J llll.>l' :i;,ir.',i^i,i( 1 iiiPlfe lllH |llll!llHttHI|timilH;Hllli OF THE IRew IPorF? State IDctcrinar^ CoUefie Digitized by Microsoft® CORNELL UNIVERSITY LIBRARY 3 1924 104 224 864 Digitized by Microsoft® This book was digitized by Microsoft Corporation in cooperation witli Cornell University Libraries, 2007. You may use and print this copy in limited quantity for your personal purposes, but may not distribute or provide access to it (or modified or partial versions of it) for revenue-generating or other commercial purposes. Digitized by Microsoft® CLINICAL LABORATORY METHODS Digitized by Microsoft® Digitized by Microsoft® CLINICAL LABORATORY METHODS A MANUAL OF TECHNIQUE AND MORPHOLOGY DESIGNED FOR THE USE OF STUDENTS AND PRACTITIONERS OF MEDICINE BY ROGER SYLVESTER MORRIS, A.B., M.D. AB30CIATE PROFEaSOH OF MEDICINE IN WASHINGTON TJNIVEHSITT, ST. LOUIS. FORMERLY ASSOCIATE IN MEDICINE, THE JOHNS HOPKINS UNIVEHSITY; ASSISTANT RESIDENT PHYSICIAN, THE JOHNS HOPKINS hospital; INSTRUCTOR IN MEDICINE AND DEMONSTRATOR OF CLINICAL UEDICINE, THE UNIVERSITY OF MICBIQAN. D. APPLETON AND COMPANY NEW YORK AND LONDON 1913 Digitized by Microsoft® Copyright, 1913, bt D. APPLETON AND COMPANY Printed in the United States of America ' /./ Digitized by Microsoft® TO WILLIAM SYDNEY THAYER AND GEORGE DOCK THIS VOLUME IS DEDICATED •WITH THE AFFECTION AND GRATITUDE OF THE AUTHOR Digitized by Microsoft® Digitized by Microsoft® PEEFACE Coincident with the improvement in medical education in this country there has been a widespread increase in the use of labora- tory methods as aids to diagnosis. Not only is this true in the case of the more recent graduates in medicine, but, what is more hopeful, the older practitioners — those whose college days pre- ceded the introduction of Clinical Pathology into the medical cur- ricula — are quite generally realizing the necessity of the laboratory in their daily work. Probably no one thing has done more to bring about this much desired result than the discovery by Wasser- mann and his co-workers of the well-l?nown serum reaction for the diagnosis of syphilis; unconsciously, perhaps, but none the less effectively, attention has been focused upon laboratory diag- nostic methods. The present volume is not a text-book of Clinical Pathology ; it is a manual of laboratory technique and morphology, dealing merely with methods and with morphological elements which are of diag- nostic importance. It attempts to give in detail the means of de- tecting the abnormal in urine, gastric contents, feces, blood, spu- tum, and puncture fluids. Unlike the text-books, the significance of the abnormal is not discussed. That there is need for such a work the author has long believed. There is much which it is absolutely essential that the student of mediciue-^graduate and undergradute — remember. He must know, for example, under what conditions albuminuria may occur, whether it be of nephritic, cardiac, toxic, physiologic, or whatever origin. He must be aware of the possible significance of a second- ary anemia, of an atypical reduction test in the urine, of Charcot- Leyden crystals in sputa, of a hydrochloric acid deficit in the gastric contents. But it is useless to try to burden the memory with the details of the various laboratory methods, by which such abnormalities are detected, and with the sources of error in the methods. Digitized by Microsoft® PREFACE No attempt lias been made to include within the present volume a multiplicity of methods; in fact, the aim of author has been to select one method or more of proved value. Nor have the more exact, time-consuming methods of physiological chemistry been drawn upon; in his daily work the average clinician has not the time, if he has the ability, to employ them. Free use has been made of the following works: Emerson's "Clinical Diagnosis," Wood's "Chemical and Microscopical Diag- nosis," Simon's "Clinical Diagnosis," Sahli's "Klinische Unter- suchungsmethoden, " Hoppe-Seyler's "Handbuch der chemischen Analyse, ' ' Hammarsten 's ' ' Lehrbuch der physiologischen Chemie, ' ' Neubauer-Huppert's "Analyse des Hams," Schmidt and Stras- burger's "Die Paezes des Mensehen," Braun's "Thierische Parasi- ten des Mensehen," Blanchard's "Traite de Zoologie Medicale," Cabot's "Clmical Pathology of the Blood," Naegeli's "Blutkrank- heiten und Blutdiagnostik, " and Tiirk's "Vorlesungen ueber klin- ische Haematol ogie." Other authors have been consulted less freely. To the more recent literature direct reference has been given in the form of footnotes ; in all instances the writer has en- deavored to give proper credit to authors. It is hoped that the volume will prove helpful to medical students who have completed a course in Clinical Pathology ^and to practitioners of medicine, or that it may serve as a supplement to a course of laboratory lectures. For the original illustrations in black and white the author is indebted to Dr. James S. Brotherhood. It is a pleasure, also, to acknowledge his indebtedness to his wife and to his mother for assistance in the preparation of the index and in other ways. Roger Sylvester Morris. St. Louis; Digitized by Microsoft® CONTENTS CHAPTER I THE UEINE PAGE Collection of the Urine 1 Preservation op the Urine 2 Macroscopic Examination op the Urine .... 3 Color op Urine ... .... 4 Quantity op Urine 4 Reaction op Urine 4 Quantitative determination of urinary acidity: Folin's method 5 Specipic Gravity 6 Urea 7 Hiifner's hypobromite method 8 Uric Acid 9 Qualitative determination of uric acid .... 9 Quantitative determination of uric acid: Method of Folin and Shaffer 10 Ammonia 12 Quantitative determination of ammonia: Folin's method — the vacuum distillation method — the formalin titra- tion method — Shaifer's modification of Schlosing's method 13-19 Nitrogen .20 Kjeldahl's method for determination of total nitrogen . 20 Chlorids 23 Qualitative test 23 Quantitative determination of chlorids: Harvey's modi- fication of Volhard's method 24 ix Digitized by Microsoft® X CONTENTS PAGE Sulphates 26 Obermayer's test 27 Jaffe's test 27 Albumin .28 Qualitative tests: Heat and acetic acid test — Heat and nitric acid test — Heller's test — Potassium ferrocyanid and acetic acid test 28-34 Quantitative determination of albumin : Tsuchiya 's mod- ification of the Esbach method — Removal of albumin from the urine 35-36 Bence- Jones' Body 36 Albumose 37 Glucose 38 Qualitative tests: Trommer's test — Fehling's test — Al- men-Nylander 's test — The fermentation test — Cippo- lina 's modification of the phenylhydrazin test . . 38-42 Quantitative estimation of glucose: Benedict's first method — Benedict's second method — Polariscopic deter- mination — Robert's specific gravity method — Measure- ment of carbon dioxid gas formed during fermentation 44-51 Levulosb . 53 Seliwanoff's test, as modified by Borehardt . 53 The phenylhydrazin test . 55 Maltose ......... . 56 Lactose . 56 Sacchaeose . 57 Pentose . 57 The phloroglucin test . 57 The orcin test . 58 Bial's modification of the orcin test . 58 Glycuronic Acid . 59 B. Tollens' test . .... . 60 Alkaptonuria . 61 Acetone . . . . . . 62 Qualitative tests: Gunning's test — Lieben's test- -Legal 's test — Lange's test ...... . 62-64 DiACETic Acid . 65 Gerhardt's test . 65 Digitized by Microsoft® CONTENTS XI DiACETic Acid {Continued) Arnold's test ^^ OxYBUTYRic Acid Black's test Hart's test Urobilinogen Ehrlich's aldehyd test Urobilin Spectroscopic determination Schlesinger 's test Jaffe's test Bile Pigments Qualitative tests: Foam test — Gmelin's test — Rosen bach's modification of Gmelin's test — Huppert's test— Hammarsten's test Hematoporphyrin Garrod's test .... Hemoglobin . . . . Spectroscopic determination . The guiac test . Heller's test .... Teichmann's hemin-crystal test The Diazo Reaction Chyluria ... LiPURIA ... Ferments in the Urine Wohlgemuth 's method for the determination of diastase Determination of lipase according to Hewlett The Urinary Sediments The unorganized sediments: The quadriurates of sodium and potassium — Uric acid — Calcium oxalate — Calcium sulphate — Monocalcium phosphate — Plippuric acid — Cholesterin — Xanthin — Hematoidin — Tyrosin — Leucin — Cystin — Tricalcium and trimagnesium phos- phates — Calcium carbonate — Ammoniomagnesium phos- phate — Ammonium biurate — Neutral magnesium phos- phate — Neutral calcium phosphate . . . . 66 67 67 68 70 70 70 71 71 72 73 73-75 76 76 77 78 81 82 82 83 84 86 86 87 88 89 91-99 Digitized by Microsoft® xu CONTENTS The Ubinaet Sediments (Continued) The organized sediments : Epithelial cells — Pus — Blood — Casts — Mucous threads — ' ' Clap threads ' ' — Gonococcus — Treponema pallidum — Bacillus tuberculosis . 100-115 Animal Parasites in Urinary Passages Trichomonas vaginalis Filaria bancrofti Dioctophyme renale . Schistosoma hematobium Prostatic Fluid Functional Diagnosis op the Kidney The phthalein test of Rowntree and Geraghty 116 116 117 117 118 119 119 120 CHAPTER II THE GASTRIC JUICE Test Breakfasts 125 Examination of the Fasting Stomach .... 127 Macroscopic Examination of the Gastric Contents . . 128 Quantity 128 Odor 128 Mucus 128 Color 129 Food 129 Chemical Examination of the Gastric Contents . . 131 Reaction 131 Hydrochloric acid: Qualitative tests for free hydrochlo- ric acid (von den Velden's methyl violet test; Giinz- berg's test; Tropeolin test; Congo-paper test; Topfer's test; Sahli's desmoid test) — Quantitative determination of gastric acidity (Topfer's method for free hydrochloric acid; Other indicators; Titration of total acidity) — The hydrochloric acid deficit . . 131-138 Lactic acid: Qualitative tests for lactic acid (Ueffel- mann's test; Strauss' test; Kelling's test) . . 141-142 Butyric acid 142 Digitized by Microsoft® CONTENTS Xlll Chemical Examination of the Gastric Contents (Continued) PAGE Acetic acid 143 Pepsin: Qualitative test for pepsin — Quantitative meth- ods (Mette's method as modified by Nierenstein and Schiff) 143-144 145 146 146 148 149 Eennin: Qualitative test for rennin Eennin zymogen Pathological enzyme in the gastric contents . Mucus MiCEOscopic Examination op the Gastric Contents CHAPTER III THE FECES Macroscopic Examination op the Feces .... 152 Amount 153 Form 153 Color 153 Mucus 154 Gallstones 155 Parasites 155 Intestinal Test Diet ... .... 155 Diet No. I 156 Diet No. II 156 Weight of Dried Feces . 157 Chemical Examination op the Feces 157 Reaction 157 Pigments • . 157 Urobilin (Hydrobilirubin) : Schmidt's test — Schlesin- ger's test — Spectroscopic determination .... 158 Bilirubin: Schmidt's test — Gmelin's test . . 158-159 Blood: Weber's test — The guiac test — Teichmann's hemin crystal test . ... . 159-161 Trypsin: Method of Gross for the determination of trypsin 162 Digitized by Microsoft® xiv CONTENTS PAGE Chemical Examination op the Feces (Continued) Amylase: Wohlgemuth 's method for the determination of amylase, as modified by liawk . . . 163 Microscopic Examination of the Feces . . . 166 Pood remnants . ...... 167 Bacteria ■ . . . 168 Cells. . . 169 Crystals 170 Intestinal parasites: Protozoa, Bhizopoda — Flagellata — Infusoria — Nematodes — Trematodes — Cestodes . 172-194 Preservation of gross specimens of cestodes and other parasites : Permanent preparations of flat worms (Method of Mink and Ebling — Boggs' method^Creosote method) . . 199-203 Accidental contaminations ...... 203 CHAPTEE IV THE SPUTUM Amount 205 Reaction 205 Character 205 Odor 205 Consistence 205 Air Bubbles 205 Dittrich's Plugs 205 Bronchial Casts 206 Curschmann's Spirals 206 Layer Formation 206 Microscopic Examination 206 Examination of fresh sputum 206 Yellow elastic tissue 208 Curschmann's spirals ....... 208 Alveolar epithelial cells 209 Dust cells . 210 " Heart- failure cells" . 210 Digitized by Microsoft® CONTENTS XV PAGE Microscopic Examination {Continued) Red blood corpuscles 210 Pus cells 211 Eosinophilic leukocytes 211 Lymphocytes 211 Chareot-Leyden crystals 211 Microorganisms in sputa: Bacillus tuberculosis (Ziehl- Neelsen method; Antiformin method for the detection of tubercle baciUi) — Diploeoccus pneumoniae (Gram's method of staining; "Welch's capsule stain) — Bacillus in- fluenzae — Bacillus diphtherias (Neisser's staining method; Beall's method) — Actinomyces bovis — Streptothrix ep- pengeri — Blastomyeetes . . ... 212-223 Animal parasites in the sputum: Entamoeba histolytica — Entamoeba tetragena — Trichomonads — Paragonimus westermanii — Echinococcua cyst .... 224-225 CHAPTER V THE BLOOD Obtaining Blood for Examination 226 Blood Stickers 226 Counting the Blood Corpuscles 227 The hemoeytometer 227 Procedure in counting the erythrocytes : Diluting fluids — - Filling the pipette — Filling the counting chamber — The enumeration of the cells — Calculation of the result — Cleaning the apparatus 229-234 Counting the leukocytes : Diluting fluid — Filling the pi- pette — Filling the counting chamber — The enumeration of the leukocytes — Calculation of the result — Biirker's modification of the Thoma counting chamber . 236-239 Counting the eosinophilic leukocytes: Dunger's method 241 Counting the blood platelets: Method of Wright and Kirmicutt 242 Digitized by Microsoft® XVI CONTENTS PAGE Hemoglobin Determinations 244 Tallqvist's method 244 Sahli's hemometer 245 The Fleisehl-Mieseher hemoglobinometer . . . 249 Haldane's hemoglobinometer 252 Dare's hemoglobinometer 252 Sulphhemoglobinemia 252 Methemoglobinemia 252 Color Index 253 Volume Index 253 Measuring the Diameter op Cells 255 Viscosity op the Blood and Other Fluids . . . 256 Method of Hess 256 The Specific Gravity op the Blood 259 The Coagulation Time of the Blood 260 Method of Brodie and Eussell, as modified by Boggs . 260 Milian's method, as modified by Hinman and Sladen . 263 The Resistance op the Red Blood Corpuscles . . . 264 The Examination op Fresh and Stained Preparations of Blood 265 The cleaning of cover glasses and slides .... 265 Examination of the fresh blood : Sealing the fresh speci- men — The preparation of dry (permanent) blood smears — Fixation of blood smears 265-271 Staining the blood: Vital staining of the blood — Vaughan's method; Method of Widal, Abrami, and Brule ; The ' ' dry ' ' method of vital staining ; The stain- ing of dried blood films — Methylene blue ; Eosin ; Hema- toxylin; Carbol-thionin. Staining mixtures of two or more stains — Ehrlich's triacid stain; The Romanowsky stains (Wilson's stain, Leishman's stain, Giemsa's stain); Jenner's stain; Methyl green-pyronin mixture of Pappenheim; The iodin reaction of the leukocytes 273-295 Differential counting of the leukocytes: Normal leuko- cytes — Pathological leukocytes .... 296-299 Digitized by Microsoft® CONTENTS xvii PAGE The Examination of Fresh and Stained Preparations of Blood (Continued) The normal and pathological red blood corpuscles: Erythrocytes — Erythroblasts — Abnormalities in the staining of the red corpuscles .... 301-302 Demonstration of protozoa in the blood: Malarial para- sites 305 Examination of the blood for animal parasites: Filaria bancrofti — Trichinella spiralis . . . 308-310 CHAPTER VI PUNCTURE FLUIDS Specific Gravity 312 Albumin Content 312 Incoagulable Nitrogen . .' 313 A Protein Peecipitable in the Cold by Dilute Acetic Acid 315 Cytology 315 Cerebrospinal Fluid 318 Cells of the cerebrospinal fluid 318 Method of counting the cells 318 Bacteriology of the cerebrospinal fluid . . . 319 Globulin content: Method of Noguchi — Method of Ross and Jones ■ 319-321 The Wassermann reaction in cerebrospinal fluid . . 321 Index 323 Digitized by Microsoft® Digitized by Microsoft® LIST OP ILLUSTRATIONS 1. — Apparatus for the quantitative determination of am- monia according to Folin .... ■Apparatus for the determination of ammonia according to Shaffer •Digesting rack for the Kjeldahl nitrogen determination ■Distilling apparatus for the Kjeldahl nitrogen deter- mination 5. — Lohnstein's areometer . . .... 6. — Lohnstein's fermentation saccharometer for undiluted 2.- 3.- 4.- FIG. PAGE 14 16 21 22 51 urine 52 7. — Absorption spectra 79 8. — The Sydenham sedimenting glass 89 9. — Treponema pallidum; Spirochseta refringens . . 112 10. — Embryo of Filaria bancrofti 117 11. — Ova of Dioctophyme renale 118 12. — The Autenrieth-Konigsberger colorimeter as modified by Rowntree and Geraghty for the determination of phenolsulphonephthalein 123 13. — Parasitic amebae 173 14. — Trichomonas intestinalis 177 15. — Lamblia intestinalis 178 16.— Balantidium coli 179 17. — Ovum of Necator americanus 181 18. — Necator americanus 185 19. — The rhabditiform embryo of Strongyloides stercorals . 186 20. — Ovum of Strongyloides stercoralis 187 21. — The rhabditiform embryo of Strongyloides stercoralis and the embryo of the hookworm .... 188 22. — Ovum of Oxyuris vermicularis 188 23. — Ovum of Trichuris trichiura 189 xix Digitized by Microsoft® XX LIST OF ILLUSTRATIONS FIG. PAGE 24. — Ovum of Ascaris lumbricoides ; the same under high focus, showing the albuminous coating . . . 190 25. — Unfertilized ovum of Ascaris lumbricoides . . 191 26. — Ovum of Schistosoma hajmatobium .... 193 27. — Ovum of Schistosoma japonicum 193 28. — Gravid proglottis of Tenia saginata; ovum of Tenia saginata; gravid proglottis of Tenia solium . . 195 29. — Ovum of Dibothriocephalus latus ..... 196 30. — Gravid proglottis of Dibothriocephalus latus . . . 197 31. — Ovum of Hymenolepis nana ...... 197 32. — Ovum of Hymenolepis diminuta ..... 198 33. — Dipylidium caninum, showing an egg capsule and a free ovum 199 34. — Tyroglyphus siro, the cheese-mite and ovum . . . 202 35. — Actinomyces hominis, showing club-shaped extremities to the rays 222 36. — Blastomycetes in sputum 223 37. — Ovum of Paragonimus westermanii from the sputum . 224 38. — Sediment from echinococcus cyst 225 39. — The Thoma-Zeiss hemocytometer 227 40. — The Neubauer ruling of the hemocjrtometer . . . 228 41. — Burker's hemocytometer 239 42. — The Sahli hemometer 245 43. — The Pleisehl-Miescher hemoglobinometer . . . 250 44. — The viscosimeter of Hess 257 45. — Boggs' modification of the coagulometer of Brodie and Russell .261 46. — ^Diagram to illustrate the movement of the cells during coagulation 262 Digitized by Microsoft® CLINICAL LABORATORY METHODS CHAPTER I THE URINE Collection of the Urine.— For chemical examination, as a general rule, the total amount of urine for the twenty- four hours should be preserved. The reason for saving all the urine is that different voidings may vary greatly in their chemical composition. In the morning, for example, an albuminuric may excrete urine which is normal chemi- cally, whereas specimens obtained after the patient has had more or less exercise may contain albumin. Thus, it becomes necessary that a mixture of all the urine passed during the twenty-four hours be. obtained in order to avoid the possibility of error, for the examination of the early morning specimen alone, in the case just cited, would be entirely misleading. Special circumstances arise at times, nevertheless, which make it desirable to break the rule and to secure one or more voidings at special hours of the day. For the purpose of quantitative chemical analysis, it is, of course, absolutely essential to have the total amount of urine for twenty-four hours collected. For microscopic examination a perfectly fresh specimen should always be insisted upon. The organized elements of a urinary sediment rapidly deteriorate, especially with high temperatures, so that within a few hours after the urine has been passed they may be unrecognizable, or com- pletely disintegrated. When it is not possible to make an 1 Digitized by Microsoft® 2 PEESBRVATION OF THE URINE immediate examination a preservative (thymol, toluol) should be added to the specimen, which is kept in an ice- chest as a further safeguard. Preservation of the Urine.— To all twelve or twenty- four-hour specimens of urine a preservative should be added to prevent decomposition through bacterial growth. The urine should also be kept on ice when possible. The receptacle for the urine must be perfectly cleaned and tightly stoppered. (1) Toluol (toluene) is, on the whole, one of the most satisfactory preservatives. It apparently interferes with none of the urinary tests. Bacterial growth is successfully inhibited by means of it. The one disadvantage in its use results from the fact that toluol floats on the urine; it is necessary to pipette or siphon the urine to obtain it with- out admixture of the preservative. The objection is a minor one. Diacetic acid may be preserved for weeks, whereas it disappears in a short time when other preserva- tives are used. In acidosis, therefore, toluol should be used as the preservative. Organized sediments are often beau- tifully preserved, but it must be remembered that casts or cells, becoming attached to droplets of toluol, rise to the surface and may be missed, when only a few are present; spontaneous sedimentation cannot be relied on if toluol is employed. The pipette used for withdrawing the sedi- ment must be wiped to remove the toluol before placing the drop on a slide for examination. (2) Chloroform is the most generally used preservative for chemical work. It is a fairly strong reducing agent, and urine preserved with it must be boiled to drive it off before any of the reduction tests for sugar are performed. If the sediment is to be examined, care should be exercised to avoid drawing up chloroform with it. Digitized by Microsoft® THE UEINB 3 (3) Thymol is very satisfactory, and with it the formed elements of the urine are often very well preserved. As Weinberger ^ has shown, many urines to which thymol has been added give a positive Heller's test, though albumin be absent, a source of error which must be kept in mind. A positive test for bile may also be obtained after thymol preservation (Emerson). Gum camphor and formaldehyde are used occasionally as preservatives. Formaldehyde, like chloroform, is a re- ducing agent. When available, toluol, chloroform, or thy- mol is to be preferred. Macroscopic Examination of the Urine.— As a general rule, normal, freshly voided urine is perfectly clear; the same is true of the majority of pathological urines. Occa- sionally, if the reaction of the urine be alkaline when voided, a turbidity may result from the precipitation of the phosphates and carbonates in the bladder, in the absence of a cystitis. Ordinarily, however, fresh urine, when cloudy or turbid, contains pathological ingredients, such as blood, pus, bacteria in large number, phosphates, etc. Normal and pathological urines will become turbid and produce a macroscopic deposit, more or less abundant, if allowed to stand for some hours. Concentrated urine often fur- nishes an abundant precipitate of urates on cooling; the urates may be redissolved by warming the specimen. More frequently bacterial decomposition is the cause of the tur- bidity. The nubecula is a translucent cloud, composed chiefly of mucin (mucous threads) enmeshing epithelial or other cells, which forms in the urine a short time after it is passed. 'Weinberger, W. "Thymol as a source of error in Heller's test for urinary protein." Jour. A. M. A., 1909, LII, 1310. Digitized by Microsoft® 4 EBACTION OP UEINB The color of the urine is usually dependent on the quan- tity of water excreted in the twenty-four hours ; the smaller the amount of urine the deeper the color, and vice versa. Normal urinary pigments in increased concentration or pathological pigments may lead to abnormal coloration of the urine (see urobilin, bilirubin, hemoglobin, hematopor- phyrin, etc.). Following the administration of certain drugs, the color of the urine may be altered, the most strik- ing change being the green color produced by methylene blue. Quantity of Urine. — The normal average amount of urine for the twenty-four hours in this country is about 900 to 1,200 c. c. The limits of the normal are said to be 800 to 3,000 c. c. (Emerson). In health the quantity de- pends chiefly upon two factors, the amount of water con- sumed and the amount lost by perspiration. In disease the quantity of urine passed in twenty-four hours may be nor- mal, increased, decreased, or nil. In certain diseases the urine is saved to advantage in twelve-hour periods, 7 A. M. to 7 P. M., and 7 P. M. to 7 A. M. In health the ratio of the quantity of the day urine is to that of the night urine as 67 :33, though it may be as 50:50, considering the total amount for twenty-four hours as 100. In disease the quantity voided during the night may exceed that for the day, as Edmunds ^ and others have shown. REACTION OF URINE The reaction of the urine is usually slightly acid, owing to the presence of an excess of dihydrogen (diacid) phos- phates. An amphoteric reaction (red litmus turned blue and blue turned red) may be encountered, due to the fact 'Edmunda, C. W. "Observations on the quantity of day and night urine." N. Y. Med. Jour., 1904, LXXIX, 245. Digitized by Microsoft® THE URINE 5 that monosodium phosphate, an acid salt, may exist in the urine in conjunction with disodiunr phosphate, which is alkaline. An alkaline reaction is produced largely by an excess of alkaline phosphates and carbonates. That the salts are not the only factor in rendering a urine acid has been shown by Folin, who finds that at times nearly half of the acidity may be due to organic acids. Litmus paper is used in testing the reaction of the urine. Unpreserved specimens, which have been allowed to stand for some time before testing, are often alkaline from am- moniacal fermentation produced by bacteria. The alkalin^ ity in this case is differentiated from that due to fixed alkali by the odor, by the fact that on boiling the specimen the steam "vpill turn blue a piece of moistened red litmus held in the neck of the test tube, or will cause a white frost of ammonium chlorid to appear on a glass rod, which has been dipped in hydrochloric acid. In disease the urine may be ammoniacal before it is voided. Quantitative Determination of Ukinaey Acidity For quantitative determination of the acidity the twen- ty-four-hour specimen is used. It is necessary to prevent decomposition by the addition of a preservative. Folin's Method.i Eeagents : ^sodium hydrate.^ . 0.5 per cent, phenolphthalein in 50 per cent, al- cohol. Potassium oxalate, neutral. 'Folin, O. "The acidity of the urine." Amer. Jour. Physiol., 1903, IX, 265. ^A normal aolution of acid or alkali should be purchased from a reliable firm. With this as a standard, the physician may easily prepare most of the remaining normal solutions required in routine work. Digitized by Microsoft® 6 SPECIFIC GRAVITY Method.-~"'Wit'h a pipette transfer 25 c. c. of urine into a small Erlenmeyer flask (capacity 200 c. c.)- Add one or, at most, two drops of phenophthalein and 15 to 20 gms. powdered potassium oxalate. Shake about one minute and titrate at once with tenth normal hydrate until a faint, yet distinct, pink coloration is produced throughout the con- tents of the flask. Shaking should be continued during the titration, so as to keep the solution as strong as possible in oxalate." The number of cubic centimeters of sodium hy- drate used multiplied by 4 gives the acidity per cent, in terms of tenth normal alkali. The inaccuracy of direct titration of the urine with sodium hydrate, as proposed by Naegeli, is pointed out by Folin. The two chief sources of error are ammoniujn salts and the occurrence of calcium in the presence of acid phos- phates. By first treating the urine with potassium oxalate each of these sources of error is practically eliminated. Normal values with this method are 25 to 30 acidity per cent. (Wood). SPECIFIC GRAVITY As a rule, the determination of the specific gravity of the urine is of real value only in the twenty-four-hour speci- men. It is usually determined by means of an urinometer. The short, small instruments designed for the purpose of taking the specific gravity of small quantities of urine are usually very inaccurate. In using the urinometer the urine is carefully poured into a glass cylinder, so that no foam is produced. Should foam collect despite the precautions, even though there be only a few bubbles, they should be removed with filter paper. The cylinder must be sufficiently wide to permit the urinometer to float freely without coming in contact with Digitized by Microsoft® THE UEINE 7 its wall. The reading is made with, the eye on a level with the bottom of the meniscus (the concave upper surface of the fluid). The instruments are standardized for use at a temperature of 15° C. ordinarily. For each 3° C. above this temperature the specific gravity is depressed one point in the third decimal place. As an example, if the specific gravity of a urine were found to be 1.015 at 24° C, the cor- rected reading would be 1.015+0.003=1.018. In case the specimen of urine furnished for examination be small, the urine which remains after the necessary tests have been performed may be diluted with water and the spe- cific gravity of the diluted specimen determined. The last two figures of the specific gravity found are multiplied by the dilution ; the result approximates the specific gravity of the undiluted urine. Normally the specific gravity of the twenty-four-hour specimen varies between about 1.010 and 1.025 ; absolute limits for the normal cannot be assigned, for so many fac- tors enter into the determination of the specific gravity that in individual instances the figures given may be passed in either direction, without necessarily signifying disease. It must be remembered that readings made with the urino- meter are not absolutely correct, but are sufficiently accu- rate for clinical purposes. Where greater accuracy is re- quired a pycnometer should be employed. An approximate idea of the amount of solids dissolved in the urine may be obtained by multiplying the last two figures of the specific gravity by 2.33 (Haser's coefficient), the result being the amount of solids in grams. UREA The normal amount of urea excreted daily in the urine varies within rather wide limits. Values between 20 and Digitized by Microsoft® 8 UREA 40 gms. are usually found. In clinical work urea deter- minations have been practically abandoned, except in the diagnosis of unilateral renal disease. Hiifner's Hypobromite Method.— The most convenient apparatus for applying this test is Heinz 's modification of the Doremus tube. It consists of a J-shaped tube mounted on a stand. A bulb is blown in the extreme end of the "tail" of the J-tube and a second tube of 2 c. c. capacity, graduated in _i_ c. c, is blown into the upright arm of the J-shaped tube. The connection between the two tubes may be cut by means of a glass cock in the 2 c. c. tube. The upper end of the J-tube is sealed. The reagent, Eice's bromin solution, is prepared as fol- lows: Sol. 1. Sodium hydrate 40.0 gm Distilled water 100.0 c. c Sol. 2. Bromin 10.0 c. c Potassium bromid 10.0 gm, Distilled water 80.0 c. c The two solutions are kept in separate bottles, and at the time of performing the test are mixed in equal volumes. Method. — Fill the small tube with the urine. The stop- cock is then opened until the urine reaches the zero mark. The excess of urine, which has run into the large J-tube, is removed from the latter by washing it with water, the up- per end of the tube containing the urine being appropri- ately sealed to prevent its escape. The J-tube is now filled with the mixed solutions, sufficient of the latter being em- ployed to completely fill the upright (all air must be dis- placed). The stop-cock is opened and the urine is slowly run into the mixed solutions. As the two fluids come in Digitized by Microsoft® THE URINE 9 contact, the hypobromite liberates nitrogen gas, which col- lects at the upper end of the large tube. The volume of gas liberated by 1 c. c. of urine is read on the scale marked on the upright arm of the J-tube, and gives the urea in grams in 1 c. c. of urine. The method is very inaccurate as a means for deter- mination of urea ; the results obtained approach more near- ly the total nitrogen of the urine. For this reason the method is inapplicable, where exact values for urea are re- quired, as, for example, in metabolism experiments. In the diagnosis of surgical affections of the kidney, where the urine from each kidney is examined separately, marked dif- ferences in the two kidneys are shown with sufficient ac- curacy, and it is in this connection that the method is used most at the present time. URIC ACID The normal quantity of uric acid in the urine in twenty- four hours lies between 0.1 and 1.25 gm., with a patient on a mixed diet. The endogenous uric acid of the urine varies between 0.1 and 0.4 gm. Qualitative Determination of Ueic Acid Qualitative determination of uric acid is made by the murexid test. A small drop of the urinary sediment or other material to be tested is dissolved in two or three drops of nitric acid in a porcelain evaporating dish. The material is evaporated to dryness, preferably on a water bath, care being exercised to avoid burning the prepara- tion. The stain which remains on the dish has a reddish color. (A yellow stain may indicate that an insufficient quantity of nitric acid was used.) The addition of ammo- Digitized by Microsoft® 10 URIC ACID nium hydrate or, better still, exposing the stain to ammonia fumes changes the color to a purplish red, which fades on heating. The reaction is given by uric acid and by its salts. (For further qualitative tests, see urinary sediments.) Quantitative Deteemination of Ukic Acid Method of Folin and Shaffer.^ Eeagents : Sol. 1. Ammonium sulphate 500.0 gm, Uranium acetate 5.0 gm Distilled water to 650.0 c. c Dissolve and then add : Acetic acid, 10 per cent 60.0 c. c Distilled water to 1,000.0 c. c Sol. 2. Ammonium sulphate 100.0 gm Distilled water to 1,000.0 c. c Sol. 3. ^ potassium permanganate. To prepare the twentieth normal permanganate solu- tion, dissolve 1.7 gm. of potassium permanganate in one liter of distilled water. The solution is boiled or auto- claved to render it more permanent. After it has cooled to room temperature, it is titrated against tenth normal oxalic acid solution (6.3 gm. pure crystals to one liter of distilled water). With a pipette, 10 c. c. of ^ oxalic acid are placed in a small Erlenmeyer flask or beaker, diluted with about 100 c. c. of distilled water, and 15 c. c. of concen- trated sulphuric acid added. The temperature of the mix- ture is raised, by the addition of the sulphuric acid, to about Tolin, O., and Shaffer, P. A. "Ueber die quantitative Bestimmung der Harnsaure im Harn. ' ' Ztsohr. f. physiol. Chem., 1901, XXXII, 552. Digitized by Microsoft® THE URINE 11 60° C. While still hot the permanganate solution is added to it from a burette, under constant stirring, until a uni- form red color appears, which persists throughout the fluid for a few seconds.^ This is the end reaction. The quan- tity of permanganate solution used is read off on the bu- rette. Since the permanganate solution has been made too strong, less than 20 c. c. of it should have been required to produce the end reaction. The permanganate solution which remains is accurately measured (with volumetric flasks and pipettes, not with cylinders), and is diluted with distilled water, so that exactly 20 c. c. will give the end re- action with 10 c. c. of tenth normal oxalic acid. When kept in a dark place, tightly stoppered, the potassium perman- ganate solution is fairly permanent for several months. The titer should, however, be determined from time to time with the oxalic acid solution. Example. — If 18.9 c. c. of permanganate solution gives the end reaction with 10 c. c. of n oxalic acid, and the remainder of the potassium permanganate solution amounts to 960 c. c, the necessary dilution to make the perman- ganate solution twentieth normal is determined by the fol- lowing equation: 18.9 :20 : :960 :x. x=l,015.8. Therefore, the amount of water necessary to add would be 1,015.8 — 960 or 55.8 c. c. Method. — To 300 c. c. of urine in an Erlenmeyer flask or beaker of 500 c. c. capacity or larger, add 75 c. c. of the uranium acetate reagent (sol.'l) to precipitate phosphates and other substances, which might interfere with the accu- racy of the method. Both urine and reagent must be accu- rately measured with volumetric pipettes. Stir the mixture 'Early in the titration, after the addition of the first few drops of permanganate, a red color, which may persist for fifteen seconds or so, may be noted, but it quickly disappears on adding more permanganate. Digitized by Microsoft® 12 AMMONIA well, and allow it to stand five minutes. Then filter through a double folded filter. Measure with a pipette 125 c. c. of the filtrate (this represents 100 c. c. of the urine originally used) into each of two beakers and add 5 c. c. of ammonium hydrate to each to convert the uric acid into ammonium urate. Mix well, and set aside for twenty-four hours ; by the end of this time the precipitate will have settled to the bottom of the beaker. The clear, supernatant fluid is de- canted, the precipitate collected on a filter (Schleicher and Schiill's No. 597) and washed with 10 per cent, ammonium sulphate, till the filtrate is almost chlorin free. (In testing for chlorids add a little nitric acid and then a few drops of dilute silver nitrate solution (10 to 15 per cent, solution) ; a white precipitate or cloud is formed if chlorids are pres- ent.) The filter paper is now pierced with a glass rod, and the precipitate washed into a beaker with about 100 c. c. of distilled water. Add 15 c. e. of concentrated sulphuric acid and titrate the mixture immediately, stirring constantly, until a pink color appears throughout the fluid and persists for a few seconds. Bach cubic centimeter of twentieth normal permanga^ nate is equivalent to 3.75 mg. of uric acid. This, multiplied by the number of c. c. of permanganate used, gives the uric acid in 100 c. c. of urine, from which the total amount for twenty-four hours is readily calculated. Since ammo- nium urate is slightly soluble in water and somewhat more so in urine, a correction of 3 mg. should be added to the final result for each 100 c. c. of urine. AMMONIA Normally the urine contains 0.6 to 0.8 gm. of ammonia daily for the average adult on a mixed diet. The limits of Digitized by Microsoft® THE URINE 13 the normal are about 0.3 to 1.2 gm. In health the ammonia nitrogen usually amounts to 4 to 5 per cent, of the total nitrogen, when a mixed diet is taken. Quantitative Determination of Ammonia (1) Folin's Method.! Eeagents : Sodium ehlorid. Anhydrous sodium carbonate. Petroleum or toluene. JL sulphuric acid ; JL sodium hydrate. One per cent, aqueous solution of alizarin red. The apparatus required includes areometer cylinders (45 cm. deep and 5 cm. in diameter), a suction pump, cal- cium ehlorid tube, doubly perforated stoppers to fit the cyl- inders, and tubing for connections. Folin's tube to secure thorough mixing of air and acid is a convenience, though not a necessity. The apparatus is connected as shown in the illustration (Fig. 1), the calcium ehlorid tube filled with' cotton being placed between the cylinders to prevent the al- kaline urine being drawn over into the acid. Method. — With a pipette, 25 c. c. of tenth normal acid are placed in cylinder B and diluted with distilled water sufficiently to cover the end of the mixing tube. Into cylin- der A, 25 c. c. of the twenty-four-hour specimen of urine are measured with a pipette. To the urine are added 8 to 10 gm. of sodium ehlorid, 5 to 10 c. c. of toluol or petroleum to prevent foaming (with blood or other fluid rich in pro- tein add some methyl alcohol also), and, finally, about 1 gm. 'Folin, O. "Eine neue Methode zur Bestimmung des Ammoniaks im Harne und anderen thierischen riiissigkeiten. ' ' Ztschr. f. physiol. Chem., 1902- 03, XXXVII, 161. 3 Digitized by Microsoft® 14 AMMONIA of anhydrous sodium carbonate. After the addition of the soda the cylinder is immediately stoppered and the air cur- rent started. Before entering the urine the air current may be passed through a wash bottle containing sulphuric acid to remove ammonia in the air, though usually this pre- caution is unnecessary. A B ^ Fig. 1. — Apparatus fob the Quantitative Determination of Ammonia According TO Folin. Cylinder A for urine, cylinder B for acid. The addition of the soda to the urine liberates the weaker base, ammonia, which is carried over by the air stream into the tenth normal sulphuric acid, by which it is neutralized. When Folin 's mixing tube is used, not a trace of the ammonia escapes neutralization by the acid, even though there remains an excess of only 5 c. c. of tenth normal acid. If ordinary glass tubing be employed to pass the air through the acid, a second cylinder, containing 10 c. c. of tenth normal acid, should be intierposed between the Digitized by Microsoft® THE URINE 15 acid cylinder and the pump to catch the ammonia which es- capes neutralization. The air pump should be capable of carrying 600 to 700 liters of air per hour through the apparatus. With a pump of this capacity working at room temperature (20 to 25° C.) all of the ammonia is carried into the acid in an hour or an hour and a half.^ When the process is completed the acid is poured into an Erlenmeyer flask or beaker and the cylinder rinsed with distilled water, which is added to the acid. The acid is now titrated with tenth normal sodium hydrate, using two drops of alizarin red to 200 to 300 c. c. of fluid. The end reaction is the appearance of a red color throughout the fluid; do not continue the titration to the appearance of a violet color. The difference between the number of cubic centi- meters of acid originally taken and that of the alkali used is the number of cubic centimeters of acid neutralized by ammonia. Since one cubic centimeter of tenth normal acid is equivalent to 0.0017 gm. of ammonia, this, multiplied by the number of c. c. of acid neutralized, gives the quantity of ammonia in 25 c. c. of urine. The quantity in the twenty- four-hour specimen is calculated from this. Determinations may be made in duplicate or triplicate by connecting two or more sets of apparatus in series. (2) The Vacuum Distillation Method.— Shaffer ^ has modified the vacuum distillation method. As described by him, it is carried out in the following manner : To 50 c. c. of urine in flask A (Fig. 2) add an excess (15 or 20 gm.) 'The efficiency of the pump and its "working time" are readily tested by taking a specimen of urine and titrating the acid at the end of an hour; add more tenth normal acid and titrate at fifteen-minute intervals, until acid is no longer neutralized. ^Shaffer, P. "On the quantitative determination of ammonia in urine." Amer. Jour. Physiol, 1903, VIII, 330. Digitized by Microsoft® 16 AMMONIA of sodium cMorid, and about 50 c. c. methyl alcohol. In bottle B place 25 or 50 c. c. ^^ acid and in B' 10 c. c. -^ acid, diluted in each case with a small amount of water. If too much water is added there will be danger of loss of acid by jumping over during the violent commotion which is set up in the acid by the rapid passage of the steam. If such a loss should occur the acid can always be recovered by rinsing out the filter flask C. When the apparatus is ready, about 1 gram dry sodium carbonate is added to the liquid in flask A, the stopper quickly put in place, and the suction started. Fig. 2. — Apparatus fob the Determination ttt-j.-^, j w,ti +Viq OP Ammonia According to Shaffer. VVltU a gOOQ pump Xne (After Shaffer.) pressurc wiU bo re- duced to about 10 mm. Hg. in two or three minutes, when, the liquid surrounding A being at 50° C, a rapid boiling will begin. The temperature is maintained, and the boiling allowed to continue for fifteen minutes. At the end of that time the ammonia will in all cases have been completely given off, and the operation may be stopped by slowly let- ting in air at the stop-cock in tube a. The acid in B and B' is titrated and the ammonia calculated. One per cent, aque- ous alizarin red is used as the indicator. (For a descrip- tion of the end-point and the calculation see the preceding method of Folin.) This method is very accurate, and consumes very little time. Shaffer found the results in all cases correct within less than 10 mg. of ammonia per liter. Where the neces- sary apparatus is available, this method or that of Folin is to be preferred. (3) The Formalin Titration Method.— This method was Digitized by Microsoft® THE URINE 17 first introduced by Eonchese.^ It is more accurate than the Schlosing method, and possesses an advantage over all other methods in the rapidity with which a determination may be completed. A number of observers have found it a satisfactory clinical method for following the ammonia excretion in acidosis, but, in its present form, it is not suf- ficiently accurate for metabolism experiments, since the re- sults are apt to be too high, owing, in part at least, to the fact that aminoacids are determined with the ammonia. The principle of the method is based on an observation of Delepine, and is as follows : The addition of formalin to a solution of an ammonium salt gives rise to the formation of hexamethylenamin, with the liberation from the salt of the corresponding acid. If, then, the acid equivalents in the urine be first neutralized with alkali and formalin subse- quently added, titration with tenth normal alkali will now reveal the acidity due to ammonium salts, and the quan- tity of alkali used indicates the amount of ammonia in the urine. (a) Method of RoncMse} — Ten c. c. of the twenty-four- hour specimen of urine are measured with a pipette and placed in an Erlenmeyer flask of 300 c. c. capacity. To the urine add 100 c. c. of distilled water, previously boiled to drive off carbon dioxid, and then 1 or 2 drops of 0.5 per cent, alcoholic phenolphthalein solution. Under constant stirring add tenth normal sodium hydrate from a burette until a pale rose color makes its appearance throughout the fluid. Now add 20 c. c. of 20 per cent, formalin (commercial formalin is 40 per cent, strength), which has been neutral- ized, if necessary, with phenolphthalein as indicator, and again add tenth normal alkali, till the same color reaction Eonchfise, A. "Nouveau proc^de de dosage de rammoniaque. " J»ur. de Fharm et de CMm., 1907, XXV (6th series), 611. Digitized by Microsoft® 18 AMMONIA is obtained. This is the end-point. To the quantity of tenth normal alkali used after the addition of the formalin a correction of 0.1 c. c. is added for each 3 c. c. required in the titration. This sum equals the ammonia content of 10 c. c. of urine expressed in c. c. of tenth normal ammonia. One c. c. of tenth normal ammonia contains 0.0017 gm. am- monia. The quantity of ammonia in the twenty-four-hour specimen is calculated. (b) Method of Bjorn-Andersen and Lauritzen} — Twenty c. c. of urine, 5 drops of 0.5 per cent, alcoholic solution of phenolphthalein, and 20 gm. of finely powdered neutral potassium oxalate are placed in an Erlenmeyer flask, shak- en vigorously about one minute, and then titrated imme- diately with tenth normal sodium hydrate under constant stirring, till a pale rose color is obtained. Now add 5 c. c. of commercial formalin (neutralized, if necessary), and the acid liberated will cause the color to disappear. Again titrate with tenth normal alkali to a pale rose color. (Add a little more formalin. If the color remains, the end-point has been reached; if it disappears, continue the titration until the color no longer fades on the further addition of a small quantity of formalin.) The quantity of alkali used to obtain the end-reaction after the addition of the for- malin represents the number of c. c. of tenth normal ammo- nia in 20 c. c. of urine. Each c. c. of tenth normal ammo- nia contains 0.0017 gm. of ammonia. The entire amount of tenth normal sodium hydrate used gives the "total acidity" of the urine. The authors find that the curves of total acidity and ammonia run parallel in health and in diabetic acidosis. •Bjorn-Andersen, B., and Lauritzen, M. "TJeber Saure- und Ammoniak- bestimmung im Ham und ihre klinische Anwendung. " Zeitschr. f. pjiysiol. Chem., 1910, LXIV, 21. Digitized by Microsoft® THE URINE 19 The quantity of ammonia found is somewhat high in the presence of aminoaeids. (4) Shaffer's Modification ^ of Schlosing's Method.— This method, which requires at least two days for its com- pletion, is more exact than Schlosing's, less accurate than Folin's, but for most purposes it meets the needs of the clinician. An advantage is the simple apparatus required. The ammonia, liberated under a bell jar or in a dessicator by the addition of stronger alkali, is neutralized by tenth normal acid. With a pipette, 25 c. c. of urine are placed in the bottom of a dessicator or dish having a diameter of 15 to 17 cm. An excess of sodium chlorid is added to pre- vent decomposition, then about 0.5 gm. of anhydrous so- dium carbonate. A second smaller dish containing 20 c. c. of tenth normal sulphuric acid is placed in the dessicator or under a bell jar with the urine. It is essential that the dish containing the urine have a perfectly flat bottom and that the depth of the liquid be not more than two mm. "For the same amount of urine, the wider the dish the more rapid will be the expulsion of the ammonia" (Shaffer). The length of the operation may be reduced to forty-eight hours by letting the apparatus stand at 38° C. On longer stand- ing at this temperature, the ammonia from decomposition becomes so great that the results are too high. At room temperature (about 25° C.) the apparatus is allowed to re- main four days. The acid is then titrated with tenth nor- mal sodium hydrate, using alizarin red (1 per cent, aqueous solution) as the indicator in the proportion of two drops to 200 to 300 c. c. of fluid, with a red color, not a violet, as the end-point. The ammonia in grams in 25 c. c. of urine is found by multiplying 0.0017 by the number of c. c. of ^ acid neutralized. 1 Shaffer, P. Loc. cit. (p. 15). Digitized by Microsoft® 20 NITROGEN NITROGEN The total nitrogen of the urine of a normal adult on a mixed diet lies usually between 10 and 16 gm., or about 0.2 gm. per kilo of body weight. Kjeldahl's Method for Determination of Total Nitrogen. Eeagents : ^ Crystalline copper sulphate. Crystalline potassium sulphate. Concentrated sulphuric acid. 40 per cent, solution of sodium hydrate. Talc powder. Ngulpliiiric acid; ^ sodium hydrate. One per cent, aqueous solution of alizarin red, or tincture of cochineal. Method. — With a pipette 5 c. c. of the twenty-four-hour specimen of urine are measured into a Kjeldahl oxidizing flask (Jena glass) of about 800 c. c. capacity. Then add about 15 c. c. of concentrated sulphuric acid and about 0.2 gm. of copper sulphate crystals, and, finally, about 10 gm. of potassium sulphate. The flask is placed under a hood * and is heated over a Bunsen burner,* with a low flame at first, until the foaming has ceased. The heating is con- tinued till the contents of the flask become clear. It may be ^ To determine whether the reagents are N-f ree, proceed with the method, substituting 5 c. c. of glucose solution for the urine. If nitrogen is found, the necessary correction is evident. ' A lead pipe, perforated with holes to receive the necks of the digesting flasks (see Fig. 3) and connecting with a flue, is better than moat hoods. If there is a good draught, the fumes are carried off perfectly. An outlet constructed of tile pipes is inexpensive and satisfactory. ' The most satisfactory form of apparatus is that designed by Folin and made by the International Instrument Co., Cambridge, Mass. The small model is shown in Fig. 3. Digitized by Microsoft® THE URINE 21 uecessar}' to remove the flask, so that all the charred mat- ter may be brought into the acid. After the fluid in the flask has l)ecome i)ale green or colorless, the heating is prolonged fifteen minutes to insure complete oxidation. All of the nitrogenous compounds have been converted to Fig. 3. — Digesting Rack for the Kjeldahl Nitrogen Determination. The necks of the flask.s extend into a perforated lead pipe, which is connected with a tile outlet. ammonia, which unites ANdth the sulphuric acid to form ammonium sulphate. The liquid is allowed to cool (it may crystallize eventually) ; about 300 e. c. of distilled water are then added, and, when solution is obtained, a heaping tea- spoonful of talc powder is placed iu the flask (to prevent bumping during the boiling). Finally, sufficient 40 per cent, sodium hydrate is added to render the solution Digitized by Microsoft® 22 NITROGEN strongly alkaline; the qnantity required must have been determined previously. It is well to incline the flask and pour the alkali down tlie side, to prevent mixing and pos- sible loss of ammonia. 1 The flask is immediately connected with a distilling apparatus (Fig. 4) provided with a Hop #:#ar lip ill*-. . \\ ' • i i \ vU- Fig. 4. — Distilling Appaeattjs for the Kjeldahl Nitrogen Determination. kins bulb or similar device to prevent alkali passing over into the acid, and its contents boiled. The distillate, con- taining tlie ammonia liberated by the stronger alkali, is received in an Erleumeyer flask in which 25 to 50 c. e. of tenth normal sul])huric acid have been placed. The distil- ' To gii.ird against siifh loss, a doulily perforated stopper may be pro- vided and the alkali introduced into the tiask by a fimnel, whose stem, pass- ing through the stopper, is jiluggeil immediately after adding the alkali. Digitized by Microsoft® THE URINE 23 lation is continued till the distillate is no longer alkaline to litmus — usually a half hour or less. The condensing tube is then washed with distilled water into the distillate. To determine the excess of acid, the contents of the flask are titrated with tenth normal alkali with alizarin red (2 drops to 200 to 300 c. c. of fluid) as the indicator. The end-point is a red color, not a violet. (Tincture of cochineal ^ is used at times as an indicator. It imparts a very pale brown color to acid solutions when added in the proportion of about four drops to 300 c. c. ; when the reaction becomes al- kaline the color changes to amethyst.) Since one c. c. of tenth normal acid is equivalent to 0.0014 gm. of nitrogen, the amount of the latter in 5 c. c. of urine and, finally, in the twenty-four-hour specimen is easily computed. If insufficient acid has been taken to receive the distil- late, the excess of ammonia may be titrated with tenth nor- mal sulphuric acid (Hoppe-Seyler, Thierfelder). Instead of distilling the oxidized material, the ammonia determination may be performed according to Folin's method. The titration and calculation of the result are performed as described above. Sodium hydrate is substi- tuted for sodium carbonate. CHLORIDS In health, with the usual mixed diet, the chlorids of the urine, usually amount to 10 to 15 gm. daily. The limits of the normal are said to be 6 and 22 gm. Qualitative Test About 10 c. c. of urine, placed in a test tube, are acidi- fied with strong nitric acid, and then one or two drops of ' Tincture of cochineal is prepared by grinding cochineal bugs in 50 per cent, alcohol in a mortar, allowing the mixture to digest a day, and filtering. Digitized by Microsoft® 24 CHLOKIDS dilute silver nitrate solution (10 to 15 per cent, aqueous solution) are added. A wMte precipitate denotes the pres- ence of chlorids. An approximate idea of the quantity of chlorids may be gained. Normally, a dense precipitate appears and quickly settles to the bottom of the tube. With great reduction in the chlorids only a cloud is seen, with- out flocculent precipitate. Quantitative Determination of Chlorids Harvey's! Modification of Volhard's Method.— This method is valuable because of its rapidity without sacri- fice of accuracy. As in the original method, the chlorids are precipitated by adding an excess of silver nitrate, the amount of which is determined by titration with ammonium thiocyanate. The chlorids are calculated as sodium chlo- rid. Albumin, if present in the urine in appreciable quan- tity, must be removed by boiling and the subsequent addi- tion of dilute acetic acid, before proceeding to the estima- tion of the chlorids. Largely in the author's words, the method follows : Reagents : (a) A silver nitrate solution containing 29.042 gm. of chemically pure, crystalline silver nitrate in one liter of distilled water ; 1 c. c. of this solution is equivalent to 0.01 gm. of sodium chlorid. (The silver solution may be stand- ardized against a weighed quantity of dry, chemically pure sodium chlorid.) (b) A solution of ammonium thiocyanate, 20 c. c. of which is equivalent to 10 c. c. of the silver nitrate solution. As this salt is very hygroscopic, it cannot be weighed with sufficient accuracy to make the solution directly. There- ^Harvey, S. 0. "The quantitative rletermination of the chlorids in the urine." Arch. Int. Med., 1910, VT, 12. Digitized by Microsoft® THE URINE 25 fore, 13 gm. of it are dissolved in one liter of distilled water, thus making a concentrated solution, whose strength is determined by titration against the silver nitrate solu- tion, and the requisite dilution made. This is done in the following manner : 10 c. c. of the silver nitrate solutioD are measured with a pipette into a beaker, diluted with about 20 c. c. of distilled water, 2 c. c. of the indicator (sol. c) added, and the whole titrated with the ammonium thio- cyanate solution. If, for example, 12 c. c. of the solution are used in the titration and the total volume of the thio- cyanate solution is 960 c. c, the volume to which if must be diluted with distilled water is determined according to the equation 12 :20 : :960 :x, in which x represents the required volume. (c) The indicator containing nitric acid. To 30 c. c. of distilled water add 70 c. c. of nitric acid (sp. gr. 1.2, or 33 per cent.). Saturate this menstruum with crystalline fer- ric ammonium sulphate and filter. This indicator is recommended, inasmuch as it substi- tutes one solution in place of the two (the ferric indicator and the acid), and insures the use of the proper amount of the acid. Moreover, it is sufficiently concentrated, so that it is necessary to use only 2 c. c, and, therefore, it may be kept in a small reagent bottle. The stopper of this bottle may be a graduated dropper, which can at the same time serve to measure and transfer the indicator. Method. — With a pipette transfer 5 c. c. of the twenty- four-hour specimen of urine (albumin-free) to a small beaker or Erlenmeyer flask, and dilute it with about 20 c. c. of distilled water.^ The chlorids in this solution are now 'When the urine is highly colored, add 8 to 10 per cent, solution of potassium permanganate a drop at a time, until the red color no longer fades rapidly, and the urine has become pale yellow. Digitized by Microsoft® 26 SULPHATES precipitated by adding 10 . c. c. of the silver nitrate solu- tion with a pipette. Next, place about 2 c. c. of the indica- tor in the mixture. The ammonium thiocyanate solution is then run in from a burette under constant stirring, until the first trace of red shows throughout the mixture. On al- lowing the precipitate to settle, the color may easily be recognized in the supernatant fluid. If, however, the mix- ture is stirred violently, the color will disappear. When the end-point appears on the addition of the first drop of ammonium thiocyanate solution (i. e., when the original 10 e. c. of silver solution is insufficient to precipitate all the chlorid), then 10 c. c. more of the silver nitrate solution are added, and the titration completed with corresponding al- lowance in the calculation. The calculation may be made as follows : As 20 c. c. of the ammonium thiocyanate solution are equivalent to 10 c. c. of the silver nitrate solution, divide the number of c. c. of thiocyanate solution used by two (2) and subtract the quotient from 10 c. c, the amount of silver nitrate origi- nally taken. The result is the number of c. c. of silver ni- trate solution actually used in the precipitation of the chlo- rids. As 1 c. c. of the silver solution is equivalent to 0.01 gm. of sodium chlorid, the number of cubic centimeters of silver nitrate solution used, multiplied by 0.01, will give the amount of the chlorids, expressed in terms of sodium chlo- rid, in 5 c. c. of urine, the quantity taken. From this the total amount of chlorid in the twenty-four-hours specimen is calculated. SULPHATES Quantitative estimation of the sulphates in the urine is of no practical value in general clinical work at the pres- ent time. Of the sulphates present in the urine, indoxyl sulphate alone is tested for in the usual examination. Digitized by Microsoft® THE URINE 27 The tests for indoxyl sulphate depend on the oxidation of indoxyl to indigo blue and its extraction in chloroform. It is necessary at times to precipitate the urine, before testing, with one-fifth volume of 20 per cent, lead acetate to remove pigments, which may interfere with the recogni- tion of the blue color. Obermayer's Test.— Equal parts of urine and Ober- mayer's reagent (0.2 per cent, ferric chlorid in concen- trated hydrochloric acid) are mixed in a test tube and al- lowed to stand a few minutes (2-3). A small amount of chloroform is added, and the test tube is inverted several times. With normal amounts of indoxyl sulphate a faint blue is seen in the chloroform ; an excess causes a dark blue .color. By using the same quantities of urine, reagent, and chloroform, and test tubes of uniform diameter, daily vari- ations in the intensity of the reaction may be followed. Jaffe's Test.— Equal quantities of urine and strong hy- drochloric acid are mixed in a test tube ; about 2 c. c. of chloroform and 1 to 3 drops of strong aqueous solution of calcium hypochlorite are added. The tube is inverted sev- eral times, and the indigo collects in the chloroform, as in the preceding test. If the patient has been receiving iodin in any form, a violet color is imparted to the chloroform in performin.'^ Obermayer's and Jaffe's tests. To destroy the color pro- duced by the iodin and bring out that of indigo blue, if present, the chloroform is transferred to a second test tube and is shaken with dilute potassium hydroxid ; or water and a small quantity of strong sodium thiosulphate solution are added to the chloroform and the whole shaken. The violet is decolorized, leaving the blue. Codein, when administered in large doses, is said to give a purplish red color to the chloroform. Digitized by Microsoft® 28 ALBUMIN ALBUMIN Normal urine contains albumin in traces too small to be detected with the usual tests. Before testing for albumin, two conditions must be ful- filled: (1) The urine must be perfectly clear, and (2) its reaction must be acid. (1) If the specimen to be examined is fresh and fairly- clear, passage through filter paper usually suffices to ren- der it transparent and clear. With urines containing abun- dant fine precipitates or many bacteria, simple filtration is not satisfactory. Such urine should be shaken with Kiesel- guhr (infusorial earth) and then passed through a folded filter. The meshes of the paper are plugged, so that the filtrate is perfectly clear, though it may be necessary to return the first few cubic centimeters of the filtrate to the filter. Minute quantities of albumin may be removed by the filtration with Kieselguhr. (2) If alkaline or neutral in reaction, the urine should be rendered slightly acid to litmus by the addition of a few drops of 3 per cent, acetic acid. Of the following qualitative tests it is advisable to use at least two in all instances to avoid error. Heller's and the heat and acetic acid tests form a satisfactory combina- tion. QxjALITATrVE TeSTS (1) Heat and Acetic Acid Test.— (a) First Method. — A test tube (18 to 20 mm. in diam.) is nearly filled with the clear, acid urine. Holding the tube by its lower end, the urine in the upper part is boiled over a Bunsen burner or spirit lamp, the cool urine in the lower part of the tube serving for comparison with the boiled portion. A cloud Digitized by Microsoft® THE URINE 29 may appear on boiling, due (1) to precipitation of calcium phosphate, or (2) to albumin, or (3) to the precipitation of both simultaneously. A few drops of 3 per cent, acetic . acid are now added. If the precipitate be due to phosphates alone, it will disappear on the addition of the acid, whereas the albumin coagulum will usually be intensified, never les- sened, unless a considerable excess of acid is added. When both phosphates and albumin are precipitated together, the cloud may be perceptibly diminished but not abolished by acidification. Very small quantities of albumin may give no cloud on heating, but the albumin may appear after the addition of the acid. Such traces of albumin are best de- tected by holding the tube against a dark background with the eye at a right angle to the source of light, for the faint cloud may be easily overlooked on casual inspection. The urine in the upper part of the tube (which has been boiled) is compared with the clear urine in the lower part of the test tube. When the urine is of very low specific gravity and, therefore, poor in salts, the test is improved by the addition of one-fifth to one-tenth volume of saturated so- dium chlorid solution to the urine. The urine is not to be boiled after the addition of the acid. The test is said to indicate albumin in a dilution of 1: 130,000 (Glaesgen). Sources of Error. — (a) There is danger in adding too much acetic acid, since the albumin may be converted into the soluble acid albumin or syntonin. That it requires a con- siderable excess of acid to redissolve the precipitate, how- ever, once it is formed, is easily demonstrated. It is help- ful to the worker to experiment with knoAvn specimens to determine the degree of latitude one can safely follow in the addition of the acid, (b) Nucleoalbumin may be pre- cipitated by heat and acetic acid; it is also thrown out of Digitized by Microsoft® 30 ALBUMIN solution by the addition of dilute acetic acid to tlie cold urine. Two tests may be performed, one on the cold, the other on the boiled, urine ; by comparison it is usually pos- sible to estimate whether part or all of the precipitate is due to the nucleoproteid. Or, the urine is treated with di- lute acetic acid, filtered to remove the precipitate of nucleo- albumin, a few more drops of the dilute acid added, and the contents of the test tube boiled; a precipitate appearing now is albumin. The test for nucleoalbumin is improved if the urine be diluted with v/ater; that for albumin is sharper after the addition of salt, (c) Following the ad- ministration of cubebs, copaiba, turpentine, etc., resinous bodies appear in the urine, and may be precipitated. After cooling the fluid the precipitate may be dissolved in petroleum benzine or in alcohol, albumin being insoluble, (d) Albumoses appear after the urine becomes cool; the precipitate redissolves on boiling, (e) The Bence- Jones' body is coagulated at about 60° C, but usually redissolves in part or wholly as the boiling point is reached. (b) Second Method. — This method, widely used in France, has recently been carefully examined and recom- mended by Glaeegen.^ The acetic acid is added before the specimen is boiled. About 20 c. c. of urine and 5 drops of 20 per cent, acetic acid are mixed in a test tube. The urine in the upper part of the tube is boiled (or the mixture may be divided between two test tubes, one to be boiled, the other to serve as a control). If the acetic acid produces a cloud in the cold (nucleoproteid), the specimen is cleared by filtration before boiling. The acidification previous to boiling prevents a precipitation of phosphates in the major- ' Glaesgen. ' ' Zur Methodik des Nachweises sehr kleiner pathologisober Eiweissmengen im Harn." Mixnchen. med. Wchnschr., 1911, LVIII, 1123. Digitized by Microsoft® THE URINE 31 ity of instances; if such a precipitate occurs, a few more drops of the acid are added to dissolve it. This will not cause the solution of a slight albuminous precipitate, pro- vided the specimen is not reboiled. With the precautions given, the presence of a cloud or precipitate indicates al- bumin. (For the detection of a very faint cloud, see the first method.) By this method Glaesgen finds that albumin may be demonstrated in a dilution of 1:180,000.^ (2) Heat and Nitric Acid Test.— The method of pro- cedure is the same as in the preceding test (first method), the urine in the upper part of the test tube being boiled. One to four drops of concentrated nitric acid^ are now added. The precipitate, which may form on boiling the urine, may be due to albumin or phosphates or to both. Thie phosphate precipitate is dissolved by the addition of the acid ; in such case a few more drops of nitric acid are added, when albumin is precipitated, if present. When more than a trace of albumin is present in the urine, the precipitate is flocculent and whitish or brownish. With a urine of low specific gravity the addition of one-fifth vol- ume of saturated sodium chlorid solution at times makes it possible to recognize a trace of albumin, which would oth- erwise be missed. The urine may remain clear after boil- ing, but a precipitate of albumin may still appear on acidi- fication, as in the heat and acetic acid test. If the cloud is faint, there is danger of missing it, unless the tube be held against a dark background with the eye at a right angle to the source of light. Do not boil after adding the acid. ' A somewhat limited experience with the second method has shown it to be quite as sensitive as the first, in the writer 's hands. ^ Nitric acid becomes yellow from the formation in it of nitrous acid. It is readily cleared by the addition of crystals of urea. Digitized by Microsoft® 32 ALBUMIN The test is said to be as delicate as the heat and dilute acetic acid test. Sources of Error. — The possibilities of error are much the same as in the heat and acetic acid test, (a) An ex- cess of acid is to be avoided, as the precipitate may be dis- solved, forming acid albumin. The proportion of acid to urine should not exceed about 1:1,000 (Simon), (b) Resi- nous bodies are distinguished as in the preceding test, (c) Uric acid may precipitate after standing a few minutes. The precipitate is crystalline, and gives the murexid test, (d) Albumose is soluble in the boiling solution, but in- soluble in the cold. The precipitate which forms may be redissolved by heating, (e) Bence- Jones* protein usually exhibits maximal precipitation at about 60° C, with par- tial or complete disappearance of the coagulum at the boil- ing point, (f ) In markedly icteric urine a green precipitate of biliverdin may be produced. This, unlike coagulated albumin, is soluble in alcohol. Finally, it may be added, the nitric acid possesses an advantage over dilute acetic acid, since its addition to boiling urine does not precipitate mu- cin, or nucleoproteid. The nitric acid must be free from nitrous acid.^ (3) Heller's Test.— In performing this test a wide test tube or, better still, a conical glass or horismascope should be used. Ten to 20 c. c. of urine are placed in a conical glass, and then, with the glass inclined, concentrated nitric acid ^ is poured slowly down its side. Being the denser fluid, the acid collects at the bottom. The glass is now brought to the vertical position very gradually, to prevent mixing of the urine and acid. If albumin is present in the urine, a white precipitate is formed at the line of contact ^This* refers to footnote'' on p. 31, beginning "Nitric Acid." ^See footnote on p. 31. Digitized by Microsoft® THE URINE 33 between urine and acid. The precipitate is acid albumin, which is insoluble in the great excess of acid. The breadth and sharpness of the ring will depend upon the quantity of albumin present, and also upon the success with which the urine and acid have been layered. When small quantities of albumin are present the ring may appear only after two or three minutes, and then may be overlooked unless the tube is examined against a dark background with the eye at a right angle to the source of light. Glaesgen ^ finds the reaction positive with albumin in a dilution of 1 :35,000. Sources of Error. — (a) Urines which have been pre- served with thymol may give a ring at the line of contact which is practically indistinguishable macroscopically from that produced by albumin.^ Below the ring there is a greenish zone extending into the acid, above it a red zone. When thymol and albumin coexist, it may be noted that the thymol ring forms just beneath that of albumin. The thymol may be removed by shaking the urine with an equal volume of petroleum ether for two or three minutes, (b) Urates may be precipitated, but the ring is % to 1 cm. above the line of contact. The ring is broader than that caused by albumin, and disappears on warming the urine, (c) Nucleoalbumin may produce a ring % to 1 cm. above the line of contact. As nucleoalbumin is insoluble in strong acid, the ring rises as the acid diffuses upward in the urine. The ring is more marked if the urine be diluted with about three parts of water, (d) Resinous acids may form a ring above the line of contact. The ring is partially cleared on heating. The precipitate, if due to resins, may be pipetted off and dissolved in ether. When resinous bodies are sus- ^ Loc. cit. ^Weinberger, W. "Thymol as a source of error in Heller's test for urinary protein." Jour. A. M. A., 1909, LII, 1310. Digitized by Microsoft® 34 ALBUMIN pected the following test may be employed: To 8 to 10 c. c. of urine add 2 to 3 drops of strong hydrochloric acid ; the resinous bodies are precipitated. Bender strongly acid with hydrochlorid acid and heat ; a red color develops, (e) Albnmose and Bence-Jones' body form a ring at the line of contact, which disappears more or less completely on heating, (f) Urea nitrate may be deposited between the fluids. It is easily recognized, as it is not compact and uni- form, but manifestly crystalline. Dilution of the urine causes its disappearance. (4) Potassium Ferrocyanide and Acetic Acid Test.— To 10 to 15 c. c. of urine in a test tube add a few drops (about 5) of strong acetic acid to render the urine markedly acid. Nucleoalbumin, if present, is precipitated and should be removed by filtration. Now add a few drops of 5 per cent, potassium ferrocyanide. A cloud or a flocculent pre- cipitate indicates albumin. Care must be exercised not to add an excess of the ferrocyanide, as the albuminous co- agulum may be redissolved. The test is positive with al- bumin in a dilution of 1:70,000 (Grlaesgen), but, like the preceding tests, its delicacy depends much on the concen- tration of the urine in salts. Sources of Error. — Albumoses and Bence-Jones' pro- tein are coagulated, but the coagulum disappears on heat- ing — completely in the case of albumose, partially with Bence-Jones' body. Numerous other tests for the recognition of albumin in the urine have been devised. Some of them, as Spieg- ler's, are too delicate. The tests given above have been thoroughly tested, and are almost imiversally employed by clinicians. Thorough familiarity with them should be suf- ficient for all practical purposes. Digitized by Microsoft® THE URINE 35 QUANXITATIVB DeTEKMINATION OF AlBTJMIN Tsuchiya's Modification of the Esbach Method.^— Tsuchiya has devised a new reagent for precipitating the coagulable protein, to be used with the Esbach tube. The formula of Tsuchiya's reagent is: Phosphotungstic acid 1.5 gm. Hydrochloric acid, cone 5.0 c. c. Alcohol, 96 per cent., to 100.0 c. c. Method. — If alkaline, the urine is acidified with a few drops of acetic acid to prevent bubbling, when the reagent is added. The Esbach tube is filled with urine to the mark U, and then the reagent is added to the mark E. The tube is corked and inverted twelve times to insure thorough and uniform mixing of the urine and reagent. (Do not shake, since bubbles clinging to the precipitate cause it to float.) The tube is placed in a vertical position for twenty-four hours at room temperature to allow the precipitate to settle, when the height of the precipitate is read on the scale marked on the tube. The figure obtained gives the quantity of albumin in grams per liter. With large quantities of albumin the urine should be diluted with water, so that the reading will be below 4 gm. per liter, for Mattice has shown that above this the error increases greatly. Tsuchiya's is a great improvement on the Esbach re- agent, and should supplant it. With Esbach 's reagent as the precipitant, the results are often not even approxi- mately correct. Some of the advantages of Tsuchiya 's re- agent over that of Esbach are: (1) that the precipitate * Mattice, A. E. "The quantitative estimation of albumin in the urine." Arch., Int. Med., 1910, V, 313. Digitized by Microsoft® 36 BENCE- JONES' BODY rarely floats, but (2) settles evenly in the bottom of the tube ; (3) the readings are much less affected by slight vari- ations in temperature; (4) the average error is very greatly reduced, amounting to less than 0.3 gm. per liter (controlled by the Kjeldahl and gravimetric methods), so that daily variations in albumin output can be followed with considerable accuracy, and (5) the reagent is clean, and does not stain hands or clothes (Mattice). Glucose in the urine does not interfere with the accuracy of the test. Normal urines usually yield a slight precipitate when treated with Tsuchiya's reagent, but the bulk of it is so small that it is not measurable, and in no way interferes with the test. Removal of Albumin from the Urine.— As albumin in- terferes with certain reactions, it is necessary at times to remove it before performing other tests. A convenient method is the heat and dilute acetic acid test. The coagu- lated protein is removed by filtration, and the filtrate tested by one of the other tests to determine whether it is al- bumin-free. BENOE-JONES' BODY This protein is of rare occurrence. There is no simple, decisive, qualitative test by which it may be recognized. Its presence may be strongly suspected, though not abso- lutely proved, by the following reactions: If alkaline or neutral, acidify the urine with dilute acetic acid ; filter the specimen, if necessary, to render it clear. (1) On heating the urine slowly in a test tube there appears a milky tur- bidity at about 52° C. ; at 60° C. the precipitate is abun- dant and sticky. After the temperature rises above 70° C. Digitized by Microsoft® THE URINE 37 the precipitate usually lessens materially, and may entirely disappear at the boiling point, though a slight cloud usu- ally persists. As the urine cools the precipitate reappears. (2) The addition of an excess of nitric acid to the cold urine causes a precipitate, which is partially or completely dissolved on boiling, but again separates as the tempera- ture becomes lower. (3) Similar reactions may be obtained with many of the tests for albumin. That all of these re- actions are influenced very greatly by the acidity and the salt content of the urine has been shown by Massini.^ The further identification of Bence- Jones' protein is more or less complicated ; the reader is referred to the larger works or to the literature. ALBUMOSE The secondary or deuteroalbumoses, though of wide oc- currence in the urine in disease, are ordinarily of little diagnostic importance. Their presence may be shown in the following manner (Simon) : Strongly acidify a few c. c. of urine with acetic acid, and then add an equal vol- ume of saturated solution of sodium chlorid. The pres- ence of albumose is indicated by the occurrence of a pre- cipitate, which disappears on boiling and reappears on cooling. Since albumin is usually present in the urine with albumose, the boiling urine should be filtered to re- move the albuminous precipitate. A cloud, which develops in the filtrate on cooling, signifies albumose. To the hot filtrate an excess of sodium hydrate is added to render it strongly alkaline, then 1 per cent, copper sulphate drop by drop, when a red color appears (the biuret test). * Massini, R. " Untersuchungen bei einem Falle von Benee- Jones 'seher Krankheit. ' ' Deutsch. Arch. f. Min. Med:, 1911, CIV, 29. Digitized by Microsoft® 38 GLUCOSE GLUCOSE (Dextrose, Grape Sugar) Glucose is present normally in the urine in traces, the quantity varying between 0.015 and 0.04 per cent, in the twenty-four-hour specimen. The amount is so small that it is not detected with the usual clinical tests. The urine to be tested should be clear. Simple filtration may be sufficient. If this fails the urine is shaken with powdered normal lead acetate, and then filtered. Qualitative Tests (1) Trommer's Test.— If more than a trace of albumin is present, it should be removed with heat and dilute acetic acid. To urine in a test tube add. one-third volume of 10 per cent, sodium or potassium hydrate, then 10 per cent, copper sulphate solution — the contents of the tube being thoroughly mixed after each addition of the copper — ^until a slight excess of cupric hydroxid remains undissolved. If sugar is present in the urine, much more copper sulphate can be added before a permanent precipitate is obtained, and the percentage of sugar may be roughly estimated in this way; the mixture turns deep blue. The upper part of the fluid is now heated just to boiling. In the presence of glucose cuprous oxid and hydroxid are formed, producing in the heated portion greenish-yellow clouds, which gradu- ally change to a brick red and diffuse throughout the fluid. The rapidity and intensity of the reaction depend upon the concentration of the glucose. With a high percentage of sugar, metallic copper may separate as a brownish-red coating on the side of the test tube (easily removed with nitric acid). The test will indicate 0.2 per cent, of dextrose. It should always be confirmed by other tests. Digitized by Microsoft® THE URINE 39 Sources of Error. — When the reduction of the copper is atypical, the interpretation of the result is in doubt. The combined glycuronates, uric acid, creatinin, creatin, are all capable of reducing copper to a certain extent. They never cause more than a dirty yellow ; the granular, red precipi- tate of cuprous oxid is missed, for ammonia, creatinin, etc., keep in solution the small amounts of cuprous oxid formed in sugar-free urines. With less than 0.2 per cent, of glu- cose, a similar result may be obtained, for the sugar itself may hold in solution a small. quantity of cuprous oxid; on cooling the red, granular precipitate may appear. The alkaptone bodies may also cause an atypical reduction. Other hexoses or pentose may be responsible for the reac- tion. Before testing with any copper solution, chloroform must be removed from the urine by boiling, as it is a fairly strong reducing agent. Urine preserved with formaldehyde may likewise give a reduction. (2) Fehling's Test. Solution (l):i Copper sulphate, cryst 34.65 gm. Distilled water to 1,000.0 c. c. Solution (2):i Eochelle salt 173.0 gm. Sodium hydrate 125.0 gm. Distilled water to 1,000.0 c. c. More than a trace of albumin should be removed from the urine before testing. Equal volumes of solutions (1)' ' If solution (1) is to be used for qualitative work only, it is not neces- sary to weigh the copper exactly on an analytical balance. In preparing solution (2), dissolve the Eochelle salt in hot water, then cool to room tem- perature, add the sodium hydrate and make up to one liter. Digitized by Microsoft® 40 GLUCOSE and (2) are mixed ^ in a test tube and boiled ; the deep blue fluid should remain perfectly clear. Now (a) add the urine in small amount, never exceeding one-half the volume of the mixed solutions originally taken. A yellow or red pre- cipitate appears at once. A second way (b) of perform- ing the test is to layer the urine over the mixed, boiled so- lutions by allowing it to run down the side of the test tube. At the line of contact a yellow precipitate, which quickly turns red and diffuses downward, is formed in the presence of glucose. The precipitate appears within a few seconds. With small amounts of glucose, the diffusion downward is lost, but the red oxid soon collects at the bottom of the tube. With the second procedure (b) there is less likelihood of confusion in interpreting the test. The test is said to reveal 0.08 per cent, of glucose. Sources of Error. — A dirty, greenish-yellow precipitate does not mean sugar in the majority of instances. The test contains all the sources of error of Trommer's test (q. v.). Chloroform, when used to preserve the urine, must be driven off by boiling. A precipitate which appears on standing means nothing. (3) Almen-Nylander's Test.— Beagent. Four grams of Eochelle salt are dissolved in 100 c. c. of warm 10 per cent, sodium hydrate. The mixture is saturated with bismuth subnitrate (add about 2.0 gm. of the latter), filtered, and placed in a dark bottle. The reagent is permanent. Albumin must be removed from the urine, since the sul- phid of bismuth, which may result from its presence, is .brown and interferes with the test. To the urine in a test tube add one-tenth volume of the reagent, mix, and place the tube in a boiling water bath ' A mixture of the two solutions is not permanent, and should, there- fore, always be freshly prepared at the time of performing the test. Digitized by Microsoft® THE URINE 41 for five mimites.^ More prolonged boiling should be avoided, otherwise sugar-free urine may reduce the bis- muth. If dextrose is present the fluid darkens, and a black precipitate of metallic bismuth separates. When the solu- tion turns dark only on cooling the test is negative. In a sugar-free urine a white precipitate of phosphate is formed. The test indicates 0.08 per cent, of glucose, maltose, or lactose, and 0.07 per cent, of levulose (Rehfuss and Hawk). Sources of Error. — Nylander's solution is not rediiced by uric acid, creatinin, the alkaptone bodies, pyrocatechin, and phosphates, and the test is, therefore, a good control of Trommer's and Fehling's tests. Pentose may cause a reduction; the same is true of hexoses. The test may be positive after eating asparagus, and also after the admin- istration of hexamethylenamin (urotropin). An excess of combined glycuronates may cause a reduction. Chloroform should be removed from the urine by boiling. Formalde- hyde, when added to the urine, reduces the bismuth. Eehfuss and Hawk agree with Kistermann that any pro- tein-free urine which gives a negative Nylander's test may safely be said to be sugar-free in a clinical sense. It is safer than either of the copper tests, and should be used more extensively than it is. (4) The Fermentation Test.— When positive, this test proves that the reducing body is a fermentable sugar. In the vast majority of instances the sugar is glucose. A piece of fresh compressed yeast about the size of a hazel nut is rubbed in a mortar with about 50 c. c. of urine, which is then filled into a fermentation tube, so that the air is completely displaced. As controls, use (a) normal urine and yeast, and (b) normal urine and yeast plus glu- Eehfuss, M. E., and Hawk, P. B. "A study of Nylander's reaction." Jour. Biol. Chem., 1909-10, VII, 273. Digitized by Microsoft® 42 GLUCOSE cose, to prove the activity of the yeast. The three tubes are set aside in a warm place (temperature 20° to 37° C.) for several hours. If the yeast is active and glucose pres- ent, alcohol and carbon dioxid gas will be evolved, the bubbles collecting at the top of the tube. No gas, or only a minute bubble, should be evolved in the control tube (a), whereas the glucose added to control tube (b) should be fermented. To lessen the danger of bacterial decomposi- tion,' the urine may be boiled before testing. The test will indicate 0.05 to 0.1 per cent, of glucose. As a further check the reduction tests may be repeated with the filtered urine after fermentation is completed. A positive test indicates the presence of a fermentable sugar. Sources of Error. — Levulose and maltose, if present, may be fermented with the evolution of gas. Before add- ing the yeast chloroform must be removed from the urine by boiling. Thymol and formaldehyde, when used as pre- servatives, may inhibit the growth of the yeast. It is said that hexamethylenamin in sufficient doses also prevents the fermentation. (5) Cippolina's ^ Modification of the Phenylhydrazin Test.— Albumin, when present, should be removed before performing the test. To 4 c. c. of urine in a test tube are added 5 drops of pure phenylhydrazin (the base) and 0.5 c. c. of glacial acetic acid (or 1.0 c. c. of 50 per cent, acetic acid) ; the mixture is boiled gently over a low flame for one minute. Now add 4 to 5 drops of sodium hydrate (sp. gr. 1.160) ; the mix- ture must still remain acid. The whole is heated a few sec- onds longer, and set aside to cool. Immediately or within 'Cippolina, A. "Ueber den Nachweis von Zucker im Harn. " Deutsche med. Wchnschr., 1901, XXVII, 334. Digitized by Microsoft® THE URINE 43 about twenty minutes, especially with a urine of low spe- cific gravity, the characteristic sheaf -like yellow needles of phenylglucosazone appear. Since their size is subject to considerable variation, high magnification is necessary at times to see them. The test is very delicate, indicating 0.05 per cent, of glucose. However, the sensitiveness of the test depends very largely upon the specific gravity of the urine. Con- centrated urines may react negatively in the presence of less than 0.2 per cent, of glucose. The reason for this is that phenylglucosazone crystals are held in solution in the presence of much urea, ammonium salts, and other nitroge- nous bodies. But with more than 0.2 per cent, of glucose typical crystals form within a few minutes, regardless of the specific gravity of the urine. In place of the characteristic needles yellow balls, which change to thorn-apple forms or rosettes, may be obtained. The latter are seen only in urine containing a pathological quantity of sugar, never in a normal urine (Cippoliha). Characteristic needles arranged in sheaves may be obtained by recrystallization from hot 60 per cent, alcohol. When the crystals are atypical the specimen should be set aside and reexamined at the end of one hour. To determine definitely that the crystals are derived from glucose and not from another sugar, it is necessary to filter them off and purify them by repeated recrystal- lization from hot 60 per cent, alcohol. (The melting point of the purified crystals may be determined ^ ; that of phenyl- glucosazone is 204 to 205° C. The melting point of levu- losazone is the same, while maltosazone crystals melt at 'For a description of methods, with critical discussion, see Menge, G. A. "A study of melting-point determinations." Bull. No. 70, Hyg. Lab., U. S. Pub. Health & Mar. Hosp. Serv., Wash., 1910. Digitized by Microsoft® 44 GLUCOSE about 207° C. The value of melting point determinations for the identification of one of the three sugars mentioned is, therefore, not great, though very helpful in differentiat- ing the osazone of pentose, melting point 168° C, somewhat less so with lactose, 200° C.) The dry, purified crystals may be identified by dissolv- ing 0.2 gm. of them in 4 c. c. of pure pyridin, to which 6 c. c. of absolute alcohol are subsequently added, and the whole well mixed. The 100 mm. tube of the polariscope is then filled with this mixture. Phenylglucosazone gives a levoro- tation of — 1° 30'. This procedure is seldom, if ever, neces- sary in clinical work. Quantitative Estimation of Glucose (1) Benedict's ^ First Method.— This is one of the best and quickest quantitative methods for the clinician. The solutions required are : Solution A: Eecrystallized copper sulphate ^ . . 69.3 gm. Distilled water to 1,000.0 c. c. Solution B: Crystalline Eochelle salt 346.0 gm. Anhydrous sodium carbonate.... 200.0 gm. Distilled water to 1,000.0 c. c. Solution C: Potassium sulphocyanide 200.0 gm. Distilled water to 1,000.0 c. c. * Benedict, S. E. "The detection and estimation of reducing sugars." Jour. Biol. Chem., 1907, III, 101; also N. T. Med. Jour., 1907, LXXXVI, 497. ' This must be accurately weighed on an analytical balance. Sols. B and C do not require exact weights of the substances. Digitized by Microsoft® THE URINE 45 For use, these solutions are mixed in equal proportions in the order in which they are given. As the mixed solu- tion (which is blue) keeps fairly well, it is practicable to prepare 300 c. c. or more, according to the demand. For measuring, pipettes or standard flasks are required. Thirty c. c. of the mixed solution are transferred with a pipette to an evaporating dish. To this are added 2.5 to 5.0 gm. of anhydrous sodium carbonate, in order to in- crease the alkalinity of the fluid. (For titrating dilute sugar solutions, the larger quantity of carbonate will be required, since the greater amount of urine which must be added will diminish the concentration of the alkaline salt.) The mixture is now placed over a Bunsen burner and is boiled, until the carbonate is dissolved. A small piece of washed absorbent cotton is added to prevent bumping. Urine is now run into the boiling solution from a burette until a heavy chalk-white precipitate of cuprous sulpho- cyanide is formed, and the blue color of the fluid begins to lessen perceptibly. The remaining portions of urine should be added in quantities of from two to ten drops (depending on the depth of color remaining and the relative strength of the sugar solution), with vigorous boiling of about one minute between each addition. The end-point is the com- plete disappearance of the blue color. The point is very sharp, and may be obtained with a single drop. If the precipitate be allowed to settle, the color in the supernatant fluid is more easily appreciated. For the complete reduction of the copper contained in 30 c. c. of the mixed solution, 0.073 gm. of glucose are re- quired. The quantity of urine used from the burette, there- fore, contains 0.073 gm. of glucose. From this value the amount of glucose in the twenty-four-hour quantity of urine is calculated. Digitized by Microsoft® 46 GLUCOSE When the urine is highly colored, its addition to the mixed solution may leave a yellowish supernatant fluid. To avoid this the urine may be decolorized by first shaking it with finely powdered normal lead acetate. The filtered urine is then almost colorless. Urine which has been preserved with chloroform may cause a precipitate of the red oxid to form in place of the white cuprous sulphocyanide. This difficulty is obviated by first boiling the urine to drive off the chloroform. It may also be overcome by substituting for solution C the follow- ing: Solution D : Potassium ferrocyanid 30.0 gm. Potassium sulphocyanid 125.0 gm. Anhydrous sodium carbonate 100.0 gm. Distilled water to 1,000.0 c. c. ■ (2) Benedict's ^ Second Method.— This method appears to be an improvement on Benedict's first method, in that the three solutions are replaced by one, which is permanent. As in the preceding method, a white precipitate of cuprous sulphocyanid is formed. Benedict's directions for the preparation of the solution and for the titration follow: Crystallized copper sulphate 18.0 gm. Anhydrous sodium carbonate ^ . . . 100.0 gm. Sodium citrate 200.0 gm. Potassium sulphocyanate 125.0 gm. Five per cent, potassium ferrocy- anid solution 5.0 c. c. Distilled water to 1,000.0 c. c. ^Benedict, S. E. "A method for the estimation of reducing sugars." Jour. Biol. Chem., 1911, IX, 57. ' 200.0 gm. of the crystallized salt may be used. Digitized by Microsoft® THE URINE 47 With the aid of heat dissolve the citrate, carbonate, and sulphocyanate in enough water to make about 800 c. c. of the mixture, and filter. Dissolve the copper sulphate sep- arately in about 100 c. c. of water, and pour the solution slowly into the other liquid, with constant stirring. Add the ferrocyanid solution cool, and dilute to exactly one liter. Of the various constituents, only the copper sulphate need he weighed with exactness. Twenty-five c. c. of the reagent are reduced by 0.050 gm. of glucose, or by 0.053 gm. of levulose. Method. — With a pipette measure 25 c. c. of the reagent into a porcelain evaporating dish (25 to 30 cm. in diam- eter) and add 5 to 10 gm. of anhydrous sodium carbonate (or twice the weight of the crystallized salt), and a very small quantity of powdered pumice stone. Heat the mix- ture to vigorous boiling over a free flame till the carbonate is dissolved, and from a burette run in the twenty-four-hour specimen of urine (diluted accurately 1:10, unless the sugar content is known to be very slight) quite rapidly, until a heavy white precipitate is produced, and the blue color of the solution begins to diminish perceptibly. From this point the urine is run in more and more slowly, with con- stant vigorous boiling, until the disappearance of the last trace of blue color, which marks the end-point. An interval of 30 seconds' vigorous boiling should be allowed between each addition of urine. The following explanatory points may be added regard- ing the solution : When ready mixed, the solution appears to keep indefinitely without any special precaution, such as exclusion of light, etc. The trace of ferrocyanid is added to prevent precipitation of red cuprous oxid, which may be caused by certain impurities. Chloroform has such a marked tendency in this respect that it must not be pres- Digitized by Microsoft® 48 GLUCOSE ent during the titration. The additional alkali is added prior to the titration in order to provide sufficient alkalin- ity to insure a sharp end-point. Should the mixture become too concentrated during the titration process, distilled water may be added to replace the volume lost by evapora- tion. (3) Polariscopic Determination.— The polariscope is an expensive instrument, and for this reason it is not as generally employed for sugar determinations as the rapid- ity and ease of its use would seem to warrant. For clinical use the instrument is supplied with two specially made tubes, 94.7 mm. and 189.4 mm. long, which permit a direct percentage reading of glucose ; the short tube is used with dark, highly colored urines, the readings obtained being divided by two. The tubes must be perfectly clean and dry before using; hot water or fluid should not come in contact with them, since the expansion of the glass against the outer brass tubing may crack the former. The twenty-four-hour specimen, acid in reaction, is fil- tered and decolorized, if necessary. This is best accom- plished by the addition of about 2 gm. of finely powdered normal lead acetate to the urine, which is then shaken vigorously and filtered. The first cloudy portions of the filtrate are returned to the filter, until the filtrate, which is almost colorless, is perfectly clear. Practically no sugar is held back by the normal lead acetate.^ The clear urine is now filled into the polariscope tube (189.4 mm. in length) until the fluid is convex above the end of the tube. The glass disc is then placed over the end of the tube and se- cured in place by screwing down the metal cap. Air bubbles ^ Neuberg, C, II. ' ' Ueber Klarung und Entf arbung. ' ' Biochem. Ztschr., 1910, XXrV, 423. Digitized by Microsoft® THE URINE 49 must be avoided, since their presence makes a satisfactory reading impossible. The tube is now placed in the polari- scope, which must be in a dark room. For illumination a sodium flame is used. After focusing, readings are made, first without the urine, to determine whether the zero point is accurate, next, after refocusing, with the tube of urine; starting at zero, the handle is rotated until the entire field is equally illuminated. At least six readings should be made. The percentage is read directly from the scale, tenths being obtained on the vernier. (In case the instru- ment is supplied only with the standard tubes of 100 and 200 mm. length, the percentage of glucose may be calcu- lated from the polariscopic readings by dividing the re- sults by 0.527.) The method gives fairly satisfactory results. When no disturbing bodies are present in the urine, the error is about 0.1 per cent, of glucose. Sources of Error. — (1) Albumin, when present, must be removed before making polariscopic determination of glu- cose, otherwise the albumin, which is levorotatory, will counterbalance the dextrorotatory glucose, in part at least. (2) Alkalinity of the urine precludes its use with the po- lariscope, since it has been shown that in alkaline media dextrose may be converted into levulose.^ The addition of a preservative to the specimen usually suffices to pre- vent an acid urine becoming alkaline. (3) p-oxyhutyric acid is levorotatory, and its presence, therefore, interferes with the accurate estimation of glucose. (4) The combined glycuronates are levorotatory, though they are generally present in such small quantity as to produce only slight rotation of the polarized light. (5) Levulose, when present ' Koenigsf eld, H. ' ' Zur Klinik und Pathogenese der Lavulosurie beim Dia- betes mellitus." Ztsehr. f. klin. Med., 1910, LXIX, 291. Digitized by Microsoft® 50 GLUCOSE in the urine with glucose, is antagonistic, and lowers the reading for glucose. (6) Maltose is occasionally present in the urine with glucose. Since it is more powerfully dextro- rotatory than glucose, the reading may give a value which is too high. From the foregoing it is apparent that the error arising from /?-oxybutyric acid may be estimated approximately by making polariscopic examination of the specimen be- fore and after fermentation with yeast. "With the combined presence of glucose and levulose, the relative proportions of each may be determined with a fair degree of accuracy by comparison of the value obtained by titration with cop- per solution and the polariscopic value. (4) Robert's Specific Gravity Method.^— This method depends upon a lowering of the specific gravity of the urine as a result of fermentation of the sugar. By obtain- ing the specific gravity of the fermented and the unfer- mented urine, the quantity of sugar may be calculated. About 2.0 gm. of compressed yeast are rubbed in a mor- tar with 50 c. c. of the twenty-four-hour specimen of urine, acidified with acetic acid if necessary. The specific gravity of the suspension is taken at once, the temperature of the mixture being noted. The mixture is set aside in a warm temperature (25 to 37° C.) in a receptacle plugged with cotton, or, better, in a large fermentation tube. When fer- mentation is complete the yeast settles to the bottom of the flask; it is well, nevertheless, to test the fluid to determine the complete disappearance of the sugar. The mixture is well stirred and a small portion removed. It is filtered and tested with Fehling's solution. If glucose still re- mains, the fermentation is allowed to continue, until there '^ Lohnstein, Th. "Ueber die densimetrische Bestimmung des Trauben- zuckers im Harne. " Arch. f. d. ges. Physiol., 1895-6, LXII, 82. Digitized by Microsoft® THE URINE 51 A is no longer a reduction of the copper. The mixture is again stirred thoroughly to restore the suspension, and the specific gravity is determined the second time. It is im- portant that the temperature of the suspension at the times of determining specific gravity does not differ by more . than 1° C. The usual urino- meters are too inaccurate for the determination of the spe- cific gravity, which should be carried to the fourth decimal place. Lohnstein's instrument (Fig. 5) is convenient and sat- isfactory for the purpose. It is an areometer, in whose stem the pan, A, is mounted. It is floated in the urine and weights are placed on the pan until the shelf, C, is exactly on a level with the surface of the fluid. The sum of the weights on the pan is the specific grav- ity of the fluid. If the pan is loaded too heavily, so that the surface, C, sinks into the fluid, it must be removed and dried. The quantity of sugar is calculated by multiplying the dif- ference in the specific gravities by the factor 234. The re- sult is glucose in grams per cent. When unfiltered urine is used (i. e., for the second determination, after fermentation is completed), the error does not exceed 5 per cent. (Lohnstein). The method permits the determination of glucose in strengths of 0.1 per cent, or more. (5) Measurement of the carbon dioxid gas formed dur- I Fig. 5. — Lohnstein's Aeeometer. A, pan for weights; C, shelf which should be level with the surface of the fluid; E, air chamber. (After Lohnstein.) Digitized by Microsoft® 52 GLUCOSE ing fermentation has been used to determine the dextrose content of urine. One of the most widely known forms of apparatus for this purpose is Ein- horn's. More accurate results have been obtained with Lohnstein's appa- ratus (Fig. 6). This consists of a J- shaped tube mounted on a stand. In the short arm of the tube a bulb is blown, the outlet of which may be closed by a glass stopper. A hole in the neck may be brought opposite a similar opening in the hollow stopper. The long arm is provided with a scale. A quantity of mercury and a pipette for measuring the urine are supplied with the apparatus. Method.^ — The mercury is poured into the apparatus. The bulb is partly filled, and mercury extends a short distance up the long arm of the tube. Then, with the pipette, 0.5 c. c. of urine and 0.1 to 0.2 c. c. of a yeast suspension^ (1 part of compressed yeast to 2 to 3 volumes of water) are placed in the, bulb on the surface of the mercury. The glass stopper (greased with vaselin 20 per cent., in yellow wax) is placed in the neck of ' Lohnstein, T. "Ueber GaTungs-Saccharometer Beschreibung eines neuen Garungs-Saceha- FiQ. 6. — Lohnstein's Fer- mentation Sacchako- meteb foe undiluted Urine. (After Wood.) nebst rometers f iir unverdiinnte Urine. ' ' Miinchen. med. WeJinschr., 1899, XLVI, 1671. ^ The quantity of yeast suspension employed depends upon the concentra- tion of glucose. With very low percentage of sugar the yeast may be rubbed up with 10 to 15 volumes of water. Digitized by Microsoft® THE URINE 53 the bulb in such a way that the openings are opposite one another; this is to avoid a positive pressure on inserting the stopper. The scale is now placed on the long arm of the tube; the zero line should correspond with the level of the mercury in the long arm. The stopper is turned, to close the bulb, and a weight (provided with the ap- paratus) is placed over it to prevent it from being blown out, as the carbon dioxid forms. The apparatus is placed in an incubator at a temperature of 32 to 38° C. for 4 to 5 hours, or at room temperature for 24 hours. The carbon dioxid formed in the bulb from fermentation of the sugar forces the mercury into the long arm of the tube. The percentage of glucose is read directly from the scale, which is provided with two columns of figures, one for the average room temperature, the other for body heat. LEVULOSE Levulose, when present in the urine, is usually asso- ciated with dextrose. Occasionally it is the only sugar in the urine, a few cases of levulosuria having been reported.^ Levulose responds to most of the tests for glucose. It is fermentable, reduces copper and bismuth, gives the phenyl- hydrazin test; there are, however, certain dissimilarities by which the two sugars may be separated. (1) Seliwanoff's Test, as Modified by Borchardt.^— Five to 10 c. c. of urine and an equal volume of 25 per cent, hy- drochloric acid (i. e., 2 parts of concentrated hydrochloric acid and one part of water) are mixed, and a few grains 'Strouse, S., and Friedman, J. C. " Laevulosuria. " Arch. Int. Med., 1912, IX, 99. "Borchardt, L. "TJeber die diabetische Lavulosurie und den qualitativen Nachweis der Lavulose im Ham." Ztschr. f. physiol. Chem., 1908, LV, 241. Digitized by Microsoft® 54 LEVULOSE of resorcin added. The mixture is boiled gently for a few seconds. A red color appears, usually followed by a brownish precipitate, if levulose is present. The fluid is now cooled, poured into an evaporating dish or beaker, and treated with sodium carbonate in substance until the reaction of the mixture becomes alkaline. It is then returned to a test tube, and shaken with acetic ether (ethyl acetate). In the presence of levulose the acetic ether is colored yellow. The test indicates 0.05 per cent, of levulose. Sources of Error. — (1) The simultaneous presence of nitrites and indican in considerable quantity may yield a positive reaction. The nitrites may be destroyed by acidi- fying the urine with acetic acid and boiling for one minute. (2) Large quantities of indican alone may interfere with the reaction by imparting a blue color to the acetic ether, making it impossible to recognize the yellow color from levulose. In such case the indican is removed by treating the urine with an equal volume of Obermayer's reagent and extracting several times with chloroform, until the lat- ter is no longer colored blue. The fluid is then poured into a fresh test tube, the chloroform being discarded, and is diluted with OQe-third volume of water, in order to reduce the strength of the hydrochloric acid to 12 to 13 per cent. A knife point of resorcin is now added, and the Seliwanoff test carried out. (3) Urorosein, when abundant in the urine, may impart a reddish- violet color to the acetic ether. To remove the pigment before applying Seliwanoff 's test, take equal quantities of urine and 25 per cent, hydrochloric acid, and extract the mixture two to three times in a sep- arating funnel with amyl alcohol. Discard the amyl alco- hol, which contains the urorosein, add resorcin, and pro- ceed with the test in the usual way. (4) It has been found Digitized by Microsoft® THE URINE 55 tliat patients taking santonin or rhubarb may give a posi- tive Seliwanoff reaction. Discontinuance of the drug causes the reaction to disappear. In performing the test prolonged boiling should be avoided. Borchardt finds no interference with the reac- tion from the presence of glucose, lactose, maltose, arabi- nose, or glycuronic acid compounds. Saccharose may yield a positive reaction, since boiling it with acid liberates levu- lose. (2) The phenyihydrazin test (see p. 42) gives crystals of phenyllevulosazone. They are indistinguishable micro- scopically from phenylglucosazone ; the melting point of each is the same. The crystals can be positively identified by determining their rotation of polarized light. They are purified by repeated crystallization from hot 60 per cent, alcohol. Then 0.2 gm. of the pure crystals are dissolved in 4 c. c. of pyridin, and 6 c. c. of absolute alcohol are added. The mixture is poured into the 100-mm. tube of the polari- scope and examined. Levulosazone gives a dextrorotation of 1° 20'. Levulosuria combined with glycosuria should be sus- pected when the quantity of glucose found on polari- scopic examination falls short of that shown by titration. A positive Seliwanoff reaction and the lack of a levo- rotatory body after fermentation practically confirm it. Pure levulosuria offers no difficulties in recognition, if • access to a polariscope may be had. The levorotation of the urine, together with positive Seliwanoff and reduction tests and the presence of a fermentable substance, makes the identification sufficiently complete, if all tests are nega- tive after fermentation. The phenyihydrazin test, as de- scribed above, removes all doubt, if positive. Levulosuria Digitized by Microsoft® 56 LACTOSE is usually unsuspected, unless the urine be examined with a polariscope. Alimentary levulosuria has been used in hepatic diag- nosis. In the absence of derangement of hepatic function an individual can take 100 gm. of levulose on a fasting stomach without the subsequent appearance of levulose, as a general rule. On the other hand, the majority of patients with liver disease exhibit levulosuria under such condi- tions.^ MALTOSE Maltose is occasionally present in the urine, usually in association with glucose. It reduces copper and bismuth solutions, and is fermentable. Maltose has about two and one-half times the dextrorotatory power of glucose, where- as its reducing power is only about two-thirds that of glu- cose. Therefore, its presence may be suspected when po- lariscopic values exceed the results found with the reduc- tion methods. LACTOSE Lactose may appear in the urine of women physiologi- cally in connection with stasis of milk in the breasts. Cu- pric salts are reduced more slowly than by glucose. Am- moniacal silver nitrate is reduced by lactose in the cold. Lactose is not fermentable by yeast. If equivocal results are obtained with the usual compressed yeast, the fermen- • tation may have been due to contaminating bacteria. With a pure culture of Saccharomyces apiculatus, lactose is not fermented. ^ For a discussion of this test, see Churchman, J. W. " The Strauss test for hepatic insufficiency. ' ' Bull. Johns Hopkins Hosp., 1912, XXIII, 10. Digitized by Microsoft® THE URINE 57 SACCHAROSE Saccharose is seldom encountered in the urine. It is dextrorotatory. After inversion (heat 75 c. c. of the urine with 5 c. c. cone. HCl for five minutes at a temperature be- tween 68 and 70° C), the fluid becomes levorotatory, since the levulose which is formed more than neutralizes the glucose. Reduction tests become positive after inversion of the sugar. PENTOSE Pentose is rarely found in the urine. Pentoses are sugars with five carbon atoms. The only one of importance in the urine — r-arabinose — is, unlike other sugars, optically inactive. It reduces copper and bismuth solutions slowly and incompletely; with Nylander's solution a grayish pre- cipitate may be obtained. Pentose does not ferment with yeast. Pentose should be suspected when the reduction tests are atypical, when they persist after attempts at fer- mentation, when the urine is inactive on polariscopic exam- ination. The following tests may also be employed : (1) The Phloroglucin Test.— To about 5 c. c. of urine in a test tube add an equal volume of concentrated hydro- chloric acid and a liberal knife-point (ca. 30 mg.) of phloro- glucin. The mixture is heated, preferably on a water bath. A red color appears, and, soon afterward, a dark precipi- tate forms. The contents of the test tube are cooled, and are then extracted with amyl alcohol. Spectroscopic exam- ination of the amyl alcohol extract reveals a band midway between D and E, a little to the right of the sodium line. Sources of Error. — Glycuronic acid compounds yield a positive phloroglucin test, including the absorption band, thus lessening greatly the value of the test. Lactose and Digitized by Microsoft® 58 PENTOSE galactose give the same color reaction as pentose, but the characteristic absorption spectrum is lacking. (2) The Orcin Test.— Equal parts of the urine and con- centrated hydrochloric acid (sp. gr. 1.19) and a small knife- point of orcin are boiled gently. If pentose is present a dark greenish color soon develops, and, finally, a turbidity, due to a dark blue or green precipitate. The contents of the test tube are cooled, until they are lukewarm, and are then extracted with amyl alcohol. The latter exhibits a dark, olive-green color, the depth of which depends largely upon the concentration of pentose in the urine. If the fluid is cold instead of lukewarm when extracted, the amyl al- cohol is reddish and the absorption bands are not so plainly visible (Salkowski). Spectroscopic examination reveals a band at D, the sodium line. Sources of Error. — The orcin test is also given by the paired glycuronic acid compounds. However, the latter react with orcin less readily than with phloroglucin, so that of the two the orcin test is to be preferred. It has been shown ^ that filter paper may contain pentose-like sub- stances, which are soluble in hydrochloric acid. The urine should, therefore, not be passed through filter paper. Glass wool or asbestos should be employed in its stead. (3) Bial's Modification 2 of the Orcin Test. Eeagent : Orcin 1.0 gm. 30 per cent, hydrochloric acid 500.0 c. c. 10 per cent, ferric chlorid 25 drops Keep the reagent in a dark bottle. ' Umber, F. • " Notiz iiber Pentosenreactionen in filtrirten Fliissigkeiten. ' ' Berlin. Tclin. Wchnschr., 1901, XXXVIII, 87. ^ Bial, M. ' ' Ueber die Diagnose der Pentosnrie mit dem von mir angege- benen Eeagens. ' ' Deutsche raed. Wchnschr., 1903, XXIX, 477. Digitized by Microsoft® THE URINE 59 Method. — Heat about 4 c. c. of tlie reagent to boiling and then add a few drops of the urine to be tested. "With pentose a green color develops immediately or in a few seconds. The quantity of urine employed should not ex- ceed 1 c. c. Performed in this way, the test reacts only with pentose, not with paired glycuronic acid compounds (Bial). The green fluid is extracted with amyl alcohol and ex- amined spectroscopically, as in the orcin test. The specificity of the test has been questioned by a num- ber of observers. GLYCURONIC ACID Grlycuronic acid (glucuronic acid) does not appear as such in the urine, but becomes paired or conjugated in the body with various substances, such as indoxyl, skatoxyl, phenol, in which form it is excreted in the urine. In small quantity it is normally met with. Glycuronic acid also combines with numerous drugs. Urochloralic acid, the chloral hydrate compound, is an example. When present in considerable amount, the glycuronates may lead to diffi- culty in analysis, since many of their reactions resemble those of glucose and other carbohydrates. With copper solutions the glycuronic acid compounds may give a slow, atypical reduction, often a greenish-yel- low precipitate — a reaction quite like that produced by pen- tose or by very weak solutions of dextrose. The Nylander test may be positive. The phloroglucin and orcin tests are given by the combined glycuronates. The glycuronates do not give the phenylhydrazin test of Cippolina, though the test may become positive if the urine be boiled previously with 1 per cent, sulphuric acid to liberate glycuronic acid; Digitized by Microsoft® 60 GLTCURONIC ACID the crystals obtained melt at 114° to 115° C. The combined glycuronates do not ferment with yeast. It happens, there- fore, that, when the urine contains abnormal quantities of both glucose and glycuronates, fermentation does not cause a complete loss of reducing power. The glycuronic acid compounds are levorotatory in acid urine (inactive if the reaction is alkaline), whereas glycuronic acid itself is dex- trorotatory. Therefore, boiling one to five minutes with 1 per cent, sulphuric acid changes a levorotation to dextro- rotation, or, if glucose be present, it may increase the dex- trorotation, provided some of the sugar is not destroyed. Pentose (r-arabinose) is optically inactive. /S-oxybutyric acid is levorotatory. To distinguish between the levorota- tion produced by this acid and that due to the combined glycuronates, the urine is precipitated with subacetate of lead and filtered. The glycuronates are precipitated, while /J-oxybutyric acid appears in the filtrate, where its presence may be indicated by polariscopic examination. Or, the fi- oxybutyric acid may be extracted by shaking the urine with ether three or four times, the glycuronates remaining in the urine. Normal urine may contain enough levorotatory sub- 'ances to produce 0.1 degree of levorotation; when the glycuronates are increased, the levorotation is 0.2 degree or more.- B. ToUens' Test.^— To 5 c. c. of urine in a test tube add a bit of naphthoresorcin about the size of a millet seed and then 5 c. c. of concentrated hydrochloric acid (sp. gr. 1.19). Boil the mixture gently about one minute, and set the tube aside for about four minutes. Now cool the contents of the ' Tollens. C. ' ' TJeber den GlvknTonsanren Na^hweis dureh die B. ToUensehe Eeaition mit Naphthoresorcin und Salzsaure. ' ' Miinchen. med. Wehnshr., 1909, LVI, 652. Digitized by Microsoft® THE URINE 61 tube under running water. Extract with an equal volume of ether. (The separation of the ether may be hastened by the addition of a few drops of alcohol.) If glycuronates are present in the urine in excess, the ether extract is dark blue to violet, while with smaller amounts a faint bluish or reddish violet color is obtained. Examined spectroscopi- cally, the ether extract shows a single dark band near the sodium line. (The examination should be made at once,^ as the substance giving rise to the band is not stable.) In place of naphthoresorcin in substance, 0.5 c. c. of a 1 per cent, alcoholic solution of naphthoresorcin may be substituted. The test is suflSciently delicate to detect the small quantities of glycuronates present in normal urine. The dark pigments formed. in this reaction by pentoses and other sugars are insoluble in ether. ALKAPTONURIA Alkaptonuria is a very rare condition, a disturbance in metabolism. Of the alkapton bodies, two, homogentisinic acid and uroleucinic acid, have been isolated. When pres- ent in the urine they may give to it the following charac- teristics : The fresh urine is markedly acid. It is normal in color when voided, but on standing oxidation quickly changes the color to a reddish-brown and, finally, to a black. The color changes occur more rapidly when the reaction of the urine is alkaline. The urine reduces copper and silver (the latter in the cold) but not bismuth. The urine does not give the phenylhydrazin test, does not rotate the plane of polarized light, and is not fermentable. 'Brooks, B. Personal communication. 6 Digitized by Microsoft® 62 ACETONE ACETONE Acetone, a ketone, occurs in normal urine in amounts as high as 10 mg. in twenty-four hours. It is a colorless, .odorless liquid, very volatile, of low specific gravity. In testing the urine for acetone, it is usually necessary to distill the specimen. Occasionally, when very large quan- tities of acetone are present, positive reactions for acetone may be obtained by testing the urine directly. But in no case do such tests, when negative, exclude an acetonuria. When tests of the urine are negative, it becomes necessary to distill a portion of it and to apply the tests for acetone to the distillate. Between 200 and 300 c. c. of urine are acidified with 1 to 2 c. c. of concentrated hydrochloric acid^ and distilled. The greater part of the acetone is contained in the first 20 or 30 c. c. of the distillate, which is used for the tests to be described. If distillation of the urine is impossible, about 50 c. c. of urine are extracted with 20 c. c. of ether in a separating funnel. The urine is then allowed to es- cape, and to the ether about 10 c. c. of water are added. The fluids are well shaken. A large part of the acetone is in the water, which is then used for the qualitative tests.^ Qualitative Tests (1) Gunning's Test.— Five c. c. of the distillate are ren- dered alkaline with 5 to 10 drops of ammonium hydrate, " Phosphoric acid may be used. The acid is added only to prevent the dis- tillation of ammonia and excessive foaming of the urine. ^Bohrisch, P. Pharm. Zentralhalle, 1907, XLVIII, 5; 184; 206; 220; 245. Cited by F. N. Schulz in Neubauer-Huppert 's Analyse des Hams, 11th Ed., p. 252. Wiesbaden, 1910. Digitized by Microsoft® THE URINE 63 and then Lugol's solution (potassium iodid, 6 gm., iodin, 4 gm., distilled water to 100 c.c.) or tincture of iodin is added until the deep black precipitate which forms no longer dissolves at once. This gradually disappears and is replaced by a yellow precipitate of iodoform crystals, rec- ognized by their characteristic odor and morphology. The crystals are often so small that the high power dry objec- tives of the microscope are required. They are hexagonal plates, often clustered in the form of six-pointed stars. When atypical, the crystals should be recrystallized from alcohol-free ether. They are colored yellow. When the test is applied directly to the urine the phosphates are pre- cipitated by the ammonia, usually in the form of crystals resembling fern leaves. With very small quantities of acetone it may be necessary to wait twenty-four hours for the crystals of iodoform to form. This test is the best qualitative test, since a positive reaction is obtained only with acetone. It is slightly less sensitive than Lieben's test. According to Bohrisch, the test should be applied only to the distillate, not to the urine directly. (2) Lieben's Test.— A few drops of sodium or potas- sium hydrate and then a little Lugol's solution are added to about 5 c. c. of the distillate, and the mixture is warmed. With large quantities of acetone, an immediate precipita- tion of yellow iodoform crystals (hexagonal plates or six- pointed stars) occurs. When the amount of acetone is small (0.01 mg. or less), a few hours may be required for the formation of the crystals, which are detected by micro- scopic examination of the sediment. Warming the tube in- tensifies the characteristic iodoform odor. The test is very delicate. Crystals may be demonstrable after twenty-four hours with as little as 0.0001 mg. of acetone. If the crys- Digitized by Microsoft® 64 ACETONE tals are atypical, the precipitate is dissolved in alcohol-free ether and recrystallized. Sources of Error. — Both alcohol and aldehyde give Lieb- en's test. (3) Legal's Test.— A few small crystals of sodium nitroprussid are dissolved in about 5 c. c. of the distillate. An excess of sodium or potassium hydrate is now added. If acetone is present, a red color develops, which soon changes to yellow. Glacial acetic acid, added in excess while the color is still red, causes a change to purplish red and finally to violet. The test indicates about 0.1 per cent, of acetone. Sources of Error. — According to v. Jaksch, paracresol gives a yellowish-red color with sodium nitroprussid and sodium hydrate ; on adding an excess of glacial acetic acid the color changes to a rose red, and may be confused with the acetone reaction. Creatinin causes the same prelimi- nary color changes as acetone, but on acidifying with gla- cial acetic acid the color gradually becomes green and then blue. When testing the distillate this difficulty is removed. (4) Lang'e's Test.^— About 15 c. c. of urine are placed in a test tube and treated with 0.5 to 1 c. c. of glacial acetic acid. After the addition of a few drops of a freshly pre- pared solution of sodium nitroprussid, ammonium hydrate is carefully layered above the urine. In the presence of acetone an intense violet ring appears at the line of contact. The quantity of nitroprussid used is unimportant, but the amount added should not be enough to color the urine. The test, which is a modification of Legal's, is sensitive to ace- tone in 1/400 per cent, solution. The reaction is not given by alcohol or aldehyde. 'Lange, F. "Eine Eingprobe auf Azeton." Miinchen. med. Wchnschr., 1906, LIII, 1764. Digitized by Microsoft® THE URINE 65 DIACETIC ACID Diacetic acid or acetoacetic acid, the precursor of ace- tone, is usually found in urines which contain abnormal amounts of acetone. When urine is allowed to stand the diacetic acid soon becomes converted into acetone, which in turn is lost within a few hours through volatilization or decomposition. Diacetic acid may, however, be kept in the urine with little loss for weeks by the addition of toluol to the specimen in a tightly stoppered bottle. Unless toluol be added, the urine should be tested for diacetic acid soon after it is voided. (1) Gerhardt's Test.— To about 20 c. c. of urine in a test tube add an excess of 10 per cent, ferric chlorid solu- tion. If a precipitate forms, it is removed by filtration. To the filtrate more ferric chlorid is added, as long as it pro- duces a perceptible darkening in color. A deep Bordeaux red color is produced by diacetic acid. The contents of the test tube are now halved, one portion being boiled, the other set aside as a control. If the color be due to diacetic acid boiling for several minutes (two or more) lessens its in- tensity very perceptibly, owing to the breaking up of the diacetic acid. The test indicates 0.04 to 0.05 per cent, of diacetic acid. Sources of Error. — After the administration of various drugs, notably salicylic acid, aspirin, diuretin, salol, phe- nacetin, acetates, formates, etc., a red color, at times indis- tinguishable from that produced by diacetic acid, may be seen. Except in the case of formates and acetates, the color does not fade after boiling a few minutes or standing several hours, as is the case when it is due to diacetic acid. When both diacetic acid and one of these drugs coexist, the urine is distilled, and the distillate tested for acetone. Digitized by Microsoft® 66 DIACETIC ACID When the disturbing body is either a formate or an acetate, the urine is aciduhited with sulphuric acid, cooled if neces- sary, and then extracted with an equal volume of ether. With a pipette the ether is transferred to another test tube and a small quantity of very dilute watery ferric chlorid solution added ; the tube is then well shaken. Diacetic acid causes a violet color in the watery layer, which changes to a Bordeaux red on the addition of more ferric chlorid. The color fades quickly on boiling the watery layer (remove ether first!). Formates and acetates do not give this reaction. (2) Arnold's Test.i Eeagents : Solution A. — 1 gm. of paramidoacetophenon is dissolved in 80 to 100 c. c. of distilled water with the aid of hydro- chloric acid added drop by drop during vigorous shaking. Acid is added till the yellow solution becomes water clear. An excess of hydrochloric acid is to be avoided. Solution B. — Sodium nitrate, 1 per cent, aqueous solu- tion. The solutions keep well. Method. — Two parts of solution A are mixed with 1 part of solution B (always prepare freshly at the time of making the test). Add an equal volume or less of the sus- pected urine and then 2 to 3 drops of strong ammonia, shaking well. All urines give a more or less intense brown- ish-red color. With excessive quantity of diacetic acid the addition of the ammonia produces an amorphous, brown- ish-red precipitate, but with smaller amounts no precipi- tate forms. A portion of the reddish fluid is placed in a wine glass or test tube, and a great excess of concentrated ' Arnold, V. ' ' Bine neue Eeaktion zum Nachweis der Aeetessigsaure im Harn." Wiener Uin. Wohnshr., 1899, XII, 541. Digitized by Microsoft® THE URINE 67 hydrochloric acid is added (to 1 c. c. of fluid add about 10 to 12 c. c. HCl). In the presence of diacetic acid the mix- ture takes on a beautiful purplish-violet color. With large amounts of diacetic acid the violet predominates, while with smaller quantities the red is more evident. With nor- mal urine (free of diacetic acid) only a yellow color is ob- tained. With small amounts of diacetic acid the reaction may fail if the urine be highly colored. In such case filter the urine through animal charcoal, and the reaction becomes positive with the water-clear filtrate. In using the filtrate add 2 to 3 parts of filtrate to 1 part of the mixed reagent. The reaction is specific for diacetic acid and its ethyl ester. It is not given by drugs and is more delicate than Gerhardt's test (Arnold). /3-OXYBUTYRIC ACID /5-oxybutyric acid, the third of the ' ' acetone bodies, ' ' is found in the urine only in the presence of acetone or dia- cetic acid, or both, though the converse of this is not true. It occurs in largest amount in certain cases of diabetes mel- litus. Its presence may be suspected when the urine is found to be definitely levorotatory after fermentation of the glucose ; such a finding is not, of course, conclusive evi- dence of the presence of this body. (1) Black's Test.i— Five or 10 c. c. of urine are concen- trated in an evaporating dish at a gentle heat to one-third or one-fourth of the original volume, which eliminates the acetacetic acid. The residue is then acidified with a few drops of concentrated hydrochloric acid, and made to a thick paste with plaster of Paris and allowed to stand until 'Black, O. F. "The detection and quantitative determination of yS-oxy- butyric acid in the urine. ' ' Jour. Biol. Chem., 1908, V, 207. Digitized by Microsoft® 68 /3-OXYBUTYRIC ACID it begins to set. It is then stirred and broken up in the dish with a blunt stirring rod. The porous meal thus ob- tained is extracted twice with ether by stirring and decan- tation. The ether extract, which contains /5-oxybutyric acid, is evaporated spontaneously or on the water bath. The residue is finally dissolved in water and neutralized with barium carbonate. .The fluid is now poured into a test tube and treated with 2 or 3 drops of commercial hydrogen peroxid, the whole being mixed by shaking. The /J-oxy- butyric acid is oxidized to diacetic acid. Now add a few drops of 5 per cent, ferric chlorid containing a trace of ferrous chlorid. On standing a few seconds a beautiful rose color develops, which slowly intensifies until it reaches a maximum, and then gradually fades, owing to the further oxidation of the acetacetic acid. Sources of Error. — Black says that the chief precau- tions to be observed in carrying out the test are to be sure that the solution is cold and nearly neutral, and to avoid a large excess of hydrogen peroxid and iron. If too much of the oxidizing agents is added, and but little yj-oxybutyric acid is present, the color developed is transitory or fails to appear. By starting with a small quantity and then add- ing more ferric chlorid at intervals of a few minutes, until no further color is produced, one is able to observe the full intensity of color, and thereby get a rough idea as to the amount of /?-oxybutyric acid present. The test is delicate. Black found that a solution con- taining 0.1 mg. per cubic centimeter, or one part in 10,000, gave an easily recognized color. (2) Hart's ^ Test.— Hart adds to 20 c. c. of the suspected urine 20 c. c. of water and a few drops of acetic acid, and ^Hart, T. S. "The detection of /S-oxybutyric acid in the urine." Amer. Jour. Med. Sc, 1909, CXXXVII, 869. Digitized by Microsoft® THE URINE 69 boils, until the volume is reduced to about 10 c. c. To this residue add water to the original volume, 20 c. c. Put this into two test tubes (B and C) of equal size, 10 c. c. in each test tube. To one of the test tubes (C) add 1 c. c. of peroxid of hydrogen, warm gently for about one minute (do not boil), and then allow the fluid to cool. Add to each test tube 0.5 c. c. of glacial acetic acid and a few drops of a freshly prepared solution of sodium nitroprussid, and mix. Overlay the solution in each test tube with 2 c. c. of ammo- nium hydroxid (Lange's test, p. 64). Allow the tubes to stand for four or five hours, and at the end of this time compare them. At the point of contact between the ammonia and the underlying fluid, B will show no ring (or a faint brown ring, if much creatinin is pres- ent) ; test tube C, to which the hydrogen peroxid was added, will show a purplish-red contact ring, if /^-oxybutyric acid was originally present ; if >8-oxybutyric acid was not pres- ent the two test tubes will not differ in appearance. If the two tubes are now shaken the difference in color will be seen throughout the fluid; this difference is intensified by allowing the tubes to stand for fifteen or twenty minutes after shaking. The oxidation of the /J-oxybutyric acid to acetone by means of the hydrogen peroxid is said to be gradual, and reaches its maximum in about four or five hours, after which the color slowly fades. When a very large amount of /J-oxybutyric acid is present the difference in the two tubes may become evident in a few minutes. The two tubes should always be prepared as above. B will show whether all preformed acetone and diacetic acid have been driven off. The presence of sugar does not interfere with the reac- tion. If albumin is present, it should be removed. Digitized by Microsoft® 70 UROBILIN The method, though simpler than Black's, does not com- pare with the latter in delicacy. Hart finds that it will cer- tainly detect /5-oxybutyric acid when present to the extent of 0.3 per cent, and probably less. UROBILINOGEN Urobilinogen is normally present in the urine in traces. It is converted into urobilin within a few hours after the urine is voided, so that it is necessary to employ fresh specimens in testing for it. In the twenty-four-hour speci- men an excess of the chromogen, though originally pres- ent, may be missed by the time the examination is made; in this case urobilin may be looked for. Ehrlich's Aldehyde Test. Eeagent : ^ Dimethylparamidobenzaldehyde .... 2.0 gm. Hydrochloric acid (5 per cent.)?. . . .100.0 c. c. Dissolve. Keep in dark brown glass bottle. About 10 c. c. of urine in a test tube are treated with a few drops of the reagent. In the presence of abnormally large amounts of urobilinogen a red color develops in the cold. In normal urine the red color appears only after heating. If the color fails to develop on heating, urobili- nogen is absent. UROBILIN Urobilin, whose chromogen is urobilinogen, is a con- stituent of normal urine. Though it be lacking in the fresh- ly voided specimen, traces of it are soon present, due to *As the reagent does Bot keep well, it should be prepared ia small quaa- tity, according to the demand. = About 14 c. c, cone. HCl diluted to 100 c. c. with water. Digitized by Microsoft® THE URINE 71 the action of light on urohilinogen. Large quantities of the pigment may impart a deep yellowish-brown color to the urine, though an excess may be present without noticeable change in the appearance of the specimen. (1) Spectroscopic Determination.— When there is a considerable excess of urobilin it may be detected by direct spectroscopic examination of the urine. A small hand spectroscope is most convenient for the purpose. A few c. c. of clear urine, previously treated with a few drops of Lugol's solution (about 1 drop to 2 c. c), and a few drops of mineral acid are examined directly with the spectro- scope. The characteristic spectrum of acid urobilin, a single baud (Fig. 7) between the green and the blue parts of the spectrum (between the lines b and F and extending a little to the right of F in the green), is seen. If the urine is very highly colored it may be necessary to dilute it before examining it with the spectroscope. On the other hand, with small amounts of urobilin the pigment should be ex- tracted from the acidulated urine with amyl alcohol. The extract then presents the band of urobilin in acid solution. The filtrate in the next two tests may also be examined spectroscopically. It must be remembered that urobilin in alkaline solution shows a band between b and F which is nearer b than that seen in acid solution. In solution with ammonia and zinc, the band is well seen. But if alkali be added to the urine for direct spectroscopic examination, fixed alkali will produce a darker band than ammonia. (2) Schlesinger's ^ Test.— This is the best test for rou- tine work. It is delicate, easily performed, and requires no special apparatus. About 10 c. c. of acid urine are treated with 5 or 6 drops Schlesinger, W. "Zum klinischen Nachweis des Urobilins." Deutsche med. Wchnschr., 1903, XXIX, 561. Digitized by Microsoft® 72 UROBILIN of Lugol's solution to convert any urobilinogen present into urobilin. The urine is now mixed with an equal quan- tity of a saturated solution of zinc acetate in absolute al- cohol and filtered. When held against a dark background and examined with transmitted light, the filtrate shows a beautiful green fluorescence, whose intensity is propor- tional to the quantity of urobilin present. The fluid may be examined spectroscopically. The least trace of eosin or other fluorescing compound on the glassware may lead to misinterpretation of the re- sult of the test. The fluorescence may be made to appear more marked if the light be focused on the tube with a small hand lens. The alcoholic zinc acetate solution precipitates other pigments, which may interfere with the reaction. How- ever, when bilirubin is very abundant it is precipitated by adding 2 c. c. of 10 per cent, calcium chlorid solution to 8 c. c. of urine. The mixture is filtered and the test ap- plied to the filtrate. The test is sensitive to 0.002 per cent, solutions of urobilin in urine, even in the presence of bile pigments. (3) Jaffe's Test.— The urine is treated with a few drops of Lugol's solution and then with an equal volume of 10 per cent, alcoholic solution of zinc chlorid. The precipi- tate which forms is removed by filtration. The filtrate is rendered strongly alkaline with ammonia. A green flu- orescence denotes the presence of urobilin. On spectro- scopic examination the spectrum of urobilin in alkaline so- lution may also be observed. The single band is a little nearer b than that seen in acid solution (Fig. 7). The test is not as delicate as Schlesinger's test. Zinc chlorid precipitates the interfering bodies less completely than zinc acetate, but alcoholic zinc chlorid is preferable to Digitized by Microsoft® THE URINE 73 the aqueous solution, which is frequently recommended (Schlesinger). When it is desired to determine whether urobilin is to- tally absent from the urine, large quantities of urine treated with Lugol's solution and mineral acid are extracted with a small volume of amyl alcohol, which may then be sub- jected to spectroscopic examination or to Schlesinger 's test. The aldehyde test of Ehrlich is equally decisive in proving the absence of the pigment if a perfectly fresh specimen of urine be employed. BILE PIGMENTS Bile pigments, never normally present in the urine, may or may not cause an appreciable alteration in its appear- ance, the result depending on the concentration of the col- oring matter. When much is present the urine has a dark brown, at times a greenish-brown, color. The pigments exist in the urine as such or as soluble combinations with the alkalies or alkaline phosphates. Thus it happens that bilirubin (hematoidin) in crystalline form is not usually seen in the urine; but in the urine of infants with small content in phosphates such crystals not infrequently form. They appear' as yellowish or brownish-red needles or as rhombic plates or prisms, the latter often with rounded angles. The crystals are insoluble in water. In chloro- form, especially if hot, they dissolve readily (1:600), im- parting their color to the solution. In dimethylanilin bili- rubin crystals are very soluble (1:100). The alkaline com- pounds of bilirubin found in the urine are insoluble in chloroform. Bilirubin may, therefore, be removed from chloroform solution by alkali. The bilirubin as found in Digitized by Microsoft® 74 BILE PIGMENTS the urine is precipitated by the addition of hydrochloric acid. From ammoniacal solution bilirubin is precipitated by barium chlorid, lead acetate, and silver nitrate. Solutions of bilirubin possess no characteristic absorp- tion bands; there is a continuous absorption from the red to the violet end of the spectrum. Qualitative Tests (1) Foam Test.— The urine is shaken vigorously and, if it contains considerable bile pigment, the foam presents a distinct yellow color. The test is practically specific, but is not very delicate. Similarly, icteric urines show a yellow staining of the sediment, and a similar color is left on filter paper, through which such urines have been passed. (2) Gmelin's Test. — The acid urine is superimposed carefully on yellow nitric acid ^ in a test tube ; the layering of the two fluids must be sharp. In the presence of bilirubin a play of colors is observed at the line of contact of the two fluids. The colors from above downward are green, blue, violet, red, yellow. A piece of white paper, held behind the test tube, with the light at the examiner 's back, aids in the recognition of the colors. The green color is the most im- portant. The reaction is said to be positive in a dilution of 1 :80,000. Albumin and urobilin do not interfere with the reaction. Much indican may lead to confusion at times; other tests should then be resorted to. (3) Rosenbach's Modification of Gmelin's Test.— The urine, slightly acidified with hydrochloric acid, is passed 'Yellow nitric aeid is quickly obtained by adding a small piece of pine or other soft wood to nitric acid. In a short time, nitrous acid, HNO^, is evolved. The aeid should be light yellow; too much nitrous acid may accelerate the oxidation to such an extent that the colors are missed entirely. Digitized by Microsoft® THE URINE 75 through a small filter paper several times. The paper is then unfolded and blotted lightly with dry paper. The stain on the paper is now touched with a drop of yellow nitric acid, when, in the presence of bile pigment, a play of colors is seen at the edge of the drop. From within outward the colors are yellow, red, violet, blue, and green. The green color, the result of oxidation of bilirubin to bili- verdin, is again the most important color; in its absence the test is negative. If the paper be allowed to dry, it should be moistened with a drop or two of water before applying the acid. The test is very delicate and practi- cally specific. (4) Huppert's Test.— In deeply pigmented urines or in those rich in indican or hemoglobin, this test is preferable to Gmelin's. The urine is made alkaline with sodium or ammonium carbonate, and then calcium chlorid solution is added as long as a precipitate forms. The mixture is passed through a small filter, the precipitate washed with water, and the precipitate and filter paper transferred to a test tube or porcelain evaporating dish. Acid alcohol (concentrated hydrochloric acid, 5 c. c, alcohol, 95 c. c.) is now added and carefully heated to boiling. In the pres- ence of bilirubin the color of the alcoholic solution changes to green or blue. The delicacy of the reaction varies be- tween a dilution of bile of 1:500,000 and 1 :1,000,000 (Ham- marsten). (5) Hammarsten's Test. Eeagent : Nitric acid (25 per cent.) 1 part Hydrochloric acid (25 per cent.) ... 19 parts The reagent may be kept for about a year. It is not ready for use until its color becomes yellow. Digitized by Microsoft® 76 HEMATOPORPHYRIN Ordinarily it is sufficient to pour a few drops of urine into 2 or 3 c. c. of the reagent. Almost immediately after shaking, the mixture takes on a green or bluish-green color, which will persist about twenty-four hours. When only traces of bile pigment are present, 10 c. c. of the acid or neutral (not alkaline) urine are treated with a 10 per cent, solution of barium chlorid, which is added as long as a precipitate forms. The mixture is now centrifugalized. The supernatant fluid is poured off and the sediment is shaken with about 1 c. c. of the reagent and again centrifu- galized. The supernatant fluid is now a beautiful green, which changes upon further addition of the reagent through blue to violet, red, and finally reddish-yellow. The green color is obtained in the presence of 1 part of bile pigment to 500,000 to 1,000,000 parts of urine. In the presence of considerable amounts of blood-coloring matter or other pigments, a 10 per cent, solution of calcium chlorid should be substituted for the barium chlorid solution. HEMATOPOEPHYRIN Hematoporphyrin, an iron-free derivative of hemoglo- bin, is a normal urinary pigment occurring in traces. When urine contains hematoporphyrin in considerable concentra- tion, its color is usually dark red. Garrod's Test.— One hundred c. c. of urine are treated with 20 c. c. of 10 per cent, sodium hydrate ; this precipi- tates the phosphates, which carry the pigment down with them. The precipitate is collected by filtration or cen- trifugalization, and is dissolved in acid alcohol (HCl, 5 c. c, alcohol, 95 c. c). The solution is examined spectro- scopically for the bands of hematoporphyrin in acid solu- tion (Fig. 7), one just to the left of D, the other — a broader Digitized by Microsoft® THE URINE 77 band — between D and E. The result may be further con- trolled by obtaining the absorption bands of hematoporphy- rin in alkaline solution. The alcoholic solution is rendered alkaline with ammonia, acetic acid is added till the precipi- tate of phosphates is dissolved, and the pigment is then ex- tracted with chloroform. The latter is examined spectro- scopically for the four bands of hematoporphyrin in alka- line solution (Fig. 7) ; the first about midway between C and D, the second at D extending to the right of it, the third at the left of E, the fourth^a broad band — beginning at b and extending almost to P. In place of sodium hydrate, Salkowski recommends that the urine be treated with a solution composed of equal parts of cold saturated barium hydrate and 10 per cent, barium chlorid, while Hammarsten prefers a solution of barium acetate. In either case the precipitate is washed and then dissolved in acid alcohol, as in Garrod's test, and the alcoholic solution is examined for the spectrum of hem- atoporphyrin in acid solution. By adding an excess of ammonia the bands of alkaline hematoporphyrin are ob- tained. In certain instances the urine contains hematoporphyrin in such concentration that direct spectroscopic examination reveals its presence. In such case the alkaline spectrum is the one usually observed, though the spectrum of hem- atoporphyrin in acid solution may be seen, and is still sharper after acidifying the urine with a mineral acid. HEMOGLOBIN Hemoglobin in the urine is always pathological. The hemoglobin is often changed to methemoglobin. If the urine contains hemoglobin in relatively high concentration Digitized by Microsoft® 78 HEMOGLOBIN it assumes a dark red color, which is imparted to the sedi- ment. The latter, however, is often dark brown. Occa- sionally crystals of hematoidin are found on examination of the sediment (see bilirubin crystals) ; when placed in a porcelain dish with a drop of yellow nitric acid, a play of colors, especially a green ring, is seen at the periphery of the drop (Gmelin's reaction). In hemoglobinuria red blood corpuscles are usually absent or present in small number. (1) Spectroscopic Determination (Fig. 7).— The urine, if neutral or alkaline, is rendered slightly acid with dilute acetic acid, and is filtered till perfectly clear. If very highly colored it may be necessary to dilute the specimen before examining it spectroscopically. (a) Oxyhemoglobin is characterized by the appearance of two bands between D and E, the narrower band being near D. (b) Reduced hemoglobin is recognized by a single broad band, extending from D toward E. (c) Methemoglobin in neutral or weakly acid solution produces a dark band in the spectrum between C and D, near C. The two additional bands seen between D and E are usually attributed to the coexistence of oxyhemoglobin in the solution. In alkaline solution methemoglobin pre- sents two absorption bands between D and E, resembling those of oxyhemoglobin, except for the fact that the nar- rower band in this case is situated at the right. (d) Hematin is found in the urine very infrequently. In acid solution its spectrum resembles closely that of methemoglobin in neutral or acid solution, consisting of a single dark band in the red, extending to the right of C. Hematin is readily differentiated from methemoglobin by the fact that the addition of ammonia and a reducing sub- Digitized by Microsoft® THE URINE 79 stance, such as ammonium sulphid, to the acid sohition con- verts the spectmmi into that of hemocliromogen. B c I Wavelength. "^H r^^ ^^O 111 I ill I nil E b F 5oa I L Oxyhemoglobin. Reduced hemoglo- bin. IMethemoglobin. Homtitin in acid so- lutiun. Reduced hematin. Hematoporphyrin in acid solution. Hematoporphyrin in alkaline solu- tion. Urobilin. Fig. 7. — Absorption Spectra. (After Seifort and Miillcr.) (e) Hemocliromogen [reduced hematin (d)] presents two dark l)ands, one ahout midway between D and E, Digitized by Microsoft© 80 HEMOGLOBIN the other to the right of E. Both bands are nearer the green end of the spectrum than those of oxyhemo- globin. In case the urine is very deeply pigmented, the spectro- scopic examination is facilitated by dilution with water, since concentrated solutions absorb the spectrum diffusely. On the other hand, with small amounts of hemoglobin, the delicacy of the spectroscopic test depends very largely upon the thickness of the layer of urine, through which the light passes to the spectroscope. Schumm ^ finds that with the usual test tube hemoglobin may be detected spectro- scopically in a dilution of 1 :2,000, whereas, if the urine be placed in the polariscope tube 10 or 20 cm. long, it is pos- sible to recognize one part of hemoglobin in about 25,000 parts of urine. Eoughly, this is equivalent to one drop of blood in the twenty-four-hour specimen. If the oxyhemo- globin has been changed to methemoglobin, Schumm recommends the following procedure : 50 c. c. of urine, 5 c. c. of glacial acetic acid, and 40 to 50 c. c. of ether are shaken together. The ether, after it has separated, is drawn off and shaken with 5 c. c. of water, which is then removed. The guiac test (p. 81) is then applied to a part of the ether extract. To the remainder add ammonia in excess (keep the mixture cool) and shake well. The ammonia layer and a ptirt of the ether are allowed to run into a glass, ammonium sulphid is added, and the bands of alkaline hemoehromogen are looked for. The following tests are applicable alike to the detection of hemoglobinuria and hematuria: '■ Schumm, O. ' ' Untersuchungen iiber den Nachweis von Blut im Ham mit Hilfe des spektroskopischen und einiger spektroskopisch-chemischer Ver- fahren." Miinchen. med. WcJmschr., 1908, LV, 1488. Digitized by Microsoft® THE UEINE 81 (2) The Guiac Test.^ Eeagents : Guiac resin, powdered. Alcohol. Hydrogen peroxid or ozonized oil of turpentine. Tincture of guiac is freshly prepared at the time of making the test by adding a knife-point of powdered guiac to about 5 c. c. of alcohol, shaking till solution occurs. Equal parts of tincture of guiac and hydrogen peroxid are mixed and are then layered above the urine by inclin- ing the test tube and pouring the mixture in very slowly. The urine, if neutral or alkaline, is acidified with acetic acid before testing. An opaque ring forms at the line of contact between the fluids; gradually a distinct blue color develops. The test is very delicate, but it is not specific for blood. Disturbing substances are much less apt to be encountered in the urine than in the stools or gastric contents. Pus, when present, gives the blue color, but the reaction occurs Avithout the addition of the peroxid. The urine may be treated with glacial acetic acid and extracted with ether (for details consult p. 159). For a complete list of the disturbing substances the reader is referred to the mono- graph of Kastle. This test is very delicate; it may be positive with 1 part of blood in 20,000 to 40,000 parts of urine. It is chiefly of value when negative. A positive test does not mean the presence of blood — it must be confirmed by other tests ; but a negative reaction is conclusive evidence of the absence ' For a full discussion of this and allied tests, see Kastle, J. H., ' ' Chemical tests for blood." Bull. No. SI, XJ. S. Pub. Health & Mar. Hosp. Serv., Wash., pp. 1-62, 1909. Digitized by Microsoft® 82 HEMOGLOBIN of blood. The activity of the guiac should be proved occasionally. Several other chromogenic substances have been used successfully in place of guiac, but they are all open to the same objection, i. e., lack of specificity. Bensidin, aloin, and phenolphthalin are the bodies most frequently substi- tuted for guiac. (3) Heller's Test.— The urine, if alkaline, is rendered neutral or slightly acid with acetic acid, and is then boiled. If much blood is present, a precipitate of albumin and hematin forms. The hot urine is now treated with so- dium or potassium hydrate. The phosphates are precipi- tated and carry down with them any hematin present. The latter colors the precipitate red, which constitutes a posi- tive reaction. In case the phosphates have already been precipitated from the urine, normal urine may be added to supply the salts in solution ; or a little calcium chlorid so- lution is added to the urine, which is then boiled, and so- dium phosphate is poured into it with the sodium hydrate (Hammarsten). To prove beyond -question that the red precipitate is caused by blood pigment, the precipitate is subjected to Teichmann's hemin crystal test. Heller's test is not very delicate, and is, therefore, less used now than formerly. (4) Teichmann's Hemin Crystal Test.— The precipitate obtained in Heller's test or from treating the urine with tannic acid is used for this test. The excess of phosphates may be removed by washing the precipitate with very dilute acetic acid. The precipitate is then dried on the fil- ter paper, and a small amount of it transferred to a clean glass slide. To it add a few small crystals of sodium chlo- rid. Crush the crystals and mix the powder with the pre- Digitized by Microsoft® THE URINE 83 cipitate. A cover glass is placed on the material, and gla- cial acetic acid is run under it. Heat the preparation just short of boiling % to 1 minute, replenishing the acid as necessary. The fluid turns brown. The specimen is al- lowed to cool a few minutes, and is then examined micro- scopically for the brown rhombic plates of hemin. It is often necessary to reheat the specimen several times be- fore the crystals are obtained. Instead of heating the speci- men, it may be set aside for twenty-four hours before ex- amining it ; in this case the crystals are usually somewhat larger. With small crystals, high magnification may be required for their recognition. The test is specific for hemoglobin. It often fails if too much sodium chlorid is added, or if the specimen is over- heated. THE DIAZO REACTION Ehrlich's diazo reaction is never given by normal urine, but is of frequent occurrence in febrile diseases, compara- tively rare in afebrile conditions. Eeagents : Solution 1: Sodium nitrite 0.5 gm. Distilled water 100.0 c. c. Dissolve. The solution does not keep well. Solution 2: Sulphanilic acid 5.0 gm. Hydrochloric acid, cone 50.0 c. c. Distilled water to 1,000.0 c. c. Dissolve. Digitized by Microsoft® 84 CHYLURIA One part of solution 1 is mixed with 50 parts of solution 2. The mixture should be prepared freshly each day, as it is not permanent longer than twenty-four hours. Equal quantities of the mixed solutions and the urine are mixed in a test tube. Ammonium hydrate is then run down the side of the tube, which is inclined, so that it forms a layer at the top. A red ring is formed at the line of con- tact of the fluids. The test tube is sealed and shaken vigor- ously. If the foam is colored pink, the reaction is posi- tive. The color fades rather rapidly. It is quite possible to misinterpret the result of the test, since a salmon or yellowish-red or brownish-red foam is not infrequently observed. It is essential to discard all results as negative unless the foam is unquestionably pink. At times the bodies causing the diazo reaction exist in the urine in such dilution that a positive reaction is not obtained. In such cases the test again becomes positive, if the urine is concentrated to a small volume on a water bath. If the sodium nitrite solution is used in greater strength than 1 :50, normal urine may give the color reaction. After the administration of atophan (phenyl-quinolin-carboxylic acid), 3 gm. daily, a positive diazo test may be given by normal urine.^ CHYLURIA The admixture of chyle causes the urine to appear more or less milky, depending partly on the proportion of chyle, but still more on its fat content. Whether parasitic (fila- ria) or non-parasitic in origin, chyluria is probably always ' Skorczewski, W., and Sohn, I. "Ueber einige im Atophanharne auftre- tende cliarakteristisehe Eeaktionen. " Wiener TcUn. Wchnschr., 1911, XXIV, 1700. Digitized by Microsoft® THE UEINE 85 due to a direct anatomical comiminication between the lymph channels and the genitourinary tract, as Magnus- Levy ^ and others have pointed out. Ureteral catheteriza- tion often reveals the fact that the chyluria is unilateral. The symptom is intermittent, as a rule, depending on the posture of the patient ; in some cases it appears during the day, in others only at night. All of the normal ingredients of chyle may be found in the urine. They are : (1) Neutral Fat. — Droplets of neutral fat are always present; they are derived from the chyle, not from the blood, and the quantity found varies directly with that in- gested in the food. The droplets vary considerably in size; some are so small that they are only seen distinctly with the oil immersion. They possess a sharp contour and are highly refractive. The addition of a drop of Sudan III or of Scharlach E (saturated solution in 70 per cent, alcohol) stains the fat droplets an orange or red- dish-yellow color. From the alkaline urine the fat may be extracted by means of the usual fat solvents, such as ether. (2) Cholesterin and lecithin are found if large quan- tities of urine are extracted with ether. Their quantity is dependent largely on the food. (3) Sugar may or may not be discovered in the urine. It has been shown that chyle contains about 0.1 per cent, sugar in hunger or on a fat or protein diet. One part of chyle in two parts of urine under these circumstances would give about 0.03 per cent, glucose — too little to detect with the usual clinical tests. On the other hand, after a large carbohydrate meal, especially a meal containing an excess of sugar, the urine may contain 0.3 to 0.4 per cent. ' Magnus-Levy, A. ' ' TJeber europaische Chylurie. ' ' Ztschr. f. TcUn. Med., 1908, LXVI, 482. Digitized by Microsoft® 86 FERMENTS IN THE URINE of glucose. With this amount glycosuria is easily recog- nized. (4) Lymphocytes are always to be seen, either in the sediment of the centrifugalized specimen or caught in the meshes of the clots, which occasionally form in the urine. (5) Albumin is generally found in the urine, unless the proportion of chyle be very small. With appropriate tests, such as fractional precipitation of the urine with ammo- nium sulphate, globulin and fibrinogen may usually be dem- onstrated. (6) Filaria Bancrofti. — In parasitic chyluria the em- bryos of Filaria bancrofti are present in the sediment or in the clot (see p. 117). LIPURIA Small quantities of fat are not unusual in the urine. When epithelial or pus cells are present it is common to find fat droplets in them, and a few droplets are found free in the urine. The term lipuria is reserved for those conditions in which the fat is so abundant that it is recog- nized macroscopically. It is important to exclude fat from external sources, such as dirty containers for the urine, the lubricant on catheters, the willful admixture of fat or milk for purposes of deception, etc. Fat is recognized by its appearance and microchemical reactions. Unless it is present in the form of an emulsion, it is seen on the sur- face of the fluid. FERMENTS IN THE URINE A number of enzymes have been discovered in the urine — pepsin, trypsin, lipase, diastase, etc. From a diagnostic standpoint diastase appears to be the most important, Digitized by Microsoft® THE URINE 87 though the determination of lipase has also been of some value in pancreatic disease. Wohlgemuth's Method for the Determination of Dias- tase.^— A 1 per cent, starch solution is prepared. Merck's or Kahlbaum's soluble starch is employed. The starch powder is stirred in cold distilled water, which is then heated about 10 minutes with constant stirring, until the solution is clear. It is cooled and is then ready for use. Into each of several test tubes 5 c. c. of the starch solu- tion are placed with a pipette. Next add varying fractions of 1 c. c. of urine to the tubes, which have been numbered, beginning with 0.2 c. c. and decreasing gradually. Add a small quantity of toluol to each tube to prevent bacterial growth, and place the tubes in the incubator at 37° C. for twenty-four hours. The tubes are then removed from the incubator and filled almost completely with ice water. To each tube add 1 drop of ^ iodin solution, mix well, and observe for the blue color of the starch-iodin reaction. The first tube which shows no blue is selected. From the known proportions of urine and starch solution in this tube, cal- culate the number of cubic centimeters of 1 per cent, starch solution which 1 c. c. of urine would convert to dextrin and sugar. Assuming the result to be 150, it is expressed as follows : D |.|.° =150. This means that the urine examined contained sufficient diastase (D) to convert 150 c. c. of 1 per cent, starch solution to dextrin and sugar, acting at 37° C. for 24 hours. Wohlgemuth employs the urine obtained at the second voiding in the morning for examination. The diastase is 'Wohlgemuth, J. (a) "Ueber eine neue Methode zur quantitative!! Be- stimmung des diastatischen Ferments." Biochem. Ztschr., 1908, IX, 1. (b) ' ' Untersuchungen ueber die Diastasen. Beitrag zum Verhalten der Diastase im Urin." IMd., 1909, XXI, 432. (c) "Beitrag zur fuuktionellen Diagnostik des Pankreas." Berlin, klin. Wchnschr., 1910, XL VII, 92. Digitized by Microsoft® 88 FERMENTS IN THE URINE greatest in the urine during fasting, and decreases three to four hours after meals. The highest normal value which he has obtained for the urine is 156. (2) Determination of Lipase According to Hewlett.^— Hewlett has adapted the ethyl butyrate method of Kastle and Loevenhart to the determination of lipase in the urine. The procedure follows, in the author's words: Five c. c. of urine are placed in each of three flasks. The urine in the second flask is then boiled. To the urine in the third flask are then added three drops of a 1 per cent, solution of phenolphthalein and tenth normal sodium hydrate is al- lowed to run in from a burette, until a faint pink color ap- pears throughout the fluid. The amount of sodium hydrate used is read off, and a like amount is added to the first and second flasks. To each of these two flasks, the first of un- boiled urine, the second of boiled urine, are then added 0.25 c. c. of ethyl butyrate and 0.1 c. c. of toluene, and they are placed in a thermostat at 38° C. for about 20 hours. The toluene is added to prevent the growth of bacteria. At the end of this time each flask is taken out, and sufficient tenth normal hydrochloric acid is added to more than neutralize the alkali previously added by 0.5 c. c. Each specimen is then shaken in a separating funnel with 50 c. c. of redis- tilled ether, and the ether is separated. After adding three drops of a 1 per cent, solution of phenolphthalein to 25 c. c. of pure alcohol, the latter is brought to the neutral point. The ether extract from the separating funnel is now added to the neutralized alcohol, and its acidity is de- termined by titrating with N-20 potassium hydrate solution (alcoholic). Any decided difference between the acidity of the ethereal extracts of the boiled and of the unboiled ' Hewlett, A. W. " On the occurrence of lipase in the urine as a result of experimental pancreatic disease. ' ' Jour. Med. Besearch, 1904, XI, 377. Digitized by Microsoft® THE URINE 89 •urine is due to the butyric acid formed by the cleavage of the ethyl butyrate; and, where the difference in acidity is at all great, the odor of butyric acid can be recognized. In normal healthy men (and dogs) the urine contains merely traces of lipase. The greatest difference found by Hewlett was 0.35 c. c. of twentieth normal potassium hy- drate — usually 0.1 or 0.2 c. c. THE URINARY SEDIMENTS The sediment may be obtained for microscopic examina- tion by allowing it to settle in a conical specimen glass ^ or, preferably, by centrifugalizing the urine. The objection to sedimentation is that cer- tain of the formed elements, especially casts, may be more or less completely digested, if the specimen be allowed to stand too long, whereas with a centrifuge the examination may be made almost as soon as the urine is passed. It is particularly in alkaline urines that casts rapidly disappear. It is very im- portant that a perfectly fresh specimen of urine be employed for microscopic examina- tion. With urine which has stood for twenty- four hours it is often impossible to gain a cor- rect impression of the sediment. Crystals, ^ voided, may have formed, while tha more im- which were not present when the urine was Fig. 8.— the Syd- enham Sedi- MENTiNG Glass. ■ The most satisfactory sedimenting glass with which the writer is familiar is one designed by Dr. J. S. Brotherhood (see Fig. 8). It permits one to take the specific gravity without transferring the urine to another receptacle, and its shape insures concentration of the sediment at the bottom. The weight of the base is an advantage, as the glass is not easily upset. It may be obtained from the Arthur H. Thomas Co., Philadelphia. Digitized by Microsoft® 90 THE UEINARY SEDIMENTS portant organized material may have become so altered that it is no longer recognizable. The sediment is removed with a pipette and several drops of it are transferred to a glass slide. A cover glass should not be placed on the specimen for the preliminary examination, though it may be required later. When the sediment is scanty the few drops of urine adhering to the outer surface of the pipette should be wiped off to prevent dilution of the specimen. On the other hand, with abun- dant sediment it is often advantageous to thin it somewhat so that the various elements are separated and their recog- nition made less difficult. The preliminary examination of a urinary sediment ^ should always be made with low inagnification and with the light cut off as much as possible. Usually this examination is sufficient. But, if all the elements in the preparation cannot be recognized in this way, a cover glass is placed on the drop of sediment, which is now examined under higher magnification. The oil immersion lens is not employed with a wet specimen, nor is it necessary. For microchemical tests a cover glass is placed on a drop of the sediment, and any excess of moisture removed with a blotter. With a pipette a drop of the reagent is placed on the slide at one side of the cover glass, while a piece of blotting paper is touched to the opposite side. The absorption of fluid by the paper creates a current, which draws the reagent under the cover glass. The effect uppn the sediment is observed with the low power objective. In case it is necessary to use the higher power, great care should be exercised that the lens escapes the reagent. ^H. Bieder's "Atlas der klinisclien Mikroscopie des Harnes" (Leipzig, 1898) is an extremely valuable and useful reference work on urinary sedi- ments. Digitized by Microsoft® THE URINE 91 The Unorganized Sediments For convenience the iinorganized sediments are divided into those which occur chiefly in acid urine, and those which are encountered mainly when the reaction is alkaline. It must be remembered that the classification is by no means absolute; it frequently happens that a deposit, usually found in an acid urine, persists after the reaction has be- come alkaline, and, again, that a sediment which is gen- erally met with in alkaline urine may make its appearance while the reaction is still acid. Sediments in Acid Urine (1) The Quadriurates of Sodium and Potassium.— The quadriurates of sodium and potassium, the "amorphous urates," are chiefly responsible for the pink, salmon-col- ored, yellow, or reddish deposits which may be seen in an acid urine. The salts are especially apt to be precipitated from concentrated specimens as they become cool. The precipitate absorbs the urinary pigments, urochrome (yel- low), and uroerythrin (red). Microscopically, the sediment is finely granular, the granules tending to collect in masses. On heating the specimen over the flame the urates go into solution, but are again precipitated, as the preparation cools. The addition of hydrochloric acid dissolves the de- posit; subsequently crystals of uric acid form. The latter are usually colorless. The rapidity with which the uric acid crystals appear varies greatly; often within ten or fifteen minutes they are numerous. Acetic acid also brings the urates into solution, but the formation of uric acid crys- tals may be somewhat delayed. The quadriurates give a positive murexid test (see p. 9). Digitized by Microsoft® 92 THE UEINARY SEDIMENTS (2) Uric Acid.— Uric acid may separate from an acid urine from the breaking up of the quadriurates into uric acid and biurates. Crystals of uric acid, usually colored reddish or yellowish-brown, are then deposited, giving rise to the so-called "brick dust'.' sediment. They may assume a great variety of form when viewed under the microscope. That most frequently encountered is the "whetstone" crys- tal. It is seen singly or in clusters, often arranged as a rosette. The "church- window" shape is not uncommon. Rhombic plates and six-sided prisms are also characteris- tic. Hexagonal plates are of less frequent occurrence, and may be colorless; morphologically they are indistinguish- able from crystals of eystin. The latter, however, do not give the murexid test. Needles of uric acid arranged in sheaves are rare as a spontaneous sediment, though not infrequently seen after the addition of hydrochloric acid to the quadriurates. Uric acid crystals are insoluble in acetic and hydro- chloric acids. They are unaffected by heating the speci- men. The crystals are soluble in sodium or potassium hy- drate, and may be repreeipitated by the addition of an ex- cess of hydrochloric acid. They give the murexid test (see p. 9). (3) Calcium Oxalate.— Calcium oxalate crystallizes most frequently in acid urine, but the crystals remain after the reaction has become alkaline. The crystals vary con- siderably in size ; it is often necessary to employ high mag- nification to recognize them. Most often they occur as small, highly refractive octahedra. Depending upon the position of the octahedron, its form resembles a square en- velope or a lozenge, with lines connecting the opposite angles. Dumbbell or hour-glass forms, at times with ra- dial striations, and spheroidal masses constitute rarer Digitized by Microsoft® THE URINE 93 shapes of calcium oxalate. The crystals are usually color- less, but in icteric urine they may be stained yellow. Cal- cium oxalate crystals dissolve in hydrochloric or other min- eral acid, but are insoluble in acetic acid. The envelope forms may be mistaken for triple phosphate; the latter, however, 'are readily soluble in acetic acid. Calcium sul- phate, calcium carbonate, and uric acid may assume the hour-glass form. Microchemical tests serve to differen- tiate them from calcium oxalate. (1) Calcium sulphate is insoluble in hydrochloric acid. (2) Calcium carbonate dis- solves in acetic acid with the evolution of bubbles of carbon, dioxid, which may be seen under the cover glass. (3) Uric acid is insoluble in hydrochloric acid and gives the murexid test. (4) Calcium Sulphate.— Calcium sulphate (gypsum) is a rare deposit in very acid urine. It occurs in the form of long, thin, colorless needles, as long, colorless prisms, often arranged in clusters, or as dumbbells or hour-glass crys- tals. Calcium sulphate is insoluble in mineral acids and in ammonia. (5) Monocalcium Phosphate.— Monocalcium phosphate, acid calcium phosphate, slender, colorless, rhombic tab- lets, usually in clusters, resembling somewhat calcium sulphate, is, like the latter, of rare occurrence, and is found in very acid urine. The two are easily distinguished by the solubility of the phosphate in acetic acid and in mineral acids. (6) Hippuric Acid.— Hippuric acid crystals are also very rare in the urinary sediment. They are seen as color- less, transparent, four-sided prisms, or as needles. They are insoluble in hydrochloric acid, which distinguishes them from triple phosphate. From uric acid, which they may resemble somewhat, the crystals are differentiated by the 8 Digitized by Microsoft® 94 THE URINARY SEDIMENTS fact that they do not give the murexid test, and that they are soluble in alcohol and ether. (7) Cholesterin.— Cholesterin crystals may be found in the urine occasionally. They present a characteristic shape, being rhombic plates, often superimposed, with the acute angle notched, as a rule. On the addition "of strong sulphuric acid (concentrated sulphuric acid, 5 parts, water, 1 part), the crystals are stained carmine, which later changes to violet. On adding the sulphuric acid with a little Lugol's solution the play of colors is violet, blue, green, and red. The crystals are soluble in ether. (8) Xanthin.— Xanthin crystals have been observed in human urine in only a few cases. They are colorless and, from their shape, may be mistaken for uric acid. They differ from the latter in that they are soluble on heat- ing. They also dissolve in ammonia and give the xanthin test. Weidel's Test. — On the water bath the crystals are evaporated to dryness in a porcelain dish, to which chlorin water and a trace of nitric acid have been added. The resi- due, when exposed to ammonia fumes, is stained reddish or purplish-violet. (9) Hematoidin.— Hematoidin (bilirubin) crystals may be found in icteric urine, particularly when it is very acid. They are differentiated from uric acid by the reactions given on page 73. With the murexid test they give a nega- tive reaction. Hematoidin occurs in amorphous masses or as needles, often gathered together to form sheaves, or as rhombs, colored yellow or yellowish-brown. (10) Tyrosin.—Ty rosin has been found in very few instances as a spontaneous urinary sediment. It is found precipitated in the form of needles gathered together in bundles like sheaves of wheat. In impure state tyrosin may Digitized by Microsoft® THE URINE 95 resemble somewhat spherules of leucin. The crystals are soluble in alkali, in ammonia, and in mineral acids, very slightly soluble in acetic acid. From ammoniacal solution tyrosin crystallizes spontaneously on evaporation. To obtain tyrosin from the urine ^ the twenty-four-hour specimen is treated with neutral, then with basic, lead ace- tate, as long as a precipitate forms. The excess of lead in the filtrate is removed by precipitation with hydrogen sul- phid. The filtrate is now concentrated to small volume on a water bath. By fractional crystallization tyrosin and leucin (which usually coexist) are separated, since it is chiefly the tyrosin which forms the crystalline deposit. The crystals are then subjected to the tests for tyrosin given below. The leucin which is in the filtrate is converted into its copper salt by boiling with freshly precipitated copper hydroxid. From the hot solution it crystallizes in the form of rhombic plates. The crystals are slightly soluble in water, insoluble in methyl alcohol. Piria's Test. — Dissolve the crystals of tyrosin in warm, concentrated sulphuric acid, permit the solution to cool, then dilute with water, and, finally, neutralize with barium carbonate. The mixture is then filtered, and to the filtrate ferric chlorid solution is added. A violet color appears. The test may fail if free mineral acid remains or if an excess of ferric chlorid be added. Morner's Modification of Deniges' Test. — To a few c. c. of a reagent (consisting of 1 volume of formalin, 45 vol- umes of water, and 55 volumes of concentrated sulphuric acid) add the tyrosin crystals or solution and boil. A beau- tiful green color develops. ' This and the following tests for tyrosin are taken from Hoppe-Seyler 'a "Handbuch der chemischen Analyse" (H. Thierf elder), Berlin, 1909, pp. 625 et seq., 8th edition. Digitized by Microsoft® 96 THE URINARY SEDIMENTS Hofmann's Test. — ^A few crystals of tyrosin are placed in a test tube partly filled with water, to which a few drops of Millon's reagent (one part of mercury dissolved in two parts by weight of nitric acid, sp. gr. 1.42, then warm gently, add two volumes of water, and, after standing sev- eral hours, obtain the clear supernatant fluid) have been added. On boiling the fluid is stained a beautiful red, and a red precipitate forms. (11) Leucin.— Leucin is not seen as a spontaneous sedi- ment in the urine. It is usually present with tyrosin, and may separate as globules resembling fat if the urine be con- centrated on a water bath. The globules, unlike those of neutral fat, are insoluble in ether. They are usually stained brown and present radial striations or concentric rings, or are hyalin. To obtain leucin from solution in the urine, see page 95. For its recognition the reader is referred to works on biological chemistry. (12) Cystin.— Cystin, a sulphur-containing amino-acid, occurs in the urine in the form of colorless, hexagonal plates. The presence of the crystals constitutes cystinu- ria, the manifestation of a rare disturbance of intermedi- ary protein metabolism. (The crystals are usually found in neutral or alkaline urine, but may be considered here for the sake of convenience.) Oftentimes the crystals are superimposed or overlap one another. Uric acid at times assumes the identical crystalline form and may be color- less. The two may be distinguished by the fact that cystin is readily soluble in hydrochloric acid and ammonia; fur- ther, by the fact that the murexid test is not given by cys- tin. Cystin crystals are insoluble in acetic acid, alcohol, and ether. When the crystals are atypical they may be re- precipitated from ammoniacal solution by the addition of Digitized by Microsoft® THE URINE 97 acetic acid. Microscopic examination should then reveal characteristic crystals. To isolate cystin in solution the urine is treated with neutral, then with basic, lead acetate (see under tyrosin, p. 95). The filtrate is concentrated on a warm bath. Cystin separates on prolonged standing or after the addi- tion of an excess of acetic acid. Qualitative Test. — Boil a portion of the urine with so- dium or potassium hydrate and lead acetate. A black color arises from the sulphid of lead which is formed. Albumin or other proteins, if present, must first be removed. Sediments in Neutral or Alkaline Urine In addition to the crystals described in acid urine, which frequently persist after the reaction has become alkaline, there are a number which are commonly found in alkaline urine. (1) Tricalcium and Trimagnesium Phosphates.— Tri- calcium and trimagnesium phosphate, the amorphous phos- phates, are recognized as white or grayish-white deposits, often very abundant, which are easily soluble in hydrochlo- ric and acetic acids. The lack of coloration and the fact that they do not dissolve on heating the preparation differ- entiate them from the quadriurates, which they resemble somewhat microscopically. The murexid test is negative, a further differential point. (2) Calcium Carbonate.— Calcium carbonate is also usually amorphous, and is generally found mixed with the amorphous phosphates. It differs from the phosphates in the fact that the addition of acid causes solution with the evolution of carbon dioxid. The salt may also appear as Digitized by Microsoft® 98 THE URINARY SEDIMENTS dumbbells or spheres with radiating lines, resembling sim- ilar forms of calcium oxalate, calcium sulphate, and uric acid. Its solubility in acids with gas formation identifies it as calcium carbonate. (3) Ammonio-magnesium Phosphate.— Ammonio-mag- nesium phosphate, "triple" phosphate, is the crystal most commonly observed in alkaline urine. For its formation it is necessary that ammonia be produced. It therefore happens that the crystals are occasionally encountered while the reaction of the urine is still acid, though their number rapidly increases with the progress of ammoniacal fermentation. The crystals belong to the rhombic system. The "coffin-lid" is the commonest form. Erosion of these produces the irregular X-shaped crystals. With good illu- mination triple phosphate crystals have a greenish tint. They vary greatly in size ; at times they are so large that they are visible with the unaided eye. Some of the smallest crystals, when perfect, resemble somewhat the envelope forms of calcium oxalate. Their solubility in acetic acid is a differential point. When the phosphates are precipitated artificially with ammonia, fern-like crystals are usually found. In a native sediment, particularly when it has stood for some time, it is customary to find the majority of the crystals imperfect. (4) Ammonium Biurate.— Ammonium biurate, like triple phosphate, is deposited only as a result of the lib- eration of ammonia in the urine. It forms balls or spheres of yellow or light brownish color, often with striations, of- tener with horny projections or processes, producing the so-called "thorn-apple" or "morning star" crystals. Their shape may be anything, depending on the number, posi- tion, and length of the projections. The crystals are so- luble in acetic and hydrochloric acids, with the subsequent Digitized by Microsoft® THE URINE 99 formation of uric acid crystals. They give the murexid test. It is not uncommon to find amorphous phosphates and carbonates, triple phosphate, and ammonium biurate com- bined in the sediment of an ammoniacal urine. (5) Neutral Magnesium Phosphate.— Neutral mag- nesium phosphate, dimagnesium phosphate, is a very rare sediment, which is met with in weakly alkaline urine. It forms long, refractive, rhombic plates. On treating it with 20 per cent, ammonium carbonate solution the crystals be- come opaque and the edges eroded. They are easily dis- solved in acetic acid, and may be reprecipitated by the ad- dition of sodium carbonate. (6) Neutral Calcium Phosphate.— Neutral calcium phosphate, dicalcium phosphate, is very infrequently met with in weakly acid, neutral, or weakly alkaline urine. It gives rise to colorless wedges or prisms, usually clumped together. The crystals are soluble in acetic acid. On treating them with 20 per cent, ammonium carbonate, balls of calcium carbonate are produced. The Microchemical Reactions of Sediments to Reagents The reactions described above may be summarized as follows : (1) Strong acetic acid dissolves calcium and magnesium phosphates, ammoniomagnesium phosphate, and calcium carbonate, the last with the evolution of gas. It does not dissolve calcium sulphate, calcium oxalate, uric acid, cys- tin, tyrosin (very slightly soluble), and xanthin. Salts of uric acid are slowly eroded, and after several hours crys- tals of uric acid are deposited. Digitized by Microsoft® 100 THE URINAEY SEDIMENTS (2) Hydrochloric acid dissolves all crystals excepting uric acid, hippuric acid, and calcium sulphate. (3) Ammonium hydrate dissolves cystin, tyrosin, and xanthin. Uric acid crystals are partially eroded with the formation of ammonium biurate. Calcium phosphate, cal- cium sulphate, and calcium oxalate, and the salts of uric acid are unaffected by ammonia. (4) Water in large amoimt dissolves calcium sulphate; but many other crystals are not wholly insoluble in water — uric acid and its salts, triple phosphate, tyrosin, and xan- thin. (5) Alcohol dissolves tyrosin, leuein, cystin, and hippu- ric acid. (6) Chloroform dissolves bilirubin (hematoidin) and fat. The Obganized Sediments (1) Epithelial Cells. — Epithelial cells are normally found in the urine, due to the fact that the cells of the genito- urinary mucosae are constantly desquamating. As a rule, the cells are few in number and the majority of them may be caught in the mucous threads of the nubecula, if the specimen be allowed to stand a short time. In the case of women, however, the urine frequently contains a macro- scopic sediment composed largely of enormous numbers of epithelial cells, derived chiefly from the vagina. A variety of form may be noted in the epithelial cells of the urine. The vaginal cells are rather large, squam- ous cells with relatively small, round or oval nuclei. Sheets of these cells are often shed en masse. Cells derived from the kidney are usually round or cuboidal, with large, vesicu- lar nucleus. The protoplasm of the epithelial cells is prone to under- Digitized by Microsoft® THE URINE 101 go fatty degeneration. The microscopic appearance is fairly characteristic. The droplets differ in size and may he few or numerous ; at times the cell is completely filled, and it may be impossible to demonstrate the nucleus. The fat droplets are stained a deep orange with Sudan III or Scharlach E. Occasionally myelin or albuminous granules are present in the protoplasm. Since it is quite generally agreed that it is impossible, from their morphology, to determine the origin of epi- thelial cells seen in the urine, detailed description of them is superfluous. Epithelial cells are distinguished from pus cells by the shape of the nucleus. The epithelial cell possesses a single round or oval nucleus; rarely, in disease, isolated multi- nucleated cells are observed. The pus cell, on the other hand, has a polymorphous nucleus. In the fresh sediment the nuclei are not easily seen ; they stand out sharply after the addition of dilute (3 per cent.) acetic acid. Staining is somewhat less satisfactory. "Heart-failure" cells have recently been described in the urine.^ Like those seen in the sputum, they are epi- thelial cells, which are laden with altered blood pigment. The pigment granules are light golden yellow in color. At times there is a diffuse yellowish staining of the cells in the absence of icterus. The cells are not uncommon with chronic passive congestion of the kidneys, but they are not diagnostic of the condition ; they may be found in hematu- rias unassociated with passive congestion.^ The cells are usually more or less swollen, of varying size, often very ' Bittorf , A. ' ' Ueber Herzfehlerzellen im Harne. ' ' Miinchen. med. Wchnschr., 1909, LIX, 1775. ^Koller, E. "Zum Vorkommen von 'Herzfehlerzellen' im Ham." Wiener Uin. Wchnschr., 1911, XXIV, 636. Digitized by Microsoft® 102 THE URINARY SEDIMENTS large, and at times a large, round nucleus is visible (Bit- torf). They are frequently much degenerated. (2) Pus.— In health the urine may contain isolated pus cells, though they are ordinarily missed altogether. An ex- ception is not infrequently met with in women with leukor- rheal discharge. The vaginal secretions become mixed with the urine and, as numerous pus cells may be present in the former, they are, of course, found in the examination of the urinary sediment. It is, therefore, necessary in such cases to thoroughly cleanse the external genitals before col- lecting the specimen or to obtain the urine by means of a catheter. The second procedure is the more accurate method and is to be preferred. The presence in the urine of abnormal numbers of pus cells gives rise to the condi- tion designated pyuria. When only a few cells are present there is no macroscopic alteration in the appearance of the urine, but marked pyuria causes a turbidity, and in ex- treme cases the urine may even appear creamy. The pus cells (polynuclear neutrophilic leukocytes) re- tain their characteristic morphology well in acid urine. When the urine becomes strongly alkaline, the pus forms a ropy, tenacious mass, in which the individual cells are swollen, often distorted, and so greatly degenerated that they may be no longer recognizable. However, in weakly al- kaline, amphoteric, or weakly acid urines the cells are gen- erally very well preserved, and may even exhibit ameboid activity. Microscopically, the protoplasm of the cells is finely granular. The majority of the granules are the neutro- philic granules of the cell, though fat droplets may be more or less abundant. In unaltered cells the diameter is about 12 micra — rather smaller than most of the epithelial cells. To determine the nature of the cells beyond question it is Digitized by Microsoft® THE URINE 103 necessary to demonstrate the typical nuclei. This is best accomplished by the addition of 3 per cent, acetic acid; the polymorphous nuclei are then sharply defined. The specimen is examined with the high power dry objective. Staining the sediment may be tried, but is less satisfac- tory. Carbol-thionin is one of the best stains for this pur- pose. In following a patient with pyuria, it may be desirable to count the pus cells in the urine from time to time. For this purpose the twenty-four-hour specimen should be used, and care must be exercised to prevent bacterial ammonia- cal fermentation, otherwise the cells become glued together, making a count impossible. The best chemical preservative for this purpose is formalin. Commercial 40 per cent, for- malin is added in sufficient quantity to give a solution of 1 to 2 per cent., the preservative being added to each por- tion of urine as it is collected. Such a procedure is pos- sible only in a liospital, as a rule ; when it cannot be car- ried out, 15 to 20 c. c. of formalin may be placed in the bottle in which the urine is collected. The formalin pre- vents bacterial growth and at the same time renders the cell nuclei more prominent. Its disadvantage lies in the fact that the cells are clumped together in certain instances. If an ice chest is available, simple refrigeration is to be preferred to any other method of preservation. The urine, if neutral or alkaline, is acidified with acetic acid. The specimen is well stirred to secure a uniform suspension of the cells, and the count is then made directly from it with the henjocytometer, employing the technique used for counting the blood. If the cells are very numerous, it will be found more convenient to use the red pipette. With the escape of a purulent exudate into the genito- urinary tract, the albumin of the exudate becomes mixed Digitized by Microsoft® 104 THE URINARY SEDIMENTS with the urine, constituting a false albuminuria, if the le- sion is extrarenal. It is often difScult to interpret findings when a false albuminuria, such as this, is met with. The question arises whether the albumin is derived entirely from the purulent exudate or in part from the kidneys (true renal albuminuria). The presence or absence of casts is of value in determining the latter; instrumental examination may be decisive. Posner ^ has recorded ob- servations which show that, with 80,000 to 100,000 pus cells per cubic millimeter of urine, only about 0.1 per cent, al- bumin is added to the urine. By comparing the cell count with the quantity of albumin, the source of the latter may be determined. The following chemical tests for pus may be applied to the urine or to the sediment: (a) The Guiac Test. — Equal parts of hydrogen peroxid and freshly prepared tincture of guiac (see p. 81), when layered over the urine, cause a blue ring to appear at the line of contact in the presence of pus. It may be necessary to wait a few minutes for the color to appear. The color disappears on boiling, unlike that caused by the presence of blood. The test is quite delicate, but is not specific. (b) Meyer's^ Guiac Test (adapted to the urine). — A drop or two of the centrifugalized sediment is transferred to a test tube about two-thirds full of water. The contents of the tube are well mixed and allowed to extract a few minutes, in order to liberate the oxidizing enzyme of the pus cells. The fluid is halved. On one portion freshly pre- pared tincture of guiac (without hydrogen peroxid) is su- ^ Posner, C. "Ueber Harntriibung. " Deutsche med. Wchnsohr., 1897, XXIII, 635. ^ Meyer, E. (a) ' ' Beitrage zur Leukocytenf rage. ' ' Miinchen. med. WchnscJir., 1903, L, 1489. (b) "Ueber die oytodiagnostische Bedeutung der Guajakreaktion. " Ibid., 1904, LI, 1578. Digitized by Microsoft® THE UEINE 105 perimposed carefully, and at the line of contact a blue ring appears, wMcli fades in the course of about a half hour. The remaining portion is boiled actively for two to three minutes, then cooled and treated with tincture of guiac in the manner just described. Boiling destroys the ferment, and the test is therefore negative. The test is delicate, and points definitely to the presence of an oxidase ; in the urine the only common source of oxidase is pus. If much albumin is present, the reaction may be inhibited.^ (3) Blood.— Red blood corpuscles are never found in normal, voided urine, excepting the admixtures of blood which occur during menstruation. Hematuria is the term used to signify blood in the urine. A small number of blood corpuscles produce no visible change in the appear- ance of the urine. With larger quantities the translucency of the urine is lost, it becomes "smoky" in appearance on agitating the specimen, and darker in color. A reddish- brown sediment composed largely of red cells may settle out. The chemical tests for blood are given in connection with hemoglobinuria (p. 80 et seq.). The microscopic examination is made with the high power dry objective. The erythrocytes may be well pre- served, and exhibit their characteristic morphology and color. If laking of the cells has occurred, the majority, or all, of the cells appear as "shadows," i. e., the color- ing matter has escaped from the red cell, and only the cell membrane remains. To detect the shadows it is essential that the light be cut off as much as possible. In concen- trated urine crenation of the red cells, giving rise to thorn- apple forms, is observed. (4) Casts.— The occurrence of casts in the urine, cylin- ' Watson, Helen. Personal communication. Digitized by Microsoft® 106 THE URINARY SEDIMENTS druria,^ is very frequent in disease, and may also be ob- served in old age and in association with so-called physio- logical albuminurias. The casts are derived from the renal tubules. They vary greatly in size, the longest measuring in the neighborhood of 1 mm. The thickness of casts is also variable, but in a given cast the width is quite uniform. (a) Epithelial casts are composed of renal epithelial cells in whole or in part. Any cast to which one or more renal epithelial cells are attached may conveniently be des- ignated epithelial (Emerson). The cells are not of equal size, some being large, others smaller. They have a round or oval nucleus, and are usually flat and polygonal. In most instances the protoplasm of the cells is degenerated, showing fat droplets, albumin granules, or, more rarely, myelin droplets. It is unusual to find true epithelial cylin- ders possessing a distinct lumen. To distinguish between epithelial and pus cells it is necessary to demonstrate the morphology of the cell nuclei by the addition of dilute acetic acid. The cast may be mixed, i. e., it may contain both epithelial and pus cells, it may be partly cellular, partly granular, etc. (b) Pus casts, like epithelial casts, consist in whole or in part of pus cells. The cells are generally smaller and rounder than the epithelial cells. Their protoplasm is fine- ly granular, but is subject to the same degenerations as that of epithelial cells. The cells are characterized by their polymorphous nuclei, which are usually visible only after treating the specimen with dilute acetic acid or a dye. At times the cells are so degenerated that the nuclei are no longer demonstrable. (c) Blood casts, when pure, are clots which form in the renal tubules. However, any cast in which one or more 'BmeTSon, C. P. " Cylindruria. " Jour. A. M. A., 1906, XL VI, 5; 89. Digitized by Microsoft® THE URINE 107 blood cells are visible is designated a blood cast. At times the red blood corpuscles are not well preserved; shadows of red cells and granules or crystals of bematoidin may be attached to the cast. (d) Fatty casts result from the fatty degeneration of the cells of epithelial casts, or, less commonly, of pus casts. Often the outlines of the original cells are preserved. The casts usually have a yellowish or even brownish tint. The droplets vary considerably in size, some being almost as large as a cell. Ether dissolves the fat droplets, and they may be stained by adding Sudan III or Scharlach R to the preparation. Occasionally fatty acid needles project from the cast. (e) Coarsely granular casts are whitish, yellow, or very dark brown in color, quite opaque, and are covered, either partly or entirely, by coarse granules, as their name indi- cates. Some of the granules dissolve in ether and stain with osmic acid or other fat stains, while others are al- buminous and are soluble in acetic acid. Occasionally granules resembling myelin droplets are observed. Coarse- ly granular casts are probably derived from epithelial and pus cell casts. All stages in transition may be seen. The casts are often partly waxy. (f ) Finely granular casts resemble the coarsely granu- lar, but they are much less opaque and the granules are much finer. Transitions from the coarsely to the finely granular are met with. The granules may cover part or all of the cast. The non-granular portion of the cast may be cellular; more frequently, it is hyalin. Fat droplets are of much less frequent occurrence than in the coarsely granular variety, and myelin droplets are exceptional. The finely granular casts are best seen with low illumination. They are one of the commonest types of cas(t in disease. Digitized by Microsoft® 108 THE URINARY SEDIMENTS (g) Hemoglobin casts are very rare and are always as- sociated with hemoglobinuria. They are covered with dark, granular pigment; less commonly needles of hematoidin are attached to the casts. (h) Waxy {colloid or amyloid) casts are opaque, very refractive, and white or yellowish in color. Their appear- ance suggests bodies made of paraffin or wax, according to the color of the cast. They are very brittle, and not un- commonly present transverse fissures or cracks. During centrifugalization they may be broken; the fragments are then seen in the sediment. Some waxy casts give the iodin reaction for amyloid when treated with Lugol's solution. It is not unusual to find the cast in the form of a spiral or corkscrew. Cells may be attached to waxy casts, or they may be granular in part. They are supposed to be de- rived from coarsely granular casts or from hyalin casts which have remained in the renal tubules for some time. (i) Hyalin casts are pale and very slightly refractive. Unless most of the light is cut off, it is impossible to see them; they are almost glassy in their translucency. They may be stained by dilute gentian violet or by Lugol's solu- tion, and are then easily found. They are usually narrow and have rounded ends. In a urine undergoing ammoniacal fermentation they disappear more rapidly than any other kind of cast. Between the glassy hyalin cast and the waxy cast there is a large intermediate group, consisting of casts which are much less opaque than waxy casts, but — though designated hyalin — possessing considerably more density than the glassy type of hyalin cast. Hyalin casts are sup- posed to represent albumin coagula from the renal tubules or a morbid, coagulable secretion of the renal cells. They are the commonest variety of cast. Upon any cast granular, amorphous urates may be de- Digitized by Microsoft® THE URINE 109 posited, producing a finely granular appearance. Bacteria in large number, attached to a cast, produce a somewhat similar picture. The uniform outline of the cast is often lost, and the artefact may be recognized as well by micro- chemical reactions. (j) Cylindroids are casts, one of whose ends tapers to a thread-like filament. They are usually hyalin, though at times granular, and have the same significance as casts. Under the name pseudocast is included anything which may be mistaken for a cast. Scratches on the glass slide, most often mislead the beginner. Particles of dust, fibers, etc., may also cause confusion. The morphology of the cast is not duplicated, and experience soon teaches one to differ- entiate. (5) Mucous Threads.— Mucous threads, sometimes in- cluded with cylindroids, with which they have nothing in common, are normally present in the urine. They make up the nubecula. They appear as long, narrow, translucent bands of mucin, of unequal thickness, often twisted or folded like a ribbon, at times branched. Nearly always a few epithelial cells and, perhaps, an occasional pus cell are attached to the threads. Unlike hyalin casts and cylin- droids, mucous threads are insoluble in acetic acid. The length of the threads is at times great; a single specimen may extend through several fields of the microscope. (6) "Clap Threads." — Clap threads {Tripp erf dden) are white or grayish-white, thread-like bodies which are seen floating in the urine, when the specimen is agitated. They are not always gonorrheal in origin, as the name sug- gests, though in the vast majority of instances associated with a chronic specific urethritis. They are % to 1 cm. or more long, and consist of a matrix of mucus, in which epi- thelial or pus cells or both are embedded. The pus cells Digitized by Microsoft® 110 MICROORGANISMS IN THE URINE may be so abundant that the thread is quite opaque and yellowish. In the gonorrheal cases it is often possible to demonstrate the presence of the gonococcus. MICROORGANISMS IN THE URINE Gonococcus.— The gonococcus is a diplococcus shaped like a biscuit or coffee bean. It is found either within the pus cells or free in the serum. It is Gram-negative. There- fore, the finding of a diplococcus in urethral pus, of charac- teristic morphology, which decolorizes with Gram's stain, makes it highly probable that the organism is the gono- coccus. To demonstrate the gonococcus the following procedure may be employed : (1) A smear of the pus is made on a glass slide. It is dried in the air and fixed by passing it through the flame of a Bunsen burner five or six times. (2) Stain 1 to 3 seconds with anilin water gentian vio- let. (Avoid overstaining. ) (To prepare anilin water gen- tian violet, 10 parts of anilin oil are thoroughly shaken with 100 parts of water, and, after standing about five minutes, the rather milky emulsion is filtered through a moistened filter paper. The filtrate should contain no large oil droplets. Now add 11 parts of saturated alcoholic solu- tion of gentian violet and 10 parts of absolute alcohol. The solution keeps not longer than eight to ten days. — Schmorl.) (3) Wash the preparation immediately in tap water and blot it till dry. (4) Add a drop of immersion oil, and examine the speci- men microscopically for diplococci. If none is found after examining three slides carefully, the chances are that diplo- cocci are not the etiological factor in the production of the purulent exudate. If suspicious organisms are seen, it be- Digitized by Microsoft® THE UEINE 111 comes necessary to determine whether they are Gram-nega- tive, i. e., whether they are decolorized after treating them with Gram's iodin solution. The further steps are: (5) Removal of the oil hy wiping the specimen with xylol. (6) The specimen is now covered with Gram's iodin solution, one to two minutes (iodin, 1.0 gm. ; potassium iodid, 2.0 gm. ; distilled water, 300.0 c. c), and then, with- out washing in water, it is transferred to — (7) Absolute alcohol, in which it is decolorized, until the specimen is colorless or yellowish-gray, excepting the thickest parts of the smear, which may still retain a little blue. Blot dry. (8) Counterstaining may be performed with Bismarck brown (vesuvin). [The stain is prepared by dissolving 2 gm. of the powdered stain in a mixture composed of 60 c. c. of 96 per cent, alcohol and 40 c. c. of distilled water. The solution is boiled carefully, and, after it has cooled, is filtered. To prevent bacterial growth, a few drops of car- bolic acid may be added (Schmorl).] The stain is allowed to act. one to two minutes. (9) Wash in water, dry, and examine. After the first staining all bacteria and cell nuclei are colored purple by the gentian violet. Decolorization re- moves the dye from all Gram-negative bacteria and from the cell nuclei. The counterstaining with Bismarck brown then stains the Gram-negative bacteria and nuclei brown, whereas the Gram-positive organisms retain the violet. Treponema Pallidum (Fig. 9).— Treponema pallidum (Spirochceta pallida), the specific parasite of syphilis, may be sought in the serum obtained from specific lesions. The surface of the lesion is first cleansed to remove Spiro- chaeta refringens and other contaminations as much as pos- Digitized by Microsoft® 112 MICROORGANISMS IN THE URINE sible, and then, if necessary, the lesion is slightly scarified or rubbed with sterile gauze. By pressure or, better still, by suction apparatus a drop of serum usually slightly blood- stained is obtained. Many red corpuscles render the exam- ination difficult and are to be avoided. The serum may be examined in the fresh state with a dark field illuminator, or preparations may be stained. Fig. 9. — Treponema pallidum (Spirocha'ta pallida) r)n the left; Spiroch^ta re- FEINGENS on thc right, (.\ftcr Emerson.) The Treponema pallidum has a length of 4 to 10 to 20 micra, is very delicate (0.5 micron or less in thickness), has a spiral form, the turns lieing numerous and close to- gether, and, in the fresh specimen, has a screw-like motion. In spite of its motility its position in tlie field remains al- most stationary. The organism stains very faintly, as its specific name indicates, and is difficult to see. Staining Methods. — Smears of the serum are prepared on glass slides or cover glasses, and allowed to dry in the air. (1) The smears may be fixed and stained with many of the modifications of the Eomanowsky stain, such as Digitized by Microsoft® THE URINE 113 Wright's, Leishman's, "Wilson's, Hasting 's.^ The tech- nique is the same as that used in staining the blood (p. 285). The Treponema pallidum is usually stained a faint blue, but occasionally has a pinkish color, while Spirochseta refringens is stained a deep blue. (2) Giemsa's stain is also a modification of the Roman- owsky stain. It has been employed extensively in search- ing for the spirochetes. Of the numerous methods of using it, the following are recommended: (a) The specimens ^ are fixed by immersion in absolute alcohol 15 to 20 minutes or by passing through the flame three times. Ten drops of Giemsa's stain (Griibler's mix- ture) are then mixed with 10 c. c. of distilled water, shak- ing after the addition of each drop of stain. (The dilution must be freshly prepared each time the stain is used.) Cover the specimen with the diluted stain, warm it till a slight steam arises, allow it to cool about 15 seconds; the stain is then poured off, and replaced by more of the diluted stain. This procedure is repeated four or five times, when the specimen is washed, dried, and mounted in balsam. The parasites are stained dark red. The slide must be free from grease, and the receptacle for the diluted stain and the staining forceps must be free from acid or precipitated stain. The water used for washing must not be acid. (b) Giemsa's^ azure-eosin staining mixture (Griibler's make) is diluted with an equal volume of pure methyl al- cohol (Kahlbaum's or Merck's) and placed in a dropping ' Geraghty, J. T. " The practical value of the demonstration of Spiro- chseta pallida in the early diagnosis of syphilis." Bull. Johns Hoplcins Hosp., 1908, XIX, 364. ^From Mallory, F. B., and Wright, J. H. "Pathological Technique." Philadelphia and London, 5th edition, 1911, p. 418. ° Giemsa, G. ' ' Ueber eine neue Schnellf arbung mit meiner Azur-eosin- losung." Miinchen. med. Wchnschr., 1910, LVII, 2476. Digitized by Microsoft® 114 -MICROORGANISMS IN THE URINE bottle. It is well to prepare only a small quantity at a time, as it is not known how permanent the solution is. The air- dried films are then placed in a small Petri dish with the specimen side up. The film is now covered with 10 to 15 drops of the alcoholic staining mixture for 1/2 minute. The preparation is thus fixed and the staining is begun. Add enough distilled water to cover the specimen (usually 10 to 15 c. c), and agitate the dish till a homogeneous mixture of the stain is secured. Allow the specimen to remain in this mixture 5 minutes. The film is now washed in dis- tilled watery dried, and mounted in balsam. The spiro- chetes are stained pink. (3) Stern's ^ Silver Impregnation Method. — The smears of serum, air-dried, are first placed in the incubator at 37° C. for a few hours. They are then transferred to a colorless glass filled with 10 per cent, aqueous solution of silver nitrate, and exposed to diffuse daylight for several hours. The preparation gradually assumes a brown color. When this has reached a certain shade (quickly learned by practice) and the film shows a metallic luster, it is removed from the silver and washed in distilled water. In a prop- erly treated specimen the spirochetes are stained deep black on a pale brown or colorless background. The organ- isms are slightly thicker than in specimens stained with Giemsa 's stain. Anomalies in staining may be encountered. At times the spirals are more deeply stained at the upper bend of the curve than at the lower, which then appears gray. Or there may be only a row of deep black granules or dots representing a spirochete. The erythrocytes are well preserved, show a delicate, black contour, and present a number of fine granules. ^ stern, M. ' ' Ueber den Naehweis der Spirochseta pallida im Austrich mittelst der Silbermethode. " Berlin. Tclin. Wchnschr., 1907, XLIV, 400. Digitized by Microsoft® THE URINE 115 The specimen should not be exposed to direct sunlight while it remains in the silver, for, though the preparation quickly becomes dark and even black, the spirochetes are unstained. (4) Bum's India Ink Method.^ — One loopful of serum is mixed on a glass slide with a loopful of India ink, ai;d spread in a thin film by means of a second slide. The slides must be perfectly clean. The film, which is allowed to dry in the air, should be dark brown or black. A drop of im^ mersion oil is placed on the specimen, which is ready for examination. Bacteria, spirochetes, blood corpuscles, etc., are unstained, and appear as refractive bodies on the dark background. According to Cohn and others, the best re- sults are obtained with Gunther- Wagner 's Chin-Ghin black pearl ink, though fair success may be had with other inks, as Carter's or Higgin's. Bacillus Tuberculosis.— The Bacillus tuberculosis is not easily recognized in the urine because of the constant presence of the smegma bacillus on the genitalia, an organ- ism whose morphology is quite similar to that of the tu- bercle bacillus, and which cannot be separated from the lat- ter with certainty by staining. It is, therefore, necessary to exclude the smegma bacillus from the urine as a prelim- inary step in the examination for the tubercle bacillus, as Young and Churchman - have shown. The technique which these authors have developed and which has proved reli- able is as follows : The foreskin, if present, is rolled back and the glans penis is washed thoroughly with green soap ' Cohn, J. S. " On the means of finding the Spirochasta pallida with spe- cial reference to the India ink method." Interstate Med. Jour., 1911, XVIII, 26. - Young, H. H., and Churchman, .J. W. ' ' The possibility of avoiding con- fusion by the smegma bacillus in the diagnosis of urinary and genital tubercu- losis." Amer. Jour. Med. ScL, 1905, CXXX, 52. Digitized by Microsoft® 116 ANIMAL PARASITES and water, using large amounts of water for the rinsing. The irrigating catheter is now introduced about six inches into the urethra (to the triangular ligament), while the patient keeps the sphincter urethrse closed to prevent fluid entering the bladder. About one quart of sterile water is employed in the irrigation of the urethra. Since the smeg- ma bacillus is not found back of the triangular ligament, the urinary tract is practically freed of this organism by the procedure just described. The patient is now in- structed to urinate into three glasses, and a portion of the urine from the third glass is centrifugalized at least five minutes at high speed. Three smears of the sediment obtained are made and stained for tubercle bacilli by the Ziehl-Neelsen method (p. 213). If a thorough examina- tion of the stained specimens reveals no acid-fast bacilli, the result of this particular examination is reported as negative. Occasionally pus is so abundant that the search for tu- bercle bacilli is very difficult. When such is the case the antiformin method may be resorted to (p. 214). The pus cells are completely dissolved. It must be remembered that all acid-fast bacteria are resistant to the action of anti- formin, so that it is necessary to observe the usual precau- tions to exclude the smegma bacillus. ANIMAL PARASITES IN THE URINARY PASSAGES (1) Trichomonas Vaginalis.— Trichomonas vaginalis,^ a flagellate closely allied to Trichomonas intestinalis, though probably not identical with it, may be found ' Dock, G. ' ' Trichomonas as a parasite of man. ' ' Amer. Jour. Med. Sci., 1896, CXXXI, 1. Digitized by Microsoft® THE URINE 117 in the vagina and occasionally in the bladder. In either locality it comes in contact with the urine, in which it may be found. It thrives only in an acid medium; this is sup- plied by the normal vagina, except during the menstrual period, when the mucosa is bathed in the bloody discharge. Trichomonas vaginalis is a pear-shaped organism with pointed extremity and, at the anterior rounded end, pre- sents four flagella. An undulating membrane is also pres- ent. It measures usually 0.015 to 0.022 mm. in length and 0.010 to 0.015 mm. in width, though larger and smaller forms occur. It appears to be a specific parasite of the female sex. (2) Filaria Bancrofti.— Filaria bancrofti (Fig. 10) is of common occurrence in tropical and sub- tropical countries. Its embryos may be found in the urine in cases of parasitic chyluria. They are either free in the urine and ac- tively motile in a fresh specimen, or caught in the clot. The em- bryos are 0.125 to 0.3 mm. long, with a thickness of 0.007 to 0.011 mm. (Blanchard). When found or suspected in the urine, the diagnosis should be confirmed by examination of the patient's blood (p. 310). (3) Dioctophyme Renale.— Dioctophyme renale (Eu- strongylus gigas), another nematode, is excessively rare in man, though not very uncommon in dogs in this country. It is the largest round-worm parasitic in man. Its habitat is the kidney. Lodging in the pelvis of the kidney, it pro- duces a pressure atrophy until, when the parasite reaches maturity, little or none of the parenchyma of the kidney re- mains. The male measures 14 to 35 cm. in length, with a Fig. 10. — Embkyo op Filaria BANCROFTI. X SO. (After Emerson.) Digitized by Microsoft® 118 ANIMAL PAEASITES thickness of 0.4 to 0.6 cm. The female is much larger— 25 to 100 cm. long and 0.4 to 1.2 cm. thick, and is bright red in color. Infection is diagnosed by finding the ova (Fig. 11) in the urine. The latter are oval, 0.064 to 0.068 mm. in their long axis by 0.042 to 0.044 mm. in the short (Blanchard). The shell is covered with an albuminous coating, which is stained brown and is thrown into ridges, making the surface of the egg appear more uneven than that of Ascaris lumbricoides, which it resembles somewhat. Fio. 11. — Ova op Dioctophyme benale. X400. (After Emerson.) The albuminous coating is lacking at the poles of the ovum and the latter appear colorless. (4) Schistosoma Hematobium.— Schistosoma hemato- bium (Bilharzia haematobia),^ a trematode, is an important urinary parasite in tropical and subtropical climates. It is especially prevalent in Egypt. The parasite lives in the veins of the urinary bladder; it deposits its ova in the veins. The ova then pass from the veins to the bladder. The ova are similar to those found in the feces (q. v.), ex- cept for the fact that the spine is terminal instead of sub- terminal (Fig. 26). As the sharp-spined ova pierce the wall of the vein, hemorrhage, of course, ensues, with the result that hematuria is a quite constant symptom of the infection. ' Lane, C. G. " Bilharziasis ; report of a case with appendicitis ; literature' since 1904." Boston Med. and Surg. Jour., 1911, CLXIII, 937. Digitized by Microsoft® THE URINE 119 PROSTATIC FLUID Prostatic fluid ^ is obtained by massage of the prostate gland per rectum, the urethra having been irrigated pre- viously. The amount of fluid obtained at a "milking" varies from a few drops to 4 or 5 c. c. The fluid is of low specific gravity, slightly tenacious, grayish-white, yellow- ish, or greenish in color, and usually has a milky turbidity from the lecithin granules contained in it. A fresh drop of the fluid is examined microscopically for the presence of motile spermatozoa. Lecithin granules vary considerably in size. The smallest are minute specks, the largest four micra or more in diameter. They are moderately refractive. Corpora amylacea, laminated bod- ies with a granular center, may be met with, especially in specimens obtained from the aged. They resemble starch granules not only in form, but also in the fact that they may be stained blue with iodin. Various kinds of epi- thelial cells may be found. In examining for epithelial and pus cells it is well to add dilute acetic acid to bring out the cell nuclei. Spermin crystals (Bottcher's crystals), transparent needles or whet-stones, are observed at times. They may resemble Charcot-Leyden crystals, but differ from the latter in that they are soluble in alkalies and in formaldehyde. FUNCTIONAL DIAGNOSIS OF THE KIDNEY Many tests, some simple, others complicated, have been introduced to measure the functional capacity of the kid- neys. All have had certain well-recognized limitations, and Trom Emerson, C. P. "Clinical Diagnosis." Digitized by Microsoft® 120 FUNCTIONAL KIDNEY DIAGNOSIS none has been particularly helpful where the two kidneys are equally involved in the. disease process, as in nephri- tis, until Eowntree and Geraghty described their phenol- sulphonephthalein test. This constitutes by far the most satisfactory and exact method of functional diagnosis, and, in the hands of a number of workers, has proved of im- mense value in the diagnosis, prognosis, and treatment of both medical and surgical diseases of the kidneys. In sur- gical affections some of the simpler tests may be used in conjunction with the "phthalein" test. The specimens ob- tained by ureteral catheterization often permit of accurate diagnostic conclusions through comparison of the micro- scopic and chemical findings from each kidney. Urea de- terminations with the hypobromite method are frequently made to advantage ; for, though the values obtained repre- sent total nitrogen more nearly than urea, nevertheless the comparative efficiency of the two kidneys may be fairly accurately determined in many instances. The information thus gained is practically always corroborated by the phthalein test, but frequently the latter will give evidence of disease when other tests are misleading. In nephritis and analogous conditions, where each kidney is involved to about the same extent, the phthalein test is the only reli- able measure of functional capacity. The Phthalein Test of Eowntree ajid Geraghty.^— Twenty to 30 minutes before starting the test the patient is given 300 to 400 c. c. of water to insure free urinary se- cretion. Then the bladder is catheterized with aseptic tech- nique, and 1 c. c. of a solution containing 6 mg. of phenol- ^ Eowntree, L. G., and Geraghty, J. T. (a) "An experimental and clinical study of the functional activity of the kidneys by means of phenolsulphone- phthalein. " Jour. Pharm. ^ Exp. Therap., 1910, I, 579. (b) "The sulphone- phthalein test for estimating renal function." Jour. A. M. A., 1911, LVII, 811. (c) "The phthalein test." Arch. Int. Med., 1912, IX, 284. Digitized by Microsoft® THE URINE 121 sulphonephtlialein ^ is administered intramuscularly into the lumbar muscles. (The solution is prepared as follows: "0.6 gm. of phenolsulphonephthalein and 0.84 c. c. of 1 sodium hydrate are diluted with 0.75 per cent, sodium chlo- rid solution up to 100 c. c. This gives the monosodium or acid salt, which is red in color, and which is slightly irri- tant locally when injected. It is necessary, therefore, to add 0.15 c. c. more of the - hydroxid, a quantity sufficient to change the color to a beautiful Bordeaux red. This preparation is non-irritant.") The catheter is retained until the dye appears in the urine, when it may be withdrawn if there be no obstruction, as from enlargement of the prostate. The urine is col- lected in a vessel, which contains one drop of 25 per cent, sodium hydrate, since the red color of the drug is appar- ent only when the reaction of the solution is alkaline. The time of appearance of the dye in the urine is noted. At the end of the first hour after administering the phthal- ein the patient urinates into a clean receptacle, and into a second receptacle at the end of the second hour. Each specimen is now rendered distinctly alkaline by the addi- tion of 25 per cent, sodium hydrate in order to elicit the maximal color. The dye is yellow or orange in an acid urine, but becomes purplish-red when the reaction is al- kaline. Place the urine (each specimen separately) in a volumetric flask of 1,000 c. c. capacity, and add distilled water to 1,000 c. c. Mix thoroughly, and filter a small por- tion for comparison with the standard solution. When the ureters are catheterized four specimens are obtained in the two hours. Each is examined colorimetrically for phthal- • The substance is supplied bj Hynson, Westeott & Co., Charles and Franklin Sts., Baltimore, Md. It is dispensed in glass ampuls. The dose is 1 c. c. Digitized by Microsoft® 122 FUNCTIONAL KIDNEY DIAGNOSIS ein content. The functional capacity of each kidney is de- termined, and the sum of the four determinations indicates the total function. The standard solution is an aqueous solution of phenol- sulphonephthalein containing 6 mg. to the liter, as de- scribed above, the solution being rendered strongly alka- line. Colorimetric determination of the quantity of drug excreted in a given specimen is made with the Duboscq colorimeter or with Eowntree and Geraghty's modification of the Autenrieth-Konigsberger colorimeter.^ Employ- ing the Autenrieth-Konigsberger instrument (Fig. 12), the standard solution is placed in the wedge-shaped glass. A filtered portion of the urine, rendered alkaline and diluted to one liter, as described, is poured into the rectangular glass. In one side of the case of the instrument there is a narrow slit, the opposite side being frosted glass. With the frosted glass held to the light, the observer looks through the slit and sees the two columns of fluid — urine and standard solution. By means of a thumb-screw, the wedge containing the standard solution is elevated or low- ered until the color intensity is alike on the two sides. The percentage of coloring matter in the urine is now read di- rectly from the position of the indicator on the scale. As a routine procedure Eowntree and Geraghty recom- mend intramuscular (lumbar) injection of the drug. They employ a Eecord syringe of 2 c. c. capacity graduated in ^ c. c. "Whereas in the case of phthalein a normal kid- ney excretes the greater part of the dye injected within two hours of the time of its administration, and then only a small trace for the next two hours, the moderately dis- 'This instrument costs about one-fifth as much as the Duboscq colorimeter and is perfectly satisfactory. The Eowntree and Geraghty modification may be had from Hynson, Westcott & Co., Baltimore, Md. It is made by Hellige in Freiburg. Digitized by Microsoft® THE URINE 123 eased kidney secretes a fair amount within the first two hours, say 50 per cent, of that excreted by tlie normal kid- ney, but, tlie concentration in the blood still being high, it continues to excrete a fair amount in the following two Fig. 12. — The AnTENRiETH-KoNiGssERGER Colorimeter as Modified by Rown- TREE AND GeRAGHTY FOR THE DETERMINATION OF PhENOLSULPHONEPHTHALEIN. hours, so tliat at the end of four hours little difference may exist in the total work accomplished. One-hour and, at the most, two-hour observations are, therefore, recommended. In cases in which only slight changes in function exist this can be most accurately demonstrated by one-hour collection following the use of an intramuscular (lumbar) injection." With intravenous injection the time of appearance and the duration of maximal elimination are shortened, but the re- sults are, on the whole, less trustworthy. Digitized by Microsoft® 124 FUNCTIONAL KIDNEY DIAGNOSIS With normal kidneys the following findings have been obtained : Time of Administration Appearance Quantity Excreted Intramuscular (lumbar) 5-11 min. 51.8-64.1% first hour 60-85% two hours Intravenous 3-5 ' ' 34-45 % in 1st 15 min. 50-65% in 1st 30 min. 63-80% in 1st 60 min. As the intensity of color of the dye gradually dimin- ishes in alkaline urine, it is necessary that the determina- tions be made within a few hours at the most. If the esti- mation of the dye must be delayed for some hours or days, the urine should be rendered distinctly acid, as the phtha- lein remains imchanged in acid solution. , Just before mak- ing the colorimetric determination an excess of alkali is then added to elicit the full strength of the color. When urine is highly pigmented, error in the colorimet- ric readings may be lessened by making up a standard solu- tion containing urine. The error from this source is, how- ever, so small as to be negligible in most instances. Digitized by Microsoft® CHAPTER II THE GASTRIC JUICE The gastric juice is obtained for analysis with the stom- ach tube, following the administration of a test breakfast or meal. The test breakfasts or meals are employed for the sake of simplicity and to obtain comparable conditions. It is because of the many unknown factors involved, such as the quality of food, the length of time it has remained in the stomach, the condition of the stomach before the food was taken, etc., that little dependence can be placed on the results of analysis of vomitus. Test Breakfasts. (1) E wold's breakfast consists of 40 gm. of bread and 400 c. c. of water or weak tea without sugar or cream. (2) Dock's breakfast is the same as the Ewald break- fast, except for the substitution of one shredded wheat bis- cuit for the bread. (3) Boas' breakfast is prepared by boiling one table- spoonful of oatmeal in 800 c. c. of water till the volume equals about 400 c. c. In this country Dock's breakfast is rapidly coming into use. This and the Boas breakfast possess a certain advan- tage over the Ewald breakfast, in that no lactic acid is con- tained in the food, a possible source of error when bread is used. Any of the breakfasts is allowed to remain in the stomach one hour, as a rule, at the end of which the stom- ach tube is introduced and the gastric contents evacuated. 10 125 Digitized by Microsoft® 126 TEST BREAKFASTS With normal gastric niotility the stomach yields 20 to 50 c. c. one hour after a test breakfast (Boas). To eliminate the- possibility of error through giving the breakfast to a patient whose stomach contains part of the previous meal, lavage may precede the breakfast, being performed prefer- ably an hour or so before giving the breakfast. In using the test breakfasts misinterpretation may follow if con- clusions are drawn from the results of a single meal. (4) The Fischer meal consists of an Ewald or Dock breakfast with three-quarters of a pound of finely chopped, lean beef, broiled and slightly seasoned. This meal, like the Eiegel dinner, excites the secretion of hydrochloric acid better than the breakfasts. It is usually allowed to remain in the stomach three hours. (5) The Riegel dinner is more appetizing than any of the preceding meals. It is composed of: One plate of meat broth. Beefsteak weighing 150 to 200 gm. (5 to 7 oz.). Mashed potatoes, 50 gm. (1% oz.). One roll. Eiegel says:^ "As a rule, I empty the stomach four hours after the meal, provided that other indications are not present that determine me to select some other time. If the stomach is found empty after four hours, I know that the motor power of the organ is good ; no conclusions, however, can be drawn in regard to its peptic powers. If the stomach is found empty after four hours, its contents should be withdrawn earlier the next day ; if, on the other hand, a large quantity of coarse and only half-digested morsels of food are found after four hours, the examina- tion on the next day should be made later. A single, exam- ' Eiegel, F. "Diseases of the Stomach" (edited by C. G. Stockton). ' ' Nothnagel 's Practice. ' ' Philadelphia and London, 1905, pp. 79 et seq. Digitized by Microsoft® THE GASTRIC JUICE 127 ination is never permissible." The dinner is usually given at the time of the midday meal. Other test meals have been proposed but are not very generally employed for purposes of gastric analysis.^ EXAMINATION OF THE FASTING STOMACH As the examination of the fasting stomach should pre- cede test meals, the results obtained may be considered be- fore passing to the examination of the gastric contents. The normal stomach empties itself in about seven hours. Passage of the stomach tube before breakfast should, there- fore, lead to the recovery of little fluid or none at all. Nor- mally, the amount rarely exceeds 50 c. c. (Emersoii). When 100 c. c. or more are obtained, there exists either a gastro- succorrhea (continuous secretion) or retention of the gas- tric contents (Boas). Normally or with hypersecretion, swallowed saliva or sputum may be seen in the fluid. With retention, food eaten the previous evening or several days before may be recognized; this should always be looked for, as it furnishes conclusive evidence of stagnation. The ease with which the food may be recognized will depend upon two factors: (1) the chemical composition of the gas- tric secretion, and (2) the nature of the food. With good acidity proteins may be well digested, whereas with a defi- ciency of acid they are little altered. Parts of food which resist the action of the gastric juice, such as the seeds of small fruit or berries, are easily detected. In fact, when defective motor power is suspected, it is a useful procedure to give raspberry jam or some similar jDreparation in the evening, and look for the seeds in the gastric contents or 'Eiegel, F. "Diseases of the Stomach" (edited by C. G. Stockton). "Nothnagel's Practice." Philadelphia and London, 1905, p. 79 et seq. Digitized by Microsoft® '128 EXAMINATION OP GASTRIC CONTENTS lavage the following morning. At times excessive quan- tities of fluid are found in the fasting stomach. The nor- mal organ has a capacity of about 1,600 c. c. (Ewald) ; a stomach which can retain more than this quantity is di- lated. In addition to the points just enumerated, the fluid ob- tained from the fasting stomach should be subjected to the examination to be described for the gastric contents. MACROSCOPIC EXAMINATION OF THE GASTRIC CONTENTS Quantity.— In the examination of the gastric contents obtained one hour after a test breakfast, the quantity of fluid recovered is measured. Boas finds that the amount usually lies between 20 and 50 c. c. with normal gastric motility. Higher amounts, however, are certainly obtained in health at times ; 80 c. c. is not unusual. When 150 to 200 c. c. are found in the stomach, hypomotility is quite defi- nitely indicated. A stomach which is repeatedly found empty one hour after a test breakfast has hypermotility, and it is then necessary to remove the contents after three- quarters or one-half hour. Odor. — The normal gastric contents are practically odorless. In disease the odor may be sour or rancid (ace- tic acid, butyric acid, etc.), putrid, fecal, etc. The odor of drugs should also be looked for. Mucus. — The presence of an excess of mucus is most easily detected by pouring the gastric juice from one re- ceptacle to another. If the amount be abnormal, the con- dition is at once recognized. Mucus from the respiratory passages floats because of the bubbles contained in it. From the pharynx and esophagus there may be a consid- Digitized by Microsoft® THE GASTRIC JUICE 129 erable quantity of mucus secreted during the passage of the stomach tube. It runs along the side of the tube, and is not aspirated through the tube, as in the case of true gastric mucus or swallowed sputum. Color.— Normally the gastric secretion is practically colorless. The regurgitation of bile from the duodenum may impart a deep yellow or green color, the intensity depending on the relative proportion of bile. Blood, when fresh, is characteristic in appearance; if it has remained in the stomach long enough to undergo change, the bright red color is lost, and is replaced by a dark brown, pro- ducing in many instances the so-called "coffee-ground" ap- pearance. The color of the gastric juice may also be al- tered by food or drugs. Food.— The state of digestion of the food is of great im- portance. After the usual test breakfasts, carbohydrate forms the bulk of the food ingested. The alterations found are due chiefly to ptyalin of the saliva. With hyperacid- ity this enzyme is quickly destroyed, with a consequent in- hibition or arrest of amylolysis. After a mixed meal, such as the Eiegel dinner, more information may be gained by inspection of the gastric contents. The appearances are well described by Eiegel.^ "In some cases a very fine, uni- form, mushy liquid mass is seen that contains no coarse elements at all ; in others, again, a mass of food containing many coarse pieces of meat that look as if they had just been swallowed; in addition, there is frequently an abun- dant admixture of mucus. In some cases there is so much mucus that the food looks like a tough mass and passes through the sound with difficulty, and is very difficult to filter. In other cases there is a large quantity of fiuid con- tents that forms three layers when kept in a glass vessel; ^ Eiegel, F. Loc. cit., p. 86. Digitized by Microsoft® 130 EXAMINATION OF GASTRIC CONTENTS at the bottom is seen a layer consisting of fine remnants of amylaceous material; above this a large layer of cloudy fluid, and on the top a foamy layer. If the fetter is pres- ent it may be considered evidence of gaseous fermenta- tion. This consistency of the stomach contents is found chiefly in cases in which there is stagnation or in which there is motor insufficiency . . . usually in cases in which there is an abundant quantity of free hydrochloric acid. ... If the food remnants obtained from the stomach in different diseases are compared, the great sig- nificance of macroscopic inspection will be understood. In many instances this method alone will give us diagnostic points which we would otherwise obtain only by compli- cated chemical examinations. There are cases, for in- stance, in which the stomach contents do not give any of the reactions for free hydrochloric acid. This shows that there is a deficit in the stomach. Sometimes, however, when free hydrochloric acid is absent, we find only a rela- tively small amount of finely distributed food residue; at other times we may see larger quantities of coarse food particles. If we limit ourselves to examining the filtrate in both these cases for free hydrochloric acid, we will prob- ably consider that the two are alike, and, as a matter of fact, they are alike in regard to their free hydrochloric acid, for in neither do we see a formation of free hydro- chloric acid. If, however, we consider the quantity and the appearance of the stomach contents in both, we shall see that in the first case the peptic power is better than in the second. The first case is functionally nearly normal, for all the albumin has been digested; at the same time there was no residue of free hydrochloric acid. In the sec- ond case it is different; here the production of acid was subnormal, as shown by the disturbed digestion of meat. Digitized by Microsoft® THE GASTEIC JUICE 131 If this case is more carefully examined, it will be found that the deficit of hydrochloric acid is large, whereas in the first case it is small. In this way macroscopic examination frequently gives us a clear picture of disturbances of func- tion. ..." The careful macroscopic analysis of the gastric con- tents, it is evident, is of the greatest value. CHEMICAL EXAMINATION OP THE GASTRIC CONTENTS Reaction.— The reaction of the gastric contents is tested with litmus paper. It is usually acid. An alkaline or neu- tral fluid may be obtained. Hydbochlobic Acid Hydrochloric acid is the most important chemical con- stituent of the gastric juice from the clinical standpoint. Normally it is present in excess, i. e., a test for free hydro- chloric acid is always obtained. Qualitative Tests for Free Hydrochloric Acid (1) Von den Velden's Methyl Violet Test.— Add a few drops of a saturated aqueous solution of methyl violet to a test tube nearly filled with water. The dilute solution of the dye should be transparent and violet or purple in color. It is divided equally in two test tubes. To the one add an equal quantity or less of gastric juice, to the other an equal volume of water. Free hydrochloric acid is indicated by a change in color from violet to blue, the portion to which water alone is added serving as a control. The test is said to indicate 0.025 per cent, of free hydrochloric acid. Ac- Digitized by Microsoft® 132 EXAMINATION OF GASTRIC CONTENTS cording to Eiegel, the test is especially valuable, since, when it is positive, it means that there is sufficient free acid for protein digestion. A second method of performing the test, which is use- ful when the amount of gastric juice at one's disposal is small, consists in spreading a thin layer of the dilute methyl violet solution in a porcelain plate, and then placing a drop of gastric juice in contact with it. Where the two fluids run together, the violet color is changed to blue in the pres- ence of free acid. Lactic acid does not interfere with the methyl violet re- action, since it is given only by 0.4 per cent, or stronger solutions, which never occur in the stomach. (2) Giinzberg's Test.— This is the standard test for free hydrochloric acid. It is positive only in the presence of a free mineral acid. Eeagent : Phloroglucin 2.0 gm. Vanillin 1.0 gm. Alcohol, absolute 30.0 c. c. Dissolve and keep in a brown bottle, tightly stoppered. As the reagent does not keep well, it is advisable to make small quantities, so that it may be renewed every few months. It is well to test the reagent from time to time with dilute hydrochloric acid to prove its re- liability. A few drops of the reagent are evaporated to dryness in a porcelain dish by warming gently over a Bunsen bur- ner. A drop of gastric contents is brought in contact with Digitized by Microsoft® THE GASTRIC JUICE 133 the yellowish-brown stain left by the reagent, and is evap- orated. If free hydrochloric acid is present, an intense red color develops, where the reagent and gastric juice have mixed. Instead of evaporating the reagent and gastric juice separately, equal quantities of the two may be mixed (one or two drops of each) and evaporated, when the color change appears. The evaporation must be performed with great care. It is easy to burn the reagent by overheating; the test then fails, even though there be an abundance of free acid pres- ent. The degree of heat may be tested by touching the bottom of the porcelain dish with the finger. The dish is held in the flame a second, removed, tested ; this procedure, repeated at intervals, accomplishes the desired result with a little practice. Blowing on the specimen when it is re- moved from the flame hastens the evaporation, and at the same time lowers the temperature. A safer method of evaporation is the use of a water bath. The test is sensitive to free hydrochloric acid in 0.01 per cent, solution. It is specific in the sense that a positive reaction is only obtained with free mineral acid ; organic • acids do not give the test. (3) Tropeolin Test.— A saturated alcoholic solution of tropeolin 00 is prepared. Three to four drops of this re- agent and a like quantity of the gastric juice are spread over the surface of a porcelain dish, and carefully evapo- rated to dryness. In the presence of free acid the color be- comes violet or blue. The test is less sensitive than either of the preceding tests. It is positive with free hydrochloric acid in a dilution of 0.03 per cent. Lactic acid solutions of 0.24 per cent, or stronger give the reaction (Ewald) ; in the stomach it is doubtful whether lactic acid ever occurs in sufficient concentration to give the test. Digitized by Microsoft® 134 EXAMINATION OF GASTEIC CONTENTS In place of the concentrated alcoholic solution of trope- din 00, Eiegel recommends a saturated aqueous solution. (4) Congo-paper Test.— Filter paper is saturated with a concentrated aqueous solution of Congo-red, and allowed to dry. It is then cut into narrow strips. A piece of the paper is moistened with the stomach contents. Free hy- drochloric acid turns the paper deep blue ; lactic acid pro- duces a much less intense blue. The test is fairly delicate, but with very dilute solutions of hydrochloric acid the color change is very slight and rather difficult to interpret. Lac- tic acid is never found in sufHcient concentration to lead to difficulty, according to Eiegel. (5) Topfer's Test.— One drop of 0.5 per cent, alcoholic solution of dimethylamidoazobenzol is added to a few c. c. of gastric juice. Free hydrochloric acid produces a bright red color. Organic acids also cause a color reaction, but the color is less brilliant — more of a brick red. The reac- tion is, therefore, not specific, and is the least reliable of the tests. Of the tests for free hydrochloric acid, the Giinzberg test is the most delicate and at the same time the most re- liable. It is a good routine test, and in any case should be employed wherever doubt exists. In certain instances where it is desirable to have infor- mation regarding the acid secretion of the stomach, contra- indications to the passage of the stomach tube exist. In such case Sahli's desmoid test may be used. (6) Sahli's Desmoid Test.^— This is a test for free hy- drochloric acid. It is based on the fact that raw catgut is soluble in hydrochloric acid-pepsin, insoluble in pancreatic and intestinal juices. ^Boggs, T. R. "Sahli's desmoid reaction in gastric diagnosis." Bull. Johns Hopkins Hosp., 1906, XVII, 313. Digitized by Microsoft® THE GASTRIC JUICE 135 Pills of the following formula are prepared: Methylene blue 0.05 gm. Iodoform 0.1 gm. Ext. glycyrrhiz q. s. The pills should not exceed 3 or 4 mm. in diameter. The iodoform may be omitted. The pill is placed in the center of a square of thin rubber dam, such as dentists use. The rubber is stretched and twisted about the pill. The twisted neck is then tied with three turns of raw No. 00 catgut, previously soaked in cold water till soft. Now trim the rubber so that a free edge of about 3 mm. width remains beyond the ligature. The cut edges of the rubber must not cohere, inclosing air, for the pill must sink in water, and it must be watertight. A pill prepared as described is given to the patient with his midday meal, and the urine, collected 5, 7, 18, and 20 hours afterward, is examined for the presence of methylene blue, iodin, or both. In the absence of the greenish color of methylene blue, the urine should be boiled with one-fifth volume of glacial acetic acid. If the chromogen of methy- lene blue exists in the urine, the color will then appear. Iodin may be looked for with Obermayer's test for indican (p. 27). If methylene blue appears in the urine within twenty hours after the administration of the pill, the test is considered positive. A positive test shows that there is sufficient free hydro- chloric acid secreted in the stomach to permit of digestion of the raw catgut and liberate the pill from its rubber capsule. If the gastric juice fails to digest the catgut, the pill passes into the intestines and is evacuated. The test is, therefore, one for free hydrochloric acid. As it is given with a regular meal, it encounters the optimal conditions Digitized by Microsoft® 136 EXAMINATION OF GASTEIC CONTENTS for acid secretion. The test is thus a useful adjuvant to the usual gastric analyses in certain cases of anacidity. Organic Acids.— When free hydrochloric acid is mark- edly diminished or entirely lacking, tests for organic acids should be made. With normal hydrochloric acid values, lactic acid fermentation does not occur. The tests for or- ganic acids are described on pages 141-142. Quantitative Determination of Gastric Aciditif In the quantitative analysis of the gastric juice the amount of free hydrochloric acid and of total acidity and the extent of the hydrochloric acid deficit are of importance clinically. Very little of diagnostic value has resulted from estimation of the loosely combined hydrochloric acid, i. e., hydrochloric acid in protein combination. Topfer's Method for Free Hydrochloric Acid.— This is the method generally employed, since it is quickly carried out and is sufficiently accurate for clinical purposes. A drop of 0.5 per cent, alcoholic solution of dimethyl- amidoazobenzol is added to 10 c. c.^ of filtered gastric con- tents, placed in a porcelain dish or in a beaker resting on a sheet of white paper for a background. The gastric juice should be measured accurately with a pipette. In the pres- ence of free hydrochloric acid, the addition of the drop of indicator produces a brilliant red color in the liquid. From a burette graduated in tenths of a cubic centimeter, tenth normal sodium hydrate is run into the mixture, a few drops at a time, with constant stirring, till the red color entirely disappears. This is the end reaction. The quantity of tenth normal hydrate required to neutralize the acid in 10 c. c. of the gastric contents is then read from the burette. ' If the quantity of gastric contents obtained is small the titration is made with 5 c. c, with a corresponding correction in the final calculation. Digitized by Microsoft® THE GASTRIC JUICE 137 The result is usually expressed as "acidity per cent.," i. e., the number of cubic centimeters of tenth normal alkali which would be required to neutralize the free acid in 100 c. e. of gastric contents. Since 10 c. c. were taken, the quan- tity of alkali used, multiplied by 10, gives the desired re- sult. Normally free hydrochloric acid varies between 20 and 40. The amount of hydrochloric acid may be calcu- lated. One c. c. of tenth normal alkali is equivalent to 0.00365 gm. HCl. If the amount of gastric juice is small, the same sample may be employed for the determination of total acidity. A drop of phenolphthalein is added and the titration con- tinued. The alkali used in neutralizing the free hydrochlo- ric acid must, of course, be included in the total acidity. Dimethylamidoazobenzol is not the ideal indicator, since it reacts with organic acids and acid salts as well as with mineral acids. The results obtained with it are, therefore, too high; they do not represent absolute values. Never- theless, the method fulfills all clinical needs, since the error introduced is relatively so small that it does not vitiate the results for diagnostic purposes. Other Indicators.— In place of dimethylamidoazobenzol Giinzberg's reagent and Congo-red are frequently employed as indicators in the titration of free hydrochloric acid. Gunzherg's reagent may be used in several ways. As the titration progresses, a small drop of the gastric juice is removed with the stirring rod from time to time, and placed on the evaporated Giinzberg's reagent. The drop is evaporated, and the red color appears at the margin as long as free acid exists. A second procedure consists in the addition of 25 to 30 drops of Giinzberg's reagent to the gastric juice, and then at intervals the removal of a minute drop, which is evaporated in the usual manner. The glass Digitized by Microsoft® 138 EXAMINATION OF GASTRIC CONTENTS stirring rod itself may be gently warmed till the fluid cling- ing to it is evaporated; it is then examined for the red color. The disadvantage in these procedures is that a small quantity of the gastric contents is lost with each test for free acid, so that the result is slightly low. Compara- tive titrations with Giinz berg's reagent and dimethylamido- azobenzol will show less free acid, as a rule, when Giinz- berg's reagent is used; occasionally the values are alike. Congo-red paper may also serve as the indicator in the titration of free hydrochloric acid. It is very convenient for night work. The tenth normal alkali is added to the gastric juice until a small drop placed on Congo-red paper no longer produces a blue color. As a control the paper should be moistened with distilled water, for the red color becomes somewhat darker when moistened. The results are usually intermediate between those obtained with Giinz- berg's reagent and those with dimethylamidoazobenzol. Titration of Total Acidity.— The total acidity comprises free hydrochloric acid, loosely combined hydrochloric acid (i. e., in combination with protein), acid salts, and organic acids, such as lactic, butyric, and aminoacids, when pres- ent. Its quantity is determined by titration with tenth normal alkali, using phenolphthalein as the indicator. With a pipette measure 10 c. c. (or 5 c. c.) of filtered gastric contents into a porcelain dish or Erlenmeyer flask placed on a sheet of white paper, and add one or two drops of 0.5 per cent, alcoholic solution of phenolphthalein as in- dicator. In an acid medium it is colorless, but it becomes pink as soon as all the acid is neutralized, leaving a slight excess of alkali. Tenth normal sodium hydrate is added from a burette under constant stirring, until the whole mix- ture takes on a faint pink color, which persists. The num- ber of c. c. of alkali used, multiplied by 10 (or by 20 in Digitized by Microsoft® THE GASTRIC JUICE 139 case 5 c. c. of gastric contents were taken), gives the total acidity per cent. Normally this varies between 40 and 60 or 70. The results obtained are again only approximately cor- rect, being too high as a rule. For diagnostic purposes the method is practicable. The Hydrochloric Acid Deficit A deficit in hydrochloric acid occurs whenever the gas- tric mucosa secretes so small a quantity of hydrochloric acid that there is not merely an absence of free acid, but an excess of bodies capable of binding or uniting with it. Such bodies are chiefly proteins and their end-products, peptids, and the aminoacids. If peptic digestion of the proteins alone occurs, the aminoacids are not concerned in the production of a deficit in hydrochloric acid, since pepsin is unable to carry the hydrolysis of the protein molecule to the aminoacid stage. But, when trypsin is re- gurgitated into the stomach, or when the proteolytic en- zyme of a malignant neoplasm is secreted into the stomach, aminoacids may be abundant in the stomach contents ; they may also be the result of bacterial decomposition, though probably not frequently. The presence of aminoacids is of significance in two directions in the quantitative analysis of the gastric contents, as Fischer ^ has pointed out. Pep- sin converts the proteins into peptids, which react alka- line toward litmus ; when united with hydrochloric acid the reaction is reversed. The hydrolysis of the peptids into their constituent aminoacids alters the conditions. The latter can bind hydrochloric acid and at the same time carboxyl groups are liberated. The result is that the total 'Fischer, H. "Zur Kenntnis des carcinomatosen Mageninhaltes. " Veutsch. ArcMv f. Min. Med., 1908, XCIII, 98. Digitized by Microsoft® 140 EXAMINATION OF GASTRIC CONTENTS acidity is increased, while the free hydrochloric diminishes. With an excess of aminoacids it is then necessary to add more or less hydrochloric acid before a reaction for free acid is obtained. Factors which play a less important role in the production of an acid deficit are alkalies introduced with the food or secreted, possibly, in disease. It is unnecessary to remark that only those specimens of gastric juice which fail to react to Giinzberg's reagent for free hydrochloric acid are suitable for the determina- tion of a deficit in acid. The method of determining the deficit in free hydro- chloric acid is as follows : From a burette add tenth nor- mal hydrochloric acid to 5 or 10 c. c. of the gastric contents with constant stirring, until a test for free hydrochloric acid is obtained. For this purpose the Giinzberg test is to be preferred. Dimethylamidoazobenzol is not well adapted to the titration, since organic acids which are of- ten present react with it ; Congo-red paper gives more sat- isfactory results than dimethylamidoazobenzol. The extent of the deficit may be expressed as "deficit per cent.", — the usual way; the number of cubic centimeters of tenth normal hydrochloric acid which would be required for 100 c. c. of gastric contents is calculated. Or the deficit may be expressed in terms of hydrochloric acid, calculated for 100 c. c. of stomach contents. Oeganic Acids Lactic Acid Of the organic acids which may be present in the stom- ach contents in disease, lactic acid is the most important and is the only one tested for in the usual routine exam- Digitized by Microsoft® THE GASTRIC JUICE 141 ination. It is odorless. Lactic acid is the result of fer- mentation of the gastric contents. The fermentation oc- curs only in the absence or very marked decrease of free hydrochloric acid. When many Oppler-Boas bacilli are present in the gastric contents, lactic acid is usually found, though the converse is not true. Lactic acid almost al- ways means stasis of the gastric contents ; it is not found in anacidity, where the motor power of the stomach is nor- mal. Quantitative estimation of lactic acid has not been found of value in diagnosis. Qualitative Tests for Lactic Acid. — (1) Ueffelmann's Test. — To 15 or 20 c. c. of 1 per cent, aqueous carbolic acid in a test tube, 10 per cent, ferric chlorid solution is added till an amethyst color is produced; usually 1 to 2 drops suffice. If necessary, the solution is diluted till it is trans- parent, and is then divided equally between three tubes. To the first a few drops of the filtered gastric contents are added, to the second a like quantity of distilled water to serve as a control, and to the third the same amount of dilute lactic acid solution for comparison with tube one. A yellowish-green (canary yellow) color denotes lactic acid or its salts. A similar color reaction may also be given by oxalic, citric, and tartaric acids, by alcohol and dextrose, but these substances can usually be excluded after an Ewald or Dock breakfast. To avoid error from disturbing bodies, it has been rec- ommended to extract the gastric contents with about ten volumes of ether, which is then evaporated; the residue is dissolved in water, to which the test is applied. (2) Steauss' Test. — To avoid the sources of error in the preceding test, Strauss employs a specially devised separating funnel, which is used to extract the gastric con- tents. Above the glass stopcock there are two marks which 11 Digitized by Microsoft® 142 EXAMINATION OF GASTRIC CONTENTS correspond to 5 c. c. and 25 c. c. The gastric contents are added to the mark 5, and then ether is poured to the mark 25. The two fluids are mixed thoroughly by shaking, and after they have separated the gastric contents are allowed to escape. Distilled water is then added till the ether again rises to the mark 25. After the addition of one drop of 10 per cent, ferric chlorid solution, shake vigorously, and wait for the fluids to separate. In the presence of lactic acid a greenish-yellow color is imparted to the watery layer. The extraction with ether separates the lactic acid from the in- terfering bodies. If lactic acid is combined with protein, the test may be negative ; but the lactic acid may be freed by the addition of dilute hydrochloric acid, until a test for the latter is given with Congo paper. The test now be- comes positive. (3) Kblling's Test. — A small portion of the gastric contents is diluted with 10 to 20 volumes of distilled water. A second test tube is filled with the same quantity of water alone. To each tube add one drop of 10 per cent, ferric chlorid. Lactic acid causes a canary-yellow color. Dilu- tions of 1:10,000 to 1:15,000 may give a positive reaction. The second tube, containing water and ferric chlorid, serves as a control. As in Uffelmann's test, the color is often perceived most easily by looking down into the test tube, which is held on a white background. Butyric Acid Butyric acid fermentation may take place in the pres- ence of considerable quantities of free hydrochloric acid. The odor of butyric acid, resembling that of rancid butter, is characteristic. Boiling the gastric contents accentuates the odor ; if a piece of moistened blue litmus paper be held Digitized by Microsoft® THE GASTRIC JUICE 143 in the mouth of the test tube, the volatile acid reddens it as it escapes during the boiling. Butyric acid also has the peculiar property of separating as a drop of oil on the ad- dition of a small piece of calcium chlorid. Acetic acid, like butyric acid, may be recognized by its odor if present in sufficient concentration. Acetic acid, after careful neutralization with sodium hydrate, with the formation of sodium acetate, gives a blood-red color on the addition of a drop of ferric chlorid. Gastric Ferments Normally pepsin and rennin, or their zymogens, are constituents of the gastric juice. Alterations in the en- zymes in disease are much less frequent and less striking than those occurring in the hydrochloric acid. Whenever the latter is present, it is practically always the case that pepsin is also found. With absence of free hydrochloric acid, tests for the enzymes should be made. Quantitative determination of pepsin has not proved to be sufficiently valuable to warrant its inclusion in the usual routine gas- tric examinations. Pepsin Qualitative Test for Pepsin.— Discs of coagulated egg albumin, ca. 1.5 mm. thick and 5 to 10 mm. in diameter, are cut with a cork-borer or goose-quill. They may be preserved in glycerin, but should be washed in water im- mediately before use to remove the excess of glycerin. A disc of the coagulated albumin is placed in a few c. c. of gastric contents, and, if necessary, dilute hydrochloric acid is added, till Congo paper gives a test for free acid. The material is then placed in an incubator at 37° C. (or in Digitized by Microsoft® 144 EXAMINATION OP GASTRIC CONTENTS the vest pocket). In one-half to one hour the albumin should be digested. Fibrin, usually obtained from ox blood and preserved in glycerin, may be substituted for the coagulated egg al- bumin. Quantitative Methods.— Quantitative methods for pep- sin may occasionally be desirable. Several have been pro- posed within the last few years. The results given by each are relative, not absolute, values. Mette's Method as Modified by Nieeenstein and ScHiFF.i — Capillary glass tubes, 1 to 2 mm. in diameter and 20 to 30 cm. in length, are filled with egg albumin by suction, the ends plugged with bread crumbs, and the tubes then placed in boiling water for five minutes. They are then sealed with paraffin or sealing wax. Bubbles appear in the albumin, but are no longer seen after three days, when the tubes are ready for use. If the albumin retracts from the wall of the tube, it should not be used for the test. Method. — One c. c. of filtered gastric contents is diluted with 15 c. c. of twentieth normal hydrochloric acid. With a file or glass scissors, cut off about 2 cm. of the capillary tube, and place two such pieces in the diluted gastric con- tents. The test tube is then corked and placed in an incu- bator at 37° C. for twenty-four hours. At the end of this time the tubes are removed and the amount of digestion of albumin in the four ends of the capillary tubes is meas- ured in tenths of a millimeter, a hand lens being useful for this purpose. An average of the four readings is taken. The square of this number represents the number of units of pepsin present in the diluted gastric contents. Multi- ' Farr, C. B., and Goodman, E. H. "The clinical value of the quantitative estimation of pepsin, with special reference to the Mette and ricin methods." Arch. Int. Med., 1908, I, 648. Digitized by Microsoft® THE GASTRIC JUICE 145 plying this by 16 gives the value for the undiluted speci- men. Since the albumin from different eggs may react differ- ently, and since the length of time the albumin is boiled affects its digestibility, the method can be relied upon only to show rather wide variations in pepsin. According to Cowie,^ the tubes need not remain in the incubator twenty-four hours. He finds that the amount of digestion varies directly as the time. He derives the fol- lowing formula for calculating the digestion : If A=the amount of egg white digested, B=the time the tubes remain in the incubator, C=the required time for the end reaction, X=the peptic value of the fluid tested, or the esti- mated value in millimeters, then it will be found that X= A>

from % to 1 inch in length, according to the species, broad at one end, tapering at the other, and usually beset with little spines or hairs, is sufficiently diagnostic" (Manson^). It is particularly im- portant that the specimen for examination be fresh for obvious reasons. Nematodes Nematodes, round worms, constitute a large class, a number of which are parasitic in man. Their anatomy and biology,- though of great interest, are touched upon in the following pages only in so far as they are of diagnostic significance. Necator Americanus.— Necator americanus (Uncinaria americana), the NeAV "World hookivorm, was first described by Stiles ^ in 1902. Together with its cousin of the Old World, it is by far the most important nematode parasitic in man in this country. It is the causative agent of the disease uncinariasis or anchylostomiasis. Diagnosis of infection with Necator americanus is made by examination of the stools for the ova of the parasite. The specimen of feces for examination should be fresh; in ^ Gilbert, N. C. " Infection of man by dipterous larvae, with report of four cases." ArcJi,. Int. Med., 1908, II, 226. (Literature.) ^ Manson, P. ' ' Tropical Diseases. ' ' London, 1900, p. 603. 'Stiles, G. W. (a) "A new species of hookworm (Uncinaria americana) parasitic in man." Amer. Med., 1902, III, 523. (b) "The significance of the recent American cases of hookworm disease (uncinariasis or anchylostomiasis) in man." Reprint, 18th ann. rep.. Bureau Anim. Industry (1901). Wash., 1902. (c) "Report on the prevalence and geographic distribution of hook- worm disease (uncinariasis or anchylostomiasis) in the United States." Bull, no. 10, Hyg. Lab., U. S. Pub. Health & Mar. Hosp. Serv., Wash., 1903, pp. 1-122. Digitized by Microsoft® THE FECES 181 older specimens — 24 to 48 hours — the eggs may have hatched, in which case the embryos, much like those of ■ Strongyloides stercoralis, may be observed. The specimen is examined with low power objective. All doubtful objects are examined with higher magnification. The ovum of the hookworm is characteristic, and, when once seen, can scarce- ly be mistaken for other bodies in the stool. The ova (Fig. 17) of Necator americanus are oval and possess a clear, colorless shell, which meas- ures 0.064 to 0.076 mm. by 0.036 to 0.040 mm. (Stiles). The outline of the Qgg is sharp and clearly de- fined. Inside the shell is the yolk. Fig. 17. — Ovum of Necatok 1 • T . J. 1 1 1 AMERICANUS. X460. which is unsegmented when de- posited by the female in the intestines, but usually .presents two, four, or eight segments or cells, sometimes more, by the time it is evacuated with the feces. The yolk is dark gray or brownish-gray and finely granular; usually a lighter area representing the nucleus may be observed near the center of the yolk cells, especially when their number is eight or less; as the cells multiply, the decrease in size makes this area less conspicuous. In examining a preparation for hookworm (or other) ova, a mechanical stage is a great convenience. About one- half the area of the usual 3xl-in. slide should be covered with the diluted feces, and, before rendering a negative diagnosis, ten such specimens should be examined (Stiles). " ... To stop after finding a few hookworm eggs is not good practice. The examination should be continued to find, if present, eggs of other parasites, which are likely to be present in small numbers, and to get some idea of the number. When less than ten female worms are pres- Digitized by Microsoft® 182 MICROSCOPIC EXAMINATION ent, there may be an average of less than one egg to a slide" (Dock and Bass ^). Special Methods.^ — ^When the eggs are very few, great- er diagnostic accuracy is attained by resorting to special methods for their detection. (1) Pepper's Method.^ — Pepper has found that hook- worm ova possess a peculiar property of sticking to glass. If the preparation for microscopic examination be allowed to settle, immersion in water will remove the greater part of the fecal matter, while the hookworm ova stick to the slide. Dock and Bass find that bet- ter results are obtained with the method if part of the fecal material is first removed by centrifugaliza- tion. (2) Stiles' Method of Washing and Sedimenting.* — "Take one to two ounces of feces, mix with water, and place in a large bottle, retort, jar, or any other receptacle; add enough water to make from a pint to two quarts, ac- cording to the amount of feces; shake or stir thoroughly and allow to settle; pour off the floating matter and the water down to near the sediment; repeat the washing and settling several times, or as long as any matter will float. The last time this is done use a bottle or graduate with a smaller diameter, and, when the material is thoroughly settled, examine the flne sediment. It will be found that ^Dock, G., and Bass, C. C. "Hookworm Disease. Etiology, pathology, diagnosis, prognosis, prophylaxis, and treatment." St. Louis, 1910. (An ex- cellent discussion of hookworm disease in all its medical aspects, to which the reader is referred.) ^ Hall, M. C. "A comparative study of methods of examining feces for evidences of parasitism." Bull. no. 135, Bureau Anim. Indust., U. S. Dept. of Agric, Wash., 1911. " Pepper, Wm. ' ' A new method of examination of the feces for the ova of uncinaria. " Jour. Med. Research, 1908, XIII, 75. *ioc. oit. (c), p. 85. Digitized by Microsoft® THE FECES 183 the eggs have settled more numerously in the fine sedi- ment than in the coarse material." (3) Centrifugalisation. — Simple centrifugalization of the diluted feces often gives disappointing results. The es- sentials of the method, as given by Dock and Bass/ follow : "The feces should be diluted and well mixed with ten or more times their bulk of water. This should be strained through two or three layers of gauze in a funnel to remove the coarse particles. The exact length of time necessary to centrifuge, in order to throw most of the eggs suspended in water to the bottom of the tube, should be determined by experimenting with a known specimen that has already been washed once or twice and contains many eggs. This must be determined with the particular centrifuge used. . . . As the first diluted feces are much thicker than the washed feces and eggs on which the working time of the centrifuge has been determined, the eggs will go down somewhat slower the first time. It is, therefore, a good plan to centrifuge double time at first. If, for example, the working time of the centrifuge is four seconds, the first centrifuging should be eight seconds. This throws to the bottom most things heavier than eggs — like crystals, sand, large vegetable cells, etc. — and all eggs present. There remain suspended in the supernatant fluid nearly all bac- teria and fine particles, and many coarse particles lighter and more irregularly shaped than eggs. If the centrifuge is run longer, many of these go down, which is, of course, undesirable. Pour off this fiuid and two-thirds, and often more, of the feces are removed by this washing. EefiU the tube to about three-fourths its capacity, shake up thor- oughly, and centrifuge again, running now only the work- ing time of the centrifuge. It is important not to centri- ^ Loo. cit., pp. 172 et seq. Digitized by Microsoft® 184 MICROSCOPIC EXAMINATION fuge longer than the working time of the centrifuge, as many fine and light particles would otherwise be thrown down. Considerable material remains suspended and may- be removed by pouring off the supernatant fluid. Again the tube is filled, shaken, and centrifuged a proper leng-th of time, and generally this will be sufficient for practical purposes. A part or all of the sediment is removed with a pipette, spread out on a slide, and examined for eggs. It consists of crystals, sand, and heavy, coarse food par- ticles, and eggs, if present. . . . Great care must be exercised to clean the centrifuge tubes before using them after they have had eggs in them. A proper centrifuge brush is serviceable. The method . . . permits the finding of eggs when less than half a dozen laying females are present, and often when only one is present. It is of additional service because it permits at the same time diag- nosis of infections with many other worms by which fewer eggs are laid, such as the tenias, oxyuris, bothriocephalus, etc." The adult worms of Necator americanus (Fig. 18) are found in the stools only after treatment. They are small, whitish, grayish, or reddish-brown in color, and have the anterior end curved dorsally to form a hook. The males measure 6 to 10 mm. in length, the females 8 to 15 mm. The stools, collected after treatment, are placed in a bucket or other suitable receptacle, stirred with several times their volume of water, and allowed to settle a few minutes, when the supernatant fluid is poured off. The washing is re- peated several times, and, finally, the sediment is trans- ferred to a plate, preferably with a black background, and the worms looked for. They may be identified by exam- ination under the microscope. Ankylostoma Duodenale.— Ankylostoma duodenale, the Digitized by Microsoft® THE FECES 185 Old World hookworm, is of frequent occurrence in this country, often associated with Neeator americanus. The methods of diagnosis are those described for Neeator amer- icanus. The ova (Fig. 17) are identical in appearance. Fig. 18. — Necatok AMERirANUs. Upper half males, l^wer lialf females. Inch measure. C\fter Dock and Bass.) Measurements show, however, that tliey are a little smaller than those of the New "World variety, measuring 0.052 to 0.061 by 0.032 to 0.038 mm. The differences are so slight that simple microscopic inspection does not suffice for the separation of the two. The aduU parasites are a trifle lar- ger than Neeator americanus. The males are 8 to 11 mm. long and 0.45 mm. wide; the females 10 to 18 mm. long Digitized by Microsoft® 186 MICROSCOPIC EXAMINATION and 0.6 mm. wide. There are certain well marked pecu- liarities by which the two species of hookworm may be differentiated. Strongyloides Stercoralis. — Strongyloides stercoralis (Strongyloides intestinalis), the parasite of Cochin China diarrhea, first reported in this country by Strong ^ and Thayer,- has proved to be widely spread through the South- FiG. 19. — The Rhabditiform Embbyo of Strongyloides stercoralis. X460. ern States, as Thayer predicted. Diagnosis of infection is made by the finding of the actively motile rhabditiform embryos in the stool. Perfectly fresh feces should always be used for exam- ination. If the specimen is kept too long, the embryos may die or may change into the filariform larvs. If the stool is formed, fluid about it may contain the embryos, or they may be looked for in a preparation of the diluted feces. Since this often fails, even with heavy infections, 'Strong, E. P. "Gases of infection with Strongyloides intestinalis (first reported occurrence in North America)." Johns Hopl-ins Hasp. Sep., 1902, X, 91. ' Thayer, W. S. " On the occurrence of Strongyloides intestinalis in the United States." Jour. Exp. Med., 1901, VI, 75. Digitized by Microsoft® THE FECES 187 it is better practice to give a saline cathartic, if necessairy, and examine the fluid stools. The embryos (Fig. 19) are 0.450 to 0.600 mm. long and 0.016 to 0.020 mm. thick (Blanchard), and have a characteristic wriggling or squirming motion in the fresh stool. They are grayish- white and quite refractive. They possess a rhabditiform or bottle-shaped esophagus. Embryos which have died are, of course, less conspicuous, but, if well preserved, are characteristic. If the in- fected stool be kept for one to two days un- der suitable conditions of light, moisture, temperature, and oxygen supply, the rhab- ditiform embryos may develop into the filariform larvae, the infecting stage of the parasite. Ova of Strongyloides stercoralis (Fig. 20) are extremely rare in the feces. They resemble the ova of the hookworm. In one of his cases Thayer found two eggs on daily examination of the stools for several months. Hookworm embryos which have developed in the stools (twenty- four to forty-eight hours after passage) may be mistaken for the embryos of Strongyloides sterco- FiG. 20. — Ovum of Strongyloides stercokalis. (Drawn withLeitz obj. no. 7, ocular no. 3.) (After Thayer.) Fig. 21. — The Rhabditiform Embryo of Strongt- yajj^g . LOIDES stercoralis (1) AND THE EmBRTO OP ' the former occur in the THE Hookworm (2). Showing the difference in never length of the buccal capsule. Diagrammatic. freshly evacuated feces. They are most readily differentiated by the fact that the buccal capsule (Fig. 21) is very short in the embryo of Digitized by Microsoft® 188 MICROSCOPIC EXAMINATION Strongyloides stercoralis, relatively long in the hookworm embryo (Stiles). In case of doubt, a perfectly fresh stool should be obtained after a saline cathartic, when ova with- out embryos are found with hookworm infection alone ; with double infection, both embryos and ova are seen. Students often confuse plant or vegetable hairs with (dead or inactive) embryos. The former are distinguished by a straight central canal with hyalin, refractive walls of quite uniform thickness and devoid of finer structure. The adult parasite, which inhabits the small intestine, is probably a parthenogenetic female, and is a rarity in the stool. It is quite minute — 2.2 mm. long and about 0.034 mm. thick. Oxyuris Vermicularis.— Oxyuris vermicularis {Ascaris vermicularis), the common pinworm or seatworm, is a small nematode, the males measuring 3 to 5 mm. long with the posterior end curved, the females about 10 mm. long and 0.6 mm. thick. The ova (Fig. 22) are flattened on one side, measure 0.050 by 0.016 to 0.20 mm. (Braun), and have a clear, thin shell. The ova are de- posited with the embryos already developed within the shell. It is necessary to recall the fact that the gravid females habitually wander from the region of the cecum and appendix to the rectum, anus, perineum, etc., in order to Fig. 22^0vtjm appreciate clearly the reasons for the methods OF Oxyuris vEEMicuLAEis. of dlagnosiug infection. The crawling of the worms over the skm causes itchmg, so that the patient usually scratches the affected part. The presence of pinworms may be determined ^ by (1) examining the feces for the adult worms (p. 184). Usually only females are found. The material for examination ' stiles, C. W. " Osier 's Modern Medicine, ' ' Vol. I, 1907, p. 601. Digitized by Microsoft® THE FECES 189 is best obtained by an enema given in the evening. (2) The worms may be seen in the crotch, especially if the child be examined during the restless period after retiring. (3) Microscopic examination of the scrapings of the skin about the anus or of dirt from the finger nails (the ova being picked up in scratching the perineum) may reveal the ova. (4) Eggs may be found in the feces. Fecal exam- ination for the eggs of the parasite is the least trustworthy of the methods of diagnosing infection. Stiles' statement, which is agreed to by experienced observers, should be borne in mind. He says: "The writer's experience is that the eggs may be found in fecal examination in some cases in which pinworm is not even suspected; but that a nega- tive examination is not of much value. ' ' Examination for the mature worms or for the ova in the scrapings of the perineum or finger nails give more reliable results. Trichuris Trichiura.— Trichuris trichiura (Trichoce- phalus dispar), the whipworm, is rarely seen in the feces. It may be found after treatment has been administered. The males are 35 to 45 mm. long, the females 35 to 50 mm., three-fifths of which is formed by the anterior filamen- tous portion (Blanchard). Diagnosis of in- fection is made by finding the ova in the feces. The eggs (Fig. 23) are oval in shape, with a relatively thick shell, which is gener- ally stained dark yellowish-brown. At either Fig. 23. — Ovum , ,. . ■ n T n 1 • 1 • OF Trichuris pole there is a space m the shell, which is trichiura. occluded by a plug, the outer, surface of which x^^o. projects slightly beyond the shell. Within the shell the yel- lowish or brownish granular yolk substance is seen. The dimensions of the eggs are 0.050 to 0.056 mm. long and 0.024 mm. wide (Blanchard). Though smaller than many 14 Digitized by Microsoft® 190 MICROSCOPIC EXAMINATION other eggs, they are, nevertheless, of sufficient size to be easily seen with the usual low magnifications. Ascaris Lumbricoides.— Ascaris lumbricoides, the or- dinary "roundworm" of man, is the largest of the com- moner parasitic nematodes. Diagnosis may be made by the discovery of the parasite in the feces or vomitus, or of its ova in the stool. The living worm has a reddish or grayish-yellow color. The males vary in length between Fig. 24. — Ovum of Ascaris lumbkicoides (1) ; the Same Under High Focus, Show- ing THE Albuminous Coating (2). X460. 15 and 25 cm., and are about 3 mm. thick. The posterior end is conical, and is curved ventrally. Females are 20 to 40 cm. long and about 5 mm. in thickness. Lateral, dor- sal, and ventral stripes run longitudinally along the body of the parasite, the first being the most prominent. The ova (Fig. 24, 1 and 2) are elliptical and have a thick, trans- parent shell, which at times appears laminated. A rough albuminous coating forms the outer surface of the egg, and is usually stained brown with the fecal pigments. The al- buminous coating may be lost in some of the eggs. The size of the ova varies between 0.040 to 0.050 by 0.050 to Digitized by Microsoft® THE FECES 191 0.070 mm. (Braun). Unfertilised ova'^ (Fig. 25) may be encountered. They are flatter — ^much less plump than the fertilized specimens — the shell is thinner, and the albumi- nous coating appears to be less abundant. The yolk is coarsely granular, in contrast to the finely granular ap- pearance in the fertilized eggs. Toxocara Canis.— Toxocara canis {Ascaris mystax), the common roundworm of dogs and cats, is rare in man.^ The adult parasite is much smaller than As- caris lumbricoides ; the males are 4 to 6 cm. long and 1 mm. thick, while the fe males measure 6 to 11 cm. in length and 1.7 mm. in thickness. The maximal length recorded is 20 cm. (Blanchard). The ova resemble those of Ascaris lumbricoides, but are more spherical, having a diameter of 0.068 to 0.072 mm. (Blanchard). Trichinella spiralis.— The adult males of Trichinella spiralis are 1.4 to 1.6 mm. long and 0.04 mm. thick ; the females Fig. 25.— Unfertilized _ . Ovum of Ascaris are larger, measuring 3 to 4 mm. in lumbricoides. length, with an average thickness of 0.06 mm. (Blanchard). They inhabit the small intestine. The feces, obtained by active purgation, are mixed thor- oughly with water, placed in a tall cylinder, and after the sediment has settled the fluid is poured off. The sediment is then placed in a dish with dark background; the thick- ness of the fecal layer should not exceed 1/12 inch. The dish is tilted, and any minute, hair-like objects are trans- ferred to a slide and examined microscopically (Stiles). 'Logan, O. T. "The little known atypical (unfertilized) egg of Ascaris lumbricoides." N. Y. Med. Jour., 1907, LXXXVI, 1164. ^ Biesele. ' ' ITeber einen Fall von Ascaris mystax beim Menschen. ' ' Munchen. med. Wchnschr., 1911, LVIII, 2391. Digitized by Microsoft® 192 MICROSCOPIC EXAMINATION The females may remain in the intestines eight weeks; the males die within a few days. The embryos may be re- covered from the blood (p. 310). Trematodes Trematodes ^ or flukeworms are fortunately rare in the United States, the cases reported being largely importa- tions from Asia and Africa. Of those whose presence in the body may be determined by finding the ova in the feces, the following are among the more important. The para- sites themselves are rarely seen during the life of the host. Opisthorchis Sinensis.— Opisthorchis sinensis, a liver- fluke, deposits dark brown, oval eggs with sharply defined operculum or cap. They gain access to the bowel by way of the biliary passages. The ova measure ^ 0.015 to 0.017 by 0.027 to 0.030 mm. Fasciola Hepatica,— Pasciola hepatica, also a liver- fluke, is common in many domestic animals (chiefly herbi- vora), though rare in man. The eggs are oval, yellowish- brown, with distinct operculum, and measure 0.130 to 0.145 by 0.070 to 0.090 mm. They contain no embryo when ovi- posited. Schistosoma haematobium, the causative agent in bil- harziasis (venous distomatosis), inhabits the branches of the portal vein of man, particularly the mesenteric veins, and also the veins of the urinary bladder and vagina. The sharp-spined ova pierce the wall of the vessel, and thus it happens that they may be found in either urine (see p. 'Stiles, C. W. "Illustrated key to the trematode parasites of man." Bull. no. 17, U. S. Pub. Health & Mar. Hosp. Serv., Wash., 1904. (Illustra- tions and full descriptions of parasites and ova are given, together with brief clinical notes, keys to the ova, etc.) ' The measurements of all trematode ova, unless otherwise indicated, are taken from Stiles {loc. oit.) and are the extremes reported by him from the literature or his own observations. Digitized by Microsoft® THE FECES 193 118) or feces, or in both. The eggs (Fig. 26) are oval or spindle-shaped and measure 0.050 to 0.073 by 0.120 to 0.190 mm. (Braun). The shell is clear, usually brown in color, and is provided with a sharp spine. The latter is usually situated laterally (subterminally) near one pole of the ovum when seen in the feces, whereas a termi- nal spine is commonly seen in the urine. The ovum contains a cili- ated embryo or miracidium. At times the latter may be seen swimming free in the preparation. The stool contains blood practi- cally without exception. Schistosoma Japonicum.— Schistosoma japonicum is endemic in Japan and in certain parts of China, and has been reported from the Philip- pines. It in- habits the portal and mesenteric veins chiefly; the bladder is apparently unaffected. Infection of the lung (ova in sputum) is rare. The ova (Fig. 27) are without spine or operculum, oval in shape, and measure 0.060 to 0.090 by 0.030 to 0.050 mm. (Braun). FiQ. 27.— ovma of Schis- Each Contains a fully developed mira- TOSOMA JAPONICUM. (From a specimen pre- cidium. served with formalin, . . _,,.. ,-, . -, obtained through the FaSClOlopSlS BUSKU.— FaSClolopSlS Loga°nT°x?60.°'^' buskii, like Schistosoma japonicum, is Fig. 26. — Ovttm op Schistosoma HAEMATOBIUM. (From a speci- men preserved with formalin.) X460. Digitized by Microsoft® 194 MICROSCOPIC EXAMINATION an intestinal fluke. It is widespread in Asia. The ova measure 0.120 to 0.130 by 0.077 to 0.080 mm. and have a delicate operculum. Paragonimus Westermanii.— Paragonimus westermanii, the lung-fluke, belongs to the class of parasites under con- sideration. Its ova may appear in the feces through swal- lowing of the sputa, if they pass the stomach intact. Liver infection has also been recorded. The eggs (Fig. 36) are oval, 0.068 to 0.118 by 0.048 to 0.060 mm. in size, possess a yellow shell, and are provided with an operculum. Cestodes Cestodes ^ or tapeworms include some of the commonest intestinal parasites in the United States. The larger worms usually disclose their presence to the infected in- dividual by segments, which appear in the feces. Micro- scopic examination of the stools oftentimes reveals ova where infection has not been suspected. Tenia Saginata.— Tenia saginata, the beef tapeworm, is a large parasite, measuring 4 to 10, even 36 m. in length when fully developed. From the mature, gravid segments (the segments are hermaphroditic), ova (Fig. 28, 2) are deposited in the feces. They are round or oval, and meas- ure 0.030 to 0.040 by 0.020 to 0.030 mm. (Braun). The shell is rather thick, radially striated, and light brown in color. Within it three pairs of booklets may be visible; to see them it is necessary to focus carefully, as it does not happen often that all are in the same plane. The mature segments or proglottides (Fig. 28, 1) are those usually seen in the feces. They are 16 to 20 mm. long and 4 to 7 mm. broad, and are characterized by the presence of a uterus ' stiles, C. W. " Illustrated key to the cestode parasites of man. ' ' Bull. No. S5, Hyg. Lab., U. S. Pub. Health & Mar. Hosp. Serv., Wash., 1906. Digitized by Microsoft® THE FECES 195 with central stem, from each side of which 25 to 30 lateral branches are given off; these lateral branches are them- selves subdivided into numerous smaller branches. The gross structure of the uterus may be determined by flat- tening the segment between two glass slides and holding (1) (2) TO Fig. 28. — (1) Gravid Proglottis of Tenia saginata (X4); (2) Ovum op Tenia FAGINATA (X460); (3) Gravid Proglottis of Tenia solium (X4). it to the light. The uterus then stands out in fairly sharp relief. Each segment is provided with a genital pore which is found at one side; the pores alternate very irregularly from side to side. The head of the parasite is cuboidal, 1.5 to 2.0 mm. thick. It is unarmed. Note. — From the ova alone it is impossible to distin- guish between Tenia saginata and Tenia solium (q. v.). The ova of Tenia saginata are quite innocuous to man, since the intermediary stage of the parasite, Cysticercus bovis, to which they give rise, develops practically only in beef — at all events, not in man. With Taenia solium the case is quite different. While the hog is the usual host of Digitized by Microsoft® 196 MICROSCOPIC EXAMINATION the Cysticercus celMosae, the latter may also occur in man, either from the introduction of the ova or the mature, gravid segments into the stomach. Obviously, then, it is very important to handle all intestinal discharges contain- ing ova like those described above with extreme care, until the presence of Tenia solium is definitely excluded. For similar reasons, the patient's excreta should be thoroughly disinfected, preferably by burning. Tenia Solium.— Tenia solium, the pork tapeworm, re^ sembles Tenia saginata in many respects. The fully grown parasite is 2 to 3 m. long. The ova are round or oval, 0.031 to 0.036 mm. in diame- ter (Braun), with brown shell,, radially striated. The oncosphere is about 0.020 mm. in diameter, and possesses six hook- lets. The egg is indistinguishable from that of Tenia saginata (Fig. 28, 2) micro- scopically. The mature segments (Fig. Fig. 29. — Ovdm op 28, 3), which are often seen in the feces, a"s™atu^^X are 10 to 12 mm. long and 5 to 6 mm. ^^°- broad (Braun). They differ from the seg- ments of Tenia saginata in that the uterus has only 7 to 10 lateral branches extending to either side, and they do not tend to rebranch. The rostellum of Tenia solium is char- acterized by a double crown of 22 to 32 booklets, large and small alternating. The head of Tenia saginata, on the other hand, is unarmed. Dibothriocephalus Latus.— Dibothriocephalus latus, the fish tapeworm, is the third large cestode frequently para- sitic in man. The mature parasite may measure 9 m. in length. The ova (Fig. 29) have a rather thin, clear, white or brownish shell, with a small operculum or cap. The latter stands out particularly well after treatment with Digitized by Microsoft® THE FECES 197 Fig. 30. — Gravid Proglottis op dibothriocephaltjs latus. x4. glycerin or dilute sulphuric acid (Blancliard). The eggs are elliptical, and present granular contents. They meas- ure 0.068 to 0.070 mm. by 0.044 to 0.045 mm. (Blanchard). The posterior segments or proglottides (Fig. 30) may be found in the feces, and at times are devoid of ova. Unlike the two preceding parasites, the majority of the segments are broader than they are long, though the reverse may be ob- served in the posterior seg- ments. The dark brown, rosette- shaped uterus, placed near the center of the proglottis, distin- guishes the parasite. The head, which is almond-shaped, is 2 to 3 mm. long and is unarmed. Booklets are lacking also in the ova. Hymenolepis Nana.^— Hymenolepis nana, the dwarf tapeworm, is a very common parasite, especially in chil- dren. Because of its small size, infection with this para- site is seldom diagnosed by the finding of segments in the feces. The fully de- veloped parasite is 10 to 45 mm. long and from 0.5 to 0.9 mm. thick. The head is round, 0.25 to 0.30 mm. in thickness, and presents a simple crown of 24 to 30 booklets. The ova are spherical or oval (Fig. 31). The shell is clear and trans- parent, at times having a light brownish or yellowish tint. It consists of tM^o distinct membranes ^ Eansom, B. H. " An account of the tapeworms of the genus Hymenolepis parasitic in man, etc." Bull. no. 18, Hyg. Lab., U. S. Pub. Health & Mar. Hosp. Serv., Wash., 1904. (A full description of the parasites, with the clini- cal aspects of infection.) Fig. 31. — Ovum of Hymenolepis nana. X460. Digitized by Microsoft® 198 MICROSCOPIC EXAMINATION separated by an intervening space, which contains a trans- parent substance, more or less finely granular. At two op- posite points, usually corresponding to the poles of the egg, there is a small, mammillated projection, often -not ap- parent. To each of these is attached a number of clear hyalin fibers, which pass out through the intermediate sub- stance toward the outer mem- brane. It frequently happens that the intermediate substance shrinks or retracts from the outer or inner membrane or from both, resulting in the appearance of a third membrane between the Fig. 32.— Ovdm op hymenolepis two ; in reality none exists. The DiMiNUTA. X460. Q^^gj. membrane is very thin, less than 0.001 mm. The innei* membrane is of about the same thickness, and closely invests the oncosphere, which pre- sents three pairs of hooks, usually directed toward one pole (Ransom). The outer dimension of the egg varies be- tween 0.036 and 0.056 mm. long and 0.032-0.042 mm. broad. Hymenolepis Diminuta. — Hymenolepis diminuta, commonly found in rats, is occasionally parasitic in man. The parasite is small, being 1 to 6 cm. long and 2.5 to 4 mm. wide. The head, which is unarmed, is 0.2 to 0.6 mm. wide. The ova (Fig. 32) resemble those of Hymenolepis nana. They are round or nearly so, and have Fig. 33. — Diptlidium caninum, showing AN Egg Capsule and a Free Ovum. (After Stiles.) Digitized by Microsoft® THE FECES 199 two membranes. The outer membrane may be radially striated. The intervening space between the two mem- branes is granular. The diameter of the eggs varies be- tween 0.054 and 0.086 mm. (Ransom). Dipylidium Caninum.— Dipylidium caninum is a com- mon parasite in the intestines of dogs and cats ; a number of instances of human infection are recorded.^ The fully developed parasite is 15 to 35 cm. long. The head is small and the rostellum club-shaped, with three to four rows of hooks, about 60 in number. The mature, gravid segments may be seen without difficulty with the unaided eye. They are 8 to 11 mm. long and 1.5 to 3 mm. broad; their color is often reddish. The genital pores are doiible, and are opposite. The ova (Fig. 33) are spherical, and have a diameter of 0.043 to 0.050 mm. The shell is thin. The uterus contains the eggs in capsules, 8 to 20 eggs being contained in each ; they may be encountered in the feces in this form (Stiles). Three pairs of hooks are to be seen in the oncosphere. Peesbevation op Geoss Specimens of Cestodes and Othee Paeasites Stools containing parasitic ova or embryos are best preserved by adding commercial formalin (40 per cent.) to a thin watery suspension of the material, so that the latter contains about 2 per cent, of formalin. The major- ity of eggs are quite well preserved, though thin-shelled ova, such as those of Hymenolepis nana, show considerable distortion. Specimens of feces containing the rhabditiform embryos of Strongyloides stercoralis may be kept for sev- eral years ; the structure may be, wanting in some of the ' Lins, J. ' ' Sechs Falle von Tsenia cucumerina beim Menschen. ' ' Wiener Tclin. Wchnschr., 1911, XXIV, 1595. Digitized by Microsoft® 200 PRESERVATION OF PARASITES embryos, but may remain characteristic in others. It is of interest that the formalin does not arrest the develop- ment of the ova of Ascaris lumbricoides.^ The writer has one specimen of feces now more than four years old, pre- served with formalin; many of the Ascaris ova contain living embryos. The adult parasites may also be preserved in formalin (2 per cent, solution). Permanent Preparations of Flatworms For purposes of study cestode and trematode material may be prepared in several ways. (1) Method of Mink and Ebeling.^— The fecal material is mixed with physiological salt solution heated to about the body temperature (37° to 40° C). The worms move about, and the smaller, such as Hymenolepis nana, are the more readily seen. With forceps the parasites are trans- ferred to a second dish of clear salt solution, in which they become free of mucus and feces. They are now transferred to one of three solutions for fixation: (1) Alcohol, 50 to 70 per cent., with or without glycerin; (2) Zenker's solu- tion (which consists of bichlorid of mercury, 5.0 gm. ; potas- ' slum bichromate, 2.5 gm. ; sodium sulphate, 1.0 gm. ; dis- tilled water to 1,000 c. c), or (3) a 2 per cent, formalin so- lution ; in any of these the material remains 14 to 16 hours. Zenker's fluid causes considerable shrinking and a yellow- ish discoloration. Formalin is best, since "the natural color ' Morris, R. S. " The viability of parasitic ova in two per cent, formalin, with special reference to Ascaris lumbricoides. ' ' Johns Hopkins Bosp. Bull., 1911, XXII, 299. ^ Mink, O. J., and Ebeling, A. H. "A method for the preparation of flat- worms for study. " U. S. Naval Med. Bull, 1909, III, 267. Digitized by Microsoft® THE FECES 201 of the parasite is preserved fairly well with little or no shrinkage. The fixative should be allowed to act not more than fifteen hours, when the parasites are transferred to the following medium: Syrup (glucose, 48 parts, water, 52 parts) .1,000.0 c. c. Methyl alcohol 200.0 c. c. Glycerin 100.0 c. c. Camphor, q. s. (to keep sterile). The specimens may be left in this solution indefinitely, though they are usually sufficiently cleared in 4 to 5 hours. The material is now placed on a slide in glycerin jelly. After the latter has hardened (24 hours or more), the cover glass is sealed with cement. (2) Boggs' Method.^— The worm is washed free of feces, and is placed in water or salt solution, in which it is allowed to die, so that it may be fixed while relaxed. It is then placed in a solution of 20 per cent, glycerin in 80 per cent, alcohol, which both fixes and clears the specimen. It is allowed to remain in this fluid in a partially covered dish until the alcohol is entirely evaporated. The specimen is then clear. It is transferred to a glass slide, and the excess of glycerin is removed by blotting paper. Glycerin jelly is then placed on the specimen, which is covered with a cover glass. In 24 to 48 hours, after the jelly has solidi- fied, the preparation is sealed with microscopical cement. To prevent curling of the specimen, it may be spread on a piece of heavy filter paper before immersing it in the glycerin-alcohol solution ; it may be necessary to put a light weight on the cover slip until the jelly hardens. ' Boggs, T. R. Personal communication, and in Emerson, C. P. " Clinical Diagnosis." 3rd Ed. Philadelphia and London, 1911, p. 444. Digitized by Microsoft® Digitized by Microsoft® THE FECES 203 Glycerin jelly is prepared as follows: Gelatin (gold mark) 14.0 gm. Distilled water (boiling) 120.0 c. c. Dissolve in the hot water and add — Glycerin 120.0 c. c. Cool to 50° C. and add the whites of two eggs. Heat gently without stirring. Strain the mixture through a fine-meshed wire sieve, and filter through cotton while still warm. Add water to make the volume 240 c. c. and 1 c. c. of pure car- bolic acid as a preservative. The jelly solidifies on cool- ing. For use, melt it by immersing the flask containing it in hot water. (3) Creosote Method.— The material is placed in 70 per cent, alcohol, and then in 95 per cent, alcohol, for 15 to 30 minutes. It is then transferred to Beechwood creo- sote, in which it remains until the tissue is cleared. The time required varies with the size of the specimen and with the degree to which water was withdrawn by the alcohol. Finally, the specimen is mounted in balsam. Accidental contaminations through food or drink may account for some of the ova found in the feces. As an ex- ample, the ova of the Tyroglyphus siro (Fig. 34) may be cited. This is the common cheese-mite, which may also be found in flour and other articles of diet. The mites may be found in the stool in addition to the ova. Measurements of the ova may serve to differentiate them from those of intestinal parasites. In cases of doubt, it is advisable for physicians to submit the material to a zoologist for deter- mination. Such examinations are made at the Hygienic Laboratory, U. S. Public Health Service, Washington, D. C. Digitized by Microsoft® CHAPTEE IV THE SPUTUM In the strict sense of the word, sputum refers to the expectorated material which arises in the respiratory pas- sages between the lung alveoli and the larynx. To obtain sputum for examination, the patient should be told to discard the nasal and pharyngeal discharges. He should be instructed as to the proper receptacle. In case the physician does not supply a sputum box or cup, the patient may conveniently use a ivide-mouthed bottle, which has been thoroughly cleansed and sterilized by boiling. In cases where a sputum examination is urgently indicated but no sputum is expectorated, expectorants, such as am- monium chlorid, may be given. Hausmann ^ advises that the fasting stomach be washed out in the early morning with a view to obtaining bronchial mucus ; he reports valu- able findings with this method. With children it is not infrequently necessary to wash out the stomach, examine the feces, or place the finger, covered with gauze, in the child's throat after a coughing spell and mop out the sputum. The importance of repeated examinations of the sputa cannot be overemphasized. Particularly when looking for the tubercle bacillus, if the physical signs or history are even suggestive, examinations should be continued. A single negative result means nothing. " Hausmann, T. ' ' Die Friihdiagnose der Lungentuberkulose durch die Mageninhaltsuntersuchung. " Deutsch. Arch. f. Tclin. Med., 1908, XCIV, 595. 204 Digitized by Microsoft® THE SPUTUM 205 Amount.— The quantity of the sputum expectorated in twenty-four hours varies greatly in disease. An approxi- mate estimate of the amount is usually sufficient. Reaction.— The reaction of fresh sputum is usually al- kaline. An old specimen or sputum which has stagnated in the body may be acid in reaction. Character.— Sputum is designated mucoid, mucopuru- lent, purulent, serous, or bloody, as the case may be. The terms are self-explanatory. Various combinations are met with. Bloody sputa may assume any of the shades seen in a bruise. In the presence of jaundice, it must be remem- bered that green sputa do not necessarily indicate a pre- vious pulmonary hemorrhage; the color is in most cases simply a manifestation of the icterus. Bacterial growth may at times account for a green color. Odor.— The odor of sputa is important chiefly in con- nection with putrid affections of the bronchi or lungs. Consistence. — The consistence of the sputum is gener- ally dependent on the quantity expectorated. With large quantities, the consistence is usually thin, though the sputa of croupous pneumonia form a notable exception. Simi- larly, when the sputum is small in amount, it is usually more or less tenacious. Air Bubbles.— Most sputa contain air bubbles. The size of the bubbles is said to indicate roughly the caliber of the bronchi from which the expectorated material is derived. More air is contained in sputum from the smaller bronchi than from the large. Dittrich's Plugs.— Dittrich's plugs are sausage-shaped casts of the bronchi, varying in size up to that of a white bean. Microscopically, they may be seen to contain fat droplets, fatty acid crystals, cell detritus, and bacteria. A few pus cells and occasionally red blood corpuscles or 15 Digitized by Microsoft® 206 MICEOSCOPIC EXAMINATION hematoidin in granules or needles may be observed, less commonly flagellates. Bronchial Casts.— Bronchial casts are observed in some diseases with considerable frequency. Their size is deter- mined by that of the bronchi giving rise to them. Casts are usually branched, and consist mainly of fibrin, in which leukocytes, red blood cells, epithelial cells, etc., may be em- bedded. If the casts are small, their isolation is facilitated by placing the sputum on a white plate, half of which has been painted black. By teasing the specimen with needles, the casts may be found, provided they are macroscopic in size. The addition of water often renders the teasing easier. Curschmann's Spirals.— Curschmann's spirals occur in various sizes. Only the larger specimens are visible to the unaided eye. The largest spirals measure about 1 mm. in thickness, and are ten to twenty-five times as long. They can only be distinguished as spirals by microscopic exam- ination. Layer Formation.— When abundant, as in the case of sputa from bronchiectatlc cavities, for example, distinct layer formation may be observed. Solid particles collect at the bottom, then a fluid portion, with frothy mucus on the surface. Frequently strands of mucus dip down from the upper layer. MICROSCOPIC EXAMINATION Examination of the Fresh Sputum.— Examination of the fresh sputum is greatly neglected. It is as important in the routine study of sputa as the various staining methods, and may furnish information which can be gained in no other way. Digitized by Microsoft® THE SPUTUM 207 For tlie examination of the fresh specimen two glass plates are required, one about the size of the stage of the microscope or a little smaller, the other of the same length or even a bit longer, but about one inch narrower. Old photographic plates, when cleansed, answer the purpose very well ; they may be cut into any size desired. In place of the second plate, a glass slide (3x1 in.) may be used. For handling the sputum steel hat-pins are useful. They are inexpensive, and are easily sterilized by heating in the flame of the Bunsen burner. Some of the sputum to be examined is transferred to the larger plate by means of the hat-pins. It should be so placed on the plate that, when spread out, all parts of the specimen will be accessible for microscopic examina- tion. After sterilizing the hat-pins ^ the second plate or the glass slide is placed over the sputum, which is spread out in a thin layer. Examination is made with the low power objective, for the thickness of the upper plate is usually greater than the working distance of the higher power dry objective. Furthermore, higher magnification is often unnecessary. The magnification may be increased, however, by selecting a strong ocular or by drawing out the tube of the microscope. After spreading the sputum between the two plates, it is examined macroscopically on a dark background, and any small, opaque masses are noted and singled out for microscopic inspection. Examination then determines whether the particle should be transferred to a slide and studied under a cover glass with higher magnification, or used for staining, or for both purposes. Curschmann's spirals, necrotic tissue fragments, etc., are looked for in •All sputa should be considered as infectious material, and handled ac- cordingly. \ '• i ' i , \ Digitized by Microsoft® , 208 MICROSCOPIC EXAMINATION this way. The macroscopic examination should always be made, for a great deal of time may be wasted, to say noth- ing of overlooking important findings, if the microscopic examination is performed aimlessly. There are, of course, specimens in which macroscopic examination reveals noth- ing, where microscopic study of the preparation is fruitful. But in the majority of instances the naked eye inspection of the thinly spread specimen is an important adjuvant to microscopic examination. Yellow Elastic Tissue.— Elastic tissue fibers are most easily found by means of the glass plate method. If pres- ent, they are often found in yellowish, opaque masses of necrotic tissue about the size of a pinhead or thereabouts. The fibers may present roughly the outline of one or more alveoli (alveolar elastic tissue), single fibers or several long fibers forming a network may be seen (bronchial), or there may be sheets of elastic tissue (arterial). Often only isolated fibers are met with. The yellow elastic fibers are readily seen, and are characterized by (1) their uniform diameter, (2) sharp outline, (3) great refractivity, (4) curl- ing ends, (5) a tendency to branch, and (6) by the fact that pressure on the slide produces no varicosities or thicken- ings in the fibers. Fatty acid crystals, which may be mistaken for elastic tissue, usually present varicosities after pressure, and are, furthermore, unlike elastic tissue in that they are soluble in ether or KOH. Again, warming the preparation trans- forms fatty acid needles into droplets. In contrast to the wavy elastic tissue fibers, fatty acid crystals usually pre- sent a single curve. Curschmann's Spirals.— Curschmann's spirals, often visible macroscopically but never recognizable as such, stand out with distinctness on microscopic examination. Digitized by Microsoft® THE SPUTUM 209 They occur in two forms ; in the one there is seen a twisted spiral consisting of delicate, thread-like filaments of mucus, in the turns of which eosinophilic leukocytes, pus cells, epi- thelial cells, Charcot-Leyden crystals, etc., are caught; in the other form there is a highly refractive central filament, about which the mantle of mucus containing eosinophiles, etc., is twisted. The spirals are subject to much variation in size. Isolated central filaments may be found. The thickness of the filaments differs in different spirals, but in a given specimen it is quite uniform. The finest fila- ments are extremely minute, while the largest may be twice as thick as a red blood corpuscle. Fairly satisfactory permanent preparations of spirals may be had by mounting them in glycerin jelly and sealing the specimen with cement after the jelly has hardened. Dust cells, "heart failure" cells, Charcot-Leyden crys- tals, and other objects may be seen on examining the spu- tum on the glass plate. The Charcot-Leyden crystals may be so small as to escape detection. It may, therefore, be advantageous to use a higher magnification in their study. For this a selected particle of sputum is transferred to a glass slide and pressed out under a cover glass. Alveolar epithelial cells, derived from the lung alveoli, are constantly present in the sputum. Their shape varies greatly, since they are possessed of ameboid motion — a fact which is readily demonstrable by examining a perfect- ly fresh specimen on a warm stage. They are relatively large cells, but are not of uniform size. The nucleus is large and oval. The protoplasm of the alveolar cells is rather coarsely granular, but soon undergoes degeneration, as a result of which fat droplets and myelin granules make their appearance in it. The fat droplets in alveolar cells are, like similar drop- Digitized by Microsoft® 210 MICROSCOPIC EXAMINATION lets elsewhere, of all sizes, usually round, refractive, and slightly greenish, especially the larger drops. Their na- ture is determined by adding a drop of alcoholic solution of Sudan III or Scharlach R to the specimen, by means of which fat is stained orange or orange-red. Myelin degeneration of the alveolar cells gives rise to the macroscopic masses in sputa resembling boiled sago. The myelin droplets may be large or small, and, un- like fat droplets, they are often quite irregular in con- tour. At times the center of a mass of myelin appears to be thinned. Myelin granules are refractive and have a greenish tint, which is more pronounced than that seen in fat droplets. Myelin is frequently found free in the spu- tum, probably the result of disintegration or mechanical rupture of the degenerated epithelial cells. It does not take the fat stains. Dust cells are found in the sputum of all who inhale coal dust. They are alveolar epithelial cells, which have phagocyted the minute particles of coal dust, which con- stantly are inspired in a smoky atmosphere. The dust appears as dark, brownish-black spots in the cell, which may be so heavily laden that nucleus and protoplasm are entirely obscured. Dust cells are easily distinguished in examining sputum by the plate method. When they are numerous, the sputum is stained more or less diffusely black. "Heart-failure cells" are alveolar epithelial cells which have taken up blood pigment. The name is a misnomer, for they appear in the sputum after a pulmonary or bron- chial hemorrhage from any cause whatever. The pigment, which is hematoidin, appears as light, golden-yellow gran- ules, which can scarcely be confused with coal dust. Red blood corpuscles are often seen in a state of preser- Digitized by Microsoft® THE SPUTUM 211 vation. If the hemorrhage is an old one, however, the cells disintegrate, and only hematoidin in amorphous masses — usually in heart-failure cells — or in needles will be discov- ered. Pus cells, polynuclear neutrophilic leukocytes, are al- ways present in the sputa microscopically. The polymor- phous nucleus in a cell about 12 micra in diameter with finely granular cytoplasm is characteristic. Occasionally fat droplets are contained in the cells, or they may take up foreign particles in the air passages. In a fresh speci- men active ameboid movements may be observed in these cells; pseudopodia are protruded, and the granules of the protoplasm are actively motile. Eosinophilic leukocytes, of the same size as pus cells and having a polymorphous nucleus, are distinguished by their protoplasmic granules. The latter are coarser than the neutrophilic granules, and are highly refractive, glis- tening bodies. Ameboid motion may also be observed in these cells. Free granules are usually present in the speci- men. Lymphocytes, or cells which are identical morphologi- cally, are present at times in large numbers. The round or oval nucleus with narrow rim of protoplasm and the small size of the cells — 7 to 12 micra — together with the non- granular cytoplasm are distinctive. Charcot-Leyden crystals are usually found in the spu- tum with large numbers of eosinophile cells. They are formed wherever eosinophile cells disintegrate. In form the crystals are long lozenges. Their edges are clean cut, the points sharp, and the crystals have a yellowish or greenish tint. They are quite fragile, and the larger crys- tals may be broken in preparing the specimen. They oc- cur singly or in clusters, and vary greatly in size; the Digitized by Microsoft® 212 MICKOSCOPIC EXAMINATION smallest are visible only with the oil-immersion objective, while large crystals are seen without difficulty with low magnification. On cross-section the crystals are hexagonal. Like the eosinophilic granules, they may be stained with eosin. They are soluble in mineral acids, alkalies, and boiling water. The crystals may be apparently lacking in sputa which contain enormous numbers of eosinophiles. This is particularly apt to be the case when a sputum is first flooded with these cells. If examinations are made from day to day the crystals are found sooner or later. Crystals of fatty acid, cholesterin, hematoidin, triple phosphate, etc., may be encountered in sputa, especially after stagnation. (For morphology and microchemical re- actions see the chapter on the urine or feces.) MiCKOOEGANISMS IN SpTJTA Only the more important microorganisms of the sputum are referred to in the following pages, and in no case are cultural methods described. For this and details of mor- phology the reader is referred to works on bacteriology. Bacillus Tuberculosis Bacillus tuberculosis may be found in any kind of spu- tum, for the gross appearance of the sputum in pulmonary tuberculosis is in no way distinctive; it may, in fact, be anything. If the plate examination shows necrotic tissue (elastic tissue), it should be selected for staining, as the bacilli are generally more numerous in such material. Oth- erwise purulent particles are most suitable for examina- tion. The preparation for examination is made by smearing the selected particle on a glass slide with a hatpin or other Digitized by Microsoft® THE SPUTUM 213 suitable object. Or it may be pressed between two slides, which are then drawn apart, so that the material is smeared in a thin layer on each of them. In the second way prep- arations of more uniform thickness are obtained, but there is usually some of the material at the edge of the slide, with which the fingers or other objects may become contami- nated. (This is referred to not because it is a valid objec- tion to the method, but because the writer has so frequently observed carelessness in this particular point. Still, one who neglects such an obvious source of infection is sure to make other more serious breaks in technique, and has no business examining infectious material, both for his own safety and, more particularly, for the safety of others.) If the sputum dries slowly, it may be hastened by warming the slide gently. The smear is then fixed in the usual way by passing it through the flame of the Bunsen burner sev- eral times. Ziehl-Neelsen Method.— The Ziehl-Neelsen method of staining is the one generally employed for the tubercle bacillus. The reagents required are: (1) Carbol-fuchsin. (a) Fuchsin 1.0 gm. Absolute alcohol 10.0 c. c. Dissolve and add — (b) 5 per cent, carbolic acid 100.0 c. c. (2) Acid alcohol. Hydrochloric acid, cone 3.0 c. c. 70 per cent, alcohol to 100.0 c. c. (3) Loffler's methylene blue. Methylene blue, saturated alcoholic sol 30.0 c. c. 0.01 per cent, potassium hydrate . . . 100.0 c. c. Digitized by Microsoft® 214 MICROSCOPIC EXAMINATION Method. — (1) Cover the specimen with carhol-fuchsin and warm it till the stain steams. Maintain this tempera- ture for five minutes.^ Or the specimen may be immersed in the cold stain for twenty-four hours. It is important to overstain the preparation with carbol-fuchsin, for at best many of the bacilli are decolorized. With light stain- ing, when only a few bacilli are present, they may be missed entirely (L. Brown). (2) Eemove the excess of stain by washing in running water. (3) Decolorize in acid alcohol, until only the thickest parts of the smear have a faint pinkish tint. (4) Again wash in water. (Eeturn to the acid alcohol if the specimen becomes pink after washing.) (5) Stain with LofiBer's methylene blue 5 to 20 seconds. (6) Wash in water, dry the preparation in the air or between sheets of blotting paper. Examine in immersion oil. The tubercle bacilli are stained red ; all else is blue. Antiformin Method for the Detection of Tubercle Ba- cilli,— In 1908 Uhlenhuth and Xylander^ made the impor- tant discovery that antiformin possesses the peculiar prop- erty of dissolving all bacteria except those which are acid- fast, to which class the tubercle bacillus belongs. Applied to the sputum, they found that, in addition to the major- ity of bacteria, the great mass of the sputum is also lique- fied. By the use of antiformin it is, therefore, possible to examine a large quantity of sputum and thus to concen- trate the tubercle bacilli present in it. The method is val- ^ The copper bar used in blood work is convenient for heating the specimen. The stain must be replenished from time to time as it evaporates, to prevent burning the specimen. ' Uhlenhuth and Xylander. ' ' Antiformin, ein bakterienauflosendes Desin- f ektionsmittel. " Berlin. TcHn. Wchnschr., 1908, XLV, 1346.. Digitized by Microsoft® THE SPUTUM 215 uaHe in those cases where the ordinary technique fails to demonstrate bacilli. A number of methods have been de- scribed for the use of antiformin, of which the following have been found serviceable: (1) Loffler's Method.^ — The quickest method for the use of antiformin with sputum, and one which is well adapted to clinical work, has been described by L6fl3er. With this procedure the examination may be completed in a comparatively short time. A quantity of sputum (5 to 20 c. c.) is measured, placed in a beaker or flask of Jena glass with an equal volume of 50 per cent, antiformin, and boiled. Solution of the sputum occurs almost at once, the fluid foaming and turning light brown. To 10 c. c. of the cooled solution, which is sterile, add 1.5 c. c. of a mixture composed of 1 volume of chloroform and 9 volumes of al- cohol. After shaking thoroughly, the specimen is centrifu- galized for about fifteen minutes (the time varies with the speed of the centrifuge). The chloroform is thrown to the bottom of the tube, and on its surface the sediment col- lects. The supernatant fluid is poured off and the sediment transferred with a clean pipette to a glass slide. The ex- cess of fluid is removed with filter paper, and a small drop of egg albumin (which may be preserved by the addition of 0.5 per cent, carbolic acid) is mixed with the sediment, which is spread on the slide, fixed, and stained in the usual manner. The original sputum may be substituted for egg albumin as the fixative; it is, indeed, preferable, since a more complete examination of the sputum is possible. The tubercle bacilli are said to be killed with this method (Loffler). 'Loffler, F. "Ein neues Anreicherungsverfahren zum farberischen Nach- weise sparlicher Tuberkelbazillen. " Deutsche med. Wchnschr., 1910, XXXVI, 1987. Also Williamson, C. S. "The value of the Loeffler method of sputum examination." Jour. A. M. A., 1912, LVIII, 1005. Digitized by Microsoft® 216 MICROSCOPIC EXAMINATION As a counterstain Loffler uses malachite green (0.1 per cent, aqueous solution). (2) Pateeson's^ Method. — Paterson adds to 10 c. c. of sputum 2.5 c.c. of antiformin,^ giving a 20 per cent, strength of the latter. If the sputum is very thick or tenacious, or insufficient in quantity, a smaller amount is diluted to 10 c. c. with distilled water .^ Solution of the sputum occurs rapidly. The mixture is poured into centrifuge tubes, which have been kept in potassium bichromate and sul- phuric acid,* and rinsed with distilled water just before using. The tubes are stoppered with unused corks, shaken vigorously, and set aside for 24 hours at room temperature or for 4 to 6 hours at 37° C. The tubes are again shaken and then centrifugalized. The supernatant fluid is poured off, the tubes refilled with sterile physiological salt solu- tion, again corked, shaken, and centrifugalized. The wash- ing is done a second time to rid the sediment of all alkali ; otherwise it does not adhere well to the glass. The sedi- ment is then transferred to a slide, smeared, fixed, and stained by the Ziehl-Neelsen method. 'Paterson, E. C. "A report on the use of 'antiformin' for the detection of tubercle bacilli in the sputum, etc. ' ' Jour. Med. Research, 1910, XXII, 315. ''The composition of antiformin, according to Paterson, is equal parts of 15 per cent, solution of sodium hydrate and of liquor sodse chlorinatge (B. P.). The latter is prepared as follows : Sodium carbonate 600.0 gm. Chlorinated lime 400.0 gm. Distilled water 4,000.0 c. c. Dissolve the sodium carbonate in 1,000 c. c. of distilled water. Triturate thoroughly the chlorinated lime in the remainder of the water. Filter. Mix the two and filter again. There is formed an alkaline, almost colorless liquid with a strong odor of chlorin. It keeps well. ' Before this is done, the existence of acid-fast bacilli in the distilled water should be excluded — a troublesome source of error at times, as shown by W. Brem (Jour. A. M. A., 1909, LTII, 909). * Eub up some potassium .bichromate with sulphuric acid for two minutes, allowing the acid to take up as much of the bichromate as it will. Pour off the acid and repeat the process. Digitized by Microsoft® THE SPUTUM 217 The washing with salt solution may be dispensed with. The sediment, which consists of debris, swollen and dis- torted cells, etc., is fixed to the slide with difficulty because of the alkali, but this may be overcome by first smearing the slide with some of the original sputum or with egg white (preserved by the addition of 0.5 per cent, pure car- bolic acid). The original sputum is to be preferred, since the other elements present in it may thus be studied. The tubercle bacilli are not killed and the sedirnent should, therefore, be handled with the usual precautions. The method is valuable for obtaining material for ani- mal inoculation or for cultures. (3) Boaedman's Method.^ — The following procedure has been found satisfactory by Boardman: Fifteen to 20 c. c. of sputum are placed in a conical specimen glass, and antiformin is added sufficient to make a 20 per cent, strength of the latter. After the solution has become homo- geneous and watery in consistence, an equal volume of 95 per cent, alcohol is added. By this means sedimentation is facilitated, since the specific gravity of the mixture is less than 1.000. After stirring, allow it to stand till sedimenta- tion is complete. The clear supernatant fluid is poured off (into a disinfecting solution), and smears are made of the sediment on a glass slide, using some of the original sputum as a fixative. The specimen is then fixed with heat and stained in the usual way. Diplococcus Pneumoniae The pneumococcus, found not infrequently in the upper respiratory passages of healthy individuals, is often con- ' Boardman, W. W. " The use of antiformin in the examination of the sputum for the tubercle bacillus." Johns Sopkins Hasp. Bull., 1911, XXII, 269. Digitized by Microsoft® 218 MICEOSCOPIC EXAMINATION spicuous in the sputum of acute lobar pneumonia and other conditions. It is a Gram-positive organism, whose capsule may be seen after staining with Gram 's method. Gram's Method of Staining. Eeagents: 1. Anilin water gentian violet. Ten c. c. of anilin oil are shaken with 100 c. c. of dis- tilled water till a milky emulsion is secured. After stand- ing five minutes, it is filtered through a wet filter. To the filtrate, which should contain no large oil drops, add 11 c. c. of saturated alcoholic solution of gentian violet and 10 c. c. of absolute alcohol. The solution keeps not more than 8 to 10 days (Schmorl). 2. Gram's iodin solution: lodin 1.0 gm. Potassium iodid 2.0 gm. Distilled water 300.0 c. c. Method.— (1) Stain the heat-fixed smear in anilin water gentian violet 1 to 3 minutes. (2) "Wash quickly in water. (3) Cover the specimen with Gram's iodin solution about 1% minutes. (4) Decolorize in absolute alcohol until the preparation has a grayish or yellowish color — usually about 5 minutes. (The specimen may now be dried and examined. If a counter stain is desired, the further steps are carried out.) (5) Wash in water. (6) Stain with 0.2 per cent, aqueous solution of Bis- marck brown 1 minute. (7) Wash in water, dry in the air or blot, and examine in immersion oil. The pneumococci and all other Gram-positive organisms Digitized by Microsoft® THE SPUTUM 219 are stained blue, the remaining bacteria and cell nuclei are brown. The pneumococcus or Diplocoecus pneumoniae grows in pairs. The long axes of the. organism are placed end to end. For demonstration of the capsules, Welch's method may be employed. Welch's Capsule Stain.— (1) Flood the fixed smear with glacial acetic acid, and immediately pour it off. (2) Wash off the acid with anilin water gentian violet. (3) Wash in 2 per cent, sodium chlorid solution, and examine the wet specimen. Bacillus Influenzce Bacillus influenzas is a very minute bacillus, which often exhibits polar staining. It may be found free in the spu- tum or within pus or epithelial cells. It decolorizes by Gram's method of staining and, therefore, takes the coun- terstain. It may be stained satisfactorily with carbol-fuch- sin. The stain is diluted 1:10 with distilled water, and allowed to act for ten minutes or longer. Cultural methods are required for the complete identification of Bacillus in- fluenzae. It grows on media containing blood. Bacillus Diphtherice Bacillus diphtherice, though not usually found in the sputum, may be considered here. In examining for it the membrane is brushed with a sterile swab, with which, after inoculating tubes of Lofiler's serum, smears may be made on glass slides. The smears may show the organism, but in any case it is better to examine a culture on blood serum which has been incubated at body temperature for 9 to 18 Digitized by Microsoft® 220 MICROSCOPIC EXAMINATION hours. From a fresh culture, of the age given above, the organisms exhibit polar staining. Usually two deeply staining bodies are seen in a bacillus at either pole, though there may be but one or as many as three. Chains are for the most part lacking; frequently the bacilli are placed with their long axes parallel. The polar staining is beau- tifully demonstrated with Neisser's method. Neisser's Staining Method. Eeagents : (1) Methylene blue 1.0 gm. Alcohol, 90 per cent 20.0 c. c. Dissolve and then add — Distilled water 950.0 c. c. Acetic acid, glacial 30.0 c. c. (2) Vesuvin (Bismarck brown) 2.0 gm. Boiling distilled water 1,000.0 c. c. Dissolve. Filter after the solution cools. Method. — (1) Stain in methylene blue solution 1 to 3 seconds. (2) "Wash quickly in water. (3) Stain in vesuvin 3 to 5 seconds. (4) Wash quickly in water, blot dry, and examine in oil. The diphtheria bacilli are slender rods, often having a slight bend. They are stained light brown, with one to three granules, which take a dark blue color. Parallel pairs of bacilli are characteristic. Chain formation is lacking. Other bacteria, which are present in the smear, are stained light brown, so that the blue polar bodies of Bacillus diphtherias are striking and characteristic. Digitized by Microsoft® THE SPUTUM 221 Beall's Method.^— The polar bodies may be demon- strated well by Beall's method. (1) Over stain, the specimen with anilin water gentian vdolet % to 1/2 minute. (2) Wash in water. (3) Decolorize with 10 per cent, glacial acetic acid. till little color remains. This is controlled under the micro- scope. (4) Wash in water, blot dry, and examine in immersion oil. All Grram-negative organisms are decolorized, and most of those which are Gram-positive. The polar staining is intense, and diphtheria bacilli stand out prominently. Practically all other organisms are decolorized more or less completely. Actinomyces Bovis Actinomyces bovis (Fig. 35), the ray fungus, which is the causative agent in "lumpy jaw" of cattle, is occasionally parasitic in man. The lungs are often the site of infection.^ The sputum is characteristic. Claypole studied the series of cases reported by Bridge, and de- scribes the sputum as follows: "In the majority of cases the sputum is characteristic and of two types: (1) glairy, mucilaginous, often quite watery; (2) purulent, more or less bloody, more or less — sometimes intensely — fetid. Both types may be found sparingly or in abundance. . . . The small granules (of the fungus), usually the size of a very small pinhead, can be picked out with a 'An unpublished method of Dr. H. K. Beall of Fort Worth, Texas, through whose kindness it is given here. ^For a review of the subject, see Bridge, N. " Streptothrieosis (actino- mycosis) of the lungs." Jour. A. M. A., 1911, LVII, 1501. IG Digitized by Microsoft® 222 MICROSCOPIC EXAMINATION needle and put on a slide for examination. They are quite tough, and can be washed free of debris by putting them in a dish of water and squirting them vigorously up and down a pipette. ... " "Under low magnification the yellow color is marked; to the naked eye the fungus is grayish-white. The edge is always darker, even shading into a brown; toward the center it grows lighter. From this light, almost homogeneous center, th3 characteristic radiations arise. Higher magnification shows the center to be a mass of pale, radi- ating threads, the mycelia, and at the edges a mass of threads and cocci. Both mycelia and cocci may be stained with methylene blue, the former frequently being banded light and dark in segments, sometimes granular throughout." Fig. 35. — Actinomyces hominis, Showing Clob-shaped Ex- tremities TO THE Rays. (Fresh preparation.) (After Wood.) StreptotJirix Eppengeri Streptothrix eppengeri is an organism related to the preceding. In the case described by Warthin and Olney ^ a filamentous, branching organism was found. Usually the mycelia were tangled and interwoven. No conical or club- shaped terminations were found, as in Actinomyces bovis. The threads, stained with carbolfuchsin, were not decol- orized after treatment with 25 per cent, nitric or sulphuric acids, though the stain was largely removed by washing in 95 per cent, alcohol. 'Warthin, A. S., and Olney, H. S. Jour. Med. Sci., 1904, CXXVl'lI, 637. 'Pulmonary streptothricosis. " Amer. Digitized by Microsoft® THE SPUTUM 223 Blastomycetes Blastomycetes (Fig. 36) have l)een found in tlie spu- tum in a number of cases. "In unstained preparations of pus and tissue tlie organisms appear as round or oval ^L , jE ^^HHi^P^^H^q^|V ^'-.i^Mki^^^m ■;^j^P;.- ;**y|w' *^-|:;'^J^ .k..^Bi'^"-i • W! '-' *i^*i ' •*«* 1 • ■ffl I ■'" ^ m "'W-iiiv l-*^- ^Hr. Ipa?^ ^•*::^ Fig. 36. — Blastomycetes in Sputum. X 1500. Photomierograph, (After E. E. Irons and E. A. Graham.) bodies witli a doulile contoured, higiily refractive capsule. Within the capsule, in many instances, granules or spore- like l)odies can be distinguished. The addition of a 1 to 10 per cent, solution of potassium hydrate to the specimen Digitized by Microsoft® 224 MICROSCOPIC EXAMINATION under examination facilitates the recognition of these bodies. In stained sections the double-contoured, homo- geneous capsule is usually separated from a finely or coarsely granular protoplasm by a clear space of varying width. Vacuoles of different sizes are found in some or- ganisms. In both pus and tissue organisms in pairs or in various stages of budding are commonly seen. The para- site, as a rule, varies in size from 7 to 20 micra, though slightly smaller and much larger forms occur in some cases." ^ Animal Parasites in the Sputum In this country animal parasites are comparatively rare in the respiratory passages, though probably more common than is generally supposed. Entameba histolytica (Fig. 12) may be encountered in the sputum as the result of rupture of an amebic liver abscess into the bronchial tree. The organism is identical with that found in the feces (q. v.). Perfectly fresh I '^^^gy sputum should be examined — when possi- L.„...- -._ J ble, with a warm stage. Entameba tetragena (Fig. 12) occurs in the sputum under the same conditions as Entameba histolytica. Trichomonads (Fig. 13) and cerco- monads are occasionally found in the sputa, usually in ma- terial which has stagnated in the lung. Paragonimus westermanii, the lung-fluke, the cause of "parasitic hemoptysis," is rare in this country, common t ' Montgomery, F. H., and Ormsby, 0. S. " Systemic blastomycosis. ' ' Arch. Int. Med., 1908, II, 1. See also Hektoen, L. ' ' Systemic blastomycosis and coccidioidal granuloma." Jour. A. M. A., 1907, XLIX, 1071. (Litera- ture.) Fig. 37. — Ovum of PAnAGONIMUS WES- TERMANII EKOM THE Sputum. X 400. (After Emerson.) Digitized by Microsoft® THE SPUTUM 225 in Japan and parts of China. Diagiiosis is made by find- ing the ova (Fig. 37) in the fresh s]intum (see p. 194). Echinococcus cyst, though common in certain parts of the worhl, is excessively rare in this country. Diagnosis ;A}, /^-^ - r;> l. J. >■;■ V D :l Fig. 38. — Skdiment fru.m Echi.smicoccus Cyst. Above and to the left are two degenerated srolices (X almut GO); to the right is a crown of hooklets (X400); below are hooklets of unusual shapes and a small mass of cholesterin crystals (X400). (After Emerson.) may be made after rnptnre of the cyst into the bronchi by (1) tlie finding of dangliter cysts, (2) scolices, (3) hook- lets, or (4) jiarts of tlie membrane in tlie sputum (Fig. 38). The material may arise from a hepatic cyst which has rup- tured through the diaphragm into the air passages. Digitized by Microsoft® CHAPTEE V THE BLOOD Obtaining Blood for Examination.— Blood for counts, etc., is obtained most conveniently from the lobe of the ear or the ball of the finger. The ear is, on the whole, more satisfactory than the finger. It is easily accessible, the flow of blood is as good, and it is much less sensitive to pain than the finger. There is also less likelihood of infection of the small wound through contact with dirty objects. For counts or hemoglobin determinations, blood should not be drawn from a part of the body which is cyanotic, because concentration of the blood may occur, producing results which are misleading (too high). Blood Stickers.— A number of satisfactory blood stick- ers are on the market, and require no special description. In default of these a Hagedorn needle may be used. Bass ^ has recently described a simple arrangement consisting of a straight surgical needle mounted in a cork. When not in use, the needle is carried in a small vial filled with al- cohol. A sharp steel pen, one of whose prongs has been broken off, may be employed as a sticker. The sticker should always be perfectly clean, in addition to being ster- ile. Dried blood on the point, even a very small amount of it, makes a sharp instrument seem dull. Method. — The skin of the ear or finger and the sticker are cleaned with alcohol or ether, which is allowed to evapo- *Bass, C. C. "A practical, inexpensive, aseptic blood-sticker." Med. Record, 1910, LXXVIII, 538. 226 Digitized by Microsoft® THE BLOOD 227 rate completely. The skin is then pierced. The point of the sticker should be held close to the skin and pushed in rather quickly ; beginners frequently make a sudden stab at the part from a distance of several inches, either missing the skin entirely or producing an unnecessarily deep wound. The wound must be such that the blood flows freely from it ; squeezing the tissues to obtain blood is not permissible, since the blood is thereby diluted with lymph. COUNTING THE BLOOD CORPUSCLES The Hemocytometer.— In blood counting the standard instrument in universal use is the hemocytometer of Thoma Fig. 39." -The Thoma-Zeiss Hemocytometer. a, the counting chamber; 6, the same in section; S, the diluting pipette. (Fig. 39). The instrument as originally designed by Thoma was not satisfactory for the enumeration of the leukocytes, and, as a result of this, numerous modifications of the Thoma ruling of the counting chamber have been brought forward.^ The change consists in an increase of * The writer learns from dealers in laboratory supplies that they are forced to keep the original counting chamber in stock, since physicians specify "Thoma-Zeiss" in ordering. This is doubtless due to the fact that the pur- chasers are unfamiliar with the much more practical rulings — modifications of the Thoma — ^which are mentioned above. Digitized by Microsoft® 228 COUNTING THE BLOOD CORPUSCLES the ruled area from 1 sq. mm. to 9 sq. mm. Neubauer, Tiirk, Zappert-Ewing, and others have devised rulings, which are designated by their names. The writer prefers the Nenbauer ruling (Fig. 40). In all the modifications the central square millimeter retains the original ruling of Thoma. The addi- tional eight square millimeters s u r - round it, and are solely for greater accuracy and con- venience in counting the white cor- puscles. The Eitling of THE Counting Chambee. — T here is, then, a ruled area 3 mm. on a side or 9 sq. mm. (Fig. 40). This area is divided into nine large squares, each of which is 1 mm. on a side or 1 sq. mm. The central square millimeter, which is used for counting the erythrocytes, is subdivided into 400 small squares, each of which is -^ mm. on a side, and has, there- fore, an area of ^^sq. mm.^ By means of double lines these smallest squares are grouped into blocks of twenty- five, a convenient unit to employ in counting. The ruling of the remaining eight large squares (1 sq. mm. each) varies according to the design selected. For the leukocyte count the entire nine square millimeters may be used. ^ This is always indicated on the glass slide of the counting chamber — .1. qmm. ' ' The depth is also given — "Tiefe 0.100 mm." Fig. 40. — The Neubatteb Ruling op the Hemo- cytometeb. Digitized by Microsoft® THE BLOOD 229 Construction of the Counting Chamber (Fig. 39). — The ruling is on a glass disc (B) which is mounted on a heavy glass slide (o). The disc is surrounded by a glass table (W), also attached to the slide, the surface of which is exactly one-tenth (0.1) of a millimeter above that of the ruled disc. A moat (r) about 2 mm. wide separates the disc from the table. When the cover glass (D), which is supplied with the apparatus, is placed upon the glass table, it thus forms a space between its under surface and the surface of the ruled disc, which is 0.1 mm. deep. The Diluting Pipettes. — Since whole blood is much too thick to permit direct enumeration of its corpuscular ele- ments, pipettes with which accurate dilutions of the blood can be made are required. Two pipettes are furnished with the complete hemocytometer, one for the red cells, the other for the white. Each consists (Fig. 39) of a capillary tube, which opens into a bulb containing a glass pearl. The capillary tube is divided into 10 equal parts. In the red pipette the bulb, when filled to the line on its upper out- let (marked 101), holds one hundred times the contents of the ten divisions of the capillary tube. It is, therefore, possible to obtain ten different dilutions of blood, if desired. Practically, only two dilutions are employed — 1:200 and 1 :100. With the white pipette lower dilutions are made. The bulb usually contains either ten or twenty times the con- tent of the capillary tube. Dilutions of 1 :10, 1 :20, etc., are generally made. Procedure in Counting the Erythrocytes (1) Diluting Fluids,— The requirement for the dilut- ing fluid is that it preserves well the red corpuscles. Nu- Digitized by Microsoft® 230 COUNTING THE BLOOD CORPUSCLES merous formulae have been elaborated. Among the better known of these are the following : (a) Hayem's solution: Bichlorid of mercury 0.5 gm. Sodium chlorid 1.0 gm. Sodium sulphate 5.0 gm. Distilled water 200.0 c. c. Dissolve and preserve in a tightly stop- pered bottle. This is the most satisfactory diluting fluid. It keeps indefinitely, and no organisms grow in it. The red cells settle evenly in it. (b) Physiological salt solution: Sodium chlorid 0.85 gm. Distilled water 100.0 c. c. Dissolve. The red cells settle slowly and often unevenly in salt solution. (c) Toisson's fluid: Sodium sulphate 8.0 gm. Sodium chlorid 1.0 gm. Glycerin (neutral) 30.0 c. c. Distilled water 160.0 c. c. Methyl violet q. s. Dissolve. (The methyl violet is added in minute amount (25 to 30 mg.), just enough to color the fluid, which should remain clear and transparent.) Digitized by Microsoft® THE BLOOD 231 Toisson's fluid is not stable and must be filtered before using. Low forms of vegetable life luxuriate in it, and it is, on the whole, an unsatisfactory fluid, its only advantage — which is usually negligible — being that leukocytic and other nuclei are stained. (2) Filling the Pipette.— The first essential is to have the blood flowing freely and to obtain a fresh drop. If it is necessary to squeeze the ear to obtain the blood, the latter will be diluted with tissue lymph, making the count too low; if the drop is not perfectly fresh, clotting will have begun, so that a uniform suspension of the cells can- not be secured. The blood is sucked cautiously into the capillary tube to the line marked 0.5. (With anemias of 2,500,000 cells or less, the blood is more conveniently drawn up to the mark 1 to secure a lower dilution, i. e., 1:100.) If the blood is accidentally sucked above the line, it may be lowered by drawing the finger across the tip of the pipette, provided the column of blood has not passed more than 1 mm. above the line; if it has extended farther, the blood adhering to the wall of the tube will be sufficient to intro- duce a serious error in the dilution. In the latter case the blood should be drawn up to the next line on the capillary tube, 0.6, quickly, and a corresponding correction in dilu- tion calculated. Bubbles of air in the column of blood must, of course, be avoided. After the tube has been ac- curately filled with blood, the end of the pipette is wiped free of blood, and is then plunged into the diluting fluid, which is sucked up to the line marked 101. While the diluting fluid is being drawn up, the pipette, held between thumb and fingers, is revolved to keep the glass pearl with- in the bulb in motion, both for the purpose of mixing the blood and diluting fluid and also to prevent bubbles adher- ing to the pearl, which would render the dilution inaccu- Digitized by Microsoft® 232 COUNTING THE BLOOD CORPUSCLES rate. The filling of the capillary tube and the subsequent dilution of the blood require rapid manipulation to prevent clotting. When the fluid reaches the line 101, the mouth- piece of the pipette is occluded with the tongue. The finger is then placed over the tip of the capillary tube, the thumb grasping the other end of the pipette. An even and uni- form suspension of the erythrocytes is now secured by shaking the pipette, held horizontally, for at least two min- utes. After the shaking is completed, several drops are blown out of the pipette to thoroughly empty the capillary tube (which contained only diluting fluid), and the count- ing chamber is then filled. Since the red corpuscles settle quite rapidly, the contents of the pipette must be perfectly mixed by shaking each time before filling the counting chamber. (3) Filling the Counting Chamber.— The counting chamber and cover glass must be perfectly clean and free from dust. A drop of the diluted blood is placed at one side of the ruled disc; the cover glass, used as a lever, is gradually lowered onto the drop, the edge of the glass table serving as the fulcrum. As the cover glass comes in con- tact with the drop, and the latter spreads over the surface of the ruled disc, there will be a tendency for one or more bubbles to form. By alternately raising and lowering the cover glass the fluid will spread evenly, and bubbles can be avoided. The size of the drop of diluted blood which is taken is important, and is learned by practice. It should be just large enough to cover the surface of the ruled disc, when the cover glass is applied. If it is so large that much of the fluid runs into the moat or that any fluid is found between the cover glass and glass table, the counting chamber must be cleaned and refilled. Before proceeding to count the erythrocytes, two conditions must be. fulfilled Digitized by Microsoft® THE BLOOD 233 in addition to those already given: (1) Newton's rings (prismatic colors) must be visible between the cover glass and glass table when looked at obliquely toward the light, since this proves that the two surfaces are in close apposi- tion and are not separated by a particle of dust, which would deepen the chamber; and (2) inspection under low magnification should show no clots and no gross inequali- ties in the distribution of the red cells over the ruled sur- face. These conditions being fulfilled, after allowing the erythrocytes to settle in the fluid two or three minutes so that all of them will be in the same focus, the enumeration • of the cells is proceeded with. In making the count it is advisable, if the worker is inexperienced, to use a high magnification (Leitz ocular 1, objective 6, or corresponding values for other makes of microscope), which will include a single block of twenty- five small squares. Ordinarily, however, a lower magnifica- tion is sufficient, such, for example, as one obtains with Leitz ocular 3, objective 3, with the tube drawn out to its full length. (4) The enumeration of the cells may be made in an al- most endless number of ways. The method described by Emerson ^ has been employed by the writer, and is as fol- lows : The cells in 200 small squares are counted. This is accomplished by counting separately eight blocks of twen- ty-five small squares, the blocks used being those at the corners of the ruled area from each of two preparations. A little more time is consumed in refilling the counting chamber, but it serves as an additional check on the accu- racy of one's technique. Beginning at the upper left cor- ner of a block of twenty-five small squares, the count is made from left to right in the upper tier of five small ' Emerson, C. P. " Clinical Diagnosis. ' ' Digitized by Microsoft® 234 COUNTING THE BLOOD CORPUSCLES squares, then from right to left in the second tier, and so on, until the entire block of twenty-five has been covered. The cells touching the line on two adjacent sides of a square are counted, while those on the line of the two remaining sides are disregarded. The total count for each block of twenty-five small squares is set down. When all eight blocks have been counted, the difference between the high- est and lowest total count should not exceed 25 cells. If this limit is exceeded, the distribution of the cells in the counting chamber has been so uneven as to make the re- sults untrustworthy .1 (5) Calculatioif of the Result.— The calculation is sim- ple. Since each small square is -^ mm. on a side, and the counting chamber is ^ mm. deep, the cubic content of one small square is -^ x -}- x -i- or —L- c. mm. As ^ 20-20 10 4000 200 such squares have been counted, it follows that the sum of all the cells counted is the number of cells contained in -2.00^ or -L c. mm. of diluted blood. Since the dilution 4000 20 used was 1 :200, the total number of cells counted must be multiplied by 20 and by 200, or by 4,000, to obtain the num- ber of cells in 1 c. mm. of undiluted blood, the desired re- sult. The normal count usually given for healthy adults is; for males, 5,000,000 ; for females, 4,500,000 cells per c. mm. These figures represent averages. The red count of nor- mal adults is not infrequently as high as 6,000,000. (6) Cleaning the Apparatus.— (a) The Counting Cham- ber. — The counting chamber should be cleaned with ivater only. After thorough rinsing, it is wiped dry with a soft, clean cloth. The diluted blood should never be permitted to dry in the counting chamber. Alcohol, ether, or similar * Following Emerson, students are required to fulfill these conditions and to make counts on two successive days, which shall not differ by more than 200,000 cells per c. mm., the limit of error of the method. Digitized by Microsoft® THE BLOOD 235 solvent should not be employed, as it may dissolve the cement, by which the ruled disc and the glass table sur- rounding it are fastened to the slide. (In case this acci- dent happens, all the parts may be returned to the maker, for it is possible at times to repair the damage.) (b) The Pipettes. — The pipettes should be cleaned im- mediately on completion of the count. If this is impossible, they should at least be emptied and refilled with water, un- til it is convenient to clean them. If allowed to stand, the diluted blood drying in the tip of the capillary may occlude it. The successive steps are as follows: (1) The pipette is emptied of its contents. (2) Distilled water or clear tap water is drawn into the pipette. After emptying, fill it with — (3) Ethyl alcohol, 95 per cent. Shake the pipette so that the water adhering to the pearl is mixed with the al- cohol, place the rubber tubing over the tip of the capil- lary tube, and blow the alcohol through. (4) Fill the pipette with ether, and shake again. Re- move the rubber tubing, and allow the ether to run out by inverting the pipette. (5) Aspirate air through the pipette till the walls of the bulb are dry and the glass bead rolls freely as the pi- pette is rotated. If the bead sticks to the wall, it means that there is still moisture remaining, or that the pipette is not clean. A suction pump is a great convenience in cleaning the pipettes. As a substitute for the pump, a stiff-walled rub- ber bulb may be employed. If blood clots in the capillary tube, it should be removed as soon as possible by means of a horse hair. For this purpose hairs from the tail or mane are washed in water and alcohol and kept in the latter. Wire should never be Digitized by Microsoft® 236 COUNTING THE BLOOD CORPUSCLES used, as it may scratch the glass, and there is also great danger of chipping the end of the pipette. At times, if the clot has become very firm or dry, it is necessary to place the pipette in a test tube with nitric acid. When a film of coagulated albumin forms over the inner surface of the bulb, the pipette may be filled with nitric acid and set aside for several hours. Counting the Leukocytes (1) Diluting Fluid.— For counting the white cells of the blood it is necessary to have a fluid which will render the erythrocytes invisible and cause the leukocytic nuclei to stand out prominently. Dilute acetic acid is universally employed for this purpose. It is used in about 1 per cent, strength. The solution is quickly prepafed by adding two drops of glacial acetic acid to 10 c. c. of distilled water. The dilute acid should be prepared freshly each day; yeast cells grow in it, if it is kept, and may lead to 'confusion, since single cells resemble the nuclei of lymphocytes, while several cells are not very unlike a polymorphous nucleus. It is convenient to have a small, wide-mouthed bottle with a file-scratch indicating 10 c. c. for preparing the dilute acetic acid. (2) Filling the Pipette.— The capillary tube of the white pipette is larger in caliber than that of the red pipette, and, therefore, a larger drop of blood will be required to fill it. The blood is sucked up to the mark 0.5, as a rule; the blood on the end of the pipette is wiped off, and the tip immediately plunged into the diluting fluid, which is sucked up to the mark 11 (or 21 in the case of the larger white pipettes). The pipette must be rotated more jerkily and the fluid sucked in more slowly than with the red Digitized by Microsoft® THE BLOOD 237 pipette, to avoid the air bubble wMcli so often clings to the glass pearl. After the pipette is filled the end is occluded, and the pipette, held horizontally, is shaken at least two minutes. In short, all the precautions requisite to a proper filling of the red pipette (to which the reader is referred) apply with equal force here. Bubbles in the column of blood or in the bulb of the pipette ruin the preparation. The column of blood must be drawn quickly and accurately to the desired mark. When the number of leukocytes is greatly increased, as is the case in extreme leukocytoses and usually in leukemia, it is often more convenient to use the red pipette for the leukocyte count in order to obtain a greater dilution. (3) Filling the Counting Chamber.— The counting chamber is filled in the manner described under the red cell count. Since the capillary tube of the white pipette is wider, the diluted blood flows out of it more rapidly, and the size of the drop is less easily controlled. A drop of the right size, Newton's rings, and an even distribution of the cells over the ruled area are essential. The magnification employed should be the same as that used in counting the erythrocytes (q. v.). Work is much more rapid with the lower power. (4) The Enumeration of the Leukocytes.— After the cells have settled until all are in the same focus (usually in two or three minutes), the leukocytes, whose nuclei stand out as refractive bodies, are counted in one square milli- meter at a time, including the cells which touch the line on two sides of the square only. Nine or ten square milli- meters are counted — nine in one preparation, or five in each of two preparations. The procedure is the same as that used in counting the red corpuscles, the only difference^ being the larger unit employed, 1 sq. mm. The difference 17 Digitized by Microsoft® 238 COUNTING THE BLOOD CORPUSCLES between maximal and minimal total count for 1 sq. mm. should not exceed 8 cells. The normal leukocyte count of adults lies between 5,000 and 10,000 cells per c. mm. — rarely 12,000 cells. If the diluting fluid is not freshly prepared, yeast cells may be counted and lead to serious error. A second source of error is the presence of a considerable number of nu- cleated red cells in the blood. Ordinarily erythroblasts are detected only when the stained blood is simultaneously examined ; and, as they are numerous only in marked path- ological states of the blood, they seldom escape notice. They cannot well be separated from the leukocytes in the counting chamber, so that the count obtained is the sum total of all nucleated cells in the blood — ^both red and white. The number of erythroblasts is determined by making a differential count of the stained blood and noting the rela- tive number of nucleated red cells as compared with the leukocytes. From the proportion of the two kinds of cells found, the correction of the white count is made. Thus, if the leukocyte count were 20,000, and differential count showed 125 nucleated reds to 500 leukocytes, the relative frequency of the two would be as 1:4. Therefore, there are present in one cubic millimeter of blood 5,000 nucleated red cells. Since these were included with the leukocytes in the total count, the latter must be corrected by deducting the nucleated reds. The leukocyte count thus becomes 15,000 instead of 20,000. (5) Calculation of the Result.— Let us assume that the dilution employed was 1:20, and that nine square milli- meters were counted. Each square millimeter has a cubic content of 0.1 c. mm., since the counting chamber is 0.1 mm. deep. The sum of the leukocytes in the nine large squares divided by 9 gives the average number of cells in 0.1 c. mm. Digitized by Microsoft® THE BLOOD 239 of diluted blood. To obtain tbe number of cells in 1 c. mm. of undiluted blood, this numlier is multiplied liy 10 and by 20 (dilution 1:20), or by 200. Biirker's Modification ^ of the Thoma Counting Cham- ber.— By substituting two wedge-shaped, ruled glass plates for the ruled disc, Biirker has modified Thoma 's design. i^^H ^^^^^fi 1 ^^^1 Fig. 41. — Burkeb's Hemocytometer. (Zeiss.) The wedges are placed with their bases opposite one another (Fig. 41), the apices extending out toward the sides of the glass slide. Two oblong tables at either side of the wedges with long axes running ])arallel replace the glass table which surrounds the ruled disc in the Thoma pattern. The cover glass, when resting on the tables,- is 0.1 mm. above the ruled surface of the wedges, as in the Thoma apparatus. The ruling is after a special design of Biirker. 3 Each wedge is ruled, and the chamber has, there- ' Biirker, K. ' ' Erfahriingon mit den iieucn Zahlkammer nebst einer wei- teren Verbesserung derselben. " Arch. f. d. ge.v. Physiol., 1907, CXVIII, 460. ' When ordered, clips are supplied with the apparatus to hold the cover glass firmly in place. ' By special order Zeiss makes the Biirker counting chamber with Neu- bauer ruling. Digitized by Microsoft® 240 COUNTING THE BLOOD CORPUSCLES fore, two ruled areas in place of the one of the Thoma chamber. In filling the Biirker hemocytometer for counting, the first step consists in placing the cover glass on the tables, Newton's rings being obtained. Then the blood is allowed to run under the cover from the pipette by capillarity. One side may be filled for the red count, the other for the white. Biirker calls attention to the following technical points which should be observed: (a) To avoid bubbles, the cover glass and chamber must be carefully cleaned. Further- more, in using pipettes having an angle at their tips, bub- bles are prevented with difficulty ; the tip should be rounded off with emery paper, (b) The drop of blood which flows under the cover glass should not be so large as to overflow into the gutter. Counting chambers should not be accepted in which the gutter between the two ruled surfaces is less than 2 mm. wide and the lateral gutters 1.5 mm. wide, (c) After allowing the red cells to settle for at least three min- utes, the evenness of distribution should be determined be- fore proceeding with the count. Biirker advises that this be accomplished in the following manner: The counting chamber is placed on the stage of the microscope, illumi- nated by the mirror with the diaphragm opened wide. By viewing the counting surface obliquely with the unaided eye one sees a film or veil formed by the erythrocytes. Ir- regularities in the distribution of the cells are shown by variations in the density of the film. When such irregu- larities are visible, the chamber must be refilled. In count- ing the leukocytes, this procedure is not applicable, for the cells are too few. Microscopic examination with low power must be made. Digitized by Microsoft® THE BLOOD 241 Counting the Eosinophilic Leukocytes The number of eosinopMlic cells is usuaUy arrived at by making a total count of the leukocytes in the ordinary way and, at the same time, preparing stained specimens. By finding the percentage of eosinophiles in a differential count, the absolute number of cells per cubic millimeter may then be calculated. Dunger ^ has devised a method by which the absolute number of eosinophiles per c. mm. may be determined di- rectly. Dunger 's Method.— The formula of the diluting fluid is: 1 per cent, aqueous solution of eosin. . 10.0 c. c. Acetone 10.0 c. c. Distilled water 90.0 c. c. The solution must be preserved in a tightly corked bottle to prevent evaporation of the acetone, and is then quite stable. A 1:10 dilution of the blood is made in the white pi- pette, and the mixture is thoroughly shaken three to five minutes. After blowing out the contents of the capillary tube, a drop is placed in the counting chamber (ruled for leukocyte count, i. e., 9 sq. mm.). Only the eosin ophile cells are well seen ; they appear as small, pink bodies. With a magnification of 120 to 150 diameters they are readily seen. The entire nine square millimeters of the chamber are counted. Ordinarily 9 to 18 eosinophile cells are found in this area; this corresponds to about 100 to 200 eosino- ' Dimger, E. ' ' Eine einf ache Methode der Zahlung der eosinophilen Leukocyten iind der praktisehe Wert dieser Untersuchung. " Miinchen. med. Wchnschr., 1910, LVII, 1942. Digitized by Microsoft® 242 COUNTING THE BLOOD CORPUSCLES philic leukocytes per c. mm. The calculation of the total number of cells is made in the way described for counting the leukocytes (p. 238). After a little practice an increase in the number of these cells is recognized at a glance. By making a leukocyte count in the usual manner simul- taneously, the percentage of eosinophiles may be deter- mined. Counting the Blood Platelets Several methods have been proposed for counting the platelets. The indirect method has given fair results, i. e., making a count of the erythrocytes in the usual way and at the same time determining the relative number of plate- lets as compared to the red cells in a fresh specimen of blood. The number of platelets is then calculated. Direct methods of counting platelets have been attempted; the only one which appears to give reliable results is that of "Wright and Kinnicutt. Method of Wright and Kinnicutt.^— T/ie diluting -fluid: Solution 1: "Brilliant cresyl blue" 1.0 gm. Distilled water 300.0 c. c. Dissolve. Keep on ice to prevent the growth of yeasts. Solution 2 : Potassium cyanid 1.0 gm. Distilled water 1,400.0 c. c. Method. — ' ' The blood is mixed with the diluting fluid in the proportion of 1 :100 by means of the pipette used for 'Wright, J. H., and Kinnicutt, E. "A new method of counting the blood platelets for clinical purposes and some of the results obtained with it." Jour. A. M. A., 1911, LVI, 1457. Digitized by Microsoft® THE BLOOD 243 counting red blood corpuscles, and the counting is done in the ordinary counting chamber with a high power dry ob- jective. In order to render the platelets more clearly vis- ible, the specially thin cover glass of Zeiss, with central excavation, is used (cover glass No. 146, Zeiss catalog^). The diluting fluid consists of two parts of the aqueous solu- tion of 'brilliant cresyl blue' (solution 1), and three parts of the aqueous solution of potassium cyanid (solution 2). These two solutions must be kept in separate bottles and mixed and filtered immediately before using. Of course, the pipette should be well shaken after withdrawing the sample for counting. After the counting chamber is filled, it is left at rest for ten or fifteen minutes, in order that the blood platelets may settle to the bottom of the cham- ber and be more easily and accurately counted. "The platelets appear as sharply outlined, round or oval or elongated, lilac-colored bodies, some of which form a part of small spheres or globules of hyalin, unstained substance. ' ' The red cells are decolorized and appear only as ' shad- ows,' so that they do not obscure the platelets. The nuclei of the white cells are stained a dark blue, the protoplasm light blue. If the technique is correct, there should be no precipitate in the preparation. "The cresyl blue solution is permanent, but should be kept on ice in order to prevent the growth of yeasts. The cyanid solution should be made up at least every ten days. It is, of course, necessary that the solution be made from pure potassium cyanid, which has not undergone decompo- sition. As already stated, the two solutions must be mixed and filtered immediately before using, because after filtra- ' This special cover glass is, however, unnecessary, if one has the usual thin cover glass, which permits the use of the high power dry objective. Digitized by Microsoft® 244 HEMOGLOBIN DETEEMINATIONS tion, if the mixture is allowed to stand exposed to the air for a short time, a precipitate will fortn in it. After the diluting fluid has been mixed with the blood in the pipette, however, no precipitate forms, and, as the platelets do not quickly break up in the mixture, the counting may be done after some hours, if necessary. For example, a count im- mediately after filling the pipette was 258,000 and another count from the same filling of the pipette made eighteen hours later was 253,000. "A proper technique yields a remarkably even distribu- tion of the platelets in the chamber. For all practical purposes, the counting of the platelets. in 100 small squares is sufficient, but fpr greater accuracy all 400 small squares should be counted, or 200 small squares in each of two fillings of the chamber." With their method "Wright and Kinnicutt find that the platelet count of normal adults varies between 226,000 and 367,000 per, cubic millimeter, the general average being 297,000. HEMOGLOBIN DETERMINATIONS Many methods for the determination of the amount or percentage of hemoglobin have been brought forward. For a description of all of them the reader is referred to the textbooks of hematology. (1) Tallqvist devised a color scale, which has been widely used. It consists of a series of ten shades of red, intended to represent the color intensity of hemoglobin from 10 per cent, to 100 per cent. Each color is perforated. A drop of blood is collected on a filter paper, supplied in the book containing the scale, and, as soon as the gloss has disappeared from the drop, it is placed under the per- foration in one of the red strips. It is moved until the Digitized by Microsoft® THE BLOOD 245 color of the drop of blood corresponds with one of the shades of red. This represents the hemoglobin percentage of the blood. With the Tallqvist scale it is possible, per- haps, to make a more accurate guess as to the percentage of hemoglobin than without it. It is well recognized that the scale is very inexact. In fact, the color scales in sep- arate books do not always match. When the blood is hydremic, the plasma runs beyond the corpuscles, which are concentrated at the center of the drop, introducing an additional error in the very cases where more exact results are desirable. If a hemoglobin determination is indicated a little more time should be spent than is required with the Tallqvist scale, in order to obtain a result of some value. (2) Sahli's Hemometer.— Sahli's hemometer is a modi- fication of the old Gower instrument. It consists (Fig. 42) of one tube containing the standard solution and a second tube of the same caliber graduated from to 140, each divi- sion representing 20 c. mm. The tubes are placed in a hard rubber stand, which has an opaque glass back. A pi- pette with a line representing 20 c. mm. is supplied with the instrument. The standard solution is one of acid hema- tin, prepared as follows :^ Blood 1 part •^ hydrochloric acid 10 parts Distilled water to 50 parts Mix and add — Glycerin 50 parts The hemoglobin is converted into acid hematin, which ' Hastings, T. W. " The estimation of hemoglobin-content of blood with modern instruments." Jour. A. M. A., 1907, XL VIII, 1749. Digitized by Microsoft® 246 HEMOGLOBIN DETERMINATIONS does not go into solution, but is in a very fine state of sus- pension. Tlierefore, the liematin settles slowh', when tlie instrument is not in use, and for this reason a glass pearl is placed in the tube to facilitate mixing- the standard fluid, "which should be done each time immediately before using. Sahli ' obtains the blood for the standard solution Fig. 42. — The Sahli Hemometeb. from young adult males having a high red cell count. This explains the fact that normal blood seldom shows more than 90 per cent, of hemoglobin with the Sahli hemonieter, when a new instrument is employed. In the course of time the standard solution fades. If the tul)e is protected from the light when the instrument is not in use, however, it may be kept as long as two years or more without se- rious deterioration. It is well to cheek the standard solu- ' Sahli, H. "Diagnostic Jrethods. " 1st Amer. Ed., Pliila. & London, 1905, p. 620. Digitized by Microsoft® THE BLOOD 247 tion from time to time with several bloods of normal adults having 5,000,000 red cells. With such a count the hemo- globin percentage should be 100. If the reading of the hemometer is too high or too low, the percentage of error is noted and the readings are then corrected correspond- ingly. It is more satisfactory to prepare the standard solution from blood with a 5,000,000 count, checking it with other similar bloods. By doing this the standard tube may be refilled, say, every six or twelve months, doing away with the necessity of corrections. The values obtained are then safe for use in determination of the color index. It is very important that the standard tube and the gradua,ted tube have the same diameter.^ If unequal, it is clear that the results obtained will be without value. Method. — The graduated tube is filled accurately to the mark 10 with tenth normal hydrochloric acid. The pipette is now filled with blood exactly to the line marked 20 c. mm. The blood is quickly discharged into the acid in the grad- uated tube, and the pipette is rinsed two or three times with the acid to remove that which adheres to the wall of the pipette. The graduated tube is immediately shaken to secure a uniform suspension of the blood before clotting will have begun. The blood quickly becomes dark brown in color from the conversion of the hemoglobin into acid hematin. The mixture of blood and acid is allowed to stand exactly one minute, and is then diluted with water, until its color matches that of the standard solution. Day- ' Tubes from uniform tubing have been made for several years for tlie writer by Eimer and Amend, Third Ave., New York City. The standard tube is made in the form of the usual test tube. When filled with the standard solution it may be sealed in the flame, though it is more convenient to use a paraflSned cork, as in this way it may be refilled an indefinite number of times. Digitized by Microsoft® 248 HEMOGLOBIN DETERMINATIONS light or artificial light may be used, since the tubes contain the same substance. When the graduated tube is inverted to secure thorough mixing, care should be exercised that none of the fluid- ad- heres to the finger, for enough may be removed in this way to cause a considerable lowering of the reading. When comparing the colors, it is well to rotate the graduated tube until the lines on it are not visible. When the colors have been accurately matched, the instrument is set aside for a couple of minutes to allow the fluid in the graduated tube adhering to the wall to run down. The height of the column is then read. This gives the hemoglobin percen- tage, the color of the standard fluid being considered as 100 per cent. In cases where the hemoglobin is extremely low it is difficult to obtain satisfactory readings. In such case 40 c. mm. of blood may be added to the acid. The final result is then divided by 2. Staubli ^ has made a critical study of this method of determining hemoglobin, using the Sahli hemometer and the Autenrieth-Konigsberger colorimeter. He finds that there is a progressive darkening of the acid hematin formed by mixing the blood with the tenth normal acid. The dark- ening is most rapid in the first few minutes after the mix- ture is prepared; plotting the values obtained, he found that the curve is a parabola. He has demonstrated that it is important to use tenth normal hydrochloric acid, not an approximate dilution, and to measure it into the grad- uated tube accurately, for the rapidity of darkening of the blood is directly proportional to the quantity and concen- ' staubli, C. "Zur Ausfuhrung der Hamoglobinbestimmung. (Unter Umwandluiig des Hamoglobins in salzsaures Hamatin.) " Miinchen. med. Wchnschr., 1911, LVIII, 2429. Digitized by Microsoft® THE BLOOD 249 tration of acid. The blood-acid mixture should be allowed to stand exactly one minute, as Sahli recommends, and should then be quickly diluted with water, which inhibits the effect of the acid. Staubli suggests that a better method of procedure with the Sahli hemometer is as follows : The blood-acid mixture is diluted at once with tenth normal hydrochloric acid, until the color is approximately that of the standard tube ; then wait for ten minutes ^ to make the final comparison. The final dilution, which will require only a few drops, may be made either with water or with the acid. This technique in his hands has yielded uniform results with all bloods. Whichever method is followed, it is absolutely essential that it be adhered to strictly in order to obtain comparable results. Aside from variations in the standard fluids and pos- sible lack of uniformity in diameter of the tubes, it is prob- able that Staubli 's findings explain to a great extent the anomalous results which many workers have obtained with this instrument. Cleaning the Hemometer. — The graduated tube is rinsed with water. The pipette is rinsed first with water, then with alcohol and with ether. Finally, air is aspirated through it to dry the pipette. (3) The Fleischl-Miescher Hemoglobinometer.— This instrument is generally considered to be the most accurate for the determination of hemoglobin. It is not well adapted to general use, since it is expensive and requires a dark room for making the readings. The instrument (Fig. 43) consists of a standard, on which a wedge of red glass is mounted, cells of 12 and 15 * Ten minutes is the time interval selected, since it was found that the darkening which occurs beyond this interval is slight. Digitized by Microsoft® 250 HEMOaLOBIN DETERMINATIONS mm. depth, and a mixing pipette. The pipette is similar to those used with the hemocytometer. The markings on its capillary tube, y2, fs, and 1, permit of dilutions of the blood of 1 :400, 1 :300, and 1 :200 respectively. Sodium car- bonate, 0.1 per cent, aqueous solution, is used as the dilut- ing fluid. The cells, 12 and 15 mm. deep respectively, are Fig. 43. — The Fleischl-Miescher Hemoglobinometbb. divided into two equal parts, one of which is filled with water, the other with the diluted blood. Each compart- ment should be filled until the surface of the fluid is con- vex above the upper level of the cell. The cell is then sealed by a glass disc, care being exercised to avoid bubbles in the fluids. Finally, a metal cap is placed over the glass disc. In the cap there is a slit, which should be so placed that its long axis is at a right angle to the partition divid- ing the cell. The cell is now placed on the stand, so that the compartment containing water is directly above the red Digitized by Microsoft® THE BLOOD 251 glass wedge. Candle light furnishes the most satisfactory illumination; it must be used in a dark room. All direct rays of light are cut off from the eye of the examiner, either with a large cone or by placing the instrument in a box with one side — at which the operator stands — open and a small hole cut in the opposite side near the bottom for illumination of the reflector. The colored prism is now moved until the shades of red in the two divisions of the cell are alike. The reading is made on the scale and re- corded. Ten such readings should be made, and the aver- age of them taken. If the cell 15 mm. deep has been used, the glass disc is removed and the diluted blood sucked back into the pipette. The 12-mm. cell is then filled, and ten readings are made with it, and the average taken. The latter should be four-fifths of the reading obtained with the 15-mm. cell, and should not vary by more than 2 per cent. The readings obtained do not represent hemoglobin percentages. They are to be used in connection with the table found in a pamphlet supplied with each instrument. (As the instruments are separately standardized, the tables often diifer, and, therefore, cannot be used interchange- ably.) From the table the hemoglobin in grams per 100 c. c. of blood is calculated according to the directions in the pamphlet. All values are to be reduced to a dilution of 1:300 with the 15-mm. cell. (For normal blood the 1:300 dilution is usually employed. With anemic blood use a dilution of 1:200, and with plethoric 1:400.) The normal hemoglobin value with the Fleischl-Miesch- er apparatus is subject to considerable variation. Emer- son ^ finds that in normal young adults the mean hemo- globin per 1,000,000 cells is 2.63 gm. In leukemia or with extreme leukocytoses readings may ' Emerson, C. P. " Clinical Diagnosis. ' ' 1st Ed., pp. 466 et seq. Digitized by Microsoft® 252 HEMOGLOBIN DETERMINATIONS be difficult, because of the opacity produced by the white cells. The leukocytes may be removed by centrifugalizing the diluted blood before filling the cell. Cleaning the Hemoglobinometer. — The pipette is cleaned in the same manner as the counting pipettes (q. v.). The cells should be taken apart, washed with water, dried, and reassembled. (4) Haldajie's Hemoglobinometer.— Haldane's hemo- globinometer is a very satisfactory instrument. Its only drawback — a minor one — is that illuminating gas is re- quired in its use. Like the Sahli hemometer, it is a modi- fication of the original Gower apparatus. (5) Dare's Hemoglobinometer.— Dare's hemoglobino- meter gives excellent results, but is fragile and expensive. Sulphhemoglobinemia,^ Methemoglobinemia.— The rec- ognition of these abnormal pigments in the blood is de- scribed as follows by Clarke and Curts:^ The blood is draAvu from a vein, or, if this is not allowed, a few drops from the finger or ear will usually suffice. It is immediately diluted with twice its volume of distilled water, before clotting has taken place, and is thoroughly shaken. After the fibrin has separated, the solution is filtered several times through one filter paper, and the clear solution looked at through the spectroscope. The solution is then diluted drop by drop with water, until the red color (of the spec- trum) stands out clearly. If there is a black absorption band in the red, either methemoglobin or sulphhemoglobin is present. If such a band persists after the addition of a drop of dilute ammonium sulphid, the pigment is sulph- hemoglobin ; if it disappears, it is methemoglobin. In the blood the two bands of oxyhemoglobin are always 'Clarke, T. W., and Curts, E. M. "Sulphhemoglobinemia, with a report of the first case in America." Med. Record, 1910, LXXVIII, 987. Digitized by Microsoft® THE BLOOD 253 visible. In addition to these sulphhemoglobin presents a band in the red (near the orange) midway between C and D. With methemoglobin the band is again in the red, but nearer to C. (Compare with Fig. 7.) COLOR INDEX The color index is the quotient obtained by dividing the percentage of hemoglobin by the percentage of red corpuscles, 5,000,000 cells per 1 c. mm. being considered as 100 per cent, of corpuscles. Normally, the color index is' about 1. When the index is less than 1 it indicates that the average corpuscle is poor in coloring matter, whereas with a high index the corpuscles are abnormally rich in hemoglobin. VOLUME INDEX The volume index of the blood was first studied by Capps.^ He introduced the term to designate the quotient of the percentage volume of the erythrocytes divided by the percentage number of these cells. Method. — To determine the volume of the red corpuscles, the hematokrit is employed. The usual form of apparatus is a hand or electric centrifuge armed with a frame for carry- ing two capillary tubes. The tubes are graduated from to 100. Of the various procedures which have been pro- posed, Capps recommends the following: The capillary tube is completely filled with blood, the distal end of the tube smeared with vaselin, and placed in the carrier of the hematokrit. "Two conditions are essential to prevent coagulation, viz., scrupulous cleanliness of the tubes and speed in operation. The latter condition requires that the ' Capps, J. A. "A study of volume index. Observations upon the volume of erythrocytes in various disease conditions." Jour. Med. Sesearch, 1903, X, 367. 18 Digitized by Microsoft® 254 VOLUME INDEX blood must be placed in the hematokrit within a few sec- onds of withdrawal. . . . It is desirable always to fill two tubes as a control of one's results. The machine should be operated for three minutes at a uniform speed of ten thousand revolutions a minute" (Capps). The tubes are now examined, and it is seen that the corpuscles have been thrown to the distal end, leaving the clear serum proxi- mally. With normal blood and a count of 5,000,000, the red corpuscles extend to the line marked 50, occupying one; half the capillary tube. This is the normal, and represents 100 per cent, volume. The erythrocytes are counted at the same time that the volume determination is made. The volume index =_^2lHE5_P5L_255i- 5,000,000 corpuscles being number per cent., considered 100 per cent. In normal blood the volume in- dex is 1. Owing to variations in the size of the erythro- cytes in anemias, the percentage volume does not run par- allel to the percentage number, as a rule.^ The volume index, then, expresses the relative volume of the average red cell as compared with the normal. In determining the volume of the red corpuscles, the leukocytes separate as a paler, grayish layer above the erythrocytes. Where their number is greatly increased, as in leukemia, determination of the volume of the red corpuscles is impossible. The hematokrit furnishes a ready means of making macroscopic examination of the blood serum. Lipemia, cholemia, and hemoglobinemia may be revealed in this man- ner, if sufficiently marked, though hemoglobinemia may be an artefact from mechanical injury to the red corpuscles. Cleaning the HematoJcrit Tubes. — Blood should be 'Capps (loc. cit.) reports extremely interesting observations on volume index compared with color index and with measurements of the erythrocytes in primary and secondary anemias. Digitized by Microsoft® THE BLOOD 255 blown out of the tubes as soon as the reading has been made. The tubes are cleaned by drawing water, alcohol, and ether through them successively. If they are not per- fectly cleaned, use acetic acid first, then the other fluids in the order given. MEASURING THE DIAMETER OF CELLS The diameter or a dimension of microscopic objects is expressed in micra (designated by the Greek letter /x), one micron being the thousandth part of a millimeter (0.001 mm.). In making measurefiients an ocular micrometer is employed. This is a glass disc, on which fifty equal divi- sions are marked by parallel lines. The upper lens of the eye-piece is unscrewed, and the micrometer is inserted in the tube of the ocular.^ The value of the divisions on the micrometer scale is now determined in the following man- ner: The magnification of the microscope is varied by three factors, namely, the objective, the ocular, and the tube length. The usual tube length employed is 160 mm. Using this, the value of the spaces on the micrometer is determined with the objective and ocular to be used by comparison with an object of known dimensions. The most convenient object for this purpose is the counting chamber. The ruled area is placed under the microscope, and the number of divisions of the micrometer scale, which fall between the opposite sides of one of the smallest squares, or, in the case of low magnifications, between the sides of a larger unit, is found. Knowing the dimensions of the ruled surface, it is a simple calculation to compute the ' In ordering an ocular micrometer, the name of the maker of the micro- scope should be given, as the micrometer of one make may not fit the ocular of another. Digitized by Microsoft® 256 VISCOSITY OF BLOOD AND OTHEE FLUIDS value of a single division of the micrometer scale. The smallest squares are -^ mm. on a side, or 50 micra. As applied to the blood, measurements are usually made on stained films. At least one hundred cells should be measured, and, where much anisocytosis exists, two hun- dred cells should be the minimal number. In the measurement of oval bodies, such as the eggs of many parasites, the two dimensions are readily obtained by rotating the ocular through ninety degrees. VISCOSITY OF THE BLOOD AND OTHER FLUIDS For clinical use, a number of instruments for detei*- mining viscosity have been described. That of Hess ^ has proved very satisfactory. It is compact and easily por- table. The determinations may be made with a little prac- tice in two or three minutes. The subject of viscosity is well discussed by Austrian.^ The viscosity is compared with that of water. Method of Hess.— The Hess viscosimeter (Fig. 44) con- sists of an opaque glass plate (H) on which two gradu- ated tubes, A and B, are mounted. At one end these tubes communicate with a T-tube, Gr, which in turn is connected by rubber tubing with the rubber bulb L. At the other end the graduated tubes connect with capillaries C and D. The latter open into tubes E and F, which have the same diam- eter as the graduated tubes A and B. Capillary tube C and tube E are made in one piece, while tube F is held in ap- position with tube D by means of the clip N. It is remov- able, and a number of similar tubes are supplied with the 'Hess, W. "Ein neuer Apparat zur Bestimmung der Viskositat des Blutes. ' ' Milnchen. med. Wchnschr:, 1907, LIV, 1590. ^Austrian, C. E. "The viscosity of the blood in health and disease." Johns HopUns Hosp. Bull, 1911, XXII, 9. Digitized by Microsoft® THE BLOOD 257 instniment. Through the valve Q it is possible to shut off the conununication of the graduated tube B with the T- tube, and, therefore, Avith the rubber bulb as well. Between the rubber bulb and the rubber tubing a short piece of glass tubing is inserted; in it a hole is blown. This is opened or closed with the finger, and permits instant re- lease of the negative pressure produced by the suction of the bulb. A thermometer is mounted on the glass plate. Method. — With a pipette, which is furnished with the FiQ. 44. — The Viscosimeter of Hess. (After Austrian-.) instrument, distilled water is placed at the opening of tube E. The valve Q is opened, and by suction from the bulb L the water is drawn into the tube E, until it reaches the capillary tube C. The pipette is then withdrawn, and the column of water is sucked further, until it reaches the mark on the scale of the graduated tube A. The valve Q is then closed, the pressure having been released by remov- ing the finger from the opening. (It is unnecessary to refill the tubes A, C, E, with distilled water for each de- termination; the water may be allowed to remain in the tubes and used repeatedly.) The tube F is then touched to a fresh drop of the blood to be examined. The blood should enter at the pointed end of the tube. When the lat- ter is about three-fourths full the tube is held so that the blood will run down to the funnel-shaped end of the tube, which is then placed in contact with the free end of the Digitized by Microsoft® 258 VISCOSITY OP BLOOD AND OTHER FLUIDS capillary tube D, and held in position by the clip N. By suction with the bulb the column of blood is then drawn to the line on the scale of the graduated tube B, when the pressure is again released. The valve Q is now opened, and by suction through the bulb the column of blood is drawn to the mark 1 on the scale. It is drawn exactly to the mark, when the pressure is removed by withdrawing the finger from the opening. The point on the scale to which the water has been drawn represents the degree of viscosity of the blood. The viscosity of the blood of nor- mal adults is about 4.55 (Austrian). If the viscosity of the blood is very great, or if the blood coagulates rapidly, the column of blood is drawn to the mark % or %> ^^^ the result obtained is multiplied by 4 or 2 respectively. The error arising from making the observations at ordinary room temperatures is negligible (Austrian). Cleaning the Viscosimeter. — As soon as the reading is made, positive pressure is exerted to expel the fluids from the graduated tubes. When the water reaches the zero line the valve Q is closed. The tube F is removed and the blood which escapes from the capillary tube D is caught on filter paper or cloth. A second tube, filled with concen- trated ammonium hydrate, is placed in the clip, and am- monia is drawn through the tubes A, D, at least 2 cm. be- yond the line 1 to which the blood extended. The ammonia is expelled and the tubes are refilled with fresh ammonia, which is allowed to remain in the tubes until the instru- ment is used again. The end of the capillary tube D is closed with a rubber cap. Immediately before using the apparatus the cap is removed and the ammonia expelled. If the pressure used to expel the ammonia is slight, only a trace remains, which is without appreciable effect on the result. It is essential that the tubes be perfectly clean. Digitized by Microsoft® THE BLOOD 259 If the apparatus is unused for some time, difficulty may be experienced in forcing the ammonia out of the tubes. This is usually due to the formation of ammonium salts at the opening of the capillary tube; they may be removed by solution in water. The valve should be lubricated with vaSelin. The tubes F, after use, may be cleaned by as- pirating water through them and then placing them in nitric acid for several hours. They are then dried by suc- cessive rinsings with water, alcohol, and ether. Erroneous results may be obtained if the tubes are dirty. The instru- ment should be tested from time to time with distilled water. If the result is not 1, the tubes are to be cleaned by drawing nitric acid into them. After an hour or so the acid is removed, the tubes rinsed twice with water, and then with ammonia. THE SPECIFIC GRAVITY OF THE BLOOD In clinical work the method usually used for determina- tion of the specific gravity of the blood is that of Hammer- schlag. A mixture of chloroform (sp. gr. 1.485) and ben- zol (sp. gr. 0.88) is placed in a cylinder. The specific grav- ity of the mixture should approximate that of normal blood (1.050 to 1.062). A capillary tube is filled with blood, which is flowing freely from the puncture wound, and a drop is allowed to fall into the mixture. If the drop sinks, its specific gravity is greater than that of the mixture, and more chloroform is added; if the reverse holds good, ben- zol is added. After each addition of chloroform or benzol the contents of the cylinder are well stirred. When a mix- ture is finally obtained in which the drop of blood neither sinks nor rises, its specific gravity is determined with an areometer. The result is approximately the specific grav- ity of the blood. Digitized by Microsoft® 260 THE SPECIFIC GRAVITY OF THE BLOOD As Naegeli suggests, a series of mixtures of varying specific gravity is a great convenience. The blood should not be permitted to drop into the chloroform-benzol mixture from a height, as it scatters. A bubble in the drop of blood may lead to serious error. Quick work is necessary to prevent the extraction of much water from the blood, and also to avoid evaporation of the mixture. After use the mixture may be filtered and kept in a brown glass bottle. The specific gravity of blood plasma or serum may be determined by the method of Hammerschlag. Normally it lies between 1.029 and 1.032. For the more accurate and time-consuming methods of determining specific gravity the reader is referred to works on hematology. THE COAGULATION TIME OF THE BLOOD The methods of determining the coagulation time of the blood are many, and the results obtained with each are more or less divergent. No perfectly satisfactory method has been brought forward. Among the best is that which employs the apparatus of Brodie and Eussell, as modified and improved by Boggs.^ Eesults almost as uniform have been published by Hinman and Sladen," using their modification of Milian's method. An essen- tial prerequisite to any method is absolute cleanliness of the apparatus and of the skin at the site of punc- ture. ^Boggs, T. E. "Some clinical aspects of blood coagulation." Internat. Climes, 1908, I (ISth series), 31. ^Hinman, F., and Sladen, F. J. "Measurement of the coagulation time of the blood and its application." Johns SopTcins Hosp. Bull, 1907, XVIII, 207. Digitized by Microsoft® THE BLOOD 261 (1) The Method of Brodie and Russell, as Modified by Boggs.— The instrument (Fig. 45) consists of a moist chamber A and a truncated glass cone B, mounted to fit into the former. The lower surface of the cone is 4 mm. in diameter. Through the wall of the chamber a metal tube C extends, the tip of which approximates the lower surface of the cone. By means of a rubber bulb, such as is used on a camera, attached to the outer end of the metal tube, a current of air may p ^ be directed tangentially to II \ "^~7''' l | ~* the lower surface of the cone. The upper surface of p— — the cone is covered by a cover glass D-E; at E there ^^^ 45._boogs' Modification or the is a pinhole. Coagulometer of Bhodie and Tij , , 7 , T n RussEijL. A, moist chamber; B, cone Met ho d.—A drop of ^j gi^,^ the lower surface of which blood is placed on the lower ^°^'^^ ^^^ '^'■op of biood; c, side tube, - . connecting with bulb; D and E, cover end 0± the glass cone, and glass; at E, a pinhole. (After Emer- the cone inserted in the ^"'^■^ chamber, which is then placed on the stage of the micro- scope. The drop is examined with the low power, and at the same time the bulb is squeezed, directing an air current against the periphery of the drop of blood. At first the corpuscles move freely in a circular direction. As clotting begins, masses of corpuscles take the place of the single cells. As coagulation progresses, the masses of corpuscles tend to become fixed in the drop. The air now displaces the masses in the direction of the current, but they spring back immediately after the air ceases to disturb them. The next stage, which is taken as the end-point, differs from the pre- ceding in that a very gentle blast of air produces "radial elastic motion, as of a rubber ball pressed in at one point and released" (Fig. 46). "When this point is reached the Digitized by Microsoft® 262 THE COAGULATION TIME OF THE BLOOD time is again taken. The cone is immediately removed and the blood is wiped off on a cloth or filter paper to confirm the existence of a clot. Occasionally a drop of blood fails to clot at one point. If this happens the result is valueless, and a second deter- mination must be made. The normal coagulation time of the blood with this in- strument is between three and eight minutes, usually about five minutes (Hinman and Sladen). Fig. 46. — Diagram to Illustrate the Movement of the Cells dueing Coagulation. D, the end-point. (After Emerson.) Boggs emphasizes the following points of technique: The blood must be flowing freely from the puncture. When a sufficiently large drop has collected, the cone is touched to it at right angles to the surface of the drop and not dipped in it. In this way a drop of constant size is ob- tained. The coagulation time begins with the appearance of the drop of blood in the wound. Pressure upon the tis- sues and congestion of the parts are to be avoided, as they tend to increase the coagulability of the blood. Absolute cleanliness of the apparatus is essential. The air current should be gentle, and should not be applied too frequently. Digitized by Microsoft® THE BLOOD 263 (2) Milian's Method, as Modified by Hirnnan and Sla- den.^^This method is extremely simple,, requiring only- clean glass slides and a millimeter scale. The ear is punc- tured, the first drop is discarded, and the time counted from the first appearance of the second drop. The lobule of the ear is held out, and the under-surface of the slide touched to the drop of blood, so that several small drops are obtained on it. The slide is then turned quickly to prevent the drops from flowing. Placing the slide over a scale, only those drops having diameters of 4 and 5 mm. are allowed to remain, others being wiped off. There are two methods of watching the drops to determine when coagulation has occurred; in each the slide is held verti- cally. In the one the profile of the drop is observed; be- fore coagulation the drops sag, assuming the shape of a tear, while the uniform convexity is preserved after co- agulation is complete. In the other method the vertical slide is examined by transmitted light. The denser por- tion will be found about the center of the drop, when coagu- lation has occurred; while the blood is still fluid the de- pendent part of the drop is the denser. The presence of a clot is then confirmed by transferring the drop to a cloth or filter paper. Compared with Boggs' method, in which a 4-mm. cone is employed, the authors find that a 4-mm. drop clots more rapidly, a 5-mm. drop more slowly. They therefore take the mean coagulation time of several drops of 4 and 5 mm. diameter, respectively. The majority of records fall be- tween five and eight minutes. Below eight minutes a rec- ord is within the limits for a normal coagulation time. •Hinman, F., and Sladen, F. J. "Measurement of the coagulation time of the blood and its application." Jbhns Hophins Hasp. Bull., 1907, XVIII, 207. Digitized by Microsoft® 264 EESISTANCE OF RED BLOOD CORPUSCLES Anything above eight minutes is delayed. When the co- agTilation time is delayed, only 5-mm. drops are used, in order to minimize the error due to evaporation, THE RESISTANCE OF THE RED BLOOD CORPUSCLES Numerous substances have been employed, against which the resistance of the red blood corpuscles has been measured. Solutions of sodium chlorid of varying strength, notably hypotonic solutions, have been most extensively used, and with them results of clinical importance have been obtained. Methods — Under aseptic precautions 2 to 5 c. c. of blood are aspirated from an arm vein, and immediately placed in five to ten times the volume of 1 per cent, sodium fluorid or 1.5 per cent, sodium citrate in 0.85 per cent, so- dium chlorid to prevent clotting. As soon as the blood is discharged into the fluid, the flask is shaken well to insure thorough mixture. The blood-fluorid mixture is now cen- trifugalized at high speed to throw down the corpuscles. The supernatant fluid, containing the greater part of the blood plasma, is poured off. The plasma is then com- pletely removed by washing the corpuscles three times in 0.85 per cent, solution of sodium chlorid. After the last washing the supernatant fluid is pipetted off, leaving the erythrocytes at the bottom of the centrifuge tube. The hypotonic solutions of sodium chlorid diminish from 0.85 per cent, by 0.03 per cent., the solutions being 0.82 per cent., 0.79 per cent., and so on, down to 0.25 per cent. They are quickly prepared by filling one 50-c. c. burette, graduated to -L c. c, mth distilled water, and 'Moss, W. L. "Paroxysmal hemoglobinuria: Blood studies in three cases." Johns EopMns Hosp. Bull., 1911, XXII, 238. Digitized by Microsoft® THE BLOOD 265 another with 1 per cent, aqueous solution of sodium chlo- rid. Thus, to prepare 10 c. c. of 0.70 per cent, sodium chlorid, take 7 c. c. of the 1 per cent, salt solution and 3 c. c. of distilled water. A series of small test tubes is appropriately marked and placed in a rack. To each there are added 3 c. c. of hypotonic salt solution and 0.03 c. c. (about one drop) of the red blood corpuscles. The salt solution and blood cor- puscles are well mixed by shaking. (The tubes are, of course, perfectly clean and sterile, and are plugged with cotton.) After all have been filled, they are placed in the ice chest to prevent bacterial growth, and are allowed to remain until the red cells have settled to the bottom. For the lower dilutions this usually requires about two hours. The supernatant fluid is now examined for free hemoglobin, the presence of which shows that there has been laking of the corpuscles. The tube of lowest dilution showing even a trace of hemoglobin in the fluid represents the so-called minimal resistance. That is, with this strength of salt solution the least resistant cells are "laked," their hemoglobin escap- ing from the cell membrane into the salt solution. The maximal resistance is found by noting the strength of salt solution in which all the red corpuscles are laked. Normally the minimal resistance, in terms of hypotonic salt solution, is about 0.47, the maximal resistance about 0.30. THE EXAMINATION OF FRESH AND STAINED PREPA- RATIONS OF BLOOD The first requisite in the preparation of fresh or dried films of blood is perfectly clean glassware. The Cleaning of Cover Glasses and Slides.— Of the vari- Digitized by Microsoft® 266 FRESH AND STAINED PREPARATIONS OP BLOOD . ous methods used to clean glassware for blood work in the author's laboratory, the following has given the most satis- factory results, and is always dependable : (1) Immerse the covers (or slides) in concentrated sul- phuric acid for about twenty-four hours. (2) Pour off the acid and wash in running water. (3) Drain off the water and cover the glassware with 95 per cent, alcohol for an hour or longer. (4) Replace the alcohol with chloroform and dry the glassware as needed. The covers should be dried with a perfectly clean cloth, free from lint. An old linen handkerchief which has been laundered many times is suitable. If the glassware is to be kept dry, it should be placed in a dust-proof receptacle. Ether may be substituted for chloroform, but is less satisfactory. For blood work %-in. square cover glasses, No. 1, are the best. The 3xl-in. glass slides should be thin, with straight, even edges, if they are to be employed in making blood films. If cover glasses are used in making the films, the finish of the slide is less important. Examination of the Pebsh Blood In the examination of the fresh blood, a procedure which is too generally neglected, the specimen is prepared in the following manner : The ear is pierced, but the punc- ture should not be so deep as to cause a very free flow of blood, since it is essential to be able to regulate the size of the drop accurately. Therefore, a small, superficial puncture is made, from which the blood will escape easily on very gentle pressure. (Pressure is to be avoided as far as possible, to prevent the dilution of the blood with tis- Digitized by Microsoft® THE BLOOD 267 sue lymph. In grasping the ear the fingers should be at least % inch from the wound. It is better, of course, to obtain the blood without any pressure whatever. ) A drop of blood about the size of a small pinhead is transferred to a cover slip, which is immediately placed upon a glass slide. The blood spreads out between the cover glass and slide in a thin film. Microscopic examination should show the individual red corpuscles separated from one another in the central portion of the film, with the thicker parts at the periphery presenting rouleaux formation. If the cells are not separated the drop of blood used was too large, provided the glassware was clean and the drop of blood fresh. Failure to obtain satisfactory specimens is usually at- tributable to one of several causes. If the drop of blood is allowed to remain on the ear an appreciable length of time before it is used, clotting may have begun ; this, of course, interferes with the proper spreading of the blood. Again, any dirt on the ear also has the same effect. Particles of dust or bits of lint on the glassware prevent even uniform spreading of the blood by elevating the cover glass from the slide. As dust frequently settles on the cover glasses or slides while preparing to secure the blood, it is a good plan to remove all such particles by blowing on the glass (avoid moisture from the breath on the glass), or by brush- ing the surfaces with a camel's hair brush. Any grease or dirt of any kind on the glass makes it impossible to obtain good specimens. It is advisable to handle the cover glasses with a pair of straight forceps, to avoid the grease, etc., of the fingers. Sealing the Fresh Specimen.— If the specimen is to be kept for any length of time, it should be sealed to prevent drying. Vaselin is convenient for this purpose. A small Digitized by Microsoft® 268 FRESH AND STAINED PREPARATIONS OF BLOOD quantity of it is taken up on the end of a match, which is then rapidly passed through a Bunsen flame. The edge of the cover glass is now lined with the melted vaselin, which hardens almost instantaneously, and effectually seals the specimen. Paraffin of low melting point may also be used. Specimens prepared in this way may be kept for a surprising length of time with little alteration in the red corpuscles. The Preparation of Dry (Permanent) Blood Smears.— (1) The Cover Glass-Forceps Method. — In the writer's ex- perience the best results are obtained by using two cover glasses. The covers are cleaned and dried as described on page 265. Any particles of dust are carefully removed from the covers just prior to making the smear. Forceps are used to avoid soiling the surfaces of the cover glasses with the fingers. With care, however, perfectly satisfac- tory films may be made with the fingers. Two pairs of forceps are needed. One is a cross-hilled forceps, which will hold a cover glass firmly. The spring should be strong and the blades perfectly parallel, so that the grip on the cover slip will be uniform. If the forceps are suitable it should be possible to lift them by grasping a cover glass caught between the blades of the forceps without changing the relative position of the cover glass. Forceps which cannot withstand this simple test usually prove to be useless. A pair of straight forceps is also re- quired. They should be fairly stiff, with blades having plain, square ends. When holding a cover slip firmly only the tips of the blades should touch it. To prepare blood films a clean cover glass is placed in the cross-billed forceps, the puncture wound is then wiped free of blood, and, when a drop of the proper size appears (about the size of a small pinhead with a normal count. Digitized by Microsoft® THE BLOOD 269 larger with anemic blood), it is taken up on a second cover slip held in the straight forceps. This is immediately placed on the first cover glass. The blood spreads out be- tween the two in a thin layer. Just before the drop will have stopped spreading between the covers, the overlap- ping edge of the second cover is grasped with the straight forceps, and the two are quickly pulled apart. It requires considerable practice to pull the covers apart in exactly parallel planes, which is necessary if the spreads are to be good. With good preparations microscopic examination will show the individual red cells well separated over one- half to two-thirds of the preparation. With a little ex- perience good smears may be selected with the unaided eye. When inspected by transmitted light, the area in which the cells are properly separated resembles an ex- tremely thin, gray veil; if the cells are grouped in little islands, the uniformity of the veil is lost. The thick parts of the smear are more dense and opaque. The films, which are allowed to dry in the air, are then ready for fixing and staining. At times, when the humidity is very high, it may be necessary to fan the films to hasten the drying. (During the fly season films should be pro- tected from the pests, as they may eat practically all the blood from a cover glass in a few seconds.) The size of the drop of blood is a matter of great im- portance in making blood smears with the cover glass method, as has been indicated above. The correct size will depend largely on the number of red corpuscles in the blood. With very anemic patients, whose blood is thin and hydremic, a relatively large drop will be needed. The general tendency of beginners is to take a drop which is too large. If this mistake is made, no part of the film is thinly spread, the erythrocytes being piled up so that 19 Digitized by Microsoft® 270 FRESH AND STAINED PREPARATIONS OF BLOOD study of the individual cells is impossible. If one waits until the blood has stopped spreading, it is often impos- sible to separate the covers, as they become sealed. Lint, dust, gritty particles, or grease on the cover glasses will make it impossible to secure satisfactory specimens. (2) The Glass Slide Method.— Many clinicians prefer glass slides to cover glasses in making blood films. ^ The method requires practically no practice, and is simpler than the cover glass-forceps method. The area of the blood film may be made much larger than that obtainable on a cover glass. The slides should be thin, and should have perfectly smooth, even edges and level surfaces.' They must, as a matter of course, be perfectly clean. Any dust which may have settled on the slides should be removed before using them. A drop of blood considerably larger than that required in the cover glass method is taken up on the end of one slide, which is then approximated to the surface of a sec- ond slide, placed on a table or other firm surface. The first slide is held at an angle of about 45 degrees to the second. The blood spreads out along the end of the first slide, which is now pushed rather rapidly along the sur- face of the second slide. The blood spreads out in a thin layer over the surface of the second slide. In making the spread, pressure on the slides is unnecessary. ' In the experience of the author, more satisfactory specimens are ob- tained with the cover glass method. As a rule, the leukocytes are more evenly distributed over the specimen. The large smear, which is obtained with the slide, is usually no advantage, for it is seldom the case that a greater area is needed than is contained in a cover glass preparation. The great value of the slide method, aside from the fact that good smears may be obtained with it, lies in the fact that the technique is easily acquired, and fair specimens may often be obtained with slides which have been cleaned only with water. All laboratory workers should, therefore, be able to employ the method, though the cover glass method is preferred. Digitized by Microsoft® THE BLOOD 271 Labeling the Blood Films. — With specimens made on glass slides, where the area of the blood film is large, a part of it may be employed for labeling the specimen. A very simple and practical method has been described by von Ezdorf.i The necessary data is written on the thick part of the film with a soft, black lead pencil. The label thus made is permanent, and is not affected by staining or washing the specimen. The black contrasts well with the usual pink color of the film. Fixation of Blood Smears.— Various methods are avail- able for fixing the blood cells to the slide. The following will be found useful : (1) Heat Fixation. — ^A triangular copper bar, first in- troduced into blood work by Ehrlich, is usually used for heat fixation. The bar is placed on a tripod with a Bun- sen flame under the tip of the bar. In- a short time, if pro- tected from strong drafts, all parts of the bar acquire and maintain a fairly constant temperature. By dropping water from a pipette onto the bar, the point farthest from the flame is determined, at which the drop of water remains spheroidal and rolls off. The temperature at this point, the "spheroidal point" for water, is about 150° C. The point is marked, and the blood films, with the specimen side up, are then placed just inside this point, i. e., toward the flame from the spheroidal point, and allowed to remain 30 to 45 seconds. This usually suffices to fix the films well. In certain instances a longer or shorter time is required, the extremes falling between 5 and 120 seconds. By placing four specimens of blood (cover glass preparations) at the spheroidal point and removing them at the end of. 30, 35, 40, and 45 seconds respectively, and staining all, the proper •Von Ezdorf, R. H. "The labeling of dried blood films." Jour. A. M. A., 1910, LIV, 125. Digitized by Microsoft® 272 FRESH AND STAINED PREPARATIONS OP BLOOD fixation time is quickly determined with the great majority of bloods. It is to be remembered that there is no one optimal fixation time applicable to all bloods. A separate determination must be made for each individual blood ex- amined. In place of the copper bar, an oven may be used. The specimens are placed in the oven, which is maintained at a temperature ranging between 110° and 120° C. for one to two hours, rarely longer. By removing and staining a specimen every fifteen minutes after the first hour, the correct fixation time is determined. This method of em- ploying heat fixation is particularly convenient, when a large number of specimens of the same blood are to be fixed. Heat fixation is always used with Ehrlich's triacid stain. In using Pappenheim's methyl green-pyronin mix- ture, heat fixation is also to be preferred. It is less use- ful for other blood stains. (2) Ethyl Alcohol. — The specimens may be fixed by im- mersion in absolute alcohol one to five minutes, or in 96 per cent, alcohol five to twenty minutes. They are then dried in the air or between blotting paper. Less expensive and about as satisfactory is the denatured alcohol of com- merce. This method of fixation is useful in connection with hematoxylin and eosin, methylene blue, etc. (3) Methyl Alcohol. — Absolute methyl alcohol, acting for one to five minutes, is an excellent fixative. (Methyl alcohol fixation is carried out as a part of the staining tech- nique in employing Leishman's, Wilson's, and Jenner's stains, as will appear below.) Methyl alcohol may be used in place of ethyl alcohol, and is usually employed with Giemsa 's stain. Digitized by Microsoft® THE BLOOD 273 (4) Acetone. — The specimens are placed in acetone five minutes, and are then dried in the air. (5) Alcohol-Formalin. — ^Futcher and Lazear have used 0.25 per cent, formalin in 95 per cent, alcohol. The solu- tion must be prepared freshly, and is obtained by adding one drop of commercial formalin (40 per cent.) to 10 c. c. of alcohol. The specimen is allowed to remain in this mix- ture one minute, and is then washed in water and blotted dry. This method is the best for staining with carbol-thionin. Staining the Blood The stains and combinations of stain for blood work are numerous. Those described below are among the most serviceable, and enable one to make all routine examina- tions. Most of the stains are applied to dried, fixed films of blood. The staining of the fresh, unfixed blood, the so- called "vital" staining of the blood, forms an exception. "Vital" Staining of the Blood Various stains and methods have been proposed for vital staining of the blood. Practically any basic dye may be used for this purpose, but the stains which have been used most extensively are Unna's polychrome methylene blue (Grtibler's), Pappenheim's methyl green-pyronin, and neutral red. Only the first is described, since the picture obtained is rather more brilliant. (1) Vaughan's ^ Method.— A small puncture is made in the ear, and over the wound, from which the blood has been wiped, a minute drop of Unna's polychrome blue is ' Vaughan, V. C, Jr. ' ' On the 'appearance and significance of certain granules in the erythrocytes of man. ' ' Jour. Med. Research, 1903-4, X, 342. Digitized by Microsoft® 274 FRESH AND STAINED PREPARATIONS OP BLOOD placed by means of a clean glass rod. A small drop of blood is now pressed out of the wound, so that it flows directly into the stain. The procedure is now the same as in the preparation of a specimen of fresh blood (q. v.). The relative proportions of stain and blood are quickly learned by experiment. There should be more blood than stain in the mixture. After the specimen has spread out between cover glass and slide, it is ready for examination. It should be sealed, if the examination is to be a prolonged one. On microscopic examination with the oil immersion ob- jective, the majority of the red corpuscles appear quite like those in a preparation of fresh blood, except where the stain is concentrated ; here the cells may show a diffuse purplish tint of varying intensity. Laking may occur in a certain number of the corpuscles. In some of the red cells granules stained bluish-purple are seen. These basophilic substances have been designated "granulo-reticulo-filamen- tous" by Sabrazes.^ There may be very few granules in a cell, or they may be extremely numerous. Often the gran- ules appear to be attached to a delicate filament, which may form a part of a reticulum in the corpuscle. Not in- frequently the granules are clustered at the center of the cell, suggesting by their position and number the remnants of a nucleus. In erythroblasts the nucleus takes a purple color, as do nuclear particles, when present. The chroma- tin of the blood platelets takes on a similar color, and may often be seen occupying a position at the periphery of a clear, unstained globule, as Vaughan observed. Leukocy- tic nuclei stain more or less intensely, depending largely ^Sabrazes, J., and Leuret, E. "Hgmaties granuleuses et polychromato- philie dans Picture des nouveaux-nes. " Gaz. hehd. d. soc. med. de Bordeaux, 1908, XXIX, 123. Digitized by Microsoft® THE BLOOD 275 on the concentration of the stain. In the protoplasm of- the polynuclear cells, stained granules may be found. The ameboid leukocytes retain their activity for some time. The colorless cell membrane may be seen extending some distance beyond the granules in the pseudopods of the neutrophilic cells. (2) Method of Widal, Abrami, and Brule.^— " Four to six drops of blood are allowed to fall into a test tube con- taining 10 drops of a basic coloring matter, which is quite isotonic, and contains in addition oxalate of potassium to prevent the coagulation of the blood. Potassium oxalate, 20 per cent, solu- tion 2.0 c. c. Unna 's polychrome methylene blue . 100 drops La=-0.60 "The fresh corpuscles are allowed to remain for 10 to 20 minutes in contact with the solution, after which the mixture is centrifugalized, the supernatant fluid is removed, and the corpuscles drawn up with a pipette and placed upon slides, upon which they are spread as an ordinary drop of blood ; the covers are then dried and fixed by heat. Such preparations may be preserved indefinitely" (Thayer and Morris). (3) The "Dry" Method of Vital Staining.— The dry method of vital staining consists in spreading a thin film of stain on a glass slide, allowing it to dry in the air, protected from dust, and then placing a cover glass with a drop of blood on the dried stain, just as in making a prepara- tion of the fresh blood. The blood spreads out between 'Widal, F., Abrami, P., and Brule, M. "Diversite de types des hematies granuleuses ; precedes de coloration. ' ' Compt. rend. Soc. de biol., Par., 1908, LXIV, 496. Digitized by Microsoft® 276 FRESH AND STAINED PEEPARATIONS OF BLOOD cover and slide, the stain dissolves in the plasma, and the result is much the same, as with other methods. "With this method there is less danger of laking the cor- puscles. Pappenheim's methyl green-pyronin mixture has been used extensively by the French, usually with the dry method. Neutral red may be employed. A dilute solution of the dye is prepared in physiological salt solution, or a smaller quantity of saturated, aqueous solution of the stain may be used. It may be substituted for polychrome methylene blue in Vaughan's method, or may be used in the dry method. In normal blood of adults less than 1 per cent, of the erythrocytes contain the granulo-reticulo-filamentous sub- stance, while in newborn infants the number is 7 per cent, or less (Vaughan). The Staining of Dried Blood Films The stains which are required for the routine examina- tion of blood are Ehrlich's triacid, Jenner's stain, and a Eomanowsky stain. For special purposes, however, other stains are required at times. (1) Methylene Blue.— (1) Fix the blood film in alcohol. (2) Stain with Loffler's methylene blue (p. 213) about 3 to 5 seconds. (3) Wash in water, blot dry, and mount in balsam. The stain is useful as a nuclear stain. For the demon- stration of basophilic granules and polychromatophilia, methylene blue is one of the most reliable stains. Nuclei are stained dark blue. The leukocytic granules are unstained, excepting basophilic granules, which take a Digitized by Microsoft® THE bLOOD 277 bluish-purple color. The basophilic protoplasm so fre- quently encountered, in lymphocytes is stained a paler blue than the nucleus, the shade varying greatly in different cells. The erythrocytes assume a pale, greenish tint. Poly- chromatophilic red cells are light blue to very deep blue, depending on the degree of polychromatophilia. Basophilic granules in the red cells are stained dark blue, almost as dark as the nuclei. Nuclear particles in the red corpuscles take the same color as the nuclei of erythroblasts, i. e., a dark blue. Blood platelets are indistinct, appearing as dirty grayish-blue masses. (2) Eosin.— Eosin may be used as a counter-stain in % per cent, aqueous solution. It is used after the methy- lene blue has been washed off the specimen. The stain is allowed to act a few seconds, the intensity of staining be- ing controlled by microscopic examination of the film in water. Slower staining is secured by diluting the stain- ing solution with water. Eosin adds little to the picture, except that it stains the eosinophilic granules, which now assume a brilliant pink or reddish-pink hue. However, slight polychromasia may be somewhat less evident, though often more striking because of the contrast. The ortho- chromatic erythrocytes are stained pink. If the specimen has been overstained with eosin, the pink color will be ap- parent in the protoplasm of the lymphocytes and neutro- philic leukocytes. (3) Hematoxylin.— Ehrlich's acid hematoxylin is pre- pared as follows : Solution A: Hematoxylin 2.0 gm. Alcohol, absolute 60.0 c. c. Dissolve. Digitized by Microsoft® 278 FRESH AND STAINED PREPARATIONS OF BLOOD Solution B: Saturated solution of alum in equal parts of glycerin and distilled water 60.0 c. c. Glacial acetic acid 3.0 c. c. The two solutions, A and B, are mixed and allowed to "ripen" in an open bottle for a week. The bottle is then stoppered. The ripened stain has a reddish-blue color. If the bottle is shaken or disturbed, the solution should be filtered before using. Method. — (a) Fix the blood films in alcohol. Heat fixa- tion may also be used. (b) Stain in hematoxylin 2 to 10 minutes or longer. Control the intensity of staining by examining the specimen in water. (c) Wash in tap water. The washing may be com- pleted in a few seconds, but the beauty of the nuclear stain- ing is greatly enhanced by prolonged washing in tap water. If the specimen has been overstained with hematoxylin, it may be cautiously decolorized in acid alcohol (HCl, 1.0 c. e., 70 per cent, alcohol, 100.0 c. c), and again washed in water. It is better to avoid overstaining by controlling the stain- ing carefully under the microscope. (d) Dry, mount in balsam. Hematoxylin is one of the best nuclear stains. For studying the morphology of nuclei it is particularly useful. Nuclei are stained a very dark blue, at times almost black. After prolonged washing, however, the blue is brighter — more brilliant. As with other nuclear dyes, the color intensity in a given nucleus depends, of course, on the amount and concentration of the chromatin. Mast-cell granules are stained dark blue, but may be lost after wash- Digitized by Microsoft® THE BLOOD 279 ing the specimen. Other leukocytic granules are unstained. Basophilic protoplasm is less intensely stained than with methylene blue. The red blood corpuscles are lightly stained, and are either gray or grayish-blue. The more marked grades of polychromatophilia are revealed by the darker blue stain of the cells. Coarse basophilic granules in the erythrocytes are fairly well demonstrated as dark blue spots ; the finer granules are unstained or indistinct, as a rule. Nuclear particles take on a very intense, dark blue, like the pyknotic nuclei of normoblasts. Blood plate- lets are dirty blue and indistinct. Eosin may again be employed as a counterstain. When the specimen is properly stained with eosin, no pink is seen in the protoplasm of the neutrophilic cells, while the eosin- ophilic granules stand out prominently. Hematoxylin and eosin are useful in cases where the relative number of eosinophilic cells is to be determined, as a differential count with this point alone in view may be made rapidly. (4) Carbol-thionin. Saturated solution of thionin in 50 per cent, alcohol 10.0 c. c. Carbolic acid, 1 per cent 100.0 c. e. (a) Fix the blood films in alcohol-formalin. (b) Stain with carbol-thionin i/i to 3 minutes. (c) "Wash in water. If the specimen is overstained, the washing may be continued, or the specimen may be decol- orized in 50 per cent, alcohol. (d) Dry, and mount in balsam. The stain is an excellent nuclear stain. All nuclei are stained dark blue. Leukocytic granules are not specifically stained with the exception of the granules of the mast cells, Digitized by Microsoft® 280 FRESH AND STAINED PREPARATIONS OF BLOOD ■wMch are purple. The red blood corpuscles are greenish- gray. Basophilic granules are dark blue, polychromato- philic red cells varying shades of blue. Nuclei and nuclear particles are dark blue. The bodies of malarial parasites are purple, contrasting well with the red blood corpuscles. The nuclei of the parasites are unstained. Blood platelets are indistinct, and have a mauve color. Preparations stained with carbol-thionin fade in the course of several months, as a rule. Staining Mixtures of Two or More Stains (5) Ehrlich's Triacid Stain.— For the sharp differenti- ation of neutrophilic granules, the triacid stain of Ehrlich is unequaled. It should always be used in the study of these cells. The mixture contains three stains, two of which, orange G. and acid fuchsin, are acid, the third, methyl green 00, basic. The three basic radicals of the methyl green are satisfied by the acid dyes, hence the name, triacid. The formula ^ given below, a slight modification of the usual one, has been found to yield uniformly good staining mixtures,^ whereas formerly it has been more or less a matter of good fortune to obtain a satisfactory solu- tion. Good staining mixtures may usually be had from Griibler. In preparing the mixture, saturated aqueous so- ' Morris, K. S. "The value of Ehrlich's triacid stain in blood work." Jour. A. M. A., 1910, LV, 501. ^Eecently (1912) we have encountered the first failures in more than five years. After numerous experiments the cause of the trouble was found to lie in the acid fuchsin. Three different lots of the powdered stain in Griibler 's original packages obtained through one firm resulted in poor staining mix- tures, while acid fuchsin secured from another firm (also Griibler 's make) gave very satisfactory results. With the poor acid fuchsin all cells were stained diffusely red. The nature of the defect in the acid fuchsin has not yet been determined. Digitized by Microsoft® THE BLOOD 281 lutions of metliyl green,^ orange G., and acid fuehsin are made separately. They must be allowed to settle for at least a week before use, and should be replenished as needed, so that a constant supply of the stock solutions may be on hand. Griibler 's stains are generally used. The formula, as modified, is: Saturated aqueous solution of orange Q 13.0 c. c. Saturated aqueous solution of acid fuehsin. . . 7.0 c. c. Distilled water 15.0 c. c. Absolute alcohol 15.0 c. c. Saturated aqueous solution of methyl green . . 17.5 c. c. Absolute alcohol 10.0 c. c. Glycerin 10.0 c. c. The fluids are mixed with the same graduated cylinder, which should not be rinsed. The receiving flask should be shaken vigorously after the addition of each constituent, which is added in the order given in the formula. It is essential to add the methyl green, second portion of alco- hol, and glycerin slowly, shaking well after each addition. The mixture is ready for use immediately, and does not deteriorate with age. After the mixture has stood a while a small amount of precipitate may form. Care should be exercised that this is not disturbed when using the stain.^ Method of Staining. — (a) Fix the blood spread by heat (p. 271). (b) Stain 5 to 10 minutes (overstaining is impossible). (c) Wash quickly in water, blot dry with filter paper, and mount in balsam. In a properly fixed specimen the neutrophilic granules ' Methyl green is used in place of methyl green 00 of the original formula. ' For blood stains, bottles with droppets, the rubber nipples of which also serve as stoppers, are indispensable. Barnes ' bottle is a very good one. Digitized by Microsoft® 282 FRESH AND STAINED PREPARATIONS OP BLOOD stand out sharply. When this is the case the erythrocytes are usually, though not always, colored deep orange or buff ; in an underfixed specimen they are stained red, while too prolonged fixation causes them to take a yellow color. The color of the red corpuscles, while a safe index of the fixation in most instances, fails at times. The final criterion by which a specimen is judged must be the staining of the neutrophilic granules. In a good specimen (plate I) the erythrocytes have, then, a buff color usually. Polychromasia is not demon- strated. Basophilic granules and Cabot's ring bodies are unstained. The pyknotic nuclei of normoblasts take a dark green color, the megaloblastic nuclei being less deeply stained. Often reddish areas are visible in the nuclei. Nu- clear particles are stained green, but are much less strik- ing than when stained with better nuclear stains, such as hematoxylin or a Komanowsky stain. Malarial and other parasites are not well stained. It is evident, therefore, that the triacid stain is very inferior for the study of patho- logical changes in the red blood corpuscles. Many of the abnormalities of the red cells are not shown at all, and none of them is as well demonstrated as with a Romanow- sky stain or with Jenner's stain. Blood platelets appear as ill-defined, indefinite, mauve- colored masses. Leukocytic granules are well differentiated, but the nu- clei are poorly stained, assuming a rather pale green or bluish-green color. All neutrophilic granules take a lilac color. There should be no blurring of them in a well-fixed specimen; the granules are sharply defined and distinct, though in the myelocytes the very fine granules stand out less prominently than in the polynuclear cells. Eosinophi- lic granules assume a brick-red or coppery color, while Digitized by Microsoft® Digitized by Microsoft® LEGEND FOR PLATE I. (All drawings made with camera lucida; X 1200. Ehrlich's triacid stain.) 1, 2. Normal red corpuscles. 3. Megaloblast. 4. Normoblast. 5. Lymphocyte. 6. Large mononuclear leukocyte. 7. "Transitional" leukocyte. 8. Polynuclear neutrophilic leukocyte. 9. Polynuclear eosinophilic leukocyte. 10. Mast cell or polynuclear basophilic leukocyte. 11. Neutrophilic myelocyte. 12. Eosinophilic myelocyte. 13. Mast myelocyte or basophilic myelocyte. 14. Myeloblast. Digitized by Microsoft® PLATE I t U |i. «;.Slts 10 II : &'■>■' ■■ ..-■ r-\ N digitized by Microsofff® R. tM. Digitized by Microsoft® THE BLOOD 283 basophilic granules are unstained, appearing as vacuoles in the cytoplasm. The protoplasm of the lymphocytes is either colorless or a faint rose-pink. The same holds true for the large mononuclear and transitional cells; their nuclei, being poor in chromatin, usually stain very faintly, so that they are easily overlooked. Because of this diffi- culty with the non-granular leukocytes, Loffler 's methylene blue has been used to stain the nuclei more intensely. It is applied to the blood film for a few seconds (3 to 5) after the staining with the triacid has been completed. The granular stain may be slightly impaired, but the nuclei are much more evident. The mast-cell granules are now stained purple. In this connection it may be added that Pappenheim has prepared a triacid mixture, substituting methylene blue for methyl green, but it has not been widely adopted. (6) The Romanowsky Stains.— The Eomanowsky stains are by far the best for the demonstration of pathological changes in the red corpuscles, and are, of course, indis- pensable in the study of such protozoa as the plasmodia of malaria, trypanosomes, etc. Eomanowsky 's original method of preparing the stain has undergone numerous modifications, with a view to sim- plification both of the preparation of the stain and of the staining technique. The essential dyes are eosin and meth- ylene azure, the latter being obtained from methylene blue. Methods of preparing the stain have been described by a number of workers in this country, among whom may be mentioned Wright, Harris, Hastings, MacNeal, Wilson. Leishman's stain, which antedates all of those mentioned, is used extensively in England. The staining mixtures may be purchased; it is much more convenient and satis- factory, however, in private laboratories of physicians 20 Digitized by Microsoft® 284 FEESH AND STAINED PREPARATIONS OF BLOOD where only moderate amounts of stain are used, to buy tablets of the powdered stain. A tablet is dissolved in a stated quantity of absolute methyl alcohol (usually 10 c. c), and the mixture is ready for use at once. In this way fresh stain may be had at frequent intervals, and there is less danger of deterioration of the mixture. Such tablets are prepared by Burroughs, Wellcome, & Co., and by Griib- ler. The method of preparation of only one of the modifi- cations of the Eomanowsky stain, "Wilson's, is given. The writer has employed it for several years with entire satis- faction. (a) "Wilson's Stain.^ — Prepare a 1 per cent, aqueous solution of methylene blue,^ which contains 0.5 per cent, of sodium carbonate and at least 0.5 per cent, of freshly precipitated silver oxid.^ The solution is boiled for twenty minutes; then remove one-third of it. After boiling an- other twenty minutes, remove one-half. Continue to boil the remaining portion twenty minutes. The three portions are now united and distilled water is added to the original volume, to compensate for the loss by evaporation. The mixture is allowed sufficient time for the precipitate to settle (about an hour). Now add an equal volume of 0.5 per cent, aqueous solution of yellowish eosin (filtered) to the methylene blue solution in a large evaporating dish. '■ Wilson, T. M. " On the chemistry and staining properties of certain de- rivatives of the methylene blue group when combined with eosin." Jour. Exp, Med., 1907, IX, 645. ' The cheaper grades of methylene blue may be used with satisfactory result. ' The silver oxid may be prepared by dissolving 2.0 gm. of silver nitrate in 15 c. c. of distilled water and adding to it 260 c. c. of calcium hydrate. Shake well, and set aside for the precipitate to settle. Decant the supernatant fluid, collect the precipitate on a filter, and wash with 20 to 25 c. c. of distilled water. Dry the precipitate at a temperature not exceeding 100° C, and place it in a brown bottle, tightly stoppered. Digitized by Microsoft® THE BLOOD 285 Mix the solutions well, and allow tlie mixture to stand one hour. Filter thrice, using a hard filter paper, such as the Schleicher and Schiill filter, No. 575, and finally wash the precipitate which has collected on the filter paper with physiological salt solution. (The precipitate which adheres to the evaporating dish is discarded.) Dry the precipitate in the thermostat, and transfer it to a dark bottle, tightly stoppered. The staining solution is then prepared by dis- solving 0.4 gm. of the powdered precipitate in 100 c. c. of absolute methyl alcohol (Kahlbaum's). The stain may be rubbed in a mortar with the alcohol to facilitate solution, or powder and alcohol are placed in a bottle, which is vigor- ously shaken a few minutes on several successive days. The staining solution should be preserved in a dark bottle with glass stopper. (Wilson advises the use of 0.3 gm. of the dry stain to 100 c. c. of denatured alcohol, but in our hands this has not given satisfaction.) It is best to make up small quantities of the stain at frequent intervals (3 to 4 months). Method of Staining. — ^As the methyl alcohol in which the stain is dissolved is apt to run over the edge of the cover glass, it is advisable to use the usual wire staining forceps; when staining on glass slides, the stain may be confined to the area of the smear by drawing lines on the glass with a blue wax pencil — the red wax is usually loos- ened by the alcohol. (a) Cover the unfixed> blood film with 5 to 6 drops of the stain for 1 minute. As the stain is dissolved in abso- lute methyl alcohol, the blood is fixed by this procedure. Precipitation of the stain through evaporation of the al- cohol will be troublesome, if too little stain is used. (b) Add to the stain an equal number of drops of dis- tilled water, and allow it to remain on the film 2 to 4 min- Digitized by Microsoft® 286 FRESH AND STAINED PREPARATIONS OF BLOOD utes. A metallic scum forms on the surface. (The exact proportion of stain and water should be determined for each new lot of stain. At times twice the quantity of water is necessary; occasionally, however, fewer drops of water than of stain are required, especially with old mixtures, which have become slightly acid.) (c) Wash with distilled water, blot dry, and mount in neutral balsam. The specimen should be held level during the washing and the stream of water directed against the surface of the cover glass, so that the metallic scum and precipitate in the fluid will be floated off. Avoid dumping the stain from the cover glass, for the precipitate adheres to the corpuscles, and cannot be removed by washing in water. If there is precipitate in the specimen, it may be removed by immersing the preparation momentarily in ab- solute methyl alcohol or ethyl alcohol, but always at the risk of decolorizing the cells too much; it is particularly, the basic stains, methylene blue and methylene azure, which are decolorized. A properly stained blood film should have a pinkish- gray or gray color when dry. If the color of the film is bright pink, the specimen is usually not satisfactory, or, rather, it is capable of being improved upon. The erythro- cytes are stained very pale pink (plate II) or mauve or grayish-pink. Polychromatophilia is denoted by varying admixtures of blue. In extreme grade a polychromato- philic red cell is dark blue, no trace of pink being discern- ible. Basophilic granules stain dark blue. Occasionally, particularly in the blood of pernicious anemia, fine gran- ules are seen which are stained violet or purple. Nuclei are stained purple. Nuclear particles stain like the nuclei, while the ring bodies are usually violet or reddish-purple. The differentiation of these abnormalities is more striking Digitized by Microsoft® Digitized by Microsoft® LEGEND FOR PLATE II. (All drawings made with camera lucida; X 1200. Wilson's stain, modified Romanowsky.) 1. Normal red corpuscle. 2. Pale or anemic corpuscle. 3. Basophilic granules in erythrocyte. 4. Nuclear particle (Howell's body) in erythrocyte. 5. Erythrocyte containing nuclear particle and basophilic granules. 6. Polychromatophilic red cell containing a Cabot's ring body. 7. Normoblast. 8. Slightly polychromatophiHc erythrocyte containing a nuclear particle, Cabot's ring body, and violet colored basophilic granules. 9. Normoblast showing an early stage of karyorrhexis. 10. Megaloblast, markedly polychromatophilic. IL.Poikilocyte, markedly polychromatophilic and exhibiting reddish basophilic granules. 12. Blood platelets. 13, 14. Small lymphocytes. 15. Large lymphocyte, exhibiting azurophilic granules. 16. Large mononuclear leukocyte with a few azurophilic granules in the cytoplasm. 17. "Transitional" leukocyte with fine azurophilic granules. 18. Polynuclear neutrophilic leukocyte. 19. Polynuclear eosinophilic leukocyte. 20. Mast cell or polynuclear basophilic leukocyte. 21. Neutrophilic myelocyte. Digitized by Microsoft® • »-■' PLATE 11 10 >iV^''.^- a 12 13 • t 14- 15 16 17 19 ^«:v*yfi A'*^ie*V^^fj3«. Digitized by Microsoft® (,S.I/^' Digitized by Microsoft® THE BLOOD 287 and brilliant with the Romanowsky stains than with any of the other blood stains. Cabot's ring bodies are usually demonstrable only with Romanowsky stains, i. e., they are stained with methylene azure. Blood platelets are well brought out with Romanowsky stains alone. The granular chromatin masses are stained reddish-purple, the body of the platelet being unstained or exhibiting varying shades of blue. All leukocytic nuclei are beautifully stained, the color being a deep reddish-purple. The morphology of the nu- cleus is well demonstrated. The leukocytic granules, on the other hand, stain poorly and very uncertainly, so that differential counting may be difficult or well-nigh impos- sible when pathological cells are present. The neutrophilic granules are stained lilac, but often only a few of the gran- ules — sometimes none of them — take the stain. Eosinophi- lic granules are pink, but stain very uncertainly; at times there may be considerable doubt as to their nature. Bas- ophilic granules are colored purple. The granules of the myelocytes usually are purple, regardless of the variety of the cell, though eosinophilic myelocytes may show a shade of pink in the granules. The cytoplasm of lymphocytes is colorless or blue — at times a very dark blue. In about one- third of the lymphocytes of normal blood purplish granules, varying much in size and number, are evident in the cyto- plasm. These granules are demonstrable only with methy- lene azure, and are, therefore, designated azurophile gran- ules. The large mononuclears and transitionals are, like the lymphocytes, more beautifully demonstrated with Ro- manowsky stains than by any other means. The cytoplasm of the large mononuclears is colorless or blue and generally non-granular, though it is now and then seen to be filled with azurophilic granules, which are for the most part very Digitized by Microsoft® 288 FEESH AND STAINED PREPARATIONS OF BLOOD fine and dust-like. In the case of the transitional leuko- cytes the protoplasm is studded with very fine azurophilic granules practically without exception. When these gran- ules are observed in a large mononuclear cell, the resem- blance it bears to a myelocyte is close at first glance. It is seen, however, that, while similar in color, the myelocytic granules are rather coarser, and close inspection will usu- ally reveal the granules over the nucleus in the myelocyte, a point which serves to differentiate them from the large mononuclears. Usually, too, the relations between nucleus and protoplasm are different in the two types of cell. (With the triacid stain and with Jenner's stain this diffi- culty never arises, since the large mononuclears are non- granular.) Besides precipitated stain in the specimen, which may be avoided as indicated above, the chief difficulty in the ap- plication of the Romanowsky stains arises in understaining with the basic components of the mixture. When this oc- curs, the erythrocytes are bright pink or red, the nuclei of the leukocytes blue instead of purple, and the chromatin of the platelets blue or unstained, while the chromatin of ma- larial or other parasites is entirely unstained. Such a con- dition may be due to one or more causes: (1) Dilution of the stain with too much water interferes with the nuclear staining. The requisite proportions of stain and water must be learned by experiment. If the nuclei are poorly stained, try less water. (2) Even a trace of acid in the water used for diluting or washing will weaken or remove the basic stains more or less completely. Staining in a room in which there are acid fumes is at times sufficient to ruin the specimen. (3) Acidity of the staining mixture itself may explain the failure of the nuclear stain. A minute quantity of acid in the bottle (or on the cork) in Digitized by Microsoft® THE BLOOD 289 which the stain is placed, or the formation of formic acid from the methyl alcohol, are possible sources of difficulty. Staining mixtures which contain acid may be made perfect, according to Peebles and Harlow,^ by the addition of a few drops of absolute methyl alcohol in which a small quantity of potassium hydrate (by alcohol) has been dissolved. In case too much alcohol has been added, as shown by over- staining with the blue, it may be cautiously titrated back with absolute methyl alcohol containing a trace of glacial acetic acid. (4) Prolonged washing may spoil the result. It can readily be demonstrated by experiment that washing in water tends to remove the basic (blue) elements of the stain, thus rendering the eosin more conspicuous. With a good staining mixture and the right proportion of stain and water, it is only necessary to wash long enough to re- move the excess of stain; the specimen is then blotted dry to prevent further decolorization. Overstaining with the blue is seldom a source of diffi- culty, except in old smears. When it does occur, it may be corrected by one of the four procedures just described. With very old blood films, it may be impossible to prevent diffuse blue staining of the red corpuscles, though Brem's method (p. 175) or Giemsa's slow method may give good results. (b) Leishman's Stain. — This stain is most conveniently obtained in tablet form from Burroughs, Wellcome, & Co. or from Griibler. Griibler also supplies the powdered stain in bulk. A tablet is dissolved in a stated quantity of abso- lute methyl alcohol, and the mixture is then ready for use. It should be kept in a tightly stoppered bottle. The staining technique is practically identical with that "Peebles, A. E., and Harlow, W. P. "Clinical observations of blood stains." Jour. A. M. A., 1909, LII, 768. Digitized by Microsoft® 290 FRESH AND STAINED PREPARATIONS OP BLOOD given for Wilson's stain. The nuclei have a little more of a reddish hue, but otherwise the picture is much the same. The advantages and limitations of the stain are those given above. (c) Giemsa's Stain. — The preparation of Giemsa's stain is difficult. Griibler & Co. supply a reliable solution. The staining mixture contains eosin, azure I, and azure II. Method of Staining. — (a) Fix the specimen in absolute methyl or ethyl alcohol. (b) Add one drop of the staining mixture to 1 c. c. of distilled water. (This must be freshly prepared.) Stain 10 to 30 minutes with this dilution of the staining mixture. (c) Wash with distilled water, blot dry, and mount in balsam. The appearance of the stained film is the usual Eoma- nowsky picture (see p. 286). The nuclei, however, are a little redder than usual, and the neutrophilic granules are even less uniformly stained than with most other Eoma- nowsky stains. In case the chromatin staining is unsatisfactory, a very dilute solution of sodium carbonate may be substi- tuted for distilled water in preparing the dilution of the stain. Old Blood Films. — Blood films which have been kept for several months before staining them do not give good results with the usual Romanowsky procedures. The diffi- culty lies chiefiy in the staining of the erythrocytes, which take a diffuse, slate-blue color. To avoid this to a great extent, the films may be stained with Leishman's or Wil- son's stain, using Br em's technique (p. 175), or with Giemsa's stain. With the latter the dilution of the stain should be one drop to five or more cubic centimeters of Digitized by Microsoft® THE BLOOD 291 water. The specimens are allowed to remain in this fluid 24 to 48 hours. They are then washed in water and mounted as usual. With this procedure it is often possible to demonstrate the chromatin of malarial parasites in specimens a year or more old. (7) Jenner's Stain ^ (the eosinate of methylene blue).— If one stain alone were to be selected for the general rou- tine examination of the blood, Jenner's would probably be the choice of most workers. It is a much better stain for nuclei and for pathologic alterations in the red cor- puscles than Ehrlich's triacid, though inferior to the Eo- manowsky stains in these respects. On the other hand, it is much superior to the Eomanowsky stains for the dem- onstration of the granules in leukocytes, though surpassed for this purpose by Ehrlich's triacid mixture. Prepaeation of the Stain. — The tablets of Jenner's stain, which have been placed upon the market by Bur- roughs, Wellcome, & Co. and by Griibler, have done away with the necessity of making the stain, and have made it possible to have on hand fresh solutions of the staining mixture. The tablets are dissolved in a stated quantity of methyl alcohol; the solution is ready for use at once. Numerous modifications of Jenner's methods of preparing the stain have been described, without, however, simplifica- tion or improvement of the original procedures. Jenner's methods are as follows: (a) First Method. — Prepare a 1.2 per cent, to 1.25 per cent, solution of Griibler 's water-soluble, yellowish eosin in distilled water, and also a 1 per cent, aqueous solution of Griibler 's medicinal methylene blue. Mix equal parts of the two solutions in an evaporating dish and, after stirring ' Jenner, L. "A new preparation for rapidly fixing and staining blood. ' ' Lancet, 1899, I, 370. Digitized by Microsoft® 292 FRESH AND STAINED PREPARATIONS OF BLOOD thoroughly, allow the mixture to stand for 24 hours. Filter through a hard filter paper (Schleicher and SchuU's No. 575), and dry the precipitate, which collects on the paper, either at room temperature or in the incubator at 37° C. The temperature may be as high as 55° C. without injuring the precipitate. The dried precipitate is removed from the filter paper, powdered in a mortar, shaken with dis- tilled water, and the precipitate again collected on a filter paper. The washings should have a dirty purplish color. Finally, the precipitate is again dried and stored in brown glass bottles. For use dissolve 0.5 gm. of the powdered precipitate in 100 c. c. of absolute methyl alcohol, filter, and preserve in a tightly stoppered bottle. The solution keeps well. (b) Second Method. — Instead of using aqueous solu- tions, Jenner found that the eosin and methylene blue may be dissolved directly in absolute methyl alcohol. The stain- ing mixture is prepared by adding 125 c. c. of a 0.5 per cent, solution of Griibler's yellowish eosin in absolute methyl alcohol to 100 c. c. of a 0.5 per cent, alcoholic solu- tion of Griibler's medicinal methylene blue. The mixture is ready for use immediately. Methods op Staining. — The following is the technique originally recommended by Jenner: (1) Cover the unfixed blood film with the stain 1 to 3 minutes. To prevent evaporation and precipita- tion of the stain, the specimen is covered with a watch glass. (b) Wash quickly in distilled water, blot dry, and mount in balsam. A second method of staining, which often gives good differentiation of the granules of the leukocytes and poly- chromatic nuclear staining, is as follows : Digitized by Microsoft® THE BLOOD 293 (a) Cover the unfixed specimen with about 8 drops of stain for 2 to 3 minutes. (b) Add to the stain about 10 drops of distilled water, and allow the mixture to remain on the preparation 1 to 2 minutes or longer. (c) Wash with distilled water (observing the precau- tions given on page 286), blot dry, and mount in balsam. With Jenner's stain the red blood corpuscles are terra cotta or pink. Polychromasia causes the cell to assume a bluish tint. Basophilic granules in the red cells are dark blue, nuclei and nuclear particles are of the same color, though usually somewhat more deeply stained. Cabot's ring bodies are unstained or pale blue. The hlood platelets are poorly stained. They are mauve in color and indistinct morphologically. With the second method, however, they are well differentiated, as with Eomanowsky stains. Leukocytic nuclei are stained dark blue (purple with the second method). The neutrophilic granules are red, often with a violet tint. Eosinophilic granules are also red, but they are distinguishable from the former by their greater size and brilliance of staining. Mast-cell granules are purple. The granular differentiation is less distinct in myelocytes than it is in polynuclear cells, as a rule, and is inferior to that obtained with Ehrlich's triacid stain. The protoplasm of malarial parasites is stained light blue; chromatin is usually unstained. ' In staining specimens on glass slides, where the area of the film is larger, more stain should be employed to prevent too rapid concentration of the stain through evaporation. The relative proportion of stain to water should be pre- served. Digitized by Microsoft® agfe JESS'S o ■S* Ph p, S o 2 "^ "3£+j 3 b^.m IBIS'S '^'^^^ S« SS =H 3 -So; 05 gag «= 66'So§o^SoS'aS -g ■S S o o! &, ■ M IS g Si^ o Scb ^ fH g § 8=^=;f -si ° .S s ■§ s 4S ;^ 'i ph S "^ ^- e-TJ M § . « 5 ■ S • -2 1=^ „; ^ & -^ ^sa:S^s|&|ig|-§|^-|| ^1 ^a -s ^ °|, M — Eg (Dri 3 '^':3oa; w^sp. 3ggg=^^ ^ g ^". ^1 d " -a' i 2 Is-s -S'S s S~ ^s a-S • ;^ ^ *^-& ^§ feS §1a§^ "& ^ -§ 3 ^ ^^ •6 si t-a C^l h .«■ C3 ■S P P. So?, ■sgS'g" ;3'g'asS^-^s, -§Pl ^" S'S-^gS >;. -s '^■f i ^,1 ".a.2 :^ 294 Digitized by Microsoft® THE BLOOD 295 Methyl Green-Pyronin Mixture of Pappenheim.^— Sat- urated aqueous solutions of methyl green and of pyronin are made. One part of the pyronin solution is added to three to four parts of methyl green. When sufficient pyro- nin has been added, the mixture begins to take on a bluish tint. It is often possible to obtain very good staining mix- tures from Griibler. Method of Staining. — (a) Fix the blood films with heat. (b) Stain 5 to 10 minutes. (c) Wash quickly in distilled water, blot dry, and mount in balsam. All nuclei are stained dark green or blue. Erythrocytes take a gray or slate color. Polychromatophilic cells are stained more or less intensely by the pyronin, and exhibit varying shades of red. Basophilic granules are stained a brilliant red, in marked contrast to the nuclei. Nuclear particles are stained dark green or blue,^ like normoblastic nuclei, or red, like the basophilic granules. The protoplasm of lymphocytes is stained red. Leuko- cytic granules are not specifically stained. The lodin Reaction of the Leukocytes.— This reaction, discovered by Ehrlich, is demonstrated as follows: (1) The air-dried blood films are placed in a mixture composed lodin 1.0 gm. Potassium iodid 3.0 gm. Distilled water 100.0 c. c. Gum arable, q. s. (to give a syrupy consis- tence). 'Pappenheim, A. " Vergleichende Untersuchungen ueber die elementare Zusammensetzung des rothen Knochenmarkes einiger Saugethiere. ' ' Virchow 's ArcUv, 1899, CLVII, 19. ^ Morris, B. S. " Nuclear particles in the erythrocytes. ' ' Arch. Int. Med., 1909, III, 93. (The observations reported here were made on old films. Sub- sequent study of fresh material shows that nuclear particles, while not infre- quently stained blue, are, nevertheless, often red.) Digitized by Microsoft® 296 FEESH AND STAINED PKEPAEATIONS OF BLOOD (2) The air-dried specimen, instead of being placed in the mixture given above, may be put in a small vessel, in which a few crystals of iodin have been placed. The re- action appears in a few minutes. The specimen is mounted and examined in syrup made of levulose. Permanent specimens cannot be made by either proce- dure. The erythrocytes are stained diffusely brown. A positive reaction consists in brown staining, of vary- ing degree, of the protoplasm of the polynuclear neutro- philes. Occasionally the lymphocytes and mast cells are stained, rarely the large niononuclears and eosinophiles, while myelocytes never give the reaction (ZoUikofer). Differential Counting of the Leukocytes For a differential count of the leukocytes the first es- sential is a well-spread and stained blood film. Ehrlich's triacid or Jenner's stain should be employed in the major- ity of instances. When myelocytes are present in the blood Ehrlich's triacid should be used, though for the usual run of cases Jenner's stain gives satisfactory differentiation. The Eomanowsky stains are unsatisfactory for differential counting, for the granular leukocytes are often poorly dif- ferentiated. The non-granular cells are, however, beauti- fully stained. For special purposes the triacid or Jenner's stain may be inferior to others. In studying eosinophilia, when it is desired simply to follow the percentage of eosinophiles from day to day, hematoxylin or methylene blue combined with eosin may be used, always at the risk, however, of missing certain abnormal cells, if present. In lymphoid leukemia, where the lymphocytes may constitute 90 per Digitized by IVIicrosoft® THE BLOOD 297 cent, or more of the leukocytes, it is advantageous to use a better nuclear stain than the triacid, and one which will differentiate between nucleus and protoplasm more effec- tively than Jenner's. The Eomanowsky stains are the best for this purpose, for the staining is sharp and clear. Hematoxylin also gives excellent results, though the pic- ture is less comprehensive, for all granules are unstained. The large mononuclears and transitionals are most satis- factorily demonstrated with the Eomanowsky stains. The differentiation between nucleus and protoplasm is clear, the morphology of the nucleus is shown in great detail, and the azurophile granules are made evident. For the differential count a mechanical stage is almost a necessity. The stained specimen is placed on the stage and examined with the iV -in. oil immersion objective and an eye-piece (such as Leitz No. 3 or No. 4), which gives a high magnification. In making the count it is, of course, essential that the cells be counted only once. This is accom- plished through the use of the mechanical stage, the speci- men being moved up and down, with a lateral shifting of the field at the end of each "row." At least 500 cells should be counted. Normal Leukocjrtes.— The leukocytes of the blood may be classified as follows: (1) Lymphocytes (pi. I, 5; pi. II, 13, 14, 15) are cells having a single round or oval nucleus — rarely a kidney- shaped nucleus — and a rather scanty rim of protoplasm. The protoplasm is non-granular, though about 30 per cent, of the lymphocytes of normal blood possess azurophile granules, which are demonstrable after staining with meth- ylene azure, but not with other stains. These granules vary greatly in number and size. Often there are many granules, more frequently only a few, in a cell. The diam- 21 Digitized by Microsoft® 298 FRESH AND STAINED PREPARATIONS OF BLOOD eter of lymphocytes varies between 7 and 11 micra. They constitute 22 to 25 per cent, of the leukocytes normally. (2) Large mononuclear leukocytes (pi. I, 6; pi. II, 16) resemble the lymphocytes, but are larger and have rela- tively more protoplasm. The nucleus is somewhat poorer in chromatin and, therefore, stains less intensely. The protoplasm may contain azurophile granules, which are either very fine, like those of the transitionals, or rarely coarser, like the granules of the lymphocytes. The cells are actively ameboid and phagocytic — the macrophages of the blood. The diameter is 12 to 20 micra. (3) "Transitional" leukocytes (pi. I, 7; pi. II, 17) differ from the large mononuclears in the shape of the nucleus, which is horseshoe-shaped, lobulated, or deeply indented, and in the constant presence in the protoplasm of numerous fine, dust-like azurophile granules. With stains other than the Eomanowsky, both cells present non-granular cyto- plasm, though exceptionally a few fine, faintly stained granules are discernible after staining with Ehrlich's tri- acid stain. These granules, when evident, are usually stained a reddish or pinkish tint. The large mononuclears and transitionals together form about 3 to 5 per cent, of the leukocytes of normal blood. The diameter is the same as that of the large mononuclears. (4) Polynuclear neutrophilic leukocytes (pi. I, 8; pi. II, 18) are cells with polymorphous nuclei, in whose cytoplasm are numerous fine, neutrophilic granules. These cells are about 9 to 12 micra in diameter and constitute 65 to 70 per cent, of the white cells under normal conditions. The cells are actively ameboid and phagocytic in the fresh blood and are designated microphages, in distinction to the ma- crophages or large mononuclears. (5) Polynuclear eosinophilic cells (pi. I, 9; pi. II, 19) Digitized by Microsoft® THE BLOOD 299 are similar to the last group (4), except for the presence of coarse eosinophilic or acidophilic granules in the proto- plasm. They resemble the neutrophiles in size and in the possession of ameboid activity. Normal blood contains about 2 to 4 per cent, of these cells. (6) Mast cells (pi. I, 10; pi. II, 20) are polynuclear basophilic leukocytes. The nucleus is usually simply in- dented or lobulated. The protoplasm contains basophilic granules, which are somewhat variable in size, the major- ity being about as coarse as the eosinophilic granules. The cells measure about 10 micra in diameter. Normally about 0.5 per cent, of mast cells are found in the blood. Pathological Leukoc3rtes.— In addition to the foregoing cells, which go to make up the leukocytes of normal blood, there appear in disease immature cells, the precursors of the ripe leukocytes of normal blood. (7) Neutrophilic myelocytes -{j)!. I, 11; pi. II, 21) are the antecedents of the polynuclear neutrophilic cells. They differ from the latter in having a round or oval or slightly indented nucleus. The protoplasm contains neutrophilic granules, which are often finer than those of the polynu- clear cells. In the older myelocytes the granules are abun- dant, while very young cells may contain only a few. The nucleus is poorer in chromatin than that of the mature polynuclear cell. The cells are subject to great variation in size. The majority lie between 12 and 20 micra in diam- eter, though larger and smaller cells are encountered now and then. In the nucleus one to four nucleoli may be visible, particularly after staining with methyl green-pyro- nin or with Eomanowsky stains. The nucleus is usually eccentrically situated. (8) Eosinophilic myelocytes (pi. I, 12) resemble neu- trophilic myelocytes in every respect aside from the dif- Digitized by Microsoft® 300 FRESH AND STAINED PREPARATIONS OF BLOOD ference in the granules. Frequently the immature eosino- philic granules exhibit basophilic tendencies, in that they are stained with basic dyes. Thus, in a Eomanowsky prep- aration, some or all of the granules may take a dark blue or purplish tint. (9) "Mast" myelocytes (pi. I, 13) are generally small and present basophilic granules in the protoplasm. (10) Metamyelocyte is a term used to designate cells whose nuclei have passed beyond the kidney shape and already present more or less deep indentations. They are transition stages between the myelocyte and the polynu- clear neutrophilic cells. They are not to be confused with the so-called transitional cells (which are, in reality, mis- branded, as they represent transition forms to no type of cell, so far as is known, though it was originally supposed that they developed into the polynuclear neutrophiles, hence the name "transitional"). They are differentiated from the transitionals by the abundance of neutrophilic granules in their cytoplasm, when stained with Ehrlich's triacid or Jenner's stain. "With Eomanowsky stains, on the other hand, the granules of the metamyelocyte and transitional may be identical in color, but those of the transitional are much finer. (11) Promyelocytes represent the earliest form of my- elocyte, the cell with very few granules in its cytoplasm. The term is superfluous. (12) Myeloblasts (non-granular marrow cells, undiffer- entiated cells of the marrow, lymphoid cells of the marrow, etc.) (pi. I, 14) are the parent cells of the myelocytes. They differ from the latter in the complete lack of cyto- plasmic granules. The nucleus is similar to that of the myelocyte. The protoplasm is basophilic. The cells vary from the size of a lymphocyte to cells 20 micra in diameter. Digitized by Microsoft® THE BLOOD 301 {13) Irritation forms (Tiirk) are cells with round or oval nucleus, like that of the myelocyte, and rather abun- dant protoplasm, which is markedly basophilic and gen- erally vacuolated. (14) Pathological lymphocytes. In disease lymphocytes may depart considerably from the normal. The size is subject to much greater variation; the protoplasm is often greatly reduced in amount and at times is not demon- strable. The nucleus may be convoluted or indented, the so-called Rieder cells. (15) Megakaryocytes, the giant cells of the bone mar- row, are very rare in the blood. The nucleus is greatly convoluted, and the cytoplasm, with Eomanowsky stains, exhibits fine, dust-like granules. The cells are very large in the bone marrow, but only the smaller examples pass the capillaries and appear in the circulating blood. The Normal and Pathological Red Blood Corpuscles Non-nucleated red cells are designated erythrocytes, the nucleated forms erythroblasts. Erythrocjrtes. — {1)N ormocytes (pi. I, 1, 2; pi. II, 1) are normal red blood corpuscles. In the fresh specimen they present the familiar form of biconcave discs, the center being paler, owing to the thinner layer of hemoglobin at this point. The normal cells stain with acid dyes (ortho- chromatic). The average diameter is 7.5 micra. (2) Microcytes are abnormally small erythrocytes. (3) Macrocytes or megalocytes are abnormally large erythrocytes. They may be pale, swollen corpuscles or ab- normally rich in hemoglobin. The diameter may amount to 25 micra. Abnormal variation in size of the corpuscles Digitized by Microsoft® 302 FEESH AND STAINED PREPARATIONS OF BLOOD is designated anisocytosis. Ameboid movements may. be observed in some of the cells.^ (4) Poikilocytes (pi. II, 11) are cells of irregular form. The shape may be practically anything. Erythroblasts. — Nucleated forms of the red blood cor- puscle may be subdivided as follows : (5) Normoblasts (pi. I, 4; pi. II, 7, 7, 9) are nucleated red corpuscles, having the diameter of the average normo- cyte. The nucleus is round, at times eccentric, and usually very dense (pyknotic) and rich in chromatin. Younger forms present nuclei with a visible chromatin network. (6) Microblasts are abnormally small, nucleated red cells. (7) Megalohlasts (pi. I, 3; pi. II, 10) are large erythro- blasts, possessing a large oval or round nucleus. The diameter of the nucleus exceeds that of a normal red cell (Emerson). In the youngest forms the nucleus exhibits a beautiful chromatin network, with markedly basophilic protoplasm. More mature megalohlasts are more nearly orthochromatic and the nucleus is more homogeneous, the network being less conspicuous or lacking. Mitoses may be observed.^ Ameboid activity may be seen in fresh speci- mens.^ (8) Intermediates is a term used to designate all those; nucleated red corpuscles which can be classified neither as normoblasts, megalohlasts, nor microblasts (Emerson). Abnormalities in the Staining of the Red Corpuscles.— (1) Polychromasia or polychromatopMlia (pi. II, 6, 8, 10, "Morris, R. S., and Thayer, W. S. "Amoeboid movements in maerocytes and megalohlasts." Arch. Int. Med., 1911, VIII, 581. ^Dock, G. "Mitosis in circulating blood." Trans. Assoc. Amer. Phys., 1902. 'Thayer, W. S. "The amoeboid activity of megaloblasts. " Arch. Int. Med., 1911, VII, 223. See also Morris, E. 8. and Thayer, W. S. Loc. cit. Digitized by Microsoft® THE BLOOD 303 11) is a condition in which the red corpuscles stain with basic dyes. Normally the red cells possess an afiSnity for acid dyes, such as eosin. Polychromasia varies greatly in degree. With slight grades the color of the acid dye still predominates, though the tint of the basic dye is visible. When polychromasia is marked there is an intense staining of the cell with the basic dye alone. It occurs in both erythrocytes and erythroblasts. (2) Basophilic granulation or stippling (pi. II, 3, 5) is seen in non-nucleated red cells and in nucleated corpuscles, often with intact nuclei. As the name implies, the cell contains granules which are stained only with basic dyes. The granules are always quite numerous in the cell and vary considerably in size. As a rule, the smaller the gran- ules the more numerous they are. With the variation in size there is also irregularity of form. Generally the larger granules are observed in orthochromatic cells, the smaller in polychromatophilic cells, as Askanazy has pointed out. (3) Fragmentation of the Nucleus. — Karyorrhexis (pi. II, 9 ) , the breaking up of the nucleus within the cell, leads to characteristic appearances. The fragmented nucleus may resemble a rosette, or the nucleus may resolve itself into a number of round or oval, often irregular, masses, which are united or separate from one another. (4) Nuclear Particles. — Nuclear particles (pi. II, 4, 5, 8) are derived from the nucleus of the red cell through atrophy of the nucleus or by karyorrhexis; it is possible that there is another mode of formation, since nuclear par- ticles, may be found in megaloblasts with active, intact nu- clei. They were first observed in the blood of the cat by Howell,^ and are known as Howell's bodies or Howell's ' Howell, W. H. " The life-history of the formed elements of the blood, especially the red corpuscles." Jour. Morphol., 1890, IV, 57. Digitized by Microsoft® 304 FRESH AND STAINED PREPARATIONS OF BLOOD nuclear particles. They occur also in human blood.^ They are small, round, sharply defined bodies, usually situated eccentrically in the cell, and generally occur singly, though as many as nine have been observed in a non-nucleated red corpuscle. They resemble miniature pyknotic nuclei mor- phologically. (5) Ring Bodies. — Ring bodies (pi. II, 6, 8) were first described by Cabot,^ and are usually designated Cabot's ring bodies. The ring may remain round or may be twisted so as to form a figure eight, etc. The rings are best seen after staining with Eomanowsky stains, which usually color them red or reddish-violet, rarely blue. It is believed by Cabot, and by all who have since studied these bodies, that they represent the nuclear membrane. (6) "Red Basophilic Granulation with Romanowshy Stains" (pi. II, 8, 11). — Naegeli^ in particular has called attention to the existence of a granulation seen in speci- mens stained with Griemsa's stain; it is demonstrable with all Eomanowsky stains. The granules ditfer from the usual basophilic granules, in that they are stained red or violet instead of blue. The granules were ob- served by Cabot in cells which also contained ring bodies. Naegeli believes that the granules originate from the nucleus, probably from the nuclear membrane, since ring bodies may be made of a series of dots similarly stained. (7) ScMffner's Granules. — Schiiffner's granules are 'Morris, E. S. (a) "Note on the occurrence of Howell's nuclear par- ticles in experimental anemia of the rabbit and in human blood." Johns Hopkins Hosp. Bull, 1907, XVIII, 198. (b) "Nuclear particles in the erythro- cytes. ' ' Arch. Int. Med., 1909, III, 93. ^ Cabot, R. C. "Ring bodies (nuclear remnants?) in anemic blood." Jour. Med. Research, 1903, IX, 15. "Naegeli, 0. " Blutkrankheiten und Blutdiagnostik. " Leipzig, 1912, 2nd Ed., p. 153. Digitized by Microsoft® THE BLOOD 305 found only in certain cases of malaria. They are seen in the infected corpuscles (see below). Demonstration of Protozoa in the Blood The blood protozoa which are of pathological impor- tance are few in number. At the present time the Plas- modia of malaria alone demand general consideration in this country. For protozoa in general, however, such as trypanosomes, Leishman-Donovan bodies, etc., the method of demonstration in the stained specimen, which is uni- versally employed, is one of the numerous modifications of the Romanowsky stain (q. v.). Malarial Parasites. — The plasmodia of malaria are characteristically stained by the Eomanowsky method. The protoplasm of the parasite is stained light blue, con- trasting well with the pink color of the red corpuscle. The nuclear chromatin of the parasite is colored a brilliant red or purplish-red, while the pigment retains its original color, being unstained. In certain of the infections with Plasmodium vivax and Plasmodium falciparum, peculiar granulations appear in the infected red corpuscles (Schiiffner's granules). They have been described by Schiiffner and others. With Eo- manowsky stains, the granules exhibit a dark, reddish tint, often quite like that of the chromatin of the parasite. Schiiffner's granules are not to be confused with the ordi- nary basophilic granules of the red cells or with the red granulations seen in certain corpuscles when stained with Eomanowsky stains. By means of vital staining Boggs ^ has adduced further proof of the non-identity of Schiiff- ^ Boggs, T. E. "Vital staining of 'stipple cells' in malarial blood." Jour. A. M. A., 1911, LVII, 150. Digitized by Microsoft® 306 FRESH AND STAINED PREPARATIONS OF BLOOD ner's with other granules. The granules may be missed in cells containing the youngest hyalin parasites, and usu- ally seem to increase in number with the age of the para- site. Schiiffner's granules have not been observed in cells infected with the parasite of quartan fever. Blood platelets have been mistaken for hyalin forms of the Plasmodia by inexperienced observers. This is apt to occur only when the platelet rests upon the red corpuscle. Differentiation is simple. The chromatin of the platelet is usually colored purple with less of the reddish tint than the chromatin of the parasite shows, but this difference may be lacking, for often the chromatin of the parasite is stained exactly the shade of that of the platelet. The im- portant differential point is found in the arrangement of the chromatin. In the platelet the chromatin is scattered in minute granules, while the chromatin of the hyalin para- site is in a compact mass, or, in the case of Plasmodium falciparum, in two or three masses, but still dense and compact. The body of the platelet is often unstained, but may take a pale blue color, very much like that of the proto- plasm of the parasite. Giant blood platelets have been mis- taken for extracellular forms of the malarial parasite. The constant presence of pigment granules in the parasite should be sufficient to differentiate, even though the dis- tribution of the chromatin in this instance be somewhat similar in the two — which is generally not the case. Staining Method of Ross.^ — Ross has devised a method for detecting the parasites which is useful when their num- ber in the blood is small. He prepares a thick smear of the blood and, before staining the film, extracts the greater part of the hemoglobin from the cells. A drop of blood of 'Eoss, E. "An improved method for the mieroseopical diagnosis of in- termittent fever." Lancet, 1903, I, 86. Digitized by Microsoft® THE BLOOD 307 about 20 c. mm. is placed on a cover glass {% in. square) and spread in the usual manner, or with a needle or lancet. It is dried in the air. The preparation contains about twenty times the amount of blood usually found in a smear. After becoming dry it is covered with 1 per cent, eosin so- lution (Eomanowsky's). The stain is placed on the speci- men with a glass rod and is allowed to act for as much as fifteen minutes. It is then washed. The washing must be done with a very gentle stream, since the unfixed blood is easily loosened. Then stain with Eomanowsky's methylene blue ^ a few seconds and again wash carefully. The speci- men is dried in the air and mounted in balsam. The hemo- globin is extracted from the red corpuscles, leaving only their stromata, together with leukocytes, platelets, and Plasmodia. The staining of the parasite is distinctive — protoplasm light blue and nuclear chromatin red. Hyalins are readily detected. For finding* larger, pigmented parasites Eoss prepares the film as described above; after it has dried, it is cov- ered with distilled water to extract the hemoglobin. The unstained specimen is then examined. Crescents and other pigmented forms stand out prominently. EuGE 's Modification ^ of the Method of Eoss. — A dis- advantage in the method of Eoss is the difficulty of wash- ing the unfixed specimen without losing the preparation. To overcome this, Euge fixes the thick films in 2 per cent, formalin containing % to 1 per cent, acetic acid. The hemoglobin is extracted from the erythrocytes, which are fixed to the cover glass at the same time. The specimen ' Nocht 's solution of methylene blue for the Eomanowsky stain consists of methylene blue (rectif. puriss. Hoechst) 1.0 gm., sodium carbonate 0.5 gm., dissolved in 100 c. c. of distilled water. ^Euge, E. "Zur Erleichterung der mikroskopisehen Malariadiagnose. " Deutsche med. Wchnschr., 1903, XXIX, 205. Digitized by Microsoft® 308 EXAMINATION OF BLOOD FOE ANIMAL PARASITES is then stained with EomanowsTcy 's stain. The formalin fixation interferes somewhat with the staining of the pro- toplasm of the Plasmodia, so that it may be necessary to restain the film with methylene blue. A certain amount of precipitate remains in the specimen, but the parasites are well stained. Examination of the fresh blood, whenever pos- sible, is the most satisfactory method of diagnosis of malaria, as the variety of the parasite is more easily recog- nized, as a rule, than in stained smears. The accompany- ing table gives the main differences between the three spe- cies of Plasmodia in fresh blood ; it may be used also, with certain obvious exceptions, in connection with stained smears. Degenerations in the red cells may be mistaken for hy- alin parasites. In form the degenerations may bear a strik- ing resemblance to ring forms or irregularly shaped para- sites. Ameboid activity is lacking, and it may be noted that the degenerations become more numerous in the speci- men as time advances. The larger round or oval degenera- tions are less confusing; their size appears to change on raising and lowering the focus. Examination of the Blood for Animal Parasites (1) Filaria bancrofti (Fig. 10), when present in the blood, is usually demonstrable by the ordinary method of examination of the fresh blood. The size of the drop should be a little larger than usual, in order to secure a moderately thick preparation. The blood should be taken during the sleeping hours — usually at night, when the embryos are Digitized by Microsoft® --. § PI >^ it • p. a ^1 bo ^2 s S ^ -S ^5 8 60 S ^ e K , 73 a, -3 S en o 1^ a a'O • s S'S a -4J ^ a ro J3 (2.^ .ft Irregu oval freq cS a Coarse erall Very s Ph 02 m w HmO t> to M g S H a w li §= s .as i |~T SSfl Bintn_, s o III I ° ll I § i ° i ^i I I "llia-g I ^ ^"M ■« « bS 5f.Q S=H s ^ S "^^ S^ ^ -S S,^ ^ i ^ -^ So .13 So »i 's a -§ g "^Is H o Ph U HoQpR 02 CO P O _ts ^ a . -a a S ■ "S S § Ol ^*' SW fli ij y a ^laap-g'Sa e i'S'^'^Sa" S a fl(»T:)<4H> © ^5Maafl^^l>. o a PJ .t .■ m a s . .5 .• -t! ^ B. ^ " ir .^.S o ^ 5^ ::::; a D4 ^ boo =3 t, o, l^d,Q O'C^ ri^of^ hookworm, 180-186 hydrobilirubin (urobilin), 153, 158 Hymenolepis diminuta, 198 , Hymenolepis nana, 197 iron, 153 Lamblia intestinalis, 17S leukohydrobilirubin, 154 methylene blue, 153 mucus, 154 muscle fibers, 167 Necator americanus, 180 adult parasites of, 184 embryo of, 181, 187 ova of, 181 special methods of finding ova of, 182-184 special methods of finding parasites of; 184 Opisthorchis sinensis, 192 Oppler-Boas bacilli, 169 Oxyuris vermicularis, 188 Paragonimus westermanii, 194 Paramecium coli (Balantidium coli), 179 Digitized by Microsoft® 330 INDEX Feces : parasites, intestinal, 172-203 preservation of feces, 199 preservation of parasites and ova in feces, 199, 200 pus, 154, 170 reaction, 157 sarcines, 169 Schistosoma hsematobium, 192 Schistosoma japonicum, 193 scybali, 153 starch granules, 168 Strongyloides stereoralis, 186 ova of, 187 rhabditiform embryo of, 186 differentiation of, from hookwbrm embryo, 187 tapeworms (eestodes), 194 Tenia saginata, 194 Tenia solium, 196 test diets, intestinal, 155-157 Toxoeara canis, 191 Triehinella spiralis, 191 Triehoeephalus dispar (Triehuris trichiura), 189 Trichomonas intestinalis, 177 Triehuris trichiura, 189 triple phosphate, 170 tubercle bacilli, 168 Tyroglyphus sire, 203 Uneinaria americana (Necator americanus), 180 urobilin, 153, 158 Schlesinger's test for, 158 Schmidt's test for, 158 spectroscopic determination of, 158 vegetable cells, 167 vegetable hairs, 168 vegetable spirals, 168 weight of dried feces, 157 yeasts, 169 Fehling's test for sugar, 39 Fermentation test for sugar, quali- tative, 41 quantitative, 50-53 Ferments in feces, 162-166 in gastric contents, 143-148 in urine, 86-89 Fibrous connective tissue in feces, 167 Filaria bancrofti in blood, 308 in urine, 117 Films, blood, preparation of, 268 Fischer's test meal, 126 Fixation of blood films, 271 Fleischl-Miescher hemoglobino- meter, 249 Folin's method for acidity of urine, 5 for ammonia, 13 Folin-Shaffer's method for uric acid, 10 Food remnants in feces, 167, 152 in gastric contents, 129, 149 ' Formalin preservation of feces, 199 of urine, 3 Formalin titration of ammonia, 16 Functional diagnosis of the kidneys, 119 of pancreas, 162-166 Futcher-Lazear fixation, 273 G Gall-stones in stools, 155 Garrod's test for hematoporphyrin, 76 Gastric contents: acetic acid, 143 acidity : free acid, 136 total acid, 138 Digitized by Microsoft® INDEX 331 Gastric contents: Balaiitidium coli, 151 blood, chemical tests for, 159 microscopic, 150 butyric acid, 142 Cercomonas hominis, 151 color, 129 crystals, 151 eosinophile cells, 150 fasting stomach, examination of, 127 fat droplets, 150 ferments : pepsin, 143 peptid-splitting ferment, 146 rennin, 145 food, 129 glyeyltryptophan test, 147 hydrochloric acid, 131-138, 139 deficit of, 139 qualitative tests for: Congo paper test, 134 Giinzberg's test, 132 methyl violet test, 131 Sahli's desmoid test, 134 Toepfer's test, 134 tropeolin test, 133 quantitative determination of, 136 lactic acid, 140 Kelling's test for, 142 Strauss' test for, 141 Ueffelmann's test for, 141 Lamblia intestinalis, 151 mucus, 128, 148 odor, 128 Oppler-Boas bacilli, 151 pus, 150 quantity of, after test meals, 128 reaction, 131 sarcines, 151 starch, 149 Gastric contents: test breakfasts and meals, 125- 127 Boas', 125 Dock's, 125 Ewald's, 125 Fischer's, 126 Eiegel's, 126 total acidity, 138 Trichomonas intestinalis, 151 yeasts, 150 Gentian violet, aniUn-water, 218 Gerhardt's test for diacetic acid, 65 Giemsa's stain for blood, 290 for Treponema pallidum, 113 Glassware, cleaning of, 265 Globulin in cerebrospinal fluid, 320 Noguchi's test for, 320 Koss-Jones' test for, 321 Glucose in urine, 38-53 in chyluria, 85 qualitative tests for, 38-44 quantitative determination of, 44- 53 Glycerin jelly, 203 Glycuronic acid in urine, 59 Glyeyltryptophan test, 147 Gmelin's test for bile, 74 Gonoeoecus in pus, 110 Gram's iodin solution, 218 Gram's stain, 218 Gross' method of determining tryp- sin, 162 Guiac test for blood in feces, 159 in gastric contents, 159 in urine, 81 Gunning's test for acetone, 62 Giinzberg's reagent, 132 H Hammarsten's test for bilirubin, 75 Hart's test for oxybutyric acid, 68 Digitized by Microsoft® 332 INDEX Harvey's modifleation of Volhard's method, 24 Haser's coefficient, 7 Hawk's modification of Wohlge- muth's method, 163 Hayem's solution, 230 Heart failure cells in sputum, 210 in urine, 101 Heller's test for albumin, 32 for blood, 82 Hematin, spectroscopic determina- tion of, 78 Hematoidin (bilirubin) crystals in sputum, 212 in urine, 94 Hematokrit, 253 Hematoporphyrin in urine, 76 Hematoxylin, Ehrlich's acid, 277 Hematuria, 80, 105 Hemin crystal test of Teichmann for blood, 82 Hemochromogen, 79 Hemocytometer, Thoma's, 227 Biirker's modification of, 239 Hemoglobin, estimation of, 244-252 in urine, tests for, 77, 81 spectroscopic determination of, 78 Hemoglobin easts, 108 Hess' viseosimeter, 256 Hewlett's method of determining lipase, '88 Hinman and Sladen's modification of Milian's method, 263 Hippuric acid in urine, 93 Hofmann's test for tryosin, 96 Hookworm, 180-186: Ankylostoma duodenale, 184 Necator americanus, 180 Howell's bodies, 303 Hiifner's method of estimating urea, 8 Huppert's test for bilirubin, 75 Hydrobilirubin (urobilin) in feces, 153, 158 Hydrochloric acid in gastric con- tents, 131 deficit of, 139 qualitative tests for, 131-135 quantitative determination of, 136 Hymenolepis diminuta, 198 Hymenolepis nana, 197 Hypobromite method of estimating urea, 8 Incoagulable nitrogen, in puncture fluids, 313 India ink method for spirochetes, 115 Indican in urine, JafEe's test for, 27 Obermayer's test, 27 Indicators : alizarin red, 13, 15 cochineal tincture, 23 Congo red, 134 dimethylamidoazobenzole (Toep- fer's reagent), 134 Giinzberg's reagent, 132 phenolphthalein, 138 Indoxyl sulphate {See Indican), 27 Influenza bacilli, 219 Intermediate nucleated red cells, 302 lodin reaction of leukocytes, 295 Iron in stools, 153 Irritation forms of Tiirk, 301 Jaffe's test for indican, 27 for urobilin, 72 Jenner's stain, 291 Digitized by Microsoft® INDEX 333 Karyorrliexis, 303 Kieselguhr, 28 Kjeldahl's method of determining total nitrogen, 20 Labeling blood films, 271 Lactic acid in gastric contents, 140 Lactose in urine, 56 Lamblia intestinalis in feces, 178 in gastric contents, 151 Lange's test for acetone, 64 Large mononuclear leukocytes, 298 azurophilic granules in, 287, 298 characteristics of, 298 staining reactions of, with Jen- ner's stain, 293, 294 with, triacid stain, 283, 294 with Wilson's stain, 287, 294 Lecithin in chyluria, 85 in prostatic fluid, 119 Legal's test for acetone, 64 Leishman's stain, 289 Leucin in urine, 96 Leukoejrtes. See also Blood classification of normal, 297 classification of pathological, 299 counting, 236 counting eosinophilic, 241 differential count of, 296 iodin reaction of, 295 measuring, 255 staining reactions of, with Jen- ner's stain, 293, 294 with triacid stain, 282, 294 with Wilson's stain, 287, 294 vital staining of, 273 Leukohydrobilirubin in feces, 154 Levulose in urine, 53 phenylhydrazin test for, 55 Levulose in urine, quantitaitive de- termination of, 46 Seliwanoff's test for, 53 Levulosuria, alimentary, 56 Lieben's test for acetone, 63 Lipase in urine, 88 Lipuria, 86 LoefBer's antiformin method, 215 Loeffler's methylene blue, 213 Lohnstein's areometer, 51 Lohnstein's fermentation saccha- rometer, 52 Lugol's solution, 63 Lymphocytes, 297 azurophilic granules in, 287, 297 characteristics of, 297 in cerebrospinal fluid, 318 in chyluria, 86 in puncture fluids, 316 in sputum, 211 pathological, 301 staining reactions of, with Jen- ner's stain, 293, 294 with methyl green-pjrronin mixture, 295 with triacid stain, 283, 294 with Wilson's stain, 287, 294 M Maerocyte, 301 Macrophage (large mononuclear leukocyte), 298 Magnesium phosphate in urine, 97, 98, 99 Malarial parasites, 305 differential table of, 309 in fresh blood, 308 Schiiffner's granules, 305 stained by earbol-thionin, 279 by Romanowsky stains, 283, 285, 290 Digitized by Microsoft® 334 INDEX Malarial parasites, stained by car- bol-thionin, by Ross' method, 306 by Ruga's modification of the method of Ross, 307 Maltose in urine, 56 Mast cells: characteristics of, 299 staining reactions of, with carbol- thionin, 279 with Jenner's stain, 293, 294 with methylene blue, 276 with triacid stain, 282, 294 with Wilson's stain, 287, 294 Mast myelocyte, 300 Measurement of microscopic ob- jects, 255 Megakaryocyte, 301 Megaloblast, 302 Megalocyte, 301 Metamyelocyte, 300 Methemoglobin in blood, 252 in urine, 78 Methyl green-pyronin stain, 295 Methyl violet test for hydrochloric acid, 131 Methylene blue in feces, 153 in urine (following the desmoid test), 135 Loeffler's, 213 Mette's method for determination of pepsin, 144 Microblast, 302 Microcyte, 301 Micrometer, ocular, 255 Microphage (polynuclear neutro- philic leukocyte), 298 Miescher-rieischl hemoglobinom- eter, 249 Milian's method of determining co- agulation time, 263 Millon's reagent, 96 Mink and Ebeling's method of clearing parasites, 200 Monoealeium phosphate in urine, 93 Mucous threads in urine, 109 Mucus in feces, 154 in gastric contents, 128, 148 Murexid test, 9 Muscle fibers in feces, 167 Myelin degeneration of epithelial cells in sputum, 210 in urine, 101 Myeloblast, 300 Myelocyte, eosinophilic, 299 mast (basophilic), 300 neutrophilic, 299 staining reactions, 294 N Necator americanus, 180 adult worms of, 184 embryo of, 181, 187 ova of, 181 special methods of finding ova of, 182 of finding parasites of, 184 Neisser's stain, 220 Nematodes, 180 Nitrogen, incoagulable, 313 total, 20 Noguchi's globulin test, 320 Normoblast, 302 Normocyte, 301 Nubecula in urine, 3 Nuclear particles, 303 Ny lander's test for sugar, 40 Obermayer's test for indican, 27 Opisthorchis sinensis, 192 Oppler-Boas bacilli in feces, 169 in gastric contents, 151 Digitized by Microsoft® INDEX 335 Ova. See the various parasites special methods of detecting, 182 : centrifugalization, 183 Pepper's .method, 182 Stiles' method, 182 Oxybutyric acid (/J), Black's test for, 67 Hart's test, 68 Oxyhemoglobin in urine, 78 Pappenheim's stain, 295 Paragonimus westermanii in feces, 194 in sputum, 224 Paramecium eoli (Balantidium coli), 179 Parasites in blood, 308 in feces, 172-199 in sputum, 224 in urine, 116 Paterson's antiformin method, 216 Pentose in urine, 57 Bial's orcin test for, 58 orcin test for, 58 phloroglucin test for, 57 Pepper's method of detecting hook- worm ova, 182 Pepsin in gastric contents, 143 Peptid-splitting enzyme in gastric contents, 146 Pericardial fluids, 312 Peritoneal fluids, 312 Phenolphthalein, 138 Phenolphthalin, 82 Phenolsulphonephthalein test, 120 Phthalein test, 120 Pinworm (Oxyuris vermicularis), 188 Piria's test for tyrosin, 95 Platelets, blood, counting, 242 Platelets, blood, staining reactions of, with Wilson's stain ( Roman owsky), 287, 294 Pleural fluids, 312 Pneumococcus in sputum, 217 Poikilocyte, 302 Polaiiscopic determination of sug- ars, 48 Polyehromasia, 302 Polychromatophilia, 302 Polynuclear eosinophils, character- istics of, 298 staining reactions of, with eosin, 277 with Jenner's stain, 293, 294 with triacid stain, 282, 294 with Wilson's stain, 287, 294 Polynuclear neutrophils, charac- teristics of, 298 staining reactions of, with Jen- ner's stain, 293, 294 with triacid stain, 282, 294 with Wilson's stain, 287, 294 Preservation of animal parasites, 199, 200 of feces, 199 of urine, 2 Promyelocyte, 300 Prostatic fluid, 119 corpora amylacea in, 119 lecithin in, 119 spermatozoa in, 119 spermin crystals in, 119 Protein preeipitable by acetic acid in cold, 315 Pseudoeast, 109 Puncture fluids (pleura), peritoneal, pericardial), 312 albumin content of, 312 Kjeldahl determination of, 313 Tsuchiya's reagent for, 312 cells in, 315 Digitized by Microsoft® 336 INDEX Puncture fluids, incoagulable nitro- gen in, 313 protein in, precepitable by acetic acid in cold, 315 serosamucin in, 315 specific gravity of, 312 tubercle bacilli in, 317 Pus in feces, 154, 170 in gastric contents, 150 in sputum, 211 in urine, 102 counting cells of, 103 false albuminuria due to, 104 guiac tests for, 104 Q Quadriui-ates of sodium and potas- sium, 91 Quantity of feces, dried, 157 in twenty-four hours, 153 of gastric contents after test meals, 128 from fasting stomach, 127 of urine in twenty-four hours, 4 E Reaction of feces, 157 of gastric contents, 131 of sputum, 205 of urine, 4 Red basophilic granulation, 304 Red blood cells. See Erythrocytes and Blood Reduced hematin. See Hemochro- . mogen Red"uced hemoglobin, 78 Removal of albumin from lirine, 36 Resistance of erythrocytes, 264 Rice's bromin solution, 8 Rieder cells, 301 Riegel's dinner, 126 Ring bodies, 304 Robert's specific gravity fermenta- tion method, 50 Romanowsky stains for blood, 285, i 289, 290 for Treponema pallidum, 112 Ronchese, formalin titration of am- monia, 17 Rosenbaeh's test for bile, 74 Ross' method of detecting malarial parasites, 306 Roundworms (nematodes), 180 Rowntree and Geraghty's phthalein test, 120 Ruge's modification of Ross' method, 307 S Saccharose in urine, 57 Sahli's desmoid test, 134 Sahli's hemometer, 245 Sarcines in feces, 169 in gastric contents, 151 Schistosoma haematobium in feces, 192 in urine, 118 Schistosoma japonicum, 193 Schlesinger's test for urobilin in feces, 158 in urine, 71 Schlosing's method of determining ammonia, 19 Schmidt and Strasburger's test diets, 156 Schmidt's test for bilirubin, 158 for urobilin, 158 Sehiiffner's granules, 305 Schumm's method of spectroscopic examination, 80 Scybali, 153 Digitized by Microsoft® INDEX 337 Seatworm (Oxyuris vermicnlaris), 188 Sediments in urine, organized, 100 unorganized, 91 Seliwanoff's test for levnlose, 53 Serosamucin, 315 Shaffer's vacuum distillation of ammonia, 15 Silver impregnation method for spirochetes, 114 Simon's method of staining amebse, 174 Smears, blood, 268 Smegma bacillus in urine, 115. See under Tubercle bacillus Specific gravity of blood, 259 of blood plasma, 260 of blood serum, 260 of puncture fluids, 312 of urine, 6 Spectra for glycuronic acid com- pounds (Tollens' test), 60 for hematin, acid, 78 for hematoporphyrin, 76 for hemochromogen, 79 for hemoglobin, reduced, 78 for methemoglobin, 78 for oxyhemoglobin, 78 for pentose (orcin test), 58 for urobilin, 71 Spermatozoa in urine, 119 Spermin crystals, 119 Sputum : Actinomyces bovis, 221 alveolar epithelial cells, 209 amount, 205 antiformin method for detecting tubercle bacilli, 214-217 Boardman's, 217 Loeffler's, 215 Paterson's, 216 blastomycetes, 223 Sputum : blood, 210 bronchial casts, 206 bubbles in, 205 Cereomonas hominis, 224 character, 205 Charcot-Leyden crystals, 211 consistence, 205 Curschmann's spirals, 206, 208 diphtheria bacilli, 219 Beall's method of staining, 221 Neisser's stain for, 220 Diplocoecus pneumoniae, 217 Dittrieh's plugs, 205 dust cells, 209, 210 echinococcus, 225 elastic tissue, yellow, 208 Entameba histolytica, 224 Entameba tetragena, 224 eosinophile cells, 211 fat droplets, 209 fresh sputum, plate method of examination, 207 heart failure cells, 210 influenza bacilli, 219 layer formation, 206 lymphocytes, 211 myelin degeneration of alveolar cells, 210 obtaining sputum, 204 odor, 205 Paragonimus westermanii, 224 plate method of examination, 207 pneumocoecus, 217 Welch's capsule stain for, 219 pus, 211 reaction, 205 streptothrix, 222 Trichomonas intestinalis, 224 tubercle bacilli, 212 antiformin methods for, 214-217 Ziehl-Neelsen stain for, 213 Digitized by Microsoft® 338 INDEX Stains : anilin-water gentian violet, 218 Beall's method of staining diph- theria bacilli, 221 Bismarck brown (vesuvin), 111 Brem's method for staining amebae, 175 for old blood films, 290 capsule stain, Welch's, 219 earbol-fuchsin, 213 carbol-thionin, 279 Ehrlich's hematoxylin, acid, 277 Ehrlich's triacid stain, 280, 294 eosin, 277 gentian violet, anilin water, 218 Giemsa's stain for blood,- 290 for old smears, 290 for spirochetes, 113 Gram's stain, 218 hematoxylin, Ehrlich's acid, 277 Jenner's stain, 291, 294 Leishman's stain, 289 Loeffler's methylene blue, 213 methyl green-pyronin, 295 methylene blue, Loeffler's, 213 neutral red, 174, 276 Neisser's stain, 220 Pappenheim's methyl green-py- ronin, 295 polychrome methylene blue, Unna'sj 273 Koma:nowsky stains for blood, 283, 285, 289, 290, 294 for old smears, 290 for spirochetes, 112 Scharlach R, 171 Simon's method of staining amebaB, 174 Sudan III, 171 triacid stain of Ehrlieh, 280 vesuvin (Bismarck brown). 111 Welch's capsule stain, 219 Stains : Wilson's stain, 285 Ziehl-Neelsen stain, 213 Starch granules in feces, 168 in gastric contents, 149 Staubli's method of detecting para- sites, 310 Stem's silver iinpregnation method, 114 Stiles' method of detecting hook- worm ova, 182 Stippling of red cells, 303 Stomach contents. See Gastric con- tents Stools. See Feces Strongyloides stercoralis, 186 embryo of, rhabditiform, 186 ova of, 187 Sulphates in urine (indican), 26 Sulph-hemoglobinemia, 252 Tallqvist's color scale, 244 Tapeworms (cestodes), 194 Teichmann's hemin crystal test, 82 Tenia saginata, 194 Tenia solium, 196 Test breakfasts and meals, 125 B6as', 125 Dock's, 125 Ewald's, 125 Fischer's, 126 Riegel's, 126 Schmidt and Strasburger's, 156 Test diet, intestinal, 155 : diet No. I, 156 diet No. II, 156 Thymol as preservative of urine, 3 Toepfer's test for hydrochloric acid, 134 Toisson's solution, 230 Toluol (toluene) as preservative of urine, 2 Digitized by Microsoft® INDEX 339 Toxocara canis, 191 Transitional leukocytes, 298 azurophilic granules in, 298 characteristics of, 298 staining reactions of, with Jen- ner's stain, 293 with triacid stain, 283 with Wilson's stain, 287 Trematodes, 192 Triacid stain of Ehrlich, 280 Triealcium phosphate in urine, 97 Triehinella spiralis in blood, 310 Staubli's method of detecting, 310 in feces, 191 Trichocephalus dispar (Trichuris trichiura), 189 Trichomonas intestinalis in feces, 177 in gastric contents, 151 in sputum, 224 Trichomonas vaginalis in urine, 116 Trichuris trichiura, 189 Trimagnesium phosphate in urine, 97 Triple phosphate in feces, 170 in gastric contents, 151 in sputum, 212 in urine, 98 Tripperfaden in urine, 109 Trammer's test for sugar, 38 Tropeolin test for hydrochloric acid, 133 Trypsin in feces, 162 Tsuchiya's method for albumin in puncture fluids, 312 in urine, 35 Tubercle bacillus in cerebrospinal fluid, 319 in feces, 168 in puncture fluids, 317 in sputum, 212 Tubercle bacillus in urine, 115 Ziehl-Neelsen stain for, 213 Tumor cells in puncture fluids, 316 Turk's irritation forms, 301 Tyroglyphus sire, 203 Tyrosin in urine, 94 Deniges' test for, 95 Hofmann's test for, 96 Piria's test for, 95 U Uneinaria americana (Necator americanus), 180 Urea, 7 Uric acid in urine, 9 crystals of, 92 microchemical reactions of, 92, 99 murexid test for, 9 quantitative determination of, 10. Urine: acetone, 62 Gunning's test for, 62 Lange's test for, 64 Legal's test for, 64 Lieben's test for, 63 acidity, 4 Tolin's method of determina- 1 tion of, 5 albumin, 28 heat and acetic acid test for, 28 heat and nitric acid test for, 31 Heller's test for, 32 potassium ferrocyanid and acetic acid test for, 34 quantitative determination of, 35 removal of, 36 albumose, 37 alkaptonuria, 61 ammonia, 12 Digitized by Microsoft® 340 INDEX Urine: ammonia, Polin's method of de- termination of, 13 formalin titration methods of determination of, 16 Schlosing's method of deter- mination of, 19 Shaffer's method of determina- tion of, 15 ammonio-magnesium phosphate, 98 ammonium biurate, 98 bacteria : gonoeoeeus, 110 smegma bacillus, 115. See under Tubercle bacillus tubercle bacillus, 115 Bence-Jones' body, 36 bile pigments (bilirubin), 73 bilirubin (hematoidin), 73 crystals of, 73, 94 foam test for, 74 Gmelin's test for, 74 Hammarsten's test for, 75 Huppert's test for, 75 Rosenbach's test for, 74 staining of sediment by, 74, 101 blood (hematuria), 80 cells of, in sediment, 105 guiac test for, 81 Heller's test for, 82 hemin crystal test for, 82 calcium carbonate, 97 calcium oxalate, 92 calcium phosphate, 93, 97, 99 calcium sulphate, 93 casts, 105 amyloid, 108 blood, 106 coarsely granular, 107 colloid, 108 epithelial, 106 Urine : casts, fatty, 107 finely granular, 107 hemoglobin, 108 hyalin, 108 pus, 106 ■waxy, 108 chlorids, 23 cholesterin, 94, 85 chyluria, 84 clap threads, 109 clearing, Kieselguhr in, 28 lead acetate in, 48 collection of urine, 1 color, 4 corpora amylacea, 119 cylinders. See Casts cylindroids, 109 cystin, 96 dextrose {See Glucose), 38 diacetic acid, 65 diazo reaction, 83 Dioctophyme renale, 117 Ehrlich's aldehyde test, 70 Ehrlich's diazo test, 83 epithelial cells, 100 Eustrangylus gigas (Diocto- phyme renale), 117 rilaria bancrofti, 117 functional diagnosis, phthalein test, 119, 120 glucose, 38 qualitative tests for: Almen-Nylander's test, 40 Fehling's test, 39 fermentation test, 41 phenylhydrazin test, 42 Trommer's test, 38 quantitative determination of: Benedict's first method, 44 Benedict's second method, 46 fermentation, gas, 51 Digitized by Microsoft® INDEX 341 Urine: gluoose, quantitative determina- tion of: fermentation, specific grav- ity, 50 polariscopic, 48 glycuronic acid, 59 ToUenB' test for, 60 gonococcus, 110 grape sugar {See Glucose), 38 Haser's coefficient, 7 heart failure cells, 101 hematin, acid, 78 reduced (see hemochromogen), 79 hematoporphyrin, 76 hemochromogen, 79 hemoglobin, 77 guiac test for, 81 Heller's test for, 82 spectroscopic determination of, 78 Teiehmann's hemin crystal test for, 82 hippuric acid, 93 indican, 26 Jaffe's test for, 27 Obermayer's test for, 27 indoxyl sulphate (See Indican), 26 lactose, 56 lecithin in chyluria, 85 in prostatic fluid, 119 leuein, 96 levulose, 53 phenylhydrazin test for, 55 quantitative determination of," 46 SeliwanofE's test for, 53 magnesium phosphate, neutral, 99 maltose, 56 methemoglobin, 78 Urine: mierochemical reactions of crys- tals, 99 microscopic examination, 89 specimens for, 1, 89 monocalcium phosphate, 93 mucous threads, 109 nitrogen, total 20 nubecula, 3 obtaining sediments, 89 oxybutyric acid, |8-, 67 Black's test for, 67 Hart's test for, 68 oxyhemoglobin, 78 parasites, animal, 116 : Dioctophjmae renale, 117 rilaria banerofti, 117 Schistosoma haematobium, 118 Trichomonas vaginalis, 116 pentose, 57 orcin test for, 58 modified by Bial, 58 phloroglucin test for, 57 phenolsulphonephthalein test, 120. See also Phthalein test phthalein test, 120 preservation of urine, 2 prostatic fluid, 119 pseudocasts. 109 pus, 102 counting cells of, 103 false albuminuria from, 104 guiac test for, 104 Meyer's modification, 104 microscopic, 102 quadriurates of sodium and po- tassium, 91 quantity for twenty-four hours, 4 reaction, 4 saccharose, 57 Schistosoma hsBmatobium, 118 sediments, 89 Digitized by Microsoft® 342 INDEX Urine: sediments : ammonio-magnesium p h o s - phate, 98 ammonium biurate, 98 bilirubin, 94 blood cells, 105 calcium carbonate, 97 calcium oxalate, 92 calcium phosphate, mono-, 93 neutral, 99 calcium sulphate, 93 casts, 105 cholesterin, 94 clap threads, 109 cylindroids, 109 ! cystin, 96 epithelial cells, 100 heart failure cells, 101 ' hematoidin, 94 hippurie acid, 93 leucin, 96 magnesium phosphate, neutral, 99 monoealcium phosphate, 93 mucous threads, 109 obtaining sediments, 89 pseudocasts, 109 pus, 102 quadriurates of sodium and po- tsissium, 91 ' tricaleium phosphate, 97 ■ trimagnesium phosphate, ,97 triple phosphate, 98 Tripperfaden, 109 ' tyrosin, 94 i uric acid, 92 xanthin, 94 ', smegma bacillus, 115. See under Tubercle bacillus specific gravity, 6 spermatozoa, 119 Urine: spermin crystals, 119 Spirochaeta pallida, 111 Spiroehffita refringens, 112 sulphates. See Indican Treponema pallidum. 111 tricaleium phosphate, 97 trimagnesium phosphate, 97 triple phosphate, 98 Tripperfaden, 109 tubercle bacillus, 115 tyrosin, 94 urea, 7 uric acid, 9 crystals of, 92 murexid test for, 9 quantitative determination of, 10 urobilin, 70 Jaffe's test for, 72 Schlesinger's test for, 71 spectroscopic determination of, 71 urobilinogen, 70 xanthin, 94 Urobilin in feces, 153, 158 in urine, 70 Urobilinogen in urine, 70 Vacuum distillation of ammonia, 15 Vegetable cells in feces, 167 Vegetable hairs in feces, 168 Vegetable spirals in feces, 168 Vesuvin (Bismarck brown). 111 Viscosimeter of Hess, 256 Viscosity of blood, 256 Volhard's method for determination of chlorids, 24 Volume index of red cells, 253 von den Velden's test for hydro- chloric acid, 131 Digitized by Microsoft® INDEX 343 W Wassermann reaction in cerebro- spinal fluid, 321 Weber's test for blood, 159 Weidel's test for xanthin, 94 Welch's capsule stain, 219 Whipworm (Trichuris trichiura), 189 Wilson's stain, 284 Wohlgemuth's method for diastase in feces, 163 in urine, 87 Wright and Kinnicutt's method of counting platelets, 242 Xanthin in urine, 94 Yeasts in feces, 169 in gastric contents, 150 Yellow elastic tissue, 208 Zenker's fluid, 200 Ziehl-Neelsen stain for tubercle bacilli, 213 (1) Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® UJ c/^ O S ^ >- -=i^ ?^ o^ S . , O -£ S oo a. 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