickness (See p. 187.) ANIMAL PARASITES 195 The embryo worm is smaller than the Filaria nocturna; it meas- ures about 0.2 mm. in length by 0.004 mm. in breadth. It has no sheath, and its caudal end is truncated and abruptly rounded. There is no hooked cephalic prepuce. Its motion is progressive. The adult form measures 70 to 80 mm. in length. The tail in both sexes is incurvated and the chitinous covering at the extreme tip split, as it were, into two minute triangular appendages. They have been found in the connective tissue, at the root of the mesen- tery, behind the abdominal aorta, and beneath the pericardium. LireRATURE.—Mosler u. Peiper, Spezielle Pathol. u. Therap., 1894, vol. vi, . 219. P. Manson, Allbutt’s System of Medicine, vol. ii. I. Guitéras, Med. News, April, 1886. I’. P. Henry, ibid., 1896. E. Opie, Amer. Jour. Med. Sci., 1901, vol. exxil, p. 251. P. Manson, Tropical Diseases, Cassell & Co., London, 1900. O53 Brg = ohh Asp, Gg ao S Zr re Fic. 55.— Male and female specimens of the human blood fluke (Bilharzia hematobia). «x 12. (After Looss.) Distomiasis (Bilharziasis).—Bilharzia hematobia (Cobbold): syn., Gynecophorus (Diesing); Distomum hzmatobium (Bilharz); Schistosoma haematobium (Weinland); Distoma capense (Harley); ‘Thecosoma (Maguin-T'andon). The Bilharzia hematobia belongs to the class of trematode platodes. According to Bilharz, the greater portion of the Fellah and Coptic population of Egypt is infected. It is abundant in South Africa, and has also been observed in Mesopotamia, and 196 THE BLOOD AO BT PU. AS apparently in Arabia. In the United States a few isolated cases— have been seen which were undoubtedly imported. From Europe | no endemic cases have been reported. ‘The parasite may give rise — to diarrhea, hematuria, and ulceration of the mucous surfaces. 7 The male is smaller but thicker than the female, measuring from | 12 to 15 mm. in length by 1 mm. in breadth. On its abdominal surface a deep groove is found with overlapping edges, which serves — for the reception of the female (Fig. 55). It has an oval and a ventral sucker placed close together. | The adult parasites are found in the blood of the portal veln, In its mesenteric and splenic branches, and in the vesical, uterine, and Fic. 56.—Bilharzia eggs from the urine: Group a was drawn to scale with B. & L. % obj., and 1 in, ocular; group b represents their appearance with B. & L. % obj. hemorrhoidal veins; they have also been found in the vena cava and may possibly occur elsewhere in the circulation. The eggs are more often seen. ‘They are oval bodies, measuring 0.16 mm, in length by 0.05 mm. in breadth, and are provided with a dis- tinct, spike-like projection which issues from one extremity or the side (Fig. 56). Infection usually takes place through unfiltered drinking water, but may also occur through the skin. ‘Through the portal system the parasite then invades the urogenital system, the anus, and rectum, and may also proliferate abundantly in the intes- tine, the liver, kidneys, ete. The diagnosis is usually made by examination of the urine, in which the ova will be found. ; Another variety of blood fluke has been described by J. Catto,” Schistosoma Cattoi; it was found in a Chinese who had died of cholera. Lirerature.—Bilharz, Wien. med. Woch., 1856, vol. vi, p. 49. Meissner, Schmidt’s Jahrbuch., 1882, vol. xx, p. 193. Riitimeyer, Verhandl. d. Cong. f inn. Med., 1822, vol. xi, p. 144. \ Anguilluliasis.—In 1895 Teissier reported a case of intermittent fever in which numerous embryos of anguillula were found in the hy 4 | | 1 Brit, Med, Jour,, January 7, 1905, ANIMAL PARASITES 197 blood. ‘They disappeared after expulsion of the parasites from the intestinal tract, and at the same time the fever ceased. Itis a question, however, whether ‘leissier’s parasite was identical with the common form described by Bavay, Normand, Grassi, and others. Unlike the embryos developing from the eggs of both parasitic and free- living generations, ‘leissier’s form did not present the characteristic double cesophageal enlargement, and he reports, moreover, that in the case of the adult male only one, instead of two, spicules was noted. This view is strengthened by the observation that after inoculation into frogs the worms developed in the intestinal canal and the lungs into giant forms, which may have been Ascaris nigrovenosa (syn., Rhabdonema nigrovenosum). LireratuRe.—Teissier, Compt.-rend. de l’Acad. des sci., 1895, vol. exxi, Dalz Arch. de méd. expér, et d’anat. path., 1895, vol. vii, p. 675; ibid., 1896, vol. viii, p. 586. CHAPTER ILI. | THE SECRETIONS OF THE MOUTH. SALIVA. NorMAL saliva is a mixture of the secretions derived from the submaxillary, sublingual, parotid, and mucous glands of the mouth. It is a colorless, inodorous, tasteless, somewhat stringy and frothy liquid, and serves the purpose of aiding in the acts of mastication, deglutition, and digestion. ‘lhe quantity secreted in twenty-four hours amounts to about 1500 grams. General Characteristics. Normal saliva has a specific gravity of 1.002 to 1.009, correspond- ing to 4 to 10 grams of solids. ‘The reaction is alkaline, the degree of alkalinity corresponding to from 0.006 to 0.048 per cent. of sodium hydrate. Normally an acid saliva is observed only in newly born infants and in sucklings. The reaction of the tongue and the mucous membrane lining the mouth is quite commonly acid early in the morning owing to the production of lactic acid by some of the bacteria which are constantly _ present in the mouth. ‘This acid corrodes the enamel of the teeth, and may ultimately produce dental caries. | Chemistry of the Saliva. In order to give an idea of the general composition of the saliva the following analyses are appended; the figures correspond to 1000 parts by weight: Water)... OL 9. ae ie Beem DOD 994.20 988 .10 Ptyalin't.“~ 5. =) ae 1.34 1.30 1.30 Mucin Epithelium sty! Aaa ee Ee ee ee 1.62 2.20 2.60 Batty matter, i. aoe eee a8 Ate 0.50 Sulphocyanides 5 210 ao an ee 0.06 0.04 0.09 Alkaline chloridess 2 its Jee &. 0.84 Disodium phosphate . . . . 0.94 2.20 3.40 Magnesium and calcium salts .. 0.04 Alkaline carbonates Rea mete oe EPR COS? Nitrites 5 37s the. gh, oe ke eT COR 1 These figures are too high, as they refer to the total precipitate obtained with alcohol. SALIVA 199 In order to demonstrate the presence of the sulphocyanides, it is usually only necessary to heat a few cubic centimeters of the pure saliva, faintly acidified with hydrochloric acid, with a dilute solu- tion of ferric chloride, when a red color will be seen to develop. If necessary, larger quantities, such as 100 c.c., are evaporated to a small volume; the test is then applied to the concentrated fluid. The test for nitrites is conducted in the following manner: About 10 c.c. of saliva are treated with a few drops of Ilasvay’s reagent and heated to a temperature of 80° C., when in the presence of nitrites a red color will develop. ‘The reagent is prepared as follows: 0.5 gram of sulphanilic acid in 150 e.c. of dilute acetic acid is treated with 0.1 gram of naphtylamin dissolved in 20 c.c. of boiling water. After standing for some time the supernatant fluid is poured off and the blue sediment dissolved in 150 c.e. of dilute acetic acid. The solu- tion is kept in a sealed bottle. Of organic matter, ptyalin, a little albumin mixed with mucin, and about 1 gram of urea pro liter are found. In neutral or slightly alkaline, but not in acid solutions ptyalin rapidly transforms boiled starch into dextrins and sugar at a tempera- ture of from 35° to 40° C. In order to test for ptyalin, a few cubic centimeters of saliva are filtered and added to a solution of starch; the mixture is placed in the warm chamber for 5 to 10 minutes, when it is tested with cupric sulphate or iodine. At first starch gives a blue color with iodine; after digestion has proceeded farther a red or violet red is ob- tained, indicating the presence of erythrodextrin, while no change in color at all results when achroéddextrin only is present. ‘The ‘maltose may be recognized by the fact that it turns the plane of polarization more strongly to the right than glucose; like glucose, it reduces Fehling’s solution. Microscopic Examination of the Saliva. If normal saliva is allowed to stand, two layers will be seen to form, viz., an upper clear and a lower cloudy layer, which latter con- tains certain morphological elements. Among these, salivary cor- puscles, pavement epithelial cells, and microdrganisms are found (Fig. 57). The salivary corpuscles resemble white corpuscles very closely, but differ in their greater size and coarser appearance. ‘The epi- thelial cells are large, irregular, polygonal cells, provided with well- defined nuclei and nucleoli; they exhibit certain irregularities in size, according to their origin, and belong to the class of pavement or stratified epithelium. 200 THE SECRETIONS OF THE MOUTH Microorganisms.'—While schizomycetes and molds are only exceptionally found in the mouth under normal conditions, bacteria are always present in large numbers, and it is not surprising that all forms which are found in the air, food, and drink may here be encoun- tered. Some of these, such as the Leptothrix buccalis innominata, Bacillus buccalis maximus, Leptothrix buccalis maxima, Llodococcus vaginatus, Spirillum sputigenum, and Spirocheta dentium, are always present. ‘logether with other bacteria, they have been found in carious teeth, in abscesses communicating with the mouth and pharynx, and in exudates on the mucous membranes of these parts. In all probability, however, they are non-pathogenic. ‘To this class also belongs the smegma bacillus, which has been encountered in the saliva, the coating of the tongue, and in the tartar of the teeth of per- Fie. 57.—-Buccal sereons (Eye-piece III, obj. Reichert, 1/5 homogeneous immersion: Abbe’s mirror, Open condensers.) a, epithelial cells; 6, salivary corpuscles; c, fat drops; d, leukocytes; e, ‘Spirocheta buccalis; f, comma bacillus of mouth; g, Leptothrix buccalis; h, 43 k, various fungi. (v. Jaksch.) fectly healthy individuals. ‘The Leuconostoc hominis also is a normal inhabitant of the oral cavity, but occurs in larger numbers in inflam- matory diseases (scarlatina, measles, and diphtheria).” In this connection it is interesting to note that, in contradistinction to the bacteria which are only temporarily found in the mouth, the majority of those which are constantly present cannot be cultivated on artificial media. Important from a practical standpoint is the fact that a number of pathogenic microérganisms may be found under normal con- ditions. ‘The Diplococeus pneumonize has thus been found in a virulent condition in from 15 to 20 per cent. of healthy individuals, and it is even claimed that in a non-virulent state it is constantly present in the mouth. Streptococci are likewise frequently observed, but usually possess but little virulence or none at all when obtained from the healthy mouth and tested upon animals. Pyogenic staphy-_ * 'W. D. Miller, Die Mikroérganismen d. Mundhdhle, 1892. ? Hlava, Folia ‘hematol., Vvol.d; Di Glz. SALIVA 901 locoeci may also be found at times, but are less common than the streptococci. Most important is the occasional occurrence of the diphtheria bacillus in the mouths of individuals who have not been exposed to contagion. Welch’ mentions that virulent organisms were found by Park and Beebe in the healthy throats of 8 out of 330 persons in New York who gave no history of direct contact with eases of diphtheria; 2 of these 8 persons later developed the disease. Non-virulent bacilli were found in 24 individuals of the same series, and pseudodiphtheria bacilli in. 27. Other pathogenic bacteria which may be found in normal mouths are the Micrococcus tetragenus, the Bacillus pneumoniz of Fried- linder, the Bacillus crassus sputigenus, and the Bacillus coli com- munis. Pathological Alterations. It has been mentioned that about 1500 grams of saliva are secreted in the twenty-four hours. ‘This quantity is, however, sub- ject to great variation. An increase is thus frequently noted in pregnancy, in various neurotic conditions, in tabes, bulbar paralysis, in inflammatory diseases of the mouth, in dental caries, following the administration of pilocarpine, in poisoning with mercury, acids, and alkalies, etc. ‘he quantity is diminished in all febrile diseases, in diabetes, and often in nephritis. ‘The effect of psychic influences upon the secretion of saliva as well as of other glands is well known, an increase or decrease in the flow being produced under various conditions. In determining whether or not salivation actually exists, the physi- ‘cian should not only be guided by the statements of the patient, but an actual estimation of the amount secreted within a definite ‘period of time should be made. Nervous individuals not infrequently complain of ‘‘salivation,’’ when a direct estimation will show that the amount is not only not increased, but actually diminished. An acid reaction has been noted in various diseases of the intestinal tract, in febrile diseases, and notably in diabetes. According to ‘Strauss and Cohn, however, an alkaline reaction is the rule even tinder pathological conditions. Among the qualitative changes may be mentioned an increase in the amount of urea, which has been repeatedly observed in nephritis. Urea may be demonstrated as follows: The saliva is extracted with alcohol, the filtrate evaporated, and the residue dissolved in amyl alcohol. ‘This is allowed to evaporate spontaneously, when i a. of urea will separate out, and may be further examined (see Urine) Bile-pigment and sugar have not been found in the saliva. ‘ ’ Dennis’ System of Surgery; Surgical Bacteriology. ————$————$— $$$ 202 THE SECRETIONS OF THE MOUTH SPECIAL DISEASES OF THE MOUTH. Tuberculosis.—In cases of lupus and the so-called benign form of tuberculosis of the mouth it is rarely possible to demonstrate the presence of tubercle bacilli, even in scrapings taken from the base of the ulcers or in the diseased tissue itself, while in cases of ulcer- ative stomatitis associated with phthisis in its advanced stages they may be frequently found in large numbers. In some cases, however, their demonstration is by no means easy. In the saliva they are only exceptionally seen. : Actinomycosis.—In cases of actinomycosis it is occasionally pos- sible to demonstrate the presence of the specific organism in or about carious teeth. More commonly, however, the patients are not seen until the primary symptoms of the disease have disappeared, when the typical kernels can no longer be found at the orzginal points of entry or have become unrecognizable owing to calcification and retrogressive changes. Usually the disease has already progressed to the formation of a distinct tumor or abscess, and it may then be necessary to make an exploratory incision, and to examine the scrapings which are brought away. ‘lhe number of kernels which may be found is at times very small, but a careful examination will probably always lead to their detection if the disease in question is actinomycosis. Catarrhal Stomatitis.—In this affection the quantity of saliva is increased. Microscopically an increased number of epithelial cells and many leukocytes are noted, their number depending upon the intensity of the morbid process. Ulcerative Stomatitis——In this condition, following mercurial poisoning or scurvy, the same appearance is noted microscopically as in simple stomatitis. In addition there may be necrotic tissue, red blood corpuscles, and innumerable leukocytes. ‘The reaction of the saliva is intensely alkaline, the color markedly brown, and its odor fetid. Gonorrheal Stomatitis—The number of cases of gonorrheal stomatitis that have thus far been recorded is small. ‘The disease, however, has received but little attention, and is probably more! common than is generally supposed. In suspected cases the exudate | which forms upon the gums, the tongue, and the palate should 4 examined for gonococci. Thrush.—Oidium albicans (Fig. 58) is most commonly seen in children, but may also occur in adults, and especially in phthisical individuals, and sometimes lines the entire mouth. If in such cases: a bit of the membrane is pulled off and examined microscopically, it will be found to consist of epithelial cells, leukocytes, and granular detritus, with a network of branching, band-like formations, which COATING OF THE TONSILS 203 oresent distinct segments. ‘The contents of the segments are clear, and usually contain two highly refractive granules—the spores, one \ Vay ey oe “a cg 4 ay Ne eT SP Swe 8 q | Fie. 58,—Oidium albicans, the vegetable parasite of thrush. (Reduced from Ch. Robin.) r / of which is situated at each pole. ‘These segments diminish in size coward the end of each band, their contents at the same time becom- ng slightly granular. _ Tartar.—lIn a bit of tartar scraped from the teeth actively moving spirochetes are seen, as well as long, usually segmented bacilli, fre- quently forming bands which are colored bluish red by a solution of odopotassic iodide. Leptothrix buccalis, shorter bacilli (which are aot colored by this reagent), micrococci, and a large number of leuko- sytes and epithelial cells which have undergone fatty degeneration, are also found. Infusoria have been found by Sternberg, P. Cohn- aeim, v. Leyden, and others. COATING OF THE TONGUE. | A brown coating of the tongue is often observed in severe infectious liseases, and consists of remnants of food and incrustated blood. Microscopically, in addition to a large number of epithelial cells, enormous numbers of microérganisms and a large number of dark, vell-like structures, probably derived from desquamated epithelial cells, are found. ‘The white coating of the tongue contains epithelial cells, many microorganisms, and a few salivary corpuscles. COATING OF THE TONSILS. Pharyngomycosis Leptothrica.—In the pyoid masses derived from che crypts of the tonsils in cases of follicular tonsillitis, and also in dersons who have had frequent attacks of tonsillitis, large numbers of lymphocytes of all sizes are seen, besides epithelial cells and long, 204 THE SECRETIONS OF THE MOUTH segmented fungi—the Leptothrix buccalis (Fig. 59)—which are colored bluish red by a solution of iodopotassic iodide. Ordinary polynuclear neutrophiles are only present in small numbers. At times patches composed of these fungi extend over a considerable area of the tonsils, so that it may be doubtful whether or not the disease is a beginning diphtheria. More extensive invasions have been described by Dubler, who noted a leptothrix mycosis involving the pharynx, esophagus, and larynx; and by Baginsky in the case of the pharynx, trachea, and nose. LITERATURE.—Frankel, Berlin. klin. Woch., 1873, p. 94. Miller, Die Mikro- | organismen der Mundhdhle, 1889, Leipzig. Stern, Miineh. med. Woch., 1893, p. 381. “Hering, Zeit. f. klin. Med., 1884, p. 358. Dubler, Virchow’s. Arch., 1891, vol. exxvi, p. 454. Baginsky, cit. by. Hering (vide supra). Fie. 59.—Leptothrix bucealis. (v. Jaksch.) Tonsillitis.—In tonsillitis a large number of bacteria have been) isolated from the pseudomembranous deposits. Among the more| important which are supposed to bear a causative relation to the dis-_ ease may be mentioned the various streptococci, staphylococci, less | commonly the pneumococcus, the Micrococcus catarrhalis, the Bacillus coli communis, the bacillus of Friedlander, the Bacillus ,septiceemie | sputi, and in a few isolated instances the Micrococcus tetragenus. In many cases in which tonsillar deposits are clinically regarded as. diphtheritic culture reveals only an abundance of the thrush fungus. Meyer,’ in v. Leyden’s clinic, succeeded in cultivating a diplo- streptococcus from the tonsils in five cases of acute rheumatism with! angina, and reports that bouillon cultures of the organism produced characteristic polyarticular arthritis in rabbits. . The same organism | apparently was also obtained by Allaria’ in Bozzolo’s clinic, and it, is interesting to note that his cases resulted from manifest contagion. | 1 Deutsch. med. Woch., 1901, vol. xxvii, p. 81. * Revista critica di clinica Medica, 1901, vol. ii, p. 805. COATING OF THE TONSILS 205 Vincent’s Angina.—In cases of Vincent’s angina (ulceromembranous angina and stomatitis) smears from the exudate will be seen to contain innumerable organisms which are essentially of two types, viz., spirilla and long, fusiform bacilli (Fig. 60). Occasionally, though exception- ally, the bacilli only may be found. ‘The spirilla usually present three or four convolutions and are generally actively motile. ‘Chey measure from 36 to 40 4 in length by 0.5 / in breadth. ‘The bacilli measure from 6 to 12 4 in length and are somewhat stouter in the middle than at the ends. They may occur in twos, joined end to end, and usually scattered uniformly throughout the preparation. ‘They are non-motile. Spirilla and bacilli are readily stained with a dilute solution of carbol fuchsin (1 to 20), which should be filtered before use. Lé6ffler’s blue and gentian-aniline water may likewise be used. The bacilli are obligate anacrobes; the spirilla may be obtained together with the bacilli in mixed cultures. Of late the opinion has been expressed that the spirilla and bacilli ‘may represent stages in the life history of a trypanosome. — Both organisms have occasionally been found associated with diphtheria bacilli. The disease seems to be more common than was first thought. The earlier cases were reported by Vincent, Bernheim, Conrad, and others. In the United States the disease has been described by ‘Mayer, Fisher, Crandall, Weaver and 'Tunnicliff, Berkeley, and others. _ LireraturE.—J. W. Byers, Lancet and Brit. Med. Journ., January 9, 1904. Weaver and Tunnicliff, Journ. of Infect. Dis., 1905 vol. ii, p. 446. Berkeley, ‘Med. News, 1905, vol. xxxvi, p. 976. Wright, 1904, July 4, p. 73. Diphtheria.—Recognizing the great importance of an early diagnosis in cases of diphtheria, an examination for Loffler’s bacillus has become just as important today as that for the bacillus of tuberculosis. ' By means of a stout platinum loop, a pair of forceps, or a cotton swab, a piece of membrane is scraped from the tonsils, the soft palate, or the pharynx. From this cultures are prepared as described below; at the same time smears are made on slides and fixed, when air dry, by being passed several times through the flame of a Bunsen burner. ‘They are then stained for five to ten minutes in Léffler’s alkaline solution of methylene blue, which consists of 30 c.c. of a concentrated alcoholic solution of methylene blue in 100 c.c. of an aqueous solution of potassium hydrate (1 to 10,000). They are then rinsed in water, dried and examined with a ;), oil-immersion lens. _ Arapid method of staining, and one which also gives satisfactory results, is suggested by Neisser. ‘The organism is grown on ox-blood serum and examined after nine to tw enty-four hours. The air-dried ‘smears are placed for one to three seconds in a solution composed of 20.¢.c. of an alcoholic solution of methylene blue (1 to 20 c.c. of 90 per cent. alcohol), 950 ¢.c. of distilled water, and 30 ¢.c. of glacial | | 206 THE SECRETIONS OF THE MOUTH acetic acid. ‘They are then washed in water, stained for three to- five seconds in a 0.2 per cent. hot and filtered aqueous solution of vesuvin, again washed off, dried in the air, and mounted in balsam, The bacilli are brown and have in their interior 2 to 4 blue granules which are usually located near the poles. The following method also may be employed, as suggested by Schauffler. The staining reagent has the following composition: Filtered solution of Léffler’s methylene blue. . . tre 2 0.05C.c8 Filtered solution of pyronin (0.5 gram to 10 e¢.c. of water) rd ay Ee Acid alcohol (3 ¢.c. of 25 per cent hydrochloric acid to 97 c.c. ot absolute/alcohol)iss- 2.) = a es ee ne eee Oscar Cover-glass specimens are stained for one minute; they are then washed in running water and mounted in balsam as usual. ‘The bacilli are stained blue, the pole bodies a bright ruby red. Pseudodiphtheritic bacilli are said to take only the blue stain with > this method. The organism grows best on Léfflers blood serum; upon this it develops so much more rapidly than other organisms which are usually present in the secretions of the mouth and throat, that, after six to eight hours’ incubation at 34° to 35° C., it often forms the only colonies that attract attention. Smears are then made and stained according to Neisser’s or Loffler’s method. In the absence of blood serum, bouillon, nutrient gelatin, nutrient agar, glycerin agar, and potato may be employed. Coagulated ege albumen, as pointed out by Booker, and milk are also good media. But it is to be noted that the “typical” staining effect with Neisser’s method is commonly only obtained if the organism has been grown on ox-blood serum, and if the growth is not older than twenty-four hours. According to Knapp the true bacilli, in contradistinction to the. pseudodiphtheria bacilli, will ferment dextrose and maltose. ‘The Bacillus xerosis will do the same. In contradistinction to the diphtheria organism the Bacillus xerosis will ferment cane sugar; the former, in contradistinetion to the xerosis, will ferment dextrin. ‘The fermentation tests must be made with the litmus serum-water media of His." Results after twenty-four hours’ growth at 37° C.: Pseudodiphtheria—none of the sugars fermented; media remain blue. Diphtheria—dextrose, mannite, maltose, and dextrin fer-— mented; media red and coagulated. Saccharose not fermented. Xerosis bacillus—dextrose, mannite, maltose, and saccharose fer- mented with acid production; media red and coagulated. Dextrin not fermented. ‘The Bacillus xerosis, moreover, forms a very thin scum or pellicle on the surface of the media which is absent with the other bacteria. | * Journ, of Med, Res., vol. xii, p. 475-478, See also Appendix: Media, / | COATING OF THE TONSILS 207 The colonies are large, round, elevated, and grayish white in color, with a centre that is more opaque than the slightly irregular periphery. ‘The surface of the colony is at first moist, but after a day or two it assumes a dry appearance. >y . 1 | | %. | Hie, 60: RiGee ol Fia. 62. Fie. 63. Fic. 60.—Spirilla and fusiform bacilli of Vincent’s angina. Fie. 61.—Characteristic forms of diphtheria bacilli from blood-serum cultures, showing clubbed ends and irregular stain. » 1100 diameters. (Park.) Fie, 62.—B. diphtheriew. Forty-eight hours’ agar culture. Thick, medium-clubbed rods and meee number of segments. One year on artificial culture media. >» 1410 diameters. ark.) Fic. 63.—Colonies of diphtheria bacilli. > 200 diameters. (Park.) The bacillus (Figs. 61, 62, and 63) is non-motile and varies in size and shape, its average length being from 2.5 to 3 y, its breadth from 0.5 to 0.8 4. Its morphological characteristics are so peculiar as to render its identification upon cover-slip preparations and in sections of the diphtheritic membrane an easy matter in most cases. 208 THE SECRETIONS OF THE MOUTH Sometimes the organism appears as a straight or shghtly curved rod; but especially characteristic are irregular and often bizarre forms, such as rods with one or both ends terminating in little bulbs and ois apparently broken at intervals, in which short, well- defined, round, oval, or straight segments can be made out. Very commonly two organisms lie together forming an obtuse angle, or numbers of them may be observed lying side by side. Some forms stain uniformly, others in an irregular manner; the most typical appearance is that of little granules near the poles of the bacillus, which stain blue with Neisser’s method, while the body of the organism is colored brown. Streptococci are also seen as a rule, and it may be said that the gravity of a case is directly proportionate to the number of strep- tococci present. It is important to note that diphtheria bacilli may still be found in the throat for weeks after all clinical symptoms have disappeared. Patients should hence be isolated until a bacteriological examination has demonstrated the absence of the organism. LITERATURE.—5S. Flexner, “The Bacteriology and Pathology of Diphtheria,” Johns Hopkins Hosp. Bull., 1895, p. 39. W.H. Welch, Amer. Jour. Med. Sci., 1894. Heubner, Schmidt’s ‘Jahrbiicher d. gesammten Med., 1892, vol. ccxxxvi, p. 270. Klebs, Arch. f. exper. Path., 1875, vol. iv, p. 207. Loffler, Centralbl. f. Bakt. u. Parasit., 1887, vol. ii, p. 105: and. 1890, vol. vii, p. 528. C. Frankel, “Die Unterscheidung d. echten u. d. falschen Diphtheriebacillen,”’ Berlin. klin. : Woch., 1897, p. 1087. W.G. Schauffler, Med. Record, December 6, 1902. Scarlatina.—According to Baginsky, streptococci are practically constantly found in the pharyngeal secretion. LITERATURE.—A. Baginsky, Deutsch. med. Woch., October 23, 1902. Glandular Fever.—According to Neumann and Comby, glandular fever generally depends upon infection with a streptococcus. In the case reported by Lande and Froin and by Hirtz’ bacteriological examination of the throat at the height of the febrile stage revealed the presence of the pneumococcus in a virulent condition. ' Lande et Froin, Rev. mensuelle des Mal. de l’Enfance, 1901, p. 78. Gp lr Ape Hy Ree LCT THE GASTRIC JUICE AND GASTRIC CONTENTS THE SECRETION OF GASTRIC JUICE. THE gastric juice is the result of the glandular activity of the Bemach, and is the only secretion of the digestive tract which pre- sents an acid reaction. As is well known, the mucous membrane of the stomach is cov- ‘ered throughout its entire extent by a single layer of cylindrical epithelium, which dips down in places to line the orifices and lar ver ducts of the numerous tubular glands with which it is beset. Of these, two kinds are described, viz., the fundus and pyloric glands, so named from the location in which they are principally found. ‘In the secretory portion of a fundus gland two sets of cells can be distinguished. ‘the one kind is small, granular, and polyhedral or eolumnar, bordering upon the narrow luinen of the tube: these are termed the chief or principal cells (Heidenhain), but are also known as the central or adelomorphous cells. ‘They stain with aniline dyes to only a slight extent. ‘lhe others, known as parietal, adelomor- phous, or oxy yntic cells, are variously situated between the adelomor- phous cells and the membrana propria; they are most numerous ‘in the necks of the glands. ‘They are larger than the chief cells, oval ‘or angular and finely granular in structure: they possess a strong affinity for the aniline dyes. ‘The pyloric glands, which are found ‘only in the region of the pylorus, on the other hand, are character- ized by the greater length of their ducts, which are also lined by the cylindric al epithelium of the mucous membrane proper. ‘lhe ‘secretory portion of these glands is represented by a single layer of short and finely granular, columnar cells, which closely resemble the chief cells of the fundus glands. In addition to these, a few isolated cells, the cells of Nussbaum, are found, which in structure and in their behavior to aniline dyes resemble the parietal cells. Upon chemical examination the gastric juice is found to consist essentially of water, free hydroc shloric acid, pepsin, rennet (a milk- curdling ferment), lipase, mucus, and certain mineral salts. Of these constituents hydrochloric acid is secreted by the parietal cells, pepsin, the mi k-curdling ferment, and lipase by the chief cells of the fundus and the pylori ic glands, while the mucus is the product of the cylindrical goblet eale lining the stomach and the 14 ee 210 THE GASTRIC JUICE AND GASTRIC CONTENTS wider portions of its glandular ducts. It should be borne in mind that the ferments do not exist in the cells as such, but as zymogens, which are transformed into the ferments through the activity of the free hydrochloric acid. According to modern investigations, more- over, the zymogens only are secreted by the cells. Until recently it was supposed that the gastric juice is secreted only upon appropriate stimulation of the nervous mechanism of the stom- ach, either directly or indirectly, and that the stomach in its quiescent state—i.e., when not digesting—is empty. ‘The researches of Schreiber and Martius, however, have rendered the correctness of this view doubtful, as they were able to obtain quantities of gastrie juice, varying from 1 to 60 c.c., from the non-digesting stomach of every normal person examined. ‘Test Meals.—As the amount of hydrochloric acid which is secreted varies with the amount and the character of the food ingested, it has been found useful for purposes of comparison to make analyses after the administration of test meals of constant composition. ‘The most important test meals are the following: The Test Breakfast of Ewald and Boas.—'l'his consists of 35 grams of wheat bread and 400 c.c. of water or weak tea, without | sugar. It is best to give this meal to the patient early in the morning, when the stomach is empty—t. e., as a breakfast, and in cases of dilatation or of marked atony, after previous lavage. ‘The gastrie contents are obtained one hour later. The Test Breakfast of Boas.—T'his consists of a plateful of oatmeal soup, prepared by boiling down to 500 c.c. one liter of water to which one tablespoonful of rolled oats has been added. A little salt may be used if desired, but nothing more. ‘The contents of the stomach are obtained one hour later. ‘This test meal was devised by Boas in: order to guard against the introduction from without of lactic acid, which is present.in all kinds of bread. ‘The meal is em- ployed in cases of suspected cancer of the stomach in which a quanti- tative estimation of lactic acid is to be made, the stomach being washed out completely the night before. | The Test Dinner of Riegel—'his consists of a plate of soup) (400 e.c.), a beefsteak (150 to 200 grams), and 150 grams of mashed potatoes. The contents of the stomach are obtained after four hours. The disadvantage of this method lies in the fact that the lumen of the stomach tube is frequently occluded by pieces of undi- gested meat, a source of annoyance which may be guarded against by using finely chopped meat. Moreover, a positive lactic acid reae- tion (referable to sarcolactic acid) is obtained in a large number of cases, and entirely irrespective of the amount of hydrochloric acid present. The Double Test Meal of Salzer.—For breakfast the patient receives 30 grams of lean, cold roast, hashed or cut into strips sufficiently THE SECRETION OF GASTRIC JUICE 211 small not to obstruct the stomach tube; 250 ¢.c. of milk; 60 grams of rice, and | soft-boiled egg. Exactly four hours later the second meal is taken, consisting of 35 to 70 grams of stale wheat bread and 300 to 400 c.c. of water. The gastric contents are withdrawn one hour later. In this manner the gastric juice is not only obtained at the height of digestion, but an idea may at the same time be formed of the motor power of the stomach. Under normal conditions the organ should contain no remnants of the first meal at the time of examination. The Stomach Tube.—The stomach tubes in general use are essentially large Nélaton catheters. They should measure from 72 to 75 cm. in length, and be provided with three fenestra, of which one is placed at the end of the tube and two laterally, as near the end as possible. For the purpose of washing out the stomach the tube is connected with a glass funnel. It is important that the tubes should be thoroughly cleansed in hot water as soon after use as possible. The advice of Boas, more- over, to have special marked tubes for tuber- ‘culous, syphilitic, and carcinomatous patients ‘should be borne in mind. Patients in whom lavage is to be practised for any length of time should provide their own instruments. _ Contra-indications to the Use of the Tube.— Of direct contra-indications to the use of the tube there should be mentioned the existence } of the various forms of valvular disease when in a state of imperfect compensation, angina pectoris, arteriosclerosis of high degree, aneu- tysm of the large arteries, recent hemorrhages from whatever cause, marked emphysema with intense bronchitis, acute febrile dis- \Pases, etc. Introduction of the Tube.—The technique of the introduction of the tube should be as simple as possible; the exhibition of complicated bottle arrangements for the purpose of obtaining the gastric juice only adds to the excitement of a nervous patient, and should be avoided. The patient’s cloth- ng and floor of the room should be pro- rected from being soiled by material that may be vomited along the sides of the tube, she dribbling of saliva, etc. For this purpose, ‘Tiirk’s rubber bib’ vith pouch may be advantageously employed. | Cocainization of the pharynx is not necessary, but may be resorted oin hyperesthetic individuals, a 10 per cent. solution being employed. , Fie. 64.—Boas’ bulbed tube, * Manufactured by G. Tiemann & Co., New York 912 THE GASTRIC JUICE AND GASTRIC CONTENTS The tube, held like a pen, is passed to the posterior wall of the, pharynx, the patient bending his head forward, and not backward, as is usually advised. ‘The patient is then told to swallow. ‘Lhe tube is pushed until resistance is felt when it meets with the floor of the stomach. At the least sign of cyanosis or of marked pallor the tube should be withdrawn at once, and the patient observed for a) day or two before a second attempt 1s made. | If the gastric juice does not flow at once, the patient is instructed | to bear down with his abdominal muscles, and, if this is insufficient, | to cough a little. Repeated attempts of this kind will usuatly bring about the desired result, unless the tube has not been introduced far enough or too far; in the latter case it will double upon itself, so that its end rises above the level of the liquid. Pressing upon the: abdomen with the hands is of no effect (Method of Expression). Aspiration must at times be employed. For this purpose Boas’) bulbed tube (Fig. 64) is convenient. ‘The manner in which it is used| is the following: ‘The proximal end of the tube, after having been) Fic. 65.—Arrangement of a bottle for aspiration of the gastric contents, introduced into the stomach, is compressed and the bulb squeezed | when the distal end is clamped and the bulb allowed to expand. A partial vacuum is thus produced, which usually has the desired effect) In the absence of such an instrument the stomach tube may be con: nected with a bottle, in which a partial vacuum has been establishec by aspiration (Fig. 65). Unless the patient is accustomed to the intro; duction of the tube, however, these more complicated procedures should be avoided as much as possible (Method of Aspiration). | In order to wash out the stomach, the funnel is filled with lukewarm water or any desired medicated solution, elevated above the head 0. the patient, and the water allowed to flow. From 500 to 1000 ¢.c. may be introduced at one time. By depressing and inverting the funne over a suitable vessel before all water has left the funnel a siphor GENERAL CHARACTERISTICS OF THE GASTRIC JUICE 218 arrangement is established and the stomach emptied. It is well to measure the returning water as well as the amount introduced. Should the flow diminish or cease before all the water has been removed, the end of the tube probably stands above the level of the liquid, and the flow can be started again by pushing the tube on farther or by with- drawing it a little, as the case may be. Washing out the stomach soon after the ingestion of a full meal is always very tedious and annoying, if not an impossible procedure, as the fenestra readily become Bpecreindl Should this occur, the funnel, filled with ws ater , 1s elevated as high as pdt with a view to overcome the obstruction by hydrostatic pressure; or, if this proves insufficient, the funnel is detached and the obstruction is lodged by means of air, for which purpose a Politzer bag or the bulb of a Boas tube 1 is very afr nent GENERAL CHARACTERISTICS OF THE GASTRIC JUICE. Pure gastric juice is an almost clear, faintly yellowish fluid, of a sour taste and a peculiar characteristic odor. Its specific gravity varies between 1.002 and 1.003, corresponding to about 0.5 per cent. af solids. Its reaction, owing to the presence of hydrochloric acid, s acid. _ Amount.—V ery little is known of the total quantity of gastric juice that is secreted in the twenty-four hours. ‘The figure given by Beau- pnt, viz., 180 grams pro die, based upon observations made upon the often-quoted Canadian hunter, Alexis St. Martin, is undoubtedly too low. ‘The amount given by Bidder and Schmidt,’ viz., that corre- sponding to about one- -tenth of the body weight, is probably more aearly correct.” It may be stated a priort that the quantity secreted varies within wide limits, being influenced by numerous factors, aotably by the degree of the appetite and the amount and character of the food taken, especially that of the proteids. “Phe age and sex of the individual, the time of day (notably in its relation to the gestion of food), the emotions, ete., all influence the glandular activity of the stomach.* _ From the non-digesting organ from 1 to 60 c.c. of gastric juice nay be obtained at one fime. ‘The amount which can be procured during the process of digestion, on the other hand, varies with the amount of liquid ingested, the time of expression, the size and motor | , Experiments and Observations on the Gastric Juice, Boston, 1834. , Verdauungssiifte u. d. Stoffwechsel, 1852. ® Griinewald’s figure—i. e., 1580 grams—I likewise regard as too low. According to my experience, the daily secretion appears to vary between 2000 nd 3000 ¢.¢ ae C. E. Simon, Physiological Chemistry, third edition, 1907, Lea Bros. «Co ” iy | | : 214 THE GASTRIC JUICE AND GASTRIC CONTENTS power of the stomach, and the degree of transudation; the process of resorption probably does not play any part, as it has been ascertained that very little water, if any, is absorbed in the stomach. As a rule from 20 to 50 c.c. of filtrate can normally be obtained one hour after the ingestion of Ewald’s test breakfast. ; | Abnormally large quantities of gastric juice are practically found only in cases of so-called hypersecretion, the “Magensaftfluss” of | the Germans, which may occur periodically or continuously. For- merly the presence of appreciable quantities of gastric juice in the non-digesting organ was regarded as conclusive evidence of the existence of this condition, but in the light of Schreiber’s researches this position can no longer be maintained. ‘The diagnosis should, hence only be made when in conjunction with the clinical symptoms of hypersecretion from 100 to 1000 c.c. of pure gastric juice can be | obtained from the non-digesting organ. ‘lo this end, the stomach should be emptied completely by the tube before retiring, and an | examination made on the following morning, no foods or liquids being allowed in the mean time. In various pathological conditions abnormally large quantities of | liquid may be obtained, which cannot be regarded as gastric juice, how- ever. Attention will be drawn to these conditions at another place. CHEMICAL EXAMINATION OF THE GASTRIC JUICE. The Acidity of the Gastric Juice is Referable to the Presence of Free Hydrochloric Acid.—It has been conclusively demonstrated / by Schmidt that the acidity of the gastric juice is due to the presence | of free hydrochloric acid and to this only. After accurately deter. | mining the amount of chlorine and all basic substances present, it was found that after the latter had been saturated a quantity of hydrochloric acid still remained, which in the dog varied between | 0.25 and 0.42 per cent., with an average of 0.33 per cent... The | amount of free acid was also determined by titration and the same | results reached as by gravimetric analysis. | While it can thus be regarded as an established fact that hydrochlo- ric acid only is found in the gastric juice, such as it is secreted, there | can be no doubt that traces of lactic acid may be found in the stomadh| contents during the process of digestion. ‘These traces, however, have | been introduced from without. The time at which hydrochloric acid will appear in the free state. depends ceteris paribus upon the quantity of albumins ingested. | With Ewald’s test breakfast it appears after thirty-five minutes and reaches its maximum between fifty and sixty minutes after eating. With Riegel’s meal the time is longer; it appears after one hundred’ and twenty to one hundred and fifty minutes in the free state and CHEMICAL EXAMINATION OF THE GASTRIC JUICE 915 ‘reaches its maximum after one hundred and eighty to two hundred and ten minutes. _ Under pathological conditions the amount of free hydrochloric acid, as will be shown, may undergo great variations, diminishing on the one hand to zero, and increasing on the other to 0.5 per cent., or even more. In other cases lactic acid and other organic acids may appear in notable amounts. Method of Determining the Total Acidity of the Gastric Con- ‘tents.—T’o this end a known quantity of gastric juice is titrated with a one-tenth normal solution of sodium hydrate, using phenolphthalein as an indicator, when the number of cubic centimeters of the one- tenth normal solution employed, multiplied by the equivalent of 1 c.c. Total acidity. Free HCl. 4 Last lavage. Stomach empty —O- Last expression (with chemical investigation). ; 15 30 45 60 75 90 95 Minutes. Fic. 66.—Course of the acidity of the gastrie juice after a test meal of 300 grams of tea and 50 grams of bread. (Schiile.) ‘of this solution in terms of hydrochloric acid, will indicate the amount ‘of acid present, from which the percentage acidity is readily cal- ‘culated. | Method.—5 or 10 c.c. of filtered gastric juice are titrated with the one-tenth normal solution of sodium hydrate, using 2 or 3 ‘drops of a 1 per cent. alcoholic solution of phenolphthalein as an indicator until a permanent rose color appears. ‘Che number of ‘cubic centimeters of the one-tenth normal solution employed multi- ‘plied by 0.00365 will indicate the acidity of the 5 or 10 c.c. of gastric juice in terms of HCl, from which the percentage acidity is calcu- lated. Example.—10 ¢.c. of gastric juice required the addition of 6.5 ‘ec. of the one-tenth normal solution; 6.5 0.00365 (7. e., 0.0237) would hence indicate the acidity of the 10 c.c. of gastric juice in terms of HCl, and 0.0237 & 10 = 0.237, the percentage acidity. _ Or the result may be expressed in terms of the number of c.c. of ) * 916 THE GASTRIC JUICE AND GASTRIC CONTENTS the ;", solution which would be necessary to neutralize 100 c.c. of stomach contents. In the example the total acidity would thus be 6.5 x 10 = 65. ‘This method of indicating results is indeed the usual. | Under normal conditions figures varying from 40 to 60 are usually obtained one hour after the ingestion of Ewald’s test breakfast, while in_ pathological conditions ereater variations are ee In acute and chronic inflammatory conditions of the stomach, well as in some of the neuroses, the acidity of the gastric content is below normal. Higher figures are met with in some cases of ulcer and in some cases of dilatation, but are especially common in neurotie conditions; a degree of acidity corresponding to 90 or even more is then not infrequently observed. Increased acidity, usually asso- ciated with hypersecretion of gastric juice, is met with in the so- called hypersecretio acida et continua of Reichmann. | Preparation of decinormal alkali solution.—A normal solution of | sodium hydrate is one containing the equ valent of its | weight in grams—1. e., 40 grams—in 1000 c.c. of distilled water; decinormal solution will, therefore, contain 4 grams in the same volume of water. ‘This quantity is dissolved in “about 900 c.c. anil the solution brought to the proper strength by titrating it with a solution of oxalic acid of known strength. From the equation 2Na0OH +.C,H,0, = C.Na,O, + 2H.0, it is seen that 2 molecules of NaOH (molecular weight 40) com- bine with 1 molecule of C,H,O, -- 2H,O (molecular weight 126), or 4 parts by weight of the former with 6.5 of the latter. 6.3 grams_ of chemically pure, cr ystalline oxalic acid (which is stable ane non- | deliqueseent) are dissolved in 1000 c.c. of distilled water; this makes. a 7%; normal solution of the acid. Were the alkali solution of the proper strength it should take just 10 c.c. to neutralize 10 c.c. of the acid. But as the alkali solution cannot be made up accurately | from the start (owing to inconstant weight from deliquescence), and as | it has been purposely made too strong, less than 10 c.c. will be re-| quired, e. g.,8 ¢.c. It is then ascertained how many such portions of | alkali solution map: are left, and then a corresponding amount of | water is added, 2. e., an amount representing the deficit found as compared with the aig solution. | In the present example, for instance, we started with 900 c.c. of | the uncorrected alkali solution, of which 8 c.c. were used in the test | titration. There are remaining then 892 c.c. For every 8 ¢.c. in| this bulk, viz., 111.5 portions, 2 e.c. of distilled water must be added; | hence 111.5 < 2 = 223 c.c. A second titration is made to ensure the correctness of the result. Since 1000 ¢.c. of the one-tenth normal solution containing 4 grams | CHEMICAL EXAMINATION OF THE GASTRIC JUICE 917 of NaOH are equivalent to 3.65 grams of HCl, as is seen from the - equation | NaOH +HCl=NaCl+H,0 40 36.5 1000 c.c. of the 10 normal solution represent 3.65 grams of HCl 100“ é : ) 0.365 Prana ops. . 10 “ce ce “c “ ce ‘e 0.0365 ce cc ce eee st pe Seeeereprencnta U.003007° “ho ff). f __ It has been pointed out that the reaction of normal gastric juice _ is always acid, owing to the presence of free hydrochloric acid, and the same may be said to hold good for the gastric contents in ceneral _ obtained from normal individuals. Pathologically an acid reaction _ is also the rule, as in those cases in which hydrochloric acid is absent fatty acids and lactic acid usually make their appearance. It is, therefore, not surprising that an alkaline, neutral, or amphoteric reaction is but rarely, or at least not commonly, observed in the gastric contents artificially obtained, and practically seen only in the so-called mucous form of chronic gastritis, or in those rare cases _ of anadeny, in which a complete destruction of the gastric glands has taken place. In vomited material, on the other hand, such observations are common, owing to the presence of large amounts of saliva. ‘The vomited material in cases of so-called vomitus _ matutinus, which is usually referable to a chronic catarrhal condition of the pharynx, generally presents an alkaline reaction, owing to _ the fact that the fluid brought up is largely unchanged saliva. _ The Amount of Free Hydrochloric Acid.—Pure gastric juice, _ according to Ewald,’ Szabo,’ and Boas,’ contains from 2 to 3 pro mille of free hydrochloric acid. In the digesting organ such amounts are met with only at the height of digestion, and after all basic affinities have been saturated. _ The time at which free hydrochloric acid can be demonstrated in the gastric contents after the ingestion of a meal will, hence, vary . with the character of the food and its amount. When but little work is to be accomplished free hydrochloric acid is found much sooner than otherwise. After Ewald’s test breakfast, it appears in thirty- _ five minutes; the point of maximum acidity is reached after from fifty to sixty minutes, and corresponds to 1.7 pro mille. Following Riegel’s meal, on the other hand, the free acid appears after one hun- dred and thirty-five minutes, a6 reaches its highest point (corre- sponding to 2.7 pro mille) in from one hundred “and eighty to two hundred and ten minutes. Clinically it is necessary to distinguish between euchlorhydnia, or the secretion of a normal amount of free hydrochloric acid (0.1 to a ' Loe. cit. _ * Zeit. f. physiol. Chem., 1877, vol. i, Bs 155: a | »* Loe. cit. See also A. rons Zeit. f. klin. Med., 1896, vols. xxviii and XXix. 218 THE GASTRIC JUICE AND GASTRIC CONTENTS 0.2 per cent.), hypochlorhydria, or the secretion of a deficient amount (less than 0.1 per cent.), hyperchlorhydria, in which more than 0.2 per cent. is found, and anachlorhydria, in which no hydrochloric acid at all is secreted. Euchlorhydria.—Kuchlorhydria, when associated with clinical symp- toms pointing to gastric derangement, is most commonly observed in gastric neuroses. A chronic gastritis can always be excluded in the presence of a normal amount of free acid. It may be associated with a certain degree of atony. It was formerly thought that a normal amount of ase would preclude the diagnosis of ulcer, but it is known that this association is quite possible. ‘The same is seen in pyloric stenosis due to a healed ulcer. Hypochlorhydria.—Hypochlorhydria is associated with all those diseases in which the secretory elements have been more or less damaged, as the result of general disease (anemia, chronic heart and renal lesions, phthisis, chronic icterus, many febrile diseases), or of local disease, as in subacute and chronic gastritis, in some cases of ulcer of the stomach or the duodenum, in incipient carcinoma, and in certain cases of dilatation and atony. ‘The withdrawal of chlorides from the food will also lead to a diminished production of hydrochloric acid. Anachlorhydria.— Not many years ago it was thought that the absence of free hydrochloric acid was pathognomonic of carcinoma of the stomach. ‘This view was soon abandoned, however, as it was shown that cases of carcinoma occur in which hydrochloric acid is not only present, but present in excessive amounts. ‘This 1s true especially of those cases in which the malignant growth has started’ upon the base of an old ulcer. It is noteworthy, moreover, that in early cases of carcinoma, even in the absence of ulcer, hydrochloric acid may at times be demonstrable and then disappear for days and weeks. It was furthermore shown that anachlorhydria exists in almost all cases of advanced chronic gastritis, in pernicious anemia (gastric anadeny), and is a fairly common occurrence in neurasthenie and hysterical individuals. In these cases periods of ana- hyper- and hypochlorhydria may alternate apparently without cause. In the acute febrile infections also anachlorhydria is not uncommon. Hyperchlorhydria. —Hyperchlorhydria (acid stomach, gastroxynsis) is very common in neurotic individuals, where it may alternate with hypo- and anachlorhydria. ‘The same is seen even normally during menstruation. Associated with a continuous hypersecretion of gastric Juice, it constitutes the neurosis known as hypersecretio acida et continua (gastrosuccorrhoea acida). Hyperchlorhydria is also of frequent occurrence in cases of gastric ulcer, and may even occur in carcinoma, notably in those cases in which, as stated above, the — new-growth has started from an old ulcer. Regarding the frequency of hyperchlorhydria i in uleus there can be no Adah ‘line this is found RR is Maa Gott «eee Reaction. LARS FON INTACT ON IRS HANES ERE RE EEE Theres NEARY + izarin < eal EH < _] A, Fig. 1.—Congo-red Test. Fig. 2.—Dimethyl Reaction. Fig. 3.—Al i SaaS SAMEERA AES statesmen mH 2 CHEMICAL EXAMINATION OF THE GASTRIC JUICE 919 in the majority of cases. Normal values, however, are by no means uncommon, and in some instances the amount of hydrochloric acid may be diminished. Hyperchlorhydria is also met with in passive congestion of the stomach (Schreiber’s so-called “stagnant stomach’), in certain types of mental disease, in the early stages of chronic gastritis, during ‘migraine attacks, etc. Test for Free Acids. The Congo-red Test.'—Congo-red is a car- mine-colored powder, while its solutions are of a peach- or brownish- red color, which changes to blue upon the addition of a free acid, but ‘remains unaffected in the presence of an acid salt. Congo-red may be employed in solution or in the form of a test paper. ‘The latter is less delicate than the solution, and indicates only the presence of 0.01 per cent. of hydrochloric acid, while a positive reaction can still be obtained with the aqueous solution in the presence of 0.0009 reent. ‘Ihe solution should be moderately dilute. ‘The test paper is prepared by soaking filter paper, free from ash, in this solution, drying, and cutting it ots suitable strips. In order to test for free vacid, it is only necessary to immerse a strip of the test paper in the filtered gastric juice, or to add a drop or two of the solution to a small amount of the juice, when in the presence of a free acid a blue color will develop, which varies from sky-blue to a deep azure according to the amount present. (Plate XII, Fig. 1.) If the result is positive, the nature of the free acid must be ascertained, and it is, therefore, ‘necessary to test for free hydrochloric acid, or in its absence for lactic acid and certain fatty acids. Tests for Free Hydrochloric Acid.—The various reagents which may be employed are given below, and are arranged according to their degree of delicacy, viz. : 1. Dimethyl-amido-azo-benzol . . . . . .:+ 0.02 pro mille. Seer niaromucin-vanillin ,; 9... ww ws ~~ 005 os RTT ere tober ot Yes A a Gr? 45 0.05 x Ree aA hs ee Ske yw - O30 PH Peete Tengen es a ese A ,00 . The Dimethyl-amido-azo-benzol Test..—This test has largely re- ‘placed the older phloroglucin-vanillin and resorcin tests in_ the routine work of the clinical laboratory. ‘The delicacy of the reagent is such that the natural yellow color of the indicator is changed to a | reddish tinge upon the addition of but 1 drop of a one- Penitii normal solution of “hydrochloric acid in 5 c.c. of distilled water. Its superior delicacy, as compared with the phlorogiicin-yanulin and. resorein tests, is apparent from the fact that 5 5 e.c. of a 0.5 per cent. solution ' Riegel, Deutsch. med. Woch., 1886, No. 35; and Boas, Diagnostik u. Thera- pie d. Magenkrankheiten. __ * Tépfer, Zeit. f. physiol. Chem., 1894, vol. xix. Hari, Arch. f- Verdauungs- krank., vol. ii, pp. 182 and 332. : 29.0 THE GASTRIC JUICE AND GASTRIC CONTENTS of egg albumen, to which 6 drops of a one-tenth normal solution of hydrochloric acid have been added, still give a positive reaction with dimethyl-amido-azo-benzol, while the phloroglucin-vanillin and resorcin reactions are negative. Organic acids, including lactie acid, yield a red color only when present in amounts exceeding 0.5 per cent. I have further ascertained that 7/f albumoses are present, a cherry-red color is not obtained even though lactic acid be present to the extent of 1 per cent. Loosely combined hydrochloric acid and salts do not produce a red color. For practical purposes a 0.5 per cent. alcoholic solution is em- ployed; 1 or 2 drops of this are added to a small quantity of the filtered gastric contents; in the presence of free hydrochloric acid a beautiful cherry red develops at once, which varies in intensity with the amount of free acid present (Plate XII, Fig 2.) In the presence of organic acids an orange color is obtained. In watery solution the color is a greenish yellow and the fluid is distinctly fluorescent. I have used 'Tépfer’s test for many years and am well satisfied with the results. In teaching students it is well to show the color which one obtains with lactic acid in the presence of albumoses; confusion as to whether or not free hydrochloric acid is present will then not occur. The Phloroglucin-vanillin Test.'—The solution employed con- tains 2 grams of phloroglucin and 1 gram of vanillin, dissolved in 30 c.c. of absolute alcohol; a yellow color results, which gradu- ally turns a dark golden red, changing to brown when exposed to hight. ‘The solution should therefore be kept in a dark-colored — bottle. Lenhartz Ese ia the use of separate solutions of phloro-_ glucin and vanillin, 1 or 2 drops of each being employed in the— test. Boas recommends a solution of the phloroglucin and vanillin, | in the proportions indicated in 100 grams of 80 per cent. alcohol, » and claims that the reagent is then still more sensitive and more. stable. If a few drops of gastric juice, or even of the unfiltered gastric contents, containing 0.05 per cent. or more of free hydro-— chloric acid, are treated with the same number of drops of the reagent, — no change in color results, but upon slow evaporation—boiling and rapid evaporation are to be avoided—a general rose tint or fine rose- | colored lines develop, which are characteristic of the presence af the free acid. | For practical purposes it is best to carry on this slow evaporation ) on a thin porcelain butter dish, the porcelain cover of a crucible, or | in a small evaporating dish of the same material. ‘The color obtained : in the presence of free hydrochloric acid is a rose color in every in- | stance, and varies in intensity with the amount of acid present. A | 1 Gunzburg, Centralbl. f. klin. Med., 1887, vol. viii, No 40. | CHEMICAL EXAMINATION OF THE GASTRIC JUICE 99] — brown, brownish-yellow, or brownish-red color always indicates that “excessive heat has been applied or that free hydrochloric acid is absent. Organic acids do not produce the reaction, nor is it interfered with by their presence, or that of albumins, peptones, or acid salts. A phloroglucin- -vanillin test paper, prepared by soaking strips of filter paper, free from ash, in the solution and drying ters may also be employed. If a strip of this is moistened with a drop of gastric juice and gently heated in a porcelain dish, the rose color will develop in the presence of free hydrochloric acid, and does not disappear upon the addition of ether. _ The Resorcin Test.'—The solution consists of 5 grams_ of -resublimed resorcin and 3 grams of cane sugar dissolved in 100 ‘grams of 94 per cent. alcohol. It is equally as delicate as the ge croglucin- -vanillin solution and has the advantage of goes: ‘stability: 5 or 6 drops of gastric juice are treated with 3 to 5 drops of the reagent and slowly evaporated to dryness over a small ‘flame, when a beautiful rose- or vermilion-red mirror will be obtained, which gradually fades on cooling. If the reagent is employed in the form of a test paper, a violet color at first develops, which upon ‘the application of heat turns brick red and does not disappear on treatment with ether. The presence of acid salts, organic acids, albumins, or albumoses does not interfere with the reaction. _ The Tropeolin Test..—'l'ropolin 00, when employed according to the method pes by Boas, is a very reliable reagent, indi- eating the presence of 0.2 to 0.3 pro mille of free hydrochloric acid : 3 or 4 drops of a saturated alcoholic solution of tropzolin 00, which has a brownish-yellow color, are placed in a small porcelain dish or cover, and allowed to spread over the surface. A like amount of gastric juice is added and likewise allowed to flow over the surface ‘of the dish; upon the application of gentle heat a beautiful lilac appears, which is said to be characteristic of free hydrochloric acid. A tropzolin test paper may also be prepared by soaking filter paper, free from ash, in the alcoholic solution, and then drying and ‘cutting it into strips. A few drops of gastric juice containing free “hydrochloric acid produce a more or less pronounced brown color upon this paper, which turns lilac or blue upon the application of ‘gentle heat. Organic acids, when present in large amounts, likewise produce a brown color, but this disappears on heating, and a lilac or blue color does not result. For ordinary purposes this test is sufficient, and recourse need only 1 Boas, Centralbl. f. klin. Med., 1888, vol. ix, No. 45. 4 Ewald, Kknik, d. Verdauungskrank., Berlin, 1888, vol. ii; and Boas, Deutsch. med, Woch., 1877, vol. xiii, p. 852. 9 222 THE GASTRIC JUICE AND GASTRIC CONTENTS be had to the more delicate reagents when a negative or a doubtful result is obtained. The Combined Hydrochloric Acid.—It has been pointed out else- where that hydrochloric acid will only appear in the free state after all basic affinities have been saturated. For this reason combined hydrochloric acid must of necessity be present after the administration of a test meal if free acid can be demonstrated. If the contents are withdrawn too early free acid will be absent, while hydrochloric acid in combined form may be present in normal amount, considering the stage of digestion. From the mere absence of free hydrochloric acid it 1s hence not justifiable to infer that no hydrochloric acid has been secreted. Under pathological conditions it may happen that while the stomach has lost the power to furnish a sufficient amount of hydrochloric acid to satisfy the albuminous affinities of a large meal and to subsequently appear in the free state, enough can be furnished to meet the demands of a small meal. In any case then, where free hydrochloric acid is not found, it is important to ascertain whether no hydrochloric acid at all has been secreted. ‘To this end the method of Martius and Liittke may be employed (see below). Quantitative Estimation of the Hydrochloric Acid of the Gas- tric Juice. Topfer’s Methoed.'—The free and combined hydrochloric acid is most conveniently estimated according to ‘T6pfer’s method, which is both simple and sufficiently accurate for clinical purposes. In this method the total acidity (a) of a given amount of gastrie juice—t. e., the acidity referable to the presence of free hydrochloric acid, combined hydrochloric acid, acid salts, and any organic acids that may be present—is first determined (lactic acid and the fatty acids, if present, need not be removed), using phenolphthalein as an indicator. ‘This is followed by a determination of the acidity refer- able to free acids and acid salts in another sample of gastric juice. (b), using alizarin (alizarm monosulphonate of sodium) as an indi eator. As this does not react with loosely combined hydrochloric acid, the difference between a and b will indicate the amount of the latter. ‘The free hydrochloric acid (c) finally is estimated with dimethyl-amido-azo-benzol as an indicator, the difference between a and b-+¢ giving the acidity referable to organic acids and acid salts. The solutions required are the following: 1. A decinormal solution of sodium hydrate. 2. A 1 per cent. alcoholic solution of phenolphthalein. 3. A saturated aqueous solution of alizarin. 4. A 0.5 per cent. alcoholic solution of dimethyl-amido-azo-benzol. Three separate portions of 5 or 10 c.c. of filtered gastric juice are measured into three small beakers or porcelain dishes. ‘To the first portion 1 or 2 drops of phenolphthalein are added, when it 1 Loc. cit. ' CHEMICAL EXAMINATION OF THE GASTRIC JUICE 923 { bis titrated with the one-tenth normal solution of sodium hydrate until a permanent pink color is obtained. To the second portion 3 or 4 drops of the alizarin solution are added, when it also is titrated with the one-tenth normal solution of sodium hydrate until a pure violet color is obtained (Piate XH, Fig. 3). In the third portion the free hydrochloric acid is titrated, after the addition of 3 or 4 drops of the dimethyl-amido-azo-benzol, until _ the last trace of red—in the presence of free hydrochloric acid—has disappeared, and the color has become distinctly greenish yellow (Plate XII, Fig. 2). The results are then calculated as in the follow- | ing example: ime 10 c.c. of gi astric juice, using phenolphthalein as .an indicator, re- quired 6 c.c. ok the one-tenth normal solution in order to bring about the end reaction, while a like amount titrated in the same manner with alizarin required 3 c.c. ‘The difference between 6 and 3 indicates / the number of cubic centimeters necessary to neutralize the amount of hydrochloric acid in combination with albuminous material. In the estimation of the free hydrochloric acid 2.3 ¢.c. of the one-tenth - normal solution were required. ' ‘The results can then be tabulated as follows: Total acidity (per 100 c.c. stomach contents) . . . . 60 Smee sewer ls ee a. 6S. 80 MunpinatrnvorocmMorgacid . iy Pe 8. ole 4 80 | REPU VRIFOG MOTIONS te Played, icy se ee 28 Total physiologically active hydrochloric acid. . . . 58 Salts in re etme ir Mh sl uy Le ce Te eee rs ow 8 60 __ If not enough gastric juice is available for three separate titrations one can estimate “the free hydrochloric acid in one portion of 5 c.c. _ with dimethyl as an indicator, and proceed at once to the total acidity in the same example. ‘lo this end phenolphthalein is added after He | primary titration and the titration continued for the total acidity a ‘usual. The first value will give the free hydrochloric acid and this _ plus the second value the total acidity. Deficit of Hydrochloric Acid.—W hen hydrochloric acid is absent ‘It is customary to indicate the deficit in terms of ,'5 hydrochloric /acid in a manner perfectly analogous to the method just now ' described, viz., 10 c.c. of gastric eg are treated with a few drops of dimethyl and then titrated with 2", hydroc eae acid until the red / hydrochloric acid reaction appe ars. a 1 ¢.c. was necessary to this ‘end the hydrochloric acid deficit would be 10. _ Estimation of Free Hydrochloric Acid (according to Sahli).—25 to 30 drops of Giinzburg’s reagent are added to 10 c.c. of gastric juice. 294 THE GASTRIC JUICE AND GASTRIC CONTENTS The mixture is titrated with a decinormal sodium hydrate solution as usual until a drop of the mixture, warmed on the stirring rod after each addition of the alkali, shows a red color. ‘The rod must be washed and cooled after every test. The Method of Martius and Luttke (modified).".—This method is equally exact, but requires a greater expenditure of time. It is based upon the fact that upon incineration of the gastric juice the free hydrochloric acid and that loosely combined with albuminous material escape, while the chlorine in combination with inorganic bases remains in the mineral ash unless a very intense heat 1s ap- plied for some time. By subtracting the amount of chlorine present in the latter form from the total Satan the quantity in combina- tion with albuminous material and that occurring as free acid will be found. ‘The total acidity of the gastric juice is then determined, and that referable to the presence of the free and combined hydro- chloric acid subtracted, the difference giving the amount of organic acids and acid salts. By determining the acidity due to the presence of free hydrochloric acid according to ‘Tépfer’s method, and deducting the amount found trom that eee abie to the presence of free and combined hydrochloric acid, the amount of the latter is obtained. Reagents required: 1. A solution of silver nitrate in nitric acid of such strength that 1 c.c. shall represent 0.00365 gram of hydrochloric acid. 2. Liquor ferri sulphurati oxydati. 3. A decinormal solution of ammonium sulphocyanide. 4. A one-tenth normal solution of sodium hydrate. 5. A | per cent. alcoholic solution of phenolphthalein. 6. A 0.5 per cent. alcoholic solution of dimethyl-amido-azo-benzol. Preparation of the solutions: | 1. The silver nitrate solution. As a solution is required of such strength that 1 c.c. shall be equivalent to 0.00365 gram of hydro aioe acid, the amount of silver nitrate that must he dissolved in- 1000 c.c. of water is ascertained in the following manner: Since 169.66 (molecular weight) parts by weight of nlyen nitrate combine with 36.5 parts of hydrochloric acid ( molecular weight), the amount. of silver nitrate required for each cubic centimeter is found from the equation 169.66 : 36.5 :: x : 0.00365; 36.5/2=0.6192590; x=0.0169. In 1 c.c. of the silver solution 0.0169 gram of silver nitrate must. thus be present, or 16.9 grams in the liter. ‘This quantity, or roughly | 17 grams, is weighed off and dissolved in 900 c.c. of a 25 per cent. solution of nitric acid. To this solution 50 ¢.c. of the liquor ferri | sulphurati oxydati are added, ‘The solution is then brought to the) ‘ Die Magensiiure des Menschen, Stuttgart, 1982. ® to = CHEMICAL EXAMINATION OF THE GASTRIC JUICE 995 proper strength by titration of a known number of cubic centi- meters of a one-tenth normal solution of hydrochloric acid and correcting as usual (see below). ‘The ammonium sulphocyanide solution. A normal solution of ammonium sulphocyanide contains 75.98 grams (molecular weight) per liter, and a decinormal solution 7.598 grams, ‘This quantity, or roughly 8 grams, is dissolved in ee 900 ¢.c. of w ater and the solution brought to the proper strength by titrating a known number of cubic centimeters of the silver tae solution, when ach cubic centimeter should correspond to 1 c¢.c. of the silver solution —. e., to 0.00365 gram of hydrochloric acid. It is corrected as described elsewhere (see below). Mernop.—1. ‘l’o determine the total amount of chlorine present: 10 c.c. of filtered gastric juice—Martius and Liittke make use of the unfiltered gastric contents—are measured into a small flask bearing a 100 c.c. mark, and treated with an excess of the one-tenth normal solution of silver nitrate. Experience has shown that 20 c.c. are sufficient. ‘The mixture is agitated and allowed to stand for ten minutes. Distilled water is then added to the 100 c.c. mark; the mixture is agitated once more and filtered through a dry filter into a dry beaker; 50 c.c. of the filtrate are titrated with the one- tenth normal solution of ammonium sulphocyanide until the blood- red color which appears upon the addition of every drop—due to the formation of ferric sulphocyanide—no longer disappears on stirring. By multiplying the number of cubic centimeters of the ammonium ‘sulphocy: anide solution used by 2 (the number of cubic centimeters that would have been necessary for the precipitation of the excess of silver in 100 c.c.) and deducting the result from the number of cubic centimeters of the one-tenth normal solution of silver nitrate employed, viz., 20, the number of cubic centimeters of the latter solution is found which was necessary to precipitate the chlorine in 10 ¢.c. of the gastric juice. As 1 c.c. of the solu- tion represents 0.00365 gram of hydrochloric acid, it is only necessary to multiply this figure by the number of cubic centimeters ased in precipitation of the chlorine. The resulting value, 7’, ex- oresses the total amount of chlorine present. As a general rule, it is not necessary to decolorize the gastric juice. If desired, however, 5 to 15 drops of a 5 per cent. solution of potassium permanganate may be added to the 10 c.c. employed, after the mixture has stood for ten minutes. _ 2. Determination of the amount of chlorine in combination with Morganic bases, /’: 10 c.c. of the filtered gastric juice are care- cully evaporated to dryness in a platinum crucible, on a water bath or upon a plate of asbestos, in order to avoid sputtering (as the teat applied in the process of incineration is not very intense, a oreclain crucible may also be employed). ‘The residue is then care- 15 996 THE GASTRIC JUICE AND GASTRIC CONTENTS fully incinerated over an open flame, the process being carried only to the point where the organic ash no longer burns with a luminous flame. Intense heat should be avoided, as the chlorides are volatil- ized upon the application of red heat. On cooling, the ash is moist- ened with a few drops of distilled water and mixed with a stirring rod, when the residue is extracted in separate portions with 100 c.¢, of hot distilled water and filtered. This amount is usually suffiel- ent to dissolve all the chlorides present. If any doubt should exist, however, it is only necessary to add a drop of the silver solution to a few drops of the last portion of the filtrate: the formation of a cloud, referable to silver chloride, will necessitate still further washing. ‘The whole filtrate is then treated with 10 c.c. of the one- tenth normal solution of silver nitrate, and the amount consumed in the precipitation of the chlorides determined by titration with the one-tenth normal solution of ammonium sulphocyanide, as de- scribed above. ‘The hydrochloric acid present in combination with inorganic bases is thus determined. ‘The difference between the amount of hydrochloric acid in combination with inorganic bases and the total amount of chlorine in terms of hydrochloric acid will then indicate the amounts of the free and of the combined hydro- chloric acid, which are termed L and C, respectively; hence 7—F =L+C. 3. The total acidity in terms of hydrochloric acid is further de- termined according to the method given elsewhere (see p. 220) and indicated by the letter A. ‘The difference between the total acid- ity and the amount of free and combined hydrochloric acid will represent the amount of organic acids and acid salts, O; henee O=A—(L+C). The free hydrochloric acid finally is determined according to the method of Tépfer. The difference between the value thus found and that expressing the amount of free and combined hydrochloric acid will indicate the amount of the latter; hence (L-|C)—L=C. Leo’s Method.\—This method is based upon the observation that calcium carbonate combines with free and combined hydrochloric acid: at ordinary temperatures to form neutral calcium chloride, while the acid phosphates are not affected. It is thus clear that by determin- ing the total acidity of the gastric juice, and deducting from this the acidity referable to acid salts, the amount of the physiologically active hydrochloric acid—t. e., of the free and combined hydrochloric acid—is obtained. | As it has been shown that in the presence of calcium chloride (formed, as indicated above, upon the addition of calcium carbonate), ‘owing to the formation of calcium monophosphate—CaHPO,, twice the quantity of sodium hydrate is taken up, it is necessary to make 1 Centralbl. f. d. med. Wiss., 1889, vol. xxvii, p. 481, CHEMICAL EXAMINATION OF THE GASTRIC JUICE 297 the first titration also after the addition of an excess of calcium chloride. Reagents required: 1. A one-tenth normal solution of sodium hydrate. 2. Al per cent. alcoholic solution of phenolphthalein. 3. A concentrated solution of calcium chloride. 4. Chemically pure calcium carbonate. The purity of the salt may be tested by stirring a small piece with water; the solution ‘should not color red litmus paper blue. A solution of the salt in ‘dilute hydrochloric acid should not yield a precipitate when treated ~with sulphuric acid. _ Mernop.—Organic acids that may be present are first removed by shaking with ether, 50 to 100 c.c. being required for each 10 c.c fof gastric juice. ‘The total acidity of the gastric juice is then de- termined in 10 c.c. of the filtered ‘liquid after the addition of 5 c.c. ‘of the concentrated solution of calcium chloride, the result being ,termed A. _ ‘The acidity referable to the presence of acid phosphates is deter- mmined as follows: 15 c.c. of filtered gastric juice are treated with a pinch of dry and chemically pure calcium carbonate; the mixture is jthoroughly stirred, and passed at once through a dry filter; 10 ec. of the filtrate, from which the carbon dioxide is expelled by means of a current of air, are then treated with 5 c.c. of the calcium chloride solution and titrated as above, the resulting value being termed P. A—P is hence equivalent to L -+C, The value of C can ithen be ascertained by determining the acidity referable to free hydrochloric acid according to Tépfer’ s method, and deducting the value found from L-+ Ge This method is sufficiently accurate for practical purposes, and has the advantage of not requiring the expenditure of much time. | | | The Ferments of the Gastric Juice and their Zymogens. | Normal gastric juice contains three ferments, viz., pepsin, chy- mosin, and lipase. _ Pepsin and Pepsinogen. —According to our present knowledge, the zymogen of pepsin, viz., pepsinogen or propepsin, and not pepsin itself, is secreted by the chief cells of the fundus glands. It is trans- formed into the ferment proper by the hydrochloric acid of the gastric juice. # _ This is not the place to enter into a detailed consideration of the various properties of pepsin, and it will suffice to say that the activity of the ferment is destroyed by even very dilute solutions of the alka- ine carbonates. ‘The same result is reached by exposing a watery solution of pepsin to a temperature of 70° C., ‘while in a dry state itl DIS THE GASTRIC JUICE AND GASTRIC CONTENTS a temperature of 100° C. will not destroy its activity; this is shown by the fact that a specimen of pepsin thus treated is, on cooling, still capable of digesting albumins in the presence of hydrochlorie acid. While pepsin is capable of digesting albumins in the presence of | other acids, viz., phosphoric, sulphuric, oxalic, acetic, lactic, and salicylic acids, the solutions must be stronger than in the case of. hydrochloric acid. With lactic acid, for example, a satisfactory | result is reached only with a concentration of from 12 to 18 pro mille, while of hydrochloric acid 2 to 4 pro mille are sufficient. Larger or | smaller amounts do not act so promptly. ! Figures expressing the exact quantity of pepsin or of its zymogen | are lacking, and inferences can hence only be drawn as to the physio- | logical activity of the ferment from the rapidity with which given | amounts of albuminous material are digested. ‘This, however, | depends to a large extent upon the nature and concentration of the’ free acid present. Under normal conditions 25 c.c. of gastric juice | | will dissolve 0.05 to 0.06 gram of serum albumin in one hour, the same amount of coagulated egg albumen in three hours, and a like | amount of fibrin in one hour and a half. | As abnormalities in the circulation and innervation of the stomach | apparently do not influence the production of pepsin, or rather of its | zymogen, a diminution in the degree of peptic activity, or its total absence, may be referred directly to disease of the stomach itself, | viz., its glandular apparatus. ‘The determination of the presence or absence and relative amount of pepsin in the gastric’ juice hence | furnishes more useful information than the recognition of the presence | or absence of free hydrochloric acid. | As pepsin is formed from pepsinogen through the agency of a free acid, its presence, in the absence of organic acids in notable quan | tities, indicates at once the presence of hydrochloric acid. It may | be said, vice versa, that if free hydrochloric acid is present in the | gastric juice pepsin also will be found. Should the zymogen alone be present, digestion will take place only upon the addition of an acid, | while an absence of digestion upon the addition of hydrochloric acid | | indicates the absence of both pepsin and its zymogen. At timelal though rarely, a “gastric juice” is met with which is capable of digesting albumin in the absence of hydrochloric acid, owing to the | presence of regurgitated pancreatic juice. | In the differential diagnosis of a chronic gastritis and a neurosis, | or a dyspeptic condition ‘referable to hyperemia of the gastric mucous, membrane, the demonstration of zymogen in the absence of hydro-| chloric acid may, at times, be very important, bearing in mind that. circulatory and nervous disturbances apparently do not influence’ the production of pepsinogen. An entire absence of the latter would, of course, warrant the diagnosis of anadeny of the stomach. | ee CHEMICAL EXAMINATION OF THE GASTRIC JUICE 999 Tests for Pepsin and Pepsinogen. ‘l‘est roR THE ENzymr.—lIf the presence of free hydrochloric acid has previously been ascertained, 25 c.c. of filtered gastric juice are set aside and kept at a tempera- ture of from 37° to 40° C, ., a bit of coagulated egg albumen, fibrin, or serum albumin being added. In order to permit of a comparison of results, the same amounts should always be taken; 0.05 to 0.06 gram of egg albumen, as has been shown, ought, ities physiolog- ‘ical conditions, to be digested in three hours. ‘Test ror tHe ZyMocEN.—Should hydrochloric acid be absent the test is made in the same manner, after the addition sh from 3 to ‘5 drops of the officinal solution of hydrochloric acid to 25 ¢.c. of the filtrate. Under such conditions usually pepsinogen alone is found. _ Quantitative Estimation of Pepsin Accurate methods for the quan- titative estimation of pepsin are unknown, and relative values only ‘can be obtained. _ Hammerschlag’s Method.'—T'wo Esbach tubes (albuminimeters) are employed. ‘lube 4 is filled to the mark U with a mixture of 10 ne. of al per cent. solution of egg albumen’ in 0.4 per cent. of hydro- shloric acid and 5 c.c. of filtered g ‘gastric juice. ‘The second tube, B, receives a mixture of the same solution and 5c.c. of water. After the cubes have been kept in the thermostat for one hour at a temperature of 37° C. Esbach’s reagent (see Urine) is added to each tube to the mark R. After standing for twenty-four hours the amount of precipi- ated albumen is read off in the two tubes. ‘The difference indicates he amount of albumen which was digested; this raised to the square tives the corresponding amount of pepsin (which of course is merely ‘elative). ‘The method suffices for practical purposes. | Mett’s Method.—Satisfactory comparative results can also be ob- ained with the method suggested by Mett. Capillary glass tubes wre prepared measuring from 1 to 2 mm. in diameter. ‘They are illed with white of egg, closed at the ends with breadcrumbs and soagulated in boiling water. After five minutes they are dried and the nds closed with melted paraffin. In this form they can be kept, vat before use they should be examined to see that the column of Jbumen has not shrunk from the sides. Any bubbles that may be yresent disappear after two days. When needed they are cut into vieces from 1 to 2 cm. long. The length of the column digested in a ‘iven length of time serves as a measure of the digestive power of the pecimen examined. In practice this column should be measured in aillimeters with the aid of a magnifying glass, or a low power of the 4icroscope, using a stage micrometer. The calculation of the corre- ponding amount of ferment is based upon the law of Schiitz and Jorrissow, viz., that the corresponding amounts of ferment in two olutions bear the same ratio toward each other as the square of the | ‘Wien. med. Presse, 1894, vol. xxxv, p. 1654. * The white of one egg diluted about 13 times will make a 1 per cent. solution. 230 THE GASTRIC JUICE AND GASTRIC CONTENTS number of millimeters of the column of egg albumen which has been dissolved in the same length of time. Nirenstein and Schiff’ have ascertained that the length of the digested cylinder of albumen is proportionate to the length of time that digestion goes on, pro- viding that the length of the cylinder does not exceed 7 mm. If it does exceed this, digestion proceeds more slowly. It is hence ad- visable in all cases to dilute the gastric juice. In this manner another difficulty also is obviated, viz., the antipeptic activity which is caused by certain substances which are normally present in solution (prod- ucts of digestion, sodium chloride). Nirenstein and Schiff ascer- tained that a sixteenfold dilution with ,%, HCl (0.18 per cent.) is sufficient and that this prevents the digestion of more than 3.6 mm. in twenty-four hours, which is a further condition to ensure reliable results. ’ Mernop.—The gastric juice is obtained after giving Ewald’s test breakfast. 1 c.c. of the filtered contents is diluted with 16 c.c. of 3, HCI; into this solution 4 Mett’s tubes are placed and the mixture is kept in the incubator for twenty-four hours. ‘The columns of digested albumen are measured and the average ascertained; this in terms of millimeters raised to the square and multiplied by ‘16 (the degree of dilution) indicates the relative amount of pepsin. If the digested | column measures more than 3.6 mm. the gastric juice must be diluted | thirty-two times. : The unit of measure is the amount of pepsin by which 1 mm. | of albumen is digested in twenty-four hours, with an acidity of 0.18 per cent. HCl. Nirenstein and Schiff in their series found variations from 0 to 256 pepsin units. Quantitative Estimation of Pepsinogen.—In order to estimate the amount of pepsinogen both Hammerschlag’s and Mett’s method can | be applied after rendering the gastric contents acid with hydrochloric acid to the extent of from 1 to 2 pro mille. The Milk-curdling Ferment and its Zymogen, viz., Chymosin (Rennin) and Chymosinogen.—The specific action of chymosin is exerted upon milk, or lime-containing solutions of casein, which are coagulated in neutral or feebly alk shee solutions. In this connection it is important to note that the addition of a few cubic centimeters of a solution of calcium chloride, or any other soluble lime salt, results in a transformation of the zymogen into the physiologically active ferment, and that hydrochloric acid, while it normally causes such transformation, is not absolutely necessary in) the presence of calcium chloride. Under physiological conditions chymosin and its zymogen are always present in the gastric juice. In disease the inferences that may be drawn from a quantitative estimation of the ferment and its) 1 Arch. f. Verdauungsk., 1903, vol. viii. CHEMICAL EXAMINATION OF THE GASTRIC JUICE 93] zymogen have been formulated by Boas,’ to whom we are indebted ie much valuable information in this connection: 1. Notwithstanding the absence of free hydrochloric acid, chymo- sin may be present, although i in minimal traces—i. ¢., demonstrable with a dilution of from 1 to 10 to 1 to 20 (see method below). 2. In the absence of free hydrochloric acid the zymogen may still be present in normal amounts—t. e., demonstrable with a dilution of from 1 to 100 to 1 to 150. he presence of the zymogen, especially when repeatedly observed, probably always permits of the conclusion that we are not dealing with an organic disease of the stomach, but with a neurosis or a hyperemic condition of the mucous membrane referable to disease of other organs. 3. ‘The zymogen may occur in moderately diminished amount, 50 per cent. only being present. ‘This is usually owing to the existence of a gastritis which has not reached its highest degree of severity. The nearer the amount of zymogen approaches the normal, the greater will be the probability of an ultimate recovery under suit- _ able treatment. 4. ‘The amount of the zymogen is greatly diminished (dilutions of 1to 10 to 1 to 25 yielding a negative result) or may be absent alto- gether. In cases of this kind a severe and usually incurable gas- | tritis exists, either primary or occurring secondarily to carcinoma, amyloid degeneration, ete. 5. In conditions 1, 2 and 3, the reéstablishment of the secretion _ of hydrochloric acid may be attempted with some prospect of success _ by means of stimulating remedies. These conclusions are based upon the employment of Ewald’s _test breakfast, and cannot be applied to observations made after | other test meals, without previous studies in this direction. _ ‘Vesting for the presence of chymosin and its zymogen is of decided _yalue in cases in which alkaline material is vomited, and where we jmay be called upon to decide whether this contains constituents of the gastric juice or not. , Tests for Chymosin and Chymosinogen. ‘lest FoR THE ENZYME.— 5 to 10 e.c. of milk are treated with 3 to 5 drops of the filtered gastric juice and kept at a temperature of 37° to 40° C. for ten to ‘fifteen minutes. If coagulation occurs during this time, it may be _ concluded that the enzyme is present. ‘Test ror THE ZymMoGEN.—The milk is treated with 10 c.c. of _ the filtered and feebly alkalinized gastric juice and with 2 or 3 c.c of a 1 per cent. solution of calcium chloride. The mixture is kept _at a temperature of from 37° to 40° C., when in the: presence of _ the zymogen the formation of a thick cake of casein will occur within ten to fifteen minutes. k | ' Centralbl. f. d. med. Wiss., 1887, vol. xxv, p. 417; and Zeit f. klin. Med., 1888, } a 1 ee. f » Ez vol. xiv, p. 240. See also J. Friedenwald, Med. News, 1895. | ‘i Diy THE GASTRIC JUICE AND GASTRIC CONTENTS Quantitative Estimation. Or THE ENzymE.—The method is based upon the fact that on gradually diluting a specimen of gastric juice a point is finally reached at which a chymosin reaction can no longer be obtained, the value being, of course, a relative one. Under physiological conditions a positive reaction can still be observed with a degree of dilution varying between 1 to 30 and 1 to 40. The gastric juice is neutralized with a very dilute solution of sodium hydrate. ‘Tubes are then prepared containing from 5 to 10 c.c. of the gastric juice, diluted in the proportion of 1 to 10, 1 to 20, 1 to 30, ete., to which an equal amount of neutral or amphoteric milk is added. ‘The tubes, properly labelled, are kept at a temperature of from 37° to 40° C., and the degree of dilution noted at which coagulation still occurs. | Or THE ZyMoGEN.—The gastric juice is rendered feebly alkaline and tubes are prepared containing equal amounts of milk and gastric juice, the latter variously diluted, as above directed; the examina- tion is then carried on in the same manner. Normally a positive reaction is obtained with a dilution varying between 1 to 150 and 1 to 100. Lipase—The presence of lipase as a normal constituent of the gastric juice has now been definitely established. Its demonstra- tion and quantitative estimation are described in the section on the Urine. It is essential that the examination be made after a thorough washing of the stomach and the administration of a test — meal which is free from fat. Analysis of the Products of Albuminous Digestion. In order to separate the various products of digestion from each other the following procedure may be employed: The filtered gastric contents are carefully neutralized with a dilute solution of sodium hydrate, using litmus paper to determine the re- action; a small drop of the mixture is placed upon the paper from time to time during the addition of the sodium hydrate until no change in color is produced either on the red or the blue paper. If syntonin is present, it will be precipitated, and can be collected on a small filter. Upon the addition of an excess of dilute acid or an alkali this precipitate will again be dissolved. ‘The filtrate is feebly acidified with dilute acetic acid, treated with an equal volume of a saturated solution of common salt, and brought to the boiling point. Any native albumin that may be present in solution is thus coagulated and can be filtered off on cooling. In the filtrate the albumoses and peptones remain. By one-half saturation of the filtrate with ammonium sulphate, viz., by adding an equal amount of a saturated solution of ammonium = CHEMICAL EXAMINATION OF THE GASTRIC JUICE 933 sulphate, the primary albumoses can be precipitated. If then the neutral filtrate is treated with one-half its \olume of a saturated solution of ammonium sulphate, which will thus give a two-third total saturation, a portion of the deutero-albumoses (fraction A) separates out on standing. ‘This is filtered off and the solution saturated with ammonium sulphate in substance; the deutero- fraction B is thus thrown down, and on acidifying the filtrate with one-tenth of its volume of a solution of sulphuric acid that has been saturated with ammonium sulphate, and of which 10 c.c. correspond in strength to 17 c.c. of a ;’, solution of sodium hydrate, the last traces of deutero-albumoses (fraction C) will separate out on standing. The filtrate contains the ‘‘peptones.” ‘l’o demonstrate these a 2 per cent. solution of cupric sulphate is added drop by drop, when in the presence of peptones a rose- to a purplish-red color will develop.’ Tests for the Products of Carbohydrate Digestion. Starch may be recognized by the fact that it strikes a blue color with a solution of iodopotassic iodide, while the same solution gives a violet or mahogany brown with erythrodextrin. ‘To this end it is only necessary to add a drop or two of Lugol’s solution to a few eubic centimeters of the filtered gastric juice. ‘The presence of achroddextrin may be inferred if no change in color occurs upon the addition of the reagent. | Maltose and dextrose, which both react with Fehling’s solution ‘and undergo fermentation, differ from each other in the fact that the former does not reduce Barfoed’s reagent on boiling. ‘his is prepared by adding 1 per cent. of acetic acid to a 0.5 to 4 per cent. solution of cupric acetate. ‘The rotatory power of maltose is about three times as strong as that of dextrose; (2) D=150.4, as com- ‘pared with 52.5. / Lactic Acid. Mode of Formation and Clinical Significance.—The normal occurrence of lactic acid in the stomach during digestion was until recently regarded as an established fact and generally ascribed to the action of lactic acid producing organisms which had been swal- lowed and which could exercise their activity so long as hydrochloric acid did not appear in the free state. __ Martius and Liittke, however, employing the method already described, found “that the accurately determined curve of acidity __? For a more detailed account of the chemistry of digestion and the analysis of the resulting products, see C. E. Simon, Physiological Chemistry, Lea Bros. & Co. 934 THE GAS’RIC JUICE AND GASTRIC CONTENTS a referable to hydrochloric acid coincided in all respects, even at the beginning of the process of digestion, with the curve referable to the total acidity, ,’ so that lactic acid as a physiological constituent could not have been present. ‘I'he researches of Boas,’ moreover, prove beyond a doubt that in physiological conditions no appreciable amounts of lactic acid are formed during the process of digestion, and that the lactic acid found after an ordinary meal has been introduced into the stomach as such. It is known that lactic acid is present in various kinds of bread and it is, hence, not permissible to make use | of any test meal containing lactic acid when the question as to its formation in the stomach is to be considered. For these reasons Boas suggests the use of simple oatmeal soup to which salt only has been added. For practical purposes this is probably not always necessary, as the small amount of lactic acid found after Ewald’s test breakfast may usually be disregarded; an increased amount can be referred directly to pathological conditions. The fact that the lactic acid disappears or is at least no longer demonstrable at the height of digestion may be due to its resorption on the one hand, or to an interference of the hydrochloric acid with the delicacy of the reagent usually employed—. e., Uffelmann’s reagent—on the other. Under pathological conditions notable amounts (1 to 4 pro mille) of lactic acid are met with when stagnation of the gastric contents occurs as a result of motor insufficiency, in the absence of or with a diminished secretion of hydrochloric acid. It is hence a common symptom of carcinoma of the stomach.’ It was indeed at one time thought that carcinoma was the only disease in which a notable lactic acid production took place, but experience has shown that the same may occur in benign cases of pyloric stenosis and gastric in- sufficiency. Such findings, however, are uncommon, and a high lactic acid value may still be regarded as strongly suggestive of malignant disease and especially when repeatedly observed. Karly in the disease it appears that periods of chlorhydria and lactic acid production may alternate and it is desirable that this phase of the problem more particularly receive attention. In cases in which carcinoma has developed upon the basis of an old ulcer, lactic acid may be absent and hydrochloric acid present in increased amount: In every case in which lactic acid is found the stomach should be — thoroughly washed out in the evening and no food allowed until the fol- J ' “Ueber d. Vorkommen vy. Milchsiure im gesunden u. kranken Magen,” Zeit. f. klin. Med., 1894, vol. xxv, p. 285. : 2 J. H.de Jong, “ Der Nachweis d. Milchsiiure u. ihre klinische Bedeutung,” Arch. f. Verdauungskrank., vol. ii, p. 53. J. Friedenwald, “The Significance of the Presence of Lactic Acid in the Stomach, ” N. Y. Med. Jour., 1895. Rosenheim u. Richter, “ Ueber Milchsiiurebildung im Magen,” Zeit. f. klin. Med., vol. xxviii, p. 505. Paleo lemes) LT: Mgnt ad Ate eer Kelling’s Test for Lactic Acid. LIBRARY eFTHE UNIVERSITY « "LLINOIS. > ene re —* s Ss “4 * CHEMICAL EXAMINATION OF THE GASTRIC JUICE 935 lowing morning. Boas’ test meal is then given and the examination repeated. If the presence of lactic acid can Shire be established on. re- peated examination, even if a normal condition or hy perc hlorhydri la can be demonstrated in the interval, an exploratory incision is justifiable. It should, finally, be mentioned that only that form of lactic acid which results from fermentative processes is of interest in this con- nection, and not the sarcolactic acid contained in meat. For this reason the demonstration of lactic acid after a meal of meat is of no diagnostic significance, so far as the question of carcinoma goes. | Kelling’s Method! (Author’s Modification).—'his test is best per- formed in the following manner: A test tubeful of water receives a drop or two of a moderately strong solution of the sesquichloride of iron, so that the liquid is barely colored. One half is then poured ‘imto a second tube and serves as control. A small amount of the gastric filtrate is added to the other specimen, when in the presence of lactic acid a distinct yellow develops at once, which appears the ‘more marked when compared with the nearly colorless control. This test is very delicate and to be preferred to the older method of Uffelmann. (Plate XIII). _ Uffelmann’s Test .’—Heretofore Uffelmann’s reagent was quite com- monly employed in testing for lactic acid, but everyone who has had occasion to make frequent use of this reagent in clinical work must have been struck with the uncertainty of the results so often obtained. In a large majority of the cases, particularly if Ewald’s test breakfast is employed, a characteristic reaction—. e., the occur- rence of a lemon or canary-yellow color—is not seen, notwithstanding ‘the presence of lactic acid, but a pale yellow, brownish, erayish white, or even gray color is ehiained instead, often leaving in doubt ‘whether lactic acid is present or not. Aside from doubtful results, ‘the value of the test is greatly diminished by the fact that glucose, acid phosphates, butyric acid, and alcohol give the same reaction, and that in the presence of such amounts of hydrochloric acid as are found at the height of normal digestion lactic acid is not indicated by the reagent. All these difficulties have long been appreciated, and in order to obviate at least some of them it was proposed to apply the test to an aqueous solution of the ethereal extract of the gastric contents: To this end 5 or 10 c.c. of the filtered gastric juice are extracted by shaking with from 50 to 100 c.c. of neutral sulphuric ether* in a ' “Rhodan im Mageninhalt; Zugleich ein Beitrag z. Uffelmann’schen Milch- ‘Sdurereagens,”’ Zeit. f. physiol. Chem., vol. xviil. ? Deutsch. Arch. f. klin Med., 1880, vol. xxvi; and Zeit. f. klin. Med., vol. Vili, p. 392. * Tf lactic acid is not present in the free state, but in combination with albumin (i. e., if the Congo-red test is negative), it is necessary to set it free by adding dilute hydrochloric acid until the Congo test is just positive, as the ether will ‘otherwise not extract it. 936 THE GASTRIC JUICE AND GASTRIC CONTENTS stoppered separating funnel for about twenty or thirty minutes; the ethereal extract is then evaporated on a water bath or the ether distilled off (no flame). ‘The residue is taken up with from 5 to 10 c.c. of distilled water and tested as follows: 3 drops of a saturated aqueous solution of ferric chloride are mixed with 3 drops of a concentrated solution of pure carbolic acid and diluted with water until an amethyst-blue color is obtained; to this solution a portion of the ethereal extract is added, when in the presence of only 0.1 per cent. of lactic acid a lemon or canary-yellow color is obtained. Strauss’ Method.'—Instead of evaporating the ether as in the above method, the ethereal extract may be directly examined by shaking with a freshly prepared solu- tion of ferric chloride, as suggested by Fleischer. Making use of this principle, Strauss has con- structed an apparatus (Fig. 67) which will be found very convenient, and which permits of roughly. determining the amount of lactic acid present. ‘lhe instrument is essentially a sepa- rating funnel of 30 c.c. capacity, bearing two marks, of which the one corresponds to 5: ¢.c., the other to 25 c.c. ‘The apparatus is filled with gastric juice to the mark 5, when ether (free from alcohol) is added to the 25 ce. line. After shaking thoroughly, the separated liquids are allowed to escape by opening the stopcock until the 5 c.e. mark is reached. Distilled water is then added to the 25 mark, and the mixture treated with 2 drops of the officinal tincture of ferric chloride, diluted in the proportion of 1 to 10. Upon shaking, the water will assume an intensely green color if more than 1 pro mille of lactic acid is present, while a pale green is obtained in the presence of from 0.5 to 1 pro mille. ‘The tincture of iron should be kept in a dark-colored dropping bottle of about 50 c.e. capacity. f * It will be observed that only large amounts of Fie. 67.—Strauss : : : : apparatus for the ap- lactic acid, which alone are of importance from Hon of lactic acid. & diagnostic point of view, are indicated by the apparatus. Small amounts, as those introduced with Ewald’s test breakfast, or referable to lactic acid fermentation in the mouth, are not indicated, so that confusion as to the presence or absence of the acid can never arise. Vournaso’s Method (Modification of Croner and Conheim).— The method has the advantage that extraction with ether is not ' “Ueber eine Modifikation d. Uffelmann’schen Reaktion,” Berlin. klin. Woch., 1895, No. 37. CHEMICAL EXAMINATION OF THE GASTRIC JUICE 937 necessary. It is based upon the formation of an isonitril on trans- forming lactic acid to iodoform and treating with an amino base. ‘The isonitril is readily recognized by its disagree sable odor. 2 grams of potassium iodide are dissolved in a few (not more than 5) c.c. of water and | gram of sublimed, pulverized iodine added. ‘The resultant solution is filtered through asbestos or glass wool and diluted to 50 ec. with distilled water; 5 ¢.c. of aniline are fin: ully added. ‘The reagent lis kept in a dark-colored bottle and must be shaken before using’; It ‘keeps for a number of months. < 1100 diameters, (Park.) conditions, on the other hand, as also in the stools of children which have been fed with cows’ milk, their number is found diminished, while the members of the coli group enter into the foreground. Beyond the stools the bacillus has been found in the outer portion of the secretory duct of the human mammary gland, in the milk, and the skin of the nipple and its immediate surroundings. It is apparently not pathogenic. The organism occurs,in the form of slight rods measuring 1.5 #4 to 2 » in length, by 0.6 » to 0.9 4 in breadth. It is non-motile. It is not decolorized by Gram’s method, but loses this property after from thirty-six hours to nine days. ‘The best growths are obtained on beer-wort bouillon and common bouillon when acidified with a mineral acid; the acidity of 10 c.c. of the medium may correspond to 10 c.c. of a decinormal solution of potassium hydrate. ‘The optimum temperature is 37° C.; between 20° C. and 22° C. no growth | 1 “Hin Beitrag zur Kenntniss der normalen Darmbacterien des Singlings,” Jahrbuch f, Kinderheilk., vol. lii. Also: “ Ueber die nach Gram farbbaren Bacillen d, Sauglingstuhles,”? Wien. klin. Woch., 1900, No. 5. BACTERIOLOGY OF THE FECES 281 occurs. On the various agar slants imperfect development takes place; on potato the organism does not grow. It is an active acid producer, but does not give rise to the formation of gas; with Escherich’s stain it is colored blue. Escherich’s Stain.—This stain is now extensively used by pediat- rists in order to ascertain any deviations from the normal in the flora of the feces. Under strictly normal conditions the bacilli which are found in the stools of breast-fed children are thus nearly all colored blue (these are essentially the anaerobic Bacillus bifidus communis, and the aérobic Bacillus acidophilus), while red bacilli (Bacillus coli communis and Bacillus lactis aerogenes) are but little numerous. In the case of infants, on the other hand, which are fed exculsively on cows’ milk, the red bacilli predominate, while in mixed feeding the blue enter into the foreground in about the proportion in which breast milk is employed. ‘The red bacilli belong to the coli group. ‘These further predominate, or may be found exclusively, if for any reason intestinal digestion is impaired. Staphylococci, streptococci, ete., when simultaneously present, are in either event stained blue. In staphylococcus enteritis the blue bacilli which normally exist in the stools of breast-fed infants are almost entirely replaced by staphylo- cocci. At the beginning of the enteritis they are not numerous, but they increase during the progress of the disease, and finally disappear when the child recovers. In staining, the following solutions are employed: 1. An aqueous solution of gentian violet (5 to 200). ‘This is boiled *for one-half hour and is then filtered; it keeps for a long time. 2. A mixture containing 11 parts of absolute alcohol and 3 parts of oil of anilin. 1 and 2 are mixed in the proportion of 8.5 to 1.5; the resulting solution keeps for from two to. three weeks, but not longer. 3. A solution of iodopotassic iodide containing | part of iodine and 2 parts of potassium iodide in 60 parts of water. 4. A mixture of equal parts of oil of aniline and xylol. 5. A concentrated alcoholic solution of fuchsin, diluted with an equal volume of absolute alcohol. A bit of the stool is spread upon a slide in as thin a layer as possible. After drying in the air the specimen is fixed by passing through the flame of a Bunsen burner. It is then stained for a few seconds with the mixture of 1 and 2, blotted, placed in the iodine solution for a few seconds, blotted again, decolorized with 4 until a notable extraction of color no longer occurs. It is washed with xylol, dried, and finally stained for a few seconds with the fuchsin solution, washed with water, blotted, and is then ready for examination. Bacillus (Proteus) vulgaris, Hauser.—This organism, while usually regarded as non-pathogenic, should be numbered among the bacteria which may at times develop pathogenic properties. Baginsky and Booker have frequently found it in the stools in cases of infantile 989, THE FECES summer diarrhea. Escherich observed it at times in the meconium. Brudzinski examined the dyspeptic and fetid stools of a number of artificially fed infants in Escherich’s clinic, and in all the cases found the proteus. Others have encountered it in inflammatory conditions of exposed surfaces, in appendicitis, in perforative peri- tonitis, and even in closed abscesses, either alone or in association ° with other bacteria (Welch). A mixed infection of the proteus with Loffler’s bacillus has also been observed. ‘The organism forms rods, measuring about 0.25 y in diameter, while their length is variable; at times a more roundish form is observed; at others rods measuring from 1.25 » to 3.75 yw in length, or even long threads. ‘They are readily stained, but are easily decolorized by alcohol or Gram’s method. Most characteristic is their growth upon nutrient gelatin. At the temperature of the room little depressions will be observed after six to eight hours, which are surrounded by a narrow zone of bacilli from which a thin, wide film, provided with irregular projections, extends over the culture medium. From this film islets become separated, which slowly extend over the gelatin and cause its lique- faction. ‘The organism is motile. It decomposes urea and causes albuminous putrefaction. ‘The nitroso-indol reaction is readily ob- tained in bouillon cultures. In boiled milk the organism grows well, while in fresh milk it develops only irregularly, and in acid milk no growth takes place at all. Bacillus pyocyaneus.—The Bacillus pyocyaneus has repeatedly been isolated from the stools of dysenteric patients, and has been proved the cause of several epidemics. ‘The organism in question is a small * motile bacillus measuring from 1 y to 2 v in length by 0.3 » to 0.5 p in breadth. It sometimes occurs in short chains, but is usually single. It is stained with the common aniline dyes, and is decolorized with Gram’s method. It grows on the usual culture media, and liquefies gelatin. In 2 per cent. glucose bouillon no fermentation takes place. Litmus milk is curdled in about forty-eight hours. Some varieties produce indol. Most characteristic is the production of certain pigments, viz., pyocyanin and a fluorescent, bluish-green pigment which is common to almost all varieties.’ The Bacillus coli communis,” while constantly present in normal feces, 1s described at this place, as modern investigations have shown that it may at times develop pathogenic properties. It has been found in the pus in cases of purulent perforating peritonitis, angio- cholitis, pyelonephritis, etc.; it is frequently found infecting the bladder and the pelvis of the kidney, and, as indicated elsewhere, at times forms the nucleus of gallstones. It occurs in the form of deli- cate or coarse rods, measuring about 0.4 yz in length, which manifest, a certain degree of motility, due to the presence of one or two polar ‘A.J. Lartigau, “ A Contribution to the Study of the Pathogenesis of the Bacillus Pyocyaneus,” etc., Jour. Exper. Med., 1898, No. 6. 2 Flugge, Die Microdrganismen. re | — BACTERIOLOGY OF THE FECES 283 — flagella. ‘Ihe organism is stained by the usual aniline dyes, and is Be lorized by Gone s method. ‘The colonies upon celatin closely resemble those of the bacillus of typhoid fever, forming small whitish specks im the gelatin, and delicate films with serrated borders wpon the same medium, which, moreover, is not liquefied. On potato the organism forms a brownish pellicle, while the growth of the typhoid bacillus is nearly transparent. As in the case of the cholera bacillus, the nitroso-indol reaction can be obtained when the organism is grown upon peptone-containing media.’ In solutions of glucose active fermentation takes place. Litmus milk is rendered acid and is coagulated. Important also is the behavior of the organism when grown on gelatin or agar that has been colored with neutral red; in contradistinction to the typhoid bacillus, the colon bacillus then causes an intense green fluorescence. The Bacillus lactis aerogenes (Escherich) closely resembles the organism just described, and may also at times develop pathogenic properties. It is seen quite constantly in the stools of sucklings, but may also be met with in those of adults. It occurs in the form of rather stout rods, which frequently lie in pairs, resembling diplococci. The organism is non-motile. Like the Bacillus coli communis, it is decolorized by Gram’s method. In plate cultures it forms a dense white film; in stab cultures a chain of white colonies resembling beads fo) is seen. In the latter, moreover, if the stab is closed, bubbles of gas will be seen to form, which rapidly increase in number and size. Milk is coagulated in large lumps in twenty-four hours; at the same time the formation of gas 1s much more intense than in the case of the Bacillus coli communis. The Comma Bacillus—The first detailed studies of the organisms found in cholera stools were made in 1883 by the members of the French and German commissions sent to Egypt to investigate the nature of the dreaded disease. ‘The results of their work were first published by Koch in his report to the Berlin Sanitary Office in 1883, and in 1884 by Strauss, Roux, Nocard, and Thuillier. The clinical recognition of cholera Asiatica has now become a simple matter since Pfeiffer has demonstrated that the blood serum of cholera patients possesses the property of causing arrest of motility and agglutination of the specific bacilli. Ordinary bouillon cul- tures, however, can usually not be employed, as particles of the film when broken up may easily be mistaken for agglutinated bacilli. It is best in every case to make use of agar cultures sixteen to twenty- four hours old, and to prepare emulsions in bouillon or normal salt solution as occasion requires. ‘lhe emulsion, moreover, should always be examined microscopically before use, so as to ensure the absence of ' The test for indol is very conveniently made by adding a few drops of Ehr- lich’s dimethyl- amino- -benzaldehyde solution (see Urine) to a culture of the organism in Dunham’s solution which has grown for four or five days. On shaking, and especially on heating, a cherry-red color develops. 284 THE FECES any conglomeration of bacilli. The blood is then diluted in the pro- portion of 1 to 10 or 1 to 15. If the test-tube method is employed, the’ tubes should be kept in the incubator (37° C.) for only one or two hours. Upon the slide the reaction is obtained in from five to twenty minutes. If no distinct agglutination is observed at the end of one hour, the diagnosis of cholera is rendered improbable. Dried blood retains its agglutinating properties for a considerable length of time, and may also be used for examination. The comma bacillus is a slightly arched or half-moon-shaped little rod, and is somewhat shorter than the tubercle bacillus (Fig. 78). Occasionally two are placed end to end with their convexities in opposite directions, thus presenting the appearance of the letter S. They are provided with flagella. Koch detected these bacilli in the intestinal contents and feces, but rarely in the vomited matter, in Asiatic cholera only. In the stools they at times occur in such Fic. 78.—Cholera spirilla preparation from gelatin-plate culture of cholera. < 800 diameters. (Park.) numbers as to constitute pure cultures. In plate cultures kept at a temperature of 22° C. white colonies with serrated borders may be observed after twenty-four hours. ‘lhe color of such a colony is slightly yellow or rose red, its central portion gradually assuming a deeper tint, and finally becoming liquefied. Upon agar plates the bacilli form a grayish-yellow, irregular, slimy coating, but do not liquefy the culture medium. In stab cultures, after twenty-four hours, a whitish color may be observed along the line of the stab; around this there is found a funnel-shaped depression, which gradually increases in size and apparently contains a bubble of gas. ‘The upper» portion of the culture medium at the same time becomes liquefied while the lower portion remains solid for days. In asuspended drop spirochete-like spirals are observed at the margins, which often pre-» sent as many as twenty distinct arches' 1-R. Koch, Berlin. klin. Woch., 1884, vol. xxi, pp. 477, 493, 509. i ANIMAL PARASITOLOGY OF THE FECES I85 Closely related to Koch’s comma bacillus is the bacillus of Finkler and Prior,’ discovered in 1884 and 1885. It is distinguished from the former by the following characteristics: it is larger and thicker than the comma bacillus; the colonies on gelatin plate cultures show equally round and sharp-edged forms, which present a granular appearance under a low or medium power, and are usually of a brown color. ‘The organism liquefies gelatin very rapidly, a penetrating, excessively fetid odor being developed at the same time. In stab cultures the bacillus of cholera Asiatica forms a funnel-shaped depres- sion, while the bacillus of Finkler and Prior forms a stocking-like depression. | Tubercle bacilli, when present in the feces, are indicative of intes- tinal tuberculosis, providing they are observed upon repeated exami- nation and there are clinical symptoms pointing to the bowels as the seat of the disease; otherwise they may be referable to swallowed sputa. ‘They may be demonstrated as described in the chapter on Sputum. , ANIMAL PARASITOLOGY OF THE FECES. The animal parasites which may be met with in the feces are classi- fied as follows: I.—Protozoa: 1. Rhizopoda, Monera, Ameoebina: Amceba coli, 2. Sporozoa, S. gregarina, Coccidia, 3. Infusoria, a. Ciliata, Holotricha: Balantidium coli. b. Flagellata. Monadina, Cercomonadina: Cercomonas. Isomastigoda. Tetramitina: Trichomonas. Polymastigina: Megastoma. II.—Vermes: Platodes, Cestodes, Teenia saginata. Teenia solium, Teenia nana. Teenia diminuta. Teenia cucumerina, Bothriocephalus latus. Krabbea grandis. 1 Finkler, Deutsch. med. Woch., Tageblatt der Naturforscherversammlung, 1884, vol. x, p. 36, and 1885, p. 438. Finkler u. Prior, Erginzungsheft z. Centralbl. f. allg. Gesundheitspflege, 1885, vol. i. 286 THE FECES Trematodes, Distoma hepaticum. Distoma lanceolatum. Distoma Buskii. Distoma sibiricum. Distoma spatulatum. Distoma conjunctum. Distoma heterophyes. Amphistoma hominis. Distoma hzematobium. Distoma pulmonale. Annelides, Nematodes, od Ascarides, Ascaris lumbricoides. Ascaris mystax. Ascaris maritima. Oxyuris vermicularis. Strongyloides, Ankylostomum duodenale. Trichotrachelides, Trichocephalus hominis, Trichina spiralis. Rhabdonema strongyloides, Anguillula intestinalis. Protozoa.—The rhizopoda are essentially characterized by the fact that locomotion does not take place by the aid of independent organs, but by means of pseudopodia, viz., protoplasmic processes which the animal is capable of protruding from any portion of its body. Six orders have been described by zodlogists, but only one, or possibly two, have thus far been found in the feces. Whether or not representatives of the monera occur in the feces of man is still an open question. If so, they are apparently of no pathological significance." : Of the amebina, on the other hand, a most important member has been found, viz., the Entamoeba dysenterize. Entamceba Byer S. Histolytica (Schaudinn): syn., Ameba Coli (Lésch).—In 1875 Lésch’ discovered in the stools of dysenterie patients actively moving cell-like bodies of a roundish, pear-shaped, oval, or irregular form. He did not regard these as the cause of the disease, however, but looked upon them as only accidentally present. Similar bodies were observed in Hong-Kong by Normand in cases of colitis; and also by v. Jaksch. Sansino found them in a case in Cairo, and Koch in East Indian dysentery. It is interesting to note that Koch was the first to suspect the existence of a definite relation between dysentery and these organisms. Cunningham claims to: have found amebas frequently in the stools of cholera patients at Cal- cutta, and Grassi in normal stools, but especially abundant in cases) 1 Nothnagel, loc. cit., p. 110. Grassi, cited by Bizzozero. v. Jaksch, Wien. klin. Woch., 1888, vol. % p. 511. 2 “ Massenhafte Entwickelung v. Amében im Dickdarm,” Virchow’s Archiv, vol. lvi. ANIMAL PARASITOLOGY OF THE FECES YALE | of chronic diarrhea. Whether all these observations are correct, and whether the organisms observed were identical in all cases, is, of course, difficult to say. So much is certain, that the subject was still in a very unsettled state when Kartulist announced “that dysentery and tropi- eal liver abscess associated with dysentery are caused by the presence of the Ameceba coli,” basing his conclusion upon an examination of 500 cases. ‘The fact that this parasite was absent in all other intestinal diseases, such as typhoid fever, intestinal tuberculosis, the ordinary forms of diarrhea, etc., speaks strongly in favor of Kartulis’ view. In perfect accord with these observations are those made at the Johns Hopkins Hospital.’ Osler’ was the first in this country to demonstrate the presence of the Amceba coli in a case of liver abscess, both in the pus of the abscess and in the stools. Stengel, Musser, Dock, and others confirmed these observations, and the pathogenic character of the Amoeba coli may now be regarded as an established fact." ‘his statement is based not only upon the few facts, more his- torical in character than otherwise, which have just been detailed, but rather upon the ensemble of collected data, among which the absence of microdrganisms other than the ameba in the pus of the liver abscesses, and the constant presence of the latter in such cases, rank among the most important. It is to be noted, however, that ‘different forms of tropical dysentery exist, and that the Amba coli is essentially associated with the more chronic form, while the acute types are of bacillary origin (see Shiga’s bacillus). The size of the amebas averages 35 y. When at rest their outline is, as a rule, circular, occasionally ovoid; but when in motion they present the extremely irregular contour of moving ameboid bodies (Plate XIV). The protoplasm can be differentiated into a trans- lucent, homogeneous ectosare or mobile portion, and a granular endosarc, containing the nucleus, vacuoles, and granules. Within ‘the endosarc the vacuoles constitute the most striking feature. Some- times the interior seems to be made up of a series of closely set, clear vesicles of pretty uniform size. As a rule, one or two larger vacuoles are present, the edges of which are not infrequently surrounded by fine, dark granules. True contractile vesicles displaying rhythmic pulsations have not been observed, although the vacuoles may at times be seen to undergo changes in size. In some the nucleus is quite distinct, while in others it may be altogether invisible. ‘The protoplasm of the amebas is strongly basophilic. * “Zur Aetiologie d. Dysenterie in Egypten,”’ etc., Virchow’s Archiv, 1885, vol. ey, and 1889, vol. exviii. Centralbl. f. Bakt. u. Parasit., 1890, vol. vii. * Councilman and Lafleur, ““Amcebic Dysentery,” Johns Hopkins Hosp. Rep., 1891, vol. ii. CC. E, Simon, Johns Hopkins Hosp. Bull., 1890. * Johns Hopkins Hosp. Bull., 1890. * For the more recent literature see especially H. F. Harris, “Amcebic Dysen- tery,” Amer. Jour. Med. Sci., 1898, p. 384. 288 THE FECES Most distinctive are the movements of these bodies. From any part of the surface a rounded, hemispherical knob will project, and_ with a rapid movement the process extends and the granules in the- interior flow toward it. In these movements the clear ectosarc seems to play the most important part. ‘The organisms are ‘actively phago-_ eytic and often contain red corpuscles, bacteria, and crystals. Repro- duction occurs by fission. Various attempts have been made to cultivate the Amoeba coli, but— on the whole the results have not been satisfactory. In every attempt in this direction adequate bacterial symbiosis must be secured. ‘The most comprehensive work in this direction has been done by Musgrave — and Clegg. ‘Che medium which they recommend has the following composition and is prepared as ordinary agar: Agar . sat i es, ait Meee emt liane chloride, Pay ke ee ee LOO Be pro liter. Beef:extract 7a see or ae ee Ou 4) oe The final product is most universally satisfactory when 1 per cent. alkaline to phenolphthalein, to which end it is recommended to start with an initial alkalinity of 1.5 per cent. Tubes of this medium are plated and the surface lightly smeared with material selected from feces containing amebas. ‘The first plates | must be watched frequently under the microscope, and as soon as it | is found that amebas have developed (twenty-four hours to four or five days) transplants must be made, as otherwise they are liable to die. For further details to this end the reader is referred to Mus- grave and Clegg’s monograph.* To demonstrate amebas in stools it has been generally suggested to_ procure bits of mucus or mucopus for examination. Musgrave and Clegg recommend that the patient be given a saline cathartic and that the examination be made from the fluid portion of the stool. Drops | of this are mounted, covered with cover-glasses, and examined with a%. ‘The diagnosis of amebiasis should then only be made if motile amebas are encountered. Resting or encysted forms may be mistaken | for epithelial cells, swollen leukocytes, ete. Not infrequently some of the organisms are found containing one or more red cells. (Plate XIV.) Staining is not at all essential for the purpose of demonstrating amebas in the stool. ‘The examination of the fresh material is much more satisfactory and far less likely to lead to errors of diagnosis. Very pretty pictures are obtained by vital staining with neutral red. (Plate XIV.) ‘To this end it is only necessary to run a drop of a dilute solution of the dye under the cover-glass, when it will be seen that the young, actively motile amebas take up the stain without 1 Amebas, Bureau of Government Laboratories. Biological Laboratory of Manila, 1904. Bia te 2 Ve Amcebze Fed with Neutral Red and Containing Phagocytes and Red Cells. ee eg es RE a ee 5 Se a +a PLATE XV. Eggs of Parasites. a, Uncinaria americana; b, Trichocephalus dispar; ec, Oxyuris vermicularis; d, Tenia saginata. ) | | | | ¢ : - Tage Pi Rhy hoe” ] 6 - ANIMAL PARASITOLOGY OF THE FECES 989 losing their motility. They can thea be readily watched in their _ movements. The preparation of stained permanent preparations is not very satisfactory. ‘hey are prepared like blood films and colored with one of the modifications of the Romanowsky dye. When older material only is available it may be difficult to arrive at a satisfactory conclusion. Sometimes it is possible to cause the amebas to move again by warming the stool in an open dish at body temperature, but more often they are dead. Attention should then be especially directed to ameba-like structures containing red blood cells. If such are found the inference that the cell is a dead ameba is usually warrantable. Entameeba coli (Schaudinn).—This is not to be confused with the Entamoeba dysenteri. It is smaller than the Entamoeba dysenterie, the size varying between 10 and 15. It is opaque, gray in color, and provided with a distinct nucleus. ‘The ectoplasm is usually not _yisible. ‘lhe movements are much more sluggish and the tendency to phagocytosis much less marked. It is considered to be non-patho- genic. In the Philippines it is apparently quite common. Craig’ finds 65 per cent. of normal individuals infected with it, but uses saline purgatives to produce diarrheal discharges, as recommended by Musgrave. Parameba hominis (Craig).—Craig’ observed organisms which apparently occupy a position intermediary between amebas and flagel- lates, in several cases of severe diarrhea occurring in the Philippine Islands. In one stage of its existence the parameba is capable of active progressive locomotion and is much larger than the trichomonas in the resting stage. In the flagellate stage it is distinguished from the corresponding stage of trichomonas by the absence of an undulating membrane, the presence of a single flagellum, and its circular form. The question of its pathogenicity has not been decided. The Flagellata s. mastigophora differ from the rhizopoda in being provided with from one to eight flagella, which serve as organs of locomotion and possibly also for the apprehension of food particles. Representatives of two orders only, viz., the monadina and isomasti- goda, have been found in the feces. Of the monadina in turn only one family, viz., the cenomonadina, and of the isomastigoda only two families, the tetramitina and polymastigina, are represented.* The cenomonadina are small, oval, frequently elongated ‘bodies, provided with one long flagellum at the anterior end, at the base of which food vacuoles are situated. At the posterior end ameboid movements may be observed, and there can be no doubt that the ‘ Amer. Med., 1905, pp. 850, 897, and 936. 2 Amer. Jour. Med. Sci., August, 1906, p. 214. 3°W. Janowski, Zeit. f. klin. Med., vol. xxxi, p. 445. 19 290 THE FECES taking up of food, to some extent at least, also occurs by the aid of | pseudopodia. ‘To this family belongs the cercomonas of Davaine | and Lambl. ‘The tetramitina are small, elongated bodies, provided | with four flagella and a lateral, undulating membrane, which was | formerly mistaken for a posteriorly directed flagellum. ‘The tail | end of the organism tapers to a point. ‘The nucleus is located at | the base of the flagella. ‘Io this family belongs the parasite which was first discovered by Donné in the vagina, and which later was found also in the feces, and which has been variously designated as Trichomonas hominis, Cercomonas coli hominis, ete. > ee The polymastigina are small, somewhat oval bodies, provided with | two or three flagella, situated either anteriorly or laterally—two or | three on each side—while at the same time two additional flagella | issue from the posterior end, which may either be rounded off or | taper to a point. ‘l’o this family belongs the Megastoma entericum | of Grassi. The question whether or not the flagellate bodies are of patho- logical importance still remains sub judice. ‘They are apparently met with only in diseases associated with diarrhea, and it appears that in some cases at least this is directly dependent upon their pres- ence; in others the impression is gained as though they merely main- | tained an already existing diarrhea referable to other causes; while in a third class of cases no relation can be discovered between their pres- cence and the disease in question. Cohnheim’ has pointed out that | living infusoria in the feces may be a symptom of a primary chronic stomach affection (gastritis, usually the atrophic form). According to the same writer, encysted infusoria may also be found in the feces of healthy individuals, but in such cases we may assume that at some time previously a gastritis or a gastro-enteritis has existed. He thinks they have no pathogenic significance, and are merely of symptomatic- diagnostic interest. Cercomonas of Davaine-Lambl: syn., Cercomonas hominis (Da- vaine); monas (Marchand); Monas lens (Grassi); Monas mono- mitina (Grassi). ‘he adult organism (see Fig. 79) 1s oval or roundish in form, and provided anteriorly with a single long flagellum and posteriorly with a tail-like appendage. Its length varies from 0.005 to 0.014 mm. ‘The younger forms are pear-shaped or S-shaped, and sometimes irregular in outline; the flagellum is then either absent or rudimentary. Upon prolonged observation it will be seen that the adult parasite loses its flagellum and may protrude a protoplasmic process instead, while vacuolation occurs at the same time, indicating approaching ~ death.’ 1 Deutsch. med. Woch., 1903, vol. xxix, p. 248. 2 Lambl, Prag. Vierteljahr., 1859, vol. lxi, p. 1. Davaine, Traité des entozo- aires, 1860, Paris. Marchand, Virchow’s Archiv, 1875, vol. Ixiv, p. 293, Zunker, — Deutsch. Arch. f. prakt. Med., 1878, ANIMAL PARASITOLOGY OF THE FECES 291 Trichomonas, Donné: syn., ‘I'richomenas vaginalis (Donné); Trichomonas hominis (Grassi); monocercomonas (Grassi); cimzeno- | Fic. 79.—Cercomonas intestinalis: a, Cereomonas of Davaine, after Leuckart; b, Cerco- | monas intestinalis, after Lambl; c, d, same, ordinary forms: e, /, same, well-developed forms; | g,h,i, same, degeneration forms; k, 1, same, abortive forms. Fic. 80.—Trichomonas intestinalis: a, a’, c, trichomonas of the urine, after Marchand; b, Trichomonas vaginalis, after Donné; d, Trichomonas intestinalis, after Piccardl; e, e, ©’, same, ameboid forms; /, /’, trichomonas of the urine. (After Dock.) ; | | monas (Grassi) ; Protorycomyces coprinarius (Cunningham and Lewis) ; _ Cercomonas coli hominis (May); ‘Trichomonas intestinalis (Leuckart 999 THE FECES and Roos); Cercomonas s. Bodo urinarius (Kiinstler). ‘The parasite (Fig. 80) is oval or spindle-shaped and measures from 0.012 to 0.03 mm. in length by 0.01 to 0.015 mm. in breadth. From its anterior pole four flagella are given off, which are almost as long as the organism itself. From this point an undulating membrane extends laterally to the posterior pole, which may be rounded off or tapers to a tail-like appendage. ‘his membrane is best seen when the move- ments of the flagella have ceased, as in specimens fixed in mercuric chloride solution (1 to 5000). ‘he nucleus is situated at the base of the flagella, but is usually visible only in stained specimens (methy- lene blue). At times the organisms may be observed to assume an ameboid form; the movements of the flagella have then ceased, and pseudopodia-like processes are protruded. ‘The parasite is identical a b Cc Fic. 81.—Megastoma entericum: a, front view; b, side view; c, organism attached to an epithelial cell. (Mosler.) with the trichomonas which has been found in the vagina and in the urine.t When present in the feces the organism is usually seen in large numbers. Not infrequently it is found associated with other intestinal parasites. Megastoma entericum, Grassi: syn., Cercomonas intestinalis (Lambl); Megastoma intestinale (Biitschli); Lamblia intestinalis (Blanchard); Dimorphus muris (Grassi). ‘The parasite (Fig. 81) is pear-shaped, and measures from 0.01 to 0.021 mm. in length by 0.0075 to 0.05 mm. in breadth. In its anterior portion a more or less well-marked depression can be made out, which constitutes the peristome or mouth-opening of the organism. It is provided with 1 Marchand, loc. cit. Zunker, loc. cit., p. 236. Mosler u. Peiper, Nothnagel’s Spez. Path. u. Therap., 1894, vol. vi. ANIMAL PARASITOLOGY OF THE FECES 998 eight flagella, grouped in pairs. ‘The first pair originates on the sides of the peristome and is directed backward. ‘The second and third air are situated somewhat posteriorly and are likewise directed backward, while the fourth pair issues from the tapering tail end of the body. In fresh specimens the eighth flagella can usually not be made out, as the third and fourth pair are frequently agglutinated. The best results are obtained when the organism has been killed with mercuric chloride solution. ‘lhe individual flagella vary from 0.009 to 0.014 mm. in length. In the anterior portion of the peristome two round, hyaline bodies can be recognized, which represent nuclei. Vacuoles are absent, and nutrition occurs through osmosis, the para- site adhering to epithelial cells by its periostome. When treated with fixing solutions the chitinous envelope can be readily recognized. In the encysted form the organism is oval and measures from 0.007 to 0.1 mm. in diameter. Fie, 82.—Balantidium coli: 1, 2, division; 3, conjugation. (After Leuckart, from Déflein.) Grassi observed the organism in mice, rats, cats, dogs, rabbits, and sheep.’ ‘The ciliata, as the term indicates, carry cilia, and of these only one member, belonging to the holotricha, is found in the feces, namely, the Balantidium coli. Balantidium coli, Stein: syn., Paramcecium coli (Malmsten). ‘The organism is oval and measures from 70 y to 110 y in length by 60 ft to 72 win breadth. It is covered entirely with fine, actively motile cilia, which are grouped most densely about the funnel-shaped mouth, while at the anus only a few are seen. An ectosare and an endosare may be distinguished, and the parasite possesses the power to change its shape, and may appear quite round. In its interior we find a large, somewhat kidney-shaped nucleus, two’ contractile vesicles, and frequently fat droplets, starch granules, ete. (Fig. 82). Grassi u. Schewiakoff, Zeit. f. wiss. Zoologie, 1888, vol. xlvi, p. 143. 294 THE FECES ‘The parasite is probably pathogenic, but comparatively uncommon outside of Sweden, Finland, and Russia. Infection occurs through the dejecta of swine. Strong and Musgrave report that in their case blood examination showed a relative increase of the eosinophiles. From 200 to 300 organisms have been encountered in a single drop of the liquid feces." The fourth class of protozoa, viz., the Gregarina or sporozoa,” 1s also said to be represented in the human feces. The coccidia and psorosperms belong to this order. ‘They are oval bodies, measuring about 0.022 mm. in length, and contain in their interior a large number of small nuclei arranged in groups. They are entirely devoid of organs of locomotion, and obtain their nutriment by a b Cc Fic. 83.—Segments of tapeworms: a, Tenia saginata; b, Bothriocephalus latus; c, Tenia sulium. endosmosis. . Reproduction occurs in a common capsule, which bursts at a certain time and sends forth a whole generation of fully developed organisms. In human pathology they have become of interest in so far as certain observers have ascribed to them a role in the etiology of neoplasms. A disease of the liver analogous to the * Malmsten, Virchow’s Archiv, 1897, vol. xii, p. 302. Sievers, “ Ueber Balanti- dium Coli im menschlichen Darmkanal,” Arch. f. Verdauungskrank., vol. v, p. 445. Janowski, “Balantidiam Coli,” Zeit. f. klin. Med., vol. xxxii, p. 303. Henschen, Arch. f. Verdauungsk., 1901, vol. vii, p. 501. Solorojew, Centralbl. f. Bacter., 1901, vol. xxix, pp. 821 and 849. A. Ehrenrooth, Zeit. f. klin. Med., 1903, vol. xlix, p. 321. 2 v. Wasielewski, Sporozoenkunde, 1896. ANIMAL PARASITOLOGY OF THE FECES 295 psorospermiasis of rabbits has also been described in man, and para- sites belonging to the same order have been observed in the skin. Cestodes. —Tenia saginata, Goeze: syn.,'l’. mediocanellata (Kiichen- meister) ; ‘I’. incruris (Huber); ‘Tl’. dentata (Nicola). ‘This parasite (Fig. 84) is the most common tapeworm in Europe and North America. Fic. 84.—Tnia saginata: a, natural size; b, head much enlarged; c, ova*muchTenlarged. Infection occurs through the ingestion of measly beef. Its length varies from 4 to 8 m. ‘The head, which is devoid of a rostellum, is surrounded by four pigmented suckers, each of which is encircled by a dark line. The individual segments are quite thick and opaque, and diminish in length as the head i is approached, the largest measur- ing from 2 to 3 cm. They are each provided with a very much 996 THE FECES branched uterus, which opens laterally, the primary branches num- bering about twenty on each side (Fig. 83). The ova are elliptical in form, of a brown color, and usually enclosed in a vitelline mem- brane (Plate XV). Upon careful observation a double contour with delicate, radiating strize can be discerned. In the interior the hook- lets of the embryos, which are lost in the adult worm, are seen em- bedded in a brown, granular material. The diagnosis is mostly made by the patient when segments are found in the stools. In doubtful cases the eggs should be looked for; they are readily seen with a low power (3 Bausch and Lomb). The larval form of 'Tzenia saginata, the so-called Cysticercus teenie saginate (Leuckart), or the Cysticercus bovis (Cobbold), has been encountered in cattle, the Rocky Mauntain “antelope,” the llama, and the giraffe. In the human being it has not been observed.* Tenia solium, Rudolphi: syn., ‘VT. cucurbitina, plana, pellucida, Goeze. ‘This parasite (Fig. 85) is far less common in this country than the ‘Tenia saginata, and may indeed be regarded as a curiosity. In Germany, also, it is only rarely met with now, while formerly it Fie. 85.—Head of T. solium. 45. (Leuckart.) was the most common tapeworm in that country. This change is undoubtedly owing to the fact that raw pork is now eaten less fre- quently. In Asia and Africa it is more common. ‘Tenia solium is usually much shorter than Tenia saginata, rarely exceeding 3.5 m. in length. Most characteristic is the head, which is: provided with four pigmented suckers and a rostellum, furnished with from twenty-four to twenty-six hooklets arranged in a double row. ‘lhe mature segments measure from 1 to 1.5 cm. in length by 6 to 7 mm. in breadth, and contain a uterus which has only five to seven branches, thus differing greatly from that of ‘Tsenia saginata. The ova are round, of a brownish color, and surrounded with a thick, radially striated membrane; in their interior the hooklets of the embryos can usually be made out. They are readily found in the feces and should be looked for in doubtful cases. ‘J. Ch. Huber, Die Darmcestoden des Menschen. Bibliograph. d. klin. Helmin- thol., Heft 3, No. 4, p. 69, Miinchen, 1892. R. Leuckart, Die Parasiten des Men- schen, etc., 2d ed., 1880, pt. i. ANIMAL PARASITOLOGY OF THE FECES 297 The larval form of this tapeworm, the Cysticercus cellulose, has been,found in swine, the wild boar, in monkeys, in the brown bear, in the dog, etc. At times, though rarely, an auto-infection with the proglottides of ‘l'senia solium has also been observed in the human being. Under such conditions the embryos of the worm are set free in the stomach, and may then migrate into various parts of the body, where they become encysted. Most commonly the cysticerci are found in the skin; they have, however, also been observed in the heart, the lymph glands, teen bones, tongue, spinal canal, the brain and the eyes. I have had occasion to observe a case of this kind at the Johns Hopkins Hospital (reported by Osler). ‘The patient, a laboring man, had never worked as a butcher or a cook, and never had a tapeworm. ‘I'he cysticercus nodules, which were situated between the skin and the fascia, were very numerous, seventy-five being counted on one day. One of these nodules was ‘removed for examination, and was shown to be reterable to the “eysticercus of ‘l’eenia solium. ‘The only subjective complaints in this case were pains and stiffness in the arms and legs. ‘The individual eysticercus was elliptical or roundish in form, measuring from 1 to 10 mm. in diameter. In its interior the characteristic hooklets were seen." Tenia nana, v. Siebold: syn., hymenolepis (Weinland). This parasite (Fig. 86) seems to be the most common tapeworm of Italy and Egypt. It has also been seen in Buenos Ayres, in Bangkok, ‘Siam, and a few isolated cases have been reported in England and in Germany. In the United States the parasite seems to be not at all uncommon, but has probably been overlooked in many cases. Stiles states that in his laboratory eighteen cases have been diagnos- ticated within a year (1902). It is found especially in young people, and often causes severe nervous symptoms. It is only 8 to 25 mm. long and 0.5 mm. broad. ‘The head is ball-shaped and provided ‘with four suckers and a rostellum, bearing twenty-four to twenty- ieight hooklets arranged in a single row along its anterior edge. ‘The individual segments are of a yellowish color and about four times as broad as long. ‘The uterus is oblong and contains numerous ova, which are colorless, oval, and surrounded by a distinct, non-striated ‘membrane. ‘They measure from 0.839 to ‘0.060 mm. in size. In their interior the embryonic worm, provided with five or six hooklets, may be distinguished. ‘The number of worms which may at times be found in the digestive tract is most astonishing; 5000 and even “more have been counted on several occasions. ‘The cysticercus stage occurs in snails, which are frequently eaten raw in Egypt and Italy. Tenia nana has been identified with the Tenia murina of rats and 1 Huber, loc. cit. Leuckart, loc. cit.; and Blanchard, Traité de Zoologie médicale, vol. iv, Paris. The Inspection’ of Meats for Parasites, Bull. No. 19, ‘Bureau of Animal Industry, Washington, 1898. | | 298 THE FECES 1 6 Fia. 86.—Tenia nana: 1, body; 2, natural size; 3, head; 4, hooklets; 5, eggs; 6, egg magnified-600 times. (From Mosler.) | other rodents.!. In doubtful cases the eggs should be looked for; they are readily seen with a low power (B. and L. 4). q 1 Grassi, Centralbl. f. Bakt. u. Parasit., 1887, vol. i, p. 97. Grassi u. Caland- : ruccio, ibid., 1887, vol. i, p. 282. Comini, ibid., p. 27. Bilharz, cited by Leuc- — kart. C W Stiles, New York Med. Jour., Nov. 7, 1903. | ANIMAL PARASITOLOGY OF THE FECES 299 Tenia diminuta, Rudolphi: syn., ‘Teenia flavapune tata Lia ae enia minima (Grassi); ‘Teenia varerina (Parona); ‘Tenia lepto- ephala (Creplin). ‘Teenia diminuta was first described in man by eidy, Grassi, and Parona. It measures 20 to 60 mm. in length, nd is armed with two suckers, but is without a rostellum. ‘The va resemble those of ‘Tenia solium. ‘The cysticercus occurs in ertain caterpillars and cocoons. In man it has been found in only ix instances." | Dipylidium caninum, Linné: syn., ‘Tenia canina (Linné); Tenia aoniliformis (Pallas); ‘Teenia cucumerina (Bloch); ‘Tenia sii sten Batsch). ‘The parasite is found almost exclusively in children; afection occurs through dogs and cats. In the United States the lisease is apparently rare. ‘he only case reported is that of Stiles.’ Che larval form is found in lice and fleas. The worm itself measures rom 15 to 35 cm. in length. ‘The head is small, globular; the rostel- um club-shaped with 3 or 4 transverse rows of hooks (about 60 ls number) of rose-thorn form; anterior hooks 15 4, posterior hooks . #; suckers relatively large, rather elliptical. Segments 80 to 120 a number; oravid segments S to 11 mm. long, 1. 5 to 3 mm. broad ; ften reddish-brown in color. Genital pores ¢ at equator or in posterior lalt of segment; uterus forms egg capsules, each containing from 8 > 20 eggs, eggs globular, 43 to 50 4 in diameter. ‘The ova contain mbryos already armed with hooklets (Stiles). In diagnosis Stiles uggests that search be made in the feces for the peculiar elongated liptical tapeworm segments (Fig. 87). Microscopic examination f the feces for eggs is less certain than in cases of infection with “‘enia saginata, ‘l’senia solium, or Dibothriocephalus latus, since Dipylidium is much smaller and less prolific than any of these three orms.° | Tenia Africana, v. Linstow.A—This parasite has been found in two istances, in the case of two native soldiers at Nyasa Lake. Like “he scolex of ‘Tvenia saginata, that of the present species is devoid of ooklets. Its length is about 1.4 m.; the number of segments about 00. ‘They are all much broader than long. ‘The uterus consists of main portion running fore and aft, from which from 15 to 24 side ranches issue, which do not branch dichotomously and are so closely acked that they cannot be recognized with the naked eye. | Tenia Madagascariensis (Grenet).—This parasite has been found in Madagascar, in Mauritius, in Bangkok, and in a Demarara Indian. “he worm attains a length of from 25 to 30 em. and is composed of | * Leidy and Parona, cited by Leuckart. |? Amer. Med., 1902, vol. v, p. 65. j "A. Hoffmann, Jahresb. f. Kinderheilk., 1887, vol. xxvi, Heft 3u.4. Kruger, t. Petersburg. med. Woch., 1887, vol. xii, p. 341. . Brandt. Centralbl. f. Bakt. u. Gent 1889, vol. v, p. 99. ‘Centralbl. f. Bakt. u. Parasit., 1900, vol. xxviii, p. 485. 300 THE FECES | from 500 to 600 trapezoid segments. ‘The rostellum is surrounded b a double row of minute hooklets. ‘The suckers are round and quite large. Blanchard suggests that the cockroach may be its interme | diary host. ) | Dibothriocephalus latus, Linné, Lueke: syn., Bothriocephalus latus, (Bremser), ‘Teenia lata (Linné); Dibothrium latum (Rudophi) (see_ Fie. 87.—a, Dipylidium caninum (taken from Stiles); b, gravid segment (after Diamare); | c, head, showing four rows of rose-thorn hooks on the rostellum and four unarmed suckers (Stiles); d, egg, showing six hooks of the embyro (Stiles). a SERERLEERREN ee TMM eESEREER SRDS EREEBR ESOC cai jiseoceusovinniepoiccssinevoskibetessinaiey oT Fie. 88.—Bothriocephalus latus: a, b, twin segments. (Wilson.) Fig. 88). This worm is usually 5 to 10 m. long and of a reddish-. gray color. Longer specimens, however, may also be encountered. | In Wilson’s case 82 feet of segments were obtained from two worms, | so that the length of each, supposing both to have been of the same | size, must have been more than 40 feet. ‘The head is almond-shaped | | ANIMAL PARASITOLOGY OF THE FECES 801 and upon its flat surfaces two distinct grooves can be discerned, which , probably act as suckers. It measures 2 to 3 mm. in length by 1 mm. in breadth. ‘The neck is very short and passes at once into the body segments. Adjacent segments can often be distinguished only by -means of the recurrence of the sexual apparatus, which appears regularly in spite of the imperfect individualization of the segments. The ripe segments are almost square in form, with the genital appa- -ratus opening in the median line. ‘The fully developed segments measure 2.5 to 4.5 mm. in length by 8 to 14 mm. in breadth. ‘The total number of segments may far exceed 3000. ‘The frequent occurrence of imperfect and abortive types of twin segments may be considered an almost distinctive feature of the bothriocephalus family (Wilson). ‘lhe uterus presents 4 to 6 convolutions on each side, which become especially distinct when the segments are placed in water or are exposed to the air. A rosette-like appearance is then noted, which is quite characteristic (Fig. 83). ‘The rosette deepens in color in proportion to the number of ova which the uterus contains, and toward the tail of the parasite, from the segments of which many or all the eggs have been discharged, the rosette tends to become light in color, and may indeed appear whiter than the surrounding parenchyma. ‘The eggs (Fig. 89) are oval, 0.06 to 0.07-mm. long and about 0.045 mm. broad; they are enclosed in a brown envelope, at the anterior end of which a little lid can be recognized. ‘Their contents consist of pro- toplasmic spherules, all of about the same size, which are lighter in color in the centre than at the periphery. In infected individuals they are constantly found in the stools. The larvzee have been found in various fresh-water fishes, such as the perch, the ling, the turbot, in various members of the trout family, but they are most commonly encountered in the pike. It is thus readily understood why the parasite is most common in lake Tegions, as in Switzerland, northern Russia, southern Scandinavia, ‘and northern Italy. It is seldom seen in middle Germany, but is so common in Ireland that Cobbold named it the Irish tapeworm. Outside of Europe it is most common in Japan. In the United States a few imported cases have been observed by Walker and Leidy, Packard, Hageestam, Riesman, Stengel, McFarland, and Wilson. _ Multiple infection has been repeatedly observed. Bo6ttcher notes a case in which 100 worms were found; Roux and Eichhorst both speak of cases with 90, Heller of one with 38, and in Wilson’s case 2 were undoubtedly present.,. When more than 1 occurs the growth of the individual is impeded, and small specimens are then usually seen (three to five feet or more). Clinically the parasite is of especial interest, as its presence in a certain percentage of cases is associated with the clinical picture of a pernicious anemia; in others, how- ever, no deleterious effect upon the red corpuscles is noted, although ‘several worms may be present in the intestinal tract. | 302 THE FECES Besides in man, the worm has been encountered in the dog, cat, the seal, and in some water birds. ‘The ovum, after being discharged in the feces, during a variable period of incubation in the water develops into the onchosphera, a ciliated larva with six hooklets (Fig. 91). ‘The larva is then liberated from the ovum by passing through the lidded end, and by means of its cilia moves rapidly through the water. If not eaten by fish, it dies; otherwise it develops into the bothrio- cephalus measle, the plerocercoid (Fig. 90), which has both head and tail. Infection of man then occurs when such fish are eaten either raw or but partly cooked. In man the cysticercus stage has not been observed." Krabbea grandis, Blanchard—This parasite has been observed in only one instance—in Japan. It is said to resemble certain bothrio- cephali which are found in seals. ‘The genital organs are double in each segment. ‘The vulva and uterus open ventrally. ‘The worm attains a length of 10 m. with a breadth of 2 cm. A Wie sutin— Ss Fic. 90. Fias. 89 and 90.—Eggs and plero- Tic. 91.—Embryo with cilia and hooklets of cerco‘d. (Braun.) Bothriocephalus latus. (Leuckart and Braun.) Trematodes. —The various forms of distoma which belong to this order are essentially hepatic parasites, and rarely occur in the feces. Distoma hepaticum, Abildgaard: syn., Fasciola hepatica (Linné) (Fig. 92). ‘This, the most common liyer fluke, is 28 mm. long and. a mm. broad; it is formed like a leaf. The leaf is provided with a sucker, and a second sucker may be found at its ventral surface. peer the two the genital opening is located, leading into a skein- shaped uterus. ‘The eggs are oval, measuring 0.13 mm. in length 1 Schaumann, Zur Kenntniss d. sogenannten Bothriocephalus-Anaemie, Berlin, | 1894. Schaumann u. Tallqvist, “Ueber d. blutkérperchenauflésenden Higenschaf- | ten d. breiten Bandwurms,’”’ Deutsch. med. Woch., 1898, p. 312. Runeberg, | Deutsch. Arch. f. klin. Med., 1887, vol. xli, p. 304. Askanazy, Zeit. f. klin. Med., | 1895, vol. xxvii, P- 492. BR. N. Wilson, C Bothriocephalus, Report of a Case of Double Infection,” Amer. Jour Med. Sci., 1902, vol. cxxiv, p 262, ANIMAL PARASITOLOGY OF THE FECES 30 and 0.08 mm. in breadth, the anterior end being provided with a lid; their color is brown. In the United States the organism is practi- eally unknown, while in Germany it is most common in sheep. In the human being it is rare in both countries. It occurs in cattle sheep, swine, cats, rabbits, etc. Infection occurs through a small i Qu Sai .) “nll maiNs A Dani i: / See ‘ f vg We Ae | ‘ay i 1 yp PASE Ss Fic. 92.—Distoma hepaticum, with male Fic. 93.—Dicrocelium (Distoma) lanceo- ind female genital apparatus. (From latum, Stil. and Hass; V. s., ventral sucker; ‘iegler, after Leuckart.) Cp, pouch of cirrus; J, intestinal furcations; V.sc., vitelline sacs; 7’, testicles; O, ovarium; ) Ms, oval sucker; Ut, uterus. ¥! snail, the Linnzeus minutus, which is found, in Germany especially, apon watercress." | Distoma lanceolatum, Mehlis, has been found in only five cases, all of which occurred in Germany (Fig. 93). It is much smaller han Distoma hepaticum, measuring 8 to 9 mm. in length, by 2 to | C. W. Stiles, Jour. Comp. Med. and Vet. Arch., 1894, vol. xv, and 1895, vol, vi, Huber, Trematoden, ibliog. d. klin, Helminthol., ‘Heft 7 u. 8, p. 283. ' 3.3 mm. in breadth. It is lancet-shaped, tapering toward the head end, but otherwise closely resembles Distoma hepaticum. “The ova are 0.04 mm. long, 0.03 mm. broad, and contain fully developed embryos. In cattle, sheep, and hogs the organism is quite common." Distoma Buski, Lankester: syn., Distoma rhatonisiu (Poirier); Distoma cranum (Busk); Fasciolopsis Buski (Lankester.) ‘The parasite has been observed in China, Sumatra, the Straits Settle- ments, Assam, and India. An imported case has been described in the United States (Moore). It is the largest distoma occurring in man, measuring over an inch in length. It probably inhabits the upper portion of the intestine and may give rise to attacks of recurring diarrhea and other signs of intestinal irritation. Infection probably occurs through certain fishes and oysters, with certain snails as intermediary hosts.” Distoma sibiricum, Winigradoff: syn., Distoma felinum (Rivolta). This parasite was found in Tomsk, by Winigradoff, in eight autop- sies out of one hundred and twenty-four. Askanazy also reports two cases of infection from eastern Prussia, in which the eggs were found in the stools. In one of the cases, which came to section, more than one hundred organisms were found in the biliary passages. Its length may reach 13 mm. ‘The ova are 0.026 to 0.038 mm. long _ and 0.010 to 0.022 mm. broad. ‘The intestine is simple and extends to the posterior extremity of the body. Its surface is smooth. | Distoma spatulatum, Leuckart: syn., Distoma sinense (Cobbold); Distoma endemicum (Balz); Distoma japonicum (Blanchard). It has been observed in India, Mauritius, Corea, Formosa, China, Tonkin, and Japan, and it appears that in the two last-named coun- tries it is quite common. It inhabits the biliary passages and gall- | bladder. It is distinctly pathogenic. ‘The ova may be found in the | stools. ‘The parasite possibly also occurs in cats. ‘The intermediary host is not definitely known; it may be some fresh-water mollusk. It is about 11.75 mm. long and 2 to 2.75 mm. broad. ‘The living | parasite is of a reddish color and translucent, so that it is possible to | distinguish all its interior organs. ‘The ova measure 0.028 to 0.030 mm. in length by 0.016 to 0.017 mm. in breadth, and are enclosed in | a colorless envelope.* | Other parasites belonging to this order are Distoma conjunctum (Cobbold), Distoma heterophyes (v. Siebold), and Amphistomum hominis (Lewis and McConnell). The last named appears to be common in elephants and has been encountered in natives of Assam, in two Indians in Calcutta, and in an East Indian immigrant m 304 THE FECES —$$——S = 1 Leuckart, loc. cit., p. 137. 2 Poirier, Centralbl. f. Bakt. u. Parasit., 1888, vol. ii, p. 186. 3 Winigradoff, cited by Braun, Centralbl. f. Bakt. u. Parasit., 1894. vol. xv, p. 2. 4 Blanchard, loc. cit. er i ANIMAL PARASITOLOGY OF THE FECES 305 British Guiana. It is quite small, measuring from 5 to 8 mm. in length by 3 to 4 mm. in breadth and is characterized by the large size of its posterior suckers. Distoma heterophyes is the smallest distoma, so far as we know, which is found in man. It occurs in Egypt and is thought to be innocuous. (Fig. 94.) Distoma conjunctum was discovered in an East Indian. Its surface is covered with minute spicules. It is not of much pathological importance. (Fig. 95.) V. sc Fie, 94.—Cotylogonimus (Distoma) heterophyes. 53 (v. Sieb.); C. g, cerebral ganglion; I, intestinal branches; Ct. g, cuticular glands; V. sc, vitelline sacs; G.c, genital cup; 7’, testes, the excretory bladder between them; L. c, Laurer’s canal; R. s, receptaculum seminis, with the ovarium in front of it; Ut, uterus; Vs, vesicula seminalis. On the left side above, an (Ee x Sad is depicted, and below it three chitinous rodlets from the genital cup. > 700. Oss, Distoma hematobium and Distoma pulmonale are described in the sections on the Blood and the Sputum, respectively. Annelides.—The annelides are very common intestinal parasites, and of these especially the nematodes. Ascaris lumbricoides, Linné (Fig. 96), is the cylindrically shaped worm so commonly seen in children and in the insane. ‘The head consists of three projections or lips, which are provided with suckers 20 206 THE FECES Ph eee Ms at Vs = Vesc V sc O ee a Z Ex Fic. 95.—Distoma conjunctum, Cobb (nec Lewis and Crum; nec McCon- nell), from Canis fulvus (Cobbold): Vs, ventral sucker; J, intestine; V sc, vitelline sacs; Hz, excretory bladder; T, testes; O, ovary; Ms, oral sucker; Ph, pharynx; Ut, uterus. Fic. 97.—Ascaris mystax. (v. Jaksch.) a, male; b, female; c, head; d, egg. Fic. 96.—Ascaris lumbricoides: A, female; B, male; C, egg; at a the female genital opening; c, the male spicules; on the enlarged cephalic extremity, with its three lips. (After Perlo, from Ziegler.) — 5 - & 9 ANIMAL PARASITOLOGY OF THE FECES 307 and fine teeth. ‘lhe male measures about 215 mm., the female about 400 mm. in length. ‘The tail end of the male is rolled up on its yentral surface like a hook, and is provided with papille. ‘The gen- ital aperture of the female is situated directly behind the anterior third of the body. ‘The eggs are yellowish brown in color, almost round, and measure 0.06 mm. by 0.07 mm. in size; they are sur- rounded by an irregular albuminous envelope, which is covered by a tough shell; the contents are coarsely granular. Ascaris lumbricoides is found in all countries, and also infests the pig and the ox. Its presence may occasion severe nervous symptoms.' Ascaris mystax, Zeder: syn., Ascaris marginata (Rudolphi); Ascaris alata (Bellingham) (Fig. 97). This worm is smaller and thinner than Ascaris lumbricoides, but otherwise very similar. ‘The head is pointed and provided with wing-like projections which constitute the main point of difference between the two. ‘The male measures 45 to 60 mm. in length, the female 110 to 120 mm. Its ova are round, larger than those of Ascaris lumbricoides, and enclosed in a membrane which is covered with numerous small depressions. ‘The worm is common in dogs and cats, but very rare in man.” Ascaris maritima, Leuckart, also belongs to this class. It has been observed in only one case—in Greenland. Ascaris Texana (Smith-Goeth).* A supposedly new species, which has been found in a single instance in ‘Texas. ‘lhe male has not yet been described. Oxyuris vermicularis, Bremser: syn., Ascaris vermicularis (Linné); Ascaris grecorum (Pallas) (Figs. 98, 99, and 100). ‘The male is 4 -mm., the female 10 mm. long. At the head three lip-like projections »with lateral cuticular thickenings may be seen. ‘The tail of the (male is provided with six pairs of papillee and the female with two juteri. ‘The eggs are 0.05 by 0.02 to 0.03 mm. in size, and covered ) with a membrane showing a double or triple contour; in the interior, which is coarsely granular, the embryos are contained. The female worm lives in the cecum, but after impregnation travels downward to the rectum. Here it causes most annoying symptoms, which are especially distressing at night, when the organ- ‘ism emerges from the anus. In doubtful cases of pruritus ani et _vulvee an examination of the feces should be made for this parasite. The ova themselves do not occur in the feces." Uncinaria duodenalis (Roilliet), Ankylostomum duodenale (Dubini): _syn., Ankylostoma duodenale (Dubini); Strongylus quadridentatus __ ? Lutz, Centralbl. f. Bakt. u. Parasit., 1888, vol. iii, pp. 553, 584, 616. Hogg, | a. Med. Jour., 1888, p. 121. Kartulis, Centralbl. f. Bakt. u. Parasit., vol. |. p. 65. *K. A. Rudolphi, Arch. f. Zool. u. Zoot., 1803, vol. iii, pt. ii, p. 1. Idem, Ento, zoorum s. vermium intestinal. historia naturalis, Amsteraedami, ii, 2. * Jour. Amer. Med. Assoc., Aug. 20, 1904, p. 542. * Lutz, loc. cit. 308 THE FECES (v. Siebold), Dochmius ankylostomum (Molin); Sclerastoma duo- denale (Cobbold) ; Strongylus duodenalis (Schneider); Dochmius duo- denale (Leuckart) (Figs. 101 to 103). This organism belongs to the family Strongyloides, and is one of the most dangerous parasites met with in the human being. It has been found in Italy, Germany, 1 ‘ 2 Fig. 99.—1, Oxyuris vermicularis: a, male; b, female; natural size. 2. Magnified. Fic. 98.—Oxyuris vermicularis: a, sexually Fia. 100.—Eggs of Oxyuris vermicularis in| mature female; b, female filled with eggs; various stages of development: a, b, c, division c, male. Magnification, 10. (After Heller, of the yolk; d, tadpole-like embryo; e, worm- from Ziegler.) shaped embryo. Magnification, 250. (After Zenker and Heller, from Ziegler.) Switzerland, Belgium, Egypt, and the West Indies. C. W. Stiles. has shown that a distinct species of the hookworm exists in the United. States as also in the West Indies, viz., in Cuba and Porto Rico, the Uncinaria Americana, and that in the sand regions of the South infection with this parasite is common. Infection occurs very largely through the skin and perhaps altogether so. C. A. Smith insists: that uncinariasis exists in all cases in which ground itch has occurred | ANIMAL PARASITOLOGY OF THE FECES 309 7 Fie, 102.—Ankylostoma duodenale, male and female. Natural size. (From Mosler.) Bh ha 1% “lity YF Ml; ttf ( Uh Ml lldd Vedayyy pe rapes Wy @ Within Fic. 1u3.— Head of Ankylostoma duodenale: a, buccal capsule; b, teeth of capsule; c, teeth of dorsal margin; d, oral cavity; e, ventral prominence; jf, muscle layer; g, dorsal groove; h, esophagus. (After Schulthess, from Ziegler.) Fic, 101.—Male Ankylostoma duodenale: a, head; b, esophagus; c, gut; d, anal glands; €, cervical glands; f, skin; g, muscular layer; h. excretory pore; i, trilobed bursa; k, ribs of bursa; l, seminal duct; m, vesicula seminalis; n, ductus ejaculatorius; 0, its groove; p, penis; %, penile sheath. Magnification, 20. (After Schulthess, from Ziegler.) : 310 THE FECES within eight years, and that the disease is rarely if ever present in those who have not had ground itch within that time. From a pathological standpoint the parasite is of special interest, as its presence may give rise to severe and fatal anemia. Gries singer was the first to point out that the so-called Egyptian chlorosis is produced by this organism. Subsequently it was shown that the same parasite was responsible for the anemia which developed among the workers on the St. Gothard tunnel, and which is com- mon among the brickmakers in certain districts in Germany. In this country the anemia of the dirt-eaters has long been known in ~ the South, and has been generally attributed to the peculiar habit. Its real cause is now manifest. In Porto Rico the disease was very common until very recently and responsible for much of the severe anemia which was so frequent among the natives. In Germany, France, and Belgium the mining Aerts have become extensively | infested and the eradication of the disease a serious problem. Outside of man the parasite is not uncommon in dogs, cattle, and sheep. The male is 6 to 11.5 mm. long, the female 10 to 18 mm. ‘The head, which tapers somewhat, is turned toward the back; the mouth capsule is hollowed out and surrounded by 4 teeth;’ the tail of the male forms a 3-lobed bursa, while that of the female tapers coni- cally; the genital opening is behind the middle of the body. Its egos have an oval form and a smooth surface, measuring from 0.05 to 0.06 by 0.03 to 0.04 mm. In their interior two or three segmenting bodies are found, which rapidly develop outside of the human body, so that after twenty-four to forty-eight hours embryos may be found in the same feces in which the eggs were observed, or fully developed ova may be found after allowing the feces to stand for only a few hours (Plate XV). When allowed to dry, the young parasites become encysted, but after remaining so even for from one to two weeks they are capable of infection. A second host for its cycle of development is, according to Leichtenstern, not necessary.” The habitat of the adult worm. is the jejunum. It is rarely found in the feces. Its eggs, however, are common, and should be looked for in every case of anemia the cause of which is not manifest, especially in miners, tunnel-workers, brickmakers, dirt-eaters, ete. Any speci- men of fecal material will answer as a rule, but it is best to procure — ‘The American species has only one dorsal, conical tooth, which projects pro- minently into the buceal cavity (Stiles). 2 Centralbl. f. klin. Med., 1885, vol. vi, p. 195; Deutsch. med. Woch., 1885, vol. xi; 1886, vol. xii; 1887, vol. xiil. Lutz, Volkmann’s Sammlung, 1885, Nos. 255 and 256. American cases: C. W. Stiles, “The Significance of the Recent: | American Cases of Hookworm Disease,’”’ Ei ehteenth Annual Report of Bureau of Animal Industry, 1901. H. F. Harris, Amer. Med., Nov. 15, 1902, p. 776. A. Js Smith, Am. Jour. Med. Sci., 1903, vol. CXXVi, p. 768. C.F. Craig, ibid., p. 798; C. A. Smith, Jour. Amer. Med. Assoc., Aug. 27, 1904. | | ; ANIMAL PARASITOLOGY OF THE FECES 311 a thin stool, as-after a purge. It is then merely necessary to mount a small drop on a slide and to examine the covered specimen with a low power; a Bausch & Lomb % is quite sufficient. A mental picture of the size of the eggs should be made, for I have known it to occur that an observer saw the eggs, but did not recognize them as such. Once seen, they are easily recognized again. To hatch the eggs artificially Smith feacmmends to mix the fecal materal with a fare amount of soil in a Petri dish, using.a sufficient amount of water for the purpose. ‘There should be just: sufficient moisture to keep the soil damp. If there is too much the cover is left off for an hour or so. Every two to three days a few drops of water are e added to replenish the moisture. Under favorable condi- tions in this respect all the eggs will hatch within twenty-four hours; otherwise several days will elapse. In such cultures the larve will remain alive for three or four months and can be observed with a 4 in the inverted dish. Trichocephalus hominis, Schwank: syn., ‘Trichocephalus dispar (Rudolphi); mastigodes (Zeder); trichuris (Biittner). This parasite, which belongs to the family T'richo- trachelides, is formed like a whip, the last end being the head end, while the tail end is very much thicker. ‘The male measures 46 mm. and the female 50 mm. in length. ‘The eggs are brownish in color, measuring 0.05 by 0.06 mm. in size, and present a doubly con- toured shell, with a depression at each end, closed by a lid. The contents are coarsely granular. The organism is said to be the most widely distributed intestinal parasite, occurring in Europe, North America, Asia, Africa, and Australia. Its habitat is the cecum. The living worm is only rarely pet, found in the feces' (Fig. 104). ; ERG LATA ISSAC anS Lvbe? ieiocla arith the Trichina spiralis, Owen, is rarely ‘metio! exteimity embgided in the mucous found in the feces. ‘The male measures 1.5 mm. in length, and is provided with four papillee between the conical lips. The female is 3 mm. long. The uterus is situated nearer the head than the ovary, which opens into it. Fertilization occurs in the intestinal canal. The eggs develop into embryos in the uterus, emerge from this, and penetrate the * Ermi, Berlin. klin. Woch., 1886, vol. xxiii, .p. 614. 312 THE FECES intestinal walls, whence they are carried by the blood current to the muscles. ‘The diagnosis of sporadic cases has been greatly facili- tated by the discovery of Brown that eosinophilia, often of high grade, is practically of constant occurrence during the acute stage of the disease. In doubtful cases a small piece of muscle tissue (biceps, gastrocnemius) may be excised and examined for young Fia. 105.—Trichina spiralis in muscle. trichinas. With the naked eye the cysts appear as minute little white — specks. ‘The worms can be rendered easily visible by placing a bit of the tissue in glycerin containing 5 per cent. of acetic acid; after a few minutes it is pressed out between two slides and examined with alow power (Fig. 105). While it is believed that trichinosis is less common in the United States than in Germany, there can be no_ doubt that it is not nearly as rare as was believed. Many light cases _. go practically unrecognized. Strongyloides intestinalis (Bavay): syn., Anguillula intestinalis (Bavay); Anguillula stercoralis (Bavay); Rhabditis _ stercoralis ANIMAL PARASITOLOGY OF THE FECES 313 (Bavay); Leptodera stercoralis (Bavay, Cobbold), Leptodera intes- tinalis (Bavay, Cobbold); Strongyloides intestinalis (Bavay, Grass1) ; Pseudorhabditis stercoralis (Bavay, Perroncito); Rhabdonema, strongyloides (Leuckart); Rhabdonema intestinale (Bavay, Blan- chard). In the feces of patients infested with the parasite in question the eggs of the mother-worm are only rarely found, and the adult worm itself probably never appears unless an anthelmintic has been admin- istered and active catharsis established. Instead we find embryos -(rhabditic form) measuring about 0.33 by 0.022 mm. in size. If the stools are kept, uncovered, at a temperature of about 37° C., their larvee undergo development and reach full growth and sexual dif- ferentiation in almost five days. ‘The length of the full-grown female is about 1 mm.; its breadth about 0.04 mm. ‘The body is eendrical, shghtly diminishing in size anteriorly and tapering to a sharp point “posteriorly. When the worm retracts forcibly, shght | Rrcverse furrows may be seen. ‘he. mouth possesses dis- ‘tinct lips and is continuous with a triangular esophagus, which beyond a constriction dilates again into a second ovoid enlarge- ment. ‘lhe intestine which follows ends in a little protrusion on one side of the body near the base of the tail. A little below the middle of the body, and on the ventral side, is the vulva, which leads to the uterus, extending from the intestinal ventricle to a point near ‘the anus. Here the eggs may be massed in varying numbers. Some- _times the young have actually broken the shell of their eggs and may _be seen free in the uterus; but more commonly the ova, on deposition, | contain well-formed motile embryos (filariform brood). ‘The male ‘is about one-fifth smaller than the female. ‘The testicle ends at the base of the tail, in two small, horn-like spicules with tapering ends, “which are curved inward. ‘These spicules contain canals; they are ‘of equal size and situated symmetrically on a transverse plan, The ‘tail is coiled in the same direction as the spicules, and is half as long as that of the female. The sexually mature and differentiated forms just described repre- ‘sent the Anguillula stercoralis of Bavay. ‘Uhey represent an inter- mediate generation, developing outside of the body, which forms a link in the chain of development of the mother-worm, the Anguil- lula intestinalis (Leuckart). _ Ordinarily infection takes place through the larvee of the sexually ‘differentiated form. ‘These filariform embryos are longer than the vhabditiform brood of Anguillula intestinalis (Fig. 106). They are provided with a cylindrical esophagus descending down to about the middle of the body, and a tail, which, instead i terminating in a fine point, is apparently truncated at its extremity. On maturation “they give rise to the Anguillula intestinalis, which is encountered throughout the upper gastro- -intestinal tract, especially in the lower | | | ha 314 THE FECES part of the duodenum and the upper part of the jejunum, though occasionally they have also been found throughout the entire jeju- num and in the upper part of the ileum. On several occasions they have been found in the stomach. Anguillula intestinalis, viz., the parasitic mother-worm, is, accord- ing to Rovelli, parthenogenetic, while Leuckart expressed the ne ; ; | ’ ' { i wee e io . eee ~ geo pe perenne areas , 4 K | SORE as RUE DOO Tee AUR Sear NORRIS Se Nal ST Oe SEE er oT AO on An NACE I Ui NPE On anne STS LN PITT Tr nT Sen ni Temnat oar SOW ner Sree anne LN ene aK Sree | Fic. 106.-Strongyloides embryo (rhabditiform variety). The stool contained many red cells. opinion that it might be hermaphroditic. Its length is about 2. mm., and its average breadth 0.03 mm. ‘The body tapers a littl anteriorly, and terminates posteriorly in a conical tail, the extremity ol which is appreciably rounded and even a trifle dilated. ‘The mouth ANIMAL PARASITOLOGY OF THE FECES 315 s without horny armature, and shows three small lips. It opens nto a cylindrical esophagus, which occupies about one-fourth of he length of the animal, and shows neither swellings nor striations. he intestine extends nearly to the posterior extremity of the body, sut is almost invisible in the middle part owing to the presence of v large, elongated ovary. The vulva is sedated in the posterior hird of the Animal: and the uterus contains usually five or six rather Jlongated ova. The anus is situated toward the base of the tail. Fic. 107.—A, egg of Strongyloides intestinalis (parasitic mother-worm); B, rhabditiform mbryo; Cc; filariform embryo, derived by direct transformation from a rhabditiform mbryo, (Taken from Thayer.) Che eggs are of a yellowish-green color, rather opaque, and appar- mtly finely granular (Bavay); in their general appearance they esemble those of the uncinaria (Fig. 107). While infection originally takes place through the filariform larvee £ Anguillula sterooralia, an auto-infection w ith the larvee may also recur Roithaut the intervention of the sexually differentiated forms, *y a direct transformation from the rhabditiform embryos of the varasitic mother-animal, and there is evidence to show that this 216 THE FECES latter cycle is indeed more common ‘There is no evidence to show that the sexually mature intermediate generation ever develops in the intestinal tract during life. The time elapsing between infection with the filariform larvee and the appearance of rhabditiform embryos in the stools is about seventeen days. | The parasite is the recognized cause of the so-called Cochin- China’ diarrhea, and is of ‘Bnaaken interest from its resemblance to Anky- lostoma eiiodeaaie with which it is not infrequently found ASsO- ciated. Excepting in very rare instances, it does not cause intestinal | ulceration, and it is supposed that the injurious effects of the para- | site are purely mechanical. It is possible, however, that these may also be owing to the irritating action of its excretory products. ‘The clinical manifestations of ha disease are mainly those of a chronie diarrhea and a comparatively mild anemia. ‘There are usually three or four pasty stools a day. The organism was first discovered in individuals who had con- tracted severe diarrhea in Cochin-China. Grassi and Parona later found the worm in Italy, and at the building of the St. Gothard tunnel it was frequently seen in association with the ankylostoma. ‘Thayer was the first to find it in the United States, and it is interest- ing to note that two of his three cases must have become infected in either Maryland or Virginia. ‘The third case may have originated in Austria; in it the anguillula was associated with amebas and the ‘Trichomonas intestinalis; it ended fatally, beg complicated by liver abscess. Since then additional cases have been reported in the United States by Moore, Price, Lamar, and others. | Other cases have been observed in Belgium, Holland, Martinique, Brazil, Sicily, the Dutch Indies, Egypt, Germany, Spain, and the Philippine Islands. LITERATURE.—Grassi, Centralbl. f. Bakt. u. Parasit., 1887, vol. ii, p. 413. Leich- tenstern, Deutsch. med. Woch., 1898, p. 118. Perroncito, Arch. p. 1. sci. méd., — 1881, No. 2. Compt.-rend. de l’Acad. des sci. , 1882, No. 1. Teissier, ibid., vole exxi, p. 171. Bavay, ibid., 1876, vol. lxxxiii, D. 694; ibid., 1877, vol. Ixxxiv, py 266. Normand, ibid., 1876, p. 316. W.S. Thayer, Jour. of Exper. Med., 1901, vol. vi, No. 1 (full literature to L901) aM LG, Price, Jour. Amer. Med. ‘Assoc, Sept. 12, 1903 (literature to date since Thayer’s paper). Chemistry of the Feces. Reaction.—The reaction of the feces is normally usually alkaline, | sometimes neutral, rarely acid, the alkalinity being due to ammoniacal fermentation, the acidity to ice and butyric Aa fermentation. In disease As the reaction of the stools is variable and of but little. clinical interest. In typhoid fever an alkaline reaction is so constantly met with that this symptom might possibly be of value in doubtful ANIMAL PARASITOLOGY OF THE FECES ee 17, eases. It may, however, also be neutral, amphoteric, or even acid. In acute infantile diarrhea an acid reaction is the rule, but exceptions also are not infrequent. Normal stools of suc klings are acid, the degree of acidity, according to Langstein, corresponding to about 2.1 to 3.7 per cent. of normal NaOH for 100 grams of the moist feces. General Composition.—'The following table, taken from Gautier, will give an idea of the composition ae fresh feces, calculated for 1000 parts by weight: Adult man, Suckling. ete ee ee tos we PS hens 153.00 851.3 tt foe ee eee «2.8207. 00 148.7 | Total organic TiNCMAby te MMe anton gs 334. THE SPUTUM MICROSCOPIC EXAMINATION OF THE SPUTUM. Under this heading it is necessary to consider leukocytes, red blood corpuscles, epithelial cells, elastic fibers, corpora amylacea, parasites, and crystals. | Leukocytes.—Leukocytes, usually polynuclear in character, are found in every sputum in considerable numbers, embedded in a homogeneous, more or less tenacious material. At times they con- tain fat droplets, or granules of pigment, such as carbon or hematoidin. Their number varies considerably, being naturally greatest in cases of perforating abscess, empyema, putrid bronchitis, ete. While the leukocytes which usually are found in the sputum are of the neutrophilic variety, eosinophiles may also be observed, and especially in asthmatic sputa, in which they predominate. Free eosinophilic granules are then also seen, and I have repeatedly observed specimens in which the spirals (see above) were literally covered with these granules (Plate XVII). The presence of eosino- philic leukocytes is, however, not characteristic of the sputa of bronchial asthma, as they may be met with in other diseases as well. ‘Teichmiiller has pointed out that they are present in a large percentage of tuberculous cases, and may be found months before tubercle bacilli can be demonstrated. He regards their occurrence as evidence of a defensive struggle on the part of the body, which is most evident in fairly strong individuals. In recovery a gradual increase in their number is noticeable, and a diminution, Teich- miiller thinks, is indicative of a relapse, or, if the diminution occurs rapidly, of florid consumption. ‘These statements, however, lack confirmation and are probably too dogmatic. Ott, Fuchs, Bett- mann, ‘Turban, and Cohn, in fact, deny the prognostic significance of the eosinophilic cells in. cases of phthisis; and Cohn states, as the result of an examination of 100 cases, many of which were com- paratively early, that the occurrence of eosinophilic leukocytes 1s fairly uncommon in tuberculous sputa. Stadelmann’ also states that he has been unable to verify Teichmiiller’s observations. On the other hand, he has been able to confirm the observation which has been repeatedly made, that large numbers of eosinophilic cells appear in the sputum following hemoptysis. ‘Teichmiiller has also described an “eosinophilic” bronchitis, which is said to differ from other forms of the disease in the abundance of eosinophilic cells which are encoun- tered. ‘T’he sputum in such cases is described as transparent, mucoid, and loose, with yellow, purulent admixtures. It is said to be mark- edly different from the tough, thick sputa of bronchial asthma. ' Discussion on tuberculosis, Deutsch, med, Woch., 1901, vol. v, p. 210, rere ak VoL. itum from Case of Bronchial Asthma, showing Large Numbers of Eosinophilic Leukocytes and Free Granules. 1 be noted that the leukocytes are all mononuclear. (Hye-piece 1, objective 1-8, Bausch & Lomb.) . MICROSCOPIC EXAMINATION OF SPUTUM 835 Typical spirals are absent, but rudimentary forms may be encountered. Charcot-Leyden crystals are present.* Very curiously the majority of the eosinophilic cells which are met with in the sputum (notably in asthma) are mononuclear; they are not myelocytes, however, but probably mononuclear histogenetic forms. Griinwald’ states that in the sputa of the most diverse diseases eells are met with which contain a hypoeosinophilic granulation, and that the granules in question may also occur outside of the cells in the absence of evidence of special cell destruction. ‘These gran- ules, in contradistinction to the true eosinophilic cells, lose their color on treating with an acid, and readily take up the blue stain on subsequent staining with methylene blue. Griinwald states, how- ever, that a sharp line of distinction does not exist between the two varieties of granules, and that intermediary conditions exist, as also transitions between oxyphilic and basophilic granules in the nature of an amphophilic granulation. To demonstrate eosinophilic leukocytes in the sputum, smears are made as usual, slightly fixed by drawing through the flame of a burner, and stained for two minutes in a (0.5 per cent. alcoholic solu- tion of eosin. ‘The preparations are then immersed in 50 per cent. alcohol to the point of decolorization, when they are counterstained with methylene blue, briefly washed with water, and dried. The eosinophilic granules and the red cells in part hold the eosin dye. Basophilic leukocytes (mast-cells) have also been observed in the sputa. Red Blood Corpuscles.—The presence of red blood corpuscles in small numbers does not, by any means, indicate serious pulmonary or cardiac disease, as they may be found in almost any sputum, and ‘especially in that of individuals who smoke much or live in a smoky ‘atmosphere; they are, without doubt, derived from the catarrhally inflamed bronchial or tracheal mucosa. Whenever they occur in large numbers, however, their presence becomes important. "They may be observed in acute bronchitis, pneumonia, edema of the lungs, bronchiectasis, abscess, gangrene—in fact, in all pulmonary diseases. "Their occurrence is most important in phthisis, and is, in fact, one of the most constant symptoms of the disease. The form of the red corpuscles will depend upon the length of time that they have remained in the lungs, and all gradations from the typical red corpuscle to its shadow, or even fragments, may be * Teichmiiller, “Die eosinophile Bronchitis,’ Deutsch. Arch. f. klin. Med., vol. (xiii, p. 444. See, also, K. Schénbrod, Ueber den gegenwiirtigen Stand der Beurtheilung der eosinophilen Zellen im Blute und im Sputum, Inaug. Diss., Erlangen, 1895. A. Hein, Ueber das Vorkommen eosinophiler Zellen im Sputum, (naug. Diss., Erlangen, 1894. Be idien uber d. Zellen im Auswurf, etc.,”’ Virchow’s Archiv, 1899, vol. elviii, D. 336 THE SPUTUM observed. In pneumonia the microscopic examination may at times be disappointing, the appearance of the sputum suggesting | that red corpuscles in large numbers are present, while, as a matter — of fact, they are almost all destroyed, the color being due to altered | pigment. It may even be necessary to depend. upon chemical methods to clear up the question. It should be remembered that the presence | of blood pigment is not always indicated by a red color, but that it may also assume a golden-yellow or even a greenish tinge, owing to | certain chemical changes which have taken place. ‘The golden-yellow and the grass-green sputa observed in cases of pneumonia dua convalescence belong to this class. To demonstrate the presence of traces of blood in the sputum, the | aloin or guajac test (see Feces) may be employed, after first boiling © the sputum with 20 per cent. caustic alkali solution and susequently neutralizing with acetic acid. Epithelial Cells —Epithelial cells are found in practically every sputum. ‘They are mostly of the pavement variety and may be derived from the mouth, pharynx, and the upper larynx. Many of — the cells are full of invading bacteria, which may lead to their — entire destruction. Cylindrical epithelial cells, providing they do _ not come from the nose, indicate in a general way an inflammatory condition of the lower larynx, trachea, or bronchi. As a rule their form is so much altered that it is often difficult to recognize them; they may thus become polyhedral, cuboidal, or even round, and can then hardly be distinguished from leukocytes. Actively moving cilia may be found only in perfectly fresh sputa, immediately after being expectorated, but are very rarely seen. Formerly much importance was attached to the so-called alveolar epithelial cells (Fig. 114) as an aid in diagnosis. Buhl thus regarded — them, particularly when undergoing fatty or myelin degeneration, — to be pathognomonic of pulmonary disease, and especially of that form of pneumonia which has been termed essential idiopathic desquamative pneumonia. Bizzozero,’ however, as well as others, have shown that these cells not only occur in almost every known pulmonary disease, but that they are present also in the so-called — “normal” expectoration which at times is obtained upon making a forcible expiration. They are round, oval, or polygonal cells varying in size from 20 4 to 50 y. ‘They may contain one, two, or three oval nuclei, which are rather small and provided mattt nucleail Usually the latter are hidden beneath numerous granules. Some of the granules are albuminous, but most of them are either pigment granules, fatty granules, or myelin granules. The myelin granules were first discovered by Virchow’, and termed myelin granules on 1 Microscopie clinique, 2d ed. Frangaise, Paris, 1885. ? Virchow’s Archiv, 1854, vol. vi, p. 562. PLATE OAV IT. BIG i; Tuberculous Sputum Stained by Gabbett’s Method. The Tubercle Baeilli are seen as Red Rods, all else is Stained Blue. (Abbott.) Heart Disease Cells, showing Alveolar Epithelial Cells, Loaded Down with Granules of Hematin. 7 s Y Si, a ‘ - A, P ' ri , 7 7 v . & ; a " 2% Ss a ‘ a | ¥ | * = : » ' + -t | a= 5 4 am * J account of their resemblance to mashed nerve matter. ‘They are distinguished from the other forms by their clear, pale, colorless appearance, and the fact that at times fine concentric striations can be detected. ‘These forms may be round, but more often they are irregular. Chemically, the myelin droplets have been shown to con- tain a considerable amount of protagon, besides traces of lecithin and cholesterin." ‘They are readily soluble in alcohol, somewhat so in chloroform and ether. ‘They swell in water and stain yellow with iodine. ‘hey are colored but little by the anilin dyes and do not turn black on treating with osmic acid. Sometimes myelin granules are found together with fatty and pig- ment granules in the same cell. The sputa of chronic bronchitis referable to heart disease are characterized by the presence of so-called heart-disease cells. ‘These MICROSCOPIC EXAMINATION OF SPUTUM 337 Fic. 114. Epithelium, leukocytes, and erystals of the sputum. (Eye-piece III, objective 8A, Reichert.) a, a’, a”, alveolar epithelium: b, myelin forms; c, ciliated epithelium; d, crystals of calcium carbonate; e, Kematoidin crystals and masses; f, f/f, f, white blood cor- puscles; g, red blood corpuscles; h, squamous epithelium. (v. Jaksch. are alveolar epithelial cells containing hematoidin granules (Plate XVIII, Fig. 2). They appear to be most numerous in cases of mitral disease, but may also occur in congestive affections of the broncho- pulmonary apparatus, even with the heart intact.’ Liver cells may at times be observed in the sputa in cases of liver _ abscess, and are easily recognized by their characteristic form. Elastic Tissue.—Much more important from a clinical stand- point are the elastic fibers and shreds of elastic tissue which may _ be found in sputa. They vary much in length and breadth, and are provided with a double, undulating contour;: they are usually curled at their ends. Very often they exhibit an alveolar arrange- ment (Fig. 115), which at once determines their origin. — _ 1A. Schmidt, “Ueber Herkunft u. chem. Natur d. Myelinformen d. Sputums,”’ Berlin. klin. Woch., 1898, p. 73. See, also, Zoja, Maly’s Jahresberichte, vol xxiv, p. 694. ae R. C. Regolo, Gaz. d. Ospedali, Milano, vol. xxii, No. 135. | 22 338 THE SPUTUM Whenever present, elastic tissue is an absolute indication that a destructive process is going on in the lungs. It is found in cases of abscess of the lungs, bronchiectasis, occasionally in pneumonia, pul- monary gangrene and infarct, and, most important of all, in phthisis, in which it is said to be present in 90 per cent. of all cases. ‘This percentage, which was obtained by Dettweiler and Setzer in 1878, is unquestionably too high in comparison with what is seen today, where the diagnosis of tuberculosis is made much earlier. In gan- grene of the lung elastic tissue is generally said to be absent, but Osler states that he has never seen a case without it, and that usually it occurs in large fragments. | In every case it is necessary to determine whether the elastic tissue has not been ntroduced from without, and it may hence be stated as a rule that it can only be regarded as absolutely characteristic when showing the alveolar arrangement. In order to demonstrate the presence of elastic tissue in the sputum the following method is very convenient: A small amount of the thick, Fic. 115.—HElastic fibers in the sputum. (Eye-piece III, objective 8 A, Reichert.) (v. Jaksch.) purulent portion of the sputum is pressed into a thin layer between two pieces of plain window-glass, 15 by 15 cm. and 10 by 10 cm. The particles of elastic tissue appear on a black background as grayish- yellow spots, and can be examined in situ under a low power. Or, the upper piece of glass is slid off till the piece of tissue is uncovered, when it is picked out and examined on a slide, first with a low and then with a higher power. At first there will be some difficulty in distinguishing with the naked eye between elastic fibers and particles of bread, or milk globules, or collections of epithelium and debris, but with practice such mistakes are rarely made, and the microscope - always reveals the difference. If only very little elastic tissue is present, it is necessary to examine large quantities of sputum with a moderately low power, and best ANIMAL PARASITOLOGY OF SPUTUM 339 after the addition of a solution of sodium hydrate. The sputum is boiled with a 10 per cent. solution of the reagent, an equal volume being added; the boiling is continued until a “homogeneous solution has been obtained: after dilution with four times its volume of water it is allowed to settle for tw enty-four hours or centrifugalized and the sediment examined at once. May’ recommends the following method of demonstrating the presence of elastic tissue in sputum: ‘The material in question is heated on a boiling water bath with an equal volume of a 10 per cent. solution of sodium hydrate until it has all apparently dissolved. The mixture is then centrifugalized and the supernatant fluid decanted. ‘lhe sediment is treated with about 2 c.c. of an orcein solution prepared according to the formula of Unna-Tanzer, viz., orcein, 1 gram; absolute alcohol, 80 c.c.; distilled water, 40 c.c.; concentrated hydrochloric acid, 40 drops. On adding the stain, owing to the remaining alkali, the color turns violet; a few drops (3 to 5) of hydrochloric acid are added until the original color of the stain returns. ‘The tube is then placed for from two to five minutes in boiling water, after which acid alcohol (concentrated hydrochloric acid, 5 c.c.; 95 per cent. alcohol, 1000 c.c.; distilled water, 250 c.c.) is added to decolorize. ‘The mixture is again centrifugalized and the sediment washed once or twice more with the acid alcohol by centrifugation and decantation. ‘The sediment is then examined directly, when the elastic tissue fibers may be recognized by their more or less intense brownish-violet color. ANIMAL PARASITOLOGY OF THE SPUTUM. Protozoa. Entamceba Dysenterie.—In cases of amebic abscess of the liver with perforation into the lung the Amceba coli may be demon- strated in the sputa. Such sputum commonly presents the anchovy sauce appearance already mentioned. As a rule the amebas are not numerous and slide after slide may have to be examined before a single organism is discovered. ‘The material should be kept at body tem- perature and the slides warmed. A Bausch and Lomb ¢ or Leitz 6 or 7 is used (see also Amebas in Feces). Only actively moving organisms are diagnostic. Trichomonads have at times been observed in cases of gangrene of the lung, and in the pus removed postmortem from lung cavities. They are identical with the Trichomonas vaginalis of Donné. Cercomonads have been found in the sputum and in the Dittrich plugs in gangrene of the lung. Beye oes. Tenia Echinococcus.—Portions of echinococcus cysts, , pieces of membrane (Fig. 114) and hooklets (Fig. 119), are ‘ Deutsch, Arch, f. klin. Med., 1900, vol, xviii, p. 427. 7 —* 340 THE SPUTUM occasionally seen when the parasite has lodged in the lungs or in the neighboring organs. ‘The disease is not common in this country. Lyon’ collected 241 cases in the United States and Canada up to July 1, 1901. 91 per cent. occurred in foreigners. In Canada a large proportion is referable to the Icelandic immigrants in Manitoba. — Fig. 116. Fig. 118. Fic. 116.—Tnia echinococcus. 50. The cirrus pouch, the vagina, uterus, ovary, shell- gland and vitellogene gland and the testicular vesicles at the sides are recognizable in the second proglottis; the uterus partly filled with eggs, as well as the cirrus pouch and the vagina. Fig. 117. Section through an echinococcus cyst with brood capsules. Fic. 118.—A piece of the wall of an echinococeus veterinorum stretched out and seen from > the internal surface. > 50. A few brood capsules with scolices directed toward the interior and exterior. (Thomas.) Thomas,’ of Adelaide, has thoroughly investigated the disease in Australia, where it is quite common. The adult parasite (Fig. 116), Tenia echinococcus (vy. Siebold), is a three- or four-segmented tapeworm, 4 to 5 mm. in length, whose 1N. Y. State Jour. Med., Oct., 1902. * Hydatid disease, 1884. nine around, and four suckers’ behind ANIMAL PARASITOLOGY OF SPUTUM 341 habitat is the intestinal canal of the dog, dingo, jackal, wolf, ete. The larval or cystic form develops in cattle, sheep, swine, rabbits, ete., and is also found in man. ‘The ova, 0. O67 mm, in diameter, are per sueee by food, water, or by ahs ‘atic m iW, dust, In the the ivesting 4 juices of the sanieal bores its “way through the intes- tinal wall, and finds a resting place in’ iti lie er, ‘hurig, oc other part of the body, there developing into the cyst e. form, that may attain enormous size. The primary or mother cyst may pr oduce Agiehier bak vineee latter granddaughter cysts, and these a third generation, often in great number; so that the cavity may be filled with cysts of varying size, formed by exogenous or endogenous growth. On the other hand, the single cyst may remain sterile—aceph- alocyst—or may produce scolices (Fig. 117) which are attached by pedicles to the lining of the vesicles or brood capsules in which they develop. Each scolex, or echinococcus head, 0.4 to 0.25 mm. in diameter, is a round or oval body with a head capable of protrusion or retraction. ‘There is a single or double circlet of hooklets the rostellum. The body is partly covered with calcareous _ particles. These scolices may ordinarily be found in hydatid-cyst contents. . Hydatid membrane (Fig. 113) ee Fie. 119.—Hooklets of echinococcus; in thickness according to the size of a, Echinococcus veterinorum: 6, Tenia echinococcus, three weeks after infection; the cyst, a mother-cyst membrane c, adult Teenia echinococcus; d, three being often § inch or thicker; the etharate acne GO ee smaller cysts have walls of greater delicacy. It is usually pearly or grayish white, opaque, and of gelatinous consistency, but the thin walls of the daughter cysts may be perfectly clear and transparent. ‘The membrane consists of two layers: (1) the ectocyst, of regular lamin of chitinous-like material, readily torn on manipulation, the innermost layers whiter and aie than the outer; (2) the delicate, soft, eranular endocyst, consisting of a mass of ae ‘ate polygonal cells without distinct nuclei. Emini this the scolices and daughter cysts are developed. ‘The ectocyst usually lies in close apposition to the fibrous adventitious capsule formed by the organ in which the hydatid is present. “The ectocyst, known also as the cubicula by Continental writers, presents under the microscope a peculiar stratified structure which is quite charac- teristic. It shows no appearance of fibers or cells, and even under 342 THE SPUTUM high magnifying powers it exhibits a nearly hyaline or at most a faintly granular appearance” (‘homas). ca When a hydatid cyst of the lung, liver, or neighboring tissue has ruptured into:thée larger or smaller divisions of the bronchi, quantities of clear, watery fluid, giving the characteristic tests for hydatid fluid (see Cystic Contents), maybe coughed up and be found to contain pethapizeyeueie ts) jcceee Hoe) ; M: (a) Small: cysts full:of clear fluid, from the size of a pin’s head upward—thecaughter or, granddaughter cysts. (b) Whitish, dot-like bodies just visible to the naked eye when single, or more evident when grouped together in colonies—the scolices, or echinococcus heads (Fig. 118). we Fig. 120. Fia, 122. Fic. 120.—Paragonimus westermanni (Kerb.). 10. (Leuckart.) Mouth, pharynx, intestinal branches; at the sides of which the vitelline sacs are observed. The genital pore is behind the ventral sucker, and next to it, at the left, the ovary; at the right, the uterus; the two testes at — the back; the excretory vessel in the middle. Fig. 121.—Paragonimus westermanni (Kerb.) (natural size). To the left, dorsal aspect; to the right, ventral aspect. (Katsurada.) Fig. 122 —-Egg of Paragonimus westermanni (Kerb.) from the sputum. 1000. (Katsurada.) (c) Some of the component parts of the cysts or scolices, viz.: 1. Collapsed cysts—the well-known “grape skins,” or pieces of the - gelatinous membrane of a mother or daughter cyst. 2. Hooklets and calcareous corpuscles from the bodies of the scolices, visible only under the microscope. Where the hydatid has suppurated before rupture, pus in large or small amount takes the place of the clear fluid or is mixed with it, the other elements being recognized on examination. Microscopic Examination of Hydatid Material—A piece of mem- brane (often yellowish and shreddy in degenerating cases) is picked — ANIMAL PARASITOLOGY OF SPUTUM 343 _ up with forceps, placed on a slide, a drop or two of water applied, _ and lightly crushed under the cover-glass. At the torn edges of the membrane the characteristic laminated structure can be readily seen with the low power (Fig. 113). It does not stain readily, but staining is unnecessary. A section may be cut with the freezing microtome and stained with carmine. Sputa may continue to be expectorated from a hydatid cavity of the lung for months or years, and are then usually of a purulent or mucopurulent character, perhaps blood-tinged. A thick smear on a slide may reveal, when examined with a low power, pieces of laminated membrane or hooklets. A piece of membrane, if seen on floating the sputa in water, should be picked out with forceps. ‘Tubercle bacilli are sometimes found in the sputa of cases of pulmonary hydatid. When a hydatid of the liver has ruptured into a bronchus the sputa may be bile-stained.* Trematodes. Distoma Pulmonale (Lung Fluke).—A form of pul- monary disease closely simulating phthisis and associated with pul- monary hemorrhage is very common in Japan, and has been shown to be referable to the presence of a parasite in the lungs, Distoma pulmonale (Balz)—syn., Distoma westermanni (Kerbert), Distoma Ringeri (Cobbold), Paragonimus westermanni. ‘The parasite is 8 to 10 mm. long, 4 to 6 mm. wide, rounded very markedly in front, less so posteriorly. ‘The color during life is a reddish brown. ‘The two sucking disks are nearly equal in size. ‘The ova are brown, with a thin shell and lidded. ‘They measure from 80 to 100 in length and 40 to 60 “in breadth. ‘The worm and its ova are found in the sputum. If the sputum is shaken in water and the water renewed from time to time, in the course of a month or six weeks (according to the tempera- ture) a ciliated embryo is developed in each ovum. When the ovum is mature, on placing it on a slide and exercising slight pressure on the cover-glass, the operculum will be forced back and the embryo will emerge and at once begin to swim and gyrate in the water (Manson). Outside of Japan the parasite has been found in Corea and Formosa. In the United States it has been found in the cat and in the dog; in the human being one case, occurring in a Japanese student, has been reported. Many Charcot-Leyden crystals are found in the sputum at the same time. LireraturE.—C. D. Stiles, “ Distoma Westermanni,’’? Johns Hopkins Hosp. Bull., 1894, p. 57. Brown, Die thierischen Parasiten, etc., Stuber, Wurzburg, 1895. Distoma Hematobium.—Manson found the ova of a species of Distoma hzmatobium in the bloody expectoration of a Chinese -who had lived for some time on the island of Formosa. ' For the above account of the component parts of hydatid material I am indebted to my friend Dr. John Ramsay, of Launceston, Tasmania. 344 THE SPUTUM BACTERIOLOGY OF THE SPUTUM. — Tubercle Bacillus.—From macroscopic examination it is impos- | sible to decide whether or not a particular sputum is of tuberculous — origin. At times a sputum may have a suspicious appearance, but — it is never possible to speak with certainty from simple inspection, as a mucoid sputum may contain tubercle bacilli in large numbers, while a mucopurulent sputum may be entirely free from them, and vice versa. Reliance should, hence, only be placed upon a careful microscopic examination. In all cases the fine, cheesy particles previously described should be carefully sought for, as they contain the largest number of bacilli. In their absence reliance should be placed upon the examination of a large number of preparations, attention being directed especially to the purulent and mucopurulent foci of the sputum. If but few bacilli are present the following procedure may be employed: About 100 c.c. of sputum are boiled with double the amount of water, to which from 6 to 8 drops of a 10 per cent. solution of sodium hydrate have been added, until a homogeneous solution has been obtained, water being added from time to time to allow for evaporation. ‘The mixture is then centrifugated or set aside for twenty-four to forty-eight hours and examined for tubercle bacilli and elastic tissue. Or, the following procedure, suggested by d’Arrigo and Stampacchia, may be employed: Four or five sputum masses are placed in a test-tube and covered with Ranvier’s acid alcohol (70 per cent. alcohol, containing 1 per cent. of concentrated hydrochloric acid), so that this fills about two-thirds of the tube. The mixture is well shaken and kept, stoppered with cotton, for twenty-four hours at 37° C. or for three hours at 50° C. ‘The acid alcohol destroys the mucus and fixes the cells and bacilli, which sink to the bottom. It is claimed that in a sediment prepared in this manner it is possible to demonstrate the tuberlce bacilli even after several years. If, notwithstanding the fact that all due precautions have been taken, no bacilli can be demonstrated in the sputum, and the clinical history and the physical signs are indefinite or negative, the proba- bilities are that we are dealing with a benign process. From an examination of the sputa alone in such cases it is utterly impossible to reach a definite conclusion. When the amount of sputum, more over, is small and contains but little pus, the absence of tuberlce bacilli in doubtful cases is less suggestive of the absence of tuber- culous disease than in cases in which the sputum is more abundant and mucopurulent. Only two bacilli are likely to be mistaken for the tubercle ba- cillus, viz., the bacillus of leprosy and the smegma bacillus. All BACTERIOLOGY OF THE SPUTUM 345 three are characterized by the difficulty with which they take up basic dyes, and the great tenacity with which they hold the dye when once stained, even upon treatment with mineral acids. (acid fastness) and alcohol. ‘This peculiarity has been generally referred to the presence of fat in the bacilli, but it appears from more recent researches that the chitin or chitinous substances in the bodies of the tubercle bacilli are primarily concerned in the reaction (Helbing).* Sata”, moreover, has shown that other bacteria, such as the anthrax bacillus, the bacillus of glanders, the Staphylococcus aureus, etc., give a fat reaction which is as intense as that of the tubercle bacillus, while these organisms are not in the least resistant to the action of acids when stained. That confusion should arise in the differentiation between the tubercle bacillus and the bacillus of leprosy is very unlikely. More important is the smegma bacillus, which is known to occur at times upon the tonsils, the tongue, and in the tartar of the teeth of per- fectly healthy individuals. In sputum coming from the lungs it has been observed by Pappenheim,’ Friinkel,* and others. Methods of Staining the Tubercle Bacillus. 1. Gabbett’s Method.—Bits of purulent or hemorrhagic material, or if present the cheesy particles referred to above, are spread on slides in thin layers. These are dried in the air and fixed by being passed a few times through the flame of a Bunsen burner or an alcohol lamp. ‘The specimens are covered with a few drops of carbol-fuchsin solution? and heated to boiling for one-quarter to one-half minute. ‘The solu- tion is composed of 1 part of fuchsin dissolved in 100 parts of a 5 per cent. solution of carbolic acid and 10 parts of absolute alcohol. ‘The excess of the staining fluid is drained off and replaced, without washing, with a solution, composed of 2 parts of methylene blue in 100 parts of a 25 per cent. solution of sulphuric acid. After a minute or two they are washed in water, dried, and examined directly in oil. It has been suggested by Pagani" to use lactic acid instead of sul- phuric acid, in order to avoid a too energetic decolorization. He claims that excellent results are obtained if the second solution of 1** Erkliirungsversuch f. d. specifische Firbbarkeit d. Tuberkelbacillen,”’ Deutsch. med. Woch., 1900, V. B. p. 133. 2“ Ueber d. F ettbildung durch verschiedene Bakterien,” ete., Centralbl. f. allg. Path. u. path. Anat. 1900, Nos. 3, 4. > ** Befund v. Smegmabacillen im menschlichen Lungenauswurf,” Berlin, klin. Woch., 1898, No. 37. aon inige ‘Bemerkungen iiber d. Vorkommen v. Smegmabacillen im Sputum,” ibid., 1898, p. 880. 5 In its place Czaplewsky recommends the use of a solution prepared by dis- solving 1 gram of fuchsin together with 5 c.c. of liquefied carboliec acid in 50 e.c. of glycerin and diluting to 100 ¢.c. with water. The solution does not oe e rise to the unsightly precipitates which are seen with the usual solution of earbol fuchsin, unless filtered. * Ref. in Centralbl. f. Path. u. path. Anat., 1901, vol. xii, p. 323. 346 THE SPUTUM Gabbet is replaced by the following: water, 50 c.c.; alcohol, 50 c.c.; lactic acid, 2.5 grams; and methyl blue to saturation. ‘The cover- glass specimens or slides are immersed in this solution for from fifteen to twenty seconds while gently agitating. Gabbet’s method of staining is very convenient, and is the one most generally employed. ‘The smegma bacillus, however, is also stained.* 2. The Weigert-Ehrlich Method.—Dried specimens are prepared, and stained for twenty-four hours with a solution of fuchsin in aniline- water. ‘lhe staining fluid is prepared as follows: A. test-tube full of water is shaken with about 20 drops of pure — aniline oil and, after standing for a few minutes, filtered through a moistened filter. ‘Io this solution a few drops of a concentrated alcoholic solution of fuchsin or of methy] violet are added until the mixture becomes slightly cloudy—~. e., until a metallic lustre is noted on the surface. After twenty-four hours the preparations are washed with water in order to remove an excess of staining fluid. ‘They are then immersed for several seconds in a dilute solution of nitric or hydrochloric acid (1 to 6, 1 to 3, or 1 to 2), and washed again with water or with absolute alcohol. At this time the specimens should have a faintly red or violet color. They are then dried, and mounted as usual. If it is desired to use a counter-stain, Bismarck brown, vesuvin, or methylene blue in watery solutions may be used. Into such a solution the specimen is placed after treatment with nitric acid and washing in water. It remains for about two minutes, and is then washed, dried, and mounted as above. 3. Ziehl-Neelsen’s Method.—A mixture of 90 parts of a 5 per cent. solution of carbolic acid and 10 parts of a concentrated alco- holic solution of fuchsin is used. The procedure is the same as that described under the Weigert-Ehrlich methd. It is usually not necessary to stain the preparations for twenty-four hours, however, and as a rule it is sufficient to place a few drops of the staining fluid upon the preparation and to heat over the free flame as described when the specimen is decolorized as before. In this manner excellent results may be obtained in a few minutes. Stained according to one of these methods, the bacilli appear as rods, measuring about 1.5 to 3.5 y in length by 0.2 in breadth (Plate XVIII, Fig. 1). Much larger specimens may, however, also be seen, up to 11 / in length. he shortest forms are commonly straight; the common types are usually slightly curved. ‘They may occur joined in chains of two or three, and branching forms . have also been observed. Occasionally one may see a couple of organisms, each bent to a crescent, linked in the form of the letter 5. 1 Friinkel, Berlin. klin. Woch., 1884, vol. xxi, p. 195; and Deutsch. med. Woch., 1887, vol. xvii, p. 552 BACTERIOLOGY OF THE SPUTUM 347 ery commonly they are beaded, and it is possible to make out from 1 to 8 clear spaces in an organism which are separated by round or rod-shaped granules, which are deeply stained and appear to lie in a lightly staining capsule. ‘The small hyaline bodies were once regarded as spores, but it is more likely that they are vacuoles. Sometimes bacilli are seen which have club- or knob-shaped enlarge- ‘ments at the extremities. ‘These enlargements likewise have been _yiewed as spores, while others look upon them as products of degenera- ‘tion. When present in large numbers, the bacilli are often seen in clumps, as though they had been agglutinated, but in every specimen isolated organisms are also found scattered through the field; or two or three in groups. Cultivation of the Tubercle Bacillus.—The cultivation of the tubercle bacillus is best accomplished on blood serum or glycerin agar (agar with 6 per cent. of glycerin added) at a temperature of 37° or 38° C. Below 30° C. and at a temperature higher than 42° C. \the organism does not grow. Primary inoculation from the tissue should be made on blood serum, as the bacillus usually does not |grow on glycerin agar when this is inoculated directly from the ‘tuberculous focus. Subcultures, however, grow readily on glycerin agar and more rapidly than on blood serum. ‘The individual colo- (nies appear like small, dry scales, which gradually coalesce and form ja wrinkled film of a dull, whitish color. Older cultures present a } brownish or gray elena color. An adequate idea may be formed of the growth of the organism after two or three weeks. Sunlight rapidly kills the tubercle bacillus. Number in Sputum. —The number of bacilli which may be found in a sputum varies greatly, and while in general it may be said that it is in direct ratio to the intensity of the disease, and may thus be con- ‘sidered of prognostic value, too much reliance should not be placed upon this statement, as in acute miliary tuberculosis, and in cases that have gone to the formation of cavities, the number may be small _or they may be absent altogether. In an incipient case, on the other | hand, in a little mucoid sputum the number may be large. If the ‘number of bacilli steadily decreases in a series of examinations at intervals sufficiently long, the patient may be regarded as improv- ing, but here the constitutional symptoms and local signs give much more accurate information. If on repeated examination large numbers of tubercle bacilli are found, the disease has in all probability advanced to cavitation (Brown). In tabulating the number of tubercle bacilli in reports one may adapt Gaffky’s scheme, modified by L. Brown as follows (,'; oil immersion; ocular 1; B. & L.): 1. Only 1 to 4 in a whole preparation. 2. Only 1 bacillus on an average in many fields. 348 THE SPUTUM 3. Only 1 bacillus on an average in each field. 4, 2 to 3 bacilli on an average to each field. 5. 4 to 6 bacilli on an average to each field. 6. 7 to 12 bacilli on an average to each field. 7. 13 to 25 bacilli on an average to each field. 8. About 50 bacilli on an average to each field. 9. 100 or more bacilli on an average to each field. 10. Enormous numbers on an average to each field. An attempt has been made to attach prognostic significance to the. form and grouping of the tubercle bacilli in the sputum. ‘To judge | from’ the experience gathered at Saranac, it appears that virulent | and attenuated forms of tubercle bacilli possess practically the same | morphology and that short bacilli usually represent a younger growth. | | Arrangement of the bacilli in clumps is more apt to be found in the | severer cases, but may occur in all (Brown). Of the variations in number and form of the tubercle bacilli during | | treatment with Koch’s tuberculin it is unnecessary to speak at this | place, as the prognostic significance attaching to such variations is questionable.* The Diplococcus Pneumonie.—The Diplococcus pneumoni of Frinkel and Weichselbaum, also commonly termed the pneumococcus, is the recognized cause of acute croupous pneumonia in the majority of cases. It is then seen in the sputum in large numbers and recog- nized by its capsule. It may, however, also occur in the mouth of perfectly healthy individuals, so that its diagnostic significance is somewhat limited. ‘lo demonstrate the organism smears on slides or cover-glasses are placed for one or two minutes in a | per cent. solution of acetic acid; they are then removed and the excess of acetic acid drawn off, when they are allowed to dry in the air; they are subsequently placed for several seconds in saturated aniline-water and gentian-violet solution, washed in water, and examined. Rod- shaped diplococci (Fig. 123), surrounded by a capsule, which latter is considered the characteristic feature of this organism, will be seen in cases of acute croupous pneumonia.” As a rule the capsule is not well shown in this way. ‘The best results are obtained with Buerger’s method.’ Smears are prepared as usual. As soon as the edges begin to dry they are covered with ANTE s fluid,* saturated with bichloride of mercury (ordinarily about 5 per cent.). The specimens are gently warmed over the flame for ‘EF. Fischel, Unters. iiber d. Morphol. u Biol. d. Tuberculose-Erregers, 1895. Gaffky, Mittl. aus. d. Kais. Gesundh. Anz., vol. xi, p. 126; L. Brown, Jour.% Amer. Med. Assoc., 1903, vol. xl, p. 514. ? Vrinkel, Zeit. f. klin. Med. 1886, vol. ii, p. 437. Weichselbaum, Wien. med. Woch., 1886, vol. xxxix, pp. 1301, 1339, 1367. * Buerger, L. Med. News., Dec. 10, 1904. * Composition of Miiller’s fluid: 2.5 grams potassium bichromate, 1 gram sodium sulphate, and 100 ¢.c. of water. ' BACTERIOLOGY OF THE SPUTUM 349 about three seconds (using cover-glass smears), rapidly washed in _ water, flushed once with Rice ‘ohol (80 to 95 per cent.), and then treated with ordinary tincture of iodine for one or two minutes. ‘The iodine in turn is thoroughly washed off with alcohol and the preparations dried in the air. “They are then stained for two to five seconds with gentian aniline-water (aniline- oil 10 ¢.c., water 100 c.c.; shake, filter, and add 5 c.c. of a saturated alcoholic solution of gentian violet; or 10 per cent. aqueous fuchsin solution, viz., saturated rik oholic axinuon of fuchsin 10 ¢.c. and water 100 .0.). Washing with a 2 per cent. aqueous solution of salt completes the process. ‘The preparations are examined in a drop of the salt solution and ringed with vaselin. With this method there is visible a refractile, deeply staining, regularly outlined, narrow, elliptical capsule membrane, separated from the diplococcus by a clear area of capsular substance which either remains unstained or takes a faint color. Fic. 123.—Pneumococcus from bouillon culture, resembling streptococcus. (Park.) If smears are to be made from cultures or from material which in itself is essentially non-albuminous, Buerger directs that a drop of blood serum diluted with an equal amount of saline solution should be placed upon the slide or cover, and that the smear be made in this. _ Epstein finds that albumen-water (egg albumen shaken with an equal _yolume of water or normal salt solution) works just as well and will keep for two or three weeks. The Bacillus of Influenza.—T‘he bacillus of influenza was discovered in 1892 by Pfeiffer. It is found in the bronchial sputum in large numbers and is essentially characterized by its minute size, measuring only 0.2 to 0.3 # in breadth by 0.5 in length (Fig. 124). The organ- isms occur for the most part singly, but may also form chains of | threes and fours. In suitably stained specimens they may at first sight appear as diplococci, owing to the fact that the poles are stained 350 THE SPUTUM ! more deeply than the intervening portion. Carbol fuchsin diluted in the proportion of 1 to 10 with water stains the bacillus very | and brings out the polar staining. | ‘The organism is non-motile and forms no spores. It can be grown on media containing blood or serum (blood agar, hydrocele agar, Loffler’s serum). Human blood and pigeon blood are the best, Growth, however, in any event is slight and occurs slowly. In order to cultivate the influenza bacillus from the sputum, this is collected in sterile cups and examined without delay. ‘The sputa are washed in sterile bouillon or sterile normal salt solution and cultures made on blood agar. (Boggst recommends pigeon-blood agar or agar to which sterile fetal blood has been added.) ‘Tiny, water-clear colonies then develop, as described by Pfeiffer. On the fetal-blood agar Boggs noted that involution forms appear earlier and in much greater num- ber than when pigeon, rabbit, or adult human blood was used. Some of these forms are so large and irregular as to give at first sight the impression of a mixed infection. From the blood the organism is rarely obtained. Influenza-like bacilli have been found in whooping-cough sputa by Spengler, Joch- mann, and Krause, and more recently by Fie. 124,—Influenza bacilli, Wollstein. ‘The organism in question has been named the Bacillus pertussis, Eppen- dorf. According to Spengler the bacillus of Czaplewski and Hensel is only a contaminating pseudodiphtheria bacillus. To cultivate the Bacillus pertussis the sputum masses coughed up after a paroxysm are washed in six successive beakers of peptone water and spread upon blood-agar plates prepared by mixing placental blood with melted agar. ‘The predominating colonies are then small, transparent, dew-drop like, and not surrounded by a hemolytic zone, as in the case of the pneumococcus and streptococcus. Microscopic- ally they appear as slightly raised, almost structureless droplets. After forty-eight hours the colonies show a slightly granular centre. ‘The bacilli also grow in bouillon to which a drop of fresh or hemolyzed blood is added. On ascitic fluid agar, glycerin agar, Léffler’s serum, plain bouillon, serum broth, milk and gelatin no growth takes place. ‘The organisms are not motile. ‘They are short, plump, ovoid, with rounded ends, lying singly or in small groups between the pus and epithelial cells of the sputum ‘They are decolorized by Gram’s method. Somewhat larger forms are found in older cultures, and Spengler speaks of very long chains. ? Amer. Jour., Nov., 1905, p. 902. N . | | | t | | | | f BACTERIOLOGY OF THE SPUTUM 351 Wollstein’ obtained agglutination with the serum of the correspond- ing child in dilutions of i to 200 and occasionally of 1 to 500. The Smegma Bacillus—In a few isolated cases the smegma bacil- lus has been encountered in the sputum, and, as I have already stated, the same organism may normally be present in the saliva, the coating of the tongue, the tartar of the teeth, ete. Like the tubercle bacillus, it resists the decolorizing action of acids when once stained, and may hence be confounded with it unless special precautions are observed (see Urine). The Typhoid Bacillus—It has been conclusively shown that the typhoid bacillus can be present in the sputum of typhoid patients, especially if there is a coexistent bronchitis or pneumonia.’ The Plague Bacillus—The plague bacillus is seen in the sputum in enormous numbers in cases of the pneumonic type of the disease. By direct observation, however, it may not be recognized immediately, and it is best in every case to resort to culture as well (Fig. 44, page 176, see Blood). ‘The organism may be found in the sputum on the first day of the disease. Micrococcus Catarrhalis.—This organism is frequently seen in the sputa and nasal discharge. It is larger than the common staphylo- cocci, but, like these, frequently occurs in lateral pairs, the contiguous sides being concave. Micrococcus Tetragenus.—'his organism is frequently seen in the sputum under the most varied pathological conditions and may also occur in the mouths of perfectly healthy individuals. It is a coccus occurring in fours, each measuring about 1 » in diameter. ‘The form which is found under normal candor in contradistinction to dis- ease, cannot be cultivated. Staphylococci and Streptococci may be found in the mouths of apparently healthy individuals, but are more commonly encountered in inflammatory conditions of the most divers kinds. Where cavity formation is going on in the lungs they are usually very numerous. Streptothrices——Within recent years there is a tendency among pathologists to abandon the older terms actinomyces, cladothrix, etc., and to speak of infections with branching mycelial organisms under the collective term streptothricosis, designating the specific variety by its special term. Up to 1902 about 100 cases of supposed cattle actinomycosis had _ been reported 1 in the United States, as occurring in man (Ewing), -but it is difficult to say how many of the older cases really belonged to this order; in the light of recent investigations it seems not unlikely that many were referable to different species. In the cattle disease yellow granules (so-called sulphur granules) may be found in the pus derived from actinomycotic tumors, in the 1 Jour. Exper. Med., 1905, vol. vii, p. 335. 7M. W. Richardsen, Boston Med. and Surg. Jour., Feb. 5, 1903. 352 THE SPUTUM sputum, and in the feces, when the disease has attacked the lungs and | intestines respectively, which measure from 0.5 to 2 mm. in diameter, — If such a granule is examined microscopically, slight pressure being applied to the cover-glass, it will be seen to consist of numerous threads which radiate from a centre in a fan-like manner and present club- shaped extremities (Fig. 125). The cattle organism is termed the Streptothrix (Actinomyces) bovis communis (Streptothrix actinomycotica, or ray fungus). It may be demonstrated in the following manner: Dried cover-glass preparations — are stained for five to ten minutes with aniline-water—gentian violet (see Weigert-Ehrlich stain for tubercle bacilli), when they are rinsed in normal salt solution, dried between filter paper, and transferred for two or three minutes to a solution of iodopotassic iodide (1 to 100 Fic. 125.—Actinomyces. (Musser.) or | to 150). ‘They are then again dried between layers of filter paper, decolorized in xylol-aniline oil (1 to 2), washed in xylol, and mounted in balsam. ‘The mycelium assumes a dark-blue color.’ ‘The organism is acid fast, but loses its color on washing with alcohol (95 per cent:). In addition to the cattle cases there exists a group of pulmonary cases which present the clinical features of tuberculosis, broncho- pneumonia, or gangrene, but in which the infecting agent is a species of streptothrix different from the cattle variety. About 30 cases of this kind have been reported (1906). Different species have been described, such as the Streptothrix eppingert (Cladothrix asteroida), Streptothrix pseudotuberculosa, Flexner; Streptothrix hominis, Fouler-_ ton, and Streptothrix isreeli. 7 The organism is found in the sputum, often in the form of small, grayish-yellow granules. ‘These are made up of a mycelium of branching organisms, which in the unstained specimen appear as fine, homogeneous, glistening threads, about two to four times as wide as a tubercle bacillus. They are acid fast, but can be decolorized with 1 R. Paltauf, Sitzungsber. d. IX. K. Gesellsch. d. Aerzte Wien, 1886. BACTERIOLOGY OF THE SPUTUM , 353 alcohol. In such specimens many of the threads present a beaded appearance e and sometimes seem to be breaking up into short rods of varying length. With Gram some varieties stain well, while others do less so. Culture yields uncertain results. F (ecier obtained no growth. Kppinger succeeded with gelatin, inspissated horse serum, maltose agar, and potato. Lirerature.—Ashton and Norris, Jour. Amer. Med. Assoc. Sept. 9, 1905. Flexner, ‘Trans. Assoc. Amer. Phys., 1898, vol. xii. Warthin and Olney, Amer. Jour., Oct., 1904. W.G. Ewing, Johns Hopkins Hosp. Bull., 1902, vol. xiii. J. Ruhrih, Annals of Surg., 1899, vol. xxx (analysis of 62 cases). Blastomycetes.—In the rare cases of systemic blastomycosis blasto- mycetes may be demonstrable in the sputum. Such a case has Fig. 126 —Blastomycetes. Smear from sputum mounted in 1 per cent. potassium hydrate solution, showing circular and budding organisms. 1200. (Eisendrath and Ormsby.) been described by Eisendrath and Ormsby.‘ For the examination of pus or sputum the writers recommend the addition of a little 10 per ‘cent. NaOH solution to the specimen and to examine unstained with a % or + objective. The refractile parasite is thus well brought out. (Figs. 126, 127, and 128.) Molds.—Of other fungi which are occasionally observed, there may be mentioned various varieties of mucor and aspergillus. Some of these organisms (Mucor corymbifer and Aspergillus fumigatus) have been found associated with cavity formation and seem to have ‘ Journal Medical Association, October 7, 1905. 23 854 THE SPUTUM pathogenic properties. ‘They may at times overgrow the saprophytic bacilli (Pneumonomycosis aspergillina, seu mucorina). ‘They are best. studied in the fresh specimen, not stained (Figs. 129 and 130). Sarcina pulmonalis has been found at times, especially in the, mycotic bronchial plugs occurring in putrid bronchitis. It is usu-. ally smaller than the Sarcina ventriculi, but larger than the variety observed in the urine; it presents the characteristic form of the latter, Fic, 127.—Blastomycetes. Smear from growth on media, five weeks old, in 1 per cent. potassium hydrate solution.. Low power. (Eisendrath and Ormsby. ) Oidium albicans may be seen in children, and is usually derived from the mouth. Crystals.—Of crystals which may occur in sputa, it will be neces- sary to consider briefly the crystals of Charcot-Leyden, hematoidin, cholesterin, margarin, tyrosin, calcium oxalate, and triple phosphates. Charcot-Leyden Crystals.'—'l"hese crystals were discovered in the sputa of patients suffering from bronchial asthma, and were supposed 1 Leyden, Virchow’s Archiv, 1872, vol. liv, p. 324. Schreiner, Liebig’s Annal., 1878, vol. exciv, p. 68. Cohn, Centralbl. f. allg. Path. u. path, Anat., vol. x, p. 940. Brown, Phila, Med, Jour., 1898, p. 1076, h BACTERIOLOGY OF THE SPUTUM 355 to stand in a causative relation to the disease. ‘This view has been abandoned, and it is known that they may occur in other diseases as well. But while their presence is almost constant in bronchial asthma at a time when Curschmann’s spirals can also be demonstrated, they are only exceptionally met with in other diseases, such as acute and chronic bronchitis, phthisis, ete. ‘They were formerly regarded as identical with Bdttcher’s sperma crystals, but it has been shown that this is not the case. ‘They are straight, hexagonal, double pyramids, | ‘and appear under the microscope as flattened needles of variable size \(Pig. 112). Some attain a length of from 40 1 to 60 4, while others are searcely visible even with a comparatively high power of the micro- scope. ‘They show a feeble, positive, double refraction, and have but one optical axis, while the sperma crystals are biaxial and strongly 1 double refracting. Their behavior to solvents is essentially the | same as that of the sperma crystals, but they differ from these in their insolubility in formol. They are colored yellow with Florence’s | é Fic. 128.— Higher magnification of Fig. 127. > 1200. 396 THE SPUTUM reagent, while the sperma crystals are stained a bluish black. Ve curiously the appearance of Charcot-Leyden crystals is closely asso- ciated with the presence of eosinophilic leukocytes, and they have hence been termed leukocytic crystals. ‘They may in fact originate within the cells. In bronchial asthma it is not uncommon to find: microscopic preparations: of the sputum literally studded with eosinophilic leukocytes and free granules. Outside the sputum they are also found in the blood, in myelogenous leukemia, and in the stools in association with animal parasites. ‘hey readily form in both) normal and abnormal red bone-marrow, and _ excellent specimens may be obtained for purposes of demonstration if a piece of a rib is allowed to remain exposed to the air for a few days. ‘The marrow then usually contains large numbers. ‘The crystals also form in decomposing viscera in general, and at times form a complete covering of old anatomical preparations.) Their occurrence may be re- garded as evidence of retrogres-| sive changes in the cellular elements of an organ. Of the’ relation which they bear to the eosinophilic leukocytes, with which they are so constantly associated, nothing is known, The Charcot-Leyden crystals can be stained with the triacid stain, with thionin, with the eosinate of methylene blue, and other dyes. essen TY Hematoidin crystals may be ee Tat Dee yan Peta feed Reis observed in the sputa following extravasations of blood into the lung. They frequently occur in the form of ruby-red columns or needles; amorphous granules, however, are also seen, enclosed in the bodies of leukocytes, in which case they are probably always indica- tive of a previous hemorrhage, while the needles are generally observed when an abscess or empyema has perforated into the lungs. The substance is derived from blood pigment, and is now known to be identical with bilirubin. | Cholesterin crystals are at times seen in the sputa in cases of phthisis, pulmonary abscess, and, in general, whenever old accumula- tions of pus have entered the lung from a neighboring organ. ‘They are readily recognized by their characteristic form and chemical properties (see Feces). 4 Fatty acid crystals are frequently observed in cases of putrid bron- chitis and gangrene of the lung, and also in cases of bronchiectasis and phthisis, ‘They occur in the form of single needles or groups BACTERIOLOGY OF THE SPUTUM 307 vw yf needles, which are long and pointed. ‘They are easily soluble in other and hot alcohol; insoluble in water and acids. Chemically, they are probably composed of the higher fatty acids, such as palmitic and stearic acids. - Tyrosin crystals have been observed in cases of putrid bronchitis, gerforating empyema, etc. Leucin is then usually also present, occur- cing in the form of highly refractive globules. For the recognition of these bodies, particularly of tyrosin, a chemical examination should always be made, as crystals of the soaps of fatty acids have frequently been mistaken for those of tyrosin (see Urine). _ Calcium oxalate crystals are rarely seen. Fiirbringer observed them in large numbers in a case of diabetes, and Unger found them | \ | na case of asthma. ‘They are readily recognized by their envelope | Fie. 130.— Aspergillus fumigatus of the lung, partly schematic: a, mycelium of aspergillus | in roset-like rays; b,sporangium. X 285. (Weichselbaum.) form and central cross, but they occur also in amorphous masses. They are soluble in mineral acids; insoluble in water, alkalies, organic acids, alcohol, and ether. _ Triple phosphate crystals also are rarely seen, but may occur in tases of perforating abscesses, etc. ‘hey are recognized by their voffin-lid shape and the readiness with which they dissolve in acetic acid. The Pneumoconioses. Anthracosis.—''’o some extent particles of rarbon may be found in the sputum of almost every individual. ‘The ‘xpectoration in such cases is of a pearl-gray color, and is brought Ip in larger or smaller masses, especially in the morning upon rising. _arger amounts are noted in miners and in those who are brought nto close contact with coal-dust. Microscopically, particles of car- } / ) 358 THE SPUTUM bon and epithelial cells, of the alveolar type, as well as eukon loaded with the pigment, are seen. Siderosis—In_ siderosis the sputum presents a brownish-black | color and contains cells enclosing particles of ferric oxide. ‘These | may be readily recognized by treating with a drop of ammonium sulphide or potassium ferrocyanide solution in the presence of hydro- : chlorie acid, when a black color on the one hand or a blue color on | the other is obtained in the presence of iron. | Chalicosis.—In chalicosis silicates are found in the sputa.* | CHEMISTRY OF THE SPUTUM. In addition to the substances described, sputum contains certain albumins, volatile fatty acids, glycogen, ferments, and various inor- ganic salts. Among the albumins may be mentioned serum albumin, and especially mucin, which is often present in large amounts. In pneu- monic and purulent sputa albumoses also have been found. In order to demonstrate the presence of serum albumin the sputa are treated with dilute acetic acid, when the filtrate is tested with potassium ferrocyanide, as described in the chapter on Urine. Serum albumin is, of course, found in notable quantities in cases of edema of the lungs. Especially interesting is the albuminous eapectoration which at times follows thoracentesis. ‘lhe amount of sputum usually varies between 200 and 900 grams, but may be much larger and may reach 2000 ¢.c. or even more. Occasionally it begins before the tap- ping is completed or immediately after. More commonly, however, an interval varying from five minutes to one or two hours elapses before the expectoration begins. Its duration is variable. Sometimes it lasts only a few minutes, more often an hour or two, and in rarer cases a whole day or two. ‘The condition is probably due to edema of the lungs:? The volatile fatty acids contained in sputa may be obtained by diluting with water, acidifying with phosphoric acid, and distilling, when the distillate is further examined as described i in the chapter on Feces. Acetic, butyric, propionic, and capronic acids have been found. The fats and fixed fatty acids are extracted from the residue with ether, and shaken with a solution of sodium carbonate in order to transform them into their sodium salts, when the ether is decanted and evaporated, leaving the soaps behind. 1 Betts, “Chalicosis Pulmonum,” Jour. Amer. Med. Assoc., 1900, No. 2. 2 In the United States cases of albuminous expectoration following thoracentesis have peen reported by Pepper, Allen, Pateck, and Riesman. See especially the paper by Riesman, in which a full account of the literature is given, Amer. Jour. Med. Sci., April, 1902, p. 620. . A > ; CHEMISTRY OF THE SPUTUM 359 Glycogen has repeatedly been demonstrated in sputa, and may be ‘detected by Ehrlich’s method (see Blood). The sputa of gangrene of the lung and putrid bronchitis have been shown to contain a ferment resembling trypsin. In order to test for this, the sputa are extracted with glycerin; the examination is then continued as described in the chapter on the Examination of Cystic Contents. The myelin granules, as I have already indicated, consist largely of protagon, lecithin, and cholesterin. CUBA SPE Bahveaye Lele THE URINE. GENERAL CHARACTERISTICS OF THE URINE. Appearance.—Normal urine, just voided at an ordinary tempera- ture, is either perfectly clear or but faintly cloudy, owing to the fact that the acid and normal salts present are all soluble in water. It may be stated, as a general rule, that whenever a urine freshly passed presents a distinct cloudiness, some abnormality exists. When allowed to stand for a time a light cloud develops, which gradually settles to the bottom, constituting the so-called nubecula of the ancients. Examined under the microscope this is found to contain a few round, granular cells, somewhat larger than normal leukocytes, the so-called mucous corpuscles, and a few pavement- epithelial cells, derived from the bladder or genital organs. Chemi- cally the nubecula probably consists of traces of mucus. When kept for twenty-four hours at an ordinary temperature, crystals of uric acid are frequently observed in addition to the above elements, usually presenting the so-called whetstone form. If, how- ever, the temperature at which the urine is kept approaches the freezing point, the entire volume becomes cloudy, owing to precipitation of - acid urates, as these are much less soluble in cold than in warm water; on standing they gradually settle to the bottom of the vessel and form what is known as a sediment, while the supernatant fluid again becomes clear. If kept still longer exposed to the air, at the temperature of the room, the entire volume of urine again becomes cloudy, owing to a diminution of its normal acidity, the result being a precipitation of ammonio-magnesium phosphate, calcium phosphate, and still later, when the urine has become alkaline, of ammonium urate. Gradually a heavy sediment, containing these salts in addition to the constituents of the primitive nubecula, forms at the bottom of the vessel; the supernatant fluid, however, remains cloudy. On micro- scopic examination it will be seen that this cloudiness is due to the presence of enormous numbers of bacteria. 7 : The changes which take place in a normal urine when allowed to stand at ordinary temperature may be tabulated as follows: GENERAL CHARACTERISTICS OF THE URINE 361 1. Urine clear, no sediment; reaction acid. 2. Urine slightly cloudy, owing to development of the nubecula; reaction acid. Mucous corpuscles, Nubecula : Pavement-epithelial cells. 3. Urine clear; the nubecula has settled; reaction acid. { Mucous corpuscles, Epithelial cells, Uric acid crystals, | A few bacteria. a | Sediment + 4, Urine cloudly, owing to the precipitation of phosphates; reaction faintly acid or alkaline. 5. Urine cloudy, owing to the presence of bacteria; reaction alkaline. ( Bacteria, Mucous corpuscles, Epithelial cells, Triple phosphates, Tricalcium phosphate, | Sediment 4 | | Ammonium urate. Color.—The color of normal urine may vary from a very light yel- low to a brownish red, the particular shade depending essentially upon the specific gravity, becoming lighter with a diminishing and darker with an increasing density. Pathologically the same rule holds good, except in diabetes, in which a very high specific gravity is generally associated with a very light color. ‘The reaction of the urine also exerts a marked influence upon its color, an acid urine being more highly colored than an alkaline urine, which can be readily demon- strated by allowing a specimen of acid urine to become alkaline, and by treating an alkaline urine with dilute hydrochloric or acetic acid. At the same time it may be said that every urine darkens slightly on standing, the reaction remaining acid. The various shades observed in normal urines may be grouped under the following headings: 1. Pale urines vary from a faint yellow to a straw color. 2. Normally colored urines are of a golden or an amber yellow. 3. Highly colored urines present a reddish-yellow to a red color. 4, Dark urines vary between brownish red and reddish brown. As these shades may occur in both normal and pathological urines, definite conclusions cannot, as a rule, be drawn from mere inspection. A very pale urine indicates an excess of water, which may be normal, but may also occur in such diseases as chronic interstitial nephritis, diabetes mellitus, diabetes insipidus, hysteria, and the various anemias; it is further seen during convalescence from acute febrile diseases, while a highly colored urine, though also occurring in health, may indicate the existence of a febrile process. The normal color of the urine is probably owing to the presence 369 THE URINE of several pigments, which are most likely closely related to each other and to hematin. In addition to these colors others may be observed at times which are either pathological or accidental—~. e., due to the presence of cer- tain drugs. ‘The former are, on the whole, of greater importance to the physician than those mentioned above, as more definite conclu- | sions can be drawn from their presence. ‘lhe most important pathological pigments are: 1. Blood-coloring matter. ‘The color in such cases may vary from a bright carmine to a jet black, the exact shade depending upon the quantity of blood-coloring matter present, upon changes that the blood may have undergone either before or after being passed, and also upon the presence of the pigment in solution or contained in red corpuscles. 2. Biliary coloring matter. ‘The color here varies from a greenish yellow to a greenish brown. Among the accidental abnormalities in color are those due to the presence of substances like carbolic acid and its congeners, santonin, etc. A milky-colored urine is observed in cases of chyluria. As the recognition of the causes of such alterations, normal, pathological, and accidental, largely depends upon a more detailed study of the individual pigments, this subject will be dealt with more fully farther on. (See Pigments and Chromogens.) Odor.—The odor of the urine is usually of little significance. Normally it resembles that of bouillon, and in some cases that of oysters; it is probably due to the presence of several volatile acids. The odor of urines undergoing decomposition is characteristic and has been termed “the urinous odor of urine,” an ill-chosen term, as this odor is always indicative of an abnormal condition. The ingestion of asparagus, onions, oil of turpentine, etc., pro- duces characteristic odors. Consistence.—Urine, while normally fluid and but slightly viscid, may in disease acquire a marked degree of viscidity, which becomes especially apparent upon attempting its filtration; the liquid passes through the paper with more and more difficulty, and finally clogs its - pores altogether. In old, neglected cases of cystitis it may be ropy and gelatinous. 3 Quantity.—The quantity of the urine is normally subject to great variations, the amount eliminated in the twenty-four hours being influenced by that of the fluid ingested, the nature and quantity of the food, the process of digestion, the blood pressure, the surrounding temperature, sleep, exercise, body weight, sex, age, etc. It is easy to understand, then, why figures given by different observers in different countries should vary considerably. Salkow- ski, in Germany, thus gives 1500 to 1700 ¢.c. as the normal amount; v. Jaksch, in Austria, 1500 to 2000 ¢.c.; Landois and Sterling, in Se GENERAL CHARACTERISTICS OF THE URINE 863 England, 1000 to 1500 ¢.c.; Gautier, in France, 1250 to 1300 ce.e. In the United States I have found an average secretion of from 1000 to 1200 e.c. in the adult male, and 900 to 1000 c.c. in the adult female. It is thus seen that the secretion of urine is greatest in Germany and Austria, where the body weight and ingestion of liquids are greater than in England, France, and the United States. Children pass less, but relatively more (considering their body weight) urine than adults. Women pass somewhat less than men. During the summer months, when a larger proportion of water is eliminated through the skin and lungs than in cold weather, less urine is voided. ‘The same occurs during repose, more urine being passed during active exercise, and hence less during the night than during the day. The amount of urine secreted in the different hours of the day varies greatly, reaching its maximum a few hours after meals. It decreases toward night, and reaches its lowest point in the first hours of the night, after which it begins to rise rapidly until 2 or 3 o’clock in the morning. The ingestion of large amounts of liquid, of course, increases the daily amount considerably, and 5000 c.c. may be passed under such conditions by an individual in good health, while it may decrease to 800 or 900 c.c. when but little liquid is taken. After the ingestion of much solid food the secretion of urine is temporarily diminished. Water containing no salts possesses distinct diuretic properties, as do also beer, wine, coffee, tea, ete. The most important medicinal diuretics are digitalis, squill, broom, spirit of nitrous ether, juniper, urea, etc. Pathologically the amount of urine varies within wide limits. In a given case, moreover, it may be exceedingly difficult to determine whether or not the secretion is within physiological limits. As a general rule, whenever less than 500 ¢.c. or more than 5000 c.c. are passed some abnormal condition exists, providing all other causes which might lead to the secretion of such an amount can be elimi- nated. Clinically we speak of polyuria and oliguria. Polyuria.—Polyuria is observed in many diseases, and is present under such varied conditions that a classification is only warrantable upon a hypothetical basis, especially as the causative factors concerned in its production are mostly unknown. As polyuria is almost invariably associated with diabetes mel- litus, its presence in any case should always excite suspicion and lead to a proper examination. ‘The quantity of fluid eliminated in diabetes is usually dependent upon the amount ingested. ‘The excre- tion of a proportionately large amount of fluid, however, does not 364 THE URINE necessarily follow the ingestion directly, and retention of a large amount may occur; it has been shown, as a matter of fact, that the diabetic patient excretes liquids with greater difficulty than the healthy subject. At the same time it should be borne in mind that — the polyuria in diabetes is not necessarily continuous, and_ that periods during which a normal or even a subnormal amount is observed may alternate with true polyuria. From 2 to 26 or even 50 liters may be passed within twenty-four hours. Intercurrent dis- eases of a febrile character may modify the quantity very materially and cause the elimination of a normal or subnormal amount. ‘The cause of the polyuria in diabetes mellitus is unknown. The polyuria associated with the resorption of large pericardial, pleural, ascitic, and subcutaneous effusions is more readily under- stood, although the primum mobile may be unknown; it depends in such cases entirely upon the presence of excessive quantities of fluid in the bloodvessels. A form of polyuria which has been termed “epicritic polyuria” is frequently observed during convalescence from acute febrile dis- eases, and is of prognostic importance. Its occurrence in a given case is regarded by many as a good omen, especially in typhoid fever; still it must not be forgotten that a polyuria may occur after subsidence of the fever, and be followed by a considerable degree of oliguria, and in some cases may precede death. A polyuria of this kind probably always indicates the elimination of waste products which have accumulated in the blood during the course of the disease, but it may, at the same time, be due to the presence of retained water. Second in constancy is the polyuria associated with granular atrophy of the kidneys. Cases have been reported in which 10,000 c.c. of urine were secreted in the twenty-four hours; 2000 to 4000 c.c. represent the usual amount. Polyuria is of frequent occurrence early in the course of renal tuber- culosis, the increase amounting to one-half of the normal amount. Very curiously, polyuria may occur also in association with mul- tiple myelomas of the bones and the presence of Bence Jones’ albumin in the urine. In one of the cases reported by Hamburger,’ which I had occasion to study in greater detail from a chemical point of view, 3500 c.c. were voided in the twenty-four hours. ‘The symptom, however, is not constant. . Polyuria, furthermore, has been observed in the most diverse diseases of the nervous system, both functional and organic. It is frequently observed both as a transitory and a more or less _per- ' manent symptom in cases of hysteria. Large quantities of a very pale urine are secreted after the occurrence of severe hysterical ! “Two Examples of Bence Jones’ Albuminuria associated with Multiple Mye- loma,” Johns Hopkins Hosp. Bull., Feb., 1901. C.E. Simon, Amer. Jour, Med. Sci., 1902, vol. cxxiii, p. 954. GENERAL CHARACTERISTICS OF THE URINE 365 seizures, but the same may be observed throughout the course of the disease. A similar condition is frequently seen in neurasthenia, migraine, chorea, and epilepsy. Generally speaking, it may be said that a paroxysmal polyuria in nervous diseases is associated with functional derangement, while a continuous polyuria appears to be connected rather with true organic changes. It has been observed in certain cases of tabes, cerebrospinal ‘and spinal meningitis; during the first stage of general paresis; in association with tumors involving the medulla, the cere- bellum, and the spinal cord; in injuries affecting the central nervous system, in Basedow’s disease, etc. Cases of. idiopathic diabetes insipidus also should probably be classified under this heading. Enormous quantities of urine may be secreted in this disease, which are equalled only by cases of diabetes mellitus, and may at times reach 45 liters per diem. Oliguria.—Oliguria is, on the whole, more frequent than polyuria, and is met with in almost all conditions associated with a lowered blood pressure. First in order stand those cases of cardiac disease in which compensation has failed, whether the cardiac weakness 1s primary or occurs secondarily to other diseases—. e., pulmonary, hepatic, and renal. The oliguria observed in the so-called continued fevers, notably typhoid fever, is probably also referable to cardiac we eakness. It should be remembered, however, that a larger proportion of water is eliminated through the skin and lungs than normally, and that a retention of fluids also undoubtedly occurs which is not due to cardiac weakness; still other factors may be concerned in its pro- duction. The oliguria occurring in acute nephritis and in chronic paren- chymatous nephritis in all probability depends largely upon mechani- cal causes, the increased intra-canalicular resistance in the form of desquamated epithelium and tube casts, as well as the pressure of the exudate upon the bloodvessels obstructing the passage of urine, while the functional activity of the diseased glandular elements is at the same time lowered. Upon mechanical causes, also, depend all those cases of oliguria which are associated with the presence of a stone or tumor pressing upon a portion of the urinary tract. Oliguria may occur as a nervous manifestation in connection with puerperal eclampsia, lead colic, hysteria, psychic depression, preced- ing and during epileptic seizures, etc. Whenever there is a diminu- tion in the amount of bodily fluids oliguria is also observed; this is particularly marked in cholera and following severe hemorrhage. Obstruction to the flow of blood in the vena cava or liver, lead- ing to an increase of venous pressure and a decrease of arterial pressure in the kidneys, likewise results in oliguria, as is seen in atrophic hepatic cirrhosis, acute yellow atrophy, thrombosis of the 366 THE URINE vena cava and the renal vein, or in cases in which pressure is exerted upon these by tumors, ascitic fluid, ete. In any case the oliguria may go on to complete anuria, which condition not infrequently precedes death. Anuria may, however, also occur independently of a preéxisting oliguria, as in hysteria. Specific Gravity.—The specific gravity of normal urine varies — between 1.015 and 1.025, corresponding to 1200 to 1500 c.c., viz., the normal amount of urine voided in twenty-four hours. Pathologically, a specific gravity of 1.002 on the one hand and 1.060 on the other may occur, depending upon the amount of solids and fluids present, increas- ing as the solids increase, the amount of urine remaining the same, and decreasing as the amount of fluid increases, the solids remaining the same. ‘The specific gravity is thus an index in a general way of the metabolic processes taking place in the body. The necessity of determining the specific gravity of the total amount of urine voided in a given case, and not that of an individual specimen passed during the twenty-four hours, becomes apparent upon considering the variations which may occur in the quantity of solids and liquids ingested during the day. ‘The ingestion of large amounts of fluid would, of course, result in the passage of a correspondingly large quantity of urine within the next few hours, containing but a small amount of solids, and hence presenting a low specific gravity. From such an observation it would be erroneous to infer a diminished excretion of solids for the day, as succeeding specimens would in all probability be passed presenting a higher specific gravity. An observation made upon a specimen taken from the collected urine of the twenty-four hours moreover, can only then convey a correct idea if the total quantity is known. From the specific gravity the amount of solids can be calculated with sufficient accuracy for clinical purposes by multiplying the last two decimal points by 2, the number obtained indicating the amount of solids in 1000 c.c. of urine. From the rule, that the specific gravity of a urine is inversely pro- portionate to the amount of fluid eliminated, it must follow that - whatever causes produce oliguria will also produce a high specific gravity, while all those causes which produce polyuria will similarly produce a low specific gravity, with the following exceptions: 1. A diminished amount of urine with a lowered specific gravity — occurs in many chronic diseases and toward the fatal termination of acute diseases, indicating a defective elimination of solids. 2. The same may be observed in certain cases of edema. 3. Following copious diarrhea, vomiting, and sweating. 4. A high specific gravity is associated with polyuria in diabetes mellitus. Unfortunately the determination of the specific gravity and the solids contained in urine does not furnish as valuable information GENERAL CHARACTERISTICS OF THE URINE 267 vo in many cases as would be expected a priori. This is largely. owing to the fact that the organic constituents of the urine have a lower specific gravity than the inorganic salts, and especially the chlorides, which are usually present in considerable amount. It thus not infre- quently happens that the nitrogenous constituents are considerably increased, while the specific gravity is relatively low, owing to the absence or a diminution in the amount of chlorides. In other words, Fie, 132.—The pyknometer. while the specific gravity may be regarded as a fair index of the total amount of solids excreted, its increase or decrease furnishes no information as to the nature of the constituents causing such a change. Determination of Specific Gravity. —The specific gravity of the urine is most conveniently determined by means of a hydrometer indicating degrees varying from 1.002 to 1.040. Such instruments, constructed especially for the examina- Re tion of urine, are termed wrinometers Bes 131 Urinometer. (Fig. 1381). A good instrument should have a stem upon which the individual divisions are at least 1.5 mm. apart, and each division should corre- spond to 0.5 degree. Urinometers may also be purchased which are provided with a thermometer. Every instrument should be carefully tested by com- parison with a standard hydrometer. In order to determine the specific gravity in a given case a cylindrical vessel is nearly filled with urine and the urinometer slowly introduced, 368 THE URINE the reading being taken at the lower meniscus as soon as the instrument has come to rest. Precautions: 1. The urinometer must be given ample room, and the reading should never be taken when the instrument touches the sides of the vessel, as owing to capillary attraction it is otherwise raised, causing the reading to be too high. 2. ‘The instrument must be perfectly dry and clean before being used, and should never be allowed to “drop” into the urine, as other- wise the weight of the instrument is increased by adhering drops of fluid, and the reading is too low. 3. Any foam upon the surface of the urine should first be removed by means of a piece of filter paper, as it interferes with the accuracy of the reading; bubbles of air adhering to the instrument, and thereby elevating it, should be removed with a feather. 4. ‘The specific gravity should always be determined in specimens taken from the twenty-four-hour urine. 5. If the quantity of urine is too small to determine its specific gravity with a urinometer, the following method may be employed: About 50 c.c. of urine are measured into a small bottle provided with a ground-glass stopper, or into a pyknometer like the one pic- tured in Fig. 132, and accurately weighed. ‘The weight of the urine divided by its volume gives the specific gravity, which must, however, be corrected for the temperature of the urine. If accuracy is re- quired, such corrections should be made in every case, as the specific gravity increases or decreases by 1° for every 3° C. above or below the point for which the instrument is registered, viz., 15° C. Determination of the Solid Constituents.—As indicated above, the amount of solids can be calculated with a degree of accuracy sufficient for clinical purposes by multiplying the last two figures of the specific gravity by 2; the number obtained indicates the amount of solids in every 1000 c.c. of urine. If greater accuracy is required, the follow- ing method may be employed: 5 ¢.c. of urine, accurately measured, are placed in a watch-crystal containing a little dry sand (sand and crystal having been previously weighed) ; this is placed over a dish con- taining concentrated sulphuric acid, and under the receiver of an air pump which has been made perfectly air-tight by thoroughly lubricat- ing the ground-glass edge of the bell with mutton tallow and applying the bell with a slightly grinding movement to the ground-glass plate. The receiver is now exhausted and the urine allowed to remain in the vacuum for twenty-four hours, when the bell is again exhausted and left for twenty-four hours longer; at the end of this time the crystal is weighed, the difference between the two weights obtained indicating the amount of solids in 5 c.c. of urine, from which the percentage and total amount are readily calculated. The slight loss of ammonia which results when this method is employed scarcely affects the accuracy of the result. GENERAL CHARACTERISTICS OF THE URINE 369 Reaction. —The reaction of the twenty-four-hour urine is, as a rule, acid; individual specimens, passed in the course of the same twenty- four hours, may be either alkaline, acid, or amphoteric. It has been generally held in the past that the acid reaction of normal urine is due to the presence of diacid phosphates. But it was assumed also that monosodium phosphate was present at the same time. Folin’ has shown that this assumption is not correct, that the phosphates in clear urine are all of the monobasic kind, and that the acidity of such urines is ordinarily greater than the acidity of all the phosphates, the excess being due to free organic acids. An alkaline urine results ‘when the alkalies exceed the acid equiva- lentsin amount. ‘Chis may occur under normal conditions (see below), and is then due to a preponderance of monacid over diacid phos- phates. An amphoteric urine (red litmus turned blue and blue litmus red) is the outcome, when the acid equivalents of diacid phosphates equal the basic equivalents of the monophosphates; this is essentially an accidental occurrence. As the alkalinity of the blood increases the acidity of the urine decreases, until an alkaline urine results. ‘The degree of the alkalinity of the blood, however, depends essentially upon the nature of the food and the secretion of the gastric Juice, viz., the hydrochloric acid. The ingestion of vegetable food, rich in salts of organic acids, which become oxidized in the body to the carbonates of the alkalies, will result in the passage of an alkaline urine, for the alkalies thus formed ~when absorbed into the blood are more than sufficient to neutralize completely all the acids present, and the elimination of neutral sodium phosphate alone takes place. In the case of animal food the reverse holds good. ‘The alkaline carbonates here formed are not sufficient to neutralize the excess of acids, and diacid phosphate of ‘sodium is hence eliminated in large quantity.’ As the alkalinity of the blood is increased during the secretion of the acid gastric juice, it may frequently happen, especially follow- ing the ingestion of a large amount of food, that an alkaline urine is voided. If this does not take place, the acidity of the urine is at ‘least diminished, but increases again during the process of resorp- tion.’ If an acid urine is allowed to stand exposed to the air for a cer- tain length of time, its degree of acidity gradually diminishes and the reaction finally becomes alkaline. At the same time the urine becomes cloudy ; and deposits a sediment, which consists of ammonio- | Magnesium phosphate, MeNH,PO,+6H,O, neutral calcium phos- | phate, Ca,(PO,),, and still later Dering ammonium urate, C,H,- NHD)N, O,, in addition to the constituents of the primitive nubecula 1 Amer. Jour. of Physiol., Feb., 1905, vol. xiii. 2 E. Salkowski u. J. Munk, Virchow’s Archiv, 1877, vol: Ixxvi, p. 500. > Quincke, Zeit. f. klin. Med., vol. vil. 24 [ ws 370 THE URINE —i. e., a few mucous corpuscles and pavement epithelial cells. ‘The. entire volume of urine, moreover, remains cloudy, owing to the: presence of innumerable bacteria. ‘The odor becomes extremely disagreeable and distinctly “urinous.” In short, “ammoniacal de- composition” has occurred. ‘This has been shown to depend upon the action of certain bacteria, notably the Micrococcus ure and the Bacterium urez, which are present in the air." ‘These organisms cause) the decomposition of the urea found in every urine, with the forma-. tion of ammonium carbonate, according to the following equations: CO(NH,), +2H.0 =(NH,).CO; (NH,).CO,;=2NH; +H,O +COs,. I An alkaline urine, the alkalinity of which is not due to ammo- niacal fermentation, however, but to other causes, as indicated above, may, of course, undergo the same change as an acid urine; but it is necessary to distinguish sharply between these two varieties of alkaline urines, as the recognition of the cause of the alkalinity 1 is. very often most important in diagnosis. ‘The distinction is readily made by fastening a piece of sensitive red litmus paper in the cork, of the bottle containing the urine. If the alkalinity of the urine is due to the presence of ammonia, the litmus paper will turn blue, but soon changes to red when exposed to the air; while a urine the alkalinity of which is due to the presence of fixed alkalies will turn red litmus paper blue only when immersed in the urine, the change in color at the same time persisting. As ammoniacal decomposition can also occur within the urinary. passages, it is important, whenever an alkaline reaction due to the presence of ammonia is observed, to test the urine at once upon being voided, or, still better, to procure a portion with a catheter. Such urines are frequently seen in cases of cystitis the result of paralysis, urethral stricture, gonorrhea, etc. In this connection it is interest-. ing to note that whereas in old, neglected cases of cystitis an alkaline reaction is frequently observed, Brown has shown that in the great, majority of cases of cystitis, both acute and chronic, and also in those of pyelitis and pyelonephritis, the urine is acid." | An intensely acid reaction is observed in almost all concentrated urines, especially in fevers, in certain diseases of the stomach asso-. ciated with a diminished or suspended secretion of hydrochloric acid, in gout, lithiasis, acute articular rheumatism, chronic Bright’s dis- ease, diabetes, leukemia, scurvy, etc. Whenever a very acid urine is secreted for a considerable length of time, the possibility of renal. irritation and the formation of concretions should be borne in mind. _An alkaline urine the alkalinity of which is not owing to the pres- | 1 W. Leube, “Ueber die ammoniakalische Harngihrung,” Virchow’s Archiv, 1885, vol. c, p. 555. 2T R, Brown, Johns Hopkins Hosp. Rep., 1901, vol. x, p. 11. Aeil GENERAL CHARACTERISTICS OF THE URINE 371 ence of ammonia, but to fixed alkali, is observed in certain cases of debility, especially in the various forms of anemia, following the resorption of alkaline transudates, the transfusion of blood, frequent vomiting, a prolonged cold bath, ete. It may also be due to the ingestion of certain drugs, viz., salts of the organe acids and alkaline carbonates, the former being transformed into the latter, as has been mentioned. An increase in the degree of acidity may similarly take place after the ingestion of mineral acids. Of interest is the observation of Pick’ that in twenty-four to forty- eight hours after the crisis in pneumonia the urine shows a marked decrease in its acidity, becoming neutral or even alkaline. ‘This phenomenon, which was observed in 3i out of 38 cases, persists fora day or a day and a half, and then the acidity returns. In all likelihood the change i is due to absorption of the large amounts of sodium which are present in the exudate. An increase in the acidity of the urine upon standing has repeat- edly been observed, and is probably due to the formation of new acids from preéxisting acid-yielding substances, such as certain earbohydrates, alcohol, etc., which have undergone fermentation. This phenomenon is frequently observed in diabetic patients. A decrease in the acidity of normal urine upon standing, however, is the rule, owing to a gradual decomposition of sodium urate by the acid sodium phosphate, acid sodium urate, and, later on, uric acid resulting, which are thrown down as a sediment in consequence ‘of the diminished acidity of the urine, and which, hence, no longer ‘influence its reaction. ‘This is shown in the equations: ‘ie NaH.PO, +C,H,Na.N,O; eT Na,HPO, +C;H;} YaN,Os. yA NaH.PO, +C;H;Na AGE ss Na,HPO, +C;H,N,QO3. Determination of the Acidity of the Urine.—Folin has shown that ithe methods of Freund, Lieblein and Nigeli, which have heretofore been largely in use, are inapplicable and has suggested the following | procedure: Folin’s Method.—'The total acidity which indicates the acidity due to diacid phosphates and free organic acids is first determined as Hollows: 25 c.c. of urine are treated with 1 or at most 2 drops of 4 ‘per cent. alcoholic solution of phenolphthalein and 15 to 20 grams of powdered potassium oxalate. ‘The solution is shaken for about a ‘minute and titrated at once with decinormal sodium hydrate solution “until a faint, yet distinct pink color is obtained. ‘The flask should be shaken during the titration, so as to keep the solution as strong as possible in oxalate. ‘The acidity i is expressed in terms of decinormal ‘sodium hydrate solution for the total amount of urine of tw enty-four hours. ‘The total acidity is termed 'T. 1“The Urine in Pneumonia,’’? Minch. med. Woch., 1898, No. 17. | In a second specimen the total phosphates are then determined, the value being termed P (see Phosphates). ‘The result is expressed in| terms of decinormal acid, viz., alkali as above (1 ¢.c. 77 =7.1 mgrms, | of P,O,). ‘TT minus P then indicates the acidity due. 2 uncombined | organic acids (O. A.), and the difference the mineral acidity (M. A,).. Tt may happen that the acidity calculated from the total phosphates, is greater than the titrated acidity; in that case practically no free organic acids are present and the titrated acidity represents the amount of phosphates present in the diacid form. Urines of this kind | are turbid, unless they are also free from calcium (Folin). As average normal value for the acidities a the total bulk of twenty- four hours’ urine Folin obtained 617 (e.c. 7/9 n. acid, viz., alkali), of which 304 was referable to mineral and 313 to organic acidity. The corresponding minimal and maximal values were ‘I’ 554, viz., 669; M. A. 204, viz., 417; O. A. 252, 'viz., 378. With this method a complete revision of all the work previously done will be necessary. The older results given above have reference, only to the old method of titration with a one-tenth normal solution of sodium hydrate. | 3792 THE URINE LirERATURE.—-Folin, Amer. Jour. of Physiol., 1903, vol. ix, p. 265; and ibid., 1905, Feb., pp. 53 and 54, and ibid., p. 102. | j Determination of the Mineral Acidity or the Excess of Mineral Acids or Bases.—Folin’s method may be employed instead of determining all the different metals and acids separately as Bunge, Magnus Levy and others have done. To 25 c.c. of urine in a platinum dish is added from 0.3 to 0.5 gram of potassium carbonate, weighed within an accuracy of two- tenths of amgrm. ‘The solution is evaporated to dryness, and the residue ignited, when perfectly dry, over a radial burner, using at first a very low heat, and at no time allowing the dish to become more: than faintly red hot. ‘The dish is heated at this temperature for one hour, then cooled, when 10 c.c. of hydrogen peroxide are added and evaporated. The dried residue is ignited as before for one hour. It is dissolved 1 in a0) e5 Crs of tenth normal hydrochloric acid and water (50 to 75 c.c. 4%, HCl), transferred to an Erlenmeyer flask, boiled to remove carbonic acid, and cooled. One or two drops of phenol- phthalein solution and a few crystals of neutral potassium oxalate (to precipitate the calcium) are added, and the solution titrated as. usual. ‘The ammonia, the acidity of the hydrogen peroxide, and the acidity of the organic sulphur (neutral and ethereal, 8 grams of which | are taken to represent 1 c.c. tenth normal acid) must be subtracted from the result given by the direct titration. ‘These values, as well as the acidimetric value of the potassium carbonate, must be separately determined. This procedure gives very reliable results, if proper care is used é | CHEMISTRY OF THE URINE 373 ‘in the evaporation and the burning of the urine. It is to be used only when the actual excess of mineral acids above that necessary for the neutralization of the mineral bases is to be estimated, or when the total amount of organic acids in urine (whether free or combined ‘with bases) is to be determined (Folin). | ( = | CHEMISTRY OF THE URINE. _ General Chemical Composition of the Urine.—A general idea of ‘the chemical composition of the urine and the quantitative variations of the individual components may be formed from the following table, which I have constructed from analyses made in my labo- ‘ratory. The individuals from which the urines were obtained were ‘adults, and their general mode of life, as regards diet, exercise, etc., was that of the average American city dweller. Th addition, the following substances may be encountered under pathological con- iditions: serum albumin, serum globulin, albumoses, mucin (nucleo- albumin), glucose, lactose, inosit, dextrin, biliary constituents, viz., ibile acids and bile pigments, blood pigments, melanin, leucin, tyro- sin, oxybutyric acid, allantoin, fat, lecithin, cholesterin, acetone, ‘alcohol, Baumstark’s substance, urocaninic acid, cystin, hydrogen ‘sulphide, and still others. ANALYSIS OF URINE. | ee ee rl, ee Bee et 20041700 grams. | a eee to; Sy aa) et eee deed ROL) | Inorganic solids . pa ete oe’ as ee er tO (ee Sulphuric acid (H, SO ee ee ie On. co bein Phosphone acid (POs). ° 6% 48.65 2k ee eB eab ys Chlorine (NaCl) ae et ae 4 ewe eek DENIER ES [iter sk Va Pa Tard GON Gh On ee ee te EN Fe es wie eh SERB ANNAN TE i mae A ees PR a 9 8 pg ed @ ae ae Bienes Wel) )2> "4 We Ae ee el ee See Osu Z bende ode et AN 3 Ee ee. aa mea ane io |r a HlGomgemmtrates, ete... Snaly yu. mm O82 * COHEN. ocd. 5 lll ae rams Ma meas 01 Os tad 0 Urea ts ee ema ote eee ee Lets Ne Per ties sk OS Ns ere en DE, oy ORAS BRTELNIT) Danes “~. s:. te. oy ieee, aioe an ae Le () ‘. RET eAgetti te oot 7 UA) 2 ick ee ee ae Pore eee (2 etl YOR 44 5 Oxalic acid... bh. meee: ne cee 1.05 ? Conjugate sulphates Mv i ies Poe TO lta Opa © BROOUTICHCIO: 0.2, 25k oe eee Pe OL Ghedhs gel Wola tatby acid cnt 2 ote a0 ae ie 0505 7 Pherarvanic sols Uneaten tai s C25 Re Quantitative Estimation of the Mineral Ash of the Urine.—In order to estimate the amount of mineral ash in the urine the follow- ing method may be employed: 50 c.c. of urine are evaporated to dryness in a weighed porcelain dish, at a temperature of 100° C., and then 314 THE URINE . heated, while covered, over the free flame until gases cease to be evolved, care being taken not to heat too strongly in order to avoid | sputtering. ‘The residue is taken up with distilled boiling water, | and, after standing, filtered through a Schleicher and Schiill filter, | the weight of the ash of which is known. ‘The dish and the contents | of the filter are well washed with hot water. Filtrate and wash- ings are set aside and the dish and filter dried in the oven at 115° C. | The filter is now placed in the > dish and slowly incinerated. So soon as the ash has turned white | the filtrate and washings are placed | I ia in the same dish, evaporated at | | 100° C., and then carefully heated | eaten crcl Wi over the free flame. Upon cool- h, | ing in the desiccator (Fig. 133) the | dish with its contents is weighed, © the difference between its present | and previous weight indicating the » Fic. 133,—Desiceator. quantity of ash contained in 50° e.c. of urine. | Precautions: 1. Care should be taken to allow the dish to become faintly red only for a moment, as some of the chlorine is otherwise | volatilized. Some phosphoric acid may also escape, and too strong. a heat, moreover, may cause the transformation of sulphates into’ sulphides, the organic material present acting as are ducing agent. | 2. If the organic ash is not completely incinerated, it is best to’ allow the dish to cool and then to moisten the ash with a few drops: of dilute sulphuric acid, when the heating is continued. ual im 5 The Chlorides. | ) The chlorides which are excreted in the urine are derived from the. food. As they are thus present in a much larger amount than all. other inorganic salts combined, and in quantity more than sufficient | to supply the needs of the body economy, the relatively large amount of chlorides found in the urine under physiological conditions, as compared with the other inorganic constituents, is readily explained. | Of the alkalies in the urine, sodium in combination with chlorine. exists in greatest amount, and for clinical purposes it is most con-: venient to calculate the total quantity of chlorides in terms of sodium chloride; a small proportion also occurs combined with potassium, ammonium, caleium, and magnesium. . From 11 to 15 grams of sodium chloride, representing the total 4 CHEMISTRY OF THE URINE 375 quantity of chlorine, are normally eliminated in the twenty-four hours, the amount depending, of course, directly upon that contained in the food ingested. If the amount of nourishment is diminished, a decrease in the elimination of the chlorides is observed. If this is earried to the point of starvation, the chlorides disappear almost entirely from the urine, the traces remaining being derived from the body fluids. ‘The latter retain tenaciously a certain amount, which differs but slightly from that normally present. If at this stage food containing sodium chloride is again taken, a portion will be retained in the body until the original equilibrium is restored. A similar retention may be observed for a few days following the ingestion of large quantities of water, which causes an increased elimination of chlorides. This tenacity on the part of the body in retaining sodium chloride is strikingly seen when the potassium salt is substituted for the sodium salt; in this case the amount of the sodium in the serum of the blood will be found to vary very slightly. It has also been shown that the excretion of sodium chloride ean be increased very materially by the ingestion of potassium salts, notably the neutral potassium phosphate (K, HPO,). ‘This is supposed to decompose the sodium chloride present in the serum, with the formation of potassium chloride and neutral sodium phosphate, which are both eliminated as foreign material; a point is finally reached, however, when the sodium chloride ceases to be excreted. This provision of the economy, in virtue of which an increase in the elimination of the salt is followed by its retention, and a pre- vious retention by an increased elimination, is supposed to be inti- mately associated with the albuminous metabolism of the body. It may be stated, as a general rule, that any increase in the amount of circulating albumin will be followed by an increased elimination of chlorides, these having been previously retained by the albuminous bodies in consequence of the great affinity which exists between them. At the same time the elimination of the chlorides is influ- enced by the quantity of urine excreted, increasing and decreasing with its volume. Pathologically the excretion of the chlorides may vary within wide limits, diminishing on the one hand to zero and _ increasing on the other to 50 grams or more in the twenty-four hours. A marked diminution, which in some cases may go on to a total absence, was formerly thought to be pathognomonic of acute croupous pneu- monia.!. More modern investigations, however, have shown that such a condition occurs to a greater or less degree in most acute ' Rettenbacher, Wien. med. Zeit., 1850, p. 373. Heller, Heller’s Archiv, 1844, wol. i, p. 23. 376 THE URINE febrile diseases, such as scarlatina, roseola, variola, typhus and typhoid fevers, recurrens, and acute yellow atrophy. Intermittent fever appears to be an exception to this rule; usually it is true the chlorides are diminished, but not to the extent seen in the other diseases mentioned. ‘They have, moreover, been found to increase during and sometimes immediately after a paroxysm, this increase being, of course, followed by a corresponding diminution. The chlorides are diminished in all acute and chronic renal dis- eases associated with albuminuria.’ In this connection it is interest- ing to note that in cases of nephritis associated with edema and other transudates the withdrawal of the chlorides from the food results in marked improvement and in some cases in the complete disappear | ance of the effusion. | In all cases of carcinoma of the stomach, and in chronic hyper-_ secretion associated with dilatation, a decrease is observed, which in- certain cases of hypersecretion and hyperacidity, the result of gastric: ulcer, may go on to a total absence.’ In anemic conditions the chlorides are likewise diminished, as also in rickets. In melancholia and idiocy a striking decrease is observed; in dementia, chorea, and pseudohypertrophic paralysis this is less marked. A total absence has been noted in pemphigus foliaceus, and a con- siderable diminution in the beginning of impetigo, as also in chronie lead poisoning. The chlorides are found in increased amount in all conditions in which retention has previously occurred, chief among these being the acute febrile diseases and cases in which a resorption of exudates and transudates, associated with an increased diuresis, is taking place. A marked increase has been noted in some cases of diabetes insipidus, in which 29 grams have been eliminated in the twenty-four hours.’ A similar increase may occur in prurigo, in which, in one instance, 29.6 grams were passed in twenty-four hours.* In cases of general paresis, during the first stage, an increased elimination goes hand in hand with an increased ingestion of food. In epilepsy the polyuria following the attacks is associated with an increase in the chlorides. Of drugs, certain diuretics, and some of the potassium salts, as has been mentioned, produce an increase: the chlorine contained in chloroform, whether administered internally or as an anesthetic, is in part excreted in the form of a chloride. Salicylic acid, on the other hand, is said to cause a temporary diminution. It is of practical importance to note that in acute febrile disease the diminution in the chlorides appears to vary with the intensity 1 Réhmann, Zeit. f. klin. Med., 1886, vol. i, p. 513. ? Gluzinski, Berlin. med. Woch., 1887, vol. xxiv, 983. * Oppenheim, Zeit. f. klin. Med., vol. vi. * v. Brueff, Wien. med. Woch., 1871, p. 552. i CHEMISTRY OF THE URINE 377 of the disease, a decrease to 0.05 gram pro die justifying the con- clusion that the case under oservation is of extreme gravity. It may at times also indicate a preceding attack of severe diarrhea or the formation of exudates of considerable extent. A continued increase, on the other hand, should lead to the conclusion that the patient’s condition is improving. The elimination of the chlorides also furnishes a fair index to the digestive powers of the patient. All other causes which might lead to an increase or decrease being eliminated, an excretion of from 10 to 15 grams indicates a fair condition of the appetite and a normal digestive power, a decrease being associated with the reverse. An increased elimination of chlorides occurring in cases of edema, and associated with the existence of serous exudates, is always of good prognostic omen, pointing to a resorption of the fluid. A continued elimination of more than 15 to 20 grams, all other causes being excluded, may be considered as pathognomonic of dia- betes insipidus. Of late attention has been directed to the ratio between the elimi- nation of the chlorides and the total nitrogen. With an ordinary diet this is as 1 to 1 (Salkowski), even though the total amount of chlo- rides may not amount to 10 to 15 grams, but may be as low as7 to 10 grams. In disease this ratio may be much disturbed owing to chlo- ride retention (1 Cl to 15 N); a change toward the normal is ceteris paribus a favorable sign. Test for Chlorides in the Urine.—The recognition of the chlorides in the urine is based upon the fact that silver nitrate causes their precipitation. ‘lhe sliver chloride thus formed is insoluble in nitric acid. The test is made in the following manner: A few cubic centimeters of urine are acidified in a test-tube with about 10 drops of pure nitric acid, and treated with a few cubic centimeters of silver nitrate solu- tion (1 to 20). The occurrence of a white precipitate indicates the presence of chlorides. An idea may be formed at the same time of the quantity present; the occurrence of a heavy, caseous precipitate - points toa large amount. Albumin, if present, must first be removed by boiling, after acidifying the urine with a few drops of dilute acetic acid. , Quantitative Estimation of the Chlorides by the Method of Salkowski-Volhard.'—When a solution of silver nitrate acidified with nitric acid is treated with a solution of potassium sulphocyanide or ammonium sulphocyanide, in the presence of a ferric salt, the potassium sulphocyanide first causes the precipitation of white silver sulphocyanide, which, like silver chloride, is insoluble in nitric acid. As soon as every trace of silver is precipitated, it combines with * E. Salkowski, Zeit. f. physiol. Chem., vol. i, p. 16, and vol. ii, p. 379, 378 THE URINE the ferric salt to form ferric sulphocyanide, which is of a blood-red color. If the potassium sulphocyanide solution is of known strength, it is possible to estimate accurately the amount of silver present in the solution, the ferric salt serving as an indicator of the end of the reaction between the silver and the potassium sulphocyanide. Application to the urine: to urine which has been acidified with nitric acid an excess of a silver solution of known strength is added, and the silver not used in the precipitation of the chlorides then esti- mated as indicated above. ‘The difference between the quantity thus found and the total amount used will be that consumed in the pre- cipitation of the chlorides, from which, knowing the strength of the silver soluion, its equivalent in terms of sodium chloride is readily determined. Reagents required: 1. A solution of silver nitrate of such strength that each cubic centimeter shall correspond to 0.01 gram of sodium chloride. 2. A solution of potassium sulphocyanide of such strength that 25 c.c. shall correspond to 10 c.c. of the silver nitrate solution. 3. A solution of a ferric salt, such as ammonioferric alum, satu- rated at ordinary temperature. 4. Nitric acid (specific gravity 1.2). Preparation of these solutions: 1. As pointed out, the silver nitrate solution is made of such strength that each cubic centimeter shall correspond to 0.01 gram of sodium chloride. The silver nitrate must be pure, and it is best to use the crystal- lized salt, and not the sticks wrapped in paper, which always contain reduced silver. In order to test the purity of the salt, about 1 gram is dissolved in distilled water, heated to the boiling point, the silver precipitated by dilute hydrochloric acid and filtered off. When evaporated in a platinum crucible the filtrate should leave either no residue at all or only a very faint one; otherwise it is necessary to recrystallize the salt until the desired degree of purity is reached. The determination of the quantity to be dissolved in 1000 c.c. of water is based upon the fact that 1 molecule of silver nitrate (mole- cular weight 170) combines with 1 molecule of sodium chloride (molecular weight 58.5) to form silver chloride and sodium nitrate. As the solution of silver nitrate shall be of such strength that 1 c.e. corresponds to 0.01 gram of sodium chloride, or 1000 ¢.c. to 10 grams, the quantity to be dissolved in 1000 e.c. is found according to the following equation: 58.5:170: : 10 2, 58.5 x=1700, «=29.059. Theoretically, then, this quantity should be dissolved in 1000 c.c. of water. It is better, however, to dissolve it in a quantity some- what less than 1000 ¢.c., such as 900 or 950 e.c., as the silver salt CHEMISTRY OF THE URINE 379 contains water of crystallization and the weighed-off quantity would not represent the exact amount required, but less, the correcting of a solution which is too strong being a much simpler matter than that of a solution which is too weak. * ‘To make this correction, or, in other words, to bring the solution to its proper strength, 0.15 gram of sodium chloride, which has previously been dried carefully by heating in a platinum crucible, is accurately weighed off, dissolved in a little distilled water, and further diluted to about 100 ¢.c. ‘To this solution a few drops of a solution of potassium chromate are added, when the mixture is titrated with the silver solution. ‘The silver nitrate will first precipitate the sodium chloride, and then combine with the potassium chromate, forming red silver chromate. ‘The slightest orange tint remaining after stirring indicates the end of the reaction. Were the solution of the silver nitrate of the proper strength, exactly 15 c.c. should have been used, as each cubic centimeter shall represent 0.01 gram of sodium chloride. As a matter of fact, less will in all probability be needed, the solution having been purposely made too strong. Its correction then becomes a simple matter, as it is merely necessary to determine the degree of dilution required. Supposing that 29.059 grams of silver nitrate were dissolved in 900 ¢.c. of water, and that 14.5 ¢.c. instead of 15 ¢.c. had been re- quired to precipitate the 0.15 gram of sodium chloride, it is evident that each 14.5 ¢.c. of the remaining solution must be diluted with 0.5 ¢c.c. of water. It is, hence, only necessary to divide the number of cubic centimeters of the silver nitrate solution remaining by 14.5; the result multiplied by 0.5 represents the amount of water which must be added in order to bring the solution to the required strength. Hence the rule for the correction of a solution which has been found too strong: N.d Gaz E bd n in which C represents the number of cubic centimeters of water which must be added to the solution remaining; N the total number of cubic centimeters remaining after titration; n the number of cubie centimeters consumed in one titration; and d the difference between the number of cubic centimeters theoretically required and that actually used in one titration. In the example given the equation would then read: 936.5 X 0.5 C= 14.5 = 32.29. 32.29 c.c. of distilled water are added to the remaining 936.5 c.c., when the strength of the solution is tested by a second titration. If the solution is found too weak, it is best to make it too strong, and then to correct as described. 380 THE URINE 2. Preparation of the potassium sulphocyanide solution: as 1 molecule of silver nitrate (molecular weight 170) combines with 1 molecule of potassium sulphocyanide (molecular weight 97), the quantity of the latter to be dissolved in 1000 c.c. of water is found from the following equation: : 1702 Ofe21 162362 2+ 170.0 = Li O230s0 i pa olG. As potassium sulphocyanide is extremely hygroscopic, a solution is made which is too strong, by dissolving about 10 grams of the salt in 900 c.c. of distilled water. In order to bring this solution to its proper strength, 10 c.c. of the silver solution are diluted to 100 c.; 4 c.c. of nitric acid (specific gravity 1.2) and 5 e.c. of the am- monioferric alum solution are added, when the mixture is titrated with the potassium sulphocyanide solution; the end reaction is recognized by the production of a slightly reddish color, which per- sists on stirring. ‘The sulphocyanide solution BBV IDE been purposely made too strong, it will be found that less than 25 c.ec. are needed to precipitate all the silver present. ‘The quantity of water necessary for dilution is ascertained, as above, according to the formula 3. The solution of ammonioferric alum is a solution saturated at ordinary temperatures, care being taken to ensure the absence of chlorides in the salt, which may be effected, if necessary, by recrys- tallization. Method as Applied to the Urine.—10 c.c. of urine are placed in a small stoppered flask bearing a 100 ¢.c. mark, diluted with 50 c.c. of distilled water, and acidified with 4 ¢.c. of nitric acid. From a burette 15 c.c. of the standard solution of silver nitrate are added. The mixture is thoroughly agitated and diluted with distilled water to the 100 ¢.c. mark. ‘The silver chloride formed is filtered off through a dry, folded filter into a dry eee 80 c.c. of the filtrate are placed in a beaker, and, after the addition of 5 ¢.c. of the ammonio- ferric alum solution, titrated with the sulphocyanide solution until the end reaction—2. e., a slightly reddish tinge—is seen. If necessary, two such titrations should be made, the sulphocyanide solution being - added 1 c.c. at a time in the first, while in the second the total number of cubic centimeters needed to bring about the end reaction, less 1 c.c., are added at once, and then 0.1 ¢.c. at a time. The amount of chlorides present in the urine is calculated as fol- lows: Example.—Total quantity of urine 600 c.c.; 6.5 ¢.c. of the sul- phocyanide solution were required to bring about the end reaction in 80 c.c. of the filtrate; this would correspond to 8.125 c.c. for the CHEMISTRY OF THE URINE 381 total 100 c.c. of filtrate, representing 10 ¢.c. of urine, as is seen from the equation 100n 5n R0 caaete in which a represents the number of cubic centimeters correspond- to 100 c.c. of the filtrate, and n the number of cubic centimeters actually used. These 8.125 c.c. were used in precipitating the silver nitrate not decomposed by the chlorides. As 25 ¢.c. of the sulphocyanide solu- tion correspond to 10 c.c. of the silver solution, the excess of silver solution in cubic centimeters is found from the equation BERNE Nos aoc cael Al eee aes elie Ve.) 25 5 in which a represents the excess of the silver solution in cubic centi- meters, and N that of the sulphocyanide solution as found according to the equation above, w in this case being 3.25 c.e. The difference between the total amount of silver solution em- ployed (2. e., 15 c.c.) and the excess (7. e., 3.25 c.c.) indicates the number of cubic centimeters necessary for the precipitation of the chlorides in 10 ¢.c. of urine. In the case under consideration 11.75 c.c. were employed. As 1 c.c. of the silver solution represents 0.01 gram of sodium chloride, there must have been present in the 10 c.c. of urine 0.1175 gram; in 100 c.c., hence, 1.175 grams, and in the total amount—~. e., 600 ¢.c. of urine—7.05 grams. The method described may be employed in the presence of albu- mins, albumoses, and sugar; the urine, however, must be fresh, so as to ensure the absence of nitrous acid. Direct Method.'—If accuracy is not required, the following method may be employed: 10 c.c. of urine are diluted with distilled water to 100 ¢.c. and treated with a few drops of a solution of potassium chromate. ‘This mixture is titrated with a one-tenth normal solution of silver nitrate until the end reaction is reached—. e., a faint orange tinge—which no longer disappears on stirring. ‘The number of cubic centimeters used multiplied by 0.01 will indicate the amount of chlorides present in 10 c.c. of urine. As uric acid, the xanthin bases, hyposulphites, sulphocyanides, and pigments are also precipitated by the silver nitrate, the end reaction is delayed; moreover, unless the urine is very pale, its recog- nition may be difficult, and the error thus caused considerable. This is especially true of febrile urines which contain only a small amount of chlorides. Should iodides or bromides have been taken, these must first be removed, as silver iodide and bromide, which are insoluble in nitric acid, would give too high a value. He o0.2 as 100; 80 2= 100 nm: z= 1, Mohr, Lehrbuch d. Titrirmethode, 1856, ii, p. 13. 389 THE URINE The Phosphates. The phosphates occurring in the urine are sodium, potassium, calcium, and magnesium salts of the tribasic acid H,PO,. The most important of these, as was pointed out in the chapter on Reaction, is the diacid sodium phosphate NaH,PO,, to which the acidity of the urine is in part due. It is owing to the presence of this salt in the urine that the calcium phosphate is held in solution; the fact, at least, that calcium and magnesium phosphate are thrown down when the urine is neutralized would point to this conclusion. ‘The composition of the phosphates is liable to considerable varia- tion, depending upon the degree of acidity of the urine. As would be expected, diacid sodium phosphate and diacid calcium phosphate are present in an acid urine; in an amphoteric urine, in addition to these there are found disodium phosphate, monocalcium phosphate, and monomagnesium phosphate, while in an alkaline urine trisodic phosphate, neutral calcium phosphate, and neutral magnesium phos- phate may be present. The alkaline phosphates normally exceed the earthy phosphates by one-third, and sodium is combined with by far the greater amount of phosphoric acid, the potassium salt normally occurring in only very small amounts. In addition to the mineral phosphates, phosphoric acid is excreted also in combination with glycerin as glycerin-phosphoric acid, which need not, however, be considered in a quantitative estimation, as it is present only in traces." As in the case of the chlorides, the greater portion of the phos- phates is derived from the food, while only a small portion is refer- able to the tissue proteids. But just as the percentage of sulphur varies in the different tissues, so also does that of phosphorus vary; nerve tissue, for example, which is very rich in lecithins and nucleins, yields relatively more phosphorus than muscle tissue. Not all the phosphoric acid ingested, however, is excreted in the urine, as one-third to one-fourth of the total quantity is eliminated in the feces. The ee of phosphoric acid seis. which normally varies between 2.5 and 3 grams, is thus largely dependent upon the amount ingested, increasing with an animal and decreasing with a vege- table diet. During starvation a considerable increase is likewise observed, referable, no doubt, to an increased destruction of bony tissue, which is very rich in the phosphates of the alkaline earths. In accordance with this view, increased amounts of calcium and 1 Lépine et Eymonnet, Comp.-rend. de la Soc. de biol., 1882. * Zilzer, Virchow’s Archiv, vol. lxvi, p. 223. CHEMISTRY OF THE URINE 383 magnesium are also seen during starvation. ‘The relation between the excretion of phosphoric acid and nitrogen, normally | to 7, changes, moreover, in such a manner that both the absolute ‘and the relative amount of phosphoric acid, as compared with the nitrogen, increases; this leads to the conclusion that in addition to the muscles some other tissue rich in phosphorus and relatively poor in N must suffer during the process, and the only one which could enter into con- sideration is bone.’ If at this time food containing phosphorus is again given, a retention will take place, so that the general rule stated in the chapter on Chlorides, that increased Simirnon is followed by a certain degree of retention, and that a previous retention is fol- lowed by an increased elimination, seems to hold good for all the mineral acids found in the urine (see also the chapter on Sulphates). An increased elimination is caused also by the ingestion of large uantities of water, which is followed by a certain degree of retention. Observations on the phosphatic excretion during muscular exercise have not given uniform results.” Mental exercise appears to cause a diminished excretion of the alkaline phosphates and an increased elimination of the earthy phosphates.’ ‘The latter also takes place during sleep. In disease the total amount of phosphates may either be increased or diminished. A diminished elimination is observed in most cases of acute febrile disease, such as pneumonia, typhoid fever, typhus fever, recurrens, during a paroxysm of intermittent fever, etc. ‘The degree of dimi- nution is usually proportionate to the severity of the disease, reaching its lowest figure as death approaches. Such a state of affairs may, at first sight, appear paradoxical in view of what has been said above of the effects of tissue destruction upon the elimination’ of phos- phates. It is necessary, however, to distinguish sharply between an increased production and an increased elimination; in all probability a retention occurs analogous to that of the chlorides, which may be observed under the same conditions. It has been supposed that the phosphates set free during the process of tissue destruction are utilized in the building up of new leukocytes, and an increase in these is actually noted in some of the diseases mentioned. A dimin- ished excretion of phosphates is, however, not always observed, and an increased elimination may occur in certain cases. In fatal cases this condition may persist even until the time of death. It is very difficult to give a satisfactory explanation of this fact at the present time. ‘The phenomenon, in typhoid fever at least, appears to be connected with the intensity of the nervous manifestations, and Robin concludes that here an increased elimination during the fastig- 1 Ziilzer, loc. cit. 2 Fleischer u. Penzoldt, Virchow’s Archiv, vol. lxxxvii, p. 210. ’ Mariet, Compt.-rend. de la Soc. de biol., 1884. 284 THE URINE ium is an unfavorable omen, while an increase during defervescence warrants a favorable prognosis. A similar decrease in the phosphates has also been observed in pulmonary phthisis associated with high fever.’ Very interesting and important is the diminshed excretion of phosphates associated with acute and, to some extent also, with chronic nephritis, amyloid degeneration of the kidneys, and the anemias, in which an actual insufficiency on the part of the kidneys in the elimination of these salts appears to exist.’ A diminished or, atleast, no increased excretion is usually seen in certain diseases of the bones, such as osteomalacia. ‘This may depend either upon a retention or an elimination through other channels. The earthy phosphates especially are found in greatly diminished amount, or may even be absent altogether in certain cases of nephritis. A similar condition is observed in acute and chronic rheumatism. The data regarding the phosphatic elimination in nervous and mental diseases are, on the whole, scanty and by no means uniform. During attacks of hysteria majo, in contradistinction to epilepsy, in which an increased elimination takes place, the phosphates are diminished, the degree of diminution being generally proportionate to the intensity of the attack, increasing again together with the other urinary constituents with the subsequent increase in the diuresis.’ In chronic lead poisoning a diminution to one-third of the normal quantity may occur. Very low figures have been noted in Addison’s disease, in acute yellow atrophy (in which even a total absence may occur), and in certain cases of hepatic cirrhosis. In gout the phos- phoric acid curve follows that of the uric acid quite closely, decreas- ing before the onset of the acute symptoms and then rising and reaching its maximum about the third day (see Uric Acid).* An increased elimination of phosphates, on the other hand, amount- ing in some cases to 7 or even to 9 grams in the twenty-four hours, has been described by Teissier, of Lyon, under the name of phos- phatic diabetes, the patient presenting various symptoms commonly seen in diabetes mellitus; sugar, however, is usually absent. Whether or not phosphatic diabetes is a disease sui generis is not certain.” In true diabetes mellitus a curious relation has been found to exist between the elimination of sugar and of phosphates, the quan- tity of the latter rising and falling in an inverse ratio to the amount of sugar. In diabetes insipidus a slight increase is at times found. 1 Edlefsen, Schmidt’s Jahresber., vol. exevi, p. 59. 2 Fleischer, Deutsch. Arch. f. klin. Med., vol. xxix, p. 129. 3 De la Tourette and Cathelineau, Centralbl. f. d. med. Wiss., 1889, vol. xlviii, ay '# iS 4'T. B. Futcher, Jour. Amer. Med. Assoc., 1902, vol. xxxix, p. 1046. > G. Rankin, “ Phosphatic Diabetes,” Lancet, March 24, 1900. ‘Teissier, Thése, Paris, 1876. CHEMISTRY OF THE URINE 385 Corresponding to the phosphatic retention observed in acute febrile diseases an increased elimination is noted during convalescence. An increase occurs in the course of cerebrospinal meningitis. In a case of pseudoleukemia an increase of 7 grams has been noted, while the number of red corpuscles fell from 2,200,000 to 800,000 in four days. ‘To judge from the very careful observations made, there could be no doubt that the high degree of phosphaturia, which was limited to the alkaline phosphates, was referable to this latter source. In leukemia also very high figures are at times observed. Magnus-Levy reports a case in which the patient eliminated about 15 grams of PO, in fifteen hours. ‘This is exceptional, but other observers have noted 5 to 7 grams on repeated occasions. Con- sidering the extensive destruction of leukocytes and hence of nucleins in leukemia an increased phosphatic excretion appears natural. In hemorrhagic purpura Edsall’ noted a large excretion of P,O,: 6.192 grams. ‘lhe same observer states that he has seen this also in chronic leukemia, as soon as x-ray treatment is begun; at least in those cases in which there was the characteristic general response, ‘while it did not occur in the negative cases. While it is apparent that important conclusions cannot be drawn, ‘on the whole, from a knowledge of the absolute phosphatic elimina- tion, unless it be from a study of the relation existing between the excretion of the alkaline and earthy phosphates, a study of the rela- ‘tive phosphatic excretion seems to promise more valuable results. (According to Ziilzer,” a definite amount of the phosphates and of the urinary nitrogen is referable to the destruction of albuminous mate- jmial, so that the relation between the phosphoric acid and the nitro- ‘gen must be constant. Another portion, however, is derived from lecithin, one of the most important constituents of nerve tissue, ‘which contains more phosphorus than the albuminous molecule. ‘Whenever, then, the lecithin-containing tissues are more involved in the general metabolism than under normal conditions the rela- tion will no longer be a stable one. ‘This relation which exists ‘between the elimination of nitrogen and phosphoric acid has been ‘termed the relative value of phosphoric acid. The relative value of phosphoric acid in the urine has been calcu- lated as varying from 17 to 20, that of the blood being 3, of muscle tissue 12.1, of brain 44, of bone 426 to 480. This value supposes ‘ithe absolute value to v: ary between 2 and 3 grams pro die. It is found according to the following equation: 1) en XO N b in which N indicates the amount of nitrogen actually observed, | P.O, the amount of phosphoric acid in the same specimen of urine, BPO. 100 Sand 7= * Amer. Jour. Med. Sci., October, 1905. 2 Loc. cit. 25 386 THE URINE and x the amount of P,O; corresponding to 100 grams of N. By observing this relative value a much better idea may be formed of the metabolic processes taking place in the body in disease than from a mere expression of the absolute phosphatic value. In acute febrile diseases the relative as well as the absolute dimi- nution of the phosphates has been ascribed to a retention, they being possibly utilized in the building up of white blood corpuscles. In the course of these diseases oscillations in the relative value are fre- quently observed; during convalescence the relative as well as the absolute value again rises. In accordance with these considerations a diminished relative ex- cretion of phosphoric acid should be expected in all cases associated with a notable elimination of leukocytes through other channels, as in pneumonia, for example, or a storing away of the same, as in cases of empyema. ‘lhe facts observed are in accord with this view. A relative decrease has further been noted in the various forms of anemia, conditions of cerebral excitation, and especially preceding an attack of epilepsy. In progressive paralysis following syphilis the relative value, at first low, rises greatly after-the administration of potassium iodide, while the excretion of the earthy phosphates is lessened. In Sheinie cerebral affections, delirium tremens, and acute hydrocephalus a relative decrease has been noted. In mania, during the period of excitement, both the alkaline and the earthy phosphates are found increased, while during the stage of depression, as also in melancholia, the alkaline phosphates are diminished and the earthy phosphates increased. On the other hand, an increase in the relative value has been noted in apoplexy (amounting to 34.3 in one case, two days after an attack), brain tumors, tabes, arthritis deformans (30), pernicious anemia (23.8 to 58), etc.’ Of drugs, bromides appear to diminish the absolute amount of phosphoric acid. Cocaine and quinine cause a decrease, and salicylie. acid an increase. A relative decrease is produced by the cerebral. excitants, such as strychnine, small doses of alcohol, phosphorus, | valerian, cold baths, salt-water baths, ete. An opposite effect is produced by the cerebral depressants, such as chloroform, morphine, — chloral, large doses of alcohol, potassium bromide, mineral and vegetable acids, prolonged cold baths, Turkish baths, low tempera- | ture,,etc. Tests for the Phosphates in the Urine.—The test for the detection — of the phosphates occurring in the urine depends upon the precipita-_ tion of phosphoric acid by means of ferric chloride as ferric phosphate, which is insoluble in cold acetic acid. The same result may be accomplished by the addition of a solution of uranyl nitrate; this ! Zilzer u. Edlefsen, loc, cit, CHEMISTRY OF THE URINE 387 gives rise to the formation of uranyl phosphate, which is also in- soluble in acetic acid. Trest.—A few cubic centimeters of urine are acidified with a few drops of acetic acid, and treated with a few drops of a solution of ferric chloride (1 part of the officinal solution to 10 parts of water), when the occurrence of a yellowish-white precipitate will indicate the presence of phosphates. Ifa solution containing an acid phosphate of the alkalies is treated ‘with an alkaline hydrate, the diacid alkaline phosphate is transformed into the monacid salt. ‘This is further changed into the normal salt. As the monacid and neutral salts are both readily soluble, the ‘solution remains clear. If at the same time, as in the urine, a soluble diacid phosphate of the alkaline earths is present, this is likewise \transformed into the monacid and finally into the neutral salt; the ‘latter, however, being insoluble, is thrown down. |) Test ror THE EartHy PuHospHatTes.—10 c.c. of urine are rendered alkaline with ammonia, when the occurrence of a flocculent precipitate will indicate their presence. ‘Test FOR THE ALKALINE PHospHates.—After having removed the earthy phosphates from 10 c.c. of urine, as just described, the clear filtrate is acidified with acetic acid and tested with ferric chlo- ride or uranyl nitrate, as shown above. The alkaline phosphates may also be detected by treating the sammoniacal filtrate with a few drops of magnesia mixture (1 part of erystallized magnesium sulphate, 2 parts of ammonium chloride, 4 parts of ammonium hydrate, and 8 parts of distilled water), when i ammoniomagnesium phosphate, which is almost insoluble in ammo- nium hydrate, will be thrown down. | Quantitative Estimation of the Total Amount of Phosphates. Principle. —When a solution of disodium phosphate acidified with acetic acid is treated with a solution of uranyl nitrate or uranyl acetate, a dirty- looking precipitate of uranyl phosphate is thrown down. It is apparent that the quantity of phosphoric acid can be estimated accurately, if the solution of uranyl nitrate or acetate is of known strength. _ Solutions required: 1. A solution of uranium nitrate of such strength that 20 c¢.c. shall orrespond to 0.1 gram of P,O,. _ 2. A solution containing sodium acetate and acetic acid. _ 3. ‘Tincture of cochineal. _ Preparation of these solutions: 1. From the equation 1 | 2UO.NO;-+Na:HPO, =(UO),HPO, +2NaNO;, tis apparent that 2 molecules of uranium nitrate combine with 1 aolecule of disodium phosphate to form uranium phosphate and | | ' * r 388 THE URINE sodium nitrate. ‘The molecular weight of uranium nitrate being 318 and that of disodium phosphate 142, it is seen that 636 parts by weight of the former combine with 142 parts by weight of the latter. As 20 ¢.c. of the solution of uranium nitrate shall correspond to: 0.1 gram of P,O,, 1000 c.c. must be equivalent to 5 grams of P,O,. In 142 parts by weight of disodium phosphate there would be present 71 grams of P,O,, equivalent to 636 parts by weight of uranium nitrate. ‘The quantity of the latter, then, to be dissolved in 1000 c.e. of water will be found from the equation: 636 : 71 ::a:5;ad a= 44.78. 44.78 grams of uranium nitrate are weighed off and dissolved in about 900 ¢.c. of water, the solution being purposely made too strong for reasons pointed out in the chapter on Chlorides. In order to bring this solution to its proper strength it is necessary to titrate with the uranium solution a solution of disodium phosphate’ of such strength that each 50 c.c. shall contain 0.1 gram of P,O,, or 1000 ¢.c. 2 grams. ‘The molecular weight of Na,HPO,+12H,O being 358, this amount of disodium phosphate in grams is equiy- alent to 142 grams of P,O,; the quantity of P,O,; corresponding to 2 grams, in terms of Na,HPO,+12H,O, is found from the equation: 358 :142::a”:2; and « = 5.042. This amount of pure, dry, and non-deliquescent Na,HPO, is dissolved in 1000 c.c. of distilled water. If non-deliquescent disodium phosphate is not at hand, about 6 or 7 grams of the salt are dissolved in 1000 c.c. of distilled water; of this solution 50 ¢.c. are evaporated in a weighed platinum dish, and the residue gently heated, the disodium phosphate being thereby trans- formed into sodium pyrophosphate, Na,P,O,. The molecular weight of Na,P,O, being 266, this corresponds to 142 grams of P,O,. If the solution is of the correct strength—7. e., containing 0.1 gram of PO, in 50 c.c. of water—the residue should weigh 0.1873 gram, as: is seen from the equation : 142 : 266: : 0.1: 2; and «=0.1873. Supe posing, however, that the residue weighs 0.1921 gram, it is manifest: that the solution is too strong, and must be diluted, the degree of dilution being ascertained according to the equation: 0.1873 : 1000: : 20.1921 : 2; and «=1025; 7. e., 1000 cc. of the solution must Be diluted to 1025 ¢.c. to make it of the proper strength. | In the case given, 50 c¢.c. were used; the 950 c.c. are then diluted: with the amount of water found from the equation: 1000 : 1025 :: 950 : 2; and «=973.75. Having thus obtained a solution of diso- dium phosphate of such strength that each 50 ¢.c. shall contain 0.1 gram of P,O,, this is titrated with the uranium solution, which has been made too strong, in order to determine the amount of { ' A solution of chemically pure crystallized monopotassium phosphate can also be used for standardization (Sutton’s Volumetric Analysis, 8th ed., p. 316), 4 ing to the formula: C=- n CHEMISTRY OF THE URINE 389 water that must be added to the latter. ‘lo this end, a burette is filled with the uranium solution; 50 c.c. of the disodium phosphate solution are treated with a few drops of the tincture of cochineal and 5 c.c. of the acetic acid mixture (see below). This mixture is heated in a beaker, and as soon as the boiling point has been reached titrated with the uranium solution until a trace of a greenish color is noticed in the precipitate which does not disappear on. stirring. This point having been accurately determined by means of a second titration, the number of cubic centimeters of distilled water with which the remaining solution must be diluted is determined accord- Nid , in which C represents the number of cubic centimeters which must be added, N the number of cubic centimeters remaining after the test titration, n the number of cubic centimeters consumed in one titration to bring about the end reac- tion, and d the difference between the number of cubic centimeters used in one titration and that theoretically required. The amount of distilled water necessary for dilution is now added _and the solution again tested, when 20 c.c. will correspond to 0.1 gram of P,O,,. 2. ‘The acetic acid mixture is prepared by dissolving 100 grams of sodium acetate in a little water, adding 30 grams of glacial acetic acid and diluting the whole to 1000 c.e. 3. ‘Tincture of cochineal. This may be prepared as foliows: A few grams of cochineal granules are digested at ordinary tempera- tures with 250 c.c. of a mixture of 3 volumes of water and 1 volume of 94 per cent. alcohol. ‘The solution is then decanted and ready for use. ‘I‘he residue may be utilized in the preparation of a fresh supply of the tincture. Application to the Urine.—50 c.c. of clear filtered urine are treated with 5 c.c. of the acetic acid mixture, the object being to transform any monacid sodium phosphate present into diacid sodium phosphate, and to neutralize any nitric acid that may be formed during the titration, as otherwise the nitric acid would cause a partial solution of the precipitated uranyl phosphate. A few drops of the tincture of cochineal are added, when the mixture is heated to the boiling point and titrated as described above. ‘Two titrations are usually required. Should it be desired to use potassium ferrocyanide as an indicator, the uranium solution must have been standardized with the same indicator, as errors will otherwise arise. The technique is simple. -Anumber of droplets of the potassium ferrocyanide solution (about / 9 per cent.) are placed on a piece of white filter paper. After every ‘addition of the uranium solution to the boiling urine a droplet of the mixture is placed upon the ferrocyanide stain. ‘The end reaction is ‘indicated by the occurrence of a brown color. 390 THE URINE The results are calculated as follows: Supposing 15 c.c. of the uranium solution to have been used, the corresponding amount of P.O, in 50 ¢.c. of urine is found from the equation: 20: 0.1 : : 15: a; | and «= 0.075. ‘The percentage amount would, hence, be 0.075 X _ = 0.15. Supposing the total amount of urine to have been 2000. c.c., the elimination of P,O,; would correspond to 3 grams. | The presence of sugar and albumin does not interfere with the method. | Separate Estimation of the Earthy and Alkaline Phosphates. — If the alkaline and earthy phosphates are to be determined separately, | the total amount of P,O; is estimated in one portion of the urine, while the P,O, in combination with the alkaline earths is determined in | another, as follows: 200 c.c. of filtered urine are made strongly alkaline with ammonium hydrate and set aside, covered, for several hours, | when the earthy phosphates thus preciptated are collected on a filter, | washed with dilute ammonia (1 to 3), and then transferred to a beaker, ' | with the aid of a little water containing a few drops of acetic acid, | by perforating the filter. ‘They are then dissolved with as little | acetic acid as possible, diluted to 50 c.c. with distilled water, and | titrated with the uranium solution as described. ‘The difference | between the total amount of P,O; and the amount thus obtained indicates the quantity of alkaline phosphates present. ! Removal of the Phosphates from the Urine.—Whenever it is necessary to remove the phosphates from the urine in the course of an | analysis, as is frequently the case, the urine is rendered alkaline by the addition of the hydrate of an alkaline earth and precipitated with a soluble calcium or barium salt. ‘They may also be precipitated by: means of neutral or basic lead acetate, in which case the excess of lead is removed by means of hydrogen sulphide or dilute sulphuric acid. The Sulphates. The sulphuric acid found in the urine is derived essentially from , the albuminous material which is constantly broken down in the body, a very small portion only of the inorganic sulphates being refer- | able to the mineral constituents of the food. As was pointed out in the’ chapter on Reaction, sulphuric acid is constantly produced in the body, and, coming into contact with the so-called neutral phosphates present in almost all the tissues, transforms these into acid phos-_ phates, both appearing in the urine. ‘The alkaline carbonates, which _ are derived from the organic salts ingested by a process of oxidation, | are also attacked by the sulphuric acid. | As the amount of food ingested is gradually diminished a point is reached when the body most tenaciously holds any alkaline salts that | may still be present. A new source for the neutralization of the CHEMISTRY OF THE URINE 29] vw acid is then found in the ammonia, which would otherwise have been eliminated as urea. While the greater portion of the sulphuric acid excreted in the urine is found in the form of mineral sulphates, about one-tenth of the total amount may be shown to be in combination with aromatic substances belonging to the oxy-group; most important among these are the salts of phenol, indoxyl, and skatoxyl. Indoxyl and skatoxyl, as will be shown later, are derived from indol and skatol, which, together with phenol, are formed during the process of intestinal putrefaction. ‘Their amount increases and decreases with the degree of putrefaction, and hence serves as an index of its intensity. The mineral sulphates have been termed preformed sulphates in contradistinction to the others, which are known as conjugate or ethereal sulphates. In the following pages the former will be desig- nated by the letter A, the conjugate sulphates by the letter B, and ' the total sulphates as A+B. The amount of A+B excreted in the twenty-four hours by normal individual varies between 2 and 3 grams, the ratio of A to B being as 10 to 1.’ From what has been said, it is apparent that the elimination of sulphates is largely dependent upon the degree of albuminous destruc- | tion taking place in the tissues and fluids of the body, and hence to a certain extent upon the quantity of proteid material ingested, the mineral sulphates occurring in such small amount in the food as scarcely to affect the quantity excreted. Secondarily, the degree of intestinal putrefaction plays a role. During starvation A+B is _ diminished, this diminution affecting A especially; in some cases B ' may be considerably increased.’ An increase in the elimination of the total sulphates is observed, as would be anticipated, in all cases in which an increased _ tissue destruction is taking place, as in acute febrile diseases. It must be remembered, however, that the quantity excreted is then not always greater than during convalescence, the diet remaining the same. Here, as elsewhere, in urinary studies, it is necessary to dis- tinguish between a relative increase and an absolute decrease. In pneumonia and acute myelitis the highest figures have been observed, the increased elimination during ie Febrile period being especially marked 3 Fever diet. Full diet. a ——— = Fever. No fever. No fever. SRPATMONIE. 0) bs, na iisen 01 2IN:,,. 1247 gm; 2.25 gm. Acute myelitis . . . + 2,.62gm. 1.52 gm. 2.33 gm. 1 vy, d. Velden, Virchow’s Archiv, vol. vii, p. 348. 2 Clare, Inaug. Diss., Dorpat, 1854. ’ Pp. Fiirbringer, Virchow’s Archiv, vol. Ixxiii, p. 39. 399 THE URINE During convalescence the excretion of the sulphates is diminished, a retention analogous to that of the chlorides and phosphates takin place. In contradistinction to the latter salts, it is in all probabilit not the mineral matter proper that is demanded by the body, but the sulphur-containing albuminous material. A considerable elimination of A-}+B has also been observed in leukemia, in which an average of 2.46 grams is excreted, as com- pared with 1.51 grams by a healthy individual receiving the same amount and kind of food. In one case of acute leukemia 5.8 grams were eliminated on the day preceding death.’ In diabetes mellitus, diabetes insipidus, esophageal carcinoma, progressive muscular atrophy, pseudohypertrophic paralysis, -and eczema an increased elimination has likewise been observed, while in chronic renal diseases a diminished excretion is the rule. A study of the elimination of the conjugate sulphates and of the relation existing between A and B in disease is still more important than that of the total sulphates; but in both cases the data available are scanty, and further observations are urgently needed. yv. Noor- den regards the elimination of more than 0.3 gram of conjugate sulphates in the twenty-four hours as excessive, the patient being on an ordinary mixed diet. The conjugate sulphates, as would be expected, are increased in all cases of increased intestinal putrefaction.* In coprostasis the result of carcinoma the ratio of the preformed to the conjugate sul- phates, normally 10, may diminish enormously. In one case, reported by Kast and Baas,’ it fell to 2, but rose to 7 and 8, and finally to 9.5 and 15 after an artificial anus had been established. I have observed a drop to 1.5 in a case of volvulus of ten days’ standing. H. Baldwin notes a case of pernicious vomiting of pregnancy in which the factor A: B was 1.9; following abortion it fell to 4 and a little later to 5.4. Biernacki* found an increase in the elimination of conjugate sulphates amounting to from 0.15 to 0.5 gram pro die in cases of chronic pareachymatous nephritis, going hand in hand apparently with a decrease in the secretion of hydrochloric acid by the stomach; the normal amount, according to his observations, varies from 0.19738 to 0.2227 gram. In one case B fell from 0.4882 to 0.1505 during the administration of hydrochloric acid, to increase again to 0.4127 upon its discontinuance. In accord with these observations are those of Wasbutzki and 1 Ebstein, Deutsch. Arch. f. klin. Med., vol. xliv, p. 346. 2 Blumenthal has called attention to the fact that this is not necessarily the case, and that an acid fermentation may occur in lieu of the formation of aromatie products. He hence suggests that at times it maybe necessary to estimate the volatile fatty acids also. 3 Minch. med. Woch., 1888. 4 Deutsch. Arch. f. klin. Med., vol. lxix. CHEMISTRY OF THE URINE 393 Kast.’ The former found an increased elimination of B in cases of intense bacterial fermentation taking place in the stomach, while hydrochloric acid was either totally absent or present in greatly diminished amount. A diminished elimination was-observed in cases of intense torular fermentation, hyperchlorhydria existing at the same time. In the absence of hydrochloric acid a normal or even a slightly diminished amount was observed in cases of intense acid fermentation, lactic acid and butyric acid being present in large quantities. By neutralizing the gastric juice with large doses of sodium bicarbonate Kast was able to bring about a macken increase in the elimination of B, the ratio A: B having fallen from 10.3 to 16.1 to 2.9 to 6.1. Personal observations eae led me to the same con- clusion.” (See also chapter on the Aromatic Bodies. ) In obstructive jaundice the excretion of B is likewise increased; it returns to the normal as soon as the permeability of the biliary passages has again become established. ‘The total sulphates were found diminished in cases of non-obstructive jaundice.* In Béhm’s* cases of catarrhal jaundice the excretion of conjugate sulphates varied between 0.4 and 0.7 gram. Of interest in this con- nection are the observations of Miiller,> who notes the elimination of 0.29, 0.24, and 0.28 gram of conjugate sulphates on three con- if i | ; secutive days in a case of total obstruction of the biliary duct in consequence of a stone. ‘The patient during this period was on a milk diet, and there can be little doubt that the low values are here refer- able to the pure lactic acid producing organisms crowding out the colon bacilli. On a meat diet the same patient passed 0.48 and {0.51 gram. Other observers have obtained less constant results in | their cases of catarrhal jaundice. In cases of hepatic cirrhosis and malignant disease of the liver Kiger’ and Hopadze’ found increased amounts of conjugate sulphates. In cases of diarrhea A +B, as well as B, is diminished, while 4: B is increased. Of drugs, large doses of morphine, potassium bromide, sodium salicylate, and antifebrin appear to cause an increased elimination of \ the total sulphates, while alcohol slightly diminishes the excretion. Most important are the observations which have established a ‘diminished excretion of the conjugate sulphates following ingestion of the terpenes and camphor, Karlsbad and Marienbad water, which 1 Kast, Festsch. z. Er6éffnung d. neuen allgem. Krankenhauses, Hamburg, 1889. Wasbutzki, Arch. f. exper. Path. u. Pharmakol., vol. xxvi. 2. E. Simon, Amer, Jour. Med. Sci., 1895, vol. ex. § Ziilzer, Unters. uber d. Semiol. d. Harns, Berlin, 1884. 4 Deutsch. Arch. f. klin. Med., 1901, vol. xxi, p. 73. 5 Zeit. f. klin. Med., 1887, vol. xii. ® Inaug. Diss., St. Petersburg, 1893. 7 Wratsch, 1893, Nos, 48 to 50. be ¥ 7 ‘ s i 394 THE URINE latter two, however, at first cause an increase. Kefir, in doses of from 1 to 1.5 liters pro die, has proved a most excellent remedy with which to combat this type of intestinal putrefaction. Injections of tannic acid and of a saturated solution of boric acid apparently produce little effect unless the dose is so large as to cause syn on of poisoning. Tests for the Sulphates in the Urine.—'The detection of the mii and the conjugate sulphates in the urine depends upon the fact that the sulphates of the alkalies are precipitated by barium chloride as insoluble barium sulphate. In the urine the addition of barium chloride at the same time causes a precipitation of the phosphates. ‘These must be kept in solution by the addition of an acid, acetic acid being employed for this purpose whenever the presence of the mineral sulphates is to be demonstrated; hydrochloric acid is inad- missible, as it would cause the decomposition of the conjugate sul- phates and set free the sulphuric acid thus held. To test for the mineral sulphates, a few cubic centimeters of urine strongly acidified with acetic acid are treated with a few drops of a_ solution of barium chloride, when in their presence a cloud or a white precipitate of barium sulphate will occur. To test for the conjugate sulphates, 25 c.c. of urine are treated with about the same volume of an alkaline barium chloride mixture (2 volumes of a solution of barium hydrate and 1 volume of a solu- tion of barium chloride, both saturated at ordinary temperatures) and filtered for a few minutes, the preformed sulphates as well as the phosphates being thus removed. ‘The filtrate is then strongly acidified with hydrochloric acid and boiled; the occurrence of a pre- cipitate is referable to conjugate sulphates. Quantitative Estimation of the Sulphates.—The principle of the method is the same as that just described, the mineral sulphates | forming an insoluble precipitate of barium sulphate directly when — treated with barium chloride, while the conjugate sulphates do so | only upon decomposition with strong hydrochloric acid under the | application of heat. In order to estimate the mineral and conjugate — sulphates, it is best to determine the total sulphates in one portion | and the conjugate sulphates in another, the difference between the | two giving the mineral sulphates. Quantitative Estimation of the Total Sulphates (Folin).—50 c.c. of — clear, filtered urine are treated with 5 c.c. of concentrated hydro- | chloric aid and 5 c.c. of a 4 per cent. solution of potassium chlorate. The mixture is boiled until it is colorless (five to ten minutes) and then treated, while still boiling, with 25 c.c. of a 10 per cent. solution | of barium chloride, drop by drop. It is kept on a hot-water bath or | on an asbestos plate hot (but not boiling) for one-half to one hour. | The precipitate is now collected on a Schleicher‘and Schiill filter, the weight of the ash of which is known (No. 589). Care should be CHEMISTRY OF THE URINE 395 taken never to allow the filter to run dry, and small amounts of hot water must be added to the last cubic centimeters remaining, the final traces being placed upon the filter with the aid of a rubber-tipped | glass rod. ‘he precipitate is washed with hot water for a half-hour, and at intervals of a few minutes hot ammonium chloride solution (5 per cent.) is substituted for the water, so that in all five or six _ additions of ammonium chloride take place in the course of the first » twenty minutes’ washing. In the end a specimen of the washings » must no longer be rendered cloudy, even on standing a few minutes, upon adding a drop of dilute sulphuric acid. _ ‘The paper filter is partially dried by folding and pressing gently | between filter paper. It is then placed in a weighed crucible, covered with 3 to 4 ¢.c. of alcohol, and the alcohol ignited. ‘The ash is heated, at first moderately, and almost completely covered with the | lid, then only half covered, for five to seven minutes, until the contents of the crucible are white. ‘he crucible, when cooled, is placed in a desiccator and weighed, the difference between the first and the + second weighing giving the weight of the barium sulphate obtained from 50 c.c. of urine. Quantitative Estimation of the Conjugate Sulphates (Kolin).—200 -e.c. of urine (diluted to a liter if necessary) are treated with 100 c.c. of a 10 per cent. solution of barium chloride, at ordinary tempera- ‘ture. ‘The mixture is set aside for twenty-four hours and the clear supernatant fluid poured into a dry beaker by decanting. ‘This pre- liminary decantation is necessary, as the barium sulphate precipitate will otherwise go through the paper. ‘The decanted liquid is filtered, 150 c.c. of the clear filtrate, representing 100 ¢.c. of urine, measured into an Erlenmeyer flask, treated with 10 to 15 e.c. of concentrated j hydrochloric acid and 10 to 15 c.c. of a 4 per cent. solution of potas- ‘sium chlorate. ‘The mixture is then heated to boiling and kept upon 1a boilimg water bath until the barium sulphate has settled and the “supernatant fluid is clear. ‘The precipitate is filtered off, washed, ‘dried, and weighed, as described above. ‘The weight thus obtained, ‘deducted from the amount found according to the first method, indi- eates the amount referable to the mineral sulphates. ‘The molecular ‘weight of BaSO, being 232.82, that of SO, 79.86, of H,SO, 97.82, and of S 32, the figure expressing the amount of H,SO,, SO,, or S, corresponding to | gram of BaSO,, is found according to the follow- ‘ing equations: . 232.82 : 79.86 :: 1: x; and x = 0.34301. .. 1 gram of BaSO, = 0.54301 gram of SO,. | go2.52 : 97.82 ::1: a4; and x = 0.42015. .. 1 gram of BaSO, = 0.42015 gram of H,SO,,. | go2.02:92:21: 2; and x =0.13744. ... 1 gram of BaSO, = '0.13744 gram of S. _ To calculate results, it is only necessary to multiply the weight of —_ —_—__s Bie. 296 THE URINE the BaSO, by 0.34301, 0.42015, or 0.13744, in order to ascertain the amount of sulphuric acid contained in 50 ¢.c. of urine, in terms of SO,, H,SO,, or S, respectively. LITERATURE.—E. Salkowski, Zeit. f. physiol. Chem., 1886, vol. x, p. 346; and Virchow’s Arch., 1888, vol. Ixxix, p. 651. °O. Folin, Amer. Jour, of. Physiology, 1902, vol. vii, p. 152. Neutral Sulphur. While the greater portion of the sulphur of the body is eliminated in an oxidized form, small amounts of non-oxidized sulphur bodies are likewise found in every urine. ‘They are collectively spoken of as the neutral sulphur of the urine, and under normal conditions constitute from 12 to 15 per cent. of the total sulphur. The rela- tion existing between the oxidized and the neutral form is, however, inconstant, and varies with the character of the diet, the degree of the proteid metabolism, ete. Of the nature of the neutral sulphur bodies which occur in nor- mal urine, comparatively little is known. At the present time we are acquainted with only two substances belonging to this order, viz., certain sulphocyanides and cystein, or a body which is closely related to it. ‘The greater portion of the sulphocyanides is undoubt- edly derived from the saliva that has been swallowed and absorbed, while a smaller amount may be referable to the trace which is said to be present in normal, uncontaminated gastric juice. ‘The amount of sulphur which is present in this form represents about one-third of the total quantity of the neutral sulphur. Cystein probably is an intermediary product of the normal metabolism of proteid material. Under normal conditions, however, the greater portion is oxidized to sulphuric acid, and traces only escape to be eliminated as such. Whether or not taurocarbaminic acid, which is a derivative of taurin, is a constant constituent of the urine remains an open question, but is very probable. We know, as a matter of fact, that the amount of neutral sulphur undergoes a distinct diminution in animals when the bile is prevented from entering the intestinal canal by establish- ing an external fistula. Under pathological conditions a correspond- ing increase is observed in cases of biliary obstruction, and the amount of neutral sulphur may then reach 40 per cent. of the total sulphur. Thiosulphates, which are normally present in the urine of dogs and cats, do not occur in human urine under normal conditions. That they may be present in disease has been shown by Striimpell, who found them in a case of typhoid fever. Further observations, however, are wanting. Another sulphur body belonging to this class, which Abel dis- CHEMISTRY OF THE URINE 397 covered in the urine of dogs, and which appears to be identical with ethyl sulphide, has not been found in the urine of man. The greatest increase in the amount of the neutral sulphur is observed under certain conditions associated with the appearance of cystin. Normally this is not present in the urine, while traces of cystein, or a closely related substance, as I have already stated, are found. Cystin is of albuminous origin, and as a matter of fact it has been ascertained that all of the loosely combined sulphur and even a portion of the firmly combined form exists in the albu- mins in the form of the cystin complex. According to Baumann and y. Udranszky, its appearance in the urine is closely connected with the formation of certain diamins, V1z., -adaverin, putrescin, and a third diamin which is probably identical with saprin or neu- ridin. As these diamins were hitherto supposed to result only from the action of certain specific bacteria upon albuminous material, cystinuria was regarded as evidence of a definite infectious pro- cess. It is to be noted, however, that cystin itself does not occur in the feces, and that diaminuria does not necessarily accompany the cystinuria. As the result of personal observations I have been led to the conclusion that a causal connection does not exist between the two conditions, and that the diamins in question can be pro- duced in the body tissues directly without the intervention ef micro- organisms. I regard cystinuria essentially as a metabolic anomaly, the result of a specific insufficiency on the part of certain tissues (liver) of the body. ‘The condition may be temporary, but as a rule it is permanent. It may occur among several members of the same family, but it is noteworthy that no case has been reported in which a parent and chiid were cystinuric. Consanguinity among parents, which is not infrequently observed in cases of alkaptonuria, is the exception in cystinuria. In this connection it is interesting to note that according to Lowy and Neuberg' the cystinuric is not able to oxidize other mono- and diamino acids when given by the mouth, and that tyrosin and aspar- tic acid will reappear as such, while lysin and arginin are eliminated as cadaverin and putrescin. Folin and I have not been able to verify this observation so far as tyrosin goes. Abderhalden’, on the other hand, found tyrosin and leucin in the urine of a cystinuric, who had not been fed any tyrosin as such. The amount of neutral sulphur which may be met with in cystin- uria is subject to wide variation, but not infrequently exceeds 30 per cent. of the total sulphur. As a general rule, the amount of cystin eliminated in the twenty-four hours is less than 0.5 gram. At times, however, larger quantites are found, and on one occasion ' Zeit. f. physiol. Chem., vol. xliii, p. 338. * Abderhalden and Schittenhelm, ibid., vol. xlv, p. 468, CC. E. Simon, ibid., vol. xlv, p. 24, 398 THE URINE I obtained more than 1 gram. Clinically it is of interst in so far as its continued production may give rise to the formation of calculi. Unless cystin occurs as a deposit, its presence will scarcely be suspected. ‘he substance, however, may occur also in solution, and it not infrequently happens that attention is first drawn toward its existence in this state owing to the marked odor of hydrogen sulphide which such urines develop on standing (see Hydrothion- uria). If acetic acid is then added in excess, the characteristic hexagonal plates may crystallize out. ‘The same result is obtained by allowing the urine to undergo ammoniacal decomposition, as cystin is insoluble in solutions of ammonium carbonate. Structurally cystin is the disulphide of cystein, which latter is g-amino-—/3-thiolactic acid. On reduction it is transformed into cystein according to the equation: CH,S— CH,.SH | | | CH.NH, CH.NH, + 2H = 2CH.NH, | | | COOH COOH COOH Cystin crystallizes in hexagonal plates which are quite characteristic, and not likely to be confounded with other crystalline elements that may be present in urinary sediments. If doubt should arise, their solubility in ammonia and hydrochloric acid, and their insolubility in acetic acid, water, alcohol, and ether, will lead to: their identifi- cation. ‘The quantitative estimation of cystin is rather unsatisfactory, as no method is known which yields reliable results. On the whole, it is perhaps best to determine the neutral sulphur, and to refer the increase beyond its normal value to the presence of cystin. | Quantitative Estimation of the Neutral Sulphur in the Urine.—In one portion of urine the oxidized sulphur, viz., the mineral and the con- jugate sulphates, are estimated as described. In a second portion the total sulphur is determined, the difference indicating the amount of the neutral sulphur. ‘To determine the total amount of sulphur the following method is most conveniently employed: | Method of Hohnel-Glaser (modified by Modrakowsky’): 1 or@ ‘grams of sodium peroxide are placed in a nickel dish, and covered with 50 cc. of urine, added drop by drop. ‘The fluid is evaporated to a syrup on a water bath, and further treated with 2 to 3 grams of the peroxide, which is added slowly while stirring. As soon as the reaction, which at first is quite vigorous, has subsided somewhat, the dish is removed from the water bath and heated with a small alcohol lamp. If necessary, 1 to 3 grams more of the peroxide are added, Zeit, f, phys, Chem,, 1903, vol. xxxviii, p, 562. CHEMISTRY OF THE URINE 399 ~The mass now forms brown drops and finally becomes thick; this ends the reaction. On cooling, the fusion is dissolved in hot water; the solution is filtered and feebly acidified with hydrochloric acid. Barium chloride is then added and the process continued as above described (Estimation of Sulphates). LireratTuRE.—E. Salkowski, Virchow’s Archiv, vol. lxvi, p. 313, and vol. exxxvii, -p. 381. Goldmann u. Baumann, “Zur Kenntniss der Schwefelhaltigen Ver- Bitnyen des Harns,” Zeit. f. physiol. Chem., vol. xii, p. 254. KE. Salkowski, Virchow’s Archiv, vol. lviii, p. 461. J. Munk, ibid., vol. xix, p. 354; and Deutsch. med. Woch., 1877, No. 46. O. Schmiedeberg, “ Ueber das Vorkommen von Unterschwefliger Siure im Harn,’ Arch. d. Heilk, vol. viii, p. 425. A. Strimpell, ibid., vol. xvii, p. 390. J. Abel, “Ueber das Vorkommen von Ethylsulfid im | Hundeharn,” etc., Zeit. f. physiol. Chem., vol. xx, p. 253. (See also Cystinuria and Hydrothionuria.) CC. E. Simon, “ Cystinuria and its Relation to Diaminuria,”’ Amer. Jour. Med. Sci., 1900, vol. cxix, p. 39. C. E. Simon and-M. W. Lewis, | “Transitory Cystinuria,”’ ibid., 1902, vol. exxiii, p. 888. C. E. Simon and D. G. | J. Campbell, “A Contribution to the Study of Cystinuria,’”’ Johns Hopkins Hospital Bull., 1904, vol. xv, p. 365. Urea. | | Urea is the most important nitrogenous constituent of the urine, and normally represents from 85 to 86 per cent. of the total amount ‘of nitrogen which is eliminated by the kidneys. Chemically, it may be regarded as carbamide—1. e., as the amide of carbonic acid—and is represented by the formula NH GO7 Lees \NH 2° \It is thus a comparatively simple substance, and the question natu- tally arises: What relation does urea bear to the complex albuminous molecule from which it is derived? According to the older concept lof the process of albuminous digestion this leads to the formation of albumoses and peptones, which latter were regarded as a unity and as very complex substances. From these bodies the reconstruction lof the albuminous molecule then supposedly took place in the intesti- inal mucous membrane, whence the resulting albumin found its way ‘into the blood and lymph. Here it existed as so-called circulating albumin, in contradistinction to the organized albumin of the cells. Voit further taught that the circulating albumin is broken down in ithe tissues at large, through the special activity of the living proto- plasm, but without becoming an integral part of the cells before its destruction. Pfliiger, on the other hand, took the contrary view according to which the circulating albumin must become part and parcel of the cells before it can undergo katabolic disintegration. In either event it was generally accepted that urea was derived from the tissues of the body at large and to a great extent from the muscles. Regarding the nature of its intermediary antecedents, Drechsel sup- } / | | 400 THE URINE posed that amino-acids first result by hydrolysis in the tissues, and that ammonia, water, and carbon dioxide are then formed from these by oxidation. Ammonia and carbon dioxide then combine and form ammonium carbamate, which is carried to the liver and is there trans- formed into urea through loss of water. ‘These stops are represented by the equations: 1. CH,.NH,.COOH +30=NH, + 2CO, + H.O. nee 1h. 2, CO, + £NH hecaCce Cate NH oC) fa : Ler, NH, \O.NH,—H,0=CO¢ - \ NH). Urea As a matter of fact it is well known that the ingestion of amino- acids leads to an increased elimination of urea and that the liver plays an important role in its final formation. But there is still much doubt whether amino-acids are formed in the tissues at large to such an extent as the older theories of Voit and Pfliiger would demand. It is rather significant that normally they are scarcely ever encoun- tered in the tissues of the mammalian organism, and for some years past there has been a growing tendency to regard ammonium para- lactate as the principal form in which the greater portion of the nitrogen leaves the tissues. In the liver this is then supposedly transformed into ammonium carbonate, from which the urea results with the inter- mediary formation of ammonium carbamate. This hypothesis has certain facts in its favor. We thus find that — after extirpation of the liver in geese the uric acid, which in birds plays the same part as the urea in mammals, disappears and is largely replaced by ammonium lactate. In diseases of the liver, moreover, in which an extensive destruction of the parenchyma is taking place, on ie some cases of acute yellow atrophy, in phosphorus poisoning, , the elimination of urea is diminished, and in its place a cor- — Ee baa ae amount of ammonia in combination with lactic acid is _ found. In dogs in which the liver has been in part excluded from the general circulation by the establishment of an Eck fistula, and in which the hepatic artery has at the same time been ligated, the elimination of urea is much diminished, while that of ammonia | increases rapidly so soon as the first symptoms of illness appear in the animals. From these observations it is apparent also that the synthesis of urea takes place in the liver. This is further proved by the fact that on transfusion of isolated livers of dogs with blood | to which ammonium carbonate or ammonium lactate has been added, urea is formed as a result. In other organs of the body this syntheal | apparently does not occur, but there is evidence to show that at least | CHEMISTRY OF THE URINE AOI a small amount of urea originates elsewhere within the body through processes of hydrolysis. ‘his amount, however, is unquestionably slight. ‘hat a fraction, moreover, is formed from uric acid, and in the last instance from the xanthin bases through processes of oxida- tion, can scarcely be doubted, but this transformation apparently also takes place in the liver.! Of late Folin’ has formulated a theory of proteid metabolism which in my judgment is more in conformity with our present knowledge of proteid digestion and more satisfactorily explains many questions connected with the subject of nitrogenous metabolism than any other. He distinguishes sharply between tissue metabolism or en- dogenous metabolism which tends to be constant, and exogenous or intermediate metabolism which is variable. As essential nitrogenous end product in the first instance he regards kreatinin, the elimination of which he finds practically constant for one and the same individual. Urea, according to his conception, is the principal nitrogenous end ‘product in the case of the exogenous metabolism. According to his idea the amino-acids which result on gastro-intestinal digestion, in so far as they are not needed immediately to make up for tissue loss ‘in nitrogen, are at once desamidized in the liver. ‘The non-nitrog- jenous remainder is then utilized in the. formation of fats and carbo- hydrates, while the amino group gives rise to the formation of urea. In this manner the presence of the large amounts of ammonium com- pounds which are found in the portal blood during digestion is well vexplained. But as Howell remarks, while a portion and perhaps a large portion of the urea arises from this early hydrolysis of the pro- teids of the food we must admit also that ammonium compounds may be formed in the tissues of the body generally, probably by a similar ‘process of hydrolysis followed by oxidation. ‘This would suggest itself especially under pathological conditions where the amount of ‘urea nitrogen may be in excess of that corresponding to the ingested food. It has been stated that 84 to 86.6 per cent. of all the nitrogen eliminated in the urine is in the form of urea, the remaining 13.4 per cent. being excreted as uric acid, hippuric a kreatinin, xanthin } 'The origin of urea: O. Schultzen u. M. Nencki, Zeit. f. Biol., 1872, vol. viii, 2. 124, E. Salkowski, Zeit, f. physiol. Chem., 1879, vol. iv, p. 100. v. KXnieriem, Zeit. f. Biol., 1874, vol. xD. Seok Salkowski, Zeit. f. physiol. Chem., 1877, ‘vol i, p. 38. Hoppe- Seyler, Physiol. Chem., 1881, p. 810. _ Drechsel, Jour. f. orakt. iy vol xv, p. 417: vol. xvi, pp. 169 and 180, and vol. xxii, p. 476. M. Hahn, V. Massen, M. Nencki, and J. Pawlow, “ La fistula d’Eck, ” ete., Arch. 'L. Sci. biol. de St. Petersburg, 1 1892, vol. i. ) Seat of formation: W. Srhréder, Arch. f. exper. Path. u. Pharmakol., 1882, Vol. xv, » p- 364. W. eeinicen Virchow’s Archiv, 1884, vol. xevii, p. 149. Min- xowski, ‘Ueber d. Einfluss d. Leberextirpation auf d. Stoffwechsel,” Arch. f. “xper: Path. u. Pharmakol., 1886, vol. xxi, p. 41, and 1893, vol. xxxi, p. 214. ?C. Voit, Physiol. d. alle. Stoffwechsels u. d. ’ Erniihrung. Hermans’ Hand- yuch d. Physiol., 1881, vol. vi, I, p. 301. O. Folin, Amer. Jour. of Physiol., 905, vol. xiii, 26 402 THE URINE bases, ete. It might hence be supposed that an accurate idea of the degree of tissue destruction could be formed from a quantita- tive estimation of urea. ‘This, however, is not the case, and especially in pathological conditions, as the quantitative relations existing between the excretion of urea and the remaining nitrogenous con- stituents are subject to wide variation. In acute yellow atrophy, for example, urea may disappear entirely from the urine, the nitrogen being eliminated in the form of other compounds (leucin, tyrosin, glycocoll, etc.). Whenever it becomes desirable, then, to gain an accurate insight into the degree of proteid destruction or proteid assimilation—in other words, into the nitrogenous metabolism—taking place in the body, it is necessary to resort to a quantitative determina- tion of the total amount of nitrogen excreted by the kidneys; the quantity found is then conveniently expressed in terms of urea. At the same time it is customary to express the amount of proteid tissue which is destroyed, as muscle tissue, as this serves as a fair type of body tissue in general. As 100 grams of lean muscle tissue contain about 3.4 grams of nitrogen, corresponding to 7.286 grams of urea, 1 gram of the latter is equivalent to 13.72 grams of muscle tissue. It is, hence, only necessary to multiply the quantity of urea eliminated in the twenty- four hours, corresponding to the total amount of nitrogen found by 13.72, in order to obtain an idea of the extent of albuminous destruction taking place in the body. If accurate results are desired, it becomes necessary to determine also the amount of nitrogen elimi- nated in the feces, a knowledge of the quantity in the food ingested being, of course, presupposed. With all these data given, the nitrogenous metabolism of the body can be accurately controlled. Example.—A patient eliminates 50 grams of urea in twenty-four hours; these 50 grams correspond to 50 13.72—. e., 686 grams of lean muscle tissue; on the other hand, he ingests an amount of © nitrogenous material corresponding to only 10 grams of urea, equiva- lent to 10 X 13.72—+. e., 137.2 grams of muscle tissue. The dif- ference between the amount ingested and that excreted in this case— _ i. e., 548.8 grams—must be referable to the destruction of organized | albumin. | When the amount of nitrogen eliminated is equivalent to that in- | gested, nitrogenous equilibrium is said to exist. A healthy person is _ approximately in this condition. | | During starvation urea is still eliminated from the body, although © in diminshed amount. ‘The question now arises, What happens if at this time an amount of nitrogenous food is given which corresponds - exactly in amount to that eliminated? Under such conditions an | increased elimination of nitrogen takes place, all of the nitrogen — ingested, in addition to that resulting from a breaking down of body CHEMISTRY OF THE URINE 403 tissues, being excreted. ‘The amount of nitrogen referable to the latter source, however, is somewhat less than that eliminated in the total absence of food. Unless starvation has been pushed too far, the body accommodates itself to the amount of food thus given and nitrogenous equilibrium is restored. If more food is allowed an increased elimination results, which again leads to a condition of nitrogenous equilibrium, different levels, so to speak, being possible. It is apparent, then, that the elimination of urea, and of nitrogen in general, is subject to great variation and depends to a great extent upon the amount ingested. A statement in figures expressing the daily elimination of urea and of nitrogen would, hence, be of very little value, especially in pathological conditions, in which the amount of nitrogen ingested is frequently very small. ‘The elimination of nitrogen should hence always be compared with the amount ingested, for which purpose the tables of Konig’ will be found most convenient. At the same time it must be remembered that not all the nitrogen taken into the body as food undergoes resorption, and that a variable _ amount, which in disease may be considerable, is eliminated with the | feces, so that in accurate work this nitrogen also must be taken into -account. In order to obviate the tedious estimation of nitrogen in the feces, it has been proposed to determine the standard amount of urea which should appear in the urine of a healthy person under different forms of diet. Such experiments, of course, presuppose the control person to be in a condition of nitrogenous equilibrium, which, from » what has been said above, is readily accomplished, as the human body adapts itself with ease to different forms of diet. In general practice, however, such a procedure would be difficult, but here approximate results can be obtained from a parallel estimation of the chlorides. In health the elimination of the chlorides may be placed at about one- half of the urea. Whenever the nitrogen resulting from tissue destruction is in excess of that referable to the proteids ingested, this | relation between the excretion of chlorides and urea will be disturbed, as the tissues of the body contain very litte sodium chloride. When- ever the amount of urea is in excess of the normal amount of chlo- rides, as indicated above, an increased tissue destruction may be in- ' ferred, and vice versa. If, on the other hand, the chlorides are present in diminished amount, the conclusion may be drawn that a retention _of albumins is taking place in the body; this is observed frequently during convalescence from acute febrile diseases. In most text-books the statement is found that the normal daily elimination of urea varies between 30 and 35 orams. This would imply that a lower amount could be viewed as abnormal. But, as _T have pointed out, the urea elimination depends essentially upon the amount of proteid food ingested, and I have long maintained that the 1 Chemie d. menschlichen Nahrungs u. Genussmittel, Berlin, 1893. 404 THE URINE consumption of such large amounts of proteids as would lead to the elimination of the quantities stated is totally unnecessary. Every clinician no doubt can recall data which would tend to support this view, and Chittenden and Folin have demonstrated the same fact by numerous observations. As Folin says: the immediate elimination of the greater part of the nitrogen contained in 118 to 130 grams of proteid (Voit’s standard) by means of the exogenous katabolism would seem to constitute very strong evidence in favor of the view that the proteid so katabolized can without harm, if not with advantage, be replaced by an equivalent quantity of carbohydrates. An increase in the amount of urea, and, as a matter of fact, of all the nitrogenous constituents, 1s observed especially in the acute febrile diseases, notwithstanding the diminished ingestion of nitrog- enous material, and is ascribed to the greatly increased tissue destruc- tion." An excretion of 50 grams or more is here frequently observed. Formerly it was thought that the fever itself was responsible for this increased elimination. But this view became untenable when it was shown that the excretion of urea in the beginning of a febrile attack is not proportionate to the height of the temperature, reach- ing its higest point only when the fever has been continuous for several days. Still larger amounts, moreover, may be eliminated when the fever is abating. Similar observations have since been made. An increased elimination of nitrogen may also be noted in almost every case of ague preceding the onset of the fever. ‘The latter, therefore, cannot be the only factor which causes the increased excretion of urea, and it has been suggested that the cells of the body have lost the power of taking up nitrogen. ‘The question, however, whether this is — dependent upon the increase in temperature or the action of certain — toxic substances circulating in the blood, or upon both, still remains » unanswered. | The large increase in the elimination of nitrogen in febrile dis- — eases is especially striking in those which end by crisis. ‘This is | notably the case in pneumonia, in which it may persist for two or three days after the occurrence of the crisis and is then no doubt largely due to the resorption of the exudate. Apparently, the only exception to the rule that the amount of urea is increased in acute febrile diseases is acute yellow atrophy, in which the excretion of urea is not only greatly diminished, but may cease altogether, its place being taken by other nitrogenous bodies, such as ammonium lactate, leucin, tyrosin, glycocoll, ete. Among afebrile diseases, in which an increased elimination of urea has been noted, may be mentioned the ordinary forms of diabetes a rE * Vogel, Zeit. f. rationelle Med., N. F., vol. iv, p. 362. Huppert, Arch. d. Heilk.; vol. vii, p. 1. Lébisch, Wien. med, Presse, 1889, vol, xxxix, p. 1521. Huppert He Riesellt, Arch. d. Heilk., vol. x, p. 329. Bauer u. Kistler, Deutsch. Arch. f. klin. Med., vol. xxiv, p. 53. j CHEMISTRY OF THE URINE AOS mellitus, in which the highest figures have been obtained, viz., 150 grams or more pro die. ‘This is, in all probability, explained, in part at least, by the ingestion of excessive amounts of proteid food by such patients, but carefully conducted experiments seem to show that a not inconsiderable portion of the urea is directly referable to increased tissue destruction. ‘The cases described by Hirschfele * however, which will be considered later on, form an exception to this rule. vy. Noorden and Lipman-Wolff have shown that anemia as such is not necessarliy associated with a pathological increase in the albu- minous metabolism. But it appears that in pernicious anemia, at least in the bothriocephalus form, there are periods in which an in- creased albuminous disintegration does occur. According to Rosen- qvist,” this is far too extensive to be dependent entirely upon the destruction of red corpuscles, but must be associated with changes in other nitrogenous tissues of the body. After the expulsion of the worms a well-marked nitrogenous retention was observed. ‘Similar results were obtained in cases of cryptogenetic pernicious janemia, where periods of markedly increased albuminous disinte- gration alternated sometimes with such of distinct nitrogenous reten- tion. Rosenqvist concludes that his observations are strongly in support of the theory that cryptogenetic pernicious anemia, like the bothriocephalus form, is also a toxic anemia. An unusually large output of nitrogen and greatly in excess of the amount ingested is apparently a common feature of acute leukemia. | Ebstein records a case in which 62 grams of urea were eliminated in twenty-four hours, and Edsall* mentions an instance in which with an intake of only 7.25 grams of nitrogen, 29.534 grams appeared in the urine. In this connection it is interesting to note that an astonishing in- ierease of the urinary nitrogen occurs on a-ray treatment in those cases of chronic leukemia, when the characteristic response so far as the effect upon the spleen and the number of the leukocytes is concerned, takes place, while in the negative cases this is not observed.’ In purpura hemorrhagica a notable increase of the urinary nitrogen oecurs, apparently without relation to the hemorrhages. Edsall men- tions an instance in which the patient, while ingesting not more than 3 to 4 grams, eliminated amounts varying between 14 and 23 grams. __ A moderate increase has been found in severe cases of chronic leu- ‘kemia, scurvy, minor chorea, and paralysis agitans. Observations made in cases of hystero-epilepsy have given rise to conflicting results. “Ueber eine neue klin. Form. d. Diabetes,” Zeit. f. klin. Med., vol. xix, pp. 294 and 325. * Berlin. klin. Woch., 1901, vol. xxxviii, p. 666. * Amer. Jour., Oct., 1905, p. 589. * Edsall and Musser, Univ. of Penn. Med. Bull. , Sept., 1905. 406 THE URINE It is claimed, on the one hand, that the excretion of urea is diminished following convulsive seizures of a hystero-epileptic nature, in contra- distinction to an increased elimination following true epileptic attacks, In cases of functional albuminuria associated with an increased elimination of uric acid or oxalic acid, I have observed an increased elimination of urea, and believe that in the treatment of these diseases a systematic study of the excretion of nitrogen is of fundamental importance. ‘The increase is here unquestionably due to the ingestion of excessive amounts of proteids. Of drugs, an increased elimination is produced by caffeine, mor- phine, codeine, ammonium chloride, sodium and potassium chlorides, lithium carbonate, following the ingestion of large amounts of water, etc. ‘The data concerning the action of quinine, salicylic acid, cold baths, etc., are conflicting. A large increase has been observed in cases of phosphorus poisoning. Electricity appears to exert a marked influence upon the excretion of urea, producing an increased elimination. The diminished elimination of urea observed in certain diseases of the liver,’ notably in acute yellow atrophy, carcinoma, cirrhosis, and even in Weyl’s disease, is of special interest, and is in perfect accord with the theory that the liver is the main seat of its production. As has been stated, urea may disappear altogether from the urine in acute yellow atrophy and also in Weyl’s disease, notwithstanding the frequently not inconsiderable degree of fever. In cirrhosis, hyperemia of the portal system has been thought to cause the dimi- nution, which may be increased further by the occurrence of ascites. In short, the factors which may be regarded as causing a diminished elimination of urea in hepatic diseases may be summarized under the following headings: 1. Destruction of hepatic parenchyma. 2. Diminished velocity of the flow of blood through the liver. 3. Insufficient excretion of bile and coincident digestive disturb-_ ances. | Whenever there is disease affecting that portion of the renal parenchyma which is concerned especially in the elimination of urea, a diminished amount will be met with, and carefully conducted observations upon the excretion of the various urinary constituents are here of considerable value from a diagnostic as well as a thera-_ peutic standpoint. However, as v. Noorden and others have pointed out, there are periods in the course of a nephritis when the urea | output is quite normal. While, as a rule, the excretion of urea is greatly increased in dia- | 1 Hallerworden, Arch. f. exper. Path. u. Pharmakol., vol. xii. Weintraud, ibid, | vol. xxxi. Stadelmann, Deutsch. Arch. f. klin. Med., vol. xxxiii. Fawitzki, ibid., vol. xlv. Frinkel, Berlin. klin. Woch., 1878 and 1892. v. Noorden, Lehrbuch d. | Path. d. Stoffwechsels, p. 287 CHEMISTRY OF THE URINE 407 betes mellitus, certain cases, which have been elaborately described by Hirschfeld," must be excepted. His researches have established beyond a doubt that the resorption of nitrogenous material from the intestines may be very much below moral: and with it the elimina- tion of urea. Upon these grounds he has advocated the recognition of a distinct form of diabetes, which is characterized by a com- paratively rapid course, the occurrence of colicky abdominal pains before or at the onset of the diabetic symptoms proper, the existence of pancreatic lesions in a certain proportion of the cases, a more moderate degree of polyuria, ete. In mental diseases a diminished excretion of urea has been ob- served in melancholia and in the more advanced stages of general paresis, while an increase is associated with the increased ingestion of food during the first stage of profound dementia. Following epileptic, cataleptic, and hysterical seizures, as well as in pseudohypertrophic paralysis, a decrease has been noted by some observers. In tetanus the elimination.of the urea nitrogen is normal or diminished. In Addison’s disease a decrease is commonly noted. All forms of chronic, non-progressive anemia are associated with a decrease, as are also osteomalacia, impetigo, lepra, chronic rheu- matism, ete. In chronic lead poisoning the elimination of urea may be greatly diminished. Little is known of the influence of drugs in bringing about a duninished excretion of urea. Properties of Urea.—Urea crystallizes in two forms, viz., in long, white needles if rapidly formed, or in long, colorless, quadratic rhombic prisms when allowed to crystallize gradually from its solutions. At 100° C. it begins to show signs of decomposition; at 130° to 132° C, it melts; and when heated still further it is decomposed into cyanic acid and ammonia, of which the former is immediately trans- ‘formed into its polymeric compound, cyanuric acid. Biuret is formed as an intermediary product during this decomposition, 2 molecules of urea yielding 1 molecule of ammonia and 1 molecule of biuret. As this substance, obtained on dissolving the residue remaining after all the ammonia has been driven off by careful heating, yields a beautiful reddish-violet color when a drop or two of a very dilute solution of cupric sulphate is added to its solution alkalinized with sodium hydrate, this reaction may be employed as a test in the detec- tion of urea (Biuret test). Very important is the behavior of urea when treated with a solu- tion of sodium hypochlorite or hypobromite, the most usual method of estimating urea being based upon this reaction, which may be represented by the equation CON,H,+ 3NaOBr=3NaBr+ 2N + CO,+ 2H,0. 1 Loe, cit, 408 THE URINE In the chapter on Reaction it was pointed out that urine gradually undergoes ammoniacal decomposition when exposed to the air; the ammonia is liberated from the urea according to the equation A CO + H,O=2NH, + CO,. \ NH 2 This decomposition may also be effected by heating a watery solu- tion of urea in a sealed tube to 100° C. Urea is readily soluble in water, fairly so in alcohol, and insoluble in anhydrous ether and benzol. ‘The aqueous solution of urea is neutral in reaction, but the substance combines with acids, bases, and salts to form molecular compounds. Urea nitrate, CON,H,.HNO,, crystallizes in two different forms: in thin rhombic or six-sided colorless plates, which are frequently observed arranged like shingles one on top of the other when rapidly = A! EE ) Ul ite Oy; ae YY lise As et Fic. 134.— Urea nitrate crystals. (Kruken- Fra. 185.—Urea oxalate crystals. (Kruken- berg, after Kiihne.) berg, after Kine.) formed (Fig. 134), while larger and thicker rhombic columns or plates are obtained if the process is allowed to proceed more slowly. Urea nitrate is readily soluble in distilled water, while in alcohol and in water containing nitric acid it dissolves with difficulty. Upon heat- ing, it evaporates without leaving a residue. Urea oxalate, CON,H,.C,H,O,, crystallizes in rhombic or six-sided prisms or plates (Fig. 135), which are less soluble in water than the nitrate; in alcohol and in water containing oxalic acid it is only impertectly soluble. With mercuric nitrate urea forms three different compounds, accord- ing to the concentration of the two solutions, viz., (CON,H,)Hg,(NOs),; (CON,H,).Hg,(NO,),, and (CON,H,),.Hg(NO,),+3HgO. ‘The lat-§ ter compound is of special importance, as Liebig’s quantitative esti- mation of urea was based upon its formation. a rubber tube attached to the reser- CHEMISTRY OF THE URINE 409 For the separation of urea from the urine the reader is referred to works on Physiological Chemistry. Quantitative Estimation of Urea. Hypobromite Method.— ‘he method most commonly used in the clinical laboratory is the one based upon the decomposition of urea into carbon dioxide and nitro- gen in the presence of sodium hypobromite. ‘lhe carbon dioxide thus formed is absorbed by an excess of sodium hydrate in the hypo- bromite solution, while the nitrogen is set free, and can be collected and measured; the determination of the corresponding amount of urea is then a simple matter. ‘The hypobromite solution is pre- pared from two stock solutions. ‘The first of these contain 125 grams of bromine and 125 grams of sodium bromide in 1000 c.c. of water. ‘The second is a 22.5 per cent. solution of sodium hydrate. Immediately before use equal portions of the two solutions are mixed and diluted with one and one-half volumes of water. The reaction which takes place may be represented by the equation 2NaOH + 2Br = NaBr+ NaOBr+ H,.O Various forms of apparatus, termed wreometers, have been sug- gested for the estimation of urea by this method. One which I have found very satisfactory is repre- sented in Fig. 136. It consists of a burette, C, with an ascending voir, B, which can be raised or lowered as required for the purpose of equalizing the pressure after col- lection of the gas. A descending Fre. 136.—The author’s ureometer. tube leads to a wide-mouthed bot- tle, A, which contains the hypobromite solution. ‘This is closed by a tightly fitting rubber stopper, to which a loop of platinum wire is attached carrying a little bucket made of glass or porcelain; this can be swung from its support by inclining the bottle. Metuop.—The rubber stopper is removed from the bottle A, and water poured into B until the system B C is filled to such an extent that the water level is visible in B above the point where the rubber tube is attached. About 25 to 30 c.c. of the hypobromite solution bi 410 THE URINE are placed in the bottle A, and 2 ¢.c. of urine in the bucket; this is then attached to the wire loop. ‘The stopper is now adjusted and the water in B and C brought to the same level, when the first read- ing is taken. A is then inclined until the bucket drops into the liquid below. ‘The nitrogen which is liberated collects in the burette C; as a consequence the water falls in C and rises in B. After twenty to thirty minutes the pressure in C is equalized by lowering B until the water in both tubes is at the same level. ‘The second reading is then taken, the difference between the two indicating the volume of nitro- gen liberated from 2 c.c. of urine at the temperature of the water in CB, which, as well as the barometric pressure, should be previously noted. As the volume of gases is influenced by the temperature, the baro- metric pressure, and the tension of the aqueous vapor, it becomes necessary, in order that the results reached shall be comparable with those obtained by other observers, to reduce the volume of nitrogen actually noted to a certain standard. ‘This has been placed at 0° C and 760 mercury millimeters pressure, in the absence of moisture. The correction is made according to the following formula: y OBOE thy ~ 760.(1+-0.00366.t)’ volume of the gas in terms of c.c., v the volume actually observed, B the barometric pressure in Hgmm., 7 the tension of the aqueous vapor at the temperature noted, t. ‘The volume of nitrogen observed being thus corrected, the calculation of the corresponding amount of urea is based upon the following considerations: From the formula CON, H, it is apparent that 2 atoms of nitrogen are contained in 1 molecule of urea; in other words, that 28 parts by weight of nitrogen correspond to 60 parts by weight of urea. ‘lhe equivalent of 1 gram of urea is then found according to the equation: 60 :28:: 1: x; and =().46666. ‘The volume corresponding to 0.4666 gram of dry nitrogen at 0° C. and 760 Hgmm. pressure is 372.7 c.c. It has been found, however, that only 354.3 ¢.c. of nitrogen are evolved from 1 gram of urea at best when the hypobromite method is employed Knowing that 354.3 c.c. of nitrogen correspond to 1 gram of urea, the amount of urea to which the volume of nitrogen actually observed is refer- able would then be found according to the equation in which V represents the corrected 1: 354.3:: a: y; and ee in which y denotes the number of cubic centimeters of nitrogen evolved from 2 c.c. of urine, and « the corresponding amount of urea. In order to ascertain the ee amount of urea it is only necessary to multiply the figure just obtained by 50. Precautions: (1) The urine must be free from albumin. (2) It should contain only about 1 per cent. of urea—i. e., not more than 0.025 i ? CHEMISTRY OF THE URINE 411 gram in 2 c.c. Whenever a greater amount is noted, therefore, the urine is diluted to the proper degree, due allowance being made in the calculation. In ordinary clinical work the barometric pressure, as well as the tension of the aqueous vapor, may be ignored. Of the many other ureometers the one devised by Doremus in the | modification of Heinz is most convenient and furnishes very satis- factory results. [ivi = =a us = BN s — OR — a) oad =C x leak eae Bie: =e = —o) = © a? = WC ; S \ Se SS Fic. 137.—Doremus-Heinz ureometer. Fia, 138.—Folin’s safety-tube. Its general construction is seen in Fig. 137.. A small amount of urine is poured into B while the stopcock (C) is closed. ‘This is then opened for a moment and again closed, so as to fill its lumen. ‘The tube A is washed out with water and filled with the hypobromite solution. ‘lhe tube B is filled with urine to the zero mark, and 1 c.c. (or less, if the urine is concentrated) is allowed to mix with the hypo- bromite solution’ in A. After all bubbles of gas have disappeared the reading is taken. Each small division corresponds to 0.001 gram ' This is prepared as described on p. 409. Rtn 412 THE URINE of urea and every ten divisions hence to 0.01 gram, for the amount of urine used, The urine must be free from albumin and should not contain more than 1 per cent. of urea. If necessary it is diluted with water. In the presence of ammonium compounds the results may be faulty, | | and in cases where this is suspected it is advisable to resort to more — accurate methods, such as that of Folin. } Method of Folin.'—This is based upon the following considera- | tions: At a temperature of about 160° C. crystallized magnesium | chloride, MgCl,.6H,O, boils in its water of crystallization. In such | a solution urea is quantitatively decomposed into ammonia and car-_ bon dioxide within one-half hour. If the process is carried out in | acid solution, the ammonia can subsequently be distilled off after | rendering the mixture alkaline, and is then titrated. ‘The correspond- | ing amount of urea is ascertained by calculation. At the same time, — however, the preformed ammonia is obtained, and it is hence necessary _ to eliminate this source of error by a separate estimation of this form. | This is conveniently done according to the method which has like-_ wise been suggested by Folin (see below). | MerHop.—3 c.c. of urine when carefully measured with a 5 c.c. pipette graduated in twentieths are placed in an Erlenmeyer flask of 200 c.c. capacity, together with 20 grams of magnesium chloride — and 2 c.c. of concentrated hydrochloric acid. (The magnesium chlo- ride usually contains a small amount of ammonia, which must be separately determined.) The flask is closed with a perforated stopper through which a specially constructed safety-tube passes (see Fig. 138).* The mixture is now boiled until the drops flowing back through the tube produce a hissing sound on coming in contact with the solution. After this point has been reached the boiling is con- tinued more moderately for about forty-five minutes. Immoderate foaming during this process and the subsequent distillation is guarded against by adding a small piece of paraffin (about the size of two coffee beans). The solution while still quite hot is carefully diluted to about 500 c.c.—at first by allowing the water to flow drop by drop through the tube; it is then transferred to a 1000 c.c. retort, treated with about 7 or 8 c.c. of a 20 per cent. solution of sodium hydrate, and the ammonia distilled off into a measured amount of a decinormal solution of sul- phuric acid. ‘The distillation may be interrupted when about 350 c.c. have passed over (viz., after about sixty minutes). The distillate is boiled for a moment to remove any carbon dioxide which may be present in solution, and on cooling is titrated to determine the excess of acid. Each cubic centimeter of the decinormal ammonia present . | | 1 Zeit. f. physiol. Chem., vol. xxii, p. 504, and vol. xxxvi, p. 333. ? The tube can be obtained from Messrs. Kimer and Amend, of New York. CHEMISTRY OF THE URINE 413 in the distillate corresponds to 0.003 gram, viz., to 0.1 per cent. of urea. From this result the amount of preformed ammonia and _ that resent in the 20 grams of magnesium chloride must be deducted. Estimation of Nitrogen.—For the purpose of estimating the total amount of nitrogen in the urine, the method of Kjeldahl is most con- veniently employed. _ Kjeldahl’s Method.’ Principle.—The organic matter of the urine Is decomposed by means of sulphuric acid, when all the nitrogen Fic. 139.—Kjeldahl’s nitrogen apparatus. ‘which is not present in combination with oxygen is transformed into ammonia. After adding sodium hydrate in excess the ammonia is distilled off and received in a known quantity of titrated acid, the excess being retitrated with sodium hydrate. In this manner the amount of ammonia and the corresponding quantity of nitrogen are ascertained, it being remembered that 17 grams of ammonia correspond to 14 grams of nitrogen. Reagents required: . 1. Gunning’s mixture. This consists of 15 c.c. of concentrated sulphuric acid, 10 grams of potassium sulphate, and 0.5 gram of * “Neue Methode zur Bestimmung des Stickstoffes in organischen Ké6rpern,” Zeit. f. analyt. Chem., 1883, vol. xxii, p. 366. 414 THE URINE cupric sulphate. In the place of Gunning’s mixture one of 500 c.c. of concentrated sulphuric acid and 100 grams of phosphoric anhy- dride may also be employed, and has the advantage that oxidation proceeds more rapidly. 2. A solution of sodium hydrate containing 270 grams in the liter (sp. gr. 1.243). 3. Pulverized taleum or granulated zinc. 4. A one-fourth norma! solution of sulphuric acid. 5. A one-fourth normal solution of sodium hydrate. Apparatus required (see Fig. 139): This consists of a retort of about 750 ¢.c. capacity (.4), which is connected with a Kjeldahl distilling tube (B), and through this with a Stideler condenser (C). ‘The ammonia is received in the nitrogen bulb at D. In addition a Kjeldahl digesting flask of 200 to 300 c.c. capacity is required. MetnHop.—Five or 10 c.c. of urine are placed in the digesting flask and treated with Gunning’s mixture. ‘To this end it is best to add the sulphurie acid and cupric sulphate first, to heat until sulphuric acid vapors are given off in abundance, and then to add the potassium sulphate. ‘The heat- ing is continued until the solution = ——s becomes entirely clear and almost _ Fig, 140.—Kijeldahl’s apparatus for the COlorless, the flask being inclined ea eM eS Crus coo u cue eect ert angle of about 45 degrees. Vigorous ebullition should be avoided. If the sulphuric acid-phosphoric anhydride mixture is to be employed, the urine is first treated with 0.4 gram of mercuric oxide, and 10 c.e. of the acid mixture added. Digestion is then carried on as described. Toward the end of digestion, in either case, it is advantageous to throw a few crystals of potassium permanganate into the fusion, so as to ensure complete oxidation. Upon cooling, the contents of the flask are transferred to the retort with the aid of a little water, and slowly treated with a moder- ate excess of the sodium hydrate solution. As a general rule, 40 c.c. for each 5 c.c. of sulphuric acid are sufficient. A little pulver- ized talcum or a few pieces of granulated zinc are finally added; the retort is connected with the condenser with the interpositon of the distilling tube and the distillation begun. The talcum or zinc serves the purpose of preventing undue frothing and bumping. ‘The dis-._ tillation is continued until about two-thirds of the solution have passed over. The distillate is received in the nitrogen bulb, which should CHEMISTRY OF THE URINE 415 contain a carefully measured quantity of the one-fourth normal solution of sulphuric acid. As a general rule, 30 c.c. are sufficient. As soon as the distillation is completed the condenser is discon- nected, washed out with a small amount of distilled water, and the washings added to the distillate. After the addition of a few drops of tincture of cochineal or dimethyl-amino-azo-benzol the excess of sulphuric acid is retitrated with the one-fourth normal solution of sodium hydrate, and the amount found deducted from the 30 c.c. used. ‘The titration should be continued until every trace of yellow (in the case of the cochineal) has disappeared and a pure rose color is Fic, 141.—Kjeldahl’s apparatus for the simultaneous distillation of six specimens: a, condenser; b,: distillation flasks; c, receivers. obtained, or, in the case of the dimethyl-amino-azo-benzol, until the last trace of red has disappeared and the solution has turned yellow. The difference multiplied by 0.0035 will indicate the amount of nitrogen present in the 5 or 10 c.c. of urine. ‘The corresponding amount of urea is found by multiplying this figure by 20. Whenever several nitrogen determinations are to be carried out daily it is convenient to make use of a special apparatus, which per- mits of such determinations being conducted at one time. ‘The general plan of the outfit is seen in the accompanying illustrations (Figs. 140 and 141). Ammonia. Every urine contains a small amount of ammonia, which normally varies but little, and corresponds to from 4.1 to 4.64 per cent. of the total amount of nitrogen, viz., to about 0.7 gram in the twenty- four hours. It is present in combination with the various acids of the urine, and in all likelihood represents a small amount of the 416 THE URINE ammonia which has not been transformed into urea, but has been utilized to saturate the affinities of a slight excess of acid, formed during the nitrogenous metabolism of the body, over the available fixed alkalies. In man an increased elimination of ammonia is observed when- ever an increased formation of acids occurs, or whenever a sufficient supply of oxygen is not available. In the latter case, no doubt, the increased elimination is owing to the fact that in consequence of the deficient supply of oxygen the synthetic formation of urea is impeded in the liver. As this organ, moreover, is the principal seat of the synthesis of urea, we can readily understand that extensive parenchymatous degeneration, as in acute yellow atrophy, in phos- phorus poisoning, etc., will lead to an increased elimination of ammonia. In any event, the relative increase of the ammonia is the essential factor, while variations in its absolute quantity are of secondary importance. Some of the results which have been obtained in various diseases are given in the following table: Per cent. Normal values” ><) 2 ¢ 577s) Oia Pe Oe Febrile diseases, 37 y. “24-20 (2 eee ae Carcinoma, of the liver: 39; = “are eee Oeste Liver abscess (actinomycosis) . . . : . . 10.60 Circulatory dyspnea =. 0 eeyhgtstee So MopaiN aitns Wyte eet Eee Laas cee Respiratory’ dyspnea 775 “iy mye ee ey das) Abnormally high absolute values are quite constantly observed in diabetes, in which a daily elimination of from 4 to 5 grams may be regarded as common. In a general way the amount of ammonia in cases of diabetes gives an idea of the amount of organic acids; but, as Herter has pointed out, we cannot detect moderate quantities of organic acids in this way. (See Oxybutyric Acid.) In cases of pernicious vomiting of pregnancy Williams' found a large increase of ammonia, up to 20 to 45 per cent., while this does not occur in nervous vomiting and in eclampsia. It is advised that in such cases the uterus be emptied, when the ammonia is said to drop at once. A slight rise occurs also in normal pregnancy and reaches its maxi- mum during labor. Very curiously a diminished elimination of ammonia is observed: in many cases of nephritis so long as symptoms of venous stasis do not exist. In a case of pernicious anemia relative amounts, varying between 3.3 and 5.6 per cent., were obtained during the days immediately preceding death. Quantitative Estimation. Folin’s Method.—10 c.c. of urine are diluted to about 45 c.c., treated with a small amount of burnt mag- 1Amer. Jour. of Med. Sci., September, 1906. ' CHEMISTRY OF THE URINE 41T nesia (0.5 gram), and boiled for forty-five minutes, the distillate being received in decinormal sulphuric acid through an absorption tube, such as the one pictured in Fig. 142. ‘This consists of a glass tube, a, measuring about 8 mm. in diameter, one extremity of which has been blown into the small bulb b. By means of a heated platinum wire five or six holes, each about 1 mm. in diameter, are made in the bulb; ¢ is a rubber stopper which fits into the second tube d. ‘This is merely a test-tube (2.5 em. in diam- lg eter) which has been cut about 7.5 em. from the ! | upper end. About 3 cm. from the upper margin wd this tube is provided with six or seven holes as in th a bulb b. ‘The entire apparatus is directly immersed in the decinormal acid and ensures the complete | absorption of the ammonia in one flask, even if this | contains only 5 to 10 c.c. of the acid. ‘The ammonia is then determined by titration as above, using alizarin red as indicator; 2 drops of a 1 per cent. solution suffice for 200 to 300 ¢c.c. The titration is carried to the red point, not to the violet. As a small amount of urea, however, is decomposed during the prolonged ebullition, it is necessary to ascertain separately the quantity of ammonia which is referable to this source. ‘To this end the retort is opened at the expiration of forty-five minutes, and an amount of water added which is approximately equivalent to that of the distillate. ‘The distillation is then con- tinued for another period of forty-five minutes; the distillate is received in decinormal sulphuric acid, - | : ee 1G, 142.—Absorp- and the ammonia referable to decomposition of the peta ies urea estimated as before. The difference between the two results indicates the amount of preformed ammonia that was originally present; 1 c.c. of the 75 sulphuric acid indicates 0.0017 gram of ammonia. This method is also applicable for the determination of ammonia in the blood. LireraturE.—Hallervorden, Arch. f. exper. Path., vol. xii, p. 237. Stadel- mann, Deutsch. med. Woch., 1889, p. 942. Michaelis, ibid., 1900, p. 276. O. Folin, Zeit. f. physiol. Chem., vol. xxxii, p. 575, and ibid., 1902, vol. xxxvii, p. 161. Uric Acid. According to our present views, uric acid, in man, is not formed during the decomposition of all albuminous substances, as was for- merly supposed, but constitutes a specific product of decomposition 27 —— A18 THE URINE of one class of albumins only, namely, the nucleins.* It appears, moreover, that the mother-substance of uric acid is confined to the true nucleins, viz., to those containing a nucleinic acid radicle, while the paranucleins, in which this is lacking, are without effect upon the elimination of uric acid. On decomposition the nucleins give rise to the appearance of the xanthin, alloxuric, or purin bases, which on oxidation are transformed to uric acid. According to Emil Fischer,’ the xanthins are derived from an hypothetical compound which he terms purin, and which he supposes to be constituted as shown in the formula (6) (Ly Nes CH | (7) (2)HC (5)C-—===NH, | | _>CH(8), (3)N c N= (4) (9) By substituting the group NH, for the H atom at 6, adenin thus results, and is hence also spoken of as 6-aminopurin: N-——C.NH, | | HC CeeINTt | | ‘ou. N C NZ Hypoxanthin, according to this conception, would be 6-oxypurin; xanthin 2, 6-dioxypurin, and guanin 2-amino-6-oxypurin, as shown by the structural formulas: HN——CO HN -CO | | | | HC C—NH CO C—NH | | cn . | |! cn, Nee Ce SNe HNa= C2 Ne Hypoxanthin. Xanthin, NH——CO Guanin, From the structural formula of purin it is also apparent that still other derivatives of this substance may exist, and as a matter of fact others are known, viz., mono-methylxanthin or heteroxanthin, di-methylxanthin or paraxanthin, tri-methylxanthin, the isomeric compounds of paraxanthin, viz., theophyllin and theobromin, and others. ‘Their relation to xanthin is shown in the formulas: 1C. E. Simon, Physiological Chemistry, Lea Bros. & Co. * Ber. d. Deutsck, chem, Ges., 1897, vol. xxx, p. 549, CHEMISTRY HN——CO ii i nrae HN O——N Xanthin, CH;.N CO | | CO c N.CH, | J cu. HN— CG N= Paraxanthin. HN——CO | | CO C—N.CH, STs, | *Scu. CH;.N RY y BE Ne Theobromine. OF THE URINE 419 HN- CO 69 pia © N.CH, | 1 aS OH HN CSN Heteroxanthin. CH;.N CO | C C-—_ NH | ! Son. CH,.N——-C——N-= Theophyllin. CH,.N——— CO | CO C——N.CH, : See CH,.N — C N= <2 Caffeine. ‘T'wo of these bodies, namely, heteroxanthin and paraxanthin, have also been found in the urine. From these basic substances, then, which are found in the nucle- inic acid radicle of the true nucleins, uric acid is supposedly derived, and there are numerous facts which go to show that this supposition is in all likelihood correct. It will thus be observed that structurally uric acid is intimately related to the bodies in ques- tion, and, like these, contains the purin radicle: HN2—CO | | CO C——NH, _ | | | CO. HN == C12 NH Urie acid. Adenin. Hypoxanthin. Guanin. ) Xanthin. Uric acid. | | | It may hence be regarded as 2, 6, 8 tri-oxypurin. _ the xanthin bases, moreover, qualitatively, all yield the same decom- b bf | J? uv _ position products when treated with fuming hydrochloric acid or | c / x ; ) J ] ; _ hydriotic acid under high pressure; only the quantitative relations vary, as shown in the equations: C,H,N,O + 7H,O =3NH,+ CO, + Uric acid and C;H,N; + 8H,O =4NH,+ CO, + CH,.NH,.COOH + 2H.COOH. Glyecocoll, Formic acid. CH,.NH,.COOH + 2H.COOH. C,H,N,O + 7H,0 =4NH, + 2CO, + CH,.NH,.COOH + H.COOH. C.H,N,O, + 6H,O =3NH, + 2CO, + CH,.NH,.COOH + H.COOH. C.H,N,O, + 5H,0 =3NH, + 3CO, + CH, .NH,.COOH In accordance with this supposed origin of uric acid we find an increased elimination following the ingestion of all substances which contain purin bases either as such or in the form of true nucleins 420 THE URINE (endogenous uric acid), At the same time it must be remembered that uric acid may also result from the nucleins of the body tissues; and we find, as a matter of fact, that during starvation uric acid does not disappear from the urine (endogenous uric acid). ‘The principal source of the uric acid under such conditions are the nucleins of the leukocytes; and, according to Horbaczewski' and others, this source is indeed more important than the nucleins of the food. According to this idea, the latter call forth an increased elimination of uric acid only in an indirect manner—. e., by stimulating more strongly than other food-stuffs the cell formation and cell destruction of the body. However this may be, there can be no doubt that the amount of uric acid eliminated in the urine depends, in the first instance, upon the amount of nucleins or purin bases as such which are ingested, and upon the degree of nuclear destruction which takes place in the body. Other factors, however, also enter into consideration. We thus know that the body is capable of transforming a certain amount of uric acid into urea. ‘This fact was pointed out long ago by Frerichs and Wohler, and has recently again been confirmed. It was found that after the ingestion of large amounts of nucleins only a certain por- tion of the nuclear nitrogen is eliminated as uric acid, and that this amount is extremely variable. Whether individual peculiarities have any part in determining this amount is unknown, but not improbable. Oxidation on the part of the body tissues must also be taken into consideration, and it unquestionably varies not only in different people, but also in the same individual at different times. ‘Then again there is evidence to show that under certain conditions uric acid may be formed synthetically in the body. ‘That this is the usual mode of formation in birds and reptiles has been shown by Minkowski,’ who found that after extirpation of the liver in geese the greater portion of the urinary nitrogen was eliminated in the form of ammonia in combination with lactic acid. In the human being very little uric acid is in all likelihood formed in this manner under normal conditions, but the possibility of its occurrence, in disease more particularly, should not be overlooked. As uric acid, moreover, may in part at least be eliminated in the feces, it is clear that the amount which appears in the urine cannot be regarded as an accurate index of the degree of nuclear destruction or of the amount which is formed in the body tissues. That retention of uric acid can further occur in the body, which may or may not be followed by increased elimination, is likewise undoubted. According to our present knowledge, uric acid is formed in all the organs of the body, including the bone-marrow, the muscles, the 1 Beitrige zur Kenntniss der Bildung von Harnsiure, etc., Monatshefte fur — Chem., 1891, vol. xii, p. 221; and Wien. Sitzungsber, vol. ec. ? “Ueber den Einfluss d. Leberextirpation auf den Stoffwechsel,” Arch, f, exper. Path u, Pharmakol., 1886, vol. xxi, p. 41. CHEMISTRY OF THE URINE 421 spleen, the liver, the kidneys, ete. Under pathological conditions it may also originate in the joints and tendons. Under normal conditions the daily elimination of uric acid varies between 0.2 and 1.5 grams, thus constituting 5!) to 7}, part of the total urinary nitrogen. It is largely influenced by the character of the diet, the amount of exercise taken, the general health of the individual, ete. After the ingestion of large amounts of food rich in nucleins, such as thymus gland, liver, kidneys,and brain, a corre- sponding increase in the amount of uric acid is observed. Generally speaking, animal food causes a greater elimination of uric acid than vegetable food, and it is supposed that this difference is essentially due to the extractives of the meat. Of special interest is the increase in the elimination of uric acid which is observed five hours after the ingestion of a full meal. ‘This increase, according to Horbaczewski,’ is associated with the disappearance of the digestive leukocytosis and consequent leukolysis. Some observers have attached much importance to the relation exist- ing between the elimination of uric acid and urea, and are inclined to assume the existence of a special wric acid diathesis when this rela- tion continuously exceeds the usual standard of 1 to 50 or 1 to 60. This question is an extremely intricate one, and we are scarcely in a position to speak definitely of the significance of such varia- tions. On the one hand, there can be no doubt that an unusually high uric acid coefficient may be met with in individuals who are apparently in good health, while in others, in whom larger actual amounts of uric acid are eliminated than are usual, normal or even subnormal values may be found. ‘The entire question of the uric acid diathesis is in a chaotic condition, and it would perhaps be well to speak of such a diathesis only when a distinct increase is continuously observed. ‘That numerous symptoms of a neurasthenic type are often seen when the uric acid coefficient is increased is a matter of daily observation, but it would be premature to regard this symptom as a causative factor of the disease in question.*’ Even in gout it can scarcely be said that uric acid has been proved the materia peccans, and our knowledge concerning the etiology of the disease is still as obscure as when Garrod* showed that an accumulation of uric acid occurred in the blood of such patients. Hitherto it has been 1A Hermann, “Abhingigkeit der Harnsiureausscheidung von Nahrungs- und Genussmitteln,” Deutsch. Arch. f. klin. Med., 1888, vol. xliii, p. 273. See also W. Camerer, Zeit. f. Biol., N. F., 1896, vol. xv, p. 140. * Harnsiiureausscheidung u. Leucocytose, Sitzungsber. d. Wiener Akad. d. Wissensch., 1891, Abth. 3. See also Léwit, Studien z. Physiol. u. Path. d. Blutes, 1892. W. Kihnau, “‘ Das Verhiltniss d. Harnsiureausscheidung zur Leucocy- tose,’ Zeit f. klin. Med., vol. xxviii, p. 534. P. F. Richter, ‘‘ Ueber Harnsiure- ausscheidung und Leucocytose,” ibid., vol. xxvii, p. 290. * C, E. Simon, Amer. Jour. Med. Sci., 1899, p. 139, and N. Y. Med. Jour., 1895, p. 330. * On the Nature and Treatment of Gout, 1847. 422 THE URINE supposed that the deposition of urates in the joints and periosteum of gouty patients is referable to a diminished alkalinity of the blood, and that acute paroxysms result whenever an increase in its alkalinity occurs, leading to a resorption of the urates previously deposited and a consequent flooding of the system with the material in question. As a matter of fact, a considerable diminution in its excretion is observed immediately preceding the attack, while during the par- oxysm and immediately following it a corresponding i increase 1s noted. Numerous investigations, however, have shown that distinct changes in the alkalinity of the blood do not occur in gout, and that an increase in the amount of uric acid in the blood may not only be observed in this disease, but in other diseases as well which are not associated with gouty symptoms. ‘The conclusion is hence justifiable that the pres- ence of uric acid in the blood per se cannot be offered as an explana- tion of the occurrence of a gouty attack.* Futcher,’ who has recently observed a number of cases of gout with modern methods, states that he almost invariably found that before the onset of the acute symp- toms the uric acid is below and often far below 0.4 gram. On the second or third day after the beginning of the acute symptoms the uric acid curve steadily rises, reaching 0.8 to 1.9 grams or even higher values. With the subsidence of the acute symptoms the curve gradually falls below the lower limit of the normal, and in the interval between the acute attacks the excretion may be only 0.1 to 0.2 gram daily. In one very marked chronic case Futcher found no uric acid excretion whatever on certain days during the interval. The phosphoric acid curve runs a course almost parrellel to that of the uric acid, which suggests quite strongly that even in gout the uric acid is derived from nucleins, and is not formed synthetically, as might possibly be imagined. The greatest increase in the elimination of uric acid is observed — in leukemia, in which the quantity may amount to over 12 grams in the twenty-four hours (case of Magnus-Levy). ‘That the increased elimination in this disease is referable to the enormous increase in the number of the leukocytes and consequent leukolysis can scarcely be doubted. In other diseases which are associated with a high grade of leukocytosis, and especially those in which the disease terminates by crisis or hastened lysis, such as erysipelas and pneumonia, a con- siderable increase is likewise observed, and is referable to the same origin. ‘This increase is especially marked immediately after crisis has occurred, but it not infrequently precedes this by several hours. 1B. Laquer, Ueber die Ausscheidungsverhiltnisse der Alloxurkérper. Berg- mann, 1906. (Full literature.) C. von Noorden, Lehrbuch d. Pathologie d. Stoffwechsels, Berlin, 1893. W. Ebstein, “‘Die Natur u. Behandlung der Gicht,” | Verhandl. d. VITI Congr. f. inn. Med., 1889, p. 133. 2“°The Occurrence of Gout in the United States,’ Jour. Amer. Med. Assoc., 1902, vol. xxxix, p. 1046. | | | | CHEMISTRY OF THE URINE 493 ~ In the other febrile diseases an absolute increase is less marked and inconstant. In diabetes a diminished amount of uric acid is usually found. Cases may be seen, however, in which, associated with a diminution or an entire disappearance of the sugar, a most marked increase occurs, amounting in some cases to 3 grams in the twenty-four hours. ‘T’o this condition the term diabetes alternans has been applied. In acute articular rheumatism an increased elimination is observed so long as the temperature remains high, while with approaching convalescence the amount returns to norm mal, and may even fall below normal. In chronic rheumatism, on ae other hand, no con- stant relations have been observed. In the ordinary forms of anemia and chlorosis the amount of uric acid is quite constantly diminished, as also in chronic inter- stitial nephritis, chronic lead poisoning, progressive muscular atro- phy, and pseudohypertrophic paralysis. According to Krainsky, Haig’ and Caro,’ a decrease in the output of uric acid ‘precedes the epileptic attack, and is subsequently followed by a rise to the same degree. Haig also noticed this in connection with attacks of migraine. Rather low amounts are reported by Edsall in a case of purpura hemorrhagica. Of special interest is the observation by Edsall that in those cases of chronic leukemia in which there is a response to a-ray treatment uric acid and purin bases are at once markedly increased. Properties of Uric Acid.—The close relation existing between uric acid and the xanthin bases has been already considered. By oxida- tion uric acid is transformed into urea or into substituted ureas, such as allantoin and alloxan, which latter in turn is closely related to parabanic acid or oxalyl-urea and barbituric acid or malonyl urea. Pure uric acid forms a white, crystalline powder which is almost insoluble in cold water (1 to 40,000), with difficulty soluble in boiling water (1 to 1800), and insoluble in alcohol and ether. In concentrated sulphuric acid it dissolves with ease, but is precipitated upon dilu- tion with water. In aqueous solutions of the alkaline carbonates and hydrates it dissolves, with the formation of neutral or acid salts, as represented by the equations: C,H,N,O, + Na,CO, =C,H,NaN,O, + NaHCo,. C,H,N,O, + 2Na,CO, =C;H,Na,N,O, + 2NaHCO,, In freshly voided urine uric acid is said to occur as a quadriurate, viz., as a compound in which one molecule of sodium is in combina- tion with two molecules of uric acid. ‘The quadriurate, however, is 1 Brain, 1896, p 194. ? Deutsch. med. Woch., 1900, No. 19. 424 , THE URINE readily decomposed with the formation of uric acid and acid urates (biurates). Its solubility in the urme depends upon the amount of water present, the reaction, and the presence of imorganic salts. When acid sodium phosphate preponderates, the biurate is precipi- tated, while free uric acid is thrown down when disodic phosphate only is present. Neutral urates cannot occur in the urine. ‘The basic substances which may occur in the urine in combination with uric acid are sodium, potassium, ammonium, and possibly also calcium and magnesium. ‘These salts may be decomposed by the addition of a sufficiently large quantity of a stronger acid, such as hydrochloric acid, when uric acid is set free. ‘lhe acid salts are soluble with great difficulty, and are hence precipitated whenever the urine is markedly acid or concentrated, and also when it is exposed to a low temperature. This holds good especially for the acid ammonium compound, and upon this fact Folin’s quantitative estimation of uric acid is based. Pure uric acid crystallizes in transparent, colorless, rhombic plates, while that which usually separates from the urine is of a reddish- brown color and may assume a great variety of forms (Fig. 143). Of these, the so-called whetstone form is the most characteristic (see Sediments). Colorless rhombic platelets may, however, also be seen. Of the compounds which uric acid forms with the heavy metals, the silver salt is especially important. When a solution of uric acid in ammonia is treated with an ammoniacal solution of silver nitrate (see below) the solution remains clear; but if calcium chloride, sodium chloride, or magnesia mixture is then added, a precipitate forms, which contains the uric acid in combinatioa with silver. Test for Uric Acid. Murexid Test.—A few crystals are dissolved by means of a few drops of concentrated nitric acid, with the appli- cation of heat, upon a porcelain plate, such as the cover of a crucible. The nitric acid is then carefully evaporated, when a yellowish-red spot will remain. Upon cooling, a drop of ammonia is placed upon this spot, when in the presence of uric acid a beautiful purplish-red color develops, owing to the formation of ammonium purpurate (murexid). If now a drop of sodium hydrate solution is added, the color changes to a reddish blue, which disappears upon heating; the reaction thus differs from the somewhat similar xanthin reaction. Folin’s Modification of Hopkins’ Method..—This is the most con- venient method for the estimation of uric acid in the urine, and as accurate as the more complicated procedure of Ludwig-Salkowski. It is based upon the precipitation of uric acid by ammonium sulphate, as ammonium urate, the decomposition of the latter by sulphuric acid, and the estimation of the liberated uric acid by titration with potassium permanganate. To precipitate the uric acid, and also to — 1O, Folin u. A. Shaffer, Zeit. f. physiol. Chem., vol. xxxii, p. 552. ' remove the small amount of mucoid substance which is found in every urine, the following reagent is employed: 500 grams of ammo- nium sulphate and 5 grams of uranium acetate are dissolved in 650 e.c. of water, to which solution 60 ¢.c. of a 10 per cent. solution of acetic acid are further added. ‘The resulting solution measures about 1000 e.c.; 75 c.c. of the reagent are added to 300 ¢.c. of urine in a flask holding 500 ¢.c. After standing for five minutes the mixture CHEMISTRY OF THE URINE 425 ~ Fia. 143.—Urie acid crystals. ie filtered through two folded filters, and thus freed from the mucoid { body, which is carried down with the uranium phosphate in acid solu- tion. The filtrate is divided into two portions of 125 c.c. each, which are placed in beakers and treated with 5 c.c. of concentrated ammonia. \After stirring a little the solutions are set aside until the next day. "The supernatant fluid is then carefully poured off through a filter ‘(Schleicher and Schiill, No. 597); the precipitated ammoniuin urate 496 THE URINE is collected with the aid of a small amount of a 10 per cent. solution -of ammonium sulphate and washed with the same reagent. ‘Traces of chlorides do not interfere with the subsequent titration, and the process of filtration and washing can be completed in from twenty to_ thirty minutes. ‘The ammonium urate is washed into a beaker, after opening the filter, using about 100 ¢.c. of water; 15 ¢.c. of con- | centrated sulphuric acid are then added, and the solution is titrated — at once with a one-twentieth normal solution of potassium permanga- nate. ‘Toward the end of the titration Folin suggests to add the per-_ manganate in portions of two drops at a time, until the first trace of a_ rose color is apparent throughout the entire fluid. Each cubic centi- meter of the reagent corresponds to 0.00375 gram of uric acid. A final correction (of 0.003 gram for every 100 c.c. of urine employed) is necessary, owing to the slight extent to which ammonium urate is soluble. Preparation of the One-twentieth Normal Solution of Potassium Permanganate.—-As_ the molecular weight of potassium permanga- nate is 157.67, one would expect that a normal solution of the salt — should contain this amount in grams dissolved in 1000 c.c. of water. But the substance generally acts in the presence of free acids, upon deoxidizing substances, by losing 5 atoms of oxygen of the 8 atoms contained in 2 molecules, as is seen in the following equation: 2KMnO, + 5H,C.O, + 3H.SO, = K,SO, + 2MnSO, + 10CO, + 8H,0. It follows that two-fifths of the molecular weight, or 63.068 grams, are the equivalent of 1 oxygen atom. But as oxygen is diatomic and the volumetric normal is calculated for monatomic values, this ° number must be divided by 2, and 31.534 grams of potassium per- manganate should therefore be present in 1 liter of normal solution. A one-tenth normal solution would hence contain 3.1534 grams, and — a one-twentieth normal solution 1.576 grams pro liter. ‘This amountis © weighed off and dissolved in 950 c.c. of water, when the solution is | brought to the proper degree of dilution by titration with a one- | tt pecibes th normal solution eat oxalic acid. A one-twentieth normal solution of oxalic acid contains 3.142 grams of the acid in 1000 | c.c. of water. One c.c. of the one-twentieth normal solution of potassium permanganate should correspond to 1 ¢.c. of the oxalie j i acid solution. ‘The titration is best conducted by diluting 10 ce. | of the oxalic acid solution to 100 c.c. with distilled water and add- ing 15 ¢.c. of concentrated sulphuric acid, so as to bring the tempera- ture of the liquid to from 55° to 65° C. ‘The potassium perman- ganate solution is then added drop by drop until the red color no | longer disappears on stirring, but persists for at least thirty seconds. For Salkowski’s method of estimating uric acid see method for estimating the xanthin bases. 4 - CHEMISTRY OF THE URINE 427 | The Xanthin Bases. ~The xanthin bases which have been found in the urine are xanthin, hypoxanthin, heteroxanthin, paraxanthin, guanin, and adenin. Con- jointly they are also spoken of as the alloxur bases, or purin bases. ‘Together with uric acid they are termed alloxur or purin bodies. Their relation to uric acid and the nucleins has already been con- | sidered. (See Uric Acid.) Unlike uric acid, they also occur as such ‘in animal as well as vegetable tissues. The amount which appears in the urine under normal conditions is very small, constituting about 10 per cent. of the uric acid. Larger quantities may be met hwith i in various diseases, and, generally speaking, an increase in the Mount of uric acid is associated with an increase of the xanthin ‘bases. This is, however, not invariably the case, and at times it may be observed that an increase of the uric acid is accompanied by ia diminution of the xanthins, and vice versa. ‘These varying rela- ‘tions can, of course, be readily understood if we remember that uric ‘acid is an oxidation product of the xanthin bases, and that their ‘ultimate origin is the same. ‘The largest quantities of xanthin bases are ‘found in leukemia; Magnus-Levy has reported a case with 0.321 gram. Individually the xanthin bases are of little clinical interest. \Xanthin has once been found in a urinary sediment, and has in ‘several instances been encountered as the principal constituent of “yesical calculi. Its normal quantity is said to vary between 0.02 and 0.03 gram. Larger quantities are found after a meal rich in nucleins, in leukemia, nephritis, pneumonia, ete. Paraxanthin and heteroxanthin are present only in traces, as is apparent from the fact that Kriiger and Salomon were able to obtain ‘but 7.5 grams of heteroxanthin from 10,000 liters of urine. Both apparently are distinctly toxic. | Xanthin sediments may be recognized by means of the following test: A small amount of the material is treated with a few drops of concentrated nitric acid on a porcelain plate, and evaporated to dry- ness. In the presence of xanthin a yellow residue is obtained, which turns a violet red upon the addition of a few drops of sodium hydrate ‘solution and the application of heat (Strecker’s test). The reaction is common to all the xanthins and should not be confused with the murexid test. _ Quantitative Estimation. Salkowski’s Method.'—600 c.c. of urine are precipitated with 200 c.c. of magnesia mixture (composed of 1 part of crystallized magnesium sulphate, 2 parts of ammonium ichloride, 4 parts of ammonium hydrate, and 8 parts of distilled water), when a 3 per cent. ammoniacal solution of silver nitrate is added to from 700 to 750 c.c. of the filtrate. The proportion should be 6 c.c. | 1 Pfliiger’s Archiv, vol. lxix, p. 268. | 428 THE URINE ) | for each 100 c.c. of urine. If the precipitated silver chloride formed in the beginning does not disappear on stirring, a little more ammo-| nium hydrate is added. A flaky precipitate next separates out, and _ is allowed to settle. In order to t:st whether enough of the silver nitrate solution has been added, a few cubic centimeters of the super-| natant fluid are acidified with nitric acid. If a distinct cloudiness, referable to silver chloride, appears, enough has been added. Other- wise the few cubic centimeters that were employed for this test are, rendered alkaline again with ammonia, poured back, and treated - with more silver solution until the required amount has been reached. | After standing for one hour the mixture is filtered and the precipitate | washed with water until all the free silver has been removed. ‘The filter is then perforated, the precipitate washed into a flask with. from 600 to 800 c.c. of water, acidified with hydrochloric acid, and. decomposed with hydrogen sulphide. ‘The excess of hydrogen sie! phide is removed by heating on a water bath, when the silver sulphide | is filtered off and the filtrate evaporated to dryness. ‘The residue is treated with from 25 to 30 c.c. of dilute sulphuric acid (1 to 100). This. solution is brought to the boiling point and is allowed to stand over night. The uric acid which has then separated out is filtered off, washed with a small amount of dilute sulphuric acid (not more than 50 e.e.), then with alcohol and ether, and weighed. ‘To the resulting | weight 0.0005 gram is added for each 10 c.c. of the acid filtrate, to’ allow for the trace of uric acid which is thus lost. After having filtered off the uric acid the filtrate is again treated with ammonia and silver solution, and the xanthin bases thus pre- | cipitated. ‘Ihe precipitate is collected on a small filter, washed with | water, dried, and incinerated. ‘The ash is dissolved in nitric acid, | and the silver estimated by titration with a solution of potassium | sulphocyanide, using ammonioferric alum as an indicator. (See Chlo-| rides.) ‘The solution of potassium sulphocyanide employed in the’ estimation of the chlorides may be used, and is of such strength | that 1 ¢.c. corresponds to 0.00734 gram of silver. As 1 atom of: silver in a mixture of the silver compounds of guanin, xanthin, | hypoxantin, etc., represents 0.277 gram of nitrogen, or 0.7381 gram | of the alloxur bases, it is apparent that 1 c.c. of the potassium sul- | phocyanide solution will represent 0.002 gram of nitrogen and | 0.00542 gram of alloxur bases. In every case an accurate record | must, of course, be kept of the amount of urine and filtrate used. The amount of alloxur bases found by Salkowski in the normal urine of twenty-four hours varied between 0.0286 and 0.0561 gram. LITERATURE.—M. Kruger u. G. Salomon, “Die Alloxurbasen d. Harns,”’ Zeit. | f. physiol. Chem., vol. xxiv, p. 364, and vol. xxvi, 343; Deutsch. med. Woch.,, | 1899, p. 97. Bondsynski u. Gottlieb, “Ueber Xanthinkérper im Harn des Leukamiker,”’ Arch. f. exper. Path. u. Pharmakol., 1895, vol. xxxvi, p. 1382 F. | Gumprecht, ‘‘Alloxurkérper u. Leukocyten,” Centralbl f. allg. Path. u. path. Anat., 1896, vol. vii, p. 820. . CHEMISTRY OF THE URINE 429 | Hippuric Acid. | | Hippuric acid is a constant constituent of normal urine, 0.1 to 1 gram being excreted in the twenty-four hours. hat it is derived, 4 “o some extent at least, from albuminous material is proved by the fact that its elimination is not suspended during starvation nor during the administration of a purely albuminous diet. Jn vitro it may ‘ve obtained from glycocoll and benzoic acid, according to the equation | + | = | + HO. | COOH COOH COOH ) Benzoic acid. Glycocoll. Hippuric acid. It has been shown that phenylpropionic acid, which differs from ‘benzoic acid by the group C,H,, and which may be regarded as ee cy formic acid, is produced during the process of intestinal outrefaction. ‘lhe relation between the two bodies is seen from the Yormulas: H C,H; CH; CH,.C,H; eee | | COOH COOH OC Hipte———=an Gis Formic Phenylformic | | acid. acid. COOH COOH Propionic Phenylpropionic | acid, acid, Phenylpropionic acid is then absorbed into the blood and there, wcording to our present ideas, transformed into phenylformic acid yw benzoic acid. When the latter comes in contact with glycocoll, which is produced during the process of pancreatic digestion, an nteraction between the two substances occurs in the body, hippuric teid resulting, as shown in the above equation. ‘This view is sup- ported by the fact that phenylpropionic acid, just as benzoic acid, Wwhen introduced into the circulation of certain animals, reappears in he urine as hippuric acid. The final proof of the possible synthesis # hippuric acid from glycocoll and benzoic acid in the body has been urnished by Bunge and Schmiedeberg,’ who obtained this substance, when arterialized blood containing glycocoll and sodium benzoate vas allowed to pass through isolated kidneys of dogs. Not all the hippuric acid eliminated, however, is referable to albu- ‘ninous decomposition, but a considerable portion is derived from denzoic acid or its derivatives, which occur in many fruits, and we transformed into hippuric acid in the body. Among those which are particularly rich in these substances may be mentioned ‘he red bilberry, prunes, coffee-beans, green gages, etc., and in all tases in which an increased elimination of hippuric acid is observed she possibility of this source must be taken into account. * Arch. f. exper, Path, u. Pharmakol., vol vi, 43 THE URINE As to the seat of the synthesis there appears to be some uncer- tainty, as 1t is apparently not the same in all animals. In the dog and the frog the kidneys, according to the researches of Bunge and Schmiedeberg, must be regarded as the principal and possibly the only organs in which this process occurs. As Salomon, however, has demonstrated the presence of hippuric acid in the muscles, liver, and blood of nephrectomized rabbits, still other organs must, in the herbivora at least, be concerned in its production. Very little is known of the pathological variations in the excre- tion of hippuric acid; this is principally owing to the fact that until recently suitable methods for its quantitative estimation were not available. It is an interesting fact that, in accordance with Bunge’s experiments in dogs, the formation of hippuric acid appears to be suspended in cases of acute as well as chronic parenchymatous nephritis, for the benzoic acid which is then ingested reappears Fic. 144.—Hippuric acid crystals. in the urine unchanged. In amyloid degeneration a marked dimi-_ nution has likewise been demonstrated. Large quantities of hippuric acid, on the other hand, have been noted in acute febrile diseases, hepatic diseases, diabetes mellitus, chorea, etc. ‘The data, how: | ever, are ienient to warrant any definite conclusions.' Properties of Hippuric Acid.—Hippuric acid crystallizes in loi ) rhombic prisms when allowed to separate from its solutions gradually, | while it forms long needles if crystallization takes place rapidly and the amount is small (Fig. 144). In cold water and ether it is soluble. with difficulty, while it dissolves readily in hot water, in alcohol, and | in aqueous solutions of the hydrates and carbonates of the alkalies, with which it forms salts, and from which the acid may again be separated and caused to crystallize out by adding a stronger acid. 1 Th. Weyl u. B. von Anerep, “Ueber die Ausscheidung der Hippursiure und Benzoesiure wiihrend des Fiebers,’’ Zeit. f. physiol. Chem., 1880, vol. iv, p. 169 5 1 | CHEMISTRY OF THE URINE 431 When hippuric acid or one of its salts is evaporated to dryness with concentrated nitric acid and the residue is heated, the odor of bitter almonds is noticed; this is due to the formation of nitrobenzol. When boiled with hydrochloric acid or dilute sulphuric acid hippuric acid is decomposed into glycocoll and benzoic acid. A similar decomposition is effected during the process of putrefaction, and hence no hippuric acid is found in decomposing urine, benzoic acid taking its place. ‘The latter is always found in the urine together with hippuric acid, but has no clinical significance. In larger amounts it has been encountered in diabetes. It crystallizes in needles or lustrous lamin, presenting ragged edges, which resemble plates of cholesterin. It is soluble with difficulty in cold water, but easily soluble in ether, alcohol, and solutions of the alkaline carbonates and hydrates, forming salts with the latter. Hippuric acid in the urine occurs in combination with sodium, potassium, calcium, and magnesium. Quantitative Estimation of Hippuric Acid—The following method may be employed for the quantitative estimation of hippuric acid: Principle-—Hippuric acid readily dissolves in solutions of the alkaline hydrates and carbonates, forming salts. ‘hese are decom- posed by means of a stronger acid, when the hippuric acid which “separates out is collected and weighed. Mernop.—500 to 1000 c.c. of fresh urine are evaporated to a ‘syrupy consistence on a water bath, care being taken to keep the urine neutral by the addition of sodium carbonate. ‘The residue is extracted with cold alcohol (90 to 95 per cent.), using about half of the quantity as that of the urine employed. ‘The mixture is \then set aside for twenty-four hours. ‘The alcoholic filtrate, which contains the salts of hippuric acid, is freed from alcohol by dis- itillation. ‘The remaining solution is strongly acidified with acetic acid and extracted with at least five times its volume of alcoholic ether (1 part of alcohol to 9 parts of ether). From the combined extracts the ether is distilled off and the remaining solution evapo- ‘rated on a water bath. ‘lhe resinous residue is boiled with water, ‘set aside to cool, and filtered through a well-moistened filter. The hippuric acid, which is easily soluble in boiling water, is thus ‘separated from other constituents which are soluble in alcohol and ether. ‘The filtrate is rendered alkaline with a little milk ‘of lime, any excess of calcium being removed by passing carbon ‘dioxide through the mixture. ‘This is then brought to the boiling point and filtered. Any impurities which may be present are re- moved by shaking with ether. ‘The calcium salts remaining in solu- tion are decomposed by means of an acid, when the solution is again extracted with ether. ‘The remaining solution is evaporated to a few cubic centimeters, when the hippuric acid will separate out on stand- 432 THE URINE ing. ‘The crystals are dried on plates of plaster of Paris, shaken with benzol or petroleum ether to remove any benzoic acid, and finally weighed. ‘These crystals may be shown to be hippuric acid by their microscopic appearance, their solubility in alcohol, and their behavior when evaporated with concentrated nitric acid, as indicated above. Hofmeister’s Method.—200 to 300 c.c. of urine are evaporated in a glass dish to one-third of the original volume, and treated with 4 grams of disodium phosphate, to transform the acid into its sodium salt. ‘The mixture is evaporated to a syrupy consistence, the resi- due treated with burnt gypsum, dried thoroughly, and pulverized together with the dish. ‘The powder is extracted in a Soxhlet apparatus with freshly rectified petroleum ether (boiling point 60°” to 80° C.) for forty-six hours, and then for six to ten hours with pure ether (free from water and alcohol). After distilling off the ether _ the residue is dissolved in boiling water and decolorized with animal charcoal, the latter being subsequently thoroughly washed with boil- ing water; the solution and washings are evaporated to about 1 or 2 | c.c, at a temperature of from 50° to 60° C., and set aside to crystallize. The crystals of hippuric acid are finally washed with a few drops of water and ether, and weighed. Kreatin and Kreatinin. The antecedents of kreatin and kreatinin are unknown. ‘Two | sources of the urinary kreatinin must be recognized, viz., the muscle tissue of the body and the muscle tissue ingested as food. ‘The tissue | kreatin is possibly transformed into kreatinin and eliminated in this _ form, while the kreatin which has been ingested does not appear in the urine as kreatinin. Its fate is not known. Folin regards kreatinin — as the essential end product of the endogenous nitrogenous katab- | olism, in so far at least as the muscle tissue is concerned, He has demonstrated the interesting fact that its absolute quantity on | a meat-free diet is a constant quantity, which is different for different | individuals, but wholly independent of quantitative changes in the total | amount of nitrogen eliminated. Its relative amount is increased when _ the urea nitrogen falls. Ona diet rich in proteids the kreatinin nitrogen — represents 3.2 to 4.5 per cent. of the total, while on one free from pro- _ teids (starch and cream) the amount may rise to 17.4 per cent. ‘The absolute amount seems to depend to a certain extent upon the body weight. Fat or corpulent persons yield less kreatinin per unit of body weight, namely, 20 mgrms. per kilo, while lean persons yield about | 25 mgrms. 1.15 to 1.6 grams may thus be regarded as average values. | The study of pathological variations in the amount of kreatinin has been greatly facilitated through the introduction of Folin’s method | CHEMISTRY OF THE URINE 433 (see below). ‘The older data are of little importance, unless the diet of the individual has been carefully considered. A diet rich in meats, it should be borne in mind, greatly increases the amount. If then in patients affected with acute febrile diseases, such as pneumonia, typhoid fever, etc., a large increase is observed, the patient being at the same time upon a milk diet, an increased destruction of muscle tissue may be inferred, as a milk diet in itself, ceteris paribus, causes a diminished elimination. A decrease would logically be expected to occur during convalescence from such dis- eases. In the various forms of anemia, marasmus, chlorosis, phthisis, etc., a diminished amount is observed.!| The same is seen in advanced cases of chronic parenchymatous nephritis, in progressive muscular atrophy, in pseudohypertrophic paralysis, and in progressive ossifying myositis. Properties of Kreatin and Kreatinin —Chemically, kreatin may be regarded as a methyl derivative of glucocyamin, which latter is guanidin in which 1 NH, group has been replaced by glycocoll. Kreatinin, on the other hand, is the methyl derivative of glucocy- amidin, which differs from glucocyamin only in the absence of 1 molecule of water, so that kreatinin is kreatin minus 1 molecule of water, both being thus theoretically derivatives of guanidin. The relation between the various bodies is shown below: oN Et O-=NH \ NH, Guanidin. /NH, /NH, Ca-NH CaN F \. NH.CH,.COOH \N(CH;).CH,.COOH Glucocyamin. Kreatin. | 7 NH “NH G=NH CN \.NH.CH,.CO \. N(CH,).CH,.CO Glucoeyamidin (glucocyamin minus water), Kreatinin (kreatin minus water). _ Kreatinin crystallizes without water of crystallization in colorless, glistening prisms. At times, when the crystals are not well devel- oped, it also appears in the form of whetstones. It is readily soluble in hot and also quite soluble in cold water and hot alcohol; in cold ‘alcohol and ether it dissolves with difficulty. It forms salts with acids, and double salts with some of the salts of the heavy metals. Among these may be mentioned kreatinin hydrochloride, C,H_N,O.- HCl, which is easily soluble in water and crystallizes in the form of transparent prisms or rhombic plates. Most important is the com- ‘pound of kreatinin with zine chloride, (C,H,N,O),.ZnCl, (Fig. 145). (This is produced when a watery or alcoholic solution of kreatinin is *C. E. Simon, Physiological Chemistry, Lea Bros. & Co., 1907. Senator, Virchow’s Archiv, 1876, vol. lxvii, p. 422. Neubauer u. Vogel, Harnanalyse, pt. ii, . 28 434 THE URINE treated with zine chloride. ‘The crystalline form of this compound depends greatly upon the purity of the kreatinin solution. When obtained from alcoholic extracts of the urine it occurs in the form of varicose conglomerations which often adhere firmly to the walls of the vessel. If the solution of kreatinin is perfectly pure, how- ever, it is seen in the form of fine needles grouped in rosettes or sheaves, Kreatinin-zine chloride is soluble with much difficulty in water and insoluble in alcohol. ‘The compound is especially impor- tant, as upon its formation and properties the quantitative estimation of kreatinin in the urine is based. Silver nitrate and mercuric chlo- ride cause a precipitation of kreatinin, and may, therefore, also be employed for the purpose of obtaining the substance from the urine. Fic. 145.—Crystals of kreatinin-zine chloride. (Salkowski.) Test for Kreatinin in the Urine.—A few cubic centimeters of urine are treated with a few drops of a very dilute solution of sodium nitroprusside and then drop by drop with a dilute solution of sodium: hydrate. In the presence of kreatinin the urine assumes a ruby-red color, which is particularly well seen in the lower portion of the tube. ‘This color disappears after a few minutes, and is replaced by an intense yellow, which on warming with glacial acetic acid in pure solutions turns to green, then to blue, and on standing a deposit of Prussian blue is obtained (Weyl’s test). The presence of albumin or sugar does not interfere with the reaction. Folin’s Method.2—This method is based on Jaffé’s reaction of kreatinin with alkaline picric acid solution. ‘The red-colored solu- tion produced in this reaction has in proper concentration and when viewed by transmitted light exactly the same shade as a potassium 1 Th. Weyl, Ber. d. deutsch. chem. Gesellsch., 1878, vol. xi, p. 217; and Jaffé, Zeit. f. physiol. Chem., 1886, vol. x, p. 399. ? The above description of the method I owe to the courtesy of Dr. Folin. CHEMISTRY OF THE URINE 435 bichromate solution. Half-normal potassium bichromate solution (containing 24.55 grams per liter) is therefore used as a standard for comparison. A high-grade colorimeter, by means of which the depths both of the unknown solution and of the bichromate an be adjusted to tenths of millimeters, is necessary for the comparison.’ The following solutions are also necessary: The half-normal potas- sium bichromate solution, 10 per cent. sodic hydrate, and a satu- rated (1.2 per cent.) picric acid solution. If to 10 mgrms. of chemically pure kreatinin dissolved in 10 c.c. of water in a 500 c.c. volumetric flask are added 15 c.c. of picric acid solution and 5 c.c. of sodic hydrate, the maximum color is ob- tained at the end of five minutes. If at the end of this time the solution be diluted to the 500 c.c. mark and at once compared with the standard bichromate solution, it will be found that 8.1 mm. of the kreatinin-picrate solution have in the colorimeter exactly the same shade and depth of color as 8 mm. of the bichromate solution. The actual determination in urine is carried out in exactly the “same way, substituting 10 c.c. of urine for the kreatinin solution. ‘The more kreatinin that is present in the 10 ¢.c. of urine the deeper will, of course, be the color of the solution obtained. Supposing the colorimetric observation shows that 7.1 mm. of the urine-picrate ‘solution are equal in color to 8 mm. of the standard, the 10 c.c. : 8.7 a of urine would then contain 10 X - jail4 mgrms. of kreatinin. The following precautions are to be observed in the determination: 1. Make first a preliminary colorimetric observation, using half- normal potassium bichromate solution in both cylinders of the colorimeter, adjusting first one to the 8 mm. mark. ‘The average of three or four readings of the other cylinder should also be 8 mm., and after the first observation no two should differ by more than 0.2 mm. ‘This preliminary observation takes only two or three minutes, and is exceedingly useful in making the eye sure of the correct point to be ascertained. 2. Exactly 8 mm. of the half-normal potassium bichromate solu- tion must be used as the standard for comparison. 16 or 24 mm., for example, cannot be substituted on the basis of the calculation given above because the kreatinin-picrate solution absorbs light at an entirely different rate from that of the bichromate solution. 3. For the reason given in the preceding paragraph it is necessary to make each determination with a quantity of urine containing not ‘less than 5 nor more than 15 mgrms. of kreatinin. Within these | limits the determination as described is correct within 0.2 mgrm. 4. Sugar and albumin do not interfere with the determination. Acetone, diacetic acid, and hydrogen sulphide do interfere. Where 1 The French instrument of Duboscg, which can be obtained through Eimer & _ Amend, is admirably suited for the purpose. 436 | THE URINE these are present the urine should be measured into a porcelain evap- orating dish and heated on a water bath with 10 ¢.c. of 1 per cent. hydrochloric acid for about half an hour. When the dish is again cooled, the reagents are added directly into the dish, and finally rinsed into the volumetric flask after five minutes. 5. ‘The color due to the urine is ordinarily of no appreciable con- sequence because of the great dilution. Urines containing bile pig- ments can, however, first be cleared by the addition of egg albumen and then removing this by coagulation (heat). The whole operation can be finished in less than fifteen minutes; indeed, it should be finished at once, as the colored product obtained by the interaction of kreatinin and picric acid is not very stable. Oxalic Acid. The origin of oxalic acid in normal urine is twofold. ‘The greater portion is supposedly derived from the ingested food, but there is evidence to show that a certain amount is also formed during the metabolism of the body tissues, as the elimination of oxalic acid does not cease during starvation. The carbohydrates and_ fats probably do not play a part in this connection; and, according to Salkowski, the albumins also do not enter into consideration per se. He rather inclines to the view that the nucleins represent the antecedent of the oxalic acid, and as a matter of fact uric acid, which, as we have seen, is itself derived from the nucleinic bases, can be readily oxidized to oxalic acid, with the intermediary formation of parabamic acid and oxaluric acid. ‘The latter has been repeatedly demonstrated in the urine, and it is conceivable that the same pro- cess may occur in the animal body. But even supposing that the oxaluric acid which is obtained from the urine is formed artificially during the lengthy process of analysis, and that the substance did not exist preformed, there is no reason for the assumptioa that uric acid may not be the normal antecedent of the oxalic acid. For Salkowski has demonstrated conclusively that on oxidation with ferric chloride in aqueous solution uric acid yields oxalic acid and urea directly. The matter, however, is not quite so simple as it appears, and an increased elimination of oxalic acid by no means always occurs when the output of uric acid is increased. After the ingestion of fairly large amounts of thymus, for example, the usual increase of uric acid is not accompanied by a corresponding increase in the amount of oxalic acid, and in those cases in which it does occur we are as yet unable to exclude the large amount of connective © tissue as the source of the oxalic acid. Connective tissue and gelatin have, as a matter of fact, been shown to increase the amount of CHEMISTRY OF THE URINE 437 oxalic acid when given in large amounts. With pure nuclein no effect has been observed, and it can be shown that in those experi- ments in which this was used by mouth an absorption from the intestinal tract had manifestly not occurred (Mohr and Salomon)." Under pathological conditions oxalic acid may also be formed in the digestive tract from the ingested carbohydrates, as a result of a peculiar fermentative process. ‘This has been well shown by Helen Baldwin in Herter’s laboratory. In some of these cases no free hydrochloric acid could:-be demonstrated in the gastric con- tents, and it was observed that inoculation of a digestive mixture, which was originally free from oxalic acid, resulted in its appearance if a few drops of such stomach contents were added. In dogs pro- longed feeding with excessive quantities of glucose together with meat was seen to lead eventually to a state of oxaluria, which was associated with a mucous gastritis and the absence of free hydro- chloric acid. Ovxalic acid could then also be demonstrated in the stomach contents. Very curiously the ingestion of quite small and non-toxic amounts of oxalic acid is followed by a fairly intense indicanuria. It does not seem likely to me, however, that as Harnack and v. d. Leyen suggest, the indicanuria is here referable to a toxic action upon the tissue albumins, and I am personally inclined to explain the phe- nomenon upon the basis of increased intestinal putrefaction. (See Indicanuria. ) The amount of oxalic acid which is normally eliminated in the twenty-four hours fluctuates with the amount ingested, and varies from a few milligrams to 2 or 3 centigrams, being usually less than 10 milligrams (Baldwin). It is influenced by the character of the diet. The ingestion of oxalates by the mouth is followed by their partial elimination only in urine and feces, so that we may conclude that to a certain extent oxalic acid is decomposed during _ its passage through the animal body; possibly this may occur in the intestinal canal as the result of bacterial action. Foods rich in oxalic acid are spinach, tomatoes, carrots, celery, string-beans, rhubarb, potato, dried figs, plums, strawberries, cocoa, tea, coffee, and pepper. Foods which contain little or no oxalic acid, on the other hand, are meat, milk, eggs, butter, cornmeal, rice, peas, asparagus, cucumbers, mushrooms, onions, lettuce, cauliflower, pears, peaches, grapes, melons, and wheat, rye, and oat flour. Before drawing conclusions as to the existence of abnormal oxaluria it is hence imperative to eliminate the possibility of an increased ingestion, by placing the patient upon a diet which contains little | or no oxalic acid. 1 Deutsch. Arch. f. klin. Med., 1901, vol. lxx, p. 486. Lommel, ibid., vol. Lxii, p 599 438 THE URINE An increased elimination is notably observed in association with various dyspeptic and nervous manifestations, and constitutes the condition commonly spoken of as the owalic acid diathesis, or as idiopathic oxaluria. Its existence as a definite pathological picture — is, however, denied by most modern clinicians. Nevertheless it must be admitted that there is a certain type of neurasthenia in which, generally in association with hyperchlorhydria, an increased. elimination of oxalic acid takes place, and in which a copious deposit of calcium oxalate crystals is frequently observed. From the mere fact of the occurrence of such deposits, of course, no inference is, as a rule, to be drawn regarding the actual elimination, but its fre- quent occurrence is in itself of importance, as in such cases a similar separation from the urine may already occur within the urinary passages, and not uncommonly in the pelvis of the kidneys. Not infrequently oxaluria of this type is associated with an increased elimination of uric acid and a mild grade of albuminuria, as has been shown by Senator, von Noorden, Da Costa, myself, and others. Whether or not the oxaluria in these cases can be explained upon the basis of abnormal fermentations in the gastro-intestinal tract, as 1s suggested by the observations of Baldwin, remains to be seen. In some this may be the case, but in others I am inclined to asso- ciate the oxaluria with the coexistent lithuria. Very interesting is the apparently vicarious oxaluria which is at times observed in diabetes. Fiirbringer has reported a case of diabetes in which the elimination of oxalic acid was described as “‘enormous,” and in which oxalic acid could also be demonstrated in the sputum (oxaloptysis). Rausch has recorded a case of mild diabetes, associated with hepatic cirrhosis, in which 1.2 grams were excreted in twenty- four hours. In most cases of diabetes, on the other hand, an in- creased oxaluria cannot be demonstrated. In cases of obesity Kisch found no abnormal degree of oxaluria. In association with jaundice increased oxaluria has been repeatedly observed, and is probably referable to biliary stasis and consequent cholemia, as Salkowski has demonstrated that the bile contains oxalic acid. In pneumonia and leukemia, in both of which we find as a rule a greatly increased elimination of uric acid, the oxalic acid is not always increased, and sometimes indeed quite low in comparison with the amount of uric acid. Properties of Oxalic Acid. —Oxalic acid occurs in the urine as cal- cium oxalate, CaC,O,, and is held in solution by the diacid sodium phosphate. It can, hence, be precipitated by diminishing the acidity of the urine by the addition of a little ammonia or by allowing it to stand exposed to the air. When allowed to crystallize out slowly, calcium oxalate occurs in the form of well-defined, strongly refract- ive octahedra, in which the principal axis of the crystals is placed at right angles to the plane of the microscopic slide (Fig. 146). ‘These CHEMISTRY OF THE URINE 439 are very characteristic. Other forms, however, are also quite com- monly observed, such as single and double dumb-bells, spheroids and prisms, etc. ‘hey are insoluble in ammonia and alcohol, almost insoluble in hot and cold water, and very slightly soluble in acetic acid, but dissolve with ease in the mineral acids. When strongly heated, the salt is decomposed into calcium oxide, carbon dioxide, and carbon monoxide. Tests for Oxalic Acid.—For the detection of calcium oxalate it is frequently only necessary to examine the sediment of the urine after standing for tw enty-four to forty-eight hours. No oxalate crystals, however, may be found even when an abnormally large amount can be demonstrated by chemical methods. In such cases it is usually possible to bring about the crystallization of the salt by carefully neutralizing the urine with a little ammonia. Should this procedure not lead to the desired end, it is best to treat the urine with one-third its volume of 95 per cent. alcohol. The mixture is set aside for twenty-four to forty-eight hours, when the sediment is centrifugal- ized and examined with the microscope. This method, Baldwin states, represents a more delicate test for oxalic acid than the complicated methods of quantitative analysis which are available. Quantitative Estimation. —Heretofore the old method of Neubauer has been in general use, but it is at best unsatisfactory. It has been replaced by the methods of Dunlop and Salkowski. Dunlop’s Method (slightly modified by 9 crystals Baldwin) .—In this case the calcium oxalate is precipitated from an acid solution by means of alcohol, instead of from an alkaline solution by calcium chloride. The urine is thymolized, and, if alkaline, acidified with a trace of acetic acid; 500 c.c. of a well-mixed specimen of the collected urine of twenty- four hours are treated with 150 c.c. of over 90 per cent. alcohol, to precipitate the calcium oxalate. ‘The mixture is set aside for forty-eight hours. It is then filtered, care being taken to ensure the entire removal of the crystals from the beaker. ‘The sediment is thoroughly washed with hot and cold water, and finally with dilute acetic acid (1 per cent. solution). The filter is placed in a small beaker and soaked in a small amount of dilute hydrochloric acid. It is then washed with hot water until the washings no longer give an acid reaction. ‘The acid solution and washings are filtered, and the filtrate evaporated to about 20 ¢.c. This is treated with a very small amount of a solution of calcium chloride, to ensure the presence of an excess of calcium. ‘The solution is neutralized with 440 THE URINE ammonia, slightly acidified with acetic acid, and treated with strong alcohol, so that the mixture contains 50 per cent. After forty-eight hours the sediment is collected on a filter free from mineral ash, and is washed with cold water and dilute acetic acid until free from chlo- rides. ‘The filter with its contents is then incinerated, first over a Bunsen burner, and afterward for five minutes in a blow-pipe flame. On cooling over sulphuric acid the ash is weighed; the result multi- plied by 1.6 represents the amount of oxalic acid in the volume of urine examined. Salkowski’s Method.—In the case of human urine of moderate concentration 500 c.c. of the non-filtered urine are evaporated to about one-third. On cooling, the liquid is acidified with 20 c.c. of hydrochloric acid (sp. gr. 1.12), and extracted three times with new portions of 200 c.c. each of a mixture of 9 to 10 volumes of ether and 1 volume of alcohol. ‘The ethereal extracts, which contain the liberated oxalic acid are carefully separated from the urine and filtered through a dry filter. ‘The ether is distilled off; the re- maining alcoholic solution, which still contains a little ether, is placed in a deep evaporating dish, diluted with 10 to 15 c.c. of water, and evaporated on a water bath. ‘The resulting milky fluid is concen- trated, more water being added if necessary, until it becomes clear and a gummy material separates out. On cooling, the liquid, which should measure about 20 c.c., is passed through a small filter. ‘This is washed once or twice with a little water, when filtrate and washings are rendered slightly alkaline with ammonia, treated with 1 to 2 c.e. of a 10 per cent. solution of calcium chloride, and acidified with dilute acetic acid. ‘The reaction should be distinctly acid, but an excess should be avoided.. An indication that a sufficient amount has been added is afforded by the dissolution of the precipitate of phosphates, which occurs after the addition of the calcium chloride solution. After standing for twenty-four hours, or, still better, forty- eight hours, the calcium oxalate that has separated out is collected on a filter free from ash, washed with hot and cold water, dried, and incinerated as usual (see above). ‘The resulting weight multiplied by 1.6 indicates the corresponding amount of oxalic acid in grams. LirrraTurE.—P. Furbringer, “Zur Oxalsiiureausscheidung durch d. Harn,” Deutsch. Arch. f. klin. Med., 1876, vol. xviii, p. 148. J.C. Dunlop, “The Elimi- nation of Oxalie Acid in the Urine,” ete., Jour. Path. and Bact., 1896 (an histori-— cal review of the subject of oxaluria is here also given). H. Baldwin, “ An Experi-- mental Study of Oxaluria,’”’ Jour. Exper. Med., vol. v, p. 27. E. Salkowski, © Berlin. klin. Woch., 1900, p. 484; and Zeit. f. physiol. Chem., vol. xxix, p. 437. EK. Harnack, “Ueber Indicanurie in Folge von Oxalsiurewirkung,” Zeit. f. physiol. Chem., 1900, vol. xxix, p. 205. Albumins. The albumins which may be met with in the urine are serum albumin, serum globulin, albumoses (peptones), the albumin of CHEMISTRY OF THE URINE 44] Bence Jones, hemoglobin, nucleo-albumin, fibrin, histon, and nucleo- histon. Of these, serum albumin is the most important from a clinical standpoint. Serum Albumin.—'he question , whether or not serum albumin occurs normally in the urine—v. e., under strictly physiological con- ditions—has been much disputed. It is claimed by some that traces may be temporarily met with in apparently healthy individuals after severe muscular exercise, cold baths, mental labor, severe emotions, during menstruation, digestion, ete. This so-called physiological albu- minuria mostly occurs in young adults, and is usually, if not always, of brief duration. ‘The urine, it is claimed, is otherwise normal— 2. €., of normal amount, appearance, specific gravity, and compo- sition, and free from abnormal morphological constituents, such as casts, red corpuscles, leukocytes, and epithelial cells.‘ However, Darling’ has shown that severe muscular exercise may produce a urinary picture which, even though temporary, closely simulates what is seen in acute nephritis. He reports 0.9 per cent. of albumin in a member of a Harvard four-oared crew after a two-mile race, and amounts varying from 0.25 to 0.5 per cent. in five others under similar conditions. "The sediment at the same time contained large numbers of hyaline and finely granular casts, many with renal cells and red blood corpuscles adherent. In many of the sediments there were also numerous red cells as such and an excess of leukocytes. The existence of a physiological albuminuria, on the other hand, is denied, and the occurrence of serum albumin at least regarded as pathological in every case. I have never been able to convince my- self of the occurrence of serum albumin in the urine under strictly physiglogical conditions, and am hardly prepared to regard severe muscular and mental labor, severe mental emotions, cold baths, ete., as physiological stimuli. ‘The albuminuria, so often observed during the first days of life, at which time sediments of uric acid and urates, mucus, epithelial cells from the different portions of the urinary tract, and even casts may also be seen—i. ¢., constituents which in adults would rightly be regarded as abnormal—has also been brought for- ward in support of the theory of a physiological albuminuria. ‘There can be no doubt, however, that this form of albuminuria is referable to the profound changes that take place in the circulatory system after birth, and to some extent perhaps also to the well-known uric acid infarctions so frequently seen in the kidneys of the newly born, so that it would probably be better and more in accord with the teachings of pathology to regard this form of albuminuria also as abnormal.’ The more closely the subject of the so-called physiological albu- 1 C. E. Simon, “ Functional Albuminuria, ’”? N. Y. Med. Jour., 1895, p. 330. ? Boston Med. and Surg. Jour., September 7, 1899, p. 231. * LL. Landi, L’albuminuria nel parto, Morgagni, 1890, vol. xxxii 449 THE URINE minuria is studied, the more improbable does its physiological nature appear, and a more detailed study of the metabolic processes, it may be confidently asserted, will ultimately lead to the conclusion that the presence of albumin in every case 1s a pathological phenomenon. The association of an increased elimination of urea and uric acid with albuminuria in apparently healthy individuals was noted many years ago, but received comparatively little attention." Personal obser- vations have led me to look upon this form of albuminuria as of common occurrence, and while in almost every case the albumin can be caused to disappear from the urine by proper diet and exercise, there can be no doubt that, if neglected, granular atrophy may ulti- mately result. An albuminuria may at times be observed in anemic children and adolescents, and particularly in masturbating boys of the mouth- breathing type, but can hardly be regarded as physiological. ‘The same may be said of the albuminuria of pregnancy and parturition. As regards the action of cold baths, Rem-Picci’ reports that albu- minuria may be considered a constant phenomenon after cold baths, but that different subjects react differently under the same con- ditions. ‘Those which show albuminuria more readily are, as a rule, the less robust and thinner individuals, such as are most sensi- tive to cold. ‘The limits of temperature necessary to produce the phenomenon are from 12° to 13° C., when the immersion is not longer than three minutes. If the temperature be from 15° to 20° C., the albumin appears only after fifteen minutes’ immersion. Above this temperature albuminuria does not occur, even if the bath lasts much longer. ‘The colder the bath, the more rapid the appearance of albumin. The degree of albuminuria is always slight, and even in the more marked cases rarely exceeds 0.25 pro mille. The sedi- ment, according to Rem-Picci, occasionally shows a few hyaline casts, and often crystals of calcium oxalate. The course which may be taken by these various forms of what should be termed functional albuminuria, in which the amount of albumin rarely exceeds 0.1 per cent., is very interesting. ‘The elimi- nation of albumin may thus be quite transitory on the one hand, as when following severe muscular exercise, cold baths, and the like. It may, however, also last for several days, or even weeks, and be followed by a disappearance of the albumin for a variable length of time, and again by its reappearance and continuance for days and weeks. ‘The term intermittent albuminuria’ has been applied to this latter type. At times the albuminuria may follow a definite course, ! Da Costa, “The Albuminuria and Bright’s Disease of Uric Acid and Oxalie Acid,” Amer. Jour. Med. Sci., 1895. 2“ On Albuminuria after Cold Baths,” Il Policlinico, 1901, vol. viii, p. 389. ’ Bull, Berlin. klin. Woch., 1886, vol. xxiii, p. 717. Mareau, Rev. de méd., 1886, vol. vi, p. 855. IXlemperer, Zeit. f. klin. Med., 1887, vol. xu, p. 168. CHEMISTRY OF THE URINE 445 disappearing and reappearing with such regularity that it has not improperly been styled cyclic albuminuria.’ In this form the albu- min generally disappears from the urine during the night or during prolonged rest in bed, and reappears during the day, the erect pos- ture apparently favoring its reappearance; the term postural or orthostatic albuminuria has hence also been suggested for this form. Oswald, who made a careful study of cyclic albuminuria in Riegel’s clinic, regards its occurrence as distinctly pathological, and as indi- cating the existence of nephritis. Remembering the importance of the subject, it may not be out of place to enumerate the reasons ‘ier led Oswald’ to this conclusion: . ‘The patients generally come to the physician complaining of Retin definite symptoms which are similar to those noted in cases of true nephritis. At times, however, no complaints are made, be- cause the patients have reasons for concealing them (as in examina- tions for life insurance), or because they are temporarily absent. 2. ‘The subjective complaints, as well as the anemia so frequently observed in such cases, generally disappear, together with the albu- min, under suitable treatment, and reappear when the anemia again becomes marked. 3. In many a history of an antecedent nephritis the result of scarlatina or diphtheria may be obtained, as in 3 cases of Heub- ner, in 14 cases out of 20 described by Johnson, etc. In some also a direct transition from an acute nephritis to the cyclic form of albuminuria has been noted. Where this was not possible the history of an acute infectious disease or an angina that had been overlooked in the clinical history must be regarded as a possible cause. 4. 'The absence of morphological elements, especially tube casts, does not exclude a nephritis. A large number of cases, moreover, have recently been observed in which casts were repeatedly found. 5. A cyclic albuminuria may be observed in many cases of chronic nephritis. 6. Marked organic abnormalities (such as heart lesions) need not be demonstrable, as they may be absent for a long period of time or may be unrecognizable. According to the researches of Erlanger and Hooker’ orthostatic albuminuria is dependent upon a lowering of the pulse pressure (being the difference between the minimum and the maximum blood pressure), which constantly occurs when the individual changes from the recumbent to the erect posture. In the true form of orthostatic albuminuria the albumin present is serum albumin. Casts are absent." ' A. Keller, Beitriige z. Kenntniss d. eyklischen Albuminurie, Diss., Breslau, 1896. 2 “Cyklische Albuminurie u. Nephritis,’’ Zeit. f. klin. Med., vol. xxvii, p. 73 3 Johns Hopkins Hosp. Reports, 1904, p. 346. * Teissier, Rev. de méd., April 10, 1905, p. 233. 444 THE URINE a | | ) It may be safely asserted that a transitory, intermittent, and cyclic albuminuria 1s not infrequently observed in apparently healthy imdi- viduals, but that the facts so far brought forward do not warrant the | assumption that such forms of albuminuria are physiological. ‘The occurrence of such albuminuria unquestionably demonstrates a cer- tain insufficiency of the renal epithelium, and I am much in favor, as Martius has proposed, of discarding the term physiological albuminuria altogether, and to speak of these various forms col- lectively as constitutional albuminuria. It would lead too far to enter into a detailed consideration of the various causes that have from time to time been suggested as an ex- planation of the fact that albumin does not occur in the urine under normal conditions. ‘There can be no doubt, however, that the integ- rity of the epithelial lining of the glomeruli and the convoluted tubules must be regarded as the principal factor which prevents the albumin of the blood from passing into the urine. When the readi- ness with which the glandular structures of the kidney respond to any abnormal stimulation is considered, it is easily understood how an albuminuria may be produced in many different ways. Aside from acute and chronic inflammatory processes in the widest sense of the word, an albuminuria may be the result of circulatory dis- turbances in the kidneys of whatever kind—~. e., the result of anemia as well as of hyperemia. In many and perhaps ‘the majority of cases of what Bamberger” terms hematogenous albuminuria, we have direct evidence of the existence of circulatory disturbances, as in cases of uncompensated valvular lesion, weak heart, emphysema, hepatic cirrhosis, ete. In other cases, however, the existence of such dis- turbances can only be surmised, and the question, whether or not the albuminuria observed in the various infectious diseases, for ex- ample, is referable to circulatory abnormalities or to a direct irrita- tive action of microbic poisons upon the renal parenchyma, must still remain open. From personal studies in connection with the functional albu- minuria of Da Costa, it seems not unlikely that in many cases in which obscure circulatory disturbances are supposed to exist and are held responsible for an existing albuminuria, this is referable rather to the strain thrown upon the kidneys by the continued elimination of abnormally large quantities of organic material, the quantity of water being at the same time proportionately small. If it is remembered, furthermore, that injuries affecting certain portions of the brain are followed by albuminuria, and that this may be artificially produced by a piquure, analogous to the glucosuric 1 y, Noorden, Deutsch. Arch. f. klin. Med., vol. xxxviil, pp. 3 and 205. Leube, | Zeit. f. klin. Med., 1887, vol. xiii, p. 1. Winternitz, Zeit. f. physiol. Chem., 1891, vol. xv, p. 189. C. E. Simon, loc. cit. * Wien. med, Woch., 1881, pp. 145 and 177. ——_— —_ CHEMISTRY OF THE URINE 445 piquure of C. Bernard, still another factor is given which may possibly enter into the causation of albuminuria. Obstruction to the outflow of urine from the kidneys has also been experimentally shown to lead to albuminuria, an observation with which clinical experience is in perfect accord. In patients actually in labor albuminuria is common, and sup- posedly due to increased blood pressure in the kidneys caused by uterine contractions and the general disturbance of the circulation. The relative frequency of its occurrence is a matter of dispute, however, and widely differing statements are made by different observers, ranging from 15 to 20 per cent. (Petit, Winckel) to 99 and 100 per cent. (Trautenroth, Pajikull). As regards the occurrence of albuminuria in pregnancy the results of different observers likewise differ, viz., from 1 to 50 per cent. In the last months of pregnancy Zangemeister’ found albumin in 10 per cent. of the cases examined, and if repeated examinations were made positive results were obtained persistently during the last three months in 40 per cent. ‘The albuminuria is supposedly referable to some metabolic disturbance and impaired excretion by the kidneys. Finally, an abnormal composition of the blood may at times cause the albuminuria. In passing on to a more detailed study of the various pathological conditions in which an elimination of albumin may be noted, an attempt will be made to classify the various forms of albuminuria in accordance with the more general considerations set forth above. It should be remembered, however, as already indicated, that it may be very difficult, if not impossible, to assign one single cause to a given clinical case, as several factors may at the same time be operative in the production of the albuminuria. 1. FuncrionaL ALBUMINURIA.—Under this heading may be ‘comprised the various forms of “physiological” albuminuria, which have already been considered. 2. THe ALBUMINURIA ASSOCIATED WITH ORGANIC DISEASES OF THE KIDNEYS, viz., acute and chronic nephritis, renal arteriosclerosis, amyloid degeneration of the kidneys.” In acute nephritis, albuminuria, usually of great intensity, is a constant and most important symptom. ‘The amount eliminated is generally proportionate to the intensity of the disease, but varies within fairly wide limits, generally from 0.3 to 1 per cent., corre- sponding to a daily excretion of from 5 to 8 grams. Much larger quantities, it is true, are at times excreted, but it may be definitely stated that the daily loss of albumin seldom exceeds 20 grams. In chronic parenchymatous nephritis the elimination of albumin is likewise constant, and the amount excreted in severe cases may 1 Arch. f. Gyn., 1902, vol. lxvi, Heft 2. ? Senator, loc. cit. 446 - THE URINE even exceed that observed in the acute form. An elimination of from 15 to 30 grams, viz., 1.5 to 3 per cent. by weight, is frequently observed. In the ordinary form of chronic interstitial nephritis the elimina- tion of albumin is, as a general rule, slight, and rarely amounts to more than 2 to 5 grams pro die. At the same time it is not unusual to meet with an apparent absence of albumin if the more com- mon tests (see below) are employed. If it is remembered that very often the diagnosis of the disease is dependent upon the demon- stration of the presence or absence of albumin, the necessity of fre- quent examinations and the employment of more delicate tests, par- ticularly of the trichloracetic acid test, as well as of a microscopic examination, is at once apparent. This is even of greater impor- tance in the renal arteriosclerosis of Senator, in which albumin by the ordinary tests is probably not demonstrable in the majority of cases, and in which even the trichloracetic acid test may not be of service, and casts be absent. Amyloid degeneration of the kidneys, in the absence of inflamma- tory processes, is accompanied by a condition of the urine closely resembling that observed in the ordinary form of chronic interstitial nephritis. A total absence of albumin, however, is less frequently noted, while an amount varying between 1 and 2 per cent. is not uncommon. It will be shown later on that in this condition con- siderable amounts of serum globulin are excreted in addition to the serum albumin; larger amounts, in fact, than are generally observed in this form of chronic renal disease; so that Senator sug- gests that such a relation, in the absence of an acute nephritis, or an acute exacerbation of a enka nephritis, may be of a certain diagnostic value. 3. FEBRILE ALBUMINURIA. "That albuminuria may occur in almost any one of the various febrile diseases is a well-known fact, but it 1s important to remember that, while such an albuminuria may at times be referable to a true nephritis developing in the course of or during convalescence from an acute febrile disease, such is the exception, and not the rule. Under this heading, only that form: will be considered which is not associated with distinct changes affecting the renal parenchyma, and which generally appears during the height of the disease only, and disappears with a return of the temperature to normal. As has been mentioned, it is often diffi- cult, if not impossible, to assign a definite cause for an albuminuria of this character, and in all probability several factors are in opera- tion at the same time. In the beginning of the disease, when the blood pressure, as a rule, is increased, the albuminuria may be 1 Leyden, Zeit. f. klin. Med., 1881, vol. iii, p. 161.° H. Lorenz, Wien. klin. Woch., 1888, vol. i, p. 119. CHEMISTRY OF THE-URINE 447 referable to an ischemia of the kidneys, as the increased pressure in fever, according to Cohnheim and Mendelson, is largely refer- able to spasm of the arterioles. Later on, or in the beginning of cases in which especially severe intoxication exists, the blood pressure may besubnormal, and the albuminuria be due to this cause—7. e., a hyper- emic condition of the kidneys. As a matter of fact, it has been experi- mentally demonstrated that both anemia and hyperemia of the kidney structure may lead to albuminuria. On the other hand, it is not unlikely that the strain thrown upon the kidneys by an excessive elimination of organic material, in the absence of a correspondingly large quantity of water, may produce albuminuria. I have repeatedly seen the functional albuminuria of the type described by Da Costa disappear during the administration of a diet relatively poor in nitro- gen, while an increased diuresis was at the same time effected by the consumption of large amounts of water. In those grave cases of typhoid fever, furthermore, which are characterized by high fever and pronounced nervous symptoms, it would appear quite likely that the albuminuria, which in these cases is particularly marked, is referable to a direct influence upon the central nervous system, and in some cases, at least, also dependent upon an irritant action upon the renal epithelium on the part of the microbic poisons circulating in the blood. ‘The character of the albu- minuria will largely depend upon the intensity of the intoxication; in other words, upon the amount of bacterial poison present at any one time in the blood. | Notwithstanding statements to the contrary, albuminuria may be regarded as a constant symptom of typhoid fever, as has been defi- nitely demonstrated by Gubler and Robin. It is difficult to say why other observers have found albumin in only a comparatively small percentage of cases, but it is not unlikely that this is owing to a lack of uniformity in methods, it being presupposed also that questions of this kind can only be decided by daily examinations. According to Robin, the trace of albumin which is at times observed during the first week of the disease is an albumose, while later on serum albumin is constantly found; the amount increases with the inten- sity of the morbid process, and the highest figures are reached in fatal cases. ‘he more severe the disease, the earlier does albumin appear in the urine, it being remembered, however, that reference is had only to those cases in which distinct renal changes are not demon- strable. ‘Toward the termination of the fastigium the amount of albumin generally undergoes a certain diminution, and may even disappear entirely. ‘This diminution, however, is only temporary, and in severe cases the albumin again increases in amount during the period of great variations in the temperature. In light cases an increased elimination also takes place at this stage, but is soon followed by a decrease, after which traces only can be demonstrated. 448 THE URINE In some cases it disappears entirely, but it is rare, according to Robin, to meet with cases in which a trace at least does not reappear during convalescence. In light cases the albuminuria rarely persists longer than the fifth or eighth day of convalescence, and Robin even goes so far as to say that a relapse may be anticipated if the albuminuria does not disap- pear at that time. and illuminating gas, large amounts of the substance may be found. According to my experience, nucleo-albumin is frequently ob- tained in cases of so-called functional albuminuria, and it is not uncommon to find that this is still present when serum albumin — and serum globulin can no longer be demoustrated, even with the trichloracetic acid test. Nucleo-albuminuria may thus exist inde- pendently of the presence of the more common forms of albumin. This observation has also been made by Strauss, who found nucleo- albumiu only in several cases of cystitis, in One case of chronic inter- stitial nephritis, and in one case of emphysema pulmonum with renal hyperemia. 1 Med. News, 1894. 2 Ueber Nucleo- -albuminausscheidung, Diss., Berlin, 1894. 3 Ueber Mucin im Harn, Diss., Berlin, 1886. * Centralbl. f. klin. Med..,. 1892, VOL paeLe CHEMISTRY OF THE URINE 461 The existence of a hematogenic form of nucleo-albuminuria has thus far not been satisfactorily demonstrated. It has been assumed that its presence indicates increased epithelial desquamation in some portion of the urinary tract—in other words, that it is of cellular origin. Matsumoto, however, has shown that even though a urine containing numerous epithelial casts, renal epithelial cells, and leu- kocytes be allowed to stand for some time, a substance which can be precipitated with acetic acid either does not occur at all or only in very small quantity. He has rendered it very probable that the substance which can be precipitated from pathological urines by means of acetic acid is largely fibrinogen and euglobulin. He adds that nucleo-albumin may be present simultaneously, but in comparison to the other two substances it is of secondary importance and is rarely seen. Histon and Nucleohiston.—Kolisch and Burian’ were able to dem- onstrate the presence of histon ina case of leukemia in which it was constantly present. More recently Krehl and Matthes’ claim to have isolated the same substance in various febrile diseases, such as acute peritonitis, following appendicitis, in croupous pneumonia, erysipelas, and scarlatina. It is an albuminous body, and was first discovered by Kossel in the red blood corpuscles of the goose. It exists in the leukocytes of human blood in combination with the acid - leukonuclein, constituting the so-called nucleohiston of Lilienfeld. It is not clear in what manner the histonuria is produced; so much, however, seems certain, that it is not solely dependent upon increased destruction of leukocytes. Nucleohiston itself has been found in the urine in a case of pseudo- leukemia, by Jolles.* Tests for Albumin.—The recognition of the various albuminous bodies which may occur in the urine is based partly upon their direct precipitation and partly upon color reactions when treated with cer- tain reagents. The number of tests which have from time to time been sug- gested is large; many of them after a brief period of use have been discarded as useless or uncertain, while others have been employed only occasionally, and have not received the recognition which they deserve, from the fact that simpler tests exist, that they do not possess sufficient delicacy, or that in some instances it is too great. In the following pages no attempt is made to describe all of these tests, and attention will be directed only to those which are generally used, and which clinical experience has proved to be of value, precedence being given to those which have been longest in use. While some of *“Ueber d. Eiweisskérper d. leukiimischen Harns,” etc., Zeit. f. klin. Med., vol. xxix, p. 374. , * “Ueber febrile Albumosurie,’’ Deutsch. Arch. f, klin. Med., vol. liv, p. 508. * Ber d. deutsch. chem. Gesellsch., vol. xxx, p. 172; Zeit. f. klin. Med., vol, XXXlv, p. 53. 462 THE URINE a these are applicable for demonstrating the presence of more than one form of albumin, special tests will also be described whereby the various albumins may be individually recognized. In every case the urine should be carefully filtered, so as to free it from any morphological elements, etc., present. ‘To this end, it is generally sufficient to pass the urine through one or two layers of Swedish filter paper. Frequently, however, a clear specimen cannot be obtained in this manner; it is then advisable to shake the urine with burnt magnesia or talcum, or to mix it with scraps of filter paper, when it is filtered as usual. Tests for Serum Albumin. ‘I'HeE Nitric Acip ‘Test’ (Plate XVIII). —The value of this test, properly applied, cannot be overestimated, as it is not only simple, but yields an amount of information that can otherwise be gained only with difficulty. Usually the student is advised to make use of a test-tube partially filled with urine, along the sides of which concentrated, chemically pure nitric acid is allowed to flow, so as to form a layer at the bottom of the tube, when in the presence of serum albumin a distinct white ring appears at the zone of contact between the two liquids (Heller’s test). ‘The pictures thus obtained cannot be compared, however, with those seen when the apparently trivial change is made of using a conical glass of about 2 ounces capacity instead of the test-tube. About 20 c.c. of urine are placed in the glass, when 6 to 10 c.c. of nitric acid are added by inclin- ing the glass and allowing the nitric acid to flow down the sides. When this is carefully done the nitric acid forms a distinct zone beneath the urine. In the presence of albumin the white ring then — appears, and varies in extent and intensity with the amount of albumin present. If now the contents of the glass are allowed to stand undisturbed—and if small amounts are present, the albumin appears on standing for a few minutes—it will be observed that the cloudiness gradually extends upward; and if much albumin is present, it may be seen to rise into the supernatant liquid in the form of small, irregular columns. ‘This appearance is possibly referable to the decomposition of uric acid by means of nitric acid, nitrogen and carbon dioxide being set free, which, rising to the surface in the form of small bubbles, carry the nitric acid upward; coming into contact with albumin in solution, this is then precipitated. In practically every urine on standing for a few minutes, a fine ring appears in the clear urine above or separated from the albuminous ring by a distinct clear layer of urine (Plate XVIII). ‘This ring has been generally ascribed to the presence of urates and in certain hospitals of Paris it was long customary to gauge the amount of uric acid by the rapidity with which it forms and its extent. For years I regarded this as an established fact, but I have convinced myself: 1 J. F. Heller, Arch. f. physiol. u. path. Chem. u. Micros., 1852, vol. v, p. 169, | A, Robin, Urologie clinique de la fiévre typhoide, Paris, 1877. | PLATE XVIII. Cold Nitric Acid Test. Albumin ring below; ‘‘ urate” ring above. | ) ae > _ = - => se «a ‘ a“ ah 1. 7 = » J « ta a . . i ~ | si . a 7 r wi a vor te =? ‘ . = aon . - fi oe . aa = s . 7 —- @ = ‘ e (*s 4 . - “ ‘ é + . | | LIBRARY eF THE UNIVERSITY sf HLUUINOIS, Fi x . A : Pas a o hid 7 CHEMISTRY OF THE URINE 463 that no relation exists between this phenomenon and the amount of uric acid, as determined by one of the standard methods. Mérner has expressed the opinion that the ring in question is not referable to urates at all, but is of a special albuminous character. Further researches in th's direction are needed. Usually the ring is fine and delicate, but at times the substance is present in large amounts and may simulate common albumin, by rapidly extending downward. Its clinical significance is not understood. Should more than 25 grams of urea be contained in a liter of the urine, an appearance like hoarfrost will be noted on the sides of the vessel, which is due to the formation of urea nitrate. Spangles of the same substance appear only in the presence of at least 45 grams; and if 50 grams or more of urea are contained in the liter, a dense mass of urea ni rate may be seen to separate out. Biliary urine, when treated with nitric acid containing a little nitrous acid, shows the color play referable to the action of nitric acid upon bilirubin. The production of the colors (red, yellow, green, blue, and violet) takes place from above downward, the green color being the most characteristic; in the absence of the latter the presence of biliary pigment may be positively excluded. ‘I‘he presence of albumin does not interfere, as the color play takes place beneath the albuminous disk. In normal urine a transparent ring is also obtained, presenting a peach-blossom red; the intensity of this may vary, however, from a - faint rose to a pronounced brick color, and is referable to normal urinary pigment. In the presence of urobilin, on the other hand, this ring presents a distinct mahogany color. Indican is indicated by the appearance of a violet ring situated above that referable to the normal urinary pigment. Its intensity varies with the amount present, from a light blue to a deep indigo. The albumin ring at the zone of contact of the two fluids may be referable not only to the presence of serum albumin, but also of globulin and albumoses, while a negative reaction indicates the absence of these bodies. Should the precipitate caused by nitric acid consist of albumoses, it will clear up more or less, to reappear on cooling, the fluid at the same time assuming a markedly yellow color. ‘The occurrence of a distinctly yellow color in the urine, moreover, which is only partially cleared upon the application of heat (and be it remembered that a much higher temperature is necessary for the solution of a precipitate referable to albumoses than of one due to urates), will indicate the existence of a mixed albuminuria—. e., the presence of coagulable albumin and albumoses. Nitric acid may also cause a precipitation of certain resinous bodies, such as those contained in turpentine, balsam of copaiba and tolu, ete. If any doubt is felt, the mixture should be shaken with alcohol, when the precipitate caused by these substances is at once dissolved. AGA THE URINE Nucleo-albumin, which is at times found in the urine, is also pre- cipitated by nitric acid, but need not occupy our at ention at this place. From what has been said, it is manifest that the employment of the nitric acid test in the manner indicated furnishes much valuable information, and the adoption of the method as described not only by hospital students, but by general practitioners as well, cannot be too strongly urged. Boiuine ‘Trest.—A few cubic centimeters of urine are boiled in a test-tube and then treated with a few drops of concentrated nitric acid, no matter whether a precipitate has occurred upon boiling or not: If albumin is present, this will separate out as a flaky precipitate, which consists of serum albumin frequently mixed with serum- globulin. It is true that albuminous urines will generally yield a precipitate on boiling alone; but it must be remembered that unless the reaction is decidedly acid a precipitation of normal calcium phosphate may occur, owing to the fact that the reaction of the urine upon boiling becomes less acid from the escape of carbonic acid held in solution. In urines presenting an alkaline or amphoteric reac- tion this is very frequently noted, and might give rise to confusion, as the precipitate due to calcium phosphate closely resembles that referable to albumin. In an alkaline medium, moreover, albumin may not be precipitated at all on boiling. Care must hence be taken to ensure a distinctly acid reaction, which is best accomplished by the addition of nitric acid, when a precipitate referable to phosphates is at once dissolved, while one due to albumin remains, and may even become more marked. ‘The quantity to be added should usually be equivalent to about 0.05 to 0.1 per cent. of the volume of the urine. Under no condition should the acid be added before boiling, nor should the urine be boiled after its addition, as small amounts of albumin will otherwise be overlooked, owing to the fact that hot nitric acid dis- solves the precipitate to a certain degree. If, after the addition of the nitric acid the urine turns a distinct yellow, and if then upon cooling a white precipitate appears, the presence of albumoses may be inferred. Uric acid will cause no confusion, as this separates out only upon cooling, and then presents a dark-brown color. As in the case of the nitric acid test, so also here, a precipitation of certain resins is noted at times which may be recognized by their solubility in alcohol. Albumoses are also precipitated upon the application of heat, but such precipitates again dissolve when the temperature approaches the boiling point. Should acetic acid be used instead of nitric acid, great care must be taken to avoid an excess, as otherwise the albumin will be dis- solved. As this danger diminishes the greater the quantity of salts contained in the urine, it 1s advisable to treat the urine first with a few drops of acetic acid until a distinctly acid reaction is obtained, and then to add one-sixth its volume of a saturated solution of CHEMISTRY OF THE URINE 465 sodium chloride, magnesium sulphate, or sodium sulphate, when upon boiling a precipitation of the albumin will occur. Carried out in this manner, the test is absolutely certain and will demon- strate even minimal amounts of albumin. If an equal volume of a saturated solution of common salt is added to the acidified urine, albumoses are also precipitated, but the precipitate dissolves on boiling. THe PoTasstuM FERROCYANIDE ‘T'est.—A few cubic centimeters of urine are strongly acidified with acetic acid (sp. gr. 1.064) and treated with a few drops of a 10 per cent. solution of potassium ferrocyanide, when, in the presence of but little albumin, a faint turbidity, or, if much albumin is present, a flaky precipitate, is noted, which is best recognized by comparison with a tube contain- ing some of the pure filtered urine, both tubes being held against a black background. v. Jaksch advises the careful addition, by means of a pipette, of a few cubic centimeters of fairly concentrated acetic acid, to which a little potassium ferrocyanide has been added, when the albumin, as in Heller’s test, is seen to form a ring at the zone of contact between the two fluids. Instead of potassium ferrocyanide, potassium platinocyanide may also be employed, and has the advan- tage that the test solution is colorless. Concentrated urines should be previously diluted with water. ‘The presence of albumoses may be inferred if the precipitate disappears upon boiling, while a partial clearing up indicates the combined presence of albumoses and coag- ulable albumin. At times the addition of acetic acid by itself is followed by the appearance of a cloud in the ure, which may be due to urates or to urinary mucin (nucleo-albumin), as already mentioned. In such eases the urine should be refiltered, diluted with water, and the test again applied; nucleo-albumin will dissolve in an excess of the acid. Tue TricnLoracetic Acip 'TrEst.’—This test is undoubtedly the most delicate of those so far described, but not so delicate that a trace of albumin or nucleo-albumin can be demonstrated in every urine. An experience based upon the examination of several thou- sand urines with this reagent warrants my speaking with a certain degree of confidence upon the subject. Very frequently it is pos- sible with this method to demonstrate albumin in urines in which the more common tests yield negative results, but in which tube casts may nevertheless be found upon microscopic examination. The test is applied as follows: By means of a pipette 1 or 2 c.c. of an aqueous solution of the reagent (sp. gr. 1.147) are carried to the bottom of a test-tube containing the carefully filtered urine, so as to form a layer beneath the urine. In the presence of albumin a white | Ting will be seen to form at the zone of contact between the two fluids, 1 F. Obermayer, Wien. med. Jahrbiich, 1888, p. 375. D. M. Reese, Johns | Hopkins Hosp. Bull., 1890. } \s 30 AG6 THE URINE varying in intensity with the amount of albumin present. So far as the test for albumin is concerned, this reagent possesses an advantage over nitric acid in that the colored rings, which are so confusing to the inexperienced, are commonly not observed. Serum albumin, serum globulin, and albumoses are precipitated, the presence of the latter being recognized, as in the previous tests, by the fact that the precipitate disappears upon boiling and reappears on cooling. A cloud, referable to uric acid (?), also appears if this is present in exces- sive amounts, but disappears upon the application of gentle heat. A previous dilution of the urine, moreover, guards against its occur- rence. Other tests have also been suggested for the detection of albumin in the urine, such as the metaphosphoric acid test, the phenol, tannic acid, and picric acid tests, that with 'Tanret’s reagent, phospho- tungstic and phosphomolybdic acids, Spiegler’s reagent, etc. Of these, only the picric acid and Spiegler’s test will be considered. Picric Acip ‘Trest.—The picric acid test is not applicable as a test for albumin as such, and is mentioned in this connection only because the same reagent is employed with Esbach’s quantitative method. It is composed of 10 grams of picric acid and 20 grams of crystallized citric acid, dissolved in a liter of distilled water. If to this solution albuminous urine is added, the mixture is rendered turbid, and after some time a sediment which consists not only of albumins, but also of uric acid, kreatinin, and other extractives, will form at the bottom of the tube. (See Quantitative Estimation of Albumin.) SPIEGLER’S ‘l'Est’ (JOLLES’ MopiricaTIon).—The reagent consists of 10 grams of mercuric chloride, 20 grams of succinic acid, and 20 grams of sodium chloride, dissolved in 500 c.c. of distilled water. The urine is acidified to the extent of 5 c.c. with 1 c.c. of dilute acetic acid (30 per cent.), then superimposed by means of a pipette upon 4 to 5 c.c. of the reagent when in the presence of albumin a distinct white ring appears at the zone of contact. On warming, the precipitate does not disappear. As mucin is precipitated by the acid, it is well in doubtful cases to use for comparison 5 c.c. of the acidified urine, diluted with 4 to 5 ¢c.c. of water. Albumoses and nucleo-albumin are also thrown down, and in the presence of iodides mercuric iodide is precipitated; the latter is soluble in alcohol. SPECIAL ‘TEST FOR SERUM ALBUMIN.—Should it be desired, for any reason, to demonstrate serum albumin alone, the urine is ren- dered amphoteric or faintly alkaline with sodium hydrate, and is then saturated with magnesium sulphate in substance, in order to remove any globulin. ‘The filtrate is rendered distinctly acid with acetic acid, when a flaky precipitate, appearing upon boiling, will indicate the: presence of serum albumin. 1 Wien. klin. Woch., 1892, vol. v,p. 26. 30 or even 50 per cent. of the quantity that is found by gravimetric analysis. ‘This is owing to the fact ‘the degree of precipitation is also influenced by the | specific gravity of the supernatant urine. CHEMISTRY OF THE URINE 467 Patein’s albumin differs from the common serum albumin in being soluble in acetic acid." Very often, as in the examination for sugar, it is necessary to remove any coagulable albumin that may be present, to which end the urine is rendered distinctly acid with acetic acid and boiled. An examination of the filtrate with potassium ferrocyanide, if the amount of acetic acid added was just sufficient, will then yield a negative result. - Quantitative Estimation of Albumin.—For the quantitative esti- mation of albumin a large number of methods have been devised, which fact in itself is sufficient to indicate that the majority of them, at least, are unsatisfactory. Old Method by Boiling.—If comparative results only are desired, a definite amount of urine is boiled after acidifying with acetic acid; the albumin is allowed to settle for twenty-four hours. For this purpose Neubauer suggests the use of glass tubes measuring one- half to three-quarters of an inch in diameter, which are closed at the lower end with a cork. Ordinary test-tubes answer perfectly well, but care should be taken that the same quantity of urine is used in each case. The tubes are corked and kept for several days for comparison. ‘The results, of course, express only the relative amount of albumin present, and it should be remembered that the error incurred may amount to as much as that sometimes the albumin separates out in large flakes, and at other times in small flakes, and that Esbach’s Method.’—'l‘he reagent is composed of ‘10 grams of picric acid and 20 grams of citric acid, dissolved in 1000 c.c. of distilled water. Special tubes, termed albuminimeters (Fig. 147), are em- ployed, which bear two marks, one, U, indicating the point to which urine must be added, and one, R, the point to which the reagent is added. The lower portion of the tube up to U bears a scale read- A ee ° cat ° 1G, 147.—Esbach’s ing from 1 to 7, corresponding to the amount of ~ albuminimeter. albumin pro mille. The tube is filled to U with the filtered albuminous urine, and the reagent added until the point R is reached. ‘The tube is closed- with a stopper, inverted twelve times, ‘and set aside for twenty-four hours. _ + Patein, “Acetosoluble Albumin in the Urine,” Compt.-rend. de l’Acad. des Sei., 1889. Coplin, Phila. Med. Jour., 1899, p. 957. * Guttmann, Berlin, klin. Woch., 1886, vol. xxiii, p. 117. 468 THE URINE At the expiration of this time serum albumin, serum globulin, and albumoses, as well as uric acid and kreatinin, will have settled, when the amount pro mille in grams may be read off from the scale. A few precautions must be observed in order to obtain as accurate results as possible. ‘The reaction of the urine should be acid, and if this is not the case acetic acid is added. Its specific gravity should not exceed 1.006 or 1.008, the proper density being obtained by dilut- ing with water. ‘The amount of albumin in the specimen should not exceed 0.4 per cent.; if more be present, as determined by a pre- liminary test, the urine should be diluted. Most important, further- more, is the temperature of the room. ‘This should be 15° C.; varia- tions from this point are apt to give rise to inaccurate results, which, according to Christensen, may amount to 100 per cent. in the case of a deviation of only 5° C. It is thus clear that as generally em- ployed in the clinical laboratory the method will only give approxi- mate results. Gravimetric Method.—If accuracy is required the amount of albu- min must be determined gravimetrically as follows: A certain quan- tity of urine, after having been acidified with an amount of acetic acid sufficient to ensure complete precipitation of all albumin, is boiled; the albumin is then filtered off, dried, and weighed. For this purpose, 500 to 1000 c.c. of filtered urine should be available. A specimen of this, if already acid, is placed in a test-tube, in boiling water, until coagulation takes place, when it is further heated over the free flame and filtered. ‘The filtrate is tested with acetic acid and potassium ferrocyanide. Should no albumin be thus demon- strable, the entire amount of urine is treated in the same manner, and requires no further addition of acetic acid. Hf, however, the test yields a positive result, it 1s apparent that the urine was not sufficiently acid. ‘The entire volume is then treated with a 30 to 50 per cent. solution of acetic acid, drop by drop, the mixture being thoroughly stirred and specimens tested from time to time, as” described. When, finally, the urine remains clear or shows only a faint turbidity, 100 c.c. or less, according to the amount of albumin present, are first heated in boiling water until the albumin begins to separate out in flakes, and then brought to the boiling point over the free flame. ‘lhe supernatant urine is decanted through a filter, which has been previously dried at 120° to 130° C. and accurately weighed, when the whole amount of the precipitate is brought upon — the filter. Any albumin remaining in the beaker is detached from — its sides by means of a glass rod tipped with a piece of rubber tubing, — and collected by the aid of hot water. The entire precipitate is_ thoroughly washed with hot water until the washings no longer - become turbid when treated with a drop of nitric acid and silver’ nitrate; in other words, until the chlorides have been removed. ‘The precipitate is further washed with alcohol and finally with ether to — CHEMISTRY OF THE URINE 469 remove any fats that may be present, when it is dried at 120° to 130° C. to a constant weight. If still greater accuracy is required, the dried and weighed precipitate is incinerated to determine the amount of mineral ash in combination with the albumin, which is then de- ducted from the total weight. ‘The most accurate results are obtained if not more than 0.2 to 0.5 gram of albumin is contained in the amount of urine employed. “Ueber d. quantitative Bestimmung d. Harnindikans,” Zeit. f. physiol. Chem., vol. xxv, p. 406. CHEMISTRY OF THE URINE 505 50 c.c. are used for the quantitative estimation, while larger amounts are taken (200 to 500 c.c.) if the reaction is of only moderate intensity or negative altogether. _ ‘The urine is precipitated with lead acetate solution, care being taken to avoid an excess. A large and accurately measured por- tion of the clear filtrate is treated in a separating funnel with an equal volume of Obermayer’s reagent and extracted with chloroform. To this end 30 c.c. are added at a time and shaken for one minute. Two or three extractions are usually sufficient to remove the entire amount of indigo. ‘The extract is placed in a small flask, and the chloroform distilled off. ‘The residue is dried for a few minutes on a water bath until traces of remaining chloroform have been re- moved. It is then washed with the alcohol-ether-water mixture to remove the reddish-brown pigment which is present together with the indigo blue. ‘The latter remains undissolved. After filtering off any particles of indigo that may be in suspension, through a small filter, this is dried and repeatedly extracted with boiling chloroform. ‘The chloroform extract is filtered into the original indigo flask, the (chloroform distilled off, the residue dried as before, and while still )warm treated with 3 or 4 ¢c.c. of concentrated sulphuric acid. ‘The entire residue should be brought into solution by careful agitation. After standing ‘or twenty-four hours the contents of the flask are ‘poured into 100 c.c. of cold water; the flask is rinsed and the wash- ‘ings added to the solution. ‘This is filtered once more and titrated with the permanganate solution. At first the blue color of the solu- tion changes but little; later it turns greenish, and finally becomes ‘yellowish or entirely colorless—not red. As a rule, the end reaction Is quite distinct, but the titration requires experience. ‘The best ‘results are obtained if from 10 to 15 c.c. of the dilute permanga- inate solution are used. ‘The resulting amount of indigo contained in the measured-off quantity of the first filtrate is then ascertained as idescribed above. Example—Amount of urine: 1780 c.c. _ The stock solution of potassium permanganate contains 3 grams ‘to the liter; 1 c.c.=0.00596 gram of oxalic acid=0.0062 gram of indigo. Diluted solution (5 to 200); 1 c.c.=0.00015 gram of indigo. 300 c.c. ‘of urine were precipitated with 25 c.c. of the lead solution; 250 c.c. ‘of the filtrate, corresponding to 230.7 c.c. of urine, treated with 250 cc. of Obermayer’s reagent. Extracted twice with chloroform. 4.3 cc. of the permanganate solution were used in the titration=0.00065 ‘gram of indigo, correspondng to 0.005 gram in the 1780 c.c., accord- ing to the equation: 230.7 : 0.00065 :: 1780: 2; x= 1.157 = 0.005, 230.7 __ Other methods for the quantitative estimation of indican which have heretofore been used, with the exception of the spectroscopic ‘é 506 THE URINE method of Miiller, are not only inaccurate, but, like this, too time consuming and complicated to be of value to the practising physician. As a consequence almost all observers have based their conclusions upon an approximative estimation only. For practical purposes this is sufficient, and even Wang’s method, though accurate and simple, will hardly find a ready entrance into the clinical laboratory, as it is still too time consuming and too expensive for daily use. Other quantitative methods are those of Ellinger’ and Strauss,’ which should be read in the original. | Urohematin.*—Urohematin appears to be the chromogen of the | red pigment of the urine, and is very likely closely related to in- doxyl. Little is known of its chemical composition or of its mode of formation. In all probability the red pigment which may be obtained from this substance is identical with other red pigments | which have been described from time to time as occurring in the | urine, such as that of Scherer, the urrhodin of Heller, the urorubin of Plosz, Schunk’s ind rubin, Bayer’s indigo purpurin, Giacosa’s pigment, and also the indigo red obtained by Rosenbach and Rosin by oxidation of the urine with nitric acid. Further investigations are necessary before this subject is fully understood; but bearing in mind the probable origin of urohematin from indoxyl, it would possibly be best to speak of the red pigment + as indigo red. In accordance with the view that urohematin is an | indoxyl derivative, its clinical significance is similar to that of indican | (which see). T'est.—TVhe presence in normal urine of urohematin—~. e., a chromo- , gen yielding a red pigment when treated with certain reagents—may | be demonstrated by shaking urine with chloroform and decanting after several days, when the addition of a drop of hydrochloric acid to the | chloroform extract will cause the appearance of a beautiful rose color; | this varies in intensity according to the amount of the chromogen present. | The purplish color so often obtained in the chloroform extract when Stokvis’ modification of Jaffé’s indican test is employed is due to a mixture of indigo blue and indigo red. Indican, however, is generally present in larger amounts than urohematin. In normal and, usually also, in pathological urines a red color is not obtained _ with the test mentioned. In a few isolated cases of ileus, peritonitis, and carcinoma of the stomach I have found more indigo red than indigo blue. ‘The so-called “Reaction of Rosenbach” is a convenient test for indigo red when this is present in increased amounts; the boiling urine is treated drop by drop with concentrated nitric acid, when in — — ——— —— a 1 Zeit. f. phys. Chem., vol. xxxviii, p. 178. 2 Deutsch. med. Woch., 1902, April, 17. *G. Harley, Verhandl. d. physik. med. Ges. z. Wiirzburg, 1855, vol. v, p. 1. * | J CHEMISTRY OF THE URINE 507 the presence of large amounts of indigo red it assumes a dark Bur- gundy color, which sometimes takes on a bluish tinge when held to the light. Owing to a precipitation of the pigment the mixture at the same time bec mes cloudy and the foam assumes a blue color. In well-marked cases the Burgundy colo’ does not appear to be changed by the further addition of nitric acid, but will sometimes suddenly change from red to yellow when 10 to 20 drops of the acid have been added. Th’s reaction Rosenbach' regarded as symptomatic of various forms of severe intestinal disease associated with an impeded resorp- tion throughout the entire intestinal tract. Ewald’ likewise noted this reaction in cases of extensive disease of the small intestine, in carci- noma of the stomach, and in acute and chronic peritonitis; but he obtained negative results in carcinoma of the colon, stricture of the esophagus, chronic diarrhea, ete. Rosenbach’s reaction should be viewed in the same light as a highly incresed elimination of indican. I have met with the reaction in all conditions associated with greatly increased intestinal putrefaction, and, like Ewald, failed to note the reaction in a few cases of occulsion of the large intestine, in which an increased elimination of indican is likewise never observed. Uroroseinogen.*—In addition to indican and urohematin, still another chromogen, which yields a rose-red pigment when treated with mineral acids, appears to occur in normal urine, although in small amounts. It is commonly regarded as a skatol derivative. The pigment, which has received the name wrorosein, or Harnrosa, appears to be identical with Heller’s urophain. Urorosein is best demonstrated by treating 5 to 10 c.c. of urine with an equal amount of concentrated hydrochloric acid, and 1 or 2 drops of a concentrated solution of sodium hypochlorite, when in the presence of much indican the mixture assumes a dark-greenish, blackish, or dark- blue color, owing to the formation of indigo. When the mixture is shaken with chloroform the supernatant fluid exhibits a beau- tiful rose color, which is due to the urorosein. ‘This may now be extracted with amyl alcohol and separated from other pigments which are present at the same time, by shaking with sodium hydrate, whereby the solution is decolorized. Upon the addition of a drop or two of hydrochloric acid to the alcoholic extract the rose color teappears. Such solutions, however, soon become decolorized upon Standing. A rose-red ring, referable to this pigment, is also fre- quently obtained in pathological urines when the ordinary nitric acid test is employed. While normally urorosein is obtained only in traces, appreciable ei. klin. Woch., 1889, vol. xxvi, pp. 5, 490, and 520, and 1890, vol. xxvii, Pp. : * Ibid., 1889, vol. xxvi, p. 953. * H. Rosin, Deutsch. med. Woch., 1893, p. 51. 4 F’ 508 THE URINE amounts are often met with in pathological conditions associated with grave disturbances of nutrition, as in nephritis, diabetes, carei- noma, dilatation of the stomach, pernicious anemia, typhoid fever phthisis, and at times in profound chlorosis, ete. A vegetable diet also appears to cause an increase in the amount of the chromogen. Pathological Pigments and Chromogens. The Blood Pigments, —The blood pigments proper which may occur in the urine have already been considered and in this connection it will only be necessary to refer briefly to the occasional presence of hematin, urorubro- hematin, and hematoporphyrin. | HEMATIN is only rarely found. In order to demonstrate its pres- ence, the urine is rendered strongly alkaline with ammonia, filtered) and the filtrate examined spectroscopically. (See Blood.) | UroRUBROHEMATIN and UroruscOHEMATIN have been observec only once by Baumstark* in the urine of a case of pemphigus leprosus complicated with visceral lepra; they appear to be closely related tc hematin. | HEMATOPORPHYRIN.—McMunn found a pigment answering the des- cription of this substance in the urine in cases of rheumatism, Addi- son’s disease, pericarditis, and paroxysmal hemoglobinuria, whick he termed urohematin, but which in all probability was hematopor- phyrin. Le Nobel found the same pigment in two cases of hepatic cirrhosis and in one case of croupous pneumonia. Others have like- wise met with hematoporphyrinuria in various forms of hepatic dis- ease, as also in phthisis, exophthalmic goitre, typhoid fever, and hydroa eestivalis; further, in association with intestinal hemorrhages, in cases of lead poisoning, and especially during long-continued use of sulphonal, trional, and tetronal. Nebelthau records the history of a female patient, the subject of congenital syphilis, who had passed dark-red urine as long as she could remember, and continued to do so while under observation. Stern mentions a case in which marked hematoporphyrinuria was associated with icterus in a glucosuric individual. Recent researches, moreover, have shown that in traces at least the substance is present in every urine. As regards the origin, of these normal traces, the evidence is in favor of the view that they are formed within the body during its normal metabolism, and most likely in the liver, whence the substance is eliminated in the bile. A portion then escapes with the feces, while a similarly small amount is resorbed and eliminated in the urine. Increased amounts, would accordingly suggest the existence of a hepatic insufficiency; and, as a matter of fact, we find that actual anatomical lesions then not infrequently occur. ‘Taylor and Sailer thus report that in their case of sulphonal poisoning widespread degeneration of the hepatic cells existed; and Neubauer was able to isolate the pigment from the ' Pfliiger’s Archiv, 1874, vol. ix, p. 568. See, also, J. W. Schultz, Diss. Greifswald, 1874. CHEMISTRY OF THE URINE 509 iver of rabbits to which sulphonal had been administered, while it vas absent in all other organs. On the other hand, it is difficult to scribe all the phenomena of such hematoporphyrinuria to hepatic changes, seeing that changes of like degree may occur without con- picuous urinary abnormality, and there is still much that is obscure n this condition. | Stokvis attributed the increased elimination of hematoporphyrin n cases of lead poisoning and following the continued use of sul- hon to the occurrence of hemorrhages into the intestinal mucosa, and suggested that the transformation of the hemoglobin into aematoporphyrin was favored by the sulphonal. But while intesti- jal hemorrhages may occur in the sulphonal cases, they are not slways observed, and, as Garrod points out, Kast and Weiss, as also Neubauer, were unable to verify the recorded experiments of 3tokvis, in which he claims to have obtained a small amount of aematoporphyrin when fresh blood was digested with pepsin-hydro- hloric acid and sulphonal at from 38° to 40° C. _ Urines which contain much hematoporphyrin are usually dark ed in color, but the shade may vary from a sherry or port-wine ‘int to a dark Bordeaux. It is noteworthy, however, that this color 3 not primarily due to the exaggerated degree of hematoporphy- inuria, but, as Hammarsten first pointed out, to other abnormal igments which are but little known, but which are probably closely ‘elated to hematoporphyrin. As Garrod says, the removal of he hematoporphyrin from such urines causes little or no change f color, and when this pigment is added to normal urine until on ‘pectroscopic examination bands of similar intensity are seen, the hange of tint produced is comparatively slight. In one such case, ‘ot due to sulphonal, he was able to isolate a purple pigment which ‘iffered i in its properties from any known urinary coloring matter, ind to which the color of the urine in question was obviously i in the ain due. Neumeister also states that in sulphonal intoxication an son-containing derivative of hemoglobin occurs in the urine, which ‘resents a reddish-violet color and shows a single band of absorption jp the blue portion of the spectrum immediately bordering on the green. Albumin is not present in uncomplicated cases of hematopor- hyrinuria, and the pigment itself does not give the albumin reactions. To demonstrate the presence of hematoporphyrin under normal mditions, or when small amounts only are present in the urine, rarrod’s method should be employed. Garrod’s Method.—Several hundred c.c. of urine (500 to 1500) are veated with a 10 per cent. solution of sodium hydrate in the propor- von of 20 c.c. of the alkali solution for 100 c.c. of urine. The pre- ‘pitated phosphates are filtered off and thoroughly washed by ’peatedly suspending them in water. Should the precipitate be of ‘reddish color, or if it shows the spectrum of hematoporphyrin in 510 THE URINE alkaline solution when examined on the filter in the moist state, we may conclude that much hematoporphyrin is present. In this case it is washed until the filtrate is colorless. If traces only are present, however, one washing must suffice. ‘lhe precipitate is then treated with alcohol, which is acidified with hydrochloric aeid to such an extent that the phospates are entirely dissolved. ‘The resulting solution should not exceed 15 to 20 c.c. in volume. ‘This is then examined in a layer, of not less than 3 to 4 cm. in thickness, for the spectrum of acid hematoporphyrin, using a spectroscope with slight dispersion. ‘The solution is now rendered alkaline with ammonia and treated with an amount of acetic acid which just suffices to redissolve the precipitated phosphates. On shaking with chloro- form this extracts the pigment, and the chloroform solution then gives the spectrum of the alkaline hematoporphyrin, since organic acids do not change the pigment to the form which yields the acid spectrum. The residue which remains after evaporating the chloroform can finally be washed with water and dissolved in alcohol, when a nearly pure solution is obtained, which is comparable with a solution of hematoporphyrin obtained from hematin. Precautions: If a preliminary test shows that the urine con- tains but little phosphates, a small quantity of calcium phosphate in acetic acid is added before the urine is rendered alkaline with the sodium hydrate solution. As hematin and chrysophanic acid are also precipitated with the phosphates, their absence must be ensured. For this reason the urine should contain no rhubarb or senna. In conclusion, it may be said that a chromogen of hematopor- phyrin is also usually present in urines containing the free pigment, which probably explains why such urines gradually become darker on standing. LITERATURE.—A complete account of the literature on hematoporphyrinuria up to 1893 is given by R. Zoja, “Su gualche pigmento di alcune urine,” ete., Arch. ital. di clin. med., 1893, vol. xxxii, p. 68. A. E. Garrod, loc. cit.; and Centralbl. f. inn. Med., 1897, No. 21. Taylor and Sailer, Contributions from the William Pepper Laboratory, Philadelphia, 1900, p. 120. O. Neubauer, Arch. f. exper. Path. u. Pharmakol., 1900, vol. xlili, p. 455. B. J. Stokvis, “ Zur-Patho- genese d. Hematoporphyrinurie,” Zeit, f. klin. Med., vol. xxviii, p. 1. Kast u. Weiss, Berlin. klin. Woch., 1896, vol. xxxiii, p. 621. Hammarsten, “Skandin. Arch, f. Physiol.,’’ 1891, vol. iii, p. 31. Neumeister, Physiol. Chem., Jena, 1897. Nebelthau, Zeit. f. physiol. Chem., 1899, vol. xxvii, p. 324. B. Ogden, Boston Med. and Surg. Jour., 1898 - Biliary Pigments.—Of the four biliary pigments, viz., bilirubin, biliverdin, biliprasin, and bilifuscin, the former alone is met with in freshly voided urines, while the others may form upon standing, being oxidation products of bilirubin. ‘The pigment is never found in normal urine, and its occurrence may be regarded as a positive symptom of disease. In health it will be remembered that BILIRUBIN is formed in the liver from blood pigment, and is eliminated into the small intestine, CHEMISTRY OF THE URINE 51] in which it is transformed into hydrobilirubin and largely excreted as such in the feces, while a small portion is reabsorbed into the blood and eliminated in the urine as urochrome or normal urobilin. When- ever, then, the outflow of bile into the intestines becomes impeded bilirubin is absorbed by the lymphatics and eliminated in the urine. Among the numerous causes which give rise to choluria under such conditions may be mentioned obstruction of the biliary ducts, and especially of the common duct, referable to simple swelling of its mucous membrane, as in the ordinary forms of catarrhal jaun- dice. It may also be due to the presence of a biliary calculus, to parasites, compression of the duct by tumors of the liver, the gall | bladder, the duct itself, and of neighboring structures, and particu- ' larly of the pancreas, stomach, and omentum. Whenever the | blood pressure in the liver is lowered, so that the tension in the smaller biliary ducts becomes greater than that in the veins, choluria | likewie results. ‘The icterus occurring under all such conditions has been termed hepatogen c icterus, in contradistinction to the form observed in cases in which the liver has either totally or partially lost the power of forming bile, be this owing to the existence of | degenerative processes affecting its glandular epithelium, as in cases of acute yellow atrophy, or to destruction of red corpuscles going on so rapidly and so extensively that the organ is incapable of transforming into bilirubin all the blood pigment which is carried to it. ‘This occurs in some cases of pernicious anemia, malarial intoxi- cation, typhoid fever, poisoning with arsenious hydride, ete. Icterus _ neonatorum is probably to a certain extent also dependent upon the latter cause. ‘To this form the term hematogenic icterus has been _applied. In such cases the occurrence of bilirubin in the urine can only be explained by assuming that a transformation of blood-color- ing matter into bilirubin has taken place in the blood itself or in other tissues of the body. As a matter of fact, it appears to be generally accepted that such a trans ormation can occur outside of the liver, as the hematoidin which may be found in old extravasations of blood ' seems to be identical with bilirubin. On the other hand, however, the existence of a hematogenic icterus is positively denied, especially by Stadelmann. In accordance with his view it may be demon- strated that in cases of pernicious anemia, malaria, etc., the urine does not contain bilirubin, but usually urobilin. In cases of this kind which I had occasion to examine, bilirubin was, as a matter of fact, never found. Further investigations are necessary to settle this question. Usually the presence of biliary pigment may be recognized by | direct inspection, as urines which contain it in notable amounts present a color varying from a bright yellow to a greenish brown. _Any morphological elements which may occur in the sediment are | stained a golden yellow, and the same color is imparted to the foam 512 THE URINE of the urine as well as to the filter paper used in the filtration. At | times, however, and particularly in cases in which the icterus is only beginning to appear, the presence of bilirubin is not infrequently | OV erlooked, and urines containing urobilin in large amounts may be | similarly mistaken for icteric urines. In doubtful cases, therefore, — whether icterus exists or not, but in which the urine presents an intense yellow color, it is necessary to have recourse to chemical _ tests. A large number of these have been devised, all of which are fairly reliable. Only those will be described which I have examined myself and which are especially delicate. Smith’s Test.\—5 to 10 c.c. of urine are placed in a test-tube and treated with 2 or 3 c.c. of tincture of iodine (which has been | diluted with alcohol in the proportion of 1 to 10) in such a manner — that the iodine solution forms a layer above the urine. In the pres- ence of bilirubin a distinct emerald-green ring is seen at the zone of contact. ‘This test can be highly recommended, as it is exceedingly simple and not surpassed in delicacy by any other. Huppert’s Test.2—10 to 20 c.c: of urine are precipitated with — milk of lime (a solution of barium chloride is, perhaps, still more — convenient), and the precipitate after filtering brought into a beaker by perforating the filter and washing its contents into the latter with a small amount of alcohol acidualted with sulphuric acid. ‘The mixture is boiled, when in the presence of bilirubin the solution assumes a bright emerald-green color. Huppert’s test is as delicate as is that of Smith, but is not so convenient for the needs of the practising physician. Gmelin’s Test (as modified by Rosenbach).*—The urine is filtered through thick Swedish filter paper, when the latter is removed and a drop of concentrated nitric acid, which has been allowed to stand exposed to the air for a short time, is placed upon its inner surface. In the presence of bilirubin a prismatic play of colors will be seen to occur around the nitric acid spot. Gmelin’s Test.A—The urine is treated with nitric acid, which is carried to the bottom of the test-tube by means of a pipette, so as to form a layer beneath the urine, when a color play, as already described (p. 463), will take place at the line of contact between the two fluids; the green color is the most characteristic. In this connection a few words may also be said of the occurrence in*the urine of biliary acids and cholesterin. Biliary Acids.—These™ ‘may usually be found in the urine when | bile pigment is present, so that their clinical significance is essen- * Dublin Med. Jour., 1876, p. 449. ?’Arch. d. Heilk., 1867, vol. viii, pp. 351 and 476. 3 Centralbl. f. d. med. Wiss., 1876, vol. xiv, p. 5. ‘Tiedemann u, Gmelin, Die Verdauung nach Versuchen, Heidelboram 1826, rt t y i,®p.,80. 4 CHEMISTRY OF THE URINE 513 tially the same as that attaching to bilirubin. ‘Their demonstration is, however, attended with much difficulty (See Feces). | Cholesterin.—Cholesterin has never been found in icteric urines, ‘and is only rarely seen in other pathological conditions. It has been observed in cases of chyluria, fatty degeneration of the kidneys, diabetes, in one case of epilepsy, in eclampsia, and in several cases ‘of pregnancy. v. Jaksch noted cholesterin crystals in a urinary sedi- ‘ment in a case of tabes and cystitis. Glinsky records a similar obser- vation. Harley found it repeatedly in cases of pyuria. Reich states that he found cholesterin crystals of the size of a dollar in the urine of ‘a case of chronic cystitis. Hirschlaff found larger quantities in the urine of a case of hydronephrosis; on one occasion 5.8 grams in 100 ‘ee. of urine. I have found cholesterin crystals in the sediment in ‘a case of acute nephritis. Giiterbock described a urinary calculus ‘obtained from the bladder of a woman which consisted almost entirely of cholesterin (see also Feces’. Langgaard noted the pres- ‘ence of the substance in a case of chyluria.* Pathological Urobilin—This pigment should not be confounded ‘with the urochrome or normal urobilin described above, to which itis closely related, but from which it may be distinguished by means ‘of the spectroscope. Gautier states that pathological urobilin may be obtained from urochrome by submitting the latter to the action of reducing agents; and, as I have already pointed out, Riva and Chiodera obtained a substance from urobilin by the action of potas- slum permanganate, which closely resembles urochrome. It is said to be identical with the stercobilin found in the feces, but differs from Maly’s hydrobilirubin in containing a much smaller percentage of nitrogen, viz., 4.11, as compared with 9.22 (Garrod and Hop- kins). While its occurrence in the urine is essentially a pathological phenomenon, it is at times also met with in normal urine, and appears to be derived from a special chromogen, wrobilinogen, from Which it may be set free by the addition of an acid. Both urobilin and its chromogen are precipitated by saturating the urine with ammonium sulphate, and both are soluble in chloroform. Accord- ing to Maly, urobilin is formed by the reduction of bilirubin in the ‘mtestine, and is then in part resorbed and eliminated in the urine. Hayem, on the other hand, proposed the hypothesis that the sub- stance originates in a diseased or disordered liver, as bilirubin does n the same organ in health, and accordingly he regards the appear- ance of much urobilin in the urine as evidence of hepatic insuf- ‘ciency. Others, again, maintain that urobilin is formed in the ‘issues at large either by the reduction of bilirubin or directly from the blood pigment. ‘The first view is notably held by Kunkel, Mya, _ 1 vy. Jaksch, Klinische Diagnostik, 4th ed., p. 339. Glinsky, Maly’s Jahresber., 1894, vol. xxiii, p. 484. Langgaard, Virchow’s Archiv, vol. Ixxxvi. W. Hirs- hlaff, Deutsch. Arch., 1899, vol. Ixii, p. 53. 33 | 514 | THE URINE Giarré, and others, while the hematogenous theory is notably represented by Gerhardt. Garrod discusses these various hypotheses at some length in his most interesting lecture on the urinary pig- ments in their pathological aspects, in which he personally inclines to the intestinal theory, as now held by Miiller, Schmidt, Esser, and others. In a work of this scope it would lead too far to discuss the various investigations which lend themselves in support of this view, and I can here quote only the following from Garrod’s paper: “The chief seat of the formation of urobilin (for it is convenient to employ this term as including both pigment and chromogen) is undoubtedly the intestinal canal. ‘This can only be gainsaid by denying the identity of the urinary and fecal pigments. ‘The quan- tity normally present in the feces is far larger than that which enters the intestine with the bile (when a small amount is found), and there is strong evidence that the urobilin in bile is itself of intesti- nal origin. ‘This being so, it is clear that theories other than the intestinal and its modifications merely attempt to trace a second source for the urobilin of the urine. It is equally clear that the substance from which the intestinal urobilin is formed is the bile pigment. Under ordinary .conditions the bile pigment is destroyed in its passage along the intestine, and does not appear as such in the feces. In its place we find large quantities of urobilin, which in its turn disappears when occlusion of the common duct prevents the entrance of bile into the intestine. Again, when under certain morbid conditions the bile pigment passes along the intestine unal-. tered, urobilin is absent from the feces. However, the conversion of bilirubin into urobilin is no mere process of reduction, but in- volves a much more radical change, with elimination of nitrogen. That the change is brought about by bacterial action there is much, evidence to show. When bile is inoculated with fecal material and. kept in an incubator a formation of urobilin rapidly takes place, and at the same time the bile pigment diminishes, and ultimately dis-. appears.” ) From its frequent occurrence in febrile urines pathological urobilin - has also received the name febrile urobilin. | Its presence is very common in hepatic cirrhosis. In 12 cases of the atrophic and hypertrophic variety v. Jaksch was able to demon- | strate urobilin in every instance, a point which may at times be of considerable diagnostic importance. I have observed urobilin in a few | cases of hepatic cirrhosis, chronic malaria, and pernicious anemia, in - all of which the skin presented a light icteric hue, and in which bile | pigment was absent from the urine. Unfortunately, an examination | of the blood was not made, and I have hence not been able to con- firm the statement of v. Jaksch that bilirubin occurs in the blood in almost every case in which urobilin is present in the urine. Syl- laba, however, has shown that in pernicious anemia, urobilinuria i CHEMISTRY OF THE URINE 515 is quite constantly associated with bilirubinemia (see the latter). Urobilin has also been noted in eases of carcinoma, scurvy, ae son’s disease, hemophilia, in eases of retro-uterine hematocele, i extra-uterine pregnancy, following intracranial hemorrhages, ae According to Bargellini, the degree of constipation in simple atony of the bowel is without influence upon the amount of urinary uro- pilin, but he states that in typhoid fever it causes an obvious increase ; whereas disinfection or emptying of the large bowel preduces a notable diminution in the amount. Urobilinuria, according to Samberger,' is common early in secondary syphilis and referable to increased destruction of red cells. In some cases the urobilinuria is only observed after the mercurial treatment has been instituted, and sub- sequently disappears. _ Urines rich in urobilin usually present a dark-yellow color which is strongly suggestive of the presence of bilirubin; even the foam in such cases may be colored, making the resemblance between the two pigments still more complete. This dark color, however, is not ‘due to urobilin, but to associated pigments. : GERHARDT’S ‘Test.—If the urine contains much urobilin, which ‘the color will indicate, 10 to 20 e.c. are extracted with chloroform by shaking, and the extract treated with a few drops of a dilute solu- ‘tion of iodopotassic iodide. Upon the further addition of a dilute ‘solution of sodium hydrate the chloroform extract is colored a yellow or yellowish brown, and exhibits a beautiful green fluorescence; this Is even more intense than that noted in the case of normal urobilin. | BRAUNSTEIN’S ''ESt.—The reagent is composed of 100 c.c. of a con- ‘centrated solution of copper sulphate, 6 c.c. of concentrated hydro- ‘chloric acid, and 3 grams of ferric chloride; 20 c.c. of urine are ‘treated with 3 to 4 c.c. of the reagent and shaken with chloroform. ‘In the presence of urobilin a rose to a red color develops. | SCHLESINGER’S TEst.—10 ¢.c. of urine are treated with an equal ‘quantity of a 1 per cent. solution of acetate of zinc in absolute alcohol. The mixture is agitated and filtered, when in the presence of urobilin ‘the filtrate will show distinct fluorescence. _ Sprecrroscopic EXAMINATION.—The urine is best examined as follows: 50 c.c. of urine are extracted in a separating funnel with ‘amyl alcohol, which takes up both the pigment and its ‘chromogen. After standing for several hours the urine is allowed to flow away by opening the stopcock, when the alcoholic extract is decanted from ‘above, and is treated with a concentrated alcoholic and ammoniacal ‘solution of zinc chloride. In the presence of urobilin the liquid shows a beautiful fluorescence, and on spectroscopic examination a single band of absorption is seen between b and F. In acid solu- aos, on the other hand, a single band is likewise obtained between b 1 Arch, f. Dermat. und Syph., 1903, vol, Ixvii, 516 THE URINE and J’, but this extends to the right beyond F’, and is much darker. Should the urine contain much urobilin, its special extraction is not necessary. In such an event the acid urine shows the acid spectrum, while the alkaline band is obtained after the addition of ammonia. (See also Bang’s "Test.) LITERATURE.—A. E. Garrod, loc. cit. A. E. Garrod and F. G. Hopkins, “On Urobilin,” Jour, of Physiol., 1898, vol. xxii, p. 451. Maly, Centralbl. f. d. med. Wiss., 1871, vol. ix, p. 849. Hayem, Gaz. hebdom., 1887, vol. xxiv, pp. 520 and 534; and Gaz. des hép., 1889, vol. lxii, p. 1314. Kunkel, Virchow’s Archiv, 1880, vol. lxxix, p. 655. Mya, Arch. ital. di clin. med., 1891, vol. xxx, p. 101; and Lo Sperimentale, 1896, vol.1, p. 71. Giuarré, ibid., 1895, vol. xlix, p. 89, and 1896, vol. 1, p. 81. F. Miiller, Schlesische Gesellsch. f. vaterlind. Kultur, Janu- ary, 1892. A. Schmidt, Verhandl. d. XIII Congress. f. inn. Med., 1895, p. 320. Esser, Untersuchungen iiber d. Entstehungsweise d. Hydrobilirubins, etc., Diss. Bonn., 1896. Bargellini, Lo Sperimentale, 1892, vol. xlvi, p. 119. v. Jaksch, Zeit. f. Heilk., 1895, vol. xvi, p. 48. D. Gerhardt, Zeit. f. klin. Med., 1897, vol. XXxi,.p. 313. Melanin and Melanogen.—lIn cases of melanotic disease it has been repeatedly observed that the urine, which usually and probably always presents a normal yellow color when voided, gradually be- comes darker upon exposure to the air, and finally turns black. Such urines generally contain melanin and its chromogen in solution; deposits of melanin granules by themselves are only occasionally seen, and are not characteristic, as they may also be found in cases of chronic malarial intoxication, ete. While the occurrence of melanin in the urine is probaly indica- tive in most cases of the existence of melanotic tumors, it should be stated that this symptom cannot be regarded as pathognomonic, as it may be absent in the case of melanotic tumors, and present in wasting diseases and inflammatory affections, and may at times, though very rarely, be associated with non-pigmented growths. Nevertheless, its occurrence should always be regarded with suspicion, and, taken in conjunction with other symptoms, will often lead to a correct diagnosis. | Tests FoR MELANIN AND MELANoGEN.—1. The presence of melanogen may be assumed if upon the addition of ferric chloride solution a black precipitate appears in the urine, which is soluble in a solution of sodium carbonate, and can be reprecipitated as a black or brownish-black powder by mineral acids. Instead of ferric chlo- ride barium hydrate may also be used. 2. A few cubic centimeters of urine are treated with bromine- water, when in the presence of melanin or melanogen a precipitate 1s obtained, which is yellow at first, but gradually turns black. LirpraturE.—T. H. EHiselt, “Die Diagnose d. Pigmentkrebses durch d. Harn,” Prag. Vierteljahrschr. f. praktische Heilk., 1858, iii, p. 190, and 1862, vol. iv, p. 26. Senator, “Ueber schwarzen Urin,’’ Charité Annal., 1891. Hoppe-Seyler, Zeit. f. physiol. Chem., 1891, vol. xv, p. 179. F. Grohe, “Zur Gesch. d. Mela- naemie,”’ Virchow’s Archiv, 1861, vol. xx, p. 306. . CHEMISTRY OF THE URINE 517 Phenol.—Phenol, according to Brieger, occurs only in very small amounts in human urine, the usual phenol reactions being largely referable to paracresol. Normally, about 0.03 gram is eliminated in the twenty-four hours, but in pathological conditions much larger quantities may be found. Remembering the origin of phenol, it is clear that an increased elimination may be observed whenever putre- factive processes are going on in the tissues and cavities of the body, or whenever there is an increase in the degree of intestinal putre- faction, though in the latter condition the indican is usually the only conjugate sulphate that is found increased. In peritonitis, diph- theria, erysipelas, scarlatina, empyema, pulmonary gangrene, putrid bronchitis, ete., an increased elimination of phenol is commonly Seen, as also in certain cases of pernicious vomiting of pregnancy. Important from a diagnostic standpoint, further, is the fact that in uncomplicated cases of typhoid fever no increase is observed, while this is common in tuberculous meningitis.!| The largest amounts, of course, are seen in cases of poisoning with carbolic acid or one of ‘its derivatives (hydroquinone, pyrocatechin, salicylic acid), where ‘the urine may darken on standing, thus simulating true melanuria. As the quantitative estimation of phenol is too complicated for the ‘purposes of the general practitioner, Salkowski’s qualitative test is ‘here also described. From the intensity of the reaction certain con- ‘clusions may be drawn as to the amount present. It is especially ‘serviceable in cases of suspected poisoning with carbolic acid. SALKOWSKI’S ‘Test.—About 10 c.c. of urine are boiled in a test- tube with a few cubic centimeters of nitric acid, and, on cooling, ‘treated with bromine-water. The development of a pronounced turbidity or the occurrence of a precipitate indicates the presence of an increased amount of phenol. — Quantirative Estimation. Principle—When potassium-phenyl ‘sulphate is treated with hydrochloric acid, phenyl sulphate results, which further takes up one molecule of water, giving rise to the formation of sulphuric acid and phenol. _ From the action of bromine-water upon phenol a yellowish-white ‘crystalline precipitae of tribromophenol results: C,H;.OH + 6Br = 3HBr + C,H,Br3.0H. As 331 (molecular weight) parts by weight of tribromophenol correspond to 94 (molecular weight) parts by weight of phenol, the amount of the latter contained in a certain volume of urine is readily determined according to the equation | 331: 94::2:y; and y = 2°" — 0.28398 1, \ ON Strasser, “Ueber d. Phenolausscheidung bei Krankheiten,’’ Zeit. f, klin. Med., vol. xxiv, p. 543. Brieger, Zeit. f, klin. Med., 1881, vol. iii, p. 468. Kast 1, Baas, Miinch. med. Woch., 1888, vol. XXxv, p. 55. 518 THE URINE in which indicates the weight of the tribromophenol found in the amount of urine employed, and y the corresponding quantity of phenol. Merriop.—From 500 to 1000 c.c. of urine are treated with one-fifth of an equivalent amount of dilute hydrochloric acid (1 to 4), and dis- tilled so long as a specimen of the distillate is rendered cloudy upon the addition of bromine-water (1 to 30), the specimens used for this pur- pose being carefully preserved. ‘The total quantity of the filtered dis- tillate, together with the specimens which have been set aside, is now treated with bromine-water, shaking the mixture after each addition of the reagent until a permanent yellow color results. Beyond this point further addition is beset with danger, as compounds will be formed which contain more bromine, the presence of which would indicate a smaller amount of phenol than that actually contained in the urine. After two or three days the precipitate is collected on a filter which has been dried over sulphuric acid, washed with water containing a trace of bromine, and then dried over sulphuric acid and weighed. Salol and salicylic acid may be recognized from the fact that such urines when treated with a solution of ferric chloride develop a. marked violet color which does not disappear on standing. ‘The reaction thus differs from that obtained with diacetic acid. | Alkapton.—Urines are at times, though very rarely, seen which, | like the phenol urines, turn dark on standing, but in which the. change in color is neither referable to the presence of phenol or its _ derivatives, nor to melanin. Such urines are of a normal color when | passed, but gradually turn reddish brown upon exposure to the | air. ‘Treated with a small amount of alkai, this change occurs | almost immediately. Fehling’s solution is reduced on the applica- | tion of heat, while bismuth is not affected. Ammoniacal silver. solution is reduced in the cold, and a temporary bluish-green color | develops when the urine is treated with a ferric salt. The fermenta-— tion test is negative, and examination with the polarimeter shows | that the substance in question is not glucose. With phenylhydrazin | no osazone is formed. Bédeker, who first observed a urine of this kind, termed the sub- | stance giving rise to the reactions just described alkapton, and sub- sequently expressed the belief that his alkapton might possibly have | been pyrocatechin. Subsequent investigators succeeded in isolating | substances from such urines which have been variously termed pyro- | catechuic acid, urrhodinic acid, glucosuric acid, uroleucinic acid, and | uroxanthinie acid. Baumann and Wolkow later were able to iso- late homogentisinic acid in pure form from the urine of such cases, — and expressed the belief that some of the substances obtained by — previous observers were in reality the same. Since that time this acid has also been found by Garrod, Ogden, Stange, Stier, and others. CHEMISTRY OF THE URINE 519 Of the origin of alkapton little is known. Baumann expressed the opinion that homogentisinic acid might be derived from tyrosin, and that the condition is referable to the activity of special micro- organisms in the upper portions of the intestines. As a matter of fact the amount of homogentisinic acid can be materially increased by the administration of tyrosin, and Mittelbach has shown that if the substance is given in frequently repeated and small doses almost the entire amount reappears in the urine as homogentisinic acid. ‘l’yrosin, however, belongs to the para-series, while homogen- tisinic acid is an ortho-compound, so that the transformation of tyro- sin into homogentisinic acid cannot be a direct process, and it has accordingly been questioned whether Baumann’s view regarding the origin of alkapton is correct. ‘There is evidence indeed to show that ‘homogentisinic acid does not originate in the intestines, viz., is not a product of bacterial activity. It has thus been found that the alkap- tonuria does not cease during starvation, and that a restriction of the putrefactive processes in the intestines by means of oil of turpentine, a kefir diet, and the administration of $-naphthol does not lead ‘to a diminished elimination of homogentisinic acid. It has never » been found in the feces, moreover, and Garrod has shown that after inoculation of common bouillon, meat juice, or tyrosin broth with alkaptonuric feces homogentisinic acid is not formed. On the other hand, Embden observed that when an alkaptonuric individual took homogentisinic acid by the mouth a far larger portion appeared in ‘the urine than when the same substance was administered to a healthy ee idual, which suggests that the alkaptonuria may be referable to impairment of the normal processes of oxidation. Very significant is the discovery that a notable increase follows the administration of phenylalanin, and that the ingestion of phenylacetic acid will increase the power of reduction and of rotation of the urine. Phenylpro- /pionic acid and benzoic acid cause no increase in the elimination of homogentisinic acid. The prevailing view is that alkaptonuria is a metabolic anomaly ‘comparable to glucosuria and cystinuria; but, unlike glucosuria, it ‘can scarcely be regarded as an expression of a pathological process. ‘It may, of course, occur in individuals, suffering from disease, and has been observed in connection with glucosuria, in acute gastro- ‘intestinal catarrh, in phthisis, acute miliary tuberculosis, in one case ‘of brain tumor, carcinoma of the prostate, ete. More frequently the ‘condition is accidentally discovered in apparently healthy individuals, and has repeatedly been confounded with glucosuria owing to the ‘positive reduction test with Fehling’s solution. | Garrod, from an analysis of all the reported cases, concludes that ‘the condition is nearly always congenital. In 32 known instances which were presumably congenital, 19 occurred in seven families. One family contained 4 alkaptonurics, three others 3, and the re- 520 THE URINE maining three 2 each. In fully 60 per cent. of the cases, it appears - from Garrod’s studies, the parents of alkaptonurics were first cousins. There is thus far only one known instance in which ihe anomaly has been transmitted by an alkaptonuric father to his son. The condition commonly persists through years and perhaps a life- time. It may also occur as a transitory abnormality, however, as is apparent from the case of Hirsch, in which the condition persisted for three days, and the case of Geyger, in which the alkaptonuria was observed on only two days. A few observers further report the occurrence of alkaptonuria shortly preceding death. Very interesting in this connection is the observation of Osler and others that the urine of patients with ochronosis will darken on stand- ing and may contain homogentisinic acid. ‘The pigmentation of the cartilages thus seemed to be a possible morphological expression of the urinary abnormality. But as Garrod has already stated, it is possible also that other substances besides homogentisinic acid may cause the blackening of the urine in ochronosis. The amount of homogentisinic acid eliminated in the twenty-four hours is variable, but usually large. Baumann found an average elimination of 4.6 grams; the largest amount in twenty-four hours was 6 grams. In Meyer’s case, a child one and one-half years old, 3.3 grams were passed pro die. Larger quantities are obtained after a liberal diet of meats than with a vegetable diet. ISOLATION AND Estimation (GARROD’s MeruHop).—The urine is -_—2 $C CU heated nearly to boiling without any preliminary treatment, and for | each 100 c.c. at least 5 or 6 grams of solid neutral lead acetate are added. As soon as the acetate is dissolved, the bulky gray precipitate which forms is removed by filtration, and the filtrate, which has a pale-yellow color, is allowed to stand for twenty-four hours in a cool place. If the urine be very rich in homogentisinic acid, or if the | flask containing the filtrate be placed upon ice, minute acicular | crystals, which are almost colorless, quickly form; but as a rule crystallization does not commence until several hours have elapsed. The crystals are then much larger, are grouped in stars or rosettes, and are more deeply colored. In summer weather it would probably be desirable to start the crystallization by artificial cooling; but although the process is greatly accelerated at a low temperature, the total yield is not materially — increased. If the formation of. the crystals be long delayed, the liquid may be warmed again and more lead acetate added. After the lapse of twenty-four hours crystals cease to form, even when the liquid is placed upon ice. The crystalline product so obtained is lead HOMnpeananes When the crystals are dissolved in hot water the solution assumes a deep- i CHEMISTRY OF THE URINE 521 brown color with alkalies; it reduces Fehling’s solution readily with the aid of heat, and yields a transitory deep-blue color with a dilute solution of ferric chloride. [rom the lead salt free homogentisinic acid may be obtained by decomposing it with hydrogen sulphide. For clinical purposes the following method also may be employed: BauMANn’s MretrHop.—S0 c.c. of urine are treated with 15 grams of ammonium chloride, which should be brought into solution by shaking, in a stoppered graduate. After standing for about twelve hours to allow the uric acid to separate out the solution is filtered and an accurately measured portion of the filtrate titrated with a decinor- mal ammoniacal solution of silver nitrate. ‘The titration is con- tinued until a further reduction of the silver solution does not occur, which is ascertained by acidifying a few drops of the filtered mixt- ure with hydrochloric acid, when in the presence of free silver a turbidity referable to silver chloride occurs. Accuracy within nar- rower limits than } c.c. is scarcely possible, as the turbidity refer- able to silver chloride can only be recognized within 0.2 to 0.3 c.c. According to Baumann, 240 to 245 c.c. of the silver solution repre- sent 1 gram of homogentisinic acid. LITERATURE.—Bédeker, Annal. d. Chemie u. Pharmakol., 1861, vol. exvii, p. 98. Baumann u. Wolkow, Zeit. f. physiol. Chem., 1891, vol. xv, p. 228. Stier, Berlin. klin. Woch., 1898, vol. xxxv, p. 185. Embden, Zeit. f. physiol. Chem., 1893, vol. xvii, p. 182, and vol. xviii, p. 304. Ogden, Zeit. f. physiol. Chem., 1895, vol. xx, p. 280. Futcher, N. Y. Med. Jour., 1898, vol. lxvu, p. 69. Garrod, Jour. Physiol., 1899, vol. xxiii, p. 512; and Med.-Chir. Trans. Royal Soc., vol. Ixxxii, p. 367. E. Meyer, Deutsch. Arch., vol. lxx, Heft 5u.6. F. Wittelbach, ibid., 1901, vol. lxxi, p. 50. Blue Urines.—Blue urines are sometimes seen, the color of which is due to indigo formed from urinary indican within the urinary passages. ‘Their occurrence can only be regarded as a medical curi- osity. One case of this kind is reported by McPhedran and Goldie,’ in which after direct extraction of the urine with ether only a faint reaction was obtained on further examination, and which probably was referable to incomplete previous extraction. Formerly, when indigo was employed in the treatment of epilepsy, blue urines were frequently seen. At the present time, when methylene blue is occa- sionally used in the treatment of malaria and chyluria, this pigment is found in the urine. Green Urines.—Green urines have also been described; the cause of the color, however, has not been ascertained. Pigments referable to Drugs.—Certain drugs may also cause changes in the normal color of urine, and in doubtful cases inquiry in this direction should be made. It has been pointed out that carbolic acid, hydroquinone, pyrocatechin, and salol cause the appearance of a dark-brown color, and that after the administration of indigo ! Transactions Association American Physicians, 1901. 522 . THE URINE fel and methylene blue blue urines are voided. Santonin, rheum, and — senna color urines a bright yellow, so that they may resemble icteric urines. ‘lhe yellow color in such cases is changed to an intense red by the addition of an alkali, and, if ammoniacal fermentation is going on at the same time in the bladder, the patient may believe himself to be suffering from hematuria. ‘The red color thus produced is due to the action of the alkali upon chrysophanic acid. When urines containing copaiba are treated with hydrochloric acid a red color results, which changes to violet upon the application of heat. During. the administration of potassium iodide, or the use of iodine in any form, a dark mahogany color is obtained when the urine is treated with nitric acid. In doubtful cases Stokvis’ modification of Jaffé’s test for indican should be employed, when in the presence of an iodide the chloroform assumes a beautiful rose-red color. For the detection of other drugs and poisons in the urine the reader is referred to special works. Ehrlich’s Diazo Reaction—Under certain pathological conditions, and especially in typhoid fever, a chromogen may be present in the urine, which, when treated with diazo-benzene-sulphonic acid and ammonia, imparts a red color to the urine, varying from eosin to a deep garnet red. ‘This reaction, which is generally spoken of as Ehrlich’s reaction, or the diazo reaction, was at one time re- garded as pathognomonic of typhoid fever. Subsequent exami- nations, however, have shown that it may also be present in other diseases. Michaelis, who has made an exhaustive study of this — question, divides into four groups the diseases in which the reac- tion has been observed. In the first group, comprising diseases of the nervous system, chronic diseases of the heart and kidneys, malignant tumors, etc., the reaction is rarely seen. When present, it usually indicates a secondary infection. The second group in- cludes those diseases in which the reaction is almost always present, namely, typhoid fever and measles. In the diseases of the third group it is often, though not invariably, observed. Under this heading are classed scarlet fever, erysipelas, pneumonia, diphtheria, pyemia, acute miliary tuberculosis, ete. ‘The fourth group comprises pulmonary tuberculosis, and includes acute caseous pneumonia. ‘The value of Ehrlich’s reaction in typhoid fever was at first overesti- mated, but is at present certainly underestimated. I have studied this problem with great care, and after many years’ experience maintain, as I did years ago, that the test is a most important diag- nostic aid in the disease in question. As a general rule the reac- tion is present as early as the fifth or sixth day, and may persist into the third week; it then disappears, but may reappear when a relapse occurs. ‘This fact is generally overlooked and should be borne in ~ mind in the differental diagnosis from acute tuberculosis. Excepting ‘in children, its absence from the fifth to the ninth day usually indicates PLATE XIX. 7 Ehrlich’s Diazo-reaction, as modified by the author. -The orange colorin the lower portion of the test tube may be obtained in any urine; the dark carmine ring indicates the presence of the reaction in a well-pronounced degree ; the colorless zone above is intended to indicate the am- monia that has been added. r a Ane” xs; 4 ie > : ; sé , ad —_ , - o a .. —_ Ly ' CHEMISTRY OF THE URINE 523 a mild case. ‘Ihis rule, however, is not without exception. When the reaction is continuously present after the third week I am inclined to suspect acute tuberculosis. It may be present as early as the fourth day of the disease. In paratyphoid, as in typhoid fever, the reaction is also fairly con- stant. Of late much attention has been paid to the occurrence of Ehrlich’s reaction in pulmonary phthisis. As a result of his investigations Michaelis concludes that its presence in such cases indicates either that the process is very extensive or that it will progress very rapidly, and that the prognosis is grave. A cure, be believes, is impossible, and improvement, if any, only temporary. Clemens notes that of 100 cases of phthisis which ended fatally 87 showed the diazo reac- tion; Riitimeyer obtained positive results in 85 cases out of 106 which died. Of 13 cases of acute tuberculous pneumonia Friinkel and 'Troje found a positive reaction in 11. Grundriss states that in his fatal cases the reaction was present without exception. Similar results have been obtained by Cnopf, Sée, Goldschmidt, and_ others. Michaelis himself reports that of 111 cases of phthisis which were | admitted to the Berlin Charité with well-marked reaction 80 died ‘in the hospital, 13 were discharged unimproved, 3 were transferred ‘to other hospitals, and 15 left improved. In other words, of these 111 cases a fatal result was known to have occurred in 72 per cent. ‘Stadelmann states that of 38 other cases with positive reaction 28 {died in the hospital—i. e., about 75 per cent. ‘The subsequent fate of the remaining cases was not ascertained; but we may well assume ‘that of these at least 50 per cent. died; so that we may formulate the general rule that a fatal result may be anticipated in about 85 per -eent. of all cases of phthisis in which a positive reaction is obtained. ‘ Michaelis, moreover, suggests that the end may be expected to occur ‘within six months from the time at which a persistent Ehrlich reac- _tion is established. Exceptions occur, but the above is the rule. In ‘Koch’s institute at Berlin patients presenting the diazo reaction are ‘not treated with tuberculin (Brieger).! In tuberculous peritonitis the diazo reaction is found in about 25 per cent. of all cases. As regards the frequency of occurrence of the reaction in diph- _theria, it appears from the observations of Rivier’ and others that ‘it is decidely uncommon. Of his own 118 cases, and 44 additional ‘ones collected from the literature, only 10 gave a positive result; and of these, 4 should be eliminated as they occurred in com- plicated cases; so that the reaction was absent in about 97 per ‘cent. ' Discussion on Tuberculosis, Michaelis, Deutsch. med. Woch., 1901, vol. v, p. 1 | gy These de Paris, 1898. Py 524 THE URINE In the scarlatiniform erythema due to serum treatment the reaction is absent, while in true scarlatina it is fairly common. | Including a number of cases collected from the literature Rivier found a positive reaction in 41 cases out of 73. He concludes that’ in the differential diagnosis between the two conditions scarlatina may be affirmed if the reaction is positive, while if negative there is strong presumptive evidence against the disease. In measles a positive reaction was obtained in 75 of 85 cases. : Riittimeyer obtained the reaction in pulmonary actinomycosis. | The reaction has been referred to the presence of alloxyproteinic | acid,’ but this is denied by Clemens. ! As the preparation of chemically pure, crystalline diazo com- pounds is a difficult process, Ehrlich uses sulphanilic acid, which, when treated with nitrous acid in a nascent state, gives rise to the formation of diazo-benzene-sulphonic acid, as is shown by the- equations : 1. NaNO, + HC = NaCl + HNO,. Ve AN 2. CoH + HINO, = CyHiC SN + 2H,0. SO,H SO, Para-amino- Diazo-benzene- benzene-sulphonie acid. sulphonic acid, _'This is the active principle in the mixture employed. Other compounds may, of course, also be used, such as meta-amino- benzene-sulphonie acid, ortho- and para-toluidin-sulphonic acid, ete.; but of all these, Ehrlich found the common sulphanilic acid the most convenient. ‘Two solutions, which must be kept in separate bottles, are employed. ‘The one is a 5 per cent. solution of hydrochloric acid, to which sulphanilic acid is added in the proportion of 1 gram for every 100 c.c. ‘The other is a 0.5 per cent. solution of sodium nitrite. The two solutions are mixed in the proportion of 40 to 1 im- mediately before using. A few cubic centimeters of urine are then treated with an equal volume of the reagent; the mixture is shaken and rendered alkaline with ammonium hydrate. This is_ best allowed to flow down the sides of the tube, so as to form a layer above the mixture. At the junction of the two fluids a colored ring” will now be observed. With urines which do not contain the chro- mogen this will be a more or less distinct orange, while in its pres- ence a red color is obtained. ‘The intensity of this color may vary from eosin to a deep garnet red. If the mixture is now agitated and the reaction is positive, the foam will likewise be colored red, and upon pouring the solution into a porcelain basin containing 1 Bondzynski u. Panek, Berlin. d. deutsch. chem. Ges., 1903, vol. xxxv, p. 951. ¥ CHEMISTRY OF THE URINE 525 much water a beautiful salmon color is obtained, even if only traces of the chromogen are present. Carried out in this manner no ques- tion will arise as to the presence or absence of the reaction. Ehr- lich states that on standing a green sediment forms in the alkalinized mixture, and he regards this sediment as especially characteristic. My experience has been that this becomes manifest only when the color reaction is well pronounced, and I am inclined to attach more ‘importance to the salmon color obtained ‘upon copious dilution. ‘With normal urines this is never obtained, and it can still be seen when inspection of the fluid in the test-tube would leave in doubt. , The older method of Ehrlich I have abandoned, as the test just described is simpler, and, in my experience, just as reliable. He advised the addition of about 50 c.c. of absolute alcohol to 10 c.e. ‘of urine, subsequent filtration, and examination of the filtrate, as just described. ? Greene states that if 1 part of the sodium nitrite solution is added to 100 instead of 40 parts of the sulphanilic acid solution, a positive ‘reaction is no longer obtained in cases of croupous pneumonia and of pulmonary tuberculosis, while in typhoid fever the reaction occurs with the same intensity. While in the absence of the chromogen, as I have already stated, a more or less pronounced orange color is usually obtained, excep- tions have been noted. Ehrlich thus records that in urines contain- ing biliary coloring matter an intensely dark, cloudy discoloration occurs at times, which upon boiling is changed to a well-marked reddish violet. In rare instances of ulcerative endocarditis, hepatic abscess, and intermittent fever, and more commonly in pneumonia ‘about the time of the crisis, Ehrlich further observed an intense yolk- ‘yellow color, before the addition of the ammonia, which becomes ‘somewhat lighter after this is added. ‘The reaction is supposedly ‘referable to urobilinogen (egg-yellow reaction). | Of interest is the observation of Burghart, that after the adminis- ‘tration of tannic acid, gallic acid, and certain iodine preparations, ‘Ehrlich’s reaction disappears from the urine. But, as Burghart himself suggests, it is likely that this inhibitory effect is not exerted upon the diazo-forming substance, but upon the reagents employed. Other factors, which may prevent the occurrence of Ehrlich’s reac- tion, in pulmonary tuberculosis at least, are the occurrence of renal complications (albuminuria). Naphthalin, after its administration by the mouth, according to my experience may cause a reaction, the ‘color of which corresponds exactly to that of the diazo reaction. Other observers have noted a similar reaction after the adminis- ‘tration of opium (morphine, heroine), alcohol in large amount, phenol, -cresol, creosote,and guaiacol. Golden, on the other hand, denies its ‘occurrence after the use of some of the substances mentioned. Fy 526 THE URINE LrreraturE.—Ehriich, Zeit. f. klin. Med., 1882, vol. v, p. 285; Charit. Annal,! 1883, vol. viii, p. 28, and 1886, vol. xi. p. 189. Goldschmidt, Minch. med) Woch., 1886, vol. xxxiii, p. 35. Rutimeyer, Corresp. Blatt. f. Schweizer Aerate | 1890, vol. xxvi. Greene, Med. Record, Nov. 14, 1896. C. E. Simon, Johns Hopkins Hosp. Bull., 1890. J. Friedenwald, N. Y. Med. Jour., 1893. “M} Michaelis, Berlin. klin. Woch., 1900, p. 274; and Deutsch. med. Woch., 18995.) 156. IR. Arneill, Amer. Jour. Med. Sci., 1900, p. 296. Ehrlich’s Dimethylaminobenzaldehdye Reaction.—Ehrlich has shown that under various pathological conditions a fine cherry-red color develops on shaking a specimen of urine with a few drops of dimethyl-| aminobenzaldehyde in acid solution, and that the resulting pigment can be in part extracted with chloroform, and almost entirely so with epi- or dichlorhydrin. With normal urines a similar reaction can bel obtained, but it is much less intense, and if done at ordinary tempera tures a distinct red color does not develop. On heating, | it appears, and can likewise be extracted with epichlorhydrin. They reaction, according to O. Neubauer, is due to urobilinogen. : As regards the occurrence of the reaction in disease I can summarize) my results as follows: (1) A direct reaction, of pathological grade, does not occur in health. (2) A positive reaction is most commonly ob- tained in cases of tuberculosis. (3) It may also be seen in non- -tuber- culous cases, both febrile and non-febrile. (4) It is not dependent: upon the presence of the body which gives rise to the diazo reaction. | (5) For its production elevation of temperature, gastro-intestinal dis- turbances, and cyanosis are not essential. (6) Common to all com seems to be an increased katabolism of the tissue albumins. My positive results include cases of pulmonary tuberculosis, tuberculosis of the hip-joint, pneumonia, typhoid fever, appendi- citis, embarras gastrique, icterus, malignant endocarditis, empyema, esophageal carcinoma, and a remarkable instance of traumatic neu- rosis, in which a loss of weight of from sixty to seventy-five pou had occurred. ! My list of negative cases, on the other hand, includes, first of all, a large number of normal or supposedly normal individuals; in madiions cases of normal labor, neurasthenia, hysteria, diabetes, aortic aneurysm, myelogenous leukemia, lymphatic leukemia, acute nephritis (scarlatinal), simple diarrhea, morphinism, valvular dis- ease, phthisis (stationary), diphtheria (before and after the use of antitoxin), typhoid fever, cases of abortion, appendicitis, influenza, chronic nephritis, cystitis, pyelitis (calculous), measles, tuberculosis of the hip-joint, cystic kidney, carcinoma of the kidney, tonsillitis, acute and chronic bronchitis, pneumonia, icterus, tuberculous perito- nitis, general erythema; varicocele; following various operations, such as nephrorrhaphy, removal of pus tubes, operations for vesico- vaginal fistula, fistula in ano, and suspension of the uterus. Exami- nation of a urine containing cystin and diamins was also negative. A comparison of the negative with the positive cases will show at 7 CHEMISTRY OF THE URINE 527 once that not all cases of pulmonary tuberculosis, tuberculous hip- joint disease, pneumonia, typhoid fever, appendicitis, and icterus give a positive result. So far as tuberculosis is concerned, however, it appears that the reaction is more likely to occur in the actively progressive cases than in those which are more or less stationary. It was also noted that the positive cases almost all gave a positive diazo reaction, while in the negative cases this was not obtained. Exceptions, however, may also occur. In my personal examinations I employed a 2 per cent. solution of dimethylparaminobenzaldehyde in equal parts of water and con- -centrated hydrochloric acid. A few cubic centimeters of urine in a _test-tube are treated with from 5 to 10 drops of the reagent; the mixture is set aside or agitated for a few minutes and the color then ‘noted. Normal urines usually turn a greenish yellow, or the normal color merely becomes intensified. At times a dark-amber color develops, though this is less common in health, unless the urine is brought to the boil before the reagent is added. In this way it is a -common experience to meet with moderate or dark-amber tints. / With these reactions, however, I have not occupied myself, and, \ like Clemens and Koziczkowsky, I have only noted the reaction as ) positive when a distinct cherry-red color developed, either immediately » on adding the reagent or after agitation or standing. LITERATURE.—Ehrlich, med. Woch., 1901, No. 15. Clemens, Deutsch. Arch., 1901, vol. xxi, p. 168. Koziezkowsky, Berl. med. Woch., 1902, vol. xxxix, \ No. 44. Simon, Amer. Jour. Med. Sci., 1903, vol. exxvi, p. 471. Acetone. _ The amount of acetone which may be found in the urine under jnormal conditions varies between 0.008 and 0.027 gram, and is | greatly influenced by the character of the diet. Whenever the car- \ bohydrates are withdrawn the quantity rapidly increases and reaches jits maximum about the seventh or eighth day. At this time from /200 to 700 mgrms. may be eliminated in the twenty-four hours. If, then, carbohydrates are again added to the diet, the acetonuria | soon disappears. ‘This result is not reached, however, if fats are sub- _ stituted for the carbohydrates. ‘The acetonuria is greatest when but little albuminous food and no carbohydrates at all are ingested, and Iiring starvation the same amounts are essentially found. Increased amounts are found in fevers, the various cachexias, in conditions associated with inanition, etc.1_ The source of the acetone in these | cases was formerly sought in the increased albuminous destruction, | ly. Jaksch, Ueber Acetonurie u. Diaceturie, Hirschwald, Berlin, 1885. Rosen- \feld, Centralbl. f. inn. Med., 1895, vol. xv. Waldvogel, “ Zur. Lehre von der ) Acetonurie,” Zeit. f. klin. Med., vol. Xxxviii, p. 506, 528 THE URINE but according to more recent research it appears that in some manner the fat metabolism is involved and that the acetonuria is the result. Most important is the diabetic form of acetonuria. It may be stated, as a general rule, that the diagnosis of diabetes mellitus is justifiable whenever sugar and notable quantities of acetone are found in the urine. ‘The amount of acetone, moreover, stands in a direct relation to the intensity of the disease, the maximum excretion being usually observed toward the fatal end.* Whether or not this form of acetonuria can always be explained upon the basis given above remains an open question. ‘There can be no doubt, however, that the threatening symptoms which are so commonly associated with a greatly increased elimination of acetone will often disappear, at least temporarily, if carbohydrates are administered in large amounts. It is certain, moreover, that diabetic coma is more apt to occur when the old-fashioned plan of excluding carbohydrates entirely from the dietary of diabetic patients is adopted. Hirschfeld’ suggests that in every case of diabetes the excretion of acetone be carefully followed, and that large amounts of carbohydrates be ad- ministered whenever the acetonuria approaches a dangerous extent. Of the febrile diseases in which acetonuria has been observed may be mentioned typhoid fever, pneumonia, scarlatina, measles, acute miliary tuberculosis, acute articular rheumatism, and septi- cemia. In those of short duration, on the other hand, even if the fever is high, as in acute tonsillitis, intermittent fever, the hectic fever of phthisis, etc., an increased elimination of acetone is rarely observed. In the continued fevers the acetonuria is largely referable to the character of the diet, as carbohydrates are usually excluded entirely, and I have repeatedly observed that a return to the normal occurred as soon as sugar was administered in amounts varying from 50 to 100 grams. In certain nervous and mental diseases, as in general paresis, melancholia, following epileptic seizures, and in tabes, acetonuria is frequently observed. During the second stage of general paresis increased amounts are indeed quite constantly found, but Hirschfeld is probably correct in stating that the psychotic form of acetonuria is largely referable to improper feeding. A notable degree of acetonuria has been observed in connection with the pernicious vomiting of pregnancy,* and in eclampsia (Bag- inski). A certain amount of acetone occurs normally during the first two days of the puerperal period, but usually disappears by the third day. 1 vy, Jaksch, Zeit. f. klin. Med., 1885, vol. x, p. 362. Lorenz, ibid., 1891, vol. eb cay ony EAE > Beobachtungen iiber d. Acetonurie u. das Coma diabeticum, Zeit. f. klin. Med., vol. xxvili, p. 176, and vol. xxxi, p. 212. ° H. Baldwin, Amer. Jour., Oct. 1905, p. 649, ) CHEMISTRY OF THE URINE 529 According to Vicarelli' acetonuria occurring in the course of preg- nancy is evidence of the death of the fetus. This is possibly the rule, but exceptions have been observed. In the primary diseases of the stomach, and notably in carcinoma, acetonuria is frequently observed, and it is possible that the acetone in these cases is, to some extent at least, formed in that organ directly from the proteids ingested. ‘The facts that in carcinoma acetone may be observed at a time when marked loss of flesh has not as yet occurred, and that larger amounts of acetone may be found in the stomach than in the urine, are certainly in favor of this view.’ An enterogenic form of acetonuria has further been described, and it has been urged that in these cases the acetone is referable to the formation of unusually large amounts of fatty acids. Acetonuria of this type is also observed following the ingestion of} fatty acids as such (alimentary form).° Acetonuria has further been observed early in the course of acute phosphorus poisoning, and may persist throughout, apparently with- out being an index of the severity of the case. After chloroform narcosis the condition is also not uncommon. Tests for Acetone. Legal’s Test.‘—This test may be applied to the freshly voided urine, but is not conclusive. Several cubic centi- ‘meters of urine are treated with a few drops of a strong solution of sodium nitroprusside and sodium hydrate; the mixture assumes a red color, which rapidly disappears, and in the presence of acetone is replaced by a purple or violet red when acetic acid is added. As a rule, it is better to distil the urine (500 to 1000 c.c.) after the addi- tion of a little phosphoric acid (1 gram pro liter), and to employ the ‘first 10 to 30 c.c. of the distillate for one or more of the following tests. Lieben’s Test..—A few cubic centimeters of the distillate are rendered strongly alkaline with caustic soda solution and treated ‘with several drops of a dilute solution of iodopotassic iodide, when in the presence even of traces of acetone a precipitation of iodoform ‘in crystalline form occurs. ‘This may be recognized by. its odor ‘when the solution is heated, as also by the form of the crystals, which occur as hexagonal or stellate platelets. If traces of acetone only are present it is necessary to let the solution stand for a number of hours before examining. Alcohol and acetic aldehyde give the same reaction. For this rea- | 1 Prager med. Woch., 1893, Bd. xxxiii und xxxv; also Knapp, Centralbl. f. | Gynik, 1897. ? H. Lorenz, loc. cit. * Waldvogel u. Hagenberg, “ Ueber alimentiire Acetonurie,” Zeit. f. klin. Med., 1900, vol. xiii, p. 443. | . , 4Le Nobel, Arch. f. exper. Path. u. Pharmakol., 1884, vol. xviii, p. 9. _ * Taniguti u. Salkowski, Zeit. f. physiol. Chem., 1890, vol. xiv, p. 476. ) 34 530 THE URINE son Dunning’s modification’ is sometimes to be preferred, although it | is not as delicate. ‘l'o thisend a small amount of Lugol’s solution is | added to the distillate and a sufficient amount of ammonia to produce ( a black precipitate (nitrogen iodide). ‘This disappears on standing | and in the presence of acetone is replaced by iodoform. | Gunning’s test, like that of Legal, may be tried with the native urine | first. | Frommer’s Test.>—This test also may be applied directly to tHe | urine, and is said to indicate the presence of 0.000001 acetone in 8 | c.c. of water. It does not react with diacetic acid. About .10 ¢.c. of urine are treated with about 1 gram of caustic! soda in substance and—without waiting for the dissolution of the soda | to occur—with 10 to 12 drops of an alcoholic solution of salicylic’ aldehyde (1 gram to 10 c.c. of absolute alcohol). ‘he mixture | is heated to 70° C. In the presence of acetone a marked purple-red | color results at the zone of contact with the alkali. | If the alkali is added in solution the fluid first becomes yellow, i later reddish, then purplish red, and finally dark carmine red. ‘The color change occurs more rapidly by heating. | Denniges’ Test (as Modified by Oppenheimer).*—The reagent is prepared as follows: 20 grams of concentrated sulphuric acid are. poured into 100 c.c. of distilled water, when 5 grams of freshly pre-| pared yellow mercuric oxide are added. ‘The mixture is allowed to i stand for twenty-four hours and is then ready for use. This reagent is added to about 3 c.c. of urine, drop by drop, until the precipitate which is thus formed no longer disappears on stirring. | When this point is reached a few more drops are added. After two | or three minutes the precipitate is filtered off. The clear filtrate is | further treated with about 2 ¢.c. of the reagent and 3 to 4 cc. of a| 30 per cent. solution of sulphuric acid, and boiled for a minute or two, or, still better, placed in a vessel with boiling water. In the | presence of an abundant amount of acetone a copious white precipi- | tate forms immediately; while in the presence of traces only (less. than 1 to 50,000), a slight cloud develops on standing for seve minutes. ‘The precipitate is almost entirely soluble in an excess of hydrochloric acid. | If albumin is present, the urine becomes turbid at once when the reagent is added. In that case the test is continued as described, | attention being directed to the coarser precipitate which occurs later. | To such urines large amounts of the reagent must be added, the idea | being to precipitate everything that can be precipitated with the | reagent, before heating. | | Oppenheimer claims that the test is as delicate as that of Lieben, 1 Jour. de pharmacol. et de chim., 1881, vol. iv, p. 30. | ? Berlin. klin. Woch., Aug. 7, 1905, p. 1008, ® Tbid., 1899, p. 828. CHEMISTRY OF THE URINE 531, \yiz., giving a well-pronounced reaction with a dilution of 1 to 20,000, ‘and being still discernible with a dilution of 1 to 60,000. As diacetie jacid yields acetone when treated with mineral acids, a positive result jis always obtained when this is present. But as diacetic acid is usually found only in association with acetone, this fact does not ‘lessen the value of the test, and is an error, moreover, which is common to the other tests as well. Quantitative Estimation of Acetone.—Tlor the purpose of estimat- ing the amount of acetone the method of Messinger, as modified by Huppert, is now employed, and is greatly to be preferred to the older procedure of v. Jaksch.* Principle—The method is based upon the observation of Lieben that acetone gives rise to the formation of iodoform when treated ‘with iodine in an alkaline solution. If then a solution of acetone is ‘treated with a known amount of iodine, it is a simple matter to determine the quantity present by retitrating the iodine which was not used in the formation of iodoform. | Solutions required: . Acetic acid (50 per cent. solution). . Sulphuric acid (12 per cent. solution). . Sodium hydrate solution (50 per cent.). . A decinormal solution of iodine. . A decinormal solution of sodium thiosulphate. . Starch solution (see Boas’ method of estimating lactic acid). Preparation of the solutions: 1. The decinormal solution of iodine is prepared as described Jalsewhere (see Boas’ method of estimating lactic acid). 2. As the molecular weight of sodium thiosulphate—Na,S,O,.- jH,O—is 248, a decinormal solution of the salt would contain 24.8 yrams to the liter. ‘This quantity is dissolved in about 950 c.c. #t distilled water and brought to the proper strength by titration vith a decinormal solution of iodine. As 1 c.c. of the thiosulphate solution should correspond to 1 c¢.c. of the iodine solution, the neces- jary amount of water which must be added to the former is then letermined. Method.—100 c.c. of urine, or less if much acetone is present, is determined by Legal’s test, are treated with 2 c.c. of the wetic acid solution and distilled until seven-eighths of the total umount have passed over. ‘The distillate is received in a retort which is connected with a bulb tube containing water. As soon 4s seven-eighths of the urine have distilled over, a small amount if the distillate of the remainder is tested for acetone according © Lieben’s method. Should a positive reaction be obtained, it vill be necessary either to repeat the entire process with less urine, — - = aor wndre 1 See Neubauer u. Vogel, Analyse des Harns, 9th ed., p. 470. 532 THE URINE diluted to about 200 e.c., or to add about 100 c.c. of water to the’ residue and to distil until all the acetone has passed over. ‘The distillate is then treated with 1 c.c. of the sulphuric acid and redis- tilled. The addition of the acetic acid and of the sulphuric acid,| respectively, serves the purpose of holding back phenol and am- monia. Should the first distillate contain nitrous acid, moreover, which is recognized by the addition of a little starch paste contain-| ing a trace of potassium iodide, when the solution turns blue, the acid is removed by adding a little urea. The second distillate is| received in a bottle provided with a well-ground glass stopper, and holding about 1 liter. The distillate is then treated with a carefully: measured quantity of the one-tenth normal solution of 1odine— about 10 c.c. for 100 ¢.c. of urine—and sodium hydrate solution until the iodoform separates out. ‘To this end a slight excess of the solution must be added. Should ammonia be present, a blackish’ cloud will be observed at the zone of contact of the sodium hydrate} and the iodine solution, and it will be necessary to repeat the entire} process. The bottle is closed and shaken for about one minute.) The solution is then acidified with concentrated hydrochloric adil} when the mixture assumes a brown color if iodine is present in| excess. If this does not occur more of the iodine solution. must! be added and the process repeated until an excess is present. ‘The! excess is then retitrated with the thiosulphate solution until the fluid’ presents a faint-yellow color. A few cubic centimeters of starch, solution are now added, and the titration continued until the last trace of blue has disappeared. The number of cubic centimeters employed in the titration is finally deducted from the total amount of the iodine solution added, and the result multiplied by 0.976. The figure thus obtained indicates the amount of acetone contained in the 100 c.c. of urine, in mgrms., as 1 ¢.c. of the thiosulphate solu- tion is equivalent to 1 ¢.c. of the iodine solution, or to 0.967 mgrm of acetone. . ; Diacetic Acid. : j The occurrence of diacetic acid in the urine must always be regarded as abnormal. Its pathological significance is identical with. that of acetonuria. It is met with especially in diabetes, in various: digestive diseases, and in febrile diseases. In the continued fevers of childhood it is almost constantly present. H. Baldwin noted its | presence in a case of pernicious vomiting of pregnancy. 4 Gerhardt’s Test.—l'o demonstrate the presence of diacetic acid @ few cubic centimeters of urine are treated with a strong solution of ferric chloride drop by drop. A precipitate of phosphates is filtered off, when more of the iron solution is added to the filtrate. If a Bordeaux red color appears, this may be due to diacetic acid. To i CHEMISTRY OF THE URINE 033 make sure another portion of urine is boiled and similarly treated. As diacetic acid is decomposed on boiling no reaction at all or only a faint reddish color should be obtained. As further proof a third portion of urine is acidified with sulphuric acid and extracted with ether. ‘The diacetic acid is thus isolated. yee. 23) 0.59 Globulins oP Re wen es ie, hat og Bed ee 13752 Serum albumin, © =.0a'.6 eee ae ee 35.94 Ethereal extract whee ee SORE. SS eee 4.02 poluble salts 24". eae Acme ke | ae ee ae a ng ey 8.60 Insoluble salia 9 40 ol” 499 et ae Sy we pine 0.66 Sodium chloride é jest, bs 2 Ble x, Ole ee eee 6.19 Sodium oxidede 510 204 aati. |a\ alae A ee: 1.09 Sugar and uric acid in small amounts are also, as a rule, found in | transudates, and in one case of hepatic cirrhosis Moscatelli succeeded F in demonstrating the presence of allantoin. y. Jaksch states that he has frequently been able to demonstrate the presence of urobilin in both transudates and serous exudates, even though red blood cor- puscles and blood-coloring matter in solution were absent. Stich also reports that in the ascitic fluid removed during life from a patient with hemorrhagic nephritis, urobilin was present. Peptone is never found; and Pajikull states that nucleo-albumin is. not present in transudates of non-inflammatory origin. Hammarsten, — EXUDATES 591 ‘together with Pajikull, could, however, demonstrate an albuminous ‘substance in transudates which was regarded as a mucoid and which is present in exudates in small amounts only. It is rich in reducing ‘substance and contains more nitrogen than the true mucins. LiIreRATURE.—Moscatelli, Zeit. f. physiol. Chem., 1889, vol. xii, p. 202. v. aksch, Zeit. f. Heilk., 1891, vol. xi, p. 440. Eichhorst, Zeit. f. klin. Med., 1881, ‘vol. ili, p. 537. Stich, Minch. med. Woch., October 29, 1901. | MICROSCOPIC EXAMINATION. | Upon microscopic examination only a few isolated leukocytes and endothelial cells from the serous surfaces and undergoing fatty degeneration are usually seen. Mast-cells and eosinophilic leukocytes ‘have been observed in the ascitic fluid in cases of myelogenous leu- \kemia. Charcot-Leyden crystals were present at the same time. /Th cases in which the transudates have been confined for a long time ‘plates of cholesterin are frequently found. They are especially Jabundant in hydrocele fluid. Amebas have been found by Miura ) , in the ascitic fluid of a woman afflicted with an abdominal tumor; ‘at the same time they were present in the stools. Leyden and Schaudin likewise met with ameboid bodies in the ascitic fluid obtained from two cases of abdominal tumor. ‘The technique which \should be employed in the microscopic examination of transudates ‘is described below. ; EXUDATES. Exudates may be serous, serofibrinous, hemorrhagic, seropurulent, ‘purulent, putrid, chylous, or chyloid. Of these, the seropurulent, purulent, and putrid types are manifestly of inflammatory origin, ‘while in the case of the serous, serofibrinous, and hemorrhagic ‘forms it may at times be difficult to determine whether the fluid ‘represents a transudate or whether it is an exudate. A detailed ‘chemical and microscopic examination may then be necessary. _ Serous exudates are clear, of a light straw color, and present a ‘specific gravity which usually exceeds 1.018 (1.012 to 1.024). ‘There is a large amount of fibrin and of albumin. If blood corpuscles are present in sufficient numbers to impart a distinct red color to the fluid, ‘it is termed hemorrhagic; the color may then vary from a light ‘pink to a dark red. On standing, even the purely serous exudates ‘generally undergo a certain degree of coagulation, which becomes “more marked in the presence of blood; exceptions, however, do occur. Most important is the microscopic examination of the exudates. Generally speaking, the same methods are here employed ‘as in the case of the blood, but the interpretation of the findings is 592 TRANSUDATES AND EXUDATES - not always easy. ‘This is largely owing to the fact that the leuko- cytes often show evidence of degeneration, and that the fluid may contain endothelial cells in addition to the morphological elements of the blood, which’ further increases the difficulties attending a proper classification. (See Pus.) The principal point at issue in the study of the cellular elements of exudates is the question as to the predominance of either lymphocytes or the polynuclear elements of the blood. Widal and his collaborators, more especially, have pointed out that whereas in exudates of non-tuberculous, acute inflammatory origin the polynuclear neutrophilic leukocytes predominate, the lym- phocytes prevail in the chronic tuberculous forms. His observations have, on the whole, been confirmed by numerous investigators, and the importance of cytodiagnosis in pleuritic effusions more especially is now well established. From the available data we may formulate the following conclusions: In the very earliest stages of tuberculosis — involving the serous membranes there is found a variable number of neutrophilic leukocytes in addition to lymphocytes and endothelial cells. Very soon, however, they diminish and in the later stages the lymphocyte is by far the predominating cell, while the neutrophilic elements are present only in very small numbers. Generally speaking the percentage of lymphocytes in tuberculous pleurisies ranges from 50 to 98, increasing as the disease continues. In pleuritic effusions due to the pneumococcus and to streptococci during the serous stages, the neutrophilic leukocytes far outnumber the lymphocytes. (Average in postpneumonic cases 71.7: variations from 58 to 92.5 per cent.)' In the pneumococcus cases, moreover, it is common to meet with large numbers of endothelial cells, some- times containing polynuclear leukocytes and red cells in their interior. In cases of traumatic and aseptic pleurisy, in association with diseases of the heart and kidneys, large endothelial cells are seen which often present most grotesque appearances, occurring either singly or in groups of two, three, four or more; while the occurrence of large numbers of such cells has been regarded as characteristic of transu- dates, Carter has shown that in these cases also there may bea lym- ~ phocytosis of from 86 to 100 per cent.; so that confusion may arise in differentiating these cases from tuberculous pleurisy. ‘The low spe- cific gravity—average about 1.008—and the small amount of fibrin and albumin in the transudates will, however, aid in arriving at a conclusion. 7 French writers also describe a pleural eosinophilia in which large~ numbers of eosinophilic cells—6 to 54 per cent.—are found in the effusion, while in the circulating blood their number is not increased. Ravaut reports 4 cases of this kind. In 1 the effusion occurred secondarily in the course of syphilis; in the second in a case of typhoid - ) ‘HH, S. Carter, Med, News, October 1, 1904 EX UDATES 593 fever; the third was a case of phthisis, while in the fourth no diagnosis was made. I have recently seen a case of this kind (probably fibers culous) with 10 per cent. of eosinophiles, 4 per cent. neutrophiles, 83 per cent. of small mononuclears, and 3.4 per cent. of large mononu- clears in the exudate, and 3.5 per cent. of eosinophiles, 42 per cent. of neutrophiles, 36 per cent. of small mononuclears, and 18 per cent. of large mononuclears in the blood. Carter’ reports 2 cases of pleural effusion, referable to pistol-shot wounds of the chest walls, in which the eosinophiles numbered 70.2 and 87.8 per cent., respectively. Mast-cells are rarely seen in pleuritic effusions, and it has been observed that their granules are then quite readily soluble in water, so that they cannot be demonstrated with aqueous solutions of the usual dyes. Wolff notes a case in which the mast-cells constituted about 10 per cent. of the total number of leukocytes. Whether or not the conclusions which have been reached regard- ing the meaning of. the prevalence of certain cell forms in pleural effusions can be directly applied in the case of ascitic fluid remains to be seen. From the available data it appears that the indications are not as direct. But generally speaking endothelial plaques control the picture in ascites of mechanical origin, while lymphocytes predomi- nate in tuberculous peritonitis and in peritoneal carcinoma. ‘The occurrence of large vacuolated cells is suggestive of a cyst accompanied by ascites (ovarian cyst). ‘The same is true of the cytological study of joint effusions. Widal reports that in 3 cases of acute rheumatism he found polynuclear leukocytes in the serous exudate, while they were absent in traumatic cases of arthritis. As the result of an examination of 30 hydroceles Marchetti’ concludes that lymphocyte and epithelial cells predominate without exception. Of the cytological findings in the cerebrospinal fluid a detailed account will be given later. Generally speaking the cytological factor does not seem to depend so much upon the anatomical localization of the morbid process as upon its duration and the character of the pathogenic agent. An acute process (pneumococci, streptococci) call forth a ly mphocytosis of brief duration, which is followed sooner or later by a granulocytosis, while a less intense stimulus, and one acting more slowly (tubercle bacillus) leads to a persistent lymphocytosis. ‘lhe possibility that a stimulus of the latter order may act with undue virulence and intensity, and that one of the first type may be exceptionally mild and delay the occurrence of granulocytosis, should, however, be borne in mind. Very important also is the study of the cellular elements which 1 Med. News, October 1, 1904. 2 Gaz. d. Ospedal. e. d. clin., 1904, No. 94. 38 594 TRANSUDATES AND EXUDATES are found in serous exudates in cases of malignant disease of the serous membranes. Difficulty may here be encountered in the in- terpretation of the cellular findings, for on the one hand it is often difficult to distinguish the endothelial cells from leukocytes, as they take on phagocytic activity and often present the most bizarre forms. ‘lhe nucleus, which is normally centrally located, takes up | an excentric position, and enclosed within the cell we may find leu- | kocytes and red cells. On the other hand, it is impossible by simple — inspection to distinguish normal endothelial cells from cancer cells, In cases of doubt it is well to ascertain whether the epithelial ele- ments give the glycogen reaction and to hunt for the presence of mitosis. Qunicke has pointed out that normal endothelial cells do not contain glycogen, and that ‘a marked iodine reaction is very suggestive of carcinoma. Wolff, however, suggests that this test is probably not specific, and cites two instances in which he obtained a positive glycogen reaction, although a tumor did not exist. More important probably is the presence of mitoses. In non-malignant exudates epithelial cells never present evidence of mitosis, while in cases of tumor they may be found. Rieder regards their occurrence as pathognomonic of malignant disease. Commonly the mitosis is atypical; the division of the nucleus is not followed by a division of the cell; the chromosomes are short and show no polar or equatorial arrangement, In cases of neoplasm Quincke has also drawn attention to the occurrence of large numbers of fat droplets in the fluid, which may attain a diameter of from 40 to 50 yw. At times, however, the fat droplets are so small and so numerous as to give a chylous appear- ance to the exudate. At other times a similar appearance is due to the presence of minute albuminous granules, which may be distin- guished from fat by their insolubility in ether and the fact that they are not stained with the common fat dyes, such as Sudan, scarlet-R, and alkanin. ‘The occurrence of numerous fatty acid crystals, arranged in groups, should also excite suspicion of a neoplasm. Should bits of tissue be obtained, a positive diagnosis of malig- nant disease may, of course, be made by the usual methods. Such particles should be placed at once in absolute alcohol or formalin. Crystalline elements are not usually seen in serous or hemorrhagic exudates; at times we meet with platelets of cholesterin. Technique.—In every case the fluid should be examined as soon after puncture as possible; if this cannot be done at once, coagula- tion may be prevented by the addition of sodium citrate. ‘The material is then placed in the ice-box until a sediment has collected or this may be obtained at once by centrifugation, new portions of — fluid being repeatedly used and the sediments combined. Cover-. glass preparations may then be conveniently made, or smears on slides exactly as in the case of blood, care being taken to do as little” BACTERIOLOGICAL EXAMINATION OF EXUDATES — 595 injury to the cellular elements as possible. ‘The smears should be yery thin, so that the specimens will dry rapidly and but little chance is given for the cells to contract beyond their usual size. Subsequent treatment will depend upon the special points which are to be elicited. Unfortunately the leukocytes are often much changed, so that their classification may be attended by considerable diffi- culties. ‘he polynuclear elements may appear mononuclear and not infrequently the neutrophilic granules can no longer be demon- strated. (See Pus.) For this reason the triacid stain is not to be recommended for routine work; the eosinate is much better and _ will furnish as satisfactory results as can be obtained with a panoptic dye. Successive staining with eosin and methylene blue sometimes gives better results than a polychrome dye. Care should be had not to diagnosticate eosinophilia from the fact that cell granules are stained red, as the neutrophilic granules of degenerating cells are com- monly amphophilic, viz., they stain both with acid and neutral dyes; account must be taken of the size of the granules and the general structure of the cell. ‘Lo differentiate pseudolymphocytes from true lymphocytes, Pappenheim’s methyl-green pyronin may be employed, though it is not absolutely specific; still it will be found that even though the protoplasm of other cellular elements may take the red color of the pyronin, the intensity is distinctly less than in the case of the lymphocytes proper. Pappenheim’s Method.'—The stain is composed of a concentrated aqueous solution of methyl green to which pyronin is added until the solution just begins to turn blue viz., about 1 part of pyronin for 3 to 4 parts of methyl green. Stained in this manner the basophilic protoplasm of the lymphocytes 1s colored a fine dark carmine red, while the protoplasm of all other cells is stained a more or less pale brownish or reddish yellow, or remains colorless. Pappenheim regards this stain as essentially specific for the lymphocytes, but admits that it also stains in a similar manner the young erythroblasts that are poor in hemoglobin. ‘The difference can be recognized from the character _ of the nuclei and the fact that the margin of the lymphocytes very ~ commonly appears shaggy, while that of the erythroblasts is smooth and homogeneous. To study mitosis, hematoxylin and eosin may be employed, or the Romanowsky method in one of its various modifications. The glycogen reaction is demonstrated as in the case of the blood BACTERIOLOGICAL EXAMINATION OF EXUDATES. In a measure the bacteriological examination of exudates has been supplanted by the cytological study, as outlined above; especially as 1 Virchow’s Archiv, 1899, vol. elvii. ' 596 TRANSUDATES AND EXUDATES the bacteriological examination has been notoriously unsatisfactory in the most important group of effusions, viz., in those of tuberculous origin. It is now known that all exudates gradually become free from bacteria, even though at first they may have been caused by bacterial activity. As a result it is no longer justifiable to conclude that a process is tuberculous because bacteriological examination of the exudate has given no positive result. If it is desired to cultivate organisms that may be present, it is well to make a bouillon culture in every case so as to eliminate the bactericidal properties of the exudate as much as possible. In any event it is well to centrifugate the fluid in a sterile tube and to use the sediment for inoculations. The organisms which are most likely to be encountered are the pneu- mococcus, the various staphylococci, streptococci, and more rarely the colon bacillus and the typhoid bacillus. Inoscopy.'—Jousset recommends the following procedure for the purpose of demonstrating tubercle bacilli in exudates: The fluid is allowed to clot spontaneously or by adding a little horse serum. The clot, which is supposed to contain most of the organisms, is pressed out, torn into fragments, and placed in about 10 c.c. of a digestive mixture of the following composition: pepsin, | to 2 grams; glycerin, 10 c.c.; 40 per cent. solution of hydrochloric acid, 15 ¢.c.; sodium fluoride, 3 grams; water, 1000 c.c. ‘The material is left in the incubator for three to four hours, then centrifugalized and smears prepared from the sediment and stained as usual. Jousset claims to have obtained very good results in this manner, while others are less enthusiastic. More recently Zebrowski’ has suggested the following method as more likely to lead to satisfactory results: Coagulation of the fluid is prevented by the addition of an equal volume of a 0.5 per cent. solution of sodium fluoride. ‘The mixture is set aside in a cool place until the following day, when it is thoroughly centrifugated and smears made from the sediment and stained as usual. With this method Zebrowski claims to have found tubercle bacilli in 83 per cent. of secondary and 55 per cent of primary pleurisies. More satisfactory than either method possibly is the animal experiment, to which end a large quantity of the fluid is centrifugalized and the sediment injected into the peritoneal cavity of a guinea-pig, as in the case of the urine (which see). LITERATURE.—Widal and Ravaut, “Cytodiagnostique des 5 cpanclerae sero- fibrineux de la plévre,”’ Trans. XIII Internat. Med. Cong. Paris, 1900. Barjou and Cade, ‘‘Etudes cytol.,” etc., Arch. gén. d. méd., August, 1902. Gulland, “Cytodiagnosis, 7) etc., Scott. Med. and Surg. Jour., June, 1902, p. 490. A, Wolff, “Transudates and Exudates,” Zeit. f. klin. Med., 1902, vol. xxii, Heft 5 u. 6. Quincke, Deutsch. Arch. f. klin. Med., 1882, vol. xxx, pp. 369 and 580. Rieder, ibid., 1895, vol. liv, p. 544. 1 La semaine méd., 1903, No. 3. * Deutsch. med. Woch., September 7, 1905. ee CHEMISTRY OF EXUDATES 597 CHEMISTRY OF EXUDATES. According to Moritz, an albumin is found in exudates that can be precipitated with acetic acid and which is absent in transudates. He regards this as serum globulin which has undergone a change as a result of the inflammatory process. According to Matsumoto, on the other hand, the substance in question represents a mixture of fibrinoglobulin, euglobulin, and a small amount of pseudoglobulin; in the filtrate, however, there is also some fibrinoglobulin (fibrinogen) and euglobulin. He suggests that this last circumstance is probably referable to the small amount of salt in exudates and that in the first instance the pseudoglobulin is probably carried down mechanically. More recently Umber has studied the body in question and arrived at the conclusion that it belongs to the mucins. ‘To its presence the mucinous character of such fluids is due. It is precipitated by the addition of acetic acid and is insoluble in an excess of the reagent unless the acid is present in great concentration. ‘The body has markedly acid properties and is not coagulated by heat. It differs from the known mucins in the presence of a very small amount of reducing substance, which can only be demonstrated by special methods. It contains about 14 per cent. of nitrogen and no phos- horus. In neutral and feebly acid solution the substance does not coagulate (thus differing from the globulins). The same body appa- rently was found by Salkowski in an exudate into the hip-joint. Umber calls this substance serosamucin. Its amount is less than 0.5 per cent. According to Umber and Stihelin the serosamucin is essentially found in exudates referable to inflammatory processes or associated with new growths. In transudates, as Runeberg already pointed out, only a very slight turbidity results upon the addition of acetic acid, and not in all cases, moreover; so that a well-marked reaction, viz., a marked precipitation upon the addition of acetic acid to the point of a distinctly acid reaction, may be regarded as a valuable sign in the diagnosis between transudates and exudates. I append some of the results obtained by Umber: ASCITES. No. of cases. Serosamucin. Bete SIE OSIS oy Nr ad oe hae hat ete ee 6 0 Hepatic cirrhosis with chronic nephritis and phthisis 1 ) BP EESIAN ey ec ei ON i Aa as tie. 0 Oe EET le Pee Cee eo A 3 0 PLEURAL EXUDATES. Degeneratio cordis and nephritis Myocarditis Hepatic cirrhosis TN fete Dae Lymphosarcoma (pleura intact postmortem) Carcinoma mammze with ear metastases Tuberculosis of pleura Pleuritis exsudativa acuta Pleuritis and pericarditis . 0 0 0 0) ae + a ae 598 TRANSUDATES AND EXUDATES For the isolation of serosamucin see Umber’s paper (see Literature below.) Of the common albumins we meet with traces of fibrinogen and with fairly large amounts of globulin and serum albumin. Their percentage may at times not appear so very large, but considering the large amount of fluid and the rapidity with which it may accumu- late it is clear that the loss of nitrogen to the body in this form may be very considerable. Umber showed that in one of his cases 5000 grams of albumin representing about 15,000 grams of muscle tissue were lost within a year. In addition to the serosamucin and the common albumins men- tioned, some exudates may possibly also contain small amounts of a a nucleo-albumin, as is suggested by the findings of Pajikull. Should. ovarian cysts have ruptured into the peritoneal cavity, we may fur- ther find both pseudomucin and paramucin (which see). Of interest further is the fact that Umber succeeded in demonstrat- ing the existence of autolytic processes in exudates. He found both albumoses and mono-amino acids, viz., leucin and tyrosin. Coriat has reported a case of polyneuritic delirium, in which pleurisy with effusion developed. In the effusion he could demonstrate a peculiar albuminous substance, which he regards as identical with Bence Jones’ albumin; in the urine this substance could not be found. LirrraTurEe.—Pajikull (Swedish ref. by Hammarsten: Jahresber. f. Thierchem., 1893). Moritz, Minch. med. Woch., 1902, No. 42. Matsumoto, Deutsch. Arch., 1902, vol. Ixxv, p. 409. Stahelin, Mtinch. med. Woch., 1902, No. 34. F. Umber, Zeitsch. f. klin. Med., 1903, vol. xlviii, p. 364. Coriat, “The Occurrence of the Bence Jones Albumin in a Pleuritic Effusion,’’ Amer. Jour. Med. Sci., 1903, vol. cCxxvl, p. 631. Pus. General Characteristics of Pus.—If pus, which usually pre- sents a color varying from yellowish gray to greenish yellow, is allowed to stand for a time, a liquid gradually appears at the top, and increases in amount until it is finally possible to distinguish two distinct layers, the one above—the pus serum; the other at the bottom—the pus corpuscles. Upon the number of the latter the consistence as well as the specific gravity of the pus is dependent. This may vary between 1.020 and 1.040, with an average of 1.031 to 1.033. Fresh pus has always an alkaline reaction, which may become neutral or slightly acid upon standing, owing to the develop- ment of free fatty acids, glycerin-phosphoric acid, and lactic acid. The color of pus serum may be a light straw, a greenish or a brownish yellow. Chemistry of Pus.—The chemical composition of pus serum and pus corpuscles may be seen from the following tables: % CHEMISTRY OF EXUDATES ’ 599 ANALYSIS OF Pus SERUM. I; IT. ee ee Pee ie A Bae y 91370 905.65 PC rtrs 2) ©00e ew rateee Fe el fb. Ls 86-380 94.35 Albumins PE fee el Oe Ee os es Toes PTO eM ai A ee 1.50 0.56 ee eg el APO ee ee ie 0.26 0.29 DATIORCOTY TA wees! Sr RMT Cy gk 1 ide 0.87 Aleoholie extract ees Ce we 1,52 0: ¢3 Aqueous extract ee ee Ge ae 8 Ll. OS 6.92 PMT SALAS 90 Se ee ne ES oe tia COLE ANALYSIS OF Pus CORPUSCLES. if, inp DeCek cae Cunt pe ir. oc eee eee” 342,37 } insoinpiematter <0 ee se eee... 4205.66 673.69 Bere ee ee, We) ne hs cewek |) el, ES 7: G2 f Lecithin 1.04 ie Pend Ga teat re es 4.2F 6143 89 se: hee at. oh eee ee ee ewe we S74 OD 72.83 Mpa, er we eee et wets)! 5199 eyes ae ee ee ewe yo 4433 102.84 Albumoses are usually present, and are derived from the pus cor- puscles. Leucin and tyrosin are likewise frequently met with in the pus of old abscesses; and fatty acids, urea, sugar, glycogen, biliary pigments and acids (in catarrhal jaundice), acetone, uric acid, xanthin bases, cholesterin, etc., have occasionally been observed." Microscopic Examination of Pus. Leukocytes.—If a drop of pus is examined with the microscope, it will be seen to contain innumer- able leukocytes, many of which in perfectly fresh pus exhibit ameboid movements. ‘lhe cells in question are usually almost altogether of the neutrophilic variety, and it may be questioned whether the lym- phocytes ever occur in true pus. Even in cases of lymphatic leukemia the predominating cell in abscesses is the polynuclear leukocyte or its degeneration forms. Mononuclear elements with basophilic pro- toplasm, however, are also met with, notably in the more chronic cases, but it is likely that they are derived from the connective-tissue cells and are not of hematogenic origin. Eosinophiles are only seen in pus under certain definite conditions, as in gonorrhea (see below), and mast-cells also are quite uncommon. In pus that is not perfectly fresh it is usually not possible to dem- onstrate the presence of neutrophilic granules. In such cells, more- over, we commonly meet with fragmentation of the nucleus, asso- ciated with marked pyknosis. ‘This was first noted by Ehrlich in a case of hemorrhagic smallpox and in various exudates, and has subsequently been described by Michaelis and Wolff. ‘Vhe degenera- tion may proceed to fragmentation of the entire cell with the conse- quent formation of mononuclear neutrophilic forms (Ehrlich’s pseudo- ‘'M. Pickardt, “Z. Kenntniss d. Chemie path. Ergisse,’”’ Berlin, klin, Woch,, 1897, p. 844, : 600 TRANSUDATES AND EXUDATES lymphocytes). On the other hand, a type of degeneration is seen in which the nucleus does not become pyknotic, but swells to a large size and stains rather faintly with basic dyes. In such cells the proto- plasm appears as a narrow rim and the impression is gained as though the cell were in reality a leukocyte; if at the same time the granules have been lost, the differentiation may indeed be impossible, unless transition forms exist between the normal polynuclear neutrophile and the type in question.’ Owing to resorption of water from accumulations of pus of long standing, such material finally assumes a caseous aspect, and the leukocytes will be seen to have greatly diminished in size, and to have assumed an angular, shrunken appearance; it is then hardly possible to demonstrate the presence of a nucleus, even after the addition of acetic acid. It is noteworthy that in cases of hepatic abscess referable to Amoeba coli it is seldom possible to demonstrate any normal leuko- cytes, and it will be seen that under such conditions the pus consists almost altogether of granular and fatty detritus, while in liver abscesses due to other causes the leukocytes usually present a fairly — normal appearance. Mast-cells are only exceptionally seen in pus. Giant Corpuscles.—So-called giant pus corpuscles, measuring at times from 30 to 40 / in diameter, have been observed in abscesses of the gum, hypopyon, and in the contents of suppurating ovarian cysts, but they do not appear to have any special significance. Upon careful examination these bodies will be seen to contain one oval nucleus, usually located eccentrically within the cell, and from one to thirty or even forty pus corpuscles.” Detritus.—Fatty and albuminous detritus in variable amount may be observed in every specimen of pus, and increases with the length of time that it has been confined within the body. The same holds good for the presence of free nuclei, which were formerly regarded as young pus corpuscles, but which have now been definitely recog- nized as originating during the disintegration of the corpuscles. Red Corpuscles.—Red blood corpuscles in variable numbers are usually seen in every specimen, their appearance depending upon the length of time they have been confined. Pus corpuscles may at times contain a red corpuscle. | Pathogenic Vegetable Parasites—Of the pathogenic organisms which are of especial interest from a clinical standpoint may be mentioned the true pus organisms, notably the staphylococci and the Streptococcus pyogenes; furthermore, the tubercle bacillus, the Actinomyces hominis, the bacillus of glanders, the bacillus of anthrax, ' L. Michaelis and A. Wolf, “Die Lymphocyten,” Deutsch. med. Woch., 1901, vol. xxvii, p. 651. ’ Bottcher, Virchow’s Archiv, 1867, vol. xxxix, p. 512. Bizzozero, loc. cit. babiekl ts cy CHEMISTRY OF EXUDATES 601 leprosy, tetanus, influenza, and Friinkel’s pneumococcus, ete. ‘The majority of these have already been described. A pathogenic lepto- thrix, named by Flexner the L. asteroides, has been found by Cozzo- lino’ in the pus of a retroperitoneal abscess. A form of streptothrix has been isolated from the pus of certain cases of mycetoma, or Madura foot.’ Vincent’s’ fusiform bacilli and spirilla have been encountered in the pus of alveolar pyorrhcea, in noma, hospital gangrene, gangrenous ulcer of the penis, in bronchiectasis, abscess of the leg, ete. In the pus of abscesses in cases of systemic blastomyces infection the corresponding organism is found. Protozoa, with the exception of the Amoeba coli, have only rarely been found. Kiinstler and Pitres* observed numerous large spores with from ten to twenty crescentic corpuscles in pus taken from the pleural cavity of a man, which closely resembled the coccidia of mice. Litten® observed cercomonads in the fluid withdrawn from a pleural cavity. ‘Trichomonads have been found in empyema in con- nection with pulmonary gangrene. Most important in this connection is the demonstration of the Amceba coli in the pus, and in cases of liver abscess an examination with this end in view should never be neglected. So far as the -oecurrence of amebas in pus is concerned, the observation of Kartulis and of Flexner, who demonstrated their presence in an abscess of the lower jaw, shows that they should not be looked for in the pus of abscesses of the liver or lung only. In smears obtained from two cases of oriental boil (tropical ulcer, Delhi boil, Aleppo boil) Marzinowsky and Bargow," on the one hand, and Wright’ on the other, found little bodies, measuring from | to 4 in diameter and apparently provided with a macronucleus and a micronucleus. ‘They are inclined to look upon these as protozoa and as parasitic. Marzinowsky and Bragow name the organism Booplasma orientale; Wright calls it the Helcosoma tropicum. According to Christofers* they are identical with the Leishmania-donovani of tropi- cal splenomegaly, which latter are known to occur in the skin ulcers of kala-azar. Vermes.—Of these, the filaria and hydatids are rarely observed in this country. Bothriocephalus linguloides has been found in the pleural cavity of a Chinese patient. 1 Zeitsch. f. Hygiene, 1900, vol. xxxiii, p. 36. ? Boyce and Adams, Jour. Exper. Med., vol. ili, p. 422. 3 Annal. de l’Institut Pasteur, 1894, vol. vili, p. 129. * Compt.-rend. de la Soc. de biol., 1884, p. 523. 5 Verhandl. d. Cong. f. inn. Med., 1886, vol. v, p. 417. 6 Virchow’s Archiv, 1904, p. 178. 7 Jour. Cut. Dis., incl. Syph., New York, June, 1904, and Jour. Med. Kesearch, December, 1903. 8 “Discussion on the Leishman-Donovan Pody,” Brit. Med. Jour., September 17, 1904. 602 TRANSUDATES AND EXUDATES Crystals.—As has been stated, crystals of cholesterin are frequently found in old pus and in exudates of long standing, but are rarely seen in recent exudates. ‘They may be recognized by their charac- teristic form and their chemical reactions, as described in the chapter on the Feces. ‘Triple phosphates, fatty acid crystals, and hematoidin are likewise frequently seen, the presence of the latter, of course, indicating a previous admixture of blood. ' The technique to be employed in the examination of pus is as a rule simple. Cover-glass preparations or smears on slides are pre- pared as in the case of the blood and are then stained according to the points that are to be elicited. For routine work the eosinate of methylene blue will be found very useful. If the pus corpuscles are still fairly fresh, the neutrophilic granules are readily stained; it will be noted, however, that very commonly they exhibit a more decided red, which is referable to certain degenerative changes which cause the granules to assume an affinity for acid dyes as well. Bac- teria that may be present are usually well shown. If the pus is older and the cells have lost their granules, Pappenheim’s pyronin- methyl green will be found of value in the study of the mononuclear forms. Gonorrheal Pus. In the very earliest stages of the disease the pus contains large numbers of eosinophilic cells besides the common polynuclear neu- trophiles.' But at the same time and throughout the course of the disease mononuclear non-granular elements, with basophilic proto- plasm, are also seen. The larger number of the latter are of the type of the large mononuclear leukocyte and transition form of Ehrlich, but a certain percentage is also represented by the lymphocytes, both of the small and large variety. Mast-cells may also occur in gonor- rheal pus; a remarkable case is reported by Neisser, in which the pus consisted practically exclusively of such elements. The neutrophilic elements in gonorrheal pus commonly present evidence of degeneration. In some a loss of granular material has manifestly taken place, and it can be demonstrated that in most of the cells the granules are no longer absolutely neutrophilic, but have become amphophilic—that is, from a neutral mixture they take up the neutral dye, but they can also be stained with acid dyes. With the triglycerin mixture, for example, they are stained red by the eosin. As regards the distribution of gonococci in the different cellular elements, it is noteworthy that they are principally found in the poly- a ee ee * Other observers do not mention the early occurrence of eosinophiles in the » pus. Esserteau states that they are increased from the second to the fourth week, ; CHEMISTRY OF EXUDATES 603 nuclear neutrophiles, while they are less commonly seen in the mono- nuclear leukocytes and transition forms. In the small lymphocytes they are not encountered, and it is uncommon to find them in the eosinophilic cells. Generally speaking numerous gonococci, eosinophiles, and a small number of lymphocytes are found in cases of recent gonorrhea, while during exacerbations of chronic processes only a few cocci and numerous mononuclear elements are encountered. ‘The common neutrophilic elements of course control the picture practically at all times, so long as there is a discharge. The gonococcus (Neisser) (Plate XXI) occurs in the form of small oval or coffee-bean-shaped granules, grouped in twos and fours resembling a German biscuit; the individual cocci measure about 1.25 yw in length by 0.7 # in diameter. As a rule they are found enclosed within pus corpuscles and epithelial cells; but they may also occur free in the pus obtained from the urethra, in the vaginal discharge, and more rarely in urinary sediments, as in cases of com- plicating prostatitis, peri-urethritis, etc. In cover-glass specimens account should be taken only of those organisms which are enclosed within cellular elements, as these alone may be regarded as charac- teristic. ‘To this end a drop of the discharge is spread in a thin layer upon a slide or a glover-glass, dried in the air, and fixed by passing three or four times through the flame of a Bunsen burner. The specimens may then be stained with any one of the basic aniline dyes. In my laboratory the eosinate of methylene blue is almost exclusively used for this purpose. ‘The organisms are thus colored blue, while the granules of eosinophilic leukocytes, which may be present at the same time, appear a bright red or a brownish red. After five minutes the excess of stain is washed off, the preparations are rinsed in water, dried with filter paper, and examined with a high power. The gonococcus is decolorized by Gram’s method and can in this manner be distinguished from certain other organisms that may be present. Of the four kinds of diplococci which may be found in ure- _thritis besides the gonococcus, only two forms are similarly decolorized, and these two are rarely seen. We may conclude that in 95 per cent. of all cases Gram’s method permits a definite conclusion as to the presence or absence of the true organism. (Gram’s method is best employed in the modification suggested by Weinrich: ‘The preparations are fixed by drawing through the flame of a Bunsen burner and are then stained for from one to two minutes in Friankel’s carbol-gentian-violet solution (10 parts of a saturated alcoholic solution of gentian violet to 90 parts of a 2.5 per cent. solution of earbolic acid). Without washing they are placed for one to three minutes in Lugol’s solution (1 gram of iodine, 2 grams of potassium iodide, and 300 c.c. of distilled water), and again without washing 604 TRANSUDATES AND EXUDATES in absolute alcohol, until the alcohol ceases to extract color (about — one and one-half minutes); they are now washed in water, counter- | stained with Bismarck brown, washed, dried, and mounted. The — Bismarck-brown solution is prepared as follows: 3 grams of the dye — are dissolved in 70 c.c. of hot water; 30 c.c. of 96 per cent. alcohol are added; the mixture is well stirred and filtered. ‘The organism grows best on blood and hydrocele agar. The sur-_ face colonies are pale, grayish, translucent, and finely granular, with finely notched borders. In bouillon and blood serum mixed it forms a membrane, while the fluid remains clear. When no discharge can be obtained from the urethra, or an exam- ination of such discharge is negative, positive results may at times still be obtained if some of the gonorrheal threads are examined, which may be found floating in the urine. In these the organisms can occasionally be demonstrated after months and even years have elapsed after primary infection. LirerRATURE.—Janowski, Arch. f. exper. Pathol., 1895, vol. xxxvi, p. 15. I, Michaelis and A. Wolff, “Die Lymphocyten,” Deutsch. med. Woch., 1901, vol. xxvil, p. 651. A. Pappenheim, Virchow’s Archiv, 1901, vol. clxix, p. 72. Neisser, Centralbl. f. d. med. Wiss., 1879, vol. xvii, p. 497. J. Plato, ‘(Ueber Gonokok-. kenfarbung mit Neutralroth,” etc., Berlin. klin. Woch., 1899, p. 1085. E. R. Owings, “The Infectiousness of Chronic Urethritis,’’ Bull. Johns Hopkins Hosp., 1897, p. 210. H. H. Young, ‘Welch Festschrift,” Johns Hopkins Press, 1900, Dp. Oe Putrid Exudates. : Putrid exudates are observed following perforation of a gangren- ous focus or of a gastric or intestinal ulcer into one of the body cavities. At other times they are encountered in cases of neoplasm, and at times even without apparent cause. The material obtained in such cases has a brown or brownish-green color, and emits an odor which in itself indicates the character of the exudate. Micro- scopically, cholesterin, hematoidin, and fatty acid crystals, as well as degenerating leukocytes, are found. In cases in which aspiration of a higher intercostal space reveals the presence of serous fluid, while putrid material is obtained at a lower point, the existence of a subphrenic abscess should be suspected. In such cases a pure cul- ture of the Bacillus coli communis has been obtained. The reaction of putrid exudates is usually alkaline, but an acid reaction may be obtained in cases of perforation of a gastric ulcer; the Sarcina ven- triculi and saccharomyces may then also be found. Chylous and Chyloid Exudates. Chylous and chyloid exudates have been repeatedly observed. They are most frequently met with in the abdominal cavity (one CHEMISTRY OF EXUDATES 605 hundred and four times out of a total number of one hundred and . fifty-five, which have thus far been reported), less commonly in the _ pleural cavity (forty-nine times), and only rarely in the pericardial sac (twice only) (1904). Among the causes which may lead to chylous ascites the following are recognized (in the order of their frequency): _ compression of the thoracic duct or the lymphatic vessels by glandular enlargements, neoplasms, ete.; non-tuberculous peritonitis; occlusion of the left subclavian; excessive pressure, strain, cough; peritoneal - carcinoma; filariasis; occlusion of the thoracic duct; occlusion of lymph vessels, external pressure; diseases of the liver, syphilis, primary disease of the lymph vessels, angioma, calculus of the receptaculum | chyh, and Hodgkin’s disease. Quincke believes that the two forms can be etiologically distinguished from one another by means of a _ microscopic examination, as the cloudy appearance in the chyloid _ form is usually referable to the presence of endothelial or epithelioid cells undergoing fatty degeneration. Later observations, however, have shown that the differentiation of the two forms cannot be made upon this basis, as the same anatomical lesion, such as carcinoma or tuberculosis, may at times give rise to the formation of a chylous exudate, at others to that of the chyloid form, and both, moreover, may coexist. An instance of this kind is described by Wilson. Senator claimed that the presence of more than traces of sugar is strongly suggestive of the chylous nature of the exudate. Possibly ‘ this observation may be of some value, but only the presence of more than 0.2 per cent. is of value. Of greater significance is the fact that in chylous fluid the melting point of the fat will depend upon the melting point of the fat which was taken in as food, while this is not _ the case in chyloid effusions. The amount of fat, moreover, which is present is influenced directly by the amount ingested in the first instance. Occasionally one can get the distinct odor of the food which has been taken, in chylous exudates, while in the chyloid type this would hardly be expected. Chylous exudates in their general appearance resemble milk, while chyloid fluid is more suggestive of pus. The turbidity in both cases is usually referable to the presence of innumerable fat globules, which are especially abundant in the chylous form. In chyloid exudates the origin of the fat from cellular elements is often appar- ent at once; but, as has been said, it is impossible to draw definite etiological conclusions from that difference. Some chyloid exudates contain no fat at all, and Lion has shown that the milky appearance in such cases is owing to the presence of a curious albuminous sub- stance, belonging to the class of nucleo-albumins. Bernert, on the other hand, claims that the substance in question belongs to the globulins, and is closely associated with certain lecithins. A similar ‘observation is recorded by Micheli and Mattirolo. i 606 TRANSUDATES AND EXUDATES Edsall (cited by Wilson) reported an instance of non-fatty pleural effusion, the opacity of which was due to altered globulins, Chemical analysis of a chylous exudate (pleural) from a case of Hodgkin’s disease, which Campbell made in my laboratory, showed the following result: Water nr pie ER, fet gti se te a Cea per cent. Solids kU a Pe Ts RE PSE ae Mineral solids 50 S— 0). oa Soa ba ee OB Organic solids dg a) LS oe ee ee Coagulable‘albumins: . 9...'..%.. 9. 7) 4680 Fate, oh ark eh. EO) Sa an gee act 0) ee CIEE E WM ee Ea ay ea fey re ee NUM The specific gravity was 1.020. The cytological formula in such exudates has as yet received but little attention. In Campbell’s case only a small number of leuko- cytes was present and most of these were of the lymphocytic type. . In Muttermilch’s case lymphocytes were said to preponderate; in addition there were small numbers of neutrophilic leukocytes, con- taining fat granules, together with eosinophilic cells and a very few red cells. In the mixed case of Wilson the lymphocytes numbered 76 per cent., and the large mononuclear cells 22 per cent. LiTERATURE.—Quincke, loc. cit. Boulengier, Schmidt’s Jahrb., 1890, vol. eexxvi, p. 28. Wilson, Amer. Jour. Med. Sci., October, 1905. Boston, Jour, Amer. Med. Assoc., February 18, 1905. Micheli and Mattirolo, Wien. klin. Woch., 1900, No. 3. Muttermilch, Zeit. f. klin. Med., vol. xlvi, p. 123. Shaw, Jour, Pathol. and Bacter., vol. vi, 1900. Examination of Syphilitic Material. Spirochete Pallida.—Through the researches of Schaudinn and Hoffmann it has been ascertained that in primary and secondary syphilitic lesions a spirochete can be demonstrated which probably represents the cause of the disease. ‘Their results have been abun- dantly verified both abroad and in the United States. ‘The organism has been demonstrated in the scrapings obtained from chancres, incised papules and condylomata, and in smears from mucous patches and the aspirated juice of the inguinal glands. Schaudinn and Hoff- mann could further demonstrate the organism in the blood obtained by puncture of the spleen in a recent case of syphilis on the day pre- ceding the eruption. Levaditi found it in the vesicular contents of pemphigus syphiliticus. Buschke and Fischer, Babes and Panea, and Levaditi found the spirochaete in the internal organs of children which had died of congenital syphilis, as also in the blood, and Metschnikoft could demonstrate it in the lesions of artificial syphilis in the ape. | The Spirochete pallida derives its name from its low refractive power and the difficulty with which it takes up aniline dyes (this PLATE XXIL < = ae a i; Spirocheetee. a, 5. refringens; 6, 8S. pallida, (Stained. with Giemsa’s stain.) we “ CHEMISTRY OF EXUDATES 607 especially in contradistinction to the Spirochete refringens). It is a very delicate structure, usually presenting 10 to 40 deep spiral incur- yations with the larger specimens, or only 2 to 4 in the smaller ones. ‘The length varies from 4 to 10 # with 7 / as an average; the width does not exceed 0.5 4. In the wet preparation it may be observed that its movements occur in an oscillatory manner about the longi- tudinal axis, and that, in contradistinction to the spirilla, the move- ments of the spirocheete are winding, bending, and whipping, while in the spirilla the longitudinal axis remains rigid. Schaudinn also demonstrated the existence of a flagellum at each end, while the other spirochetas have an undulating membrane. (See Plate XXII and Fig. 171). Fig. 171.—Spirochete pallida. Staining Methods.—Excellent results are obtained with Goldhorn’s stain (which see). ‘lo this end the smears, on slides or covers, are covered with the dye for three or four seconds, when the excess is drained off. ‘lhe specimens are then introduced slowly into clean water with the film side down, permitting in this manner an interaction between the film of adhering dye and the water. ‘The slide is held in this slanting position for another four or five seconds and is next shaken in the water so as to wash off the excess of the dye. ‘The pallida appears of a violet color, which may be changed to bluish black by flooding the preparation for fifteen to twenty seconds with Gram’s iodine solution, pasting and drymg as usual. ‘I'he examination is conducted with a zy or to immersion lens. The material should in all cases be obtained by curettage, this being carried so far until a small amount of serum and blood appears, 608 TRANSUDATES AND EXUDATES and preferably at the edge of the lesion. ‘The serous fluid is then spread upon slides or covers in the usual manner. ‘The organisms are most numerous in moist papules and chancres (when the curettage is carried out at the edge of the lesion). In roseolar scrapings the search is frequently disappointing. Giemsa’s method also furnishes excellent results. I have recom- mended a dilute alcoholic solution of Victoria blue. Keidel finds that steaming the specimens with this solution for a minute or two furnishes good results. LITERATURE.—Schaudinn and Hoffmann, Arbeiten aus d. kais. Gesundheits- amte, 1905, vol. xxii, p. 527; Deutsch. med. Woch., 1905, No. 18, p. 711; Berlin. klin. Woch., 1905, May 29, p. 673. Metschnikoff and Roux, Le bulletin méd., May 17, 1905, p. 441. Levaditi, La semaine méd., May 24, 1905, p. 247. Hoff- mann, Berlin. klin. Woch., 1905, No. 22, p. 673. Fanoni, Med. News, October, 1905. Babes and Panea, Berlin. klin. Woch., 1905, No. 28, p. 865. Mulzer, ibid., No. 36, p. 1144. Goldhorn, Jour. Exper. Méd., 1906, vol. viii, No. 3. | : , — CORAM Dy Ree TX”. THE CEREBROSPINAL FLUID. ACCORDING to our present knowledge, the cerebrospinal fluid is secreted by the choroid plexuses into the lateral ventricles. Passing through the foramina of Monro, the third ventricle, and the aque- duct of Sylvius, on the one Head it reaches the fourth ventricle and enters the cystern-like subarachnoid spaces at the base of the brain, through the foramen of Magendie and the lateral clefts of the fie Benitricle. On the other ents a certain portion of the fluid reaches the same destination directly through the cleft in the descending horn of each lateral ventricle. ‘he larger portion of the fluid then passes upward through the subarachnoid spaces along the convexity of the brain to the Pacchionian granulations, while the smaller portion enters the vertebral canal through the subarachnoid spaces of the spinal arachnoid membrane. Within recent years puncture of the vertebral canal has been frequently resorted to, both for therapeutic and diagnostic purposes. The practical value of this method of diagnosis is now beyond ques- tion, and it is to be hoped that ere long physicians will resort to spinal puncture in obscure cases of cerebrospinal disease with as little hesitancy as puncture of the thoracic and abdominal cavities is now practised. - The operative method to be employed is the following: With the patient placed upon his left side—some_ observers prefer the sitting posture—and the body bent well forward, a long aspirating needle is introduced upon a level with the lower fherd of lie third or fourth lumbar spinous process, and about 1 cm. to the side of the median line, the needle being directed slightly upward and inward. ‘The depth to which it is necessary to puncture will, of course, vary with the age of the patient. In a child two years of age the vertebral canal may be reached at a depth of 2 cm., while in the adult it is necessary to insert the needle for a distance of from 4 to 8 cm. As soon as the subarachnoid space is reached cerebrospinal fluid will flow from the needle. Aspiration should always be avoided. Some writers-have advised that the operation be performed under 1H. Quincke, Verhandl. d. X Cong. f. inn. Med., 1891. A. Hand, “ A Critical Summary of the Literature on the Diagnostic and Therapeutic Value of Lumbar Puncture,’ Amer. Jour. Med. Sci., 1900, vol. exx, p.463. A. Stadelmann, “Klin- ische Erfahrungen mit d. Lumbalpunction,” Deutsch. med. Woch., 1897, p. 745. 39 610 THE CEREBROSPINAL FLUID narcosis; and without doubt this may be necessary at times, particu- larly when contracture of the dorsal muscles exists. In the majority of cases, however, it is not necessary and local anesthesia will suffice, Amount.—So far as I have been able to ascertain, no observations have been made regarding the amount of fluid which may be obtained by puncture in normal individuals. In all probability, however, this is small. Under pathological conditions the amount may vary from a few drops to 100 c.c., and even more. In general terms it may be stated that the amount is directly proportionate to the degree of intracranial pressure. Exceptions, however, are frequent. Small amounts of cerebrospinal fluid or none at all may thus be obtained when, owing to the formation of a thick exudate or the existence of a cerebral tumor, communication between the basilar subarachnoid spaces of the brain and those of the spinal cord has been interrupted. Whenever, then, symptoms of intracranial pressure exist, while no fluid or minimal amounts only can be obtained by puncture, the conclusion will usually be justifiable that we are dealing with a purulent meningitis or with a tumor of the brain, and more especially of the cerebellum. It should be remembered, however, that the same result may be obtained in cases of obliteration of the aqueduct of Sylvius, or when sclerotic processes involve the foramen of Magendie, which is occasionally observed in certain forms of hydro- cephalus. Adhesions of the pia mater to the arachnoid and the dura mater may, by interfering with the flow of cerebrospinal fluid, also lead to the formation of hydrocephalus, but in these cases a tumor can usually be excluded, as the changes in question always develop as sequels to a meningitis. A serous or tuberculous menin- gitis, as well as acute hydrocephalus and tetanus, can, however, always be excluded when only minimal amounts of fluid are obtained by puncture. ‘The largest amounts, on the other hand, are seen in cases of serous meningitis, tuberculous meningitis, and cerebral tumors, which do not interfere with the circulation of the cerebrospinal fluid. In the epidemic type of meningitis 70 to 80 ¢.c. can usually be obtained very readily. In epilepsy Pellagrini usually obtained amounts varying between 10 and 15 c.c.*. Donath gives rather higher figures, up to 60 c.c., and in a tabes case 85 c.c. Appearance.—Normal cerebrospinal fluid, as well as that obtained in cases of serous meningitis, tuberculous meningitis, hydrocephalus, and tumors of the brain, is perfectly clear, and as a rule colorless. unless a small bloodvessel has been punctured, when the fluid may present a slightly reddish tinge. More or less pronounced yellow shades are, however, at times observed. Important from the stand- point of diagnosis is the fact that in cases of hemorrhage into the ventricles pure blood is obtained, while such a result is, of course, a ‘ La Riforma med., 1901, Ann. 17, vol. ii, p. 638. THE CEREBROSPINAL FLUID 611 mechanical impossibility in cases of epidural hematoma. In subdural hematoma, on the other hand, blood may also find its way into the subarachnoid space, but the amburit is always small, and cannot be compared with that seen in cases of ventricular hemorrhage. When- ever, then, as in traumatic cases with severe cerebral symptoms, the surgeon is confronted with the question whether or not to trephine, puncture of the subarachnoid space may furnish much valuable information. If in such cases no blood at all is found, it may be inferred that an epidural hematoma or a subdural hematoma of slight extent only exists; an operation may then be performed. If, however, pure blood is encountered, it would be justifiable to assume the existence of extensive injury to the brain substance proper, or, in cases in which the history is obscure, an intracerebral hemorrhage with rupture into the ventricles. In such cases the idea of an oper- ation would, of course, be entertained only under exceptional con- ditions. If, further, the fluid is only tinged with blood, a subdural hematoma probably exists, and an operation should be advised. Accidental hemorrhage, viz., hemorrhage referable to the puncture itself, can be readily recognized, as the first few drops only are then ! tinged with blood, or the blood appears only after the flow has been definitely established: the amount, moreover, is insignificant. Cloudy fluid is obtained in all cases of purulent meningitis unless the disease is limited to a very small area. In the epidemic type, however, it may be quite clear, or but slightly cloudy. Cases of ab- scess of the brain or sinus thrombosis occur again and again in which the question as to the advisability of operative interference is largely dependent upon the presence or absence of a complicating purulent meningitis. In certain instances a satisfactory conclusion may, of course, be reached without puncture; but in many others this is impossible, and Lichtheim’s dictum, that an operation should never be undertaken in such cases unless the integrity of the meninges has been established by spinal puncture, should be borne in mind. The degree of cloudiness naturally varies in different cases, and while in some instances the character of the fluid is seropurulent, pure, creamy pus may be found in others. Generally speaking, a cloudy fluid indicates the existence of an acute inflammatory process or an exacerbation of a chronic process. Important, furthermore, is the fact that the fluid in non-inflam- matory diseases of the brain, such as tumor or abscess, rarely under- goes coagulation, while this is the rule in all inflammatory diseases. In tuberculous meningitis the coagula are very delicate, and may be well compared with spider-webs extending throughout the fluid, while in purulent meningitis the coagula are somew hat firmer. Specific Gravity. —The specific gravity of cerebrospinal fluid normally varies between 1.005 and 1.007, corresponding to the pres- ence of from 10 to 15 pro mille of solids. Under pathological con- i? 612 THE CEREBROSPINAL FLUID ditions variations from 1.003 to 1.012 may be observed, the specific: gravity, generally speaking, being higher in the inflammatory than in the non-inflammatory diseases of the brain. From a diagnostic standpoint, however, the determination of the specific gravity is of little value, as numerous exceptions occur to the above rule. ‘The reaction is always alkaline. CHEMICAL COMPOSITION OF CEREBROSPINAL FLUID. An idea of the chemical composition of the cerebrospinal fluid may be formed from the following analyses, taken from Gautier and Zdarek : Per cent. Watery (Gti? Be aS take. Shee. = a 2 a ee) Albumin, (3s 220 Soon 7 a ae a a 1516 Fat ot tae 0.09 Cholesterin ee ee Tey ped tM Ss. 0.21 Alcoholic and aqueous extract, minus wet 2.75 Wout, lAChALE 9 ds seated eee eee ; ‘ Chlorides ee 6.14 Earthy phosphates 0.10 Sulphates . . 0.20 ZDAREK’S ANALYSIS. Water 2 oe Oto" ek Re ee Solids 45 Organic solids Mineral ash Albumins : Ethereal residue Aqueous residue oe Sulphuric acid (SO,) Chlorine eas Carbon dioxide Potassium oxide Sodium oxide al 2a. Ne Ye Mineral ash, insoluble in water Glucose . i SCOROOCKOCOWOCDOCOONS S a In addition, urea is at times found, as also a substance which reduces Fehling’s solution and gives rise to a brown color when boiled with caustic potash, but which neither undergoes fermentation nor forms an osazone when treated with phenylhydrazin. The sub- stance in question is generally regarded as pyrocatechin. Its amount varies between 0.002 and 0.116 per cent. According to C. Ber- nard, glucose may also be present, but it is questionable whether this is the case under normal conditions (see below). Nawratzki discovered a reducing substance in his cases, which was demon- strated to be glucose; his subjects, however, were unfortunately not normal, but general paretics with fever. Pyrocatechin was absent. Zdarek* reports a recent case of anterior meningocele in an otherwise. ’ Zeit. f. phys. Chem., 1902, vol. xxxv, p. 202 : normal individual in which the fluid reduced Fehling’s solution and gave a glucosazone with phenylhydrazin. ‘The substance in question was dextrorotatory, the amount equalling 0.1 per cent. of glucose. Lichtheim claims to have found glucose—by means of the phenyl- -hydrazin test—in all cases of tumor which he examined. In cases of tuberculous meningitis, on the other hand, a positive result was only exceptionally obtained. Quincke also reports that he was able _to demonstrate the presence of sugar whenever the liquid obtained was sufficient in amount for the necessary tests. Unfortunately, however, he does not detail his cases. Concetti found no sugar in hydrocephalic fluid. The experience of other observers does not agree with that of Lichtheim and Quincke; and Fiirbringer,’ who has thus far reported the largest number of spinal punctures, found sugar in only 2 cases of diabetes associated with tuberculosis. So far as the albuminous bodies are concerned which may be found in the cerebrospinal fluid, serum albumin is said to be present only under exceptional conditions, while normally a mixture of globulin and albumoses is found. ‘The question whether or not mucin may also be present is still undecided.’ Under pathological conditions the amount of albumin may vary considerably, and is of diagnostic importance. According to the majority of observers, the figure given in the above analysis i is too high, and it is doubtful whether 1 pro mille may be regarded as normal. ‘The lowest values have been obtained in cases of chronic hydrocephalus (traces only), meningitis serosa (0.5 to 0.75 pro mille), and tumors of the brain (traces to 0.8 pro mille); while the largest amounts have been found in chronic hydrocephalus the result of hyperemia (1 to 7 pro mille), and in tuberculous meningitis (1 to 3 pro mille). Nawratzki in recent examinations found amounts vary- ing between 0.047 and 0.170 per cent., but the subjects of his investi- gation had fever at the time. Mott and Halliburton® found three times the normal amount of albumin in paralytics, as also some nucleo-albumin, which does not occur in health. ‘The latter they sup- pose to come from broken-down Nissl bodies. Cholin.—According to Gumprecht, the normal cerebrospinal fluid also contains traces of cholin. Donath obtained positive results (using 10 to 20 ¢.c.) in 15 cases of genuine epilepsy out of 18, three times in 3 cases of Jacksonian epilepsy, once in a case of syphilitic epilepsy, twice in 8 cases of dementia paralytica, once in 2 cases of taboparalysis, ten times in 15 cases of tabes dorsalis, three times in 3 CHEMICAL COMPOSITION OF CEREBROSPINAL FLUID 6183 1 Verhandl. d. XV Cong. f. inn. Med., 1901. ? Stadelmann, Mitth. a.d. Grenzgebieten d. Med. u. Chir., vol. ii. Comba, Clin. med., 1899 (cited in Arch. d. méd. d. enfants, 1900). Lenhartz, Verhandl. d. XIV Cong. f. inn. Med., 1900. * The Lancet, April, 1901. ie 614 THE CEREBROSPINAL FLUID cases of cerebral syphilis, twice in 2 cases of cerebral abscess, once in a case of encephalomalacia, once in a case of spina bifida, once in a case of compression myelitis, once in a case of alcoholic polyneuritis, once in 3 cases of neurasthenia, and once in 3 cases- of hystero- epilepsy. Negative results were obtained in 2 cases of hysteria and in multiple cerebrospinal sclerosis. Quantitative estimations were made in 10 cases; the amounts varied between 0.021 and 0.046 per cent. Method.—According to Donath,’ the cerebrospinal fluid (10 to 30 ¢.c.) is collected in test-tubes, feebly acidified with dilute hydro- chloric acid, and evaporated to dryness on the water bath.. The dark (orange yellow to dark brown) residue is extracted with absolute alcohol (99 per cent. is not sufficient), and the filtered solution treated with a solution of platinum chloride in absolute alcohol. On standing the chloroplatinate of cholin separates out. This can be identified by its ready solubility in cold water (as contrasted with the very slight solubility of potassium and ammonium platinochloride) and its very characteristic crystals. ‘These are usually serrated and lan- ceolated or leaf-wreath or rosette shaped, the latter with three or four leaves. Occasionally they are radiate needles, or needles arranged in sheaves (obliquely cut prisms) or hexagonal or rhombic platelets. They are commonly tinged yellow, but if very thin (especially the needles) they appear colorless. ‘The crystals are best obtained by allowing a few drops of their aqueous solution to evaporate on a slide. The alkaline platinochlorides appear as octohedra or tetrahedra, which may have blunt angles; but according to Donath they are never seen with the method as above outlined (using absolute aleohol— alcohol dehydrated with anhydrous copper sulphate and kept over this). Another delicate reagent for cholin in aqueous solution is phos- photungstic acid. In dilute solutions a white precipitate will form which appears under the microscope as composed of small hexagonal plates or rhomboids. As chloride of potassium and ammonium will also give a precipitate with phosphotungstic acid, the extract in absolute alcohol (see above) should be filtered, the alcohol evapo- rated, and the residue dissolved in water. The physiological test for cholin, viz., fall in blood pressure follow- ing its intravenous injection in aqueous solution, is usually unnecessary. Coriat® found cholin invariably present in general paresis, also in 1 case of central neuritis, in 2 alcoholic cases with polyneuritis, in 1 of senile dementia, in 1 of senile dementia associated with a tumor in the corpus callosum, in 1 of traumatic organic dementia, also associated with a tumor of the corpus callosum. The largest amounts were found in paresis. Lecithin was found twice by Donath, once in a tabes case and once in Jacksonian epilepsy. 1 Med. News, January 21, 1905. 2 Amer. Jour. Insanity, 1904, No. 4. MICROSCOPIC EXAMINATION 615 MICROSCOPIC EXAMINATION. Cytology.—Normal cerebrospinal fluid contains either no morpho- logical elements at all or only a small number of lymphocytes (three to eight to a field, with a medium power). Deviations from this normal condition, as has been first shown by Widal, Ravaut, Sicard, and others, may be of marked diagnostic value. Aside from tuberculous meningitis in which lymphocytosis is practic- ally constant an increased number of lymphocytes has been observed in syphilitic lesions of the central nervous system (general paresis, tabes, cerebrospinal syphilis, syphilitic hemiplegia), in certain cases of herpes zoster, sciatica, and parotitis. Of these the syphilitic cases are most important, but it is to be noted that the increase may be inter- mittent and paroxysmal. Asaruleitiswellmarked. Lymphocytosis also occurs in lead intoxication and in saturnine encephalopathy it may be quite intense. ‘The same has been noted in African sleeping sickness. Negative results have been obtained in poliomyelitis, syringomyelia, the hemiplegia of old age, polyneuritis, functional neuroses, compression myelitis, cerebral tumors, and epilepsy. According to Niedner, lymphocytosis is quite constant in syphilitic hemiplegia, while it is inconstant in tabes. Of 9 cases reported by Niedner and Mamlock,! lymphocytosis occurred in 5. In general paresis lymphocytosis is very common. In the epidemic form of cerebrospinal meningitis the predominat- ing cell is the polynuclear neutrophile, excepting in chronic cases where lymphocytes may prevail. This cell also enters into the foreground as recovery occurs. Donath summarizes his results in 98 cases as follows: In acute and purulent meningitis polynuclear leukocytes prevail; in chronic or less intense processes, especially in tuberculous meningitis, lymph- ocytes predominate. In the differential diagnosis of syphilitic men- ingitis, the early stages of tabes and of general paresis, from neurotic conditions and other malignant processes, lymphocytosis points to the first group. In tetanus a large number of polynuclear neutro- philes may also occur. While in cerebrospinal meningitis referable to the Diplococcus pneumoniz polynuclear leukocytosis is probably the rule, exceptions occur. Goggia” thus reports a fatal case in which daily examina- tions showed a predominance of the small mononuclear elements throughout the course of the disease. In connection with cerebral hemorrhage (especially hemorrhage into the ventricles) Sabrazés and Muratet* have described the occur- 1 Zeit. f. klin. Med., 1904, Heft 1 and 2. 2 Gaz. d. Osped. e. d. clin., 1905, No. 13. 8 Soe. d. biol., 1903, pp. 1226 and 1435. 616 THE CEREBROSPINAL FLUID rence of large, round, oval, or polyhedral cells, either singly or in plaques, provided each with a single oval nucleus containing several nucleoli. ‘These cells commonly contain red blood corpuscles, often in large numbers, as also crystals and amorphous particles of hema- toidin, leukocytic nuclear debris and vacuoles. These cells are macro- phages, derived undoubtedly from the endothelial lining of the subarachnoid spaces. Besides, granular structures may be met with which may contain globules of fat, nuclear debris, globules of myelin, red cells, and blood pigment. What these latter cells are is not known. Sabrazés inclines to view them as neuroglia cells. The technique employed in the cytological study of the cerébro- spinal fluid is the same as in the case of pleural exudates. Bacteriology.—Very important from a diagnostic standpoint is the fact that pathogenic microérganisms may be found. Lichtheim, Fiirbringer, Freyhan, Dennig, Friinkel, and many others since, were thus able to demonstrate the presence of tubercle bacilli in a fairly large number of cases of tuberculous meningitis. Some observers, it is true, have been less fortunate, but the fact that Fiirbringer found tubercle bacilli in 30 cases out of 37 is certainly significant. Schwarz states that he obtained positive results in 16 out of 22 cases; Slawyk and Manicatide found bacilli in all of 19 cases (sixteen times by direct microscopic examination and three times by the animal experiment) and Koplik found them in 13 out of 14 cases, using centrifugalized material. In order to examine for tubercle bacilli, the fluid should be placed on ice for from six to twenty-four hours, until a slight coagulum has formed, when the fine, spider-web-like threads of fibrin are transferred to a cover-slip, spread in as thin a layer as possible, and stained as described in the chapter on the Sputum. If a centrifugal machine is available, the examination may, of course, be made at once; the chances of finding the bacilli are then also much greater. In every case a large number of speci- mens should be prepared before the search is abandoned. Only a positive result, however, is of value, and in doubtful cases recourse should be had to the animal experiment. In the diagnosis of epidemic cerebrospinal meningitis lumbar puncture is of signal value, as the Diplococcus meningitidis intracellu- laris (meningococcus) of Weichselbaum-Jiger can be demonstrated ina large percentage of cases. Councilman thus states that during a recent epidemic of the disease in Boston lumbar puncture was performed in 55 cases, and that in the fluid obtained the diplococci were found — on microscopic examination or in culture in 38 cases. The organism was present in all the acute cases, but rarely found in those which ran a more chronic course. ‘The average time from the onset of the disease before spinal puncture was made was seven days in the posi- tive cases and seventeen days in the negative cases. The longest time after the onset in which a positive result was obtained was twenty- | | | MICROSCOPIC EXAMINATION 617 nine days. Similar results have also been reached by other observers. Koplik ‘thus found the organism within the first twenty-four hours after the onset of the disease and as late as the fifteenth week. In chronic cases, however, as Councilman also found, it may escape detection, especially in those of the posterior basic type. The organism in question is a diplococcus, each half being of about the same size as the ordinary pathogenic micrococci (Fig. 172). It is readily stained with the usual dyes, and decolorized by Gram’s method. Short chains of from four to six may at times be seen, as also tetrads and peculiarly swollen forms which are much larger than the usual forms. Cultivation is difficult and the organism quickly dies out. It grows best upon Loffler’s blood-serum mixture, forming round, whitish, shining, viscid-looking colonies, with smooth, sharply defined outlines, which may attain a feet of from 1 to 14 mm. in twenty-four hours. Their cultivation upon plain agar, Fig, 172.—Diplococcus meningitidis intracellularis. glycerin agar, and in bouillon is less reliable. I have obtained excel- lent results by placing a few c.c. of the cerebrospinal fluid in blood- serum tubes and found that the organisms multiplied far more actively in the fluid over the medium than in any other way. In order to obtain the best results, it is necessary to use large amounts of the exudate, and to make a number of cultures, as many of the organisms are usually dead, or at least will not grow. In ordinary cover- -slip preparations they are often numerous, and are found enclosed in the polynuclear leukocytes. Their number then varies considerably. On the one hand, only one or two may be present in a cell, while in others they may be so closely packed as to obscure the nucleus. During the past winter I examined a specimen in which the organism was present in groups composed of hundreds, but this 1 is rare. 618 THE CEREBROSPINAL FLUID Mixed infections are not uncommon in epidemic cerebrospinal meningitis. Councilman thus found the pneumococcus in 7 cases and Friedlinder’s bacillus in 1. ‘Terminal infections with staphy- lococci and streptococci also occur. In other forms of purulent meningitis a large variety of organisms has been found. Wolf gives the following figures, resulting from an analysis of 174 cases, in which epidemic cerebrospinal meningitis is, however, included: in 44.23 per cent. the pneumococcus was found; in 34.48 per cent. the Diplococcus meningitidis intracellularis; in 3.45 per cent. staphylococci; in 8.03 per cent. streptococci; in 1.13 per cent. the bacillus of Friedlander; in 2.87 per cent. the Bacillus typhosus; in 1.72 per cent. the bacillus of Neumann-Schaffer, and in 2.87 per cent. the Bacillus coli communis, the Bacillus pyogenes foetidus, the Bacillus aérogenes meningitidis, and the Bacillus mallei; while no bacteria were found in 1.15 per cent. of the cases. In 2 cases Pfeiffer’s influenza bacillus has also been encountered in the cerebrospinal fluid during life. In the African sleeping sickness trypanosomes are commonly found in the cerebrospinal fluid, obtained by lumbar puncture. Castellani obtained the organism in 20 cases of 34, and Bruce found it in all of 38 cases (see Blood). ‘The results of these earlier observers have been abundantly confirmed. In many cases, however, the para- sites never find their way into the cerebrospinal fluid. ‘They are more frequently found toward the termination of the disease. Large numbers are rare, but if they do occur there is usually an access of temperature. When present, the leukocytes are apt to be increased. There is no relation between the number present in the blood and in the spinal fluid. Toxicity While normal cerebrospinal fluid possesses distinct toxic properties, it has been found that in disease the toxicity may be markedly increased. Bellisari has thus shown that the fluid of individuals suffering from general paresis is more toxic than that of normal individuals, and that this toxicity is at its maximum after an epileptic seizure. Pellegrini further could demonstrate that the cerebrospinal fluid of epileptics is markedly toxic, and that that obtained immediately after a convulsion has a toxic and convulsive power much greater than that obtained at periods far removed from paroxysms. Similar results have been obtained by Dide and Laquepée. LireERATURE.—W. T. Councilman, ‘‘Cerebrospinal Meningitis,” Johns Hopkins Hospital Bull., 1898, p. 27; and Phila. Med. Jour., 1898, p. 937. W. T. Council- man, F. B. Mallory, and J. H. Wright, ‘‘Epidemice Cerebrospinal Meningitis,” Amer. Jour. Med Sci., 1898, p. 252. W. Osler, “The Cavendish Lecture on the AKtiology and Diagnosis of Cerebrospinal Fever,” Phila. Med. Jour., 1899, p. 26. E. Stadelmann, ‘‘Meningitis Cerebrospinalis,’”’ Zeit. f. klin. Med., vol. xxxviii, p. . 46. R. Neurath, Centralbl. f. d. Grenzgebiete d. Med. u. Chir., 1897, vol. i. J. Langer, Jahrb. f. Kinderheilk., 1901, vol. iii, p. 91. Pellegrini, Riform. med., 1901, No. 55. Dide and Laquepée, Soc. d. neurol. de Paris, April, 18, 1901. | | | . . CHAPTER X. THE EXAMINATION OF CYSTIC CONTENTS. CYSTS OF THE OVARIES AND THEIR APPENDAGES. THE material obtained from cysts of the ovaries or their appen- dages varies greatly in character. On the one hand, it may be fluid, clear, of low specific gravity, and contain little albumin; while, on the Bier. it may be dense, viscid, and of colloid appearance. The specific eravity varies between 1.018 and 1.024, owing to the presence of a large amount of albumin. In addition to smaller amounts of serum albumin and serum globulin the fluid of ovarian cysts contains a considerable quantity of another albuminous substance, which is termed metalbwmin (Scherer) or pseudomucin (Hammarsten). Like Hammarsten’s mucoid of transudates, it cannot be directly precipitated with acetic acid, but must be isolated as follows: The fluid in question is freed from coagulable albumins by boiling after acidifying with acetic acid; the filtrate is precipitated with alcohol, the precipitate dissolved in water, dialyzed, and then treated with acetic acid, when the pseudo- mucin separates out. ‘he substance contains about 30 per cent. of -glucosamin. Paramucin is another albuminous substance which is found in colloid cysts and belongs to the mucinoid bodies. Like the true mucins and the body which occurs in exudates the paramucin is also precipitated by dilute acetic acid. According to Mitjukoff, it contains at least 12.5 per cent. of a reducing substance.’ Test for Pseudomucin.—The fluid is mixed with three times its volume of alcohol and set aside for twenty-four hours, when it is filtered and the precipitate suspended in water. ‘This is again filtered and the filtrate tested in the following manner: (1) A few cubic centimeters are boiled, when in the presence of metalbumin the liquid will become cloudy, without the formation of a precipitate. (2) With acetic acid no precipitate is obtained. (3) Upon the appli- cation of the acetic acid and potassium ferrocyanide test the liquid * Literature dealing with pseudomucin and paramucin: Pseudomucin: Ham- marsten, Zeit. f. phys. Chem., 1882, vol. vi, p. 194. Pfannenstiel, Arch. f. Gynik., 1890. Zingerle, Miinch. med. W och., 1900. Paramucin: Mitjukoff, Arch. f. Gynik., 1895. Panzer, Zeit. f. phys. Chem., 1899, vol. xxviii. Leathes, Arch. f. exper. Path. u. Pharmak., 1899, vol. xlii. 620 THE EXAMINATION OF CYSTIC CONTENTS becomes thick and assumes a yellowish color. (4) When boiled with | Millon’s reagent a few cubic centimeters of the filtrate will yield a bluish-red color, while the addition of concentrated sulphuric acid, without boiling, gives rise to a violet color. | The color of cystic fluids may vary from a light straw to a reddish brown, or even a chocolate; the latter color may be observed when | hemorrhage has taken place into the cyst. | Of morphological elements, ovarian cysts contain red blood cor-' puscles, leukocytes, and at times fatty granules in large numbers, crystals of cholesterin, hematoidin, and fatty acids. Most im- portant, however, from a diagnostic standpoint is the presence of cylindrical or prismatic, ciliated epithelial cells, derived from the Fia. 173.—Contents of an ovarian cyst: a, squamous epithelial cells; b, ciliated epithelial cells: c, columnar epithelial cells; d, various forms of epithelial cells; e, fatty squamous eeubelel cells; f, colloid bodies; g, cholesterin crystals, (EHye-piece III, obj. 8 A, Reichert.) (v. Jaksch.) internal lining of the cyst, in the presence of which the diagnosis may be definitely made (Fig. 173). At times such cells cannot be demonstrated, as they may have undergone fatty degeneration; moreover, if the epithelium lining the cyst is squamous in character, it may be difficult, if not impossible, to arrive at a satisfactory con- clusion from an examination of the morphological elements alone. Colloid concretions, which may vary in size from several micromil- limeters to 0.1 mm., are occasionally observed, and more particu- larly in colloid cysts. They may be recognized by their irregular form, homogeneous appearance, slightly yellow color, and delicate outlines. In dermoid cysts, epidermal cells and occasionally hairs are ob- served, ' ) HYDATID CYSTS 621 The differential diagnosis of ovarian, parovarian, and fibrocystic (uterine) cysts cannot always be made from the character of the fluid withdrawn by puncture, but at times it is possible. ‘The most im- portant points of difference are here given: (1) The fluid in ovarian cystomas is usually more or less viscid, and often contains non- nucleated granular corpuscles of about the size of leukocytes, the granules of which do not dissolve in acetic acid nor disappear when ‘treated with ether. In all probability they are free nuclei; in the : } | United States they are often called Drysdale’s corpuscles. (2) In parovarian cysts the fluid is thin, watery, of low specific oravity ‘(under 1.010), and contains very few morphological elements. | Cylindrical epithelium is very rarely found during life in the fluid withdrawn by aspiration from either ovarian or parovarian cysts. (3) The fluid from fibrocystic tumors of the uterus is thin, watery, and coagulates spontaneously, while that from ovarian and paro- varian cysts never coagulates spontaneously unless blood is present. Fibrocystic tumors of the uterus have no epithelial lining. Of special interest are those cases of ovarian cysts in which in the course of typhoid fever infection of the cystic contents occurs with the corresponding organism.’ HYDATID CYSTS. The normal fluid in hydatid cysts is clear like water, neutral (some- times faintly acid or alkaline), of a specific gravity of 1.000 to 1.015, and rich in sodium chloride. By transmitted light it is faintly opales- cent. It contains no albumin or only a trace of it. Suecinic acid or sugar may be present in small amount. Sodium chloride may be recognized by evaporating a drop of the liquid on a slide, when the characteristic crystals of the salt will be found. Succinic acid may be demonstrated by acidifying a small amount of the fluid with hydro- chloric acid, and evaporating to dryness. ‘The residue is extracted with ether and the ether evaporated; the aqueous solution of the second residue, in the presence of succinic acid, will yield a rust- colored, gelatinous precipitate when treated with a few drops of a solution of ferric chloride. A sediment, if present, is composed chiefly of scolices, debris of parenchyma, calcareous particles, and hooklets. Hematoidin crystals may be found if blood has entered the cyst. Where tapping or exploratory puncture has been employed, albumin may afterward be found in greater quantity, as also in degenerating and suppurating cases. With the death of the hydatid, changes of a degenerative nature take place, the fluid altering greatly in character. It becomes more turbid, fatty globules may be found with granular 1M. J. Lewis and R. G. Le Conte, Amer. Jour. Med. Sci., 1902, vol. exxiv, p. 590. a 622 THE EXAMINATION OF CYSTIC CONTENTS cells, and typical crystals of cholesterin. ‘The contents may become of putty-like consistence and greasy, containing the remains of the gelatinous membranes which may be floated out in water. Should calcification ultimately occur, hooklets may be found on rubbing up the material with water in a mortor. | When suppuration takes place, polymorphonuclear leukocytes are first found between the cyst and its adventitious capsule; the cysts ultimately may become softened and burst, membranes, scolices, and hooklets floating about in the pus. (See also Sputum.) HYDRONEPHROSIS. The diagnosis of hydronephrosis can usually be made without diffi- culty if a sufficient amount of fluid can be obtained; the presence of urea and uric acid in notable quantities, as well as of renal epithelial cells, which latter especially should be sought for, is quite character- istic. Small amounts of uric acid, however, may also be present in ovarian cysts. 7 PANCREATIC CYSTS. These cysts may be recognized by the fact that the fluid possesses the power of digesting albumin in alkaline solution. A small amount of the liquid is added to a few c.c. of milk, when after precipitation of the casein the biuret test is applied; a positive reaction indicates the presence of trypsin. Unfortunately, however, the test does not always yield positive results, even if the fluid in question is derived from a pancreatic cyst, as the trypsin is apparently destroyed in the course of time. ‘The larger the cyst, the less likely will it be pos- sible to obtain the reaction. A positive result is hence only of value, while a negative result does not exclude the existence of the disease. * Karewski, Deutsch. med. Woch., 1890, vol. xvi, pp. 1035 and 1069. Hof- meister, Prag. med. Woch., 1891, vol. xvi, pp. 365 and 377 (see Gussenbauer). v. Jaksch, Zeit. f. Heilk., 1888, vol. ix, p. 126 (see Wolfler). CH sAth Be XI. THE SEMEN. ‘THE ejaculated semen is a mixture of the secretions furnished by the testicles, the prostate gland, the seminal vesicles, and the glands of Cowper. GENERAL CHARACTERISTICS. Semen is white or slightly yellowish in color, semifluid, sticky, and of an opaque, non- -homogeneous, milky appearance, which is due to the presence of white, opaque islets floating in the otherwise clear fluid; these consist almost entirely of the specific morphological elements of the semen, the spermatozoa. Its odor, which strongly resembles that of ftesh glue, is characteristic, and is owing to the presence of spermin. It is generally attributed to an admixture of prostatic fluid, as the semen obtained from the vasa deferentia is odorless. According to Robin, however, this odor is produced only at the moment of ejaculation, and cannot be ascribed to any single one of the secretions present. ‘The reaction of human semen is slightly alkaline, and its specific gravity greater than that of water, in which it sinks to the bottom. CHEMISTRY OF THE SEMEN. Accurate analyses of human semen or of mammalian semen do not exist, and only the old analyses of Vauquelin and Kolliker can be given: Man, Horse. Ox. Water é Sas, Ve See eee Le 81.90 82.10 Albuminous material ( vate Loca Extractives 6 16.45 Ethereal extract sag 2.20 Mineral material vA or Go A 6) 2.60 The mineral matter consists largely of calcium phosphate. If semen is kept, or if it is slowly evaporated, crystals of phos- hate of spermin separate out, which are commonly known as Batt- cher’s crystals, and which were long regarded as identical with the so-called Charcot- Leyden crystals that are found in the sputum of bronchial asthma, in ‘the blood of leukemia, in the stools in cases of helminthiasis, ete. : ada 624 THE SEMEN Spermin is a basic substance, and, according to Ladenburg and Abel, is closely related to, if not identical with, diethylene diamin (piperazin) : GH Ol The phosphate crystallizes in the form of monoclinic four-sided spindles or prisms, which appear as flattened needles of variable size. Some are scarcely visible even with a fairly high power of the microscope, while others attain the length of 40 4 to 60 4. The sub- stance is soluble in formalin, thus differing from the Charcot-Leyden crystals. In water it dissolves with difficulty; it is slowly soluble in acids and alkalies, even in ammonia, while it is insoluble in alcohol, ether, chloroform, and dilute saline solution. Florence’s reagent (see below) colors the crystals a bluish black. According to Cohn, the Bottcher crystals are formed exclusively in the prostate gland, the gland itself furnishing the basic component, while the necessary phosphoric acid is derived from other portions of the reproductive apparatus." MICROSCOPIC EXAMINATION OF THE SEMEN. Upon microscopic examination normal semen is seen to contain innumerable, actively moving, thread-like bodies, measuring from 50 #4 to 60 in length—the spermatozoa. ‘These consist of an egg-_ shaped head, when seen from above, which is from 3 / to 5 yp in length, the broader end being dircted anteriorly; a middle portion, 4” to 6 / in length, with which the head is united by its smaller end; and a posterior piece or tail, into which the middle piece grad-_ ually fades (Fig. 170). In addition to the spermatozoa a few hyaline bodies are seen which are derived from the seminal vesicles; further, numerous small, pale granules of an albuminous nature (lecithalbumin), some testicular and urethral epithelial cells, lecithin corpuscles, and so-called pros- tatic or amyloid corpuscles, which at first sight resemble starch granules in appearance, owing to their concentric striations. A few leukocytes and occasionally a few red corpuscles may also be found. PATHOLOGY OF THE SEMEN. The study of the semen has received little attention from clin- iclans, and gynecologists frequently hold the wife responsible for ‘Th. Cohn, ‘‘Zur Kenntniss d. Spermas,” Centralbl. f. allg. Path. u. path. Anat., — vol. x, pp. 940 and 949. Pao THE RECOGNITION OF SEMEN IN STAINS 625 sterility when an examination of the husband’s semen would— _ according to Kehrer,* in 40 per cent.—reveal an absence of sperma- tozoa, constituting the condition usually spoken of as azodéspermatism. | ‘This may be temporarily observed following venereal excesses, when the fluid finally ejaculated is almost entirely of prostatic origin; their absence then possesses no significance, but persistent azodsper- matism must of necessity be associated with sterility.’ Cases have been recorded in which, notwithstanding the presence of spermatozoa and apparently normal sexual conditions in both husband and wife, sterility existed nevertheless, but in which it was observed that the spermatozoa lost their motile power almost imme- diately after ejaculation. Under normal conditions, following inter- course actively moving spermatozoa may be found in the vagina after hours, days, and even weeks. ——————— ee _ “Whenever it is deemed advisable to make an examination of the semen, this should be done immediately following ejaculation, or as soon as possible thereafter. ‘The material should be placed in a test tube and this immersed in lukewarm water until it can be examined. Note should then be taken, not only of the presence, but also of the degree of motility of the spermatozoa, a drop of the semen being examined directly with the microscope. _ Bloody semen, constituting the condition spoken of as hemo- spermia, has been observed on several occasions. It may follow excessive sexual indulgence, but may also occur in connection with gonorrheal epididymitis. ‘The blood is readily recognized upon micro- scopic examination.” THE RECOGNITION OF SEMEN IN STAINS. In medicolegal cases the physician may be called upon to decide whether or not certain stains on body-linen are caused by spermatic fluid, whether or not a rape has been committed, etc. In such cases it is frequently only necessary to examine a drop of the vaginal fluid in order to arrive at a positive result at once. At other times recourse must be had to the following method: A fragment of the linen or scrapings from the vulva or vagina are placed in a watch- crystal and allowed to soak for at least one hour in from 27 to 30 per cent. alcohol, when a bit of the material is teased in a solution of eosin in glycerin (1 to 200), and examined. ‘The heads of the spermatozoa are thus stained a deep red, while the tails, which are often broken, exhibit a pale-rose tint, and can readily be distinguished from vegetable fibers, which do not take the stain at all. A positive 1 Beitriige z. klin. u. exper. Gyniik., 1879, vol. ii, Giessen, 2 Firbringer, Zeit. f. klin. Med., 1881, vol. ui, p. 310. a $ Feleki, Centralbl. f. Krankh. d. Harn- u. Sexualorgane, 1901, vol. xii, p. 506. 40 al 626 THE SEMEN statement can thus be made in every case, even after months and years, as spermatozoa not only resist the action of reagents, but also the process of putrefaction; this is probably owing to the large pro- portion of mineral matter which enters into their composition, and which ensures the preservation of their form. Instances have been recorded in which it was possible to demonstrate spermatozoa in stains after eighteen years. The semen test of Florencet has attracted much attention, and may be recommended in doubtful cases; only a negative result, however, is of value (see below). It is based upon the observation that very characteristic crystals of zodospermin are formed when spermatic fluid is treated with a solution of iodopotassic iodide containing 1.65 grams of pure iodine and 2.54 grams of potassium iodide, dissolved in 26 c.c. of water. When a drop of this solution is added to a drop of spermatic fluid or an aqueous extract of a seminal - stain, dark-brown crystals of iodospermin separate out at once, and may be readily recognized under the microscope. ‘They occur in the form of long rhombic platelets or fine needles, often grouped in rosettes, but also occurring singly or as twin crystals. The exami- nation with the microscope should be made at once after the addition of the reagent, as the crystals disappear on standing, As the reaction may also be obtained in cases of azodsperma- tism, and with pure prostatic secretion, while a negative result is obtained with the fluid from spermatoceles, it is manifest that the test 1s not applicable for the determination of the presence or ab- sence of spermatozoa per se. Posner’ states that he obtained similar crystals when the test was applied to a glycerin extract of ovaries. More recently Richter*? has shown that Florence’s reaction is also obtained with a decomposition product of lecithin, viz., cholin, which would explain the observation that better results are commonly obtained with dried semen than with fresh material. But it follows — also that the reaction cannot be a specific semen reaction, and Richter accordingly concludes that a negative result only is of value, and indicates that the material under examination is not semen. He states that he obtained positive results with vaginal and uterine mucus, with decomposing brain substance, and other organs as well. In confirmation of Richter’s results, Bocariust has demonstrated that the so-called iodospermin is in reality an iodized product of cholin— and not of spermin. * Du sperme et des taches de sperme en médecine legale, Arch. d’Anthrop. crimin., vols. x and xi. > “Die Florence’ sche Reaktion,” Berlin. klin. Woch., 1897, p. 602. * “1D. mikrochemische Nachweis v. Sperma,’’ Wien. klin. Woch., 1897, p. 569. * Zeit. f. phys. Chem., 1902, vol. xxxiv, p. 339. GibicAr HRs val T. VAGINAL DISCHARGES. GENERAL CHARACTERISTICS. T's secretion which is normally furnished by the vaginal glands is small in amount, and just sufficient to keep the mucous mem- brahe moist. It is a clear or somewhat milky-looking, semiliquid material, in which numerous epithelial lamine may be found. It has been stated that the reaction of the vaginal secretion in virgins is invariably acid, while an alkaline reaction is the rule in the déflorées. During pregnancy, however, the secretion is probably always acid. In 500 cases which Krénig examined in this direction an alkaline reaction was never observed. According to Zweifel,’ the vaginal secretion contains traces of trimethylamin, to which its peculiar odor is probably due. Microscopically, numerous epithelial cells, mucous corpuscles, a few large mononuclear leukocytes, cellular detritus, and bacteria are found. Déderlein’ has described a non-pathogenic bacillus or a group of bacilli which are characterized by the fact that they give rise to marked acid fermentation of sugar, and he regards these organisms as the only ones which are constantly present in the nor- mal vagina. Kroénig and Menge, however, state that they are often absent. ‘These observers have found, on the other hand, that under normal conditions there are various bacilli and cocci present which belong to the class of obligatory anaérobes, and are likewise non- pathogenic. Unfortunately they have not described these organisms in detail. Near the outlet they found bacteria which may be culti- vated upon alkaline aéorbic culture media, but which are usually absent in the upper portion of the vagina. It is important to note that various diplococci may also be found under normal conditions, and care should be taken not to confound these with gonococci. Like the gonococci, they are decolorized by Gram’s method. If the characteristics of the former be borne in mind, however, mistakes may probably always be avoided; in mar- ried women and in children it is best to make the diagnosis of gon- orrhea only when the gonococcus has been isolated by cultivation. The question whether or not pathogenic bacteria may occur in the normal vagina of pregnant or non-pregnant women, may be 1 Arch. f. Gynik., 1881, vol. xviii, p. 359. 2 Tbid., 1887, vol. xxxi, p. 412. | / 628 VAGINAL DISCHARGES answered in the affirmative; but with the exception of the gono- coccus they are not often seen.t Bergholm? thus examined the vaginal secretion of 40 pregnant women, and was unable to obtain organisms pathogenic for animals in a single case. ‘There were no pyogenic staphylococci, no streptococci, and no colon bacilli. The vaginal secretion has been shown to possess powerful bac- tericidal properties, so that pathogenic organisms, even when arti- ficially introduced into the vagina, are rapidly killed. Krénig thus found that the Bacillus pyocyaneus disappears from the vagina of pregnant women in from ten to thirty hours, the s aphylococci in from six to thirty-six hours, and the Streptococcus pyogenes within six hours. Important from a practical standpoint is the fact that the bacteria disappeared less rapidly when irrigation of the vagina with water or even antiseptics was employed. Of animal parasites, the Trichomonas vaginalis is occasionally encountered in the vaginal discharge. ‘The organism is identical with the trichomonas found in the feces and in the urine. In the United States it is not so common as among the peasant population of Central Europe. As far as is known, the organism is of no patho- logical significance. From a medicolegal standpoint, however, its presence may not be unimportant, as cases are on record in which trichomonades have been confounded with spermatozoa. In my judgment, however, such a mistake can only occur if the observer is totally without training in microscopy. The possible presence of the Anguillula aceti in the vaginal dis- charge has been pointed out by Billings, Miller, and Stiles. Stiles has suggested that it may be introduced into the vagina by injections of vinegar-water taken with the object of preventing conception, 4 VAGINAL BLENNORRHEA. r In physiological conditions an increased vaginal secretion is ob- served during sexual excitement, just preceding and at the beginning of menstruation, and during pregnancy, when a profuse blennorrhea is frequently seen, which sometimes assumes a virulent character. The secretion under such conditions readily becomes purulent. When not dependent upon a gonorrheal infection the secretion is _ thicker than normal, white, and creamy. At times also the vaginal — catarrh observed in pregnancy is complicated with mycosis, when — white or yellowish-gray patches may be seen at the orifice of the— vagina; the latter may, indeed, be filled with particles which consist entirely of fungi. : 1 Déderlein, Das Scheidensecret, Leipzig, 1892. J. W. Williams, Amer. Jour, ; Obstet., 1898, vol. xxxviii; Trans. Amer. Gyn. Soe., 1898; Amer. Jour. Obstet., » 1898. 2 Arch. f. Gynik., 1902, vol. Ixvi, Heft 3. Ba i VULVITIS AND VAGINITIS §29 MENSTRUATION. At the beginning of menstruation, as has been pointed out above, an increase in the amount of vaginal secretion is observed, in which leukocytes, prismatic epithelial cells coming from the uterus, as well as the usual vaginal cells, may be seen upon microscopic exami- nation. Later the secretion becomes sanguineous in character, and finally only epithelial cells, leukocytes, and granular detritus are encountered, the cells usually showing evidence of fatty degenera- tion. ‘The amount of blood lost at each menstrual period amounts to about 200 grams in perfectly healthy females. THE LOCHIA. The lochia during the first day following parturition are red in color—the lochia rubra—and emit the characteristic sanguineous odor. At this time a microscopic examination will reveal an abun- dance of red corpuscles, some leukocytes, and a variable number of epithelial cells, which are almost exclusively of vaginal origin. On the second and third days the number of red corpuscles dimin- ishes, while the leukocytes increase in number. Still later the dimi- nution in the red and the increase in the white corpuscles become more marked, and the discharge at the same time assumes a grayish or white color, until about the tenth day the red corpuscles have almost entirely disappeared, while the leukocytes and epithelial cells are abundant. Finally, the secretion becomes thicker, mucoid, and milky white in color—the lochia alba—which condition may persist for from three to four weeks in nursing women, and still longer in those who do not nurse, until finally the normal secretion is again established. Numerous bacteria are encountered in the lochia, and it is curious to note that among these pus organisms are quite con- stantly present without giving rise to symptoms. When a portion of the placenta or membranes have been retained the lochia soon give off a fetid odor, and assume a dirty brownish color; the reten- tion of blood clots alone may also produce this result. In such cases the lochia swarm with bacteria of all kinds.” VULVITIS AND VAGINITIS. In cases of vulvitis and vaginitis a marked increase is observed in the number of the leukocytes and epithelial cells, the character of the latter depending essentially, of course, upon the portion of the ian genital tract affected. Red corpuscles are also met with at times; their number generally stands in a direct relation to the intensity of 1 Déderlein, loc. cit. Thomen, Centralbl. f. d. med. Wiss., 1890, vol. xxviii, p. 537; and Arch. f. Gyn., 1889, vol. xxxvi, p. 231. 630 VAGINAL DISCHARGES the inflammatory process. In some instances epithelial casts of the entire vagina have been observed, constituting the condition termed vaginitis exfoliativa. ‘The condition, however, is rare. In mycotic vaginitis leptothrices have been found by v. Herff.’ The discharge of large amounts of pure pus through the vagina points to perforation of an abscess of the genital organs or of the neighboring structures into the uterus or the vagina; it is of rare occurrence. Much more common is the discharge of fecal matter or of urine through this channel, indicating the existence of a vagino- rectal or vaginovesical fistula. MEMBRANOUS DYSMENORRHEA. While ordinarily, during menstruation, shreds of desquamated uterine lining are frequently encountered, it is rare to meet with large pieces or complete casts of the uterus, the elimination of which is usually associated with the symptoms of a severe dysmenorrhea, constituting the condition spoken of as membranous dysmenorrhea. CANCER. While the diagnosis of malignant growth of the uterus is probably never based upon a microscopic examination of the vaginal discharge alone, it may be mentioned that in advanced cases this is possible, as fragments of an epithelioma of the cervix, for example, may frequently be detected upon microscopic examination. In sus- _ pected cases small pieces of tissue should be excised and examined according to usual histological methods.’ GONORRHEA. In suspected cases of gonorrhea an examination of the vaginal and urethral discharge for the presence of gonococci is important, as it is practically impossible to diagnosticate this condition positively in any other manner. Care should be taken, however, not to con- found the diplococci which may be normally present in the urethra _ and vagina with gonococci. (See chapter on the Urine.) Unfortunately, however, excepting in fairly acute cases, these examinations are- rather unsatisfactory. ‘here can be no doubt that in many cases, which unquestionably are gonorrheal, the ordinary microscopic examination is negative. Better results may possibly be reached if the examination is made twenty-four hours following the injection of — gonococcus vaccine (dose, 10,000,000 organisms). 1 Centralbl. f. Bakter., 1895, p. 750. 2T. 8. Cullen, Cancer ‘of the teers Appleton & Co., 1900. ABORTION 631 ABORTION. : In cases of abortion it is often possible to discover chorion villi in the expelled blood-clots which present the characteristic capillary network (Fig. 174), and often manifest signs of advanced fatty Fie. 175.—Decidual cells. degeneration. Important also from a diagnostic point of view is the presence of decidual cells (Fig. 175), which are characterized by their large size, their round, polygonal, or spindle-like form, and their characteristic nuclei and nucleoli. CHAPTER XIII. THE SECRETION OF THE MAMMARY GLANDS, THE SECRETION OF MILK IN THE NEWBORN. A SECRETION from the mammary glands of the male is observed only in the newborn, if we except those rare cases in which adult males were known to suckle infants. The fluid in question, which may also be obtained from the female infant, is termed ‘‘ Hexenmilch” (witches’ milk) by the Germans. Qualitatively it has the same com- position as milk, but may manifest considerable quantitative varia- tions. COLOSTRUM. Aside from those curious instances in which a secretion of milk has been observed in non-pregnant women, mammary activity is essentially connected with the physiological phenomena of pregnancy Fic. 176.—Colostrum of a woman in sixth month of pregnancy. (Eye-piece III, obj. 8 A, Reichert.) (v. Jaksch. ) and parturition. Often as early as the third month a small drop of | a serous-looking fluid can be obtained from the nipple by pressure upon the breasts. Immediately after delivery a variable amount of — fluid is secreted, which is watery, semi-opaque, mucilaginous, and of a yellowish color. ‘To this secretion, as well as to that observed during pregnancy, the term colostrum has been applied. It is dis-— tinguished from true milk by its physical characteristics and by the presence of a greater proportion of sugar and salts. The fluid, moreover, coagulates upon boiling. An idea may be formed of its composition from the appended table: HUMAN MILK 633 / 4 weeks before birth. 47 days be-| 9 days be- | 24 hours 2 days ee ik” | fore birth, | fore birth. | after birth. | after birth. Seater) ou 945.2 ¥ 852.0 1) 851.7 |. 858.8 | 843.0 867.9 meds... % 54.8 1a8 0 AS 3h 141.9 157.0 1820 1 Casein ESE el oe! De aoe ot ald “ae 21.8 Mmibumin’. .| 28.8 O02) ve 7428 a eo Fat yee io 41.3 30.2 23.5 | Nea et 48.6 Lactose . . 17.3 Soon we tae. ra We A Oe sor 61.0 Mates ‘y 5 te ‘ URBANA 2 ° z — = a ° 77 x wi 2 _ = ee we SS Ste oe ae en ae an Seah tava at ators ENED Daag