BOUGHT WITH THE INCOME FROM THE SAGE ENDOWMENT FUND THE GIFT OF Hem's W. Sase 189X .4^/AkA.n/f /..Sr/.S/./..fM.3. 5474 Cornell University Library RB 37.S59 1902 A manual of clinical diagnosis by means o 1QO/1 ni2 462 747 The original of tiiis book is in tine Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/details/cu31924012462747 A MANUAL CLINICAL PIAGNOSIS BY MEANS OF MICROSCOPICAL AND CHEMICAL METHODS, FOR STUDENTS, HOSPITAL PHYSICIANS, AND PRACTITIONERS. BY CHAELES E. SIMON, M.D., Of Baltimore, Md. FOURTH EDITION, THOBOUOIILY REVISED. ILLUSTRATED WITH 139 ENGRAVINGS AND 19 PLATES IN COLORS. LEA BROTHERS & CO., PHILADELPHIA AND NEW YORK. 1902. Entered according to Act of Congress, in the year 1902, by LEA BROTHERS & CO., In tlie Ofiloe of the Librarian of Congress, at Washington. All rights reserved. TO HIS WIFE, WHO HAS SO FAITHFULLY AIDED IN ITS PEEPAEATION, THIS VOLUME IS ArSECTIONATELY DEDICATED BY THE AUTHOR. PREFACE TO THE FOURTH EDITION. WiTHiiir the five years spanned by the four editions of this book the practical importance of laboratory methods in clinical diagnosis has come to be widely appreciated. What share this work may have had in effecting such an advance is not for the author to. say, but he construes the growing demand for it as evidence that he has measur- ably succeeded in his effort to provide a plain and straightforward guide to those modern methods which at once facilitate and simplify the only certain path to success in practice, namely, accurate diag- nosis. With such methods at his command the obligation becomes binding, both legally and morally, to use them. Research, while constantly extending this field of knowledge, is also simplifying it, so that laboratory equipment is now not only a necessary but also a practicable part of every office. The physician can readily acquire a working grasp of precise diagnosis, and the student is finding it included in the greatly extended curriculum of a rapidly increasing number of colleges. To the needs of graduates and undergraduates alike this book is addressed. It endeavors to state the best methods clearly and simply, with all necessary instructions. Every effort has been made to render the book as modern and practical as possible. In preparing a new edition I have taken special pains to keep the volume thoroughly up to date, and have accordingly added much new matter which appeared to be of value. At the same time I have complied with the request of many medical friends to supply references to the literature on the subject. This has involved much labor, and has contributed materially to increase the size of the volume. To furnish a complete bibliography was, of course, out of the question, and I have necessarily been obliged to omit reference to many valuable papers. My endeavor has been to refer to articles which treat of the individual subjects in as exhaus- tive a manner as possible, and in which more detailed references to the literature can be found. These additions, I trust, will prove of vi PREFACE TO THE FOURTH EDITION. value to the original worker and to the student, as well as to the teacher. One new colored plate and several engravings have been added. The plate, prepared by Mr. A. Horn, one of the artists of Johns Hopkins Hospital, illustrates in a very satisfactory manner the various forms of leucocytes of the blood, and notably the deviations from the commonly pictured types of non-granular mononuclear cells which have so often seemed confusing. In conclusion, I must thank the profession for the kind reception which has exhausted the large third edition in a fraction over a year. To the Publishers I desire to express appreciation of the painstaking care bestowed upon every detail of typography and illustration, as well as for many acts of courtesy. CHARLES E. SIMON. 1302 Madison Avenue, Baltimoee, Md. PREFACE TO THE FIRST EDITION. It is curious to note that, notwithstanding the great importance of clinical chemistry and microscopy, but little attention is paid to these subjects, either by hospital physicians or by those engaged in general practice. This lack of interest is referable primarily to the fact that a systematic study of these branches has hitherto been greatly neglected, not only in American medical schools, but also in those of Europe. It is no rarity to hear physicians in general practice claim that they are too busy to conduct careful examinations of the urine, sputum, blood, gastric juice, etc. Would it not be reasonable to "Suppose, however, that a physician who is overwhelmed with work to such an extent that he cannot find the time to make use of aids in diagnosis which are quite as important as the stethoscope, the laryngoscope, or the ophthalmoscope, would be in a position to employ an assistant in his laboratory ? The younger practitioner is certainly not placed in such a dilemma, and it is a fair assump- tion that he could successfully compete with his more experienced colleague, in matters of diagnosis at least, were he to familiarize himself sufficiently with laboratory methods of diagnosis. The time is at hand when the practice of medicine is becoming what it was long ago, but then unjustly, called, a true science and art. No continuing success can be built on empiricism or upon the proportion of guesswork which is inseparable from dependence upon " the experienced eye." " Diagnosis " is now the password in med- ical science. A knowledge of electro-diagnosis, of ophthalmoscopy, of laryngoscopy, etc., is at the present day a sine qua non for accu- rate diagnosis. Equally important at all times, and frequently even more important, is a knowledge of clinical chemistry and micros- copy. It is inconceivable that a physician can rationally diagnos- ticate and treat diseases of the stomach, intestines, kidneys, and liver, etc., without laboratory facilities. viii PREFACE TO THE FIRST EDITION. It has been the author's aim to present to students and physicians those facts in clinical chemistry and microscopy which are of practi- cal importance. With the hope of exciting interest in these unjustly neglected subjects, he has not confined himself to bare statements of facts, which must in themselves be dry and uninteresting, but he has attempted to point out the reasons which have led up to the conclusions reached. Chemical and microscopical methods are described in detail, so that the student and practitioner who has not had special training in such manipulations. will be enabled to obtain satisfactory results. The subject-matter covers the examination of the blood, the secre- tions of the mouth, the gastric juice, feces, nasal secretion, sputum, urine, transudates, exudates, cystic contents, semen, vaginal dis- charges, and milk. In every case a description of normal material precedes the pathological considerations, which latter in turn are followed by an account of the methods used in examination. A glance at the table of contents will furnish an idea of the various subjects considered under each heading. In conclusion, it is the agreeable duty of the author to express his sincerest thanks to his wife for assistance without which this volume could not have been written, and likewise for those illustra- tions which are original ; to Dr. William H. Welch for his kind- ness in placing the former Hygienic Laboratory of the Johns Hopkins Hospital at his disposal during the years 1892 and 1893 ; to Dr. W. Milton Lewis for much valuable aid in the correction of the manuscript and proof-sheets ; and to Messrs. Lea Brothers & Co. for the typographical excellence of the work, the extremely satisfactory reproduction of the drawings, and for many acts of courtesy. CHAELES E. SIMON. Baltimore, Md., 1896. CONTENTS. CHAPTER I. THE BLOOD General considerations ... .... General characteristics of the blood .... color . . odor . .... specific gravity . . . . '. . determination according to Eoy . determination according to Hammerschlag . . determination according to Schmaltz and Peiper indirect estimation of the hsemoglobin . . estimation of the solids of the blood reaction . . . estimation of the alkalinity according to Landois-v. Jaksch estimation of the alkalinity according to Lowy . estimation of the alkalinity according to Engel Chemical examination of the blood ... general chemistry of the blood blood-pigments . . . haemoglobin ... oxyhsemoglobin .... estimation of haemoglobin with Fleischl's hsemometer . estimation of ha;moglobin with Gowers' haemoglobinometer estimation of blood-iron with Jolles' ferrometer haemoglobinaemia ... carbon monoxide haemoglobin . . nitric oxide haemoglobin . hydrogen sulphide haemoglobin carbon dioxide haemoglobin .... ... haematin .... . .... ... hsemin methsemoglobin haematoidin hasmatoporphyrin the spectroscope the proteids of the blood the carbohydrates sugar PAGE . 17 . 17 . 17 . 18 18 . 18 19 . 19 20 . 20 . 20 . 21 . 23 . 24 . 25 . 25 . 29 . 29 29 32 . 35 . 36 . 40 41 . 42 42 . 42 43 44 44 . 45 . 45 . 46 47 . 49 . 49 X CONTENTS. Chemical examination of the blood — Continued. rKois. estimation of the sugar in the blood 50 Williamson's diabetic blood test 50 glycogen .... 51 cellulose . 52 urea . 52 uraemia . 53 ammonia . > ■ 53 uric acid and xanthin-bases 53 fat and fatty acids 55 lactic acid - 56 biliary constituents • 57 acetone . ... .... 68 Microscopical examination of the blood . ..... 58 the red corpuscles . ... .... 58 variations in the size of the red corpuscles . 58 variations in the form of the red corpuscles . 59 variations in the number of the red corpuscles 60 variations in the color of the red corpuscles 62 behavior toward anilin dyes . . 63 granular degeneration . .... 65 nucleated red corpuscles 67 the leucocytes . ... 69 general diff^entiation of the various forms of leucocytes 69 the anilin dyes .... . .70 differentiation of the leucocytes according to their behavior toward anilin dyes . . 71 variations in the number of the leucocytes . . . 80 leucocytosis . . . ... .... 80 polynuclear neutrophilic hyperleucocytosis 81 polynuclear eosinophilic hyperleucocytosis . . . 89 mixed hyperleucocytosis 91 passive hyperleucocytosis (lymphocytosis) . ... 93 hypoleucooytosis (leukopenia) . . . 94 the drying and staining of blood .... ... 96 staining with eosinate of methylene-blue (Jenner's stain) ... 99 staining with Ehrlich's tri-acid stain ... . 100 staining with Aronaohn and Philip's modified tri-acid stain . . . 100 Neusser's stain ... . . 101 staining with hsematoxylin-eosin, or orange-G solution 101 staining with Chenzinsky's eosin-methylene-blue solution 101 staining with Ehrlich's tri-glycerin mixture . 102 staining with Ehrlich's neutral mixture . . ... . 102 staining with eosin . . . . 102 basic double staining; . 102 staining with eosin-methylal and methylene-blue . 103 special staining of basophilic leucocytes .... . 103 Michaelis' eosin-methylene-blue stain 103 distribution of the alkali in the blood 104 the plaques . .... 104 CONTENTS. XI Microscopical examination of the blood — Contimied. page the hsemokonia, or dust particles of Miiller ... 105 the enumeration of the corpuscles of the blood by the method of Thoma-Zeiss 105 enumeration of the red corpuscles ... 106 enumeration of the white corpuscles . . . 108 indirect enumeration of the leucocytes . 109 differential enumeration of the leucocytes 110 enumeration of the plaques . 110 the haematokrit .... .... . 110 Bacteriology and parasitology of the blood 113 typhoid fever 114 Widal's serum test 114 pneumonia ■ . ... 118 sepsis 119 anthrax 121 acute miliary tuberculosis ... '. 121 glanders 122 influenza 122 relapsing fever 123 Malta fever 124 yellow fever 124 malaria 125 filariasis - 135 •distomiasis 136 anguilluliasis 137 CHAPTER II. THE SECEETIONS OF THE MOUTH. Saliva 138 general characteristics . 138 chemistry of the saliva 138 microscopical examination of the saliva 140 pathological alterations 1^2 Special diseases of the mouth 143 tuberculosis of the mouth 143 actinomycosis 1*3 catarrhal stomatitis • l'*3 ulcerative stomatitis • • ■ 1*3 gonorrhoea! stomatitis ■ 1*'' thrush 14* Tartar 1** Q)ating of the tongue Coating of the tonsils 1^ pharyngomycosis leptothrica tonsillitis ^^^ 145 glandular fever '■^ diphtheria ^^^ xii CONTENTS. CHAPTER III. THE GASTEIC JUICE AND THE GASTRIC CONTENTS. PAGE The secretion of gastric juice . . 148 Test-meals 149 the test-breakfast of Ewald and Boas . .... 149 the test-dinner of Riegel . 150 the double test-meal of SaJzer . . . 150 the test-breakfast of Boas . .... . . . . 150 The stomach-tube . ..... 150 contraindications to the use of the tube . . . 151 introduction of the tube . 151 General characteristics of the gastric juice . . . 153 amount . . . . 153 Chemical examination of the gastric juice 1 54 chemical composition of tlie gastric juice 154 the acidity of the gastric juice . 155 determination of the acidity of the gastric juice 157 source of the hydrochloric acid . . 159 significance of free hydrochloric acid 160 the amount of free hydrochloric acid 162 euchlorhydria . . 162 hypochlorhydria - ... 162 anachlorhydria ... . . 162 Iiyperchlorhydria . ... 163 test for free acids 163 test for free hydrochloric acid .... 1 64 the dimethyl-amido-azo-benzol test . . . . 164 the phloroglucin-vanillin test ... 164 the resorcin test .... 165 tlie tropsBolin test . . 166 Mohr's test 166 the benzopurpurin test 166 the combined hydrochloric acid ] 67 quantitative estimation of the hydrochloric acid 168 Topfer's method . 168 Martius and Liittke's method . . 170 Leo's method ... 172 the ferments of the gastric juice and their zymogens . . . . .173 pepsin and pepsinogen . 173 tests for pepsin and pepsinogen .... . . 175 quantitative estimation , ... 176 chyniosin and chymosinogen .... . . . . . 176 tests for chymosin and chymosinogen ... 178 quantitative estimation .... 178 the products of gastric digestion 178 digestion of the native albumins 178 digestion of the proteids 179 digestion of the albuminoids 179 digestion of the carbohydrates 179 CONTENTS. xiii Chemical examination of the gastric juice— Contmuei. p^eg analysis of the products of albuminous digestion 181 tests for the products of carbohydrate digestion 182 lactic acid , 03 mode of formation and clinical significance 183 tests for lactic acid . . . Ig5 Uffelmann's test 185 Selling's test Igg Strauss' test 186 Boas' test ... . .... I87 quantitative estimation of lactic acid according to Boas' method ... 188 the fatty acids 190 mode of formation and clinical significance 190 tests for butyric acid 191 tests for acetic acid . 192 quantitative estimation of the fatty acids ] 92 quantitative estimation of the organic acids 192 gases 193 acetone I95 ptomains and toxalbumins I95 vomited material igg food-material igg mucus . 197 gastrosuccorrhoea mucosa . 197 saliva . igg tile igg pancreatic juice 198 blood 198 test of Miiller and Weber 198 Donogany's method I99 pus 199 stercoraoeouB material I99 parasites 200 odor 200 Microscopical examination of the gastric contents . . . 200 the Boas-Oppler bacillus 201 sarcinse . . . 201 shreds of mucous membrane 202 tumor particles . 203 Examination of the motor power of the stomach 203 Leube's method . ... . . 204 the salol test of Bwald and Sievers 204 Examination of the resorptive power of the stomach 204 Indirect examination of the gastric juice 205 Giinzburg's method 205 Simon's method 206 xiv CONTENTS. CHAPTER IV. THE FECES. FA^GE Examination of normal feces .... 207 general characteristics 207 number of stools 207 amount 207 consistence and form 207 odor . . 208 color 208 macroscopical constituents 208 alimentary detritus 208 foreign bodies 209 microscopical constituents 209 constituents derived from food 209 morphological elements derived from the alimentary canal 210 crystals • . 210 parasites 212 vegetable parasites 212 fungi 212 Bchizomycetes 212 bacteria 213 chemistry of normal feces 214. reaction ... 214 general composition 214 phenol, indol, and skatol 216 fatty acids 217 cholesterin 218 the biliary acids 219 pigments . 220 Pathology of the feces 221 general characteristics 221 number of stools 221 consistence and form 222 amount 222 odor 222 reaction ... 223 color 223 macroscopical constituents 225 alimentary constituents 225 mucus and mucous cylinders 226 biliary and intestinal concretions ... 227 analysis of gall-stones . 228 microscopical examination 228 technique 228 remnants of food 229 epithelium 230 red blood-oorpusoles 230 mucus 230 CONTENTS. XV Pathology of the feces — Continued. paqe leucocytes 231 crystals . ... . 231 animal parasites 231 protozoa . . . . 232 Amoeba coli . 233 Cercomonas hominis 236 Trichomonas intestinalis 236 Megastoma entericum 237 Balantidium coli 239 vermes . . . . 239 Taenia saginata 240 Taenia solium 241 Taenia nana 242 Taenia diminuta 243 Taenia cucumerina 243 Bothriocephalus latus 243 Krabbea grandis 245 Distoma hepaticum 245 Distoma lanceolatum 246 Distoma Buskii . 246 Distoma sibiricuin . 246 Distoma spatulatum 246 Distoma conjunctum 246 Distoma heterophyes . . 246 Amphistomum hominis 246 Ascaris lumbricoides ... 246 Ascaris mystax . . . 247 Ascaris maritima 248 Oxyuris vermicularis 248 Anchylostomura duodenale . 249 Trichocephalus hominis 250 Trichina spiralis 251 Anguillnla intestinalis ... 251 insecta 252 vegetable parasites . . 252 bacillus of cholera . . . . 252 Finkler- Prior bacillus . . 253 typhoid bacillus . . . . 254 tubercle bacillus . . . 256 Bacillus coli communis . • . 256 Bacillus lactis aerogenes 257 Bacillus pyocyaneus 257 Bacillus acidophilus ... . . 257 Proteus vulgaris • . ... 258 Bacillus dysenteriae 259 Chemistry of the feces . . ... 260 ptomains . . . • . ■ • 262 The feces in various diseases of the intestinal tract 262 acute intestinal catarrh 262 XVI CONTENTS. The feces in various diseases of the intestinal tract — Continued. chronic intestinal catarrh ... . cholera nostras ... summer diarrhoea of infants PAGE ... 263 .... 263 ... 263 dysentery 264 amoebic dysentery 264 cholera Asiatica 265 typhoid fever 265 Meconium 265 CHAPTER V. THE NASAL SECRETION. Physiology and pathology of the nasal secretion . . . 267 CHAPTER VI. THE SPUTUM. General technique . ... , 269 General characteristics of the sputa . ... . . ... 270 amount . . . . 270 consistence . . . . 270 color . . . 271 odor . . . . 271 specific gravity . . . . .... .... 272 configuration of sputa . . . . 272 Macroscopical constituents of sputum . . .... . . 273 elastic tissue ... . . . 273 fibrinous casts ... . . . 273 Curschmann's spirals . . .... . 275 echinocooous membranes . . . 276 concretions . 276 foreign bodies . . 276 Microscopical examination 277 leucocytes 277 red blood-corpuscles . 278 epithelial cells 278 elastic tissue 280 animal parasites 281 Taenia echinoeoccus 281 Distoma pulmonale 283 vegetable parasites 283 pathogenic organisms 283 the tubercle bacillus 283 methods of staining 285 Pappenheim's method 285 Gabett's method 286 CONTENTS. xvii Microscopical examination — Ckmiinued. p^qj, Weigert-Ehrlich method 286 Ziehl-Neelsen method 287 the Diplococcus pneumouise 287 the bacillus of influenza 288 the bacillus of whooping-cough 288 the smegma bacillus . . 288 actinomycosis 289 non-pathogenic organisms 290 Oidiura albicans . 290 Sarcina pulmonalis 290 crystals ... . 290 Charcot-Leyden crystals . 291 hsematoidin 291 cholesterin 292 fatty acid crystals 292 leucin and tyrosin 292 calcium oxalate 292 triple phosphates . . 292 Chemistry of the sputum 292 The sputum in various diseases .... 293 acute bronchitis ..... 293 chronic bronchitis . 293 putrid bronchitis and pulmonary gangrene 294 fibrinous bronchitis 294 bronchial asthma . 294 pulmonary abscess . . ... 294 abscess of the liver with perforation into the lung . 295 pneumonia . 295 phthisis pulmonalis 295 oedema of the lungs . . . 296 heart-disease . . . . . 296 the pneumoconioses . 296 anthracosis . . . . 297 siderosis . 297 chalicosis . . . . 297 stycosis . 297 CHAPTER VII. THE UBINE General considerations . . . . 298 General characteristics of the urine 299 general appearance 299 color . . 300 odor . . .301 consistence 301 quantity ' 301 polyuria . 302 oliguria 305 B xvm CONTENTS. General characteristics of the urine — Continued. page specific gravity . . 305 determination of the specific gravity 308 determination of the solid constitaents 310 Eeaction ... . 3)0 determination of the acidity of the urine 314 Frennd's method .314 Chemistry of the urine • . 315 general chemical composition of the urine 315 quantitative estimation of the mineral ash of the urine 316 the chlorides . . . . . . . 317 test for chlorides in the urine . . 320 quantitative estimation of the chlorides by the method of Salkowski- Volhard . . . . 320 direct method . 324 estimation of the chlorides after incineration (according to Neubauer and Salkowski) • 325 the phosphates 325 test for the phosphates in the urine 330 quantitative estimation of the total amount of phosphates 331 separate estimation of the earthy and alkaline phosphates . . . 334 removal of the phosphates from the urine 334 the sulphates ... . . . 334 test for the sulphates in the urine 337 quantitative estimation of the sulphates 338 quantitative estimation of the total sulphates 338 quantitative estimation of the conjugate sulphates 339 neutral sulphur . 340 quantitative estimation 342 urea 343 properties of urea 343 urea nitrate 352 urea oxalate 353 separation of urea from the urine 354 quantitative estimation of urea 355 estimation of nitrogen according to Kjeldahl 364 estimation of nitrogen according to Will-Varrentrapp 366 ammonia 368 quantitative estimation 369 Schlosing's method 369 Folin's method . 370 uric acid . . 370 properties of uric acid . . 375 tests for uric acid ... 377 quantitative estimation of uric acid 377 xanthin-bases 383 quantitative estimation 334 hippuric acid . . 385 properties of hippuric acid 386 quantitative estimation of hippuric acid 386 PAOE 388 389 390 CONTENTS. Chemistry of the urine — Continued. kreatin and kreatinin . properties of kreatin and kreatinin .... test for kreatinin in the urine ... quantitative estimation of kreatinin in the urine 890 oxalic acid . ... . . 3g2 properties of oxalic acid . . 394 tests for oxalic acid ..... . . 395 quantitative e.stimation of oxalic acid 395 Albumins 3gg serum-albumin 393 Patein's or aceto-soluble albumin 409 serum-globulin 409 albumoses (peptones) 409 Bence Jones' albumin 411 haemoglobin • 412 fibrin . . 414 nucleo-albumin 414 histon and nucleohiston 415 tests for albumin . . 415 tests for serum-albumin _ 41g nitric acid test . . . 4ig boiling test 419 potassium ferrocyanide test 420 trichloracetic acid test 420 picric acid test . . .... 421 Spiegler's test 421 special test for serum-albumin 421 quantitative estimation of albumin 422 old method of boiling . . . . 422 volumetric method of Wassiliew . 422 Esbach's method .... 423 differential density method . . . 423 gravimetric method . . . . 424 test for serum-globulin and its quantitative estimation 424 tests for albumoses ... .... . 425 Salkowski's method . . 425 Bang's method . ... 426 tests for TBence Jones' albumin . 427 tests for (mucin) nucleo-albumin . .... 428 tests for haemoglobin .... . . .... . 428 Heller's test . 429 the guaiacum test . ■ • • 429 Donogany's test . . 430 test for fibrin . ... 430 test for histon 430 Carbohydrates . . . 430 glucose ... 430 tests for sugar 438 Trommer's test 439 XX CONTENTS. Carbohydrates — Continued. rAGu Fehling's test 439 Bottger's test with Nylander's modification 440 fermentation test . .... 440 phenylhydrazin test . .... 441 Kowarsky's modification . . . ... 442 polarimetrie test . . ■ 442 quantitative estimation of sugar ... ... 444 Fehling's method . .... 444 Knapp's method ... 446 differential density method . . ... . . 447 Einhorn's method 447 Lohnstein's method • 448 polarimetrie method 449 Bremer's diabetic urine test 451 lactose 452 levulose 452 maltose 452 dextrin 452 laiose ... . 453 pentoses _ 453 ToUens' orcin test 453 ToUens' phloroglucin test 453 animal gum . .... 454 Glucuronic acid . . ... 454 Inosit .... . . . 455 Urinary pigments and chromogens 455 normal pigments 455 nrochrome 455 uroerythrin 457 normal chromogens 458 indican 458 tests for indican 461 quantitative estimation 462 urohsematin 464 uroroseinogen 465 pathological pigments and chromogens 466 blood -pigments 466 hseraatin ... 460 urorubrohffimatin and urofuscohsematin 466 urohsematoporphyrin 466 biliary pigments .... 469 Smith's test 470 Huppert's test 470 Gmelin's test (as modified by Kosenbach) 471 Gmelin's test 471 biliary acids 471 cholesterin 471 pathological urobilin 471 melanin and melanogen 474 CONTENTS. xxi Urinary pigments and chromogens— ComJinaei. pase phenol urines ... 475 alkapton . . . 475 homogentisinic acid 476 blue urines . . . 478 green urines ... . . . . . 478 pigments referable to drugs . . 478 Ehrlicb's reaction 479 Conjugate sulphates 482 skatoxyl 482 phenol ... 483 Salkowski's test 483 quantitative estimation .... 483 pyrooatechin . . 484 Acetone . . 484 tests for acetone 486 Legal's test . ... . 486 Lieben's test 486 Eeynolds' test . 486 Dennigfe's test .... 486 quantitative estimation . . . 487 Diacetic acid . 489 Arnold's test . 489 Oxybut;^ic acid 490 Lactic acid 490 Volatile fatty acids 491 Fat 492 chyluria 492 galacturia. 492 Ferments 493 Gases 493 hydrothionuria 493 Ptomains ... .... 494 method of examination for ptomains . . 495 Sediments ... . . . . 496 Microscopical examination of the urine 498 non-organized sediments . . . . 500 sediments occurring in acid urines 500 uric acid . . . 500 amorphous urates 502 calcium oxalate . . . . . 502 ammonio-magnesium phosphate . . 504 monocalcium phosphate 505 neutral calcium phosphate . 505 basic magnesium phosphate 505 hippurio acid 506 calcium sulphate 506 cystin . • .... . . 507 leucin and .tyrosin . 508 xanthin . . . . • oil xxii CONTENTS. Microscopical examination of the urine — Continued. page soaps of lime and magnesia ... 51 2 bilirubin and hsematoidiu . . . 512 fat 512 sediments occurring in alkaline urines . . 513 basic phosphate of calcium and magnesium 513 ammonium urate 613 magnesium phosphate . • 513 ammonio-magnesium phosphate . • 514 calcium carbonate . .... . 514 indigo ... 514 organized constituents of urinary sediments . . . 515 epithelial cells . 515 leucocytes ... . • 518 red blood-corpuscles . . . 522 tube-casts. ... 525 true casts . 526 hyaline casts 526 waxy casts 526 pseudo-casts . .... . . ... . . 531 cylindroids . ... 531 formation of tube-casts . 531 clinical significance of tube-casts . 532 spermatozoa ... . .... 535 parasites . . . .... 536 vegetable parasites . ... 536 animal parasites . . 542 tumor particles 543 foreign bodies . 543 CHAPTER VIII. TEANSUDATES AND EXUDATES. Transudates 545 general characteristics .... 545 specific gravity . 545 chemistry of transudates 548 microscopical examination 548 Exudates ... 548 serous exudates . 549 hemorrhagic exudates 549 tuberculosis 549 cancer ... . 550 putrid exudates 550 pus 550 general characteristics of pus 550 chemistry of pus 651 microscopical examination of pus 551 leucocytes 551 CONTENTS. xxiii Exudates — Continued. p^qj, giant corpuscles 552 detritus . . . . 552 red blood-corpuscles 552 pathogenic vegetable parasites 553 protozoa ... 553 vermes . . 553 crystals . 553 chylous and chyloid exudates 554 CHAPTEE IX. THE EXAMINATION OF CYSTIC CONTENTS. Cysts of the ovaries and their appendages . . 555 test for metalbumin 555 Hydatid cysts . 557 Hydronephrosis •. . 557 Pancreatic cysts . 557 CHAPTEE X. THE CEKEBEOSPINAL FLUID. Definition ... 55g Amount . 559 Appearance . ; . 559 Specific gravity 560 Eeaction i 661 Chemical composition 561 Microscopical examination 562 Bacteriology 562 CHAPTEE XL THE SEMEN. General characteristics .... 564 Chemistry of the semen 564 Microscopical examination of the semen 565 Pathology of the semen . 566 The recognition of semen in stains 566 CHAPTEE XII. VAGINAL DISCHARGES. General description 569 Bacteriology 570 Vaginal blennorrhoea 571 xxiv CONTENTS. PAGE Menstruation 571 The lochia 571 Vulvitis and vaginitis 572 Membranous dysmenorrhoea 572 Cancer 572 Gonorrhoea . . ... . . 572 Abortion 573 CHAPTER XIII. THE SECRETION OF THE MAMMARY GLANDS. The secretion of milk in the newly bom 575 Colostrum 575 The secretion of milk in the adult female 576 Human milk . . 576 The milk in disease 577 determination of the specifie gravity 578 estimation of the fat . . 580 estimation of the proteids 580 CLINICAL DIAGNOSIS. CHAPTER I. THE BLOOD. GENERAL CONSIDERATIONS. If 'blood is allowed to flow directly from an artery into a vessel surrounded by a freezing-mixture, and containing one-seventh its volume of a saturated solution of sodium sulphate, or a 25 per cent, solution of magnesium sulphate (1 volume to 4 volumes of blood), it will be observed that after some time a sediment, presenting the color of arterial blood, has formed at the bottom, which is covered by a layer of clear, straw-colored fluid — the blood- plasma. Upon microscopical examination the sediment will be seen to contain : a. Numerous homogeneous, non-nucleated, circular, biconcave disks. These measure on an average 7.5 /i in diameter, and are of a faint greenish-yellow color when viewed through a microscope, while en masse they present the color of arterial blood — ^the erythro- cytes or red corpuscles of the blood. b. Roundish or irregularly shaped nucleated cells which are far less numerous than the red corpuscles, and devoid of coloring-matter — the leucocytes, colorless or white corpuscles of the blood. c. Minute colorless disks, measuring less than one-half the di- ameter of a red corpuscle — ^the so-called blood-plaques, or blood- plates of Bizzozero. GENERAL CHARACTERISTICS OF THE BLOOD. The Color. Chemical examination of the blood shows that its color is ref- erable to the presence of an albuminous, iron-containing substance — haemoglobin — in the bodies of the red corpuscles, which is characterized by its great avidity for oxygen, and forms a compound therewith, known as oxyhaemoglobin. The relatively larger amount of the 2 17 18 THE BLOOD. latter encountered in the arteries, as compared with the veins, causes the difference in the appearance of arterial and venous blood, the former presenting a bright scarlet-red, the latter a dark-bluish color. A bright cherry-red color is noted in cases of poisoning with carbon monoxide, while a. brownish-red or chocolate color is observed in cases of poisoning with potassium chloratCj anilin, hydrocyanic acid, and nitrobenzol. A milky appearance is frequently seen in cases of well-marked leukaemia. In chlorosis and hydraemic con- ditions, as would be expected, the blood is pale and watery. The Odor. The peculiar odor of the blood, which varies in different animals, the halitus sanguinis of the ancients, is due to the presence of certain volatile fatty acids, and may be rendered more distinct by the addition of concentrated sulphuric acid. The Specific Gravity. The specific gravity of the blood in healthy adults varies between .1.058 and 1.062, being higher on an average in men, 1.059, than in women, 1.056, and children — boys 1.052, girls 1.050. It is diminished to a certain extent by fasting, the ingestion of solids and liquids, gentle exercise, pregnancy, etc. The specific gravity, moreover, depends upon the bloodvessel from which the specimen is taken, being higher, generally speaking, in venous than in arterial blood. Under pathological conditions the specific gravity may vary between 1.025 and 1.068. In nephritis, chlorosis, the anaemias in general, and in cachectic conditions (pulmonary phthisis, carcinoma of the stomach, etc.) it may diminish to 1.031. An increased specific gravity is met with in febrile diseases (typhoid fever, 1.057 to 1.063), conditions associated with pronounced cyanosis (emphysema, fatty heart, uncompensated valvular disease, 1.054 to 1.068), and obstructive jaundice, 1.062. Methods of determining the Specific Gravity of the Blood. ^ Roy's Method. — A number of test-tubes are filled with a mixt- ure of glycerin and water in different proportions, so that the specific gravity in the different tubes varies between 1.025 and 1.068. Blood is then drawn from the tip of a, finger, or the lobe of the ear, into a capillary tube connected with an ordinary hypodermic syringe, pressure being avoided. A drop of blood is placed in each tube, in which it will sink as long as the specific gravity of the glycerin mixture is lower than that of the blood, while it will remain sus- GENERAL CHARACTERISTICS OF THE BLOOD. 19 pended in a mixture the specific gravity of which is equivalent to its own. Roy states that it is important for the purpose of comparison to make such examinations in each case at the same hour, as the spe- cific gravity of the blood has been shown to undergo diurnal varia- tions. Hammerschlag's Method. — A cylinder, measuring about 10 cm. in height, is partly filled with a mixture of chloroform (sp. gr. 1.526) and benzol (sp. gr. 0.889), having a specific gravity of 1.050 to 1.060. Into this solution a drop of blood is allowed to fall directly from the finger, pressure being avoided, and care taken that the drop does not come in contact with the walls of the vessel. The drop, moreover, should not be too large, as otherwise it will separate into droplets, giving rise to inaccurate results. Should the drop sink to the bottom, it is apparent that the specific gravity of the mixture is lower than that of the blood, necessitating the addition of chloroform. This should be added drop by drop while the mixture is thoroughly stirred. If, on the other hand, the drop should tend toward the surface, it is best to add an amount of benzol sufficient to cause the blood to sink to the bottom, and then to bring it to the proper degree of suspension by the subsequent addi- tion of chloroform. As soon as the drop remains suspended the mixture is filtered, and its specific gravity ascertained by means of an accurate areometer registered to the fourth decimal. The figure obtained is the specific gravity of the blood. The chloroform- benzol mixture may be kept indefinitely. With practice, results sufficiently accurate for clinical purposes may thus be obtained with an expenditure of very little time. Schmaltz and Peiper's Method. — Where delicate scales are avail- able the method of Schmaltz and Peiper may be employed, and is certainly the most accurate : a capillary tube, measuring about 12 cm. in length and 1.5 mm. in width, with its ends tapering to a diameter of 0.75 mm., is filled with blood and carefully weighed, when the weight of the blood, divided by the weight of an equiva- lent volume of distilled water, will indicate the specific gravity. As the result of numerous investigations it may now be regarded as an established fact that with the exception of nephritis, circulatory disturbances, leuksemia, and possibly also post-hemorrhagic ansemia and that resulting from inanition, the specific gravity of the blood varies directly with the amount of haemoglobin. A simple method is thus given by means of which haemoglobin estimations can usually be made in the absence of more expensive instruments. In the follow- ing table the specific gravities, as obtained with Hammerschlag's method, and that of Schmaltz and Peiper, are given, with the corre- sponding amounts of haemoglobin. The figures, however, are prob- ably not quite accurate : 2U THE BLOOD. Specific gravity Specific gravity according to Haemoglobin. according to Haemoglobin. Hammerschlag. Schmaltz and Peiper. 1.033-1.035 . . 25-30 per cent. 1.030 . ... 20 per cent. 1.035-1.038 . . 30-35 " 1.035 30 " 1.038-1.040 . . . 35-40 " 1.038 35 " 1.040-1,045 . . . 40-45 " 1.041 40 " 1.045-1.048 . . . 45-55 " 1.0425 . ... 45 " 1.048-1.050 . . . 55-65 " 1.0455 50 " 1.050-1.053 . . . 65-70 " 1.048 .... 55 " 1.053-1.055 . . 70-75 " 1.049 60 " 1.055-1.057 . . . 75-85 " 1.051 ... 65 " 1.057-1.060 . . . 85-95' " 1.052 70 " 1.0535 . . .75 " 1.056 ... .80 " 1.0575 .... 90 " ].059 100 " LiTEBATUKE. — Schmaltz, Deutsoh. Arch. f. klin. Med., vol. xlvii. p. 145 ; and Deutsch. med. Woch., 1891, No. 16. Stintzing n. Gumprecht, Deutsch. Arch. f. klin. Med., vol. liii. p. 265. Siegl, Prag. med. Woch., 1892, No. 20; and Wlen. med. Woch., 1891, No. 33. Hammerschlag, Ibid., 1890, p. 1018; and Zeit. f. klin. Med., 1892, vol. xxii. p. 475. Schmaltz, Deutsch. Arch. f. klin. Med., 1890, vol. xlvii. p. 145 ; and Deutsch. med. Woch., 1891, vol. xvii. p. 555. Direct Estimation of the Solids of the Blood. A few drops of blood (0.2 to 0.3 gramme), obtained by means of a fairly deep incision or puncture into the tip of a finger, moderate pressure being made upon the middle phalanx if necessary, are col- lected in a watch-crystal. This is at once covered with its fellow and weighed. The specimen (open) is then dried at a temperature of from 60° to 70° C. for twenty-four hours, and again weighed,, the weight of the solids being thus ascertained. In healthy adults the following values were obtained by Stintzing and Gumprecht : Average. Maximum. Minimum. Average water. In men 21.6 23.1 19.6 78.4 per cent. In women . . . 19.8 21.5 18.4 80.2 In conditions associated with chronic anaemia the solids, as would be expected, are always much diminished. In leukaemia, on the other hand, owing to the large number of leucocytes present, a rela- tive increase is observed. The Reaction. The reaction of the blood during life, owing to the presence of disodium phosphate and sodium carbonate, is alkaline, the degree of alkalinity in terms of sodium hydrate under normal conditions cor- responding to 182 to 218 mgrms. for every 100 c.c. of blood, v. Jaksch gives 260 to 300 mgrms. as the normal, and Canard 203 to 276 mgrms. GENERAL CHARACTERISTICS OF THE BLOOD. 21 The alkaline reaction of the blood may be demonstrated by re- peatedly drawing a strip of red litmus-paper, thoroughly moistened with a concentrated solution of common salt, through the blood, and rapidly washing off the corpuscles with the same solution, when, as a general rule, the alkaline reaction can be clearly made out. Small plates of plaster of Paris or clay, stained with neutral litmus solution, may be similarly employed, the blood in this case being washed off with water. Generally, the allcalinity of the blood is lower in women and children than in men, and is, furthermore, influenced by the proc- ess of digestion, exercise, etc. At the beginning of digestion, when hydrochloric acid is being freely secreted, the alkalinity of the blood increases ; while later on, when both hydrochloric acid and peptones are reabsorbed, the alkalinity in turn diminishes. Higher values are usually found during pregnancy than in the non- pregnant state. A decrease is observed following violent muscular exercise, such as forced marches by soldiers, owing, in all probability, to an exces- sive production of acids in the muscles. Under pathological conditions a diminished alkalinity of the blood is frequently observed. This is particularly marked 'n\ cases of severe anaemia (108 to 145 mgrms. of NaOH), and increases as the number of red corpuscles and the amount of Wmoglobin diminish. In chlorosis, however, the diminution in the number of red corpus- cles is accompanied by a normal, or but slightly diminished, alkalinity of the blood as a whole. In leukaemia, pernicious anaemia, nephritis when accompanied by uraemia, various hepatic affections, diabetes, carcinoma, the various profound cachexise, pseudoleukaemia, poison- ing with carbon monoxide and acids, and finally in highly febrile conditions, as in typhoid fever, and toxic processes in general, the alkalinity of the blood is diminished, the lowest value found corre- sponding to 108 mgrms. of NaOH. A similar decrease follows the prolonged use of acids, while an increase is brought about by the ingestion of alkalies. An increase in the alkalinity of the blood occurs after a cold bath, and it is interesting to note that this is apparently associated with an increase in the bactericidal power of the blood. Possibly the beneficial effect of cold baths in fever may be explained upon this basis. The supposition that in gout a diminished alkalinity exists between the attacks, and that this increases beyond the normal during the attack, has been proved unfounded. V. Jaksch employs the following method, a modification of that originally devised by Landois : eighteen watch-crystals are prepared, each containing a mixture of a concentrated solution of sodium sulphate and a yutt ^^^ ^ TBinT normal solution of tartaric acid, in varying proportions, so that crystal 22 THE BLOOD. No. Co. c.c. I. Shall contain 0.9 of the tJtj norm. sol of the acid, and 0.1 II. 0.8 (( i 11 11 " 0.2 III. 0.7 " 1 11 11 " 0.3 IV. 0.6 " ' (1 11 " 0.4 V. 0..5 n ' 11 u " 0.5 VI. 0.4 n 1 11 11 " O.G VII. VIII. 0.3 0.2 (( 1 11 .1 11 11 " 0.7 " 0.8 IX. 0.1 (( ') Poikiloeytosife. Unstained Specimen taken from a Case of Pernicious Antemia. (Per- sonal Observations.) o L. Schmidt iecii 4^ j§b >- Q^ ^ The Various Elements of the Blood Stained with Ehrlieh's Tri-acid Stain. I, Small Mononuclear Leucoc;-tes ; 2, Large Mononuclear Leucocytes ; 3, Transition Form ; 4, Neutrophilic Leucocytes; 5, Myelocyte; 6, Eosinophilic Leucocyte; 7, Melaniferous Leucocyte; S, Normo- blast ; g, Megaloblast ; 10, Normal Red Corpuscles. (Personal Obser\ ation.) PLATE III. The Blood of Pernicious Anasmia. Note fa) the variations in the size aiui fonn of the red corpuscles ; (/•) the existence of polychromato- philic ( I) a and granular /^ {2) defeneration in some of the red corpuscles ; (<:) the presence o( nucleated red corpuscles, both of the normoblastic (3) and megaloblastic type {4); {if) the presence of free nuclei (5), derived from nucleated red corpuscles. (Bausch and Lomb, Eye-piece i inch, objective i-i2th.) MIOROSCOPICAL EXAMINATION OF THE BLOOD. 59 Under normal conditions variations in the size of the red corpus- cles are observed, and Hayem ' distinguishes between corpuscles of average size, measuring from 7.2 (i to 7.8 /z in diameter, small cor- puscles, presenting an average diameter of from 6 /i to 6.5 fi, and large corpuscles, measuring from 8.5 // to 9 //. In certain diseases which are accompanied by a marked oligo- cythsemia both abnormally small and large corpuscles are encoun- tered, which have been termed microeytes and maorocytes, respectively. The former measure from 3.5 ;« to 6 /jl; the latter, from 9.5 /i to 12 /i in diameter. Still larger forms, the megaheytes, or giant corpuscles of Hayem, are also seen at times, of which the diameter measures from 10 // to 16 //. These latter are of especial interest, as their presence in large numbers appears to be confined almost entirely to the blood of pernicious anaemia. In chlorosis they are far less common (Plate III.). The terms miaroeythcemia and maeroeythcemia have been applied to conditions in which the smaller or the larger forms, respectively, predominate in the blood. While there appears to be no doubt that a true macrocythsemia exists in the circulating blood in various forms of anaemia, and while microeytes also may occur as such in the cir- culating blood, these are only exceptionally met with, the ordinary microcythsemic condition, according to Hayem, being artificially produced during the preparation of the specimen, so that this term really conveys a wrong impression, and should be discarded. Al- though admitting the correctness of Hayem's view to a certain degree, there can be no doubt that under pathological conditions abnormally small red corpuscles are quite constantly met with in large numbers, be they pre-existent as such in the circulating blood or produced artificially during the preparation of the specimen. They are thus seen accompanying the condition of macrocythsemia, in pernicious anaemia, leukaemia, the pseudoleukaemic condition of children, the various severe forms of anaemia in general, and even in chlorosis. Variations in the Form of the Red Corpuscles. — Going hand in hand with variations in the size of the red corpuscles are varia- tions in form which affect not only the microeytes and macrocytes, but also the corpuscles of normal size (Plate II., Fig. 1, B). Cor- puscles are thus seen which resemble a flask, a kidney, a biscuit, a boat, a balloon, a dumb-bell, an anvil, etc.; while others, again, are so irregular in appearance that it is impossible to compare them with any object.. Very characteristic also are the oval red corpus- cles so commonly seen in pernicious anaemia. Especially inter- esting is the fact that such corpuscles may manifest certain move- ments in the fresh prep^-ration, and that they have been mistaken at times for amoebae and similar organisms. ' Hayem, Le sang, Paris, 1891. 60 THE BLOOD. The term poikUooytosis has been applied to alterations both in the size and in the form of the red corpuscles. This condition may be observed in the various forms of anaemia, and is especially pro- nounced in pernicious anaemia, of which disease it was once thought to be pathognomonic. In chlorosis, poikilocytosis is usually seen only in the most severe cases, and particularly in those manifesting a tendency toward thrombosis and embolism. Variations in the Number of the Red Corpuscles. — The num- ber of red corpuscles in the blood of healthy individuals is quite constant, and the statement generally found in text-books that 5,000,000 to 5,500,000 are contained in every cbmm. of blood in the adult male and 4,500,000 in the adult' female is fairly accurate. A somewhat higher average is found among people living at a considerable elevation above the sea-level, and it is interesting to note that an increase in the number occurs whenever a change in the habitation is made from a lower to a higher level. This increase is frequently marked, as is apparent from the following table, taken from Ehrlich : ^ Altitude. Increase of. 561 meters 800,000 700 " 1,000,000 1800 " 2,000,000 4392 " 3,000,000 A corresponding diminution occurs when a change is made from a higher to a lower level. An apparent increase in the number of red corpuscles may be met with in all conditions in which a concentration of the blood as a whole occurs, as in profuse diarrhoea, vomiting and sweating, in connection with the rapid accumulation of serous effu- sions, during starvation, viz., the withdrawal of liquids, etc. In such cases counts of 6,000,000 and more may be obtained. There are other conditions, however, in which an apparent increase in the number of the red corpuscles occurs, and in which this increase is not due to a concentration of the blood as a whole. This is notably the case in diseases of the adrenal glands, in which counts of 6,000,000 and 7,000,000 have repeatedly been obtained, although the color index of the individual corpuscles was distinctly subnormal. The supposition is that in such cases a stasis of large quantities of blood occurs in the abdominal viscera, leading to oligaemia of the peripheral organs. But in consequence of the fact that the amount of plasma which is available for the nutrition of these parts corresponds to a smaller amount of blood, a localized concentration occurs, of which the polycythsemia is the outcome. Stengel ^ states that in chronic heart disease, with continued inade- ' P. Ehrich u. A. Lazarus, " Die Anaemie," Nothnagel's Speoielle Path. u. Theiap., vol. viii. Part 1. 2 A. Stengel, Proo. Path. Soo. Phila., 1899. MICROSCOPICAL EXAMINATION OF THE BLOOD. 61 quacy of the circulation of slight degree, polycythsemia is frequently observed, while in congenital heart disease the number of the red cells may reach 8,000,000 per cbmm. According to Grawitz, this is due to loss of liquid from the blood, owing to the continued low blood-pressure and vascular dilatation. Stengel, on the other hand, believes it to be due to a disturbance in the normal distribution of the corpuscles. Oertel and Grawitz ' further have pointed out that a polycythsemia occurs in conditions which are associated with chronic stasis, cyanosis, and cedema, and is more marked in the capillaries than in the arter- ies and veins. An increase is observed also in diabetes, but is not dependent upon a concentration of the blood, as it may also be seen following an increased ingestion of fluids, as well as while fasting. While there can thus be no doubt that a polycythsemia may occur, experi- ments have demonstrated almost conclusively that such a condition does not exist in what is generally spoken of as true plethora, and that the various symptoms of plethora formerly attributed to an increase in the total amount of blood or of the red corpuscles are referable more likely to vasomotor disturbances. A diminution in the number of red corpuscles, on the other hand, is more frequently observed ; it may be temporary or permanent. An oligocythsemia may occur in all forms of anaemia, of whatever origin, and the number may fall to 360,000 and even lower in fatal cases. In pernicious anaemia the lowest figures have been noted, and Quincke^ cites a case in which just before death only 143,000 red corpuscles were counted in the cbmm. When the anaemia is progressive the body apparently becomes habituated to the diminution in the number of red corpuscles, and it is surprising to find individuals attending to the duties of everyday life with a blood-count of only 2,000,000, or even less. It is not uncommon to meet with cases of pernicious anaemia in hospitals in which the patients with only 600,000 corpuscles have not been obliged to go to bed. Nevertheless, it must be admitted that when- ever the number falls below this figure recovery is probably out of the question. A sudden reduction in their number to 1,000,000, moreover, is usually followed by a fatal result. In chlorosis the oligocythsemia is generally not marked. Cabot ^ thus found 4,050,000 red corpuscles per cbmm. as the average in his series of seventy-seven cases — in other words, nearly normal values. At times, however, cases are met with in which the dim- inution of the red corpuscles almost keeps step with the diminution 1 Oertel, Dejitsch. Arch. f. klin. Med., vol. 1. p. 293. 2 Quincke, "Ueber perniclose Anaemie," Centralbl. f. d. med. Wiss., 1877, No. 47; and "Weitere Beobachtungen liber progressive perniciose Anaemie," Deutsch. Arch, f. klin. Med., vol. xx. ' E. C. Cabot, Cainical Examination of the Blood. Wm. Wood & Co., 1897. 62 THE BLOOD. in the amount of hsemoglobin. Hayem thus mentions an instance of chlorosis in which only 937,360 red corpuscles were counted in the cbmm. Such cases, of course, are rare. In leuksemia a more than moderate oligocythsemia is likewise not the rule, and is more common in the lymphatic than in the myelogen- ous form. The average figures which Cabot ' gives are 2,730,000 and 3,120,000, respectively. In Hodgkin's disease a marked diminution is also unusual. In the secondary anaemias, even in advanced cases, the oligocy- thsBmia may not be very marked, excepting the ansemias of infancy and early childhood, following profuse hemorrhages, in malaria, and in acute septicaemia. The post-typhoid ansemia is, as a rule, not very severe, but ex- ceptional cases occur in which the diminution in the number of the red corpuscles is considerable. Osier thus cites an instance in which the number fell to 1,300,000 per cubic millimeter. In acute gastritis and usually in chronic gastritis, also, the num- ber of the red corpuscles is not diminished, while in carcinoma a marked oligocythsemia occurs at some time in the course of the disease. In the earlier stages, however, this is often but little marked, and at times even an apparent increase of the red cells is noted. Later a diminution is probably always found. In the severer forms of chronic gastritis a diminution is also fairly constant, but rarely so marked as in carcinoma, if we except those cases of gastric anadeny which present the clinical picture of a pernicious ansemia. In the diiferential diagnosis between carcinoma of the stomach and per- nicious ansemia a count below 1,000,000 points to the latter disease. In ulcer of the stomach normal values are found unless hsematemesis has recently occurred or unless the disease is associated with pro- found chlorosis. In acute endocarditis Stengel ^ has noted a rapid fall of the red corpuscles, often to 50 or 40 per cent. Variations in the Color of the Red Corpuscles. — As the inten- sity of the color of the individual corpuscle depends upon the amount of haemoglobin which it contains, and is more marked along the periphery than in the centre, a deficiency of haemoglobin may be recognized at once. In a moderate grade of anaemia the entire corpuscle will thus appear paler than normally, and the pallor will naturally be more marked in the centre. In the severer forms this becomes still more apparent, and corpuscles may then be met with in which only a narrow rim of hsemoglobin can be discerned along the periphery, while the centre appears colorless. Such forms have very appropriately been compared to pessaries, and are hence spoken of as " pessary forms." This appearance can readily be made out 1 Loc. cit. 2 Loc. cit. MICROSCOPICAL EXAMINATION OF THE BLOOD. 63 upon examination of a fresh specimen, but is especially marked in stained preparations. Curiously discolored red corpuscles, presenting a bronzed appear- ance, are frequently observed in malarial blood. Their appearance should always excite suspicion, and lead to a careful examination for malarial organisms. The discoloration is in all probability evidence of a degenerative process. Behavior toward Anilin Dyes. — Under normal conditions the red corpuscles are stained only with acid dyes, such as eosin, orange G, and others. In various forms of anaemia, on the other band, this property is lost to a greater or less extent, while a certain affinity for basic stains becomes manifest. This is readily seen in blood specimens which have been taken from cases of chronic anaemia, and have been stained with hsematoxylin-eosin, eosin-methylene-blue, or the eosinate of methylene-blue (see pages 99-103). In such preparations the majority of the red corpuscles will be stained red,- but individual corpuscles will also be seen in which a blue tint is more or less apparent. In some this can be made out only indis- tinctly, while others show a very manifest bluish-red color, others a reddish blue, and still others a violet or even a deep, pure blue. A brownish color, moreover, is at times seen in severe forms of anaemia (see Plates' III. and VI.). Similar pictures are obtained with Ehrlich's tri-acid stain, but are not so well defined. This altered behavior of the red corpuscles toward the anilin dyes has been ascribed to certain degenerative processes which take place in the red blood-corpuscles, and the phenomenon has hence been termed ancemlo or polychromatophilio degeneraMon. As I have already indicated, this degeneration is observed in various forms of anaemia, and may affect not only the non-nucleated, but also the nucleated red corpuscles, and especially the megaloblasts. The peculiar coppery tint of some of the red corpuscles which is observed so frequently in malarial blood is probably also refer- able to such degenerative changes. According to some observers, the polychromatophilia of the red cells is not referable to degen- erative changes, however, but is the expression of blood regen- eration, the polychromatophilio cells representing the youngest red cells of the blood. This view is essentially based upon the observation that polychromatophilio cells are normally encountered in the embryo, and that they are especially numerous in the circu- lating blood shortly after severe hemorrhages and in other condi- tions in which an active blood regeneration is going on. Such cells have also been found in the marrow of various healthy domestic animals, and I have myself seen them in the blood of the squirrel, the sea gull, and the frog. Literature. — Ehrlich, Charity Annalen, vol. x. p. 136. Engel, Deutsch. med. Woch., 1899, p. 209. Gabritschewsky, Arch. f. exp. Path., vol. xxviii. p. 83 ; Zeit. f. klin. Med., vol. xxvii. p. 492. Askanazy, Ibid., vol. xxl. p. 415. Maragliano and Castellino, Ibid., vol. xxl. p. 415. 64 THE BLOOD. Very interesting and important is the observation of Bremer, that a distinct difference exists in the aifinity of diabetic blood for certain anilin dyes, as compared with non-diabetic blood. For, whereas non-diabetic blood is readily stained with Congo-red, methyl-blue, eosin, etc., diabetic blood is distinctly refractory, while such dyes as Biebrich-scarlet, which readily stain the diabetic blood, do not color non-diabetic blood. Upon this peculiarity in the behavior of the red corpuscles Bremer's diabetic blood test is based. Method. — A drop of blood of moderate size is mounted on a slide and spread out in a wave-like manner, using the edge of a second slide for this purpose. A number of such preparations are made, as also an equal number with normal blood for control. These are then placed on the tray of a drying-oven at a distance of 12 cm. from the bottom. The bulb of the thermometer is fixed at the same level. The temperature is then rapidly raised to about 130° C, when the flame is removed. Care should be taken that the temperature thereafter does not exceed 140° C; the optimum lies at about 135° C. The apparatus is then allowed to cool until the preparations can be conveniently handled, when a specimen of the diabetic blood is placed back to back with a control-specimen, and both are immersed in the staining fluid. A 1 per cent, aqueous solution of Congo-red, which should always be made up freshly when required, is advantageously employed. After exposure for from one and a half to two minutes the specimens are rinsed in water and dried with filter-paper. It will then be seen that the non-diabetic blood is stained the color of Congo-red, while the diabetic blood is either not stained at all or presents merely an orange color. Other stains may also be employed, such as a 1 per cent, aqueous solution of methyl-blue or Biebrich-scarlet, or Ehrlich's tri-acid stain, the eosinate of methylene-blue, and others. When using methyl-blue analogous results are obtained as with Congo-red. With Biebrich-scarlet, on the other hand, the diabetic blood takes up the color, while the non-diabetic specimen proves refractory. If Ehrlich's stain is employed, an exposure to the stain for from two to five minutes is necessary ; the diabetic specimen is stained orange, the non-diabetic blood violet. Very satisfactory results are obtained also with the following method : the preparations are first stained for from one and a half to two minutes in a 1 per cent, aqueous solution of methyl-green. Upon washing, it will be seen that both specimens are colored green, but the diabetic blood more markedly so than the other. Both are then immersed for from eight to ten seconds in a 0.12 per cent, aqueous solution of eosin, when the diabetic blood remains green, while the non-diabetic specimen is colored eosin. Analogous results are obtained with methylene-blue and eosin. MICROSCOPICAL EXAMINATION OF THE BLOOD. 65 Success in these examinations depends essentially upon the proper degree of temperature during the process of fixation. But care should also be had not to leave the specimens in the staining solu- tion longer than indicated, and to rinse quickly in water and dry. I have used this method in some ten cases of diabetes Mith very satisfactory results, and have likewise obtained a positive reaction at times when the sugar had temporarily disappeared from the urine. As a control to the urinary examination, the method is certainly of value. Regarding the nature of the substance in diabetic blood which is responsible for this peculiar behavior, little is known, but it appears certain that the reaction is not dependent upon the presence of glu- cose nor upon the degree of alkalinity of the blood, as suggested by L6pine and Lyonnet. Bremer's claim that the reaction is pathog- nomonic of diabetes and glucosuria, and may even yield positive results in the pre-diabetic stage of the disease, and when the sugar has temporarily disappeared from the urine, has been confirmed in all essential points, both in this country and abroad. A few inter- esting exceptions, however, have been noted. In animals, for example, in which glucosuria has been artificially produced by means of phlorhizin, the reaction does not occur, whereas in phloro- glucin-diabetes positive results are obtained. In Bremer's entire series of diabetic cases a negative result was obtained but once, and in this instance he believes that the diabetes was of the renal type, and analogous to the phlorhizin-diabetes of animals. He suggests that it may thus be possible to differentiate this form from the hsematogenic variety, using the latter term in its widest sense. Lupine and Lyonnet report a positive result in one case of leukae- mia, but Bremer believes this to have been due to faulty technique. Hartwig believes that Bremer's reaction is constant in diabetes, but states that it may occur at times in other conditions. Literature. — L. Bremer, ' ' An Improved Method of Diagnosticating Diabetes- from a Drop of Blood," N. Y. Med. Jour., 1896; Centralbl. f. inn. Med., 1897, p. 521. Le Goff, Eeact. ohrom. du sang diabet., Paris, 1897. Lepine and Lyonnet, Lyon med., vol, Ixxxii. p. 187. Hartwig, Deutsch. Arch. f. klin. Med., vol. Ixii. p. 287. Granular Degeneration of the Red Corpuscles. — In certain forms of anaemia, notably in pernicious anaemia, in leuktemia, in pseudoleukaemia, in cases of chronic lead poisoning, in the cachexias associated with severe septic infection, with malaria, syphilis, car- cinomatosis, and the final stages of tuberculosis, red corpuscles may be encountered which contain basophilic granules in variable num- bers. These granules are readily stained with methylene-blue, with the eosinate of methylene-blue, with Ehrlich's haematoxylin-eosin solution, etc. Methyl-green, on the other hand, which is a strong nuclear dye, does not stain the granules. Their size and form are somewhat variable. While the majority are round, others are rod- 66 THE BLOOD. like or biscuit-shaped, and frequently arranged in pairs, resembling gonococci. As a general rule, they are seen in the interior of red blood-corpuscles, but I have found them also free in the plasma (Plates III. and IV.). They may occur in cells of normal size and coloration, in poikilocytes, and even in nucleated red blood-corpus- cles, both of the normoblastic and megaloblastic type. Not infre- quently they are seen in cells which are undergoing polychroma- tophilic degeneration. Engel, Ehrlich, and others have suggested that these granules are essentially karyolytic products ; but Grawitz maintains, and I think rightly so, that they are not of nuclear origin. They may be found at a time when nucleated red corpuscles cannot be demonstrated in the blood ; nucleated cells in which no sign of karyolysis can be discerned may be studded with the granules ; unlike the nuclei of such cells, the granules show no affinity for methyl-green, and, infne, examination of the bone-marrow does not show evidence of the existence of karyolytic processes. Granular red cells are not found in the marrow even when they are numerous in the circulating blood. Grawitz hence regards their presence as indicative of a degenerative change in the hsemoglobin, and has termed the phe- nomenon "granular degeneration." Schmauch has observed similar appearances in the blood of cats. Engel has described such granular corpuscles in the blood of early cat embryos, and I have found them in that of the squirrel. Their significance under such conditions is unknown. In man they are never seen under normal conditions, and their presence may always be regarded as a symptom of haemolysis of a grave type. I have found them in pernicious anaemia, in malaria, and in a case of lymphatic leukaemia. In chlorosis and in the anaemia of chronic nephritis they are absent. In a case of v. Jaksch's anaemia, in which nucleated red corpuscles were quite numerous, I also obtained nega- tive results. In one case of pernicious anaemia in which they were especially numerous many of the cells presented a well-marked polychromatophilia ; but, like Grawitz, I do not believe that the polychromatophilia represents an early stage of granular degenera- tion. Especially important is the presence of such corpuscles in cases of chronic lead poisoning, in which they may be found at a time when clinical symptoms are not as yet manifest. With improvement of the general condition they gradually disappear, and the same holds good for those cases of pernicious anaemia which develop upon the basis of an auto-intoxication referable to the gastro- intestinal tract. In the early stages of phthisis granular degeneration is not observed, but it may occur when a septic condition has supervened. In white mice Grawitz was able to produce granular degeneration PLATE IV, The Blood of Myelogenous Leukasniia. Nole the large increase of the leucocytes, and the presence of nucleated red corpuscles of the nor- moblastic type (r). In addition to the leucocytes, found in iinrnial blood, viz., (2) mononuclear leuco- cytes, devoid of granules, and of polynuclear neutrophilic (3) and eosinophilic leucocytes (4), myelocytes, both of the neutrophilic (5) and eosinophilic (6) variety, are also seen. {Bausch and Lomb, Eye-piece i inch, objective i-i2th.) MICROSCOPICAL EXAMINATION OF THE BLOOD. 67 of the red corpuscles by prolonged exposure of the animals to a temperature of from 37° to 40° C. He therefore suggests that the analogous results which were obtained by Plehn in the case of Europeans after a brief sojourn in the tropics may possibly be referable to the high temperature. LlTEEATUEE. — E. Grawitz, "Ueber Kornige Degeneration d. rothen Blutzellen," Deutsch. med. Woch., 1899, No. 36, p. 585; "Klinische Bedeutung u. experiment. Er- zeugung korniger Degeuerationen," etc., Berlin, klin. Woch., 1900, p. 181; "Granular Degeneration of the Erythrocytes," etc.. Am. Jour. Med. Sci., 1900, vol. cxx. p. 277. Bloch, Deutsch. med. Woch., 1899, V. B. p. 279. Litten, Ibid., No. 44. Behrendt, Ibid., No. 44. " Granular Degeneration of the Erythrocyte," Am. Jour. Med. Sci., 1901, vol. cxxii. p. 266. Nucleated Red Corpuscles. — Three varieties of nucleated red corpuscles may be seen. For their study, however, dried and stained preparations are indispensable, as the nuclei can scarcely be made out in fresh specimens. 1. Normoblasts. — These are nucleated red corpuscles of the size of an ordinary red corpuscle, and appear to be identical with those normally found in the bone-marrow of adults. The nucleus, which frequently shows signs of undergoing division, is usually located centrally, although an excentric position may also occur. They are further characterized by the great avidity with which the nuclei take up the nuclear dyes (Plate II., Fig. 2 ; Plates III. and In specimens of blood in which normoblasts are numerous, as in myelogenous leuksemia, many cells are seen in which the protoplasm surrounding the nucleus is much diminished in amount, and pre- sents a ragged outline. Such cells, in my experience, always pre- sent evidence of polychromatophilic degeneration, and are at times mistaken for poorly stained lymphocytes. They are manifestly undergoing destruction. Free nuclei, which undoubtedly are derived also from normoblasts, may likewise be seen in the blood. Normoblastic red corpuscles are quite constantly found in all forms of severe anaemia, whether this be the result of traumatism, of inanition, or organic disease. In the acute anaemias they are apt to be most numerous ; but even in the chronic forms and in cachectic conditions specimens of blood may be obtained in which one or more are seen in almost every field. In his recent work on Ancemia Ehrlich * cites a case of hemorrhagic anaemia, reported by v. Noorden, in which temporarily the normoblasts were so numer- ous, while hyperleucocytosis existed at the same time, that the blood closely resembled that seen in myelogenous leuksemia. As this condition was associated with an increase of the red corpuscles to almost double their original number, v. Noorden very aptly termed it a "blood-crisis." • Loc. cit., p. 41. 68 THE BLOOD. For the accurate determination of a blood-crisis the following examinations are necessary : a. A determination of the absolute number of the red corpuscles. b. A determination of the ratio between the white and red cells. c. A determination of the ratio between the nucleated red and white cells, in dried specimens, with the aid of the quadratic ocular diaphragm. Example. — Supposing that in a given case 3,500,000 red corpus- cles are found in the cbmm., while the ratio of the white to the red corpuscles is 1 : 100, and that of the nucleated red to the white 1 : 10 ; 3500 nucleated red corpuscles must hence be present in each cbmm. of blood — i. e., one for each thousand of normal red corpuscles. Whenever the number of red corpuscles falls below 1,500,000 and normoblastic cells are not present, the disease will probably end fatally. 2. Megaloblasts. — These bodies are from two to four times as large as the normal red corpuscles, measuring from 10 /i to 20 /i in diameter. They are provided with a large nucleus, which, accord- ing to Ehrlich, never shows signs of undergoing division and does not stain nearly so deeply as the normoblastic nucleus (Plate II., Fig. 2, and Plate III.). In some specimens, indeed, the affinity for nuclear dyes is so little marked that at first sight a nucleus can scarcely be discerned. At times abnormally large megaloblasts are seen — the giganto- blasts of Ehrlich. Under normal conditions megaloblasts are found in the blood of very young infants, but they are present only in small numbers. In contradistinction to the normoblasts, megaloblasts are never found in traumatic anaemia, and even in the chronic anssmias of the severest grade they are hardly ever seen. Even in leuksemia they are usually absent. I have seen a few megaloblasts in a case of V. Jaksch's anaemia infantum pseudoleuksemia, and, together with Dr. Amberg, observed the blood in a case of severe infantile diar- rhoea referable to amoebic dysentery, in which a few isolated cells of this type were encountered. In cancer of the stomach, according to Osier and McCrae,^ mega- loblasts rarely, if ever, occur. In pernicious anfemia, even in the early stages of the disease, megaloblasts are quite constantly present, although they are usually not numerous. As they are found normally only in fcetal bone- marrow, Ehrlich views their presence in the blood as a symptom of retrogressive metamorphosis, and of grave prognostic import. The only exception to this general rule is that form of pernicious anaemia which at times is observed in association with the presence of both- > Osier and MoCrae, N. Y. Med. Jour., May 19, 1900. MICROSCOPICAL EXAMINATION OF THE BLOOD. 69 riocephali in the intestinal tract. In one case of this kind, reported by Askanazy,' the megaloblastic type of blood regeneration dis- appeared after expulsion of the parasites — sixty-seven in number, — and was replaced by the normoblastic type, the case ending in re- covery. From this observation Askanazy concluded that a material difference does not exist between normoblasts and megaloblasts, and that the former develop from the latter. But, as Ehrlich ^ maintains, it is certainly more likely that the megaloblastic degeneration of the bone-marrow is referable directly to the action of certain toxins, and that such a relation between the normoblasts and megaloblasts, as Askanazy assumes, does not exist. 3. MiCROBLASTS. — These are unusually small nucleated red cor- puscles, and only rarely observed. They have been found in cases of traumatic anasmia, but have attracted little attention. The Leucocytes. The leucocytes, or white corpuscles of the blood, as seen in freshly prepared specimens, are roundish or irregularly shaped cells, and mostly larger than the red corpuscles. They are nucleated, and many are distinctly granular in appearance, so much so, in fact, that the nuclei are often hidden from view (Plate II., Fig. 1, A). In a carefully spread specimen some leucocytes will be met with which are endowed with the power of locomotion, creeping over the field of vision by throwing out pseudopodia in a manner analogous to that seen in amcebse. In their general mode of living the motile leuco- cytes, moreover, closely resemble amcebse, and it is most interesting to observe the manner in which these little bodies take up cellular debris, and even obnoxious organisms that may be present in the blood. In malarial blood, for example, in which, as will be shown later, cer- tain amoebic parasites are present, one is frequently able to observe leucocytes approach these bodies and take them up into their interior (Fig. 15). Metschnikoff regards this function of the leucocytes as their most important one. Those leucocytes which possess this power of removing foreign matter from the blood he has termed phagocytes, and, according to his views, the outcome (jf a bacterial invasion, figuratively speaking, will depend upon the buperiority of the organisms engaged in warfare. The term phagocytosis has been applied to the destruction of micro-organisms by leucocytes. General Differentiation of the Various Forms of Leucocytes. — Upon ordinary microscopical examination three varieties of leuco- cytes can be distinguished (Plate II., Fig. 1, A). Some are round, smaller than the red corpuscles, and provided with a large round nucleus, which is surrounded with a very narrow rim of non-granular • Askanazy, " Ueber Bothrioceplialus-Anaemie," etc., Zeit. f. klin. Med., lSfl.5. vol- xxvii. ^Ehrlicli, Die Anaemie, p. 43. 70 THE BLOOD. protoplasm. Others are met with which are likewise round, of the size of an ordinary red corpuscle or somewhat larger, and contain a large single nucleus which is surrounded by a wider zone of non- granular protoplasm. The largest cells, the bodies of which are filled with granular material, often hiding the nucleus from view, are representatives of the third variety. Fig. 15. Phagocytosis. Upon further examination differences may also be demonstrated in the character of the granulations. Some leucocytes will thus be observed in which they are very fine, giving the entire body of the cell a somewhat cloudy appearance, and usually obscuring the nucleus. This may be brought into view, however, by treating the preparation with a drop or two of a 1 per cent, solution of acetic acid. On the other hand, very coarse granulations may be observed in certain leucocytes, while still others, as already pointed out, are apparently non-granular. Ehrlich,' in studying these various granulations in their behavior 1 Ehrlich divides the acid dyes derived from coal-tar into two large groups — i. e., into dyes which color certain granulations even when employed in concentrated solutions of glycerin, and into those which can be employed only in aqueous solutions. The first group comprises : (1) The highly acid bodies belonging to the fluorescin series, viz., eosin, methyl- eosin, erythrosln, coccin, pyrosin .1 and E ; (2; the highly acid nitro-bodies, such as PLATE V, \ Note the size of the N'arious leucocytes, as compared with the red corpuscles at 15. Figs, I, 2 and 6 represent the most coininon forms of the small type of lymphocytes ; 3 and 5 belong to the same group, but are manifestly atypical ; 3 shows the knob-like projections, described in the text ; 4 represents the large type of the lymphocyte, and shows the \'acuolated appear- ance of the protoplasm, which is so Gommonly seen. The metachromatism of the protoplasm, however, does not appear here as in nature. 7 and 8 are representatives of the large variety of mononuclear leucocytes ; 9 maybe classed as a transition form, which is as yet devoid of gran- ules ; 13 represents a neutrophilic myelocyte, 14 an eosinophilic myelocyte. 10 a neutrophilic polynuclear leucocyte, 11 an eosinophile of the same type, and 12 a typical basophilic leucocjle. The preparations were stained with the eosinate of methylene-blue and drawn to scale. (Bausch and Lomb, Eye-piece i inch, objective i-i2lh.) PLATE VI. '^h^'- ^ o If^isjchmidl.fpf, The Blood of Lymphatic LeukEemia. Note the targe increase of the lymphocytes. Two of the red corpuscles are undergoinft granular degeneration (i) ; in a few others polychromatophilia (2) can be discerned ; at 3 an eosino- philic leucocyte is seen with scattered grounds. (Bausch and Lomb, Eye-piece i inch, objective i-i2th.) MICROSCOPICAL EXAMINATION OF THE BLOOD. 71 toward anilin dyes, found that different chemical affinities exist be- tween these minute particles of protoplasm and the reagents em- ployed. Some are thus colored only by acid dyes, others again only by those of a basic nature, while still others are stained only by neutral dyes. These observations are of the greatest importance from a clinical standpoint, and have indeed revolutionized the entire field of hsematology. Differentiation of the Leucocytes according to their Behavior toward the Anilin Dyes. — According to their behavior toward the anilin dyes, Ehrlich^ has divided the granular leucocytes of the blood into eosinophilic or oxyphilic, basophilic, and neutrophilic leucocytes. In this manner the following forms can be distinguished (Plate II., Fig. 2 ; Plates III., IV., V., and VI.) : 1. Small Mononuclear Leucocytes. — These are mostly smaller than the red corpuscles or of equal size. They are devoid of granular matter, each cell being provided with a large, homo- geneous and uniformly staining nucleus, which is surrounded by a narrow rim of protoplasm. In the larger forms, especially, a faint areola may sometimes be seen between the nucleus and the proto- plasm, which is probably owing to artificial retraction. Nucleus and protoplasm both are basophilic, but with certain dyes the protoplasm is colored much more deeply than the nucleus. Within the nucleus one or two nucleoli may sometimes be seen. The pe- riphery of the larger forms is usually shaggy in appearance, and it is not uncommon to find particles of protoplasm in the circulating blood which have manifestly separated from this peripheral margin. In stained specimens the origin of these particles may be recognized from their color, which coincides with that of the parent-corpuscles. As the protoplasm of the small mononuclear leucocytes has no affinity for acid or neutral dyes, these elements appear merely as faintly stained, apparently free nuclei in specimens which have been colored with the tri-acid stain (see page 100). The reaction of the proto- plasm, as shown with the erythrosin method, is strongly alkaline. It contains no glycogen. At times an invagination of the nacleus may be observed, indicating beginning division of the cell. The nuclear figures which result, however, differ materially from those seen in the true polynuclear elements. Stained with methylene-blue or the eosinate of methylene-blue, the protoplasm often appears aurantia ; (3) the two groups of sulpho-acids— «. e., indalin, bengalin, and nigrosin, on the one hand, and the azo-stains, tropseolin, Bordeaux, and Ponceau, on the other. The second group comprises : (1) Fluorescin and chrysolin; (2) ammonium picrate and naphthylamin-yellow ; (3) orange and true yellow. Eepresentatives of the basic stains are : fuchsin (rosanilin), the methyl derivatives of rosanilin, viz., methyl-violet, methyl-green, etc., the phenyl derivatives of rosanilin, rosonaphthylamin, cyanin, safranin, etc. As an example of a neutral stain may be mentioned the picrate of rosanilin. lEhrlich, Farbenanalytische Untersuchungen z. Histologie u. Klinik d. Blutes, Berlin, 1891. 72 THE BLOOD. coarsely granular. This appearance, however, is not due to the presence of free protoplasmic granules, but to nodal thickenings of the reticulum. Similar appearances are seen in the nucleus. With Ehrlich's stain, on the other hand, both nucleus and proto- plasm appear perfectly homogeneous. Abnormally large forms arc notably seen in lymphatic leuksemia, but occur also in normal blood. Ehrlich states that it is scarcely likely that their true nature will be overlooked, if the characteristics just described are borne in mind. I must confess, however, that the diiferentiation of these large lymphocytes from the large mononuclear leuco- cytes proper is not always an easy matter, and I have often been puzzled to classify properly cells of this type. In typical speci- mens, in which the protoplasm is stained intensely by the basic component of the eosinate of methylene-blue, and in which an apparent vacuolization of the entire protoplasmic portion of the cell is not uncommonly seen, there is, of course, no difficulty ; but there are others in which these characteristics are not so apparent, and then the protoplasm and nucleus are both about evenly and imperfectly stained. These forms, I think, may readily be con- founded with the large mononuclear elements proper, and possibly represent transition-forms between the two groups of cells. Together with Dr. Amberg, I observed the blood of a very anaemic child in which the condition was apparently secondary to a protracted attack of amcebic dysentery, and in -which the large lymphocytes, as they are perhaps best termed, were quite numerous. But in adults also I have usually seen representatives of this group, even under normal conditions, where the eosinate of methylene-blue was used as a stain. With this dye the protoplasm commonly stains metachromatieally, and in this respect they differ from the small variety. Ortho- chromatism, however, also occurs (see Plate V.). As the small mononuclear leucocytes are formed practically only in the lymph-glands, they have been termed lymphogenie leucocytes or lymphocytes. Under normal conditions the lymphocytes constitute from 22 to 25 per cent, of the total number of the leucocytes. At birth, however, from 50 to 60 per cent, of the total number belong to this order ; larger numbers are met with also during childhood than in adults, and the normal proportion is usually not reached before the fourteenth year. Under pathological conditions the greatest absolute as well as rela- tive increase is observed in cases of lymphatic leukfemia. A relative increase alone occurs in healthy infants, in various diseases of infancy, notably those affecting the gastro-intcstinal tract, in chlorosis, per- nicious ansemia, secondary syphilis, in the late stages of typhoid fever, in certain cases of Basedow's disease, hasmpphilia, goitre, etc. (see also page 93). MICROSCOPICAL EXAMINATION OF THE BLOOD. 73 2. Large Mononuclear Leucocytes. — These are from two to three times as large as the red corpuscles, and provided with a large single nucleus, which is oval or elliptical in form and surrounded by a wide zone of non-granular protoplasm. In specimens stained with the tri-acid stain beginners are very apt to overlook this form, as the nucleus and particularly the protoplasm are often very faintly stained. The nucleus and protoplasm are both basophilic, but the latter, in contradistinction to the protoplasm of the lymphocytes, less so than the nucleus. In these cells also the protoplasm and nucleus do not appear perfectly homogeneous when stained with methylene- blue or the eosinate of methylene-blue, but show evidence of the existence of a reticulum which is coarser in the nucleus than in the protoplasm (Plate V.). These forms are by some thought to represent a later stage in the development of the small mononuclear variety, but Ehrlich ' still maintains their independent origin from the bone-marrow, and to some extent perhaps also from the spleen. They occur in increased numbers in cases of chronic malaria, in measles, in the late stages of scarlet fever, apd in many of the diseases in which the small mononuclear elements are increased. I have met with a considerable relative increase of this variety in one case of Addison's disease shortly before death. In one of my patients, a woman aiged sixty-three, attention first was directed to the existence of a large sloughing epithelioma of the neck by the discovery of 21 per cent, of the large mononuclear leucocytes. Under normal conditions the percentage varies between 1 and 2. 3. Transition-forms. — These develop directly from the large mononuclear leucocytes. They are still mononuclear, but the nucleus is greatly invaginated, indicating approaching division. As a general rule, no granules are observed, but at times they do occur, when they are neutrophilic in character. In specimens stained with the tri-acid stain the nucleus is colored somewhat deeper than in the second variety. Together with the large mononuclear elements they constitute from 2 to 4 per cent, of the total number of leucocytes. 4. The Neutrophilic Polynuclear Leucocytes. — These cells are a little smaller than the large mononuclear leucocytes and the transition-forms, and are filled with very fine neutrophilic granules, the e-granulation of Ehrlich. The protoplasm itself is finely re- ticulated, but this is generally overlooked unless the specimen is especially stained with methylene-blue or a similar stain. The nucleus is a long body, which is twisted upon itself into irregular forms, some- times resembling the letters S, Y, E, Z. At other times it presents a broken appearance, conveying the impression that several nuclei are present. Hence their original name — polynuclear leucocytes. As 1 Ehrlich, Die Anaemie, p. 49. 74 THE BLOOD. Ehrlich has suggested, however, the polynuclear appearance is prob- ably referable to post-mortem changes, the condition of the nucleus being in reality polymorphous. In accordance with this view, they are hence also spoken of as polymorphonuclear neutrophilic leu- cocytes. The nucleus stains readily with all nuclear dyes, while the protoplasm shows a marked affinity for the greater number of acid dyes. Its reaction, as tested with acid erythrosin, is alkaline, but less so than the protoplasm of the lymphocytes. In health a glyco- gen reaction is not obtained. The nucleus is coarsely reticulated, and generally shows evidence of a central nodal thickening in its lobes. According to some observers, the polymorphonuclear neutrophilic leucocytes represent a later stage in the development of the small and large mononuclear cells. Ehrlich,' however, insists that the greater number enter the blood from the bone-marrow, where they develop from the mononuclear neutrophilic leucocytes — the myelo- cytes — or bone-marrow cells proper (see page 78), but he admits that a small number may be derived directly from the transitional forms in the blood-current. In this connection it is especially interesting to note that while basophilic and oxyphilic granules are found in the blood of all ver- tebrate animals, the neutrophilic granulation occurs only in man and the ape. Of late, a diminution in the number of the neutrophilic granules has attracted some attention in association with the acute hyperleu- cocytoses. Ewing^ states that this abnormality may progress till very few granules are left, while their complete absence is seen principally in chronic leuksemia. He adds that this phenomenon is associated usually with marked nuclear changes of a degenerative character, which he describes as follows : in fresh or dry specimens the nuclei stain less densely with basic dyes, their outlines are irregular, and the lobes shrunken. The degeneration may follow the type of karyolysis with swelling and loss of chromatin, or of karyorrhexis with hyperchromatosis and subdivision of lobes. In acute leucocytosis the former type is more usual, but in leukse- mia the latter form is seen abundantly. While the lobes of the normal polynuclear leucocytes are almost invariably connected by a thread of chromatin, many of the cells in severe acute leucocytosis show complete subdivision of the nucleus into three to six separate segments. This fragmentation of the nucleus was noted first by Ehrlich in a case of hemorrhagic smallpox, and is of frequent occur- rence in fresh exudates. I have observed the same together with Dr. Amberg in a case of infantile ansemia associated with amoebic dys- entery, in which extensive hyperleucocytosis, however, did not exist. The loss of neutrophilic granules in the polymorphonuclear leuco- ' Ehrlich, loc. cit., p. 71. ' Ewlng, loc. oit., p. 113. MICROSCOPICAL EXAMINATION OF THE BLOOD. 75 cytes in cases of chronic leuksemia is sometimes most striking, and 1 was much interested to note in a recent case that while the poly- nuclear elements were mostly devoid of granules, the neutrophilic myelocytes (see below) were quite normal in appearance. In other diseases deficiency of granules is in my experience not necessarily associated with acute hyperleucocytosis, but may occur under the most diverse conditions, which as yet admit of no classification. Normally the polynuclear neutrophilic leucocytes constitute from 70 to 72 per cent, of all leucocytes. The most common forms of hyperleucocytosis are referable to an increase in the number of these elements (see page 81). All pus-corpuscles, moreover, according to Ehrlich, belong to this class. 5. The Oxyphilic or Eosinophilic Leucocytes. — In size and general appearance these cells resemble the polynuclear neutro- philic leucocytes. But they differ from these in the absence of neu- trophilic granules, and the presence, instead, of large ovoid or roundish, highly refractive, fat-like granules — ^the a-granulation of Ehrlich. These granules are stained only with acid dyes, such as eosin and acid fuchsin, and such leucocytes have hence been termed oxyphilic or eosinophilic leucocytes. Barker,' moreover, has shown that these granules contain iron. Like the polynuclear neutrophilic leucocytes, they are also phagocytic. The nucleus is usually bilobed and coarsely reticulated. According to some observers, the eosinophilic leucocytes represent the senile stage in the development of the small mononuclear leuco- cytes. But Ehrlich still regards them as independent bodies formed in the bone-marrow from mononuclear eosinophilic cells, analogous to the formation of the polynuclear neutrophilic leucocytes from the mononuclear neutrophilic cells. Normally the percentage of eosinophilic leucocytes varies between 2 and 4. An absolute increase in their number is observed in all uncompli- cated cases of myelogenous leukaemia, while a relative increase is inconstant. Statements to the contrary have been made by many observers, but, as Ehrlich suggests, this is undoubtedly owing to a confusion between the terms absolute and relative. According to Zappert,^ 50 to 100 eosinophilic leucocytes in the cbmm. of blood should be regarded as the lowest normal values, 100 to 200 as the average, and 200 to 250 as the highest normal figures. Supposing then that in a given case the percentage of eosinophiles is 3.5 ; this would, of course, be a perfectly normal percentage. But if at the same time the total number of leucocytes is 400,000, it is apparent at once that we are dealing with a considerable absolute 1 L. F. Barker, Johns Hopkins Hosp. Bull., 1894, p. 93. 2 J. Zappert, " Ueber d. Vorkomnien d. eosinophilen Zellen im anaemischen Blut," Zeit. f. klin. Med., vol. xxiii. 76 THE BLOOD. increase, corresponding in this case to 14,000 eosinophilic leucocytes — i. e., an increase of fifty-six times the maximum number observed under normal conditions. It may be stated as a general rule that whenever an absolute increase in the number' of the eosinophilic leuco- cytes is not found in a case of supposed myehgenous leukcemia this diagnosis may be abandoned, providing that complications, such as septic processes, do not exist at the same time. In sepsis the number of eosinophilic leucocytes is materially diminished, and in some cases they may be altogether absent. Exceptions, however, occur, and Ehrlich cites a case in which the total number was still from 1400 to 1500 in the cbmm., although the percentage had diminished from 3.5 to 0.43. Aside from myelogenous leukaemia, an increase in the number of the eosinophilic leucocytes has been observed in various other dis- eases ; but it is scarcely likely that any of these would be mistaken for leukaemia, especially if the other blood-changes which occur in this disease are borne in mind (see page 89). Eosinophilia has thus been noted in bronchial asthma, in certain diseases of the bones, the skin, the nervous system, in the helminthiases, in trichinosis, in malignant disease, in the post-febrile period of many of the acute infectious diseases, in gonorrhoea, etc. In diminished numbers the eosinophilic cells are found during the process of digestion, in pneu- monia, in the course of most of the acute infectious diseases, follow- ing castration, etc. (see also Eosinophilia). 6. Basophilic Leucocytes. — In normal blood these rarely exceed 0.5 per cent, of the total number of leucocytes. The size of the cells varies from 3.5 /i to 14 /i in diameter. They are usually rounded or oval, but may also be oblong, and may then have a length of 22 //. The cell substance is clear and homogeneous, but imbedded within there are granules, the y- and o -granulations of Ehrlich, which ap- pear to be identical with those observed in the so-called mast-celh, found in connective tissue especially. The same term has hence been applied to this variety in the blood. The individual granules vary somewhat in size and distribution, and are characterized by their affinity for basic dyes. They do not take on a pure color, however, but stain metachromatically. With cresyl-violet R, for example, they are colored almost a pure brown, and with eosinate of methylene-blue they take on a violet color. The nucleus is stained with great difficulty. It is lobulated, and almost always placed excentrically. It is oval or rounded, and has an almost uniform diameter of 4 fi. When the cell itself is of a less diameter, the nucleus comprises almost the entire cell and is covered by only a very thin layer of granules. In specimens stained with carbol- toluidin-blue, and differentiated with glycerin-ether, the granules appear dark red in color, while the nucleus is colored blue. In specimens stained with the tri-acid stain the granules are colorless, MICROSCOPICAL EXAMINATION OF THE BLOOD. 11 and the cells hence appear as light polymorphonuclear formations, which are apparently devoid of granulations. According to Harris/ the granules are composed of mucin, and the cells elaborate the mucin of the connective tissue. Ehrlich supposed that the mast- cells originated from the connective-tissue cells as a result of hyper- nutrition, but it is more likely, as Harris suggests, that they are derived from the large mononuclear leucocytes. An increase in the number of mast-cells is almost exclusively observed in myelogenous leukaemia, and is hence of great diagnostic importance. This increase is constant and absolute, and may even exceed the increase of the eosinophilic leucocytes. Ewing ^ states that he constantly failed to find mast-cells in the better class of healthy subjects, while in hospital and dispensary cases with minor ailments they appeared to be more numerous. Formerly I also found mast-cells only exceptionally, but since I have used the eosinate of raethylene-blue as a matter of routine in my laboratory I rarely meet with specimens of blood, no matter from what class of patients, in which they are absent. Neusser's Perinuclear Basophilic Granules. — A few years ago Neusser ^ drew attention to the fact that basophilic granules are not infrequently seen arranged about the nuclei of the mononuclear and polynuclear leucocytes. The presence of these granules he, as well as Kolisch,* regarded as characteristic of the so-called uric acid diath- esis. As tubercular disease, moreover, is usually not seen in such cases, Neusser regards their presence in cases of phthisis as a favorable symptom. Futcher,^ on the other hand, was unable to confirm these conclusions, and my own observations likewise are opposed to those of Neusser. I was able to demonstrate the pres- ence of these granules both in health and disease in almost every case, and was even led to the conclusion that their absence in a sup- posedly healthy individual may be regarded as presumptive evidence of the existence of some morbid process. Whether this conclusion will be borne out by further investigations remains to be seen. But it appears to be certain that in malignant disease the granules are either absent or present in greatly diminished numbers. In two cases of gastric ulcer and in one of acute gonorrhoea, however, I was likewise unable to find them." In suitably stained specimens the granules appear as greenish- black or entirely black little dots, which are irregularly scattered over the surface of the nucleus. Their size varies considerably. Specimens are thus encountered in which the granules are as fine as ' H. T. Harris, " Histology and Microchemic Eeactions of Some Cells to Anilin Dyes," Phila. Med. Jour., 1900, p. 757. ■^ Ewing, On the Blood, Lea Bros., Phila., 1901, p. 143. s Neusser, Wien. kiln. Woeh., 1894, p. 71. * Kolisch, Ibid., 1895, p. 797. 5 Futoher, Johns Hopkins Hosp. Bull., May, 1897. * C. E. Simon, " On the Presence of Neusser's Perinuclear Basophilic Granules in the Blood," Am. Jour. Med. Sci., vol. cxvii. p. 139. 78 THE BLOOD. ordinary neutrophilic granules, while in others they are much larger, and in some cases droplets may be seen which cover nearly the entire nucleus. They may be found in all forms of leucocytes described, but are most numerous in the polymorphonuclear and small mononuclear cells. Ehrlich has expressed the view that these granules are arte- facts, and states that they are observed ,only exceptionally when strictly pure solutions, made from the crystalline dyes of the Actien- gesellschaft fiir AnilinfarbstofPe in Berlin, are used. Whether this view is correct I am not prepared to say, as my examinations were made with the Griibler stains. A relation between their presence and the elimination of uric acid or xanthin-bases does not exist. 7. Neutrophilic Myelocytes. — These are essentially large mononuclear leucocytes, the protoplasm of which contains more or less numerous neutrophilic granules. Their size, however, is subject to considerable variation. On the one hand, they may be larger than all other elements of the blood — the so-called myelocytes of Cornil ; while others are observed which are scarcely larger than an ordinary red corpuscle — the so-called myelocytes of Ehrlich. The nucleus is large, usually centrally located, and possesses only a feeble affinity for dyes. Unlike the polynuclear neutrophilic leucocytes, they are never amoeboid. According to the school which regards the polynuclear neutro- philic leucocytes as the mature forms of the lymphocytes, the neutrophilic myelocytes represent an arrested or perverted form of development of the large mononuclear cells. Ehrlich, on the other hand, regards the neutrophilic myelocyte as the bone marrow- cell proper, and as the young form of the polynuclear neutrophilic leucocyte. Under normal conditions they are never found in the blood, and Ehrlich teaches that their presence in considerable numbers may be regarded as indicating the existence of myelogenous leukaemia. In smaller numbers they have been found in a case of lymphosarcoma with metastases in the bone-marrow ; further, in severe post-hemor- rhagic anaemia, in a case of poisoning with mercury, in the pseudo- leukemia of infants, in torpid scrofula, and, what is especially im- portant, in some of the acute infectious diseases. Engel ^ thus found that in diphtheria occurring in children myelocytes can often be demonstrated in the blood, and that a high percentage, viz., 3.6 to 16.4, of the total leucocytes is observed only in severe cases, and ren- ders the prognosis unfavorable. In mild cases they are not often seen, and when present occur only in small numbers. In pneumonia myelocytes are either absent or present only in small numbers at the beginning of the disease, while at the time of the crisis or imme- ' Engel, Deutsoh. med. Wooh., 1897, yol. xxiii. Ko. 8. MICROSCOPICAL EXAMINATION OF THE BLOOD. 79 diately thereafter they become more numerous, and in some cases may number 12 per cent, of all neutrophilic cells. Small numbers of myelocytes may further be seen in the various anaemias from whatever cause. They have been noted in pernicious anaemia, in the anaemia of syphilis, and, as I have stated, in the pseudoleukaemia of v. Jaksch, in association with malignant growths, etc. In the child, already referred to, in which a notable anaemia developed as the result of amoebic dysentery. Dr. Amberg counted 9 per cent. Neusser also records the presence of neutrophilic myelo- cytes in anaemia, asphyxia, acute mania, etc. ; and Ewing states that they have been found in considerable numbers in rickets, osteomyelitis, and osteomalacia. 8. Eosinophilic Myelocytes. — These represent the eosino- philic homologue of the form just described. Their size also may vary very much, and specimens may be met with which are a great deal larger than the polynuclear variety. According to Ehrlich, they are likewise formed in the bone-marrow, and represent an earlier stage in the development of the polynuclear eosinophilic leucocyte. Their presence is confined largely to the blood of myelogenous leu- kaemia and the pseudoleukaemia of infants. Mendel ' found them in one case of myxcedema, Tiirck ^ reports that they are occasionally seen in some of the infectious diseases, and Bignami claims to have seen them in pernicious malaria. 9. Small Neutrophilic Pseudolymphocytes. — These bodies, according to Ehrlich, are produced by direct division of the poly- nuclear neutrophilic leucocytes. They are about as large as the small lymphocytes, and provided with a single deeply staining nucleus. The narrow zone of protoplasm which surrounds the nucleus con- tains neutrophilic granules. They may be distinguished from the small forms of myelocytes by the greater intensity with which the nucleus takes up the nuclear dyes and the smaller amount of proto- plasm. Ehrlich ' states that he first saw these bodies in a case of hemorrhagic smallpox, but that they are found also in recent pleural effusions. He suggests that their study may be of importance in deciding the origin of the transitory hyperleucocytoses, which ac- cording to some are due to a destruction of leucocytes, and accord- ing to others to an altered distribution. 10. Irritation-poems. — These are mononuclear, non-granular cells, the protoplasm of which is stained a rich brown by the tri- acid mixture. The nucleus is round, often excentrically located, and colored a bluish green. The smallest forms are somewhat larger than the lymphocytes. According to Tiirck,^ who first de- ' K. Mendel, "Ein Pall von myxcedematosem Cretinismus," Berlin, klin. Woch., 1896, No. 45. 2 Tiirck, Klinische Untersnchungen iiber d. Verhalten d. Blutes bei akuten Infec- tionskrankheiten, Wien u. Leipzig, 1898. ' Ehrlich, Die Anaemie, loc. cit. * Loc. cit. 80 THE BLOOD. scribed these cells, they are met with under the same conditions as the myelocytes. Possibly they represent an early stage in the development of the nucleated red corpuscles. In addition to the above, still other forms of leucocytes have been described, especially in leuksemic blood, but so little is known of these that it is unnecessary to enter into their description at this place. > Variations in the Number of the Leucocytes. — While the number of red corpuscles is subject to very slight variations under physiological conditions, that of the leucocytes varies within fairly wide limits, being influenced by the age and sex of the individual, pregnancy, the process of digestion, the bloodvessel from which the specimen is taken, etc. According to Osier, the number of leucocytes per cbmm. of blood, obtained from the finger or the ear, normally varies between - 5000 and 7000, so that taking 5,000,000 as the average number of red corpuscles per cbmm., the ratio between the two would vary between 1 : 714 and 1 : 1000. But, as Cabot points out, the actual number may be still lower than 5000 and higher than 7000 without there being symptoms of definite illness. Generally speaking, lower figures are met with in persons who are somewhat ill-nourished, while higher figures are encountered in persons of special vigor and good nutrition. Before concluding then that in a given case the number of leucocytes is below or above the normal, an idea should, if possible, be formed of what constitutes the normal for that particular individual. It would hence be better to extend the normal limits to 3000 on the one hand, and 10,000 on the other. An increase in the number of leucocytes, to which condition the term leucocytosis was first applied by Virchow, is met with under both physiological and pathological conditions. As Goldscheider rightly suggests, it would be better, however, to restrict the term leucocytosis to indicate the number of leucocytes in a general way, and to speak of an increase in their number as hyperleucocytosis, and of a diminution in their number as hypoleuoocytosis. According to Ehrlich, furthermore, it is necessary to distinguish between active and passive hyperleucocytosis, meaning by active hyperleucocytosis that form in which the increase in the number of the leucocytes principally affects the phagocytic elements, viz., the polynuclear leucocytes, while the mononuclear elements only are increased in the passive form. Ehrlich further subdivides the active hyperleucocytoses into the following groups : 1. The polynuclear hyperleucocytoses. a. Polynuclear neutrophilic hyperleucocytosis. b. Polynuclear eosinophilic hyperleucocytosis. MICROSCOPICAL EXAMINATION OF THE BLOOD. 81 2. The mixed hyperleucocytoses in which the granule-bearing mononuclear elements take part — myelaemia. Polynuclear Neutrophilic Hyperleucocytosis. — In this form of hyperleucocytosis, as the term indicates, the increase in the num- ber of the leucocytes principally affects the polynuclear neutrophilic elements. Exceptionally it may be associated with a polynuclear eosinophilic hyperleucocytosis, as well as with a lymphocytosis, but as a general rule both eosinophilic leucocytes and lymphocytes are much diminished. This diminution, moreover, may not only be relative, but even absolute. Under this heading the following forms may be considered : Physiological Hyperleucocytosis. — An increase in the number of leu- cocytes, occurring in health, is noted during the process of digestion, in pregnancy, following cold baths, after severe muscular exercise, etc. In infancy also a hyperleucocytosis is observed quite constantly, and, according to Hayem,' is most pronounced during the first eighty hours of life, when about 18,000 leucocytes are found on an aver- age in the cbmm. of blood. This number, however, soon dimin- ishes, and during the first month about 8000 leucocytes may be regarded as the normal. In children aged from several months up to the first year this figure further drops to about 6000. Owing to the intensity*.with which the blood of infants generally reacts to all manner of stimuli, however, it is difficult to set down definite fig- ures to express the normal. It is thus not uncommon to observe a hyperleucocytosis corresponding to the first months of life even as late as the first and second year in feebly developed children, but which in other respects may be quite healthy. The process of diges- tion, moreover, as will be shown later, very materially influences the degree of leucocytosis, so that at this time of life one should be very careful in drawing inferences from the blood-count alone as to the existence of disease. Associated with an absolute increase in the number of the poly- nuclear neutrophilic leucocytes we find in the leucocytoses of infants quite constantly also a relative lymphocytosis. An idea of the marked increase in the number of the leucocytes occurring during the process of digestion, constituting the physio- logical digestive leucocytosis of Virchow, may be formed from the accompanying diagram (Fig. 16). It is especially pronounced after a preliminary period of fasting, and following a meal rich in proteids. Occasionally it is not seen, even in health, but such cases are ex- ceptional. In infants the highest grades are observed, and Cabot cites a case, reported by Schiff,^ in which 19,500 leucocytes were counted one hour after birth, 27,625 after the first meal, and 36,000; after the fourth meal. 1 Hayem, Compt. rend, de la soc. de biol., 1867, p. 270. 2 Schiff, Zeit. f. Heilk., vol. xi. p. 30, and 1890, p. 1. 82 THE BLOOD. Under pathological conditions, and notably in gastro-intestinal diseases, this form of hyperleucocytosis is frequently absent. This is notably the case in carcinoma, and for a time it was thought that the absence of a digestive hyperleucocytosis could be regarded as a valu- able symptom in the differential diagnosis between carcinoma and other diseases of the stomach.' Unfortunately, further investigations MiU. Fig. 16. Red corpuscles in 1 cbmm. of blood. A.M. P.M. 10 12 2 4 6 8 10 12 2 A.M. 4 6 5,5 5,' 5,3 5,2 5,1 5,0 =^^^.^ —* V ^ ^^ z^ i ^-"V 7^^-="^^^^ I \ / \ t ^^^ A^ \2 t ^^ V Hb. In 1 cbmm. of blood. Gms. — ~~ — ~~ — . ■*" n — — — — — — — ' — — \ 0,135 / \ ^ \ / \ 0,130 ^ \ " \ \ / 0,125 ^ / Leucocytes n 1 cbmm. of blood 8000 / \ / \ / / \ / ^ / \ / \ s / \, / S f\ \ / \ 1 \, / \ / \ / \ -6000 / "" / 5500 1 / Knnn ( Showing the diurnal variationa in the number of red oorpuscles, the amount of htemoglobin, and the number of leucocytes. (Taken from Reinekt.) have shown that cases of cancer may occur, on the one hand, in which digestive hyperleucocytosis does occur, while, on the other, it may be absent in other diseases, both functional and organic. In ' .T. Schneyer, "Das Verlialten d. Verdauungsleukooytose bei ulcus rotundum u. car- cinoma ventriouli," Zeit. f. kiln. Med., vol. xxvii. p. 249. E. Miiller, Zeit. f. Hellk., 1890, p. 213. MICROSCOPICAL EXAMINATION OF THE BLOOD. 83 anaemic individuals, from whatever cause, a digestive hyperleucocy- tosis may thus not be observed unless an especially large meal has been ingested, and in such cases indeed a subnormal number of leucocytes may be encountered.' The question of digestive hyper- leucocytosis is, however, nevertheless a most interesting one and calls for further investigation. According to Marchetti, it depends essentially upon the digestive and absorptive power of the stomach, and its occurrence or non-occurrence in carcinoma is thus essentially an index of the degree of functional impairment of the organ. In its study certain precautions must be observed : a. The first blood-count should be made after the patient has fasted for about seventeen hours. h. After this period he receives a test-meal, consisting of from 200 to 1000 c.c. of milk, and one or two eggs, the amount varying with the condition of the patient. e. Further blood-counts are made one, two, and three hours later. d. The existence of a digestive hyperleucocytosis should only be regarded as proved if an increase of at least 1500 cells occurs, pro- viding that maximal amounts of food have been taken. If smaller amounts have been given, an increase of 100 cells is sufficient to establish its existence, provided that the same result is observed on repeated examination. In the digestive hyperleucocytoses the increase in the number of the leucocytes not only affects the polynuclear neutrophilic elements, but also the lymphocytes, while the eosinophilic leucocytes are, rela- tively at least, much diminished. A very marked hyperleucocytosis is also frequently noted after a cold bath. According to Thayer,^ this may amount to even 284.6 per cent. In twenty cases of typhoid fever he found on an aver- age 7724 leucocytes before, and 13,170 after the usual Brand bath. In his own person, while in health, on one occasion the leucocytes, which numbered 3250 before the bath, rose to 12,500 twenty min- utes later. A prolonged cold bath, on the other hand, diminishes their number. Hot baths have exactly the opposite effect, viz., those of short duration produce a decrease, those of long duration an increase. Violent muscular exercise, as well as massage, likewise produces a temporary hyperleucocytosis. " In all these cases the increase affects both lymphocytes and the polymorphonuclear leucocytes. The physiological hyperleucocytosis observed in pregnancy is par- ticularly marked during the last five months, and appears to occur quite constantly in primiparse, while in multiparse exceptions are common. In an analysis of thirty-one cases Rieder ^ noted a hyper- 1 Eieder, Beitrage zur Kenntniss d. Leukocytose, 1892. 2 Thayer, Johns Hopkins Hosp. Bull., April, 1893. 'Eieder, loc. cit. 84 THE BLOOD. leucocytosis in twenty, the number of leucocytes varying between 10,000 and 16,000, with an average of 13,000 per cbmm. This increase in the number of the leucocytes continues for a variable period after parturition, and is apparently connected with the function of lactation. It is especially interesting to note that a digestive hyperleucocytosis does not occur, while that referable to pregnancy exists, and it is quite likely, as Cabot ' suggests, that this form is in reality a prolonged digestive hyperleucocytosis. The increase affects both lymphocytes and the polyuuclear neutrophilic leucocytes. Pathological Hyperleucocytosis. — In disease an increase in the number of the polynuclear neutrophilic leucocytes is observed very frequently, and is often a matter of great importance in differential diagnosis. In the acute infectious diseases hyperleucocytosis is the rule. Gen-- erally speaking, the increase in the number of the leucocytes is here directly proportionate to the intensity of the infection and the power of resistance on the part of the individual patient It may thus happen that no hyperleucocytosis occurs at all when the infection is extremely virulent, and the power of resistance practically nil, in consequence of pre-existing disease; or similar influences, even though the disease is one in which hyperleucocytosis generally occurs. This is seen especially well in pneumonia, in which death almost in- variably occurs when a hyperleucocytosis does not develop, unless indeed the infection has been so mild as not to cause an increased invasion of leucocytes. The development of a well-marked hyper- leucocytosis in diseases in which this, is the rule is no guarantee, however, that the patient will recover, although his chances are certainly much better. In pnemnonia the increase in the number of the leucocytes is usually marked. According to Cabot,^ it amounts on an average to about 24,000 above the normal. The hyperleucocytosis sets in ■quite early and persists until the time of the crisis, when it rapidly disappears. When the disease terminates by lysis the return to the normal is more gradual. A pseudocrisis is not accompanied by a fall in the number of the leucocytes. When resolution is delayed or complications occur, the hyperleucocytosis persists. In erysipelas, as in pneumonia, the leucocytosis is generally pro- portionate to the intensity of the morbid process, and also terminates by crisis. The increase in the number of the leucocytes, according to Rieder,' amounts to about 15,000 above the normal. In diphtheria a well-marked increase is the rule, and, with the ex- ception of very mild or extremely severe cases, is of constant occur- rence. It is interesting to note that excepting a temporary diminution immediately after the injection the leucocytosis is in no wise influ- ' Cabot, Clinical Examination of the Blood, Wm. Wood & Co., 1897. ' Cabot, loc. cit. ' Eieder, Die Leukocytose, 1892. MICROSCOPICAL EXAMINATION OF THE BLOOD. 85 enced by the antitoxin treatment. Besredka/ tiowever, states that the grade of the polymorphonuclear neutrophilic hyperleucocytosis, after the administration of the serum, indicates the prognosis. Thus, if one or two days after the injection the percentage of these cells is 60 or above, the prognosis is good ; with a higher temperature and 50 per cent., it is bad ; while if below 50 per cent., the disease is fatal. In septic conditions of whatever origin hyperleucocytosis is of constant occurrence unless the infection is very mild or very severe. As in pneumonia and diphtheria, absence of hyperleucocytosis may usually be regarded as a symptom of grave prognostic signifi- cance. The degree of increase may vary widely, and is always directly proportionate to the extent and the intensity of the inflamma- tory reaction. In suspicious cases a careful examination of the blood should always be made. It is equally as important in such cases as the examination of the sputum in cases of suspected phthisis or of the tonsillar coating in cases of suspected diphtheria. Especially important also is the study of the leucocytosis in appendicitis. In a recent article on blood examination as an aid to surgical diagnosis Bloodgood ^ states the following : Observed within forty-eight hours the number of white blood-cells is in the majT)rity of instances of great value in pointing to the extent of the inflammatory condition of and about the appendix. Cases of recurrent appendicitis or of appendicitis suffering from the first attack, first observed practically at the end of the attack when the clinical symptoms are subsiding, rarely show an increase in the white cells. In a few instances, first observed within forty-eight hours after the beginning of the attack, but when the symptoms are subsiding, there have been a few leucocyte-counts of 15,000, which have fallen rapidly within a few hours to 10,000 and 7000. In the cases admitted within forty-eight hours with acute symptoms, if on account of the clinical picture operation has been delayed, a falling leucocytosis has always been observed. These patients have recovered, and at a later operation the appendix was found to be the seat of a diffuse inflammation, but there was no evidence of pus outside the appendix. In one case admitted sixteen hours after the beginning of the attack the leucocytes fell in ten hours from 17,000 to 13,000, and in twenty-four hours to 11,000, associated with disappearance of the symptoms. With one exception, the highest first leucocyte-count in this group has been 17,000, falling in a few hours to 12,000, 9000, or even lower. A patient admitted twenty hours after the beginning of the acute attack had a leucocytosis of 22,000 ; the clinical symptoms, however, were not very marked. 1 Besredka, Ann. de I'Inst. Pasteur, 1898, vol. xii. 5, p. 305. Cited by T. E. Brown, Maryland Med. Jour., April, 1901. ^ J. C. Bloodgood, " Blood Examinations as an Aid to Surgical Diagnosis," American Medicine, 1901, p. 306. 86 TSE BLOOD. The patient was observed eight hours ; during this period the leuco- cytes fell to 16,000 and the local symptoms practically disappeared. Within the next twenty-four hours the leucocytes were 11,000, then 8000, 7000, and 6000. Although this patient with a leucocytosis of 22,000 at the end of twenty hours, recovered, and there is every reason to believe that the inflammatory condition about the appendix subsided, nevertheless it is an exception to the general rule, and it would be safer, I believe, to operate in those cases of acute appendicitis observed within the first forty-eight hours with a leuco- cytosis of 20,000. In acute diffuse appendicitis with operation and recovery the highest count observed was 25,000 thirty-six hours after the beginning of the attack. At operation in this case intense inflammation and a large amount of exudate were found about the appendix. In gangrenous appendicitis with operation and recovery the leuco- cytosis is higher (25,000-35,000) and rises more rapidly. As Blood- good says, the study of the leucocytosis is here of the greatest importance in the early recognition of a grave inflammatory condi- tion of the appendix, which without doubt would lead to general peritonitis and death unless early operation be instituted. A very high leucocytosis within forty-eight hours after the beginning of the attack is suggestive, but not at all positive, of beginning peritonitis. The leucocyte-count, however, does not seem to help in such cases with regard to prognosis. After the second day in cases in which the peritonitis has been present longer Blood- good never has observed recovery with a low leucocyte-count. If the leucocytosis remains still high at this period, how^ever, the prognosis seems better for ultimate recovery after operation. In intestinal obstruction an increase of the leucocytes associated even with very slight symptoms is of the highest importance in the early recognition of the lesion. Bloodgood states that in a large group of cases the leucocyte-count may rise to . 20,000 within twelve hours after the beginning of the obstruction. Within the first twelve to twenty -four hours a few observations would demonstrate that if the leucocyte-count rise above 25,000 or 30,000, the proba- bilities are that one will find gangrene of the obstructed loops or beginning peritonitis. If observed on the second or third day after the beginning of the symptoms, it is difficult to make a difler- ential diagnosis with regard to gangrene or peritonitis. After the third day, in cases in which there is no gangrene, and no peritonitis, or in which the auto-intoxication is not yet very grave, the leucocytes still remain high — 15,000—23,000 — according to the de- gree of obstruction : complete, higher ; partial, lower. In the presence of gangrene-peritonitis or grave auto-infection, the leucocytes begin to fall. If the patient is admitted after the third or fourth day, with a history of intestinal obstruction, and still has a high leuco- MICROSCOPICAL EXAMINATION OF THE BLOOD. 87 cyte-count, the prognosis is good for operation. If the count is low, and especially if it is below 10,000, the probabilities are that on operation extensive gangrene-peritonitis will be found ; or the patient will be so depressed by auto-intoxication that reaction does not follow relief of the obstruction. In acute articular rheumatism the degree of hyperleucocytosis is proportionate to the severity of the attack. The average increase beyond the normal, according to Cabot, amounts to about 16,800 cells. In scarlatina an increase in the number of the leucocytes may be observed as early as the sixth day before the appearance of the rash. The maximum, an increase of from 10,000 to 25,000 beyond the normal, is noted usually on the second or third day after the appear- ance of the eruption. The hyperleucocytosis persists for from twenty to thirty days.' In smallpox a hyperleucocytosis is observed only in the severer cases, and at a time when pustulation takes place. In the milder forms no increase occurs. In tubercular affections hyperleucocytosis is observed only when secondary infection with pus-organisms has taken place, while in pure cases the number remains normal. But as the chances for a secondary infection are more favorable in some parts of the body than in others, such as the lungs and kidneys, hyperleucocytosis is commonly present when these parts are involved. In Malta fever a marked increase in the polymorphonuclear leu- cocytes may occur just before the onset of the fever. In a case observed in the United States by Musser and Sailer,^ 11,564 leuco- cytes were counted, all varieties, however, being in normal pro- portion. It is thus seen that a hyperleucocytosis of greater or less degree occurs in the majority of the infectious diseases, and may be re- garded as the rule. There are, however, a number of very inter- esting and important exceptions. In uncomplicated cases of typhoid fever and in measles no hyperleucocytosis occurs, and the number of the leucocyte's may indeed be diminished. The importance of this fact from the standpoint of differential diagnosis is self-evident. As regards the other forms of leucocytes in the acute infectious diseases, it is known that with a return to the normal of the poly- nuclear neutrophilic elements a temporary increase in the number of the eosinophiles often occurs. With the decline of the hyper- leucocytosis, moreover, mononuclear neutrophilic leucocytes and irritation-forms frequently appear in small numbers. The lympho- cytes remain practically uninfluenced. The toxic hyperleucocytoses likewise belong to this order. An • Van der Berg, Arch. f. Kinderheilk., vol. xxv. p. 321. 2 Musser and Sailer, Phila. Med. Jour., 1898, p. 1408, and 1899, p. 89. 88 THE BLOOD. increase in the number of the polynuclear neutrophilic elements is thus observed in cases of poisoning with potassium chlorate, arseni- ous hydride, and illuminating-gas, after the administration of atro- pin and quinin, and also following the prolonged administration of chloroform and ether. Numerous observations show that in marked ansBmia when the percentage of haemoglobin is low, general anaesthesia, especially if prolonged, is dangerous. Mikulicz holds that no general ansesthetic should be given under any circumstances if the hsemoglobin is below 30 per cent., and Da Costa and Kalteyer ' believe that 40 per cent, is probably the lowest justifiable limit. As the hyperleucocytosis which follows the administration of ether is but slight, and disap- pears within twenty-four to thirty -six hours, a sudden rise in the number of leucocytes would indicate some post-operative complication. Under the heading of the toxic hyperleucocytoses Cabot also groups those forms which may be observed in certain cases of rick- ets, gout, acute yellow atrophy, advanced cirrhosis of the liver (especially when associated with jaundice), acute gastro-intestinal disorders (ptomains), acute and chronic nephritis, hydronephrosis, following the injection of tuberculin, the administration of thyroid extract, and even after the infusion of normal salt solution, and also after the ingestion of salicylates. A hyperleucocytosis affecting the polynuclear neutrophilic ele- ments is further observed in various forms of acute and chronic anaemia. This is especially marked after hemorrhages referable to traumatism, where the number of leucocytes may increase to 30,000 and more. Generally speaking, the degree of hyperleucocytosis here is proportionate to the amount of blood lost and the re- cuperative power of the individual. In the human being Eieder noted a leucooytosis of 15,000 after a pulmonary hemorrhage in phthisis ; 32,600 after hemorrhage from uterine carcinoma; and 26,500 in a case of gastric ulcer. In the primary forms of anaemia, if we except the myelogenous type of leukaemia, in which an absolute increase is associated with a relative decrease, hyperleucocytosis referable to the polynuclear neutrophilic leucocytes is not met with in uncomplicated cases. In the secondary anaemias, on the other hand, though usually of mod- erate degree, it is quite common. An ante-mortem hyperleucocytosis has further been described by Litten ^ and others in moribund individuals, in which previously no increase of the leucocytes had existed. We finally recognize a cachectic hyperleucocytosis which is ob- served in malignant disease, phthisis, etc' Ewing* states, however, that in the great majority of cases of tertiary syphilis, tuberculosis, ' Da Costa and Kalteyer, American Medicine, 1901, p. 306. ' Litten, Berlin, klin. Woch., 1877, No. 51. > Eieder, loc. cit. * Ewing, On the Blood, Lea Bros,, 1901, p. 130. MICROSCOPICAL EXAMINATION OF THE BLOOD. 89 nephritis, in a large proportion of carcinomata, and in a rather smaller proportion of sarcomata, cachexia is unaccompanied by hyperleucocytosis unless there ip distinct local inflammation, necro- sis, or hemorrhage. He suggests that marked hyperleucocytosis in the course of cachexia should lead to a search for one of these com- plications. Polynuclear Eosinophilic Hyperleucocytosis (Eosinophilia). — Aside from the increase of the eosinophilic leucocytes which may be observed in children under normal conditions, eosinophilia is essen- tially a pathological phenomenon. While a relative increase of the eosinophilic leucocytes may or may not occur in myelogenous leukaemia, the absolute number is always increased in uncomplicated cases. Where septic processes supervene, however, this increase may not occur, and the absolute as well as the relative number is then usually much diminished. For a while eosinophilia was thought to be pathognomonic of this form of leuksemia. But we now know that a polynuclear eosino- philic hyperleucocytosis occurs also in other diseases. Its constant occurrence in myelogenous leukaemia should nevertheless be borne in mind, and the diagnosis discarded whenever such an increase cannot be demonstrated (see also page 75). In bronchial asthma an increase of the eosinophilic leucocytes is observed quite constantly about the time of the paroxysm, and may amount to from 10 to 53.6 per cent.' Its occurrence is of value in diiferential diagnosis, as renal and cardiac asthma are not associated with eosinophilia. An increase of the eosinophilic cells has been noted in scarlatina by Zappert,^ Kotschetkoif,' and others, and is regarded as a favor- able prognostic sign. In measles, on the other hand, the number of the eosinophiles is either normal or diminished. In many diseases of the skin, notably in pemphigus, prurigo, psoriasis, and urticaria, a marked eosinophilia may be observed, which in some cases (urticaria) may amount to 60 per cent, of the total leucocytes. Its degree is apparently proportionate to the amount of tissue involved. The largest actual number, viz., 4800 per cbmm., was noted in a case of pemphigus. In leprosy percent- ages varying between 8.48 and 61 have been obtained. Especially interesting, furthermore, is the increase of the eosino- philic leucocytes which is observed in association with the presence of intestinal parasites. According to Leichtenstern, it is especi- ally pronounced in those cases in which Charcot-Leyden crystals are numerous in the feces. The greatest increase is found in ankylostomiasis, where 72 per cent, were counted in one case. In the presence of oxyurides Biickler* found 16 per cent. Nine- 1 Billings, N. Y.'Med. Jour., vol. Ixv. p. 691. "^ Loc. cit. * Kotschetkoff, Centralbl. f. Path.. 1892. * Biickler, Munch, med. Woch., 1894, Nos. 2 and 3. 90 THE BLOOD. teen per cent, were counted in association with ascarides, and Leich- tenstern reports one case of Taenia raediocanellata with 34 per cent. In a fatal infection with Balantidium coli Strong and Musgrave observed a relative increase, and it appears that in cases of amoebic colitis eosinophilia is likewise not uncommon. Of great practical importance is the observation, first made by T. R. Brown,' at the Johns Hopkins Hospital, that trichinosis, in its acute stage at least, is associated with a very remarkable increase in the number of the eosinophilic leucocytes. In the four cases reported by him the eosinophiles reached 68.2 per cent, of the total leucocytes, in the first; in the second, 42.8 per cent.; in the third, 49 per cent.; and in the fourth, 48 per cent.; while the total number of leucocytes per cbmm. was 35,000, 13,000, 17,000, and 18,000, respectively. As the disease is much more common than is generally believed, and as the diagnosis, except in the most marked cases or in the epidemic form, is impossible without an examination of the blood, it is advisable to make such examina- tions in febrile conditions of doubtful origin, as well as in all cases with indefinite intestinal and muscular symptoms. Whenever an eosinophilia of marked grade is discovered under such conditions, a small bit of muscle-tissue should be excised and examined for trichinae directly. Brown's results have been fully confirmed by Thayer, Cabot, Gwyn, Blumer, Neuman, and others.^ As I have pointed out, the eosinophilic leucocytes are rela- tively diminished, and may disappear altogether in the great majority of the acute infectious diseases, with the exception of scar- latina perhaps, while hyperleucocytosis referable to the poly- nuclear neutrophilic cells exists. In the post-febrile period, how- ever, the upper limit of the normal and even a well-marked eosino- philia are often observed. Turck ' thus found an epicritic eosinophilia of 5.67 (430 absolute) in a case of pneumonia, and after an attack of acute articular rheumatism 9.37 per cent. (970 absolute). Zap- pert * reports a case of malaria in which on the day following the last attack 20.34 per cent. (1486 absolute) were found. In the disease in question a moderate eosinophilia is in my experience the rule. Similar observations have been made after the injection of tuber- culin, where a febrile reaction has taken place. In one case reported by Grawitz the eosinophilia reached its highest point, viz., 41,000 per cbmm., three weeks after the injections had been stopped. ' T, E. Brown, "Studies on Trichinosis, ■with Especial Eeference to the Eosinophilic Cells in the Blood," etc.. Jour. Exper. Med., vol. iii. p. 315; Johns Hopkins Hosp. Bull., 1897. 2 W. S. Thayer, Phila. Med. Jour., vol. i. p. 654. E. Cabot, Boston Med. and Surg. Jour., vol. cxxxvii. p. 67fi. Blumer and Neuman, Am. Jour. Med. Sci., vol. cxix. p. 14. A. D. Atkinson, Phila. Med, Jour., 1899, p. 1'243. Osier and Cabot, Am. Jour. Med. Sci., 1899. 8 Loc. oit. • Zappert, Zeit. f. klin. Med., vol. xxiii. p. 227. MICROSCOPICAL EXAMINATION OF THE BLOOD. 91 In malignant disease eosinophilia apparently occurs in only a relatively small percentage of cases, and when present is usually of moderate grade — i. e., not exceeding 7 to 10 per cent. Occasion- ally, however, the increase is most remarkable. Reinbach thus cites a case of lymphosarcoma of the neck with metastases in the bone-marrow, in which 60,000 eosinophilic leucocytes were counted on one occasion. The eosinophilia which is observed in certain cases of gonorrhoea has been carefully studied by Owings in my laboratory. From an analysis of his forty-two cases it appears that with an extension of the inflammatory process to the posterior urethra the number of cases increases in which an increased percentage of eosinophiles is found in the blood, and in cases of prostatitis eosinophilia is the rule. During the iirst week of the disease the blood is apparently always normal. In the second and third weeks it is normal in only 33 per cent, of all oases, and after two months' duration an increased number is still observed in 40 per cent. Occasionally the eosinophilia is associated with an increase of the polynuclear neutro- philic leucocytes. After extirpation, as also in chronic tumors of the spleen, eosino- philia has been repeatedly observed. Miiller and Rieder ' report three cases of tumor, referable to congenital syphilis, hepatic cirrhosis, and neoplasm of the cranial cavity, in which 12.3, 7, and 6.5 per cent., respectively, were found. After extirpation of the organ an eosinophilia is not immediately observed, but develops only after many months, and is of moderate grade. An eosinophilia referable to drugs has also been described, but has attracted little attention. Two cases are reported by v. Noorden, who observed an increase of the eosinophiles to 9 per cent. Both were cases of chlorosis, and in both the eosinophilia followed the internal administration of camphor. Similar observa- tions have been made in animals after poisoning with carbon dioxide. Mixed Hyperleucocytosis. — This term is applied by Ebrlich to that form of active hyperleucocytosis, in the production of which granule-bearing mononuclear leucocytes also play a part. This con- dition is practically found in only one disease, viz., the myelo- genous form of leuksemia. Mononuclear neutrophilic leucocytes, it is true, are also found in other diseases which are associated with hyperleucocytosis, but the quotum which they furnish toward the general increase is there so slight, probably never amounting to more than 1000 per cbmm., as scarcely to affect the total number. Formerly, a sharp line of distinction between simple hyperleu- cocytosis and myelogenous leuksemia did not exist, and leukwmia was regarded as a hyperleucocytosis in which the ratio between the white and red corpuscles exceeded a definite proportion, which 1 Miiller and Rieder, Deutsch. Arch. f. k]in. Med., vol. xlviii. p. 105. 92 THE BLOOD. was generally placed as 1 : 50. As a matter of fact, there is probably no other disease in which so great an increase in the number of the leucocytes is observed, and even at the present day the diagnosis of leuksemia is practically proved when such a pro- portion can be shown to exist. The absolute number of the leuco- cytes may actually exceed that of the red corpuscles. In his series of thirty cases Cabot found 438,000 on an average per cbmm. His highest ratio was 1 : 2, and the lowest 1 : 37. There are exceptional cases of myelogenous leuksemia, however, in which the hyperleuco- cytosis is not so extreme, and in which the ratio may not exceed 1 : 200. While the enumeration of the total number of leucocytes is thus of unquestionable value in the diagnosis of myelogenous leuksemia, it alone is not the determining factor. We must know, on the other hand, what particular elements contribute toward the total increase. In the lymphatic form of leuksemia, as will be shown more specifically later on, the hyperleucocytosis is thus de- pendent upon an increase of the non-granular mononuclear elements. In contradistinction to this form, the hyperleucocytosis of myelo- genous leukaemia is essentially a hyperleucocytosis referable to leu- cocytes which are not seen in the blood under normal conditions, viz., the mononuclear neutrophilic leucocytes. As these elements are the bone-marrow leucocytes proper, we have in myelogenous leu- ksemia a true myelcemia. The number of neutrophilic mononuclear leucocytes met with in such cases is often most remarkable, and a count of from 50,000 to 100,000 in the cbmm. is by no means exceptional. In 18 cases reported by Cabot, the average percentage was 37.7, corresponding to a total number of 162,000 per cbmm. ! In addition to the neutrophilic myelocytes the eosinophilic mono- nuclear leucocytes, which normally are likewise found only in the bone-marrow, appear also in the blood, and constitute the majority of the eosinophilic cells seen in this form of leuksemia. The poly- nuclear eosinophilic elements are at the same time absolutely in- creased, but their relative percentage may be normal. This absolute increase is so invariable in uncomplicated cases that we must regard it as one of the constant symptoms of the disease. Important, further, is the invariable increase of the mast-cells, which is absolute. As a general rule, their number is about one-half that of the eosino- philes, but occasionally they are equally numerous, and exception- ally even more so. Ehrlich holds that from a diagnostic point of view they are perhaps even more important than the eosinophilic leucocytes, for the reason that in contradistinction to these we know of no other condition in which the mast-cells are materially increased. The polynuclear neutrophilic cells and the lymphocytes, although absolutely increased, are relatively much diminished. Of the latter, only 7.6 per cent, are thus found on an average, and of the MICROSCOPICAL EXAMINATION OF THE BLOOD. 93 former, 49.2 per cent., as compared with 20 to 30 and 60 to 70 per cent., respectively. The occurrence of dwarfed forms of both eosinophilic and neu- trophilic polynuclear and mononuclear leucocytes in leukaemic blood has already been mentioned. Occasionally cells in which mitoses can be observed are also seen, but they are of no special interest. The above considerations have reference to uncomplicated cases of leukaemia. When septic complications occur the blood condition may undergo great changes. Thus, in proportion to the degree of infection the myelsemic picture gradually disappears, and is replaced by that seen in simple septic conditions. The polynuclear neu- trophilic leucocytes may then increase to 90 per cent., and even higher. A very rare complication is further described by Ehrlich in which in the terminal stage of the disease the bone-marrow apparently loses its power of producing neutrophilic material, and in which, as a result, non-granular myelocytes, so to speak, appear in the blood. In one case of this kind which he reports the great majority of the mononuclear elements, which numbered 70 per cent, of the total number of the leucocytes, were entirely free from neutrophilic granules. (See also page 75.) Passive Hyperleucocytosis (Lymphocytosis). — Lymphocytosis is observed whenever an increased circulation of lymph occurs in more or less extensive lymphatic districts, the lymphocytes being mechanically washed into the blood-current. In a mild form it is thus seen in certain types of the so-called physiological hyperleucocy- tosis (see page 81), in which the increase in the number of the lympho- cytes is associated with a corresponding increase of the polynuclear neutrophilic elements. To a more marked degree it is seen in vari- ous diseases of the gastro-intestinal tract and in the infectious diseases of children. A well-pronounced lymphsemia is thus observed in whooping-cough, in which an increase to four times the normal number may occur during the convulsive stage. The polynuclear cells are at the same time increased, but not to the same degree. De Amicis and Pacchioni ' found the average number of leucocytes in whooping-cough to be 17,943. They state that the hyperleucocy- tosis is present on the first day of the disease, that it reaches its height in the convulsive stage, and is still demonstrable some time after cessation of the typical symptoms. The small mononuclear elements are said to be most numerous during the first and second stages of the disease, and the large mononuclear cells in the third stage. Rickets also is almost invariably associated with a well-marked lymphocytosis, which is both relative and absolute. A relative lymphocytosis is noted in typhoid fever ; it begins about the end of the first week, and reaches its highest point in the stage of 1 De Amiois and Pacchioni, Clin. med. ital., 1899, No. 1. 94 THE BLOOD. defervescence. (See below.) Ewing' states that he has found a uniform relation in this disease between the lymphocytosis of the blood and the grade of lymphatic hyperplasia found at autopsy. He records an instance in which the examination of the blood led to a strong suspicion of lymphatic leukaemia, and in which at autopsy the mesen- teric glands were of unusually large size, and the edges of the partly necrotic intestinal ulcers rose 1.5 cm. above the mucosa. Following the injection of tuberculin lymphocytosis is occasionally observed, and Waldstein claims to have produced a marked increase by hypodermic injections of pilocarpin. Important from a diagnostic standpoint is the fact that in malig- nant lymphoma lymphocytosis is constantly observed, and may be of very high grade. In v. Jaksch's pseudoleuksemia of infants the increase of the leucocytes principally affects the large mononuclear cells. The highest grade of lymphocytosis, however, if we except malig- nant lymphoma, is met with in lymphatic leukaemia. As in mye- logenous leukaemia, the total number of the leucocytes is here also very much increased, but never to the same degree. The average proportion between the white and red corpuscles thus scarcely ever exceeds 1 : 40, corresponding to 141,000 leucocytes per cbmm. The highest count in Cabot's series was 220,000, and the lowest only 40,000. Of this number, about 90 per cent, are lymphocytes. Myelocytes and eosinophilic leucocytes are scanty. When septic processes develop in such cases, the total number of the leucocytes, as in the myelogenous form of leukaemia, likewise undergoes a con- siderable diminution, but the lymphocytes still remain relatively increased. In one case of Cabot's, in which, as the result of septicae- mia, the total number of leucocytes fell to 471 per cbmm., the per- centage of lymphocytes still was 94.7. Hypoleucocytosis (Leukopenia). — In the foregoing pages it has repeatedly been pointed out that a qualitative diminution in the num- ber of the leucocytes may occur under the most diverse conditions. A quantitative diminution, on the other hand, viz., a diminution of the total number of leucocytes, is observed only in comparatively few diseases. Most important from a diagnostic standpoint is the hypoleucocy- tosis which in uncomplicated cases of typhoid fever is so commonly seen as to constitute one of the most important symptoms of the disease. Exceptions to this rule occur, but are not common. In the initial stage of the disease, owing to a concentration of the blood, resulting from starvation and diarrhoea, higher counts are sometimes observed, but as the disease progresses the number soon diminishes, and in the later weeks is practically always markedly below the nor- mal. Not uncommonly less than 2000 are counted in the cbmm., ' Loo, cit. MICROSCOPICAL EXAMINATION OF THE BLOOD. 95 and in some instances less than 1000 are seen. Whenever an in- crease in the number of the leucocytes is observed in a case of sus- pected typhoid fever it is more than probable that some complication exists or that the diagnosis is wrong. Nageli/ who made a careful study of the blood in fifty cases of typhoid fever, arrived at the fol- lowing results. In typhoid fever systematic blood-counting is valu- able both for diagnosis and prognosis. The alterations in the numbers (not necessarily the percentages) of the polymorphonuclear neutrophiles, eosinophiles, and lymphocytes are characteristic in the different stages of the disease, and are produced by the action of the typhoid toxins upon the bone-marrow, hindering the production of polymorphonuclear neutrophiles and eosinophiles. The changes in the first stage of the disease (steadily rising temperature) are : a neutrophilic hyperleucocytosis of moderate degree, rapidly de- creasing until the neutrophiles are diminished ; a disappearance of the eosinophiles, and a moderate decrease of the lymphocytes. In the second stage (continued fever) the polymorphonuclear neutro- philes and lymphocytes are still further decreased, although toward the end of this stage the latter tend to increase again. In the third stage (remission) the neutrophiles become fewer, the lymphocytes continue to increase, and a few eosinophiles appear. In the fourth stage (defervescence) the neutrophiles reach their minimum, the lym- phocytes are greatly increased, and the eosinophiles gradually return to their normal number. As soon as the fever disappears the neutro- philes begin to increase again, and there is often for some time a considerable lymphocytosis. All these blood-changes are more pro- nounced in children. Favorable indications are the early appear- ance of the eosinophiles, a moderate diminution in the polymorpho- nuclear neutrophiles, and the extreme increase of the lymphocytes. Uncomplicated cases of tuberculosis are likewise not associated with hyperleucocytosis. But as it is very much more common to meet with cases in which secondary infection has taken place, lead- ing to hyperleucocytosis, its absence is often of value in differential diagnosis. According to Cabot and Warthin, a subnormal number of leucocytes may also be observed in acute miliary tuberculosis, though Kolner ^ thinks the leucocyte count important in distinguish- ing between typhoid fever and the latter disease. Important, furthermore, is the hypoleucocytosis of measles, which is commonly observed in uncomplicated cases, and may aid in dis- tinguishing the disease from scarlatina. In severe cases of anaemia the occurrence of hypoleucocytosis is always a grave symptom, as it indicates an inability on the part of the bone-marrow to produce a sufficient number of blood -corpuscles. 1 Nageli, Deutsch. Arch. f. klin. Med., vol. Ixvii., Parts 3 and 4. CSted by T. E. Brown, Maryland Med. Jour., April, 1901. 2 Kolner, "The Blood-changes in Typhoid Fever," Deutsch. Arch. f. klin. Med., vol. Ix. p. 221. 96 THE BLOOD. Ehrlich supposes that in such cases the fatty marrow of the long bones is not transformed into red marrow, and he has observed two cases in which the correctness of this supposition was demon- strated at the post-mortem table. A hypoleucocytosis of this order was observed by Descatelle and Hof bauer ' in five cases of pernicious anaemia, in four of chlorosis, in two of post-hemorrhagic anaemia, in two of liver abscess, one of phthisis florida, one of sepsis with severe anaemia, in three severe anaemias of unknown origin, in two cases of pseud oleukaemia and two of splenic anaemia. In uncomplicated cases of influenza the number of the leucocytes , is commonly diminished. It may, however, be normal. When increased, some complication probably exists.^ While the hypoleucocytosis in the diseases mentioned is rarely extreme, most extraordinary instances of leukopenia are at times encountered. Ehrlich thus cites the case of a well-built young man, in whom brief epileptiform seizures occurred, and in one of which the patient died. The post-mortem examination was entirely nega- tive. During the three days of observation preceding death two examinations of the blood were made. On the first day not a single leucocyte could be demonstrated in ten blood films, and on the second day but one was found in the same number of specimens. Of drugs, atropin, camphoric acid, tannic acid, picrotoxin, agari- cin, menthol, sulphonal, and several other antihydrotics cause a marked decrease of the leucocytes.^ The Drying and Staining of Blood. In order to obtain the best results, cover-glasses of the finest grade, measuring not more than 0.08 to 0.10 mm. in thickness, are indispensable. They should be cleansed with special care. To this end, Ehrlich's method may be employed : the glasses are first placed in a tray with ether for half an hour, care being taken that they are well separated from one another. They are then dried with fine linen, or so-called Joseph's paper, placed in absolute alco- hol for a few minutes, dried again, and kept in dust-proof glass dishes until needed. When once cleansed, the cover-glasses should be handled only with forceps, as the moisture of the hands is in itself sufficient to cause post-mortem changes in the red corpuscles. For this purpose, especially constructed instruments, such as those suggested by Ehrlich, will be found most serviceable. One cover- glass is grasped with the flat-bladed forceps, provided with a sliding lock (Fig. 17) and held in the left hand. The second cover is taken up with the other forceps, which should have a light spring 1 Descatelle and Hof bauer, Zeit. f. klin. Med., vol. xxxlx. p. 488. 2 Eieder, Miinch. med. Wooh., 1892, p. 511. Head, Pffidiatrics, Feb. 1, 1900. ' K. Bohland, " On the Effect of the Hydrotics and Antihydrotics upon the Num- ber of Leucocytes in the Blood," Centralbl. f. inn. Med., 1899, No. 15. MICBOSCOPICAL EXAMINATION OF THE BLOOD. 97 and need not be provided with a lock (Fig. 18), and is brought in contact with the drop of blood, and then immediately placed upon the first. Providing that the glasses are of the proper quality and clean, the drop of blood will spread out in a uniform layer. Ehrlich now recommends that the top cover be slid from the lower cover with the fingers, by grasping the former tightly and drawing it away in a plane parallel to the other. But it seems to me that at this stage forceps should also be employed. Fig. 17. Ehrlich's cover-glass forceps. The drop of blood may be obtained from the tip of a finger or the lobe of the ear, after careful cleansing with soap and water, and, whenever possible, also with ^alcohol and ether. Under no consid- eration should the drop be so large that the top cover floats upon the blood. I have myself abandoned the use of cover-glasses altogether for the purpose of spreading the blood, and greatly prefer slides. These are cleansed in the same careful manner. A fair-sized drop of blood Fig. 18. Linsley's cover-glass forceps. is mounted near the end of one slide and spread out, with an even sweep, with the edge of a second slide, which is held almost ver- tically to the first (Fig. 19). Better spreads are thus obtained than with cover-glasses, and a sufficient number of leucocytes is present, in even one normal specimen, for the purpose of a differential count if a mechanical stage is available. After drying in the air the specimens are placed between layers of filter-paper, and may then be examined at leisure. If for any rea- son it is desired to preserve the specimens for a long time — i. e., for months or years — it is best to coat the blood films with a thin layer 7 98 THE BLOOD. of paraffin, which is later dissolved by immersion in toluol. In this manner especially valuable and rare specimens may be kept almost indefinitely without change ; but even without this precaution the blood films will remain in good condition for a long time. Before staining, it is often necessary to fix the albuminous bodies of the blood. To this end, different methods may be em- ployed. Immersion in absolute alcohol for from five to thirty minutes, or in a mixture of equal parts of absolute alcohol and ether for two hours, furnishes good results. There can be no doubt, however, that the finest pictures are obtained when the speci- mens have been fixed by heat. For ordinary purposes it is only necessary to expose the air-dried blood films to a temperature of from 100° to 120° C. for from one-half to two minutes, while in spe- cial cases a more prolonged exposure or a higher temperature may be required. For fixing by heat, Ehrlich recommends the use of the Fig. 19. Method of making blood smears. so-called Victor-Meyer apparatus in a slightly modified form. This is essentially a small copper kettle, covered with a thin plate, which is perforated for the reception of the boiling tube. If a small amount of toluol is boiled in this kettle for a few minutes, the cop- per plate is soon heated to a temperature of from 107° to 110° C, and retains this temperature sufficiently long for ordinary purposes. In the absence of such an instrument, a small coal-oil stove, upon which a copper plate measuring 10 by 40 cm. is placed, will answer the purpose. Upon this plate the line corresponding to the desired tem- perature is ascertained by means of a series of drops of water, tol- uol (boiling-point 110° to 112" C), xylol (boiling-point 137° to 140° C), etc., and noting the line at which ebullition occurs. Once properly regulated, the apparatus, which may be advantageously placed in a box, so as to guard against currents of air, will be found MICROSCOPICAL EXAMINATION OF THE BLOOD. 99 to furnish a fairly constant temperature. A drying-oven provided with a thermostat and thermometer may, of course, be used for the same purpose. Of late, formol has also been much lauded as a fix- ing agent, and may be used in connection with the tri-acid stain, haematoxylin and eosin, thionin, etc. A 1 per cent, alcoholic solu- tion is employed. This is prepared by diluting one part of formol, which is a solution of 40 per cent, of formaldehyde, in methyl alco- hol and water, with nine times its volume of water, and one part of the resulting solution with nine times its volume of alcohol. Fixation is completed in one minute, and for practical purposes it is necessary merely to cover the blood film with a few drops of the solution, which is then drained off and replaced with the staining reagent directly. When fixed according to one of the methods described, the dried specimen is ready for staining. For this purpose a number of so- lutions may be employed, the selection of the special mixture de- pending upon the points to be elicited. Staining with Eosinate of Methylene -blue ' (Jenner's Stain). — I now use this stain as a matter of routine in my laboratory, and much prefer it to all others. It furnishes excellent results and yields more information than Ehrlich's tri-acid stain, which for many years was the standard stain in blood-work. The reagent is prepared as follows : equal parts of a 1.2 to 1.25 per cent, aqueous solution of Grubler's eosin (yellow shade), and of a 1 per cent, aqueous solution of methylene-blue are mixed in an opan basin, thoroughly stirred and set aside for twenty-four hours. The resulting precipi- tate is filtered off, dried, powdered, washed with water, again filtered, and dried. Of the dye which has thus been prepared, a 0.5 per cent, solution in pure methyl alcohol is made, to which I further add about 10 per cent, of glycerin. "With this solution the cover- glass specimens or, as I prefer, the slides, are stained for about five minutes without previous fixation ; the excess of stain is rapidly poured off, and the specimen rinsed until the film presents a pink color. It is then dried in the air, rapidly passed though the flame of a Bunsen burner, and mounted in balsam or oil of cedar ; or, if slides are used, the specimens may be examined in oil of cedar directly. The red corpuscles are stained a terra-cotta color, the nuclei of the leucocytes are blue, the plaques mauve, the neutrophilic granules a purphsh red, the eosinophilic granules a bright red, and the basophilic granules a dark violet. Malarial organisms and bacteria can be demonstrated at the same time ; they are colored blue. The basophilic granules which are encountered in granular degeneration of the red corpuscles are likewise blue, while red corpuscles which are under- going polychromatophilic degeneration present a tint in which the 'C. E. Simon, "Eosinate of Methylene-blue," Maryland Med. Jour., April, 1900. 100 THE BLOOD. terra-cotta color becomes less and less distinct, and the blue color more and more predominant (Plate III.). Staining with Ehrlich's Tri-acid Stain. — This method is like- wise one of the most useful and convenient for all practical pur- poses. The information which it offers is not so complete, however, as that furnished by the eosinate of methylene-blue. Great care, moreover, is necessary in the preparation of the stain, and chemically pure dyes are absolutely essential. Ehrlich recommends the crystal- line dyes prepared by the Actiengesellschaft fur Anilinfarbstoffe in Berlin. In my experience I have found the well-known prepa- rations of Dr. G. Griibler & Co. of Leipzig entirely satisfactory. Saturated aqueous solutions of orange-G, acid fuchsin, and methyl- green are first prepared, and allowed to clear by standing for at least one week. The various ingredients are then mixed in the order given below, in one and the same measuring glass. After the addition of the methyl-green solution the mixture should be thoroughly stirred until the final ingredients have been added. When completed, trial specimens are stained in order to ascertain whether the requisite amounts of acid fuchsin and methyl-green have been added. Should the neutrophilic granules be insufficiently stained, a few drops more of the acid fuchsin or methyl-green, or of both, are added, as the case may be. Orange-G solution . . 13-14 c.c. Aoid fuchsin solution . 6-7 c.c. Distilled water 15 c.c. Alcohol . 15 c.c. Methyl-green solution 12.5 c.c. Alcohol . 10 c.c. Glycerin 10 c.c. The solution is ready for use at once and improves with age.' If properly prepared, the nuclei of the leucocytes will be stained greenish, the eosinophilic granules a copper color, and the neutro- philic granules violet. The nuclei of the basophilic leucocytes are stained a pale green, while the surrounding protoplasm remains colorless. Ordinarily the red corpuscles are stained orange, but in cases of chronic aneemia, more especially, individual corpuscles may be seen which do not take on a pure orange tint, but a mixed tint, in which the fuchsin predominates to a greater or less degree. This altered susceptibility on the part of the red corpuscles to different dyes has been designated as anaemic or polychromatophUie degenera- tion (see page 63). Staining with Aronsohn and Philip's Modified Tri-acid Stain, — Saturated solutions of orange-G, acid rubin, and methyl-green are prepared, and the various ingredients mixed in the following pro- portions : ' A reliable tri-acid stain is sold by Messrs. Hynson & Westcott, of Baltimore, Md., from whom the eosinate of methylene-blue may likewise be procured. MICROSCOPICAL EXAMINATION OF THE BLOOD. 101 Orange-G solution 55 c.c. Acid rubin solution 50 c.o. Distilled water 100 c.c. Alcohol 50 c.c. To this mixture are added : Methyl-green solution 65 c.c. Distilled water . 50 c.c. Alcohol 12 c.c. The mixture should stand for from one to two weeks before being used. A drop of the reagent added to a Petri dishful of water is used for staining purposes. The specimens must be carefully fixed by heat. Exposure to the stain for twenty-four hours is required. They are then rinsed in water and absolute alcohol, cleared in ori- ganum oil, and mounted. The various elements are stained as with Ehrlich's stain. Neusser's Stain. — In order to stain the basophilic perinuclear granules of Neusser, the following modification of Ehrlich's tri-acid stain should be employed : Saturated aqueous solution of acid fuchsin 50 c.c. Saturated aqueous solution of orange-G 70 c.c. Saturated aqueous solution of metliyl-green 80 c.c. Distilled water ... . 150 c.c. Absolute alcohol . 80 c.c. Glycerin . . 20 c.c. The specimens require only a slight degree of fixation, and are stained as with Ehrlich's tri-acid stain. Staining with Ehrlich's Hsematoxylin-eosin, or Orange-G Solution. — The solution is prepared by dissolving 2 grammes of hsematoxylin in a mixture of 100 grammes each of distUled water, alcohol, and glycerin. To this solution 10. grammes of glacial acetic acid and an excess of alum are added. After exposure to the sunlight for from four to six weeks about 0.5 gramme of eosin or orange-G is added. The specimens are fixed in absolute alcohol, or by heat (a brief exposure only is necessary). They are then left in the stain, in the sunlight, for from one-half to two hours, when they are thoroughly washed in water, dried, and mounted. The red corpuscles and eosinophilic granules are colored a bright red, the nuclei of normoblasts and megaloblasts a deep black, the bodies of the leucocytes a light lilac, and their nuclei a dark lilac. The bodies of the lymphocytes, however, are scarcely stained at all, while their nuclei appear only a shade lighter than those of the nucleated red corpuscles. Staining with Ohenzinsky's Eosin-methylene-blue Solution. — 102 THE BLOOD. This consists of 40 c.c. of a concentrated aqueous solution of methylene-blue, 20 c.c. of a 0.5 per cent, solution of eosin in 70 per cent, alcohol, and 40 c.c. of distilled water. The solution keeps fairly well, but should always be filtered before using. A slight degree of fixation only is necessary. The specimens are stained for from six to twenty-four hours in air-tight watch-crystals at a temperature of from 37° to 40° C. The red corpuscles and eosinophilic granules are stained a bright red, the nuclei and basophilic granules a deep blue, and the malarial organisms a light sky-blue. The stain is very useful in studying nuclei, and the eosinophilic and basophilic granules. Staining with Efarlich's Tri-glycerin Mixture. — This is com- posed of 2 grammes each of eosin, aurantia, and nigrosin in 30 grammes of glycerin. These constituents are brought into solution by keeping the mixture in the warm chamber (37° to 40° C.) for about one week. The specimens must be well fixed, an exposure to a temperature of about 110° C. for at least two hours being best. They are then allowed to remain upon the stain for from sixteen to twenty-four hours, when they are rinsed in water, dried, and mounted as usual. The red corpuscles are colored orange, the bodies of the leucocytes a dirty gray, with dark nuclei, and the eosinophilic granules a bright red. Staining with Ehrlich's Neutral Mixture. — This consists of five volumes of a saturated aqueous solution of acid fuchsin, to which one volume of a saturated aqueous solution of methylene-blue is slowly added, while shaking. The mixture is treated with five volumes of distilled water and filtered, after having stood for several days. The specimens are stained for from five to twenty minutes. Only a slight degree of fixation is necessary. The red corpuscles are stained the color of fuchsin, their nuclei, as well as those of the leucocytes, are black, or a light lilac, the eosinophilic granules red, and the neutrophilic granules violet. Staining with Eosin. — It is most convenient to use a 0.25 to 0.5 per cent, alcoholic solution, with which the specimen is stained for about one minute. If a 0.1 to 0.5 per cent, aqueous solution is employed, an exposure for from ten to twenty minutes is necessary. The fixation need only be slight. The red corpuscles are stained a bright red, the protoplasm of the leucocytes a faint red, while the eosinophilic granules are deeply colored. Basic Double Staining. — A saturated aqueous solution of methyl- green is treated with a small amount of an alcoholic solution of fuchsin. After brief fixation the specimens are stained for five minutes. The nuclei appear green, the red corpuscles red, and the protoplasm of the lymphocytes the color of fuchsin. The stain is MICROSCOPICAL EXAMINATION OF THE BLOOD. 103 especially serviceable for demonstration purposes, in cases of lym- phatic leukaemia. Staining with Eosin-methylal and Methylene-blue. — The re- ageut consists of 10 c.c. of a 1 per cent, aqueous solution of eosin, to which 8 c.c. of methylal and 10 c.c. of a saturated aqueous solu- tion of chemically pure methylene-blue have been added. The mixture is ready for use at onbe, and furnishes very good results. Unfortunately, however, it is very unstable and had better be pre- pared in small quantities as needed. The best results are obtained if the specimens have been previously carefully heated for about two hours. Staining for one or two minutes is sufficient. The baso- philic granules are colored a pure blue, the eosinophilic granules red, and the neutrophilic granules a reddish blue. Special Staining of Basophilic Leucocytes. — The staining fluid consists of 100 c.c. of distilled water, to which 50 c.c. of a saturated alcoholic (absolute) solution of dahlia are added. This solution, upon clearing, is mixed with 10 to 12.5 c.c. of glacial acetic acid. The specimens are stained for from five to ten minutes. A saturated aqueous solution of methylene-blue may be used for the same purpose and in the same manner. With the exception of bacteria, only the basophilic leucocytes are stained, while the neutrophilic leucocytes are but faintly tinged. As I have indicated, good results are also obtained with the eosin- ate of methylene-blue, and I no longer make use of a special stain in order to demonstrate the basophilic granules. Michaelis' Eosin-methylene-blue Stain.* — Two solutions are prepared, viz., one containing 20 c.c. of absolute alcohol and 20 c.c. of a 1 per cent, aqueous solution of chemically pure methylene- blue, the other consisting of 28 c.c. of acetone and 12 c.c. of a 1 per cent, aqueous solution of chemically pure eosin. The two solu- tions are kept in separate bottles, and are mixed in equal proportions immediately before using. The mixture is placed in a watch-crystal and covered without delay. The blood films are fixed by. heat or by immersion in absolute alcohol for from one to twenty-four hours, and are then placed in the stain, face downward, for from one-half to ten minutes, the time varying with each preparation. The staining should be stopped as soon as the blue color, which is first observed, has turned to red, as otherwise the nuclei of the leucocytes will be decolorized. Should the leucocytes, moreover, be numerous, it is best to stop even before this point has been reached. If, on the other hand, the blue stain has acted too energetically, the specimen is stained a little longer. The preparations are finally rinsed in water, thoroughly dried, and mounted as usual. The various elements of the blood are stained as with the eosinate of methylene-blue. ' L. Michaelis, " Eine Uniyersalf arbemethode f. Blutpraparate," Deutsch. med. Woch., 1899, p. 490. 104 THE BLOOD. Distribution of the Alkali in the Blood. A very good idea of the distribution of the alkali in the blood may be formed by making use of the following method, suggested by Ehrlich : a drop of blood is carefully spread between two cover- glasses, when the air-dried specimens are immediately placed in a watch-crystal, containing a solution of the free staining acid of ery- throsin in chloroform. In a few minutes the specimens have assumed a bright-red color, when they are transferred for a minute or two into a crystal containing chloroform. While still moist they are then imbedded in Canada balsam. Prepared in this manner, the alkaline elements of the blood are colored red. The plasma presents a distinctly red color, while the red corpuscles have not taken up the stain. The protoplasm of the leucocytes and especially of the lymphocytes, as also the plaques, the fibrin-filaments, and the bits of protoplasm derived from the leucocytes are all stained a deep red, while the nuclei of the leucocytes remain colorless. If mala- rial organisms are present, these are likewise stained. In order to prepare the stain, the following procedure may be em- ployed : a saturated aqueous solution of erythrosin (tetra-iodo-fluor- escin) is acidified with dilute hydrochloric acid, and the staining acid, which is thus precipitated, collected on a filter, after having been washed with distilled water. The precipitate is dissolved in chloroform, to which it imparts an orange color. This solution is employed for staining. In every case care should be had that the glass utensils which are used are freed from adherent alkali, by wash- ing with concentrated acids and then with distilled water. The Plaques. In addition to the leucocytes and the red corpuscles large num- bers of small, roundish elements, measuring about 3 fi in diameter, are encountered in the blood, which are free from coloring-matter and may be frequently observed collected into small heaps, resem- bling bunches of grapes ; they stain lightly with both acid and basic dyes. These are the blood-plates or plaques of Bizzozero. Accord- ing to Hay em, they represent ordinary red corpuscles in an early stage of development, and have hence been termed hcematoblasts. This opinion, however, is not shared by many hsematologists, and it is more likely that they are derived from the red corpuscles and take some part in the coagulation of the blood. According to Howell and others, they represent fragments of the nuclei of disin- tegrated leucocytes. According to Osier, their number varies under normal conditions between 200,000 and 500,000 per cbmm. Brodie and Russell, how- ever, claim that this number is too small, and state that if their im- proved method of counting is used, an average of 635,300 will be MICROSCOPICAL EXAMINATION OF THE BLOOD. 105 found in the cbmm. The ratio between the plaques and the red corpuscles would thus be 1 : 7.8, accepting 5,000,000 red corpuscles as the average normal number for the red. A large increase is ob- served in post-hemorrhagic ansemia and in chlorosis, coincidehtly with an increased coagulability of the blood ; while in purpura, in which this is always much diminished, a corresponding diminution of the plaques has been noted. In malaria and various febrile dis- eases smaller numbers than usual are also said to occur. Hayem's statement that they occur in greatly diminished numbers in the blood of pernicious ansemia lacks confirmation. Owing to the rapidity with which the plaques tend to agglutinate after the blood has been drawn, it is usually not possible to study the individual bodies in fresh specimens, mounted in the ordinary way. Various methods have hence been devised to overcome this difficulty. One of the best is to place a drop of Hayem's fluid (see page 107) upon the finger, and to puncture this through the drop. For ordinary purposes this method will suffice, but if it is desired to count the plaques, the procedure of Brodie and Russell should be employed (see page 110). Literature. — Bizzozero, "Ueber einen neuen rormbestandtheil d. Blutes," etc., Tirchow's Archiv, vol. xc. Howell, "The Life-history of the Formed Elements of the Blood," eto.^ Jour. Morphol., 1891, vol. iv., p. 57. Brodie and Russell, "The Enu- meration of the Blood-platelets," Jour. Physiol., 1897, Nos. 4 and 5. The Dust Particles or Haemokonia of Miiller. These may be seen in any fresh specimen of blood mounted in the usual manner. They are small, generally round, sometimes dumb-bell-shaped, colorless, highly refractive granules, which mani- fest very active molecular movements. They occur in the plasma of the blood, and are apparently not connected with the process of coagulation. Miiller found them abnormally numerous in a case of Addison's disease, while they were diminished during starvation and in various cachectic conditions. Stokes and Wegefarth regard these granules as identical with the neutrophilic and eosinophilic granules of the leucocytes. They suppose, moreover, that the bactericidal power of the leucocytes of the blood, and of the serum of man and many animals, is due to their presence. LiTEKATUBE. — H. F. Miiller, "Ueber einen bisher nicht beachteten Formbestand- theil d. Blutes," Centralbl. f. allg. Path. u. path. Anat., 1896, p. 929. W. E. Stokes and A. Wegefarth, " The Presence in the Blood of Free Granules, etc.. and their Pos- sible Relation to Immunity," Bull. Johns Hopkins Hosp., 1897, p. 246. E. B. San- gree, "Leucocytio Granules," etc., Phila. Med. Jour., 1898, p. 472. The Enumeration of the Corpuscles of the Blood by the Method of Thoma-Zeiss. Of the various instruments employed for the enumeration of the blood-corpuscles, that of Thoma-Zeiss is the most satisfactory (Fig. 20). 106 THE BLOOD. It consists of a capillary pipette {8), having a bulb in its upper third, the lower end being graduated in parts numbered from 0.1 to 1, while above the bulb a mark bearing the number 101 is placed. With this goes a counting-chamber {B) measuring exactly 0.1 ram. Fig. 20. 0.100 mm. rfr mm. Thoma-Zeiss blood-counting apparatus. in depth, the floor of which is ruled into sets of 16 small squares, each small square underlying a space of x^-^ cbmm. Enumeration of the Red Corpuscles. — In order to count the red corpuscles with this instrument, the tip of a finger or the lobe of the ear is punctured with a sharp-pointed scalpel, after having been carefully cleansed with soap and water, alcohol, and finally with ether. The exuding blood is drawn into the capillary tube to a given mark, generally to 1 or 0.5, according to the degree of dilu- tion desired, care being taken that no pressure is made upon the finger, and that the tip of the instrument comes in contact with the blood only. The point of the tube is then rapidly wiped, and the blood diluted with a 3 per cent, solution of common salt, which is drawn into the pipette to the mark 101. Toison's fluid is still more convenient as a diluent, as the leuco- cytes are stained by the methyl-violet, and are thus rendered more easily visible. Its composition is the following : Distilled water 160 grammes. Glycerin 30 " Sodium sulphate . 8 " Sodium chloride 1 gramme. Methyl-violet 0.025 " Other solutions such as a 15-20 per cent, solution of magnesium sulphate, a 5 per cent, solution of sodium sulphate, Hayem's or Pa- cini's fluid, may also be employed for the same purpose. MICROSCOPICAL EXAMINATION OF THE BLOOD. 107 Formula of Hayem's fluid : Mercuric chloride 0.5 gramme Sodium sulphate 5.0 grammes Sodium chloride 2.0 " Distilled water 200.0 " Formula of Pacini's fluid ; Mercuric chloride 2.0 grammes Sodium chloride 4.0 " Glycerin 26.0 " Distilled water 226.0 " The contents of the bulb are now thoroughly mixed by shaking, in which the glass bead (jB) contained in the bulb aids very ma- terially. The contents of the capillary tube are then cautiously expelled, as this contains only the diluting fluid. A drop of the mixture is now placed on the counting-chamber, and the cover-slip (r) adjusted, bubbles of air being carefully excluded. When properly prepared, Newton's colored rings should be seen at the margin of the drop. After allowing the corpuscles to settle — from three to five minutes are generally sufficient — they are counted. At least one whole field, or, if special accuracy is required, two whole fields, should be gone over* — i. e., 200 or 400 small squares, respectively, when counting the red, and at least four whole fields when counting the white. It is convenient to count the red corpuscles in sets of four small squares, lying side by side in a horizontal direction, note being Fig 21. o e° '•°t •V •* i JA " o' V-" o'"S ', , - ■ t ' o" 1.0° I'.'l. (a o *D '« "i'u Y: l< » a */• l\ \ 'I ' ' ' o ° :•; " o' = ; " n" ** " ' • \ ' '/ ■•? " V O" 0. o * ■ V 'J ' o'l" a ,0 ':• ■/.• D " .'" •; ' .' " 'o"" o' " •> ; = - '■ "" ""> X "' r „'■ D '" (" p • • ■v. lli •:• "o " •'.' '••:'• a" a* s Appearance of blood in the Thoma-Zeiss cell taken of every corpuscle that touches the upper and left boundary- lines of the large squares, no matter whether the body of the cell lies inside or outside of these lines ; those touching the lower and right lines are ignored. It will be noted that every large square is separated from its neighbor, both horizontally and vertically, by a row of small squares traversed by a mesially placed line, which 108 THE BLOOD. serves as a guide to the next large square (Fig. 21). As a general rule, it will be found most convenient to ignore these intermediary- squares, account being taken only of the large ones. In order to calculate the number of red corpuscles contained in one cbmm. of blood the total number noted is divided by the num- ber of small squares counted, the result giving the average number contained in one small square — i. e., in -^-^^ cbmm. One cbmm. of the diluted blood will then contain 4000 times this number, and one cbmm. of undiluted blood the product of this figure and the degree of dilution. Example. — Supposing that 1200 red corpuscles were counted in 400 small squares, the average number contained in one — i. e., in f-^^ cbmm. of diluted blood — ^would be 3, corresponding to 12,000 corpuscles for each cbmm.; supposing, further, that the blood was diluted 200 times, there would be 2,400,000 in 1 cbmm. of the un- diluted blood. Enumeration of the White Corpuscles. — The leucocytes when present in increased numbers may also be counted with this instru- ment, but not less than four whole fields should be covered in the examination. With an approximately normal number of leucocytes, however, it is necessary to resort to special pipettes, which are constructed for a dilution of 1 : 10 or 1 : 20. But with the diluting fluids men- tioned above, it would be impossible to count the leucocytes in a mixture of this proportion, as a large number would be concealed by the red corpuscles. A 0.3-0.5 per cent, solution of acetic acid is therefore used, which dissolves the red corpuscles and renders the nuclei of the white more distinct. In the absence of a special pipette, an ordinary 1 cbmm. pipette accurately graduated in tenths may be employed. 0.9 c.c. of the acetic acid solution is placed in a watch- crystal and there mixed with 0.1 c.c. of blood, when the counting- chamber is filled and covered as described. In order to obtain greater accuracy, the entire field of the microscope is now counted, a lower power being employed with which the rulings are just visible. The cubic contents of the field of vision are now determined accord- ing to the formula Q^ nr^ X 0.1. Q represents the cubic contents to be determined ; r, the radius, which is readily ascertained by noting the number of vertical lines which cross the field, bearing in mind that the distance between two of these is equivalent to ^ mm. (the area of each small square being ^-^ mm.), and dividing the transverse distance by 2 ; the value t: is constant, 3.1416 ; 0.1 rep- resents the depth of the chamber. If n represents the number of white corpuscles contained in the field, the cubic contents of which are Q, the number of corpuscles, N, contained in 1 cbmm. of the diluted blood is ascertained accord- ing to the equation MICROSCOPICAL EXAMINATION OF THE BLOOD. 109 As the blood has been diluted ten times, the value of N for the non-diluted blood will be —-, where n represents the total number of leucocytes and/ the number of iields counted. Exanvph. — Supposing the number of leucocytes found in 50 fields to have been 600, and the cubic contents of each field 0.03925 cbmm., the total number of leucocytes contained in 1 cbmm. of un- diluted blood would be 3057, as ascertained by the equation ^^10.™_ 10X600 f.q 50 X 0.03925 Special care should be taken to keep the pipette in a clean condi- tion. After use, it should be rinsed with : (1) the diluting fluid, (2) distilled water, (3) absolute alcohol, and (4) ether. If dust or coag- ulated blood adheres to the pipette, it should be removed by repeated rinsings with strong acids or alkalies, assisted if necessary by a bristle. Indirect Enumeration of the Leucocytes. The numbef of leucocytes may also be ascertained in an indirect manner by accurately counting the number of red corpuscles and leu- cocytes in dried and stained specimens with a Zeiss net-micrometer, the ratio between the two varieties being thus ascertained. With the Thoma-Zeiss apparatus the number of red corpuscles contained in 1 cbmm. of blood is then determined, when the corresponding number of leucocytes is found according to the equation I: r : : L : R, and i = — > where I and r represent the number of leucocytes and red corpuscles, respectively, as counted in the dried specimens, and where L indi- cates the unknown number of leucocytes and R the number of red corpuscles in 1 cbmm. of blood, as determined with the Thoma- Zeiss instrument. Example. — Supposing that 700 red corpuscles and only 1 leuco- cyte were counted ■ in the dried specimen, and that an estimation of the red corpuscles with the Zeiss apparatus indicated the presence of 5,000,000 in 1 cbmm. of blood, the corresponding number of leucocytes would be 7142, as is apparent from the calculation : J _ /J? _ 1 X 5000000 _y^^^ r 700 Notwithstanding the apparent simplicity of the process of blood- counting, a great deal of experience is required in order to obtain 110 THE BLOOD. results which are free from unavoidable errors. In using the Thoma- Zeiss apparatus errors of more than 2 to 3 per cent, should not occur. Differential Enumeration of the Leucocytes. — A differential enumeration of the various forms of leucocytes can be carried out only in specimens which have been stained so as to bring out the different granulations. Ehrlich's tri-acid stain has heretofore been employed almost exclusively for this purpose. It gives good results if it has been prepared carefully, but it does not color the basophilic granules. During the past two years I have used the eosinate of methylene-blue almost exclusively, and have come to the conclusion that in many respects it is better than Ehrlich's stain. The neutro- philic granules are well shown and the stain can be prepared without difficulty. In making a differential count of the leucocytes I go over the preparation as thoroughly as possible, beginning at the left upper corner. A movable stage is, of course, very convenient, but is not a necessity. The individual leucocytes are classified as they are met with, and the percentages finally calculated. To obtain accurate results, at least 1000 should be counted. Enumeration of the Plaques. Method of Brodie and Russell. — The method is an indirect one. First the red corpuscles are counted in the usual manner. A drop of the staining fluid, composed of equal parts of a 2 per cent, solu- tion of common salt and a saturated solution of dahlia in glycerin, is then placed upon the finger, when this is punctured through the drop and the blood allowed to mix with the reagent. In this mixture the ratio between the plaques and the red corpuscles is ascertained, and the total number of plaques contained in 1 cbmm. of blood determined by calculation. The plaques are stained the color of dahlia and can readily be counted. Rapid work, however, is essential, as the staining fluid soon attacks the red corpuscles. Ehrlich suggests the enumeration of the plaques in air-dried specimens which have been stained with acid erythrosin. Owing to the relatively large amount of alkali which the plaques contain, they are stained an intense red with this reagent (see page 104). Rosin proposes that the air-dried specimens be fixed for twenty minutes by exposure to the vapors of osmic acid, and then stained in a concentrated aqueous solution of methylene-blue. The Hsematokrit. Within late years the centrifugal machine has also been applied to blood-counting, but has not become very popular in the clinical laboratory. MICROSCOPICAL EXAMINATION OF THE BLOOD. Ill Daland's latest modification of the instrument, originally devised by Hedin, is represented in the accompanying illustrations. It consists essentially of a metallic frame (Fig. 23), supported upon a Fig. 22. Fig. 23. Daland's haematokrlt. spindle which can be rotated at high speed, one single revolution of the large handle causing 134 revolutions of the frame. Two glass tubes 50 mm. in length and having a diameter of 0.5 mm. accompany the instrument. Each tube (Fig. 25) bears a scale 112 THE BLOOD. ranging from to 100, the individual divisions of which are ren- dered easily visible by a lens-front. The outer ends of the tube fit into small, cup-like depressions, the bottoms of which are covered with thin rubber. The inner extremities are held in position by Fig. 24. Fig. 25. Daland'B haematokrit. springs. The instrument should be secured firmly to a solid table and oiled daily when in use. To examine the blood, a rubber tube, provided with a mouth- piece (Fig. 26), is slipped over the end of one of the glass tubes, and the tube filled completely by suction from a drop of blood obtained from the finger or the ear. The blunt point of the tube BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD. 113 is covered quickly with the finger and the tube fixed in the frame. This is rotated at a speed of 10,000 revolutions for two or three minutes, when the volume of the red corpuscles is directly read off. In healthy individuals the volume of the red corpuscles is about 50 per cent., so that in a given case a proportionate expression of the percentage of corpuscles, as compared with the normal, can be ob- tained by multiplying the figure upon the scale by 2. As it has been ascertained that 1 per cent, by volume represents about 100,000 red corpuscles, it is only necessary to add five ciphers to the percentage-volume found in order to obtain the number of red corpuscles in 1 cbmm. of blood. ' Fig. 26. Suution-tube of Daland's haematokrit. Example. — Supposing that in a given case the reading was 35 ; by multiplying this figure by 100,000, 3,500,000 would represent the number of red corpuscles contained in 1 cbmm. of blood. If normal blood is examined with the hsematokirt, the leucocytes will be seen to form a narrow white band at the central end of the column of red corpuscles ; a hyperleucocytosis is thus readily recognized. I am personally not an enthusiast, as regards the use of the hsBmatokrit in blood-work. The instrument in my laboratory is a hand centrifuge, and I freely confess that I am in fear of an accident whenever the attempt is made to rotate the attachment at the pre- scribed rate of speed. This, moreover, is a feat in itselfi Others, who are using electric centrifuges, speak more favorably. BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD. It is generally admitted that micro-organisms do not normally occur in the blood ; in conditions which may be said to stand mid- way between health and disease, however, they are met with at times. In patients suffering from furuncles, for example, bacteria may be found in the skin, in the lymph-glands, and even in the blood of neighboring tissues, other symptoms of disease being absent. To this condition the term "latent microbism" has been applied by Verneuil. Under truly pathological conditions, on the other hand, micro-organisms are not infrequently found, and an examination with this view will often lead to a correct diagnosis. For ease of reference, the various organisms that may be met with 114 THE BLOOD. in the blood in disease will be described under the headings of the respective diseases in which they are found. Typhoid Fever. Recent researches have shown 'that in typhoid fever the specific organism (Plate XII., Fig. 3) can be isolated from the blood di- rectly in a fairly large percentage of cases and at a time when the Widal reaction (see below) may not as yet be obtainable. Schott- miiller thus found the organism in forty cases out of fifty, Castellani in twelve out of fourteen, and Auerbach and Unger in seven out of ten. Neuhaus, Neufeld, Curschmann, Rumpf, and others had previously shown that the bacillus may at times be cultivated from the blood taken from the roseolar spots. The blood is withdrawn by means of a sterilized syringe from one of the superficial veins of the arm ; 300 c.c. of bouillon are inoculated with 30 drops, of the fresh blood and examined after from eighteen to twenty -four hours. If a negative result is obtained in the hanging drop, a further examination is made twenty-four hours later. At first the bacilli are but little active, but on further cultivation and reinoculation their motility increases. For purposes of identification they are grown on agar slant, in milk, bouillon, glu- cose, and further tested with an actively agglutinating serum (see below). Positive results have in this manner been obtained thirty- six hours after the first inoculation. Literature. — Neuhaus, Berlin. Win. Woeh., 1886, Nos. 6 and 24. SchottmuUer, Deutsch. med. Woeh., 1900, No. 32. Castellani, cited in Presse m6d., June, 1900. Auerbach u. Unger, Deutsch. med. Woeh., 1900, No. 29. Cole, Johns Hopkins Hosp. Bull., 1901, p. 203. Widal Serum Test. — Of greater practical utility than the culti- vation of the typhoid bacillus from the blood is the fact that the blood-serum of patients affected with typhoid fever possesses the property of causing arrest of motility and agglutination of the spe- cific bacilli. This observation, originally made by Pfeiffer, was first utilized for diagnostic purposes by Widal, in 1896. The method which bears his name has now been quite generally adopted in the clinical laboratory, and must be regarded as a most valuable aid in the diagnosis of typhoid fever. The reaction occurs in over 95 per cent, of undoubted cases, and may appear as early as the first day of the disease, meaning thereby the first day that the patient spends in bed or the fifth day of general malaise. Such instances, however, are very uncommon, and, as a general rule, a positive result is ob- tained only after the fifth or sixth day in bed. In a small number of positive cases, on the other hand, the patient may pass through the entire course of the disease, and present typical clumping only dur- ing convalescence or a subsequent relapse. In every case, therefore, in which no reaction is obtained upon first trial, the test should be BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD. 115 repeated at regular intervals throughout the disease untU a definite result is obtained. Intermittence of the reaction, moreover, is very- common, and emphasizes still further the necessity of frequent exami- nations in apparently negative cases. While in some instances the reaction disappears very soon after the temperature reaches normal, and even earlier, it generally con- tinues into convalescence, and may be observed for months and years after the attack. Cases have thus been recorded in which a positive reaction, could be obtained as long as thirty-seven years after in- fection. The question, whether or not Widal's reaction is a specific reac- tion of the typhoid organism, can, I think, be answered in the affirmative, notwithstanding the facts that at times cases of true typhoid fever are seen in which no clumping is obtained, and that the reaction has been observed in cases which were apparently non- typhoid. Such exceptions, no doubt, are due in part to faulty tech- nique, viz., to too low a degree of dilution of the serum, the use of old or impxire cultures, too long a time-limit of observation, single negative tests, etc. On the other hand, there can be no doubt that typhoid bacilli are at times present in the body without giving rise to symptoms of typhoid fever. In a case of cholelithiasis, reported by Gushing, typhoid bacilli were thus found in the gall-bladder, and distinct clumping was observed with a dilution of 1 : 30, although no history of typhoid fever could be obtained. There can further be no doubt that individuals exist who are naturally immune against typhoid fever, and that some of the positive results which have been obtained in perfectly healthy individuals who have never had typhoid fever may be explained in this manner. While the reaction may hence be regarded as a specific infectious reaction of the typhoid organism, nevertheless its value in diagnosis is limited. This is owing largely to the fact that in many cases a positive result is not obtained before the end of the second or third week, and may even be delayed until a relapse occurs. Its per- sistence for years after infection is also an obstacle to its general utility, not to speak of its occurrence in apparently healthy individ- uals and in diseases in which an association with the typhoid organ- ism is not apparent. WidaVs test is a most valuable aid in the diagnosis of typhoid fever, but cannot be relied upon to the exclusion of other symptoms. Technique. — The method is based upon the fact that typhoid serum will cause arrest of motility and agglutination of the specific bacilli even when diluted, whereas clumping of the same organism is obtained only with sera from other diseases and healthy individuals when these are used in a more concentrated form. The time-limit at which clumping occurs is likewise an important factor, as non- typhoid sera are at times met with in which, notwithstanding a cer- 116 THE BLOOD. tain degree of dilution, agglutination occurs, providing that the speci- men is kept for a long time. Both factors, viz., the degree of dilu- tion necessary to eliminate the agglutinating power of non-typhoid sera, as also the time-limit of observation, have been arbitrarily de- termined. Widal originally advised a dilution of 1 : 10, and Griiber a time-limit of one-half hour. At the present time there is a ten- dency, among German physicians especially, to increase the degree of dilution to 1 : 40, and even 1 : 50, and the time-limit to from one to two hours. Generally speaking, a positive reaction is of greater value the greater the degree of dilution at which it can still be obtained. A uniform standard, however, is necessary in order to allow a strict comparison of results, and I am personally inclined to favor the German standard. In any event, only a full-virulent, fresh bouillon culture of the typhoid bacillus, viz., one not older than sixteen to twenty-four hours, should be used. The further technique is simple : 1 volume of blood-serum is diluted with the requisite amount of the bouillon culture, viz., to 10, 20, 30, 40, or 50 volumes, as the standard may ■ be. Of this mixture, one drop is mounted on a slide, covered, and examined with a moderately high power. If the case in question is one of typhoid fever, it will be observed that after a variable length of time the individual bacilli, which at first actively dart about the field of vision, become quiescent and tend to gather in distinct clumps, while the interspa9es become entirely free from ba- cilli or very nearly so. After one-half hour, or one or two hours, according to the degree of dilution, all motion has ceased. "When the time-limit has expired' and loss of motility and agglutination have not occurred the result is negative. In such an event further examinations should be made on the following days. In every case it is well to make a control-test with the simple bouillon culture, so as to insure the absence of preformed clumps and the virulence of the organism ; of the latter, the degree of motility is the best index. In order to secure the necessary degree of dilution, various meth- ods have been suggested. The simplest and the one generally em- ' ployed in municipal bacteriological laboratories, is to receive a large drop of blood upon a slide or slip of glazed paper, and allow it to dry. A drop of distilled water is then placed on- the blood and allowed to remain for several minutes, when it is washed off and intimately mixed with the requisite number of drops of the bouillon culture, and examined as* described. The principal advantages of this method are its simplicity and the fact that the dried blood retains its agglutinating properties for weeks and months. The results, however, are less reliable than with the use of liquid blood. If this is to be employed, properly graduated capillary pipettes are prepared, similar to the pipettes accompanying the Thoma-Zeiss hsemocytometer. Blood is first drawn up to a given mark and BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD. 117 expelled into a small watch-crystal ; the requisite amount of the bouillon culture is then obtained with the same pipette and imme- diately mixed with the blood, and a drop of the mixture is ex- amined under the microscope. Sterilization of the apparatus used is unnecessary, and each pipette is destroyed after use. If it is desired to keep the liquid blood for any length of time, similar pipettes may be used with a small bulb blown in the middle. These are first sterilized by heat and sealed at the ends. Before use, one end is broken off, the bulb heated in a spirit flame, and filled by capillary attraction. It is then again sealed, when the blood may be kept indefinitely. Another method, which is said to be even more reliable than those mentioned, is the following : After careful disinfection of the arm, 5 or 6 c.c. of blood are withdrawn from one of the superficial veins, by means of a sterilized hypodermic syringe, and placed in a sterilized test-tube measuring from 10 to 12 cm. in length. The blood is allowed to stand until the serum has separated from the clot, which may be hastened by loosening the coagulum from the walls of the tube with a platinum needle. Eight drops of the serum are added to 4 c.c. of nutrient bouillon, which should be as nearly neutral as possible, when the mixture is inoculated with 1 oese (platinum loopful) of a fresh bouillon culture of the typhoid bacillus not more than twenty-four hours old. The tube is kept at a temperature of 37° C. for twenty- four hours. At the end of this time, and frequently earlier, the bouillon will be absolutely clear, or very nearly so, while little flakes, composed of the bacilli, will be seen at the bottom and adhering to the sides of the tube, if the case under observation is one of typhoid fever ; otherwise the bouillon becomes uniformly cloudy and ' a true sediment is not formed. A pseudo-reaction also may occur at times, which should not be confounded with the one just described. Innumerable microscopical, dust-like particles will then be seen scattered throughout the fluid, which can readily be distinguished from the cloudy appearance of non-typhoid specimens. It has been suggested that this result is obtained in cases of intense infection with the Bacillus coli communis. Should doubt arise, it is only necessary to keep such tubes for a few hours at a temperature of 37° C, when it will be noticed that the dust-like aspect has given place to the ordinary cloudy appearance observed in cases which are not typhoid fever. Of the nature of the substance or substances which cause agglu- tination — agglutinins — little is known that is definite. It appears that in the blood they are intimately associated with fibrinogen and globulin, as plasma from which these two bodies have been removed no longer possesses agglutinating properties. As chemical differ- ences, however, apparently do not exist between normal globulin and globulin obtained from typhoid blood, it seems likely that the 118 THE BLOOD. substances in question do not form an integral part of the globulin molecule, but perhaps are thrown down mechanically when the proteid substances are precipitated. This view is rendered probable by the fact that typhoid urine free from albumin may liJsewise cause arrest of motility and agglutination of typhoid bacilli. Attempts to separate the agglutinins from the proteids of the blood have thus far not been successful. The milk of immunized animals or of typhoid patients acts like the blood, and in it the agglutinins are apparently associated with casein. Exposure of such milk to a temperature of 80° C. destroys its agglutinating power. Very interesting is the observation of Malvoz, that very dilute solutions of safranin and vesuvin act upon the typhoid bacilli as typhoid serum does, and upon these bacilli only. Literature. — Pfeiffer, Zeit. f. Hyg., vol. xviii. p. 1. Pfeiffer u. Kolb, Dentsch. med. Woch., 1896, p. 185. Griiber ii. Durham, Miinch. med. Woch., 1896, pp. 206 and 285. Widal, Soc. m^d. des H&p., 1896, p. 561 ; and Presse m^d., 1897, i. p. o. Biggs and Park, Am. Jour. Med. Sci.. vol. cxiii. p. 274. Stewart, Trans. Am. Pub. Health Assoc, vol. zxiii. p. 151. Forster, Zeit. f. Hyg., vol. xxiv. p. 500. Da Costa, N. Y. Med. Jour., 1897. Anders and MoFarland, Phila. Med. Jour., 1899, pp. 778 and 832. Pneumonia. Recent research has brought to light the interesting fact that in fatal cases of acute croupous pneumonia the specific diplococcus is quite frequently present in the blood, while in cases ending in recovery it is encountered only exceptionally. I have found, as a matter of fact, that a positive result is obtained in more than 89 per cent, of the fatal cases. The invasion of the blood usually occurs twenty-four to forty-eight hours before death, but may take place at an earlier date or be delayed. From the standpoint of prognosis a bacteriological examination of the blood may thus be of considerable importance. It should be remembered, however, that while a positive result is always a symptom mali ominis, there are cases on record in which recovery occurred notwithstanding the presence of diplococci in the blood. In such cases metastatic infection probably has occurred. Prochaska, working under Eich- horst's direction, reports that he found pneumococci in the blood in each of ten cases examined. The examination, which should be repeated every day, is conducted as follows : after disinfection of the arm one of the superficial veins is compressed with a finger and punctured with an ordinary hypo- dermic syringe which has previously been sterilized in boiling water. Five c.c. of blood are aspirated and agar-tubes — liquefied at 40° C. — inoculated, each with 1 c.c. of the blood. Plates are then pre- pared and kept at a temperature of from 35° to 37° C. The colonies number from 2 to 200, and appear as small, round, grayish, jelly-like drops, which are quite characteristic. During their growth they cause a greenish discoloration of the blood-agar. Other bacteria BACTERIOLOGY AND PABASITOLOOY OF THE BLOOD. 119 possess the same property, but to a less marked degree than the Diplococcus pneumoniae. The organism also grows on gelatin with- out causing its liquefaction. The individual organism (Plate XIY., Fig. 2) is capsulated, and usually occurs in pairs arranged end to end or in short chains. At times, however, the chains are quite long, and then it may be difBcult to distinguish it from streptococci. It is easily stained with the common anilin dyes. In order to differentiate the capsule the follow- ing method, suggested by Welch, is best employed : spread and dried cover-glass preparations are treated first with glacial acetic acid, which is allowed to drain off, and is replaced (without washing in water) with anilin-gentian-violet solution. The staining solution is added repeatedly until all the acid is replaced. The specimen is now washed in a weak salt solution (about 2 per cent.), and examined in this, and not in balsam. The capsule and coccus can thus be differentiated. LiTERATUKE. — Sittmann, Deutsoh. Arch. f. klin. Med., vol. liii. p. 323. Kohn, Deutsch. raed. Woch , 1897, p. 136. James and Tattle, N. Y. Presbyterian Hosp. Eep., vol. iii. p. 44. Goldscheider, Deutsch. med. Woch., 1892, No. 14. Sepsis. The impoijance of a careful bacteriological examination of the blood in cases of septic infection has now been established definitely. Large quantities of blood are, however, necessary, and reliance should never be placed upon a microscopical examination of a single drop. In doubtful cases it is best to cup the patient and to inocu- late agar-plates and bouillon-tubes with the serum. The animal ex- periment, viz., the injection of 0.5 to 2 c.c. into the peritoneal cavity of white mice, will also be found most valuable. Petruschky has shown that in severe cases of septic infection it is almost always possible to find streptococci in the blood, while in the milder cases a negative result is reached. He has found, moreover, that while as a general rule the presence of streptococci will justify a grave prognosis quoad vitam, death does not necessarily occur in every case. His results are tabulated below : Negative Results. Deaths. 5 cases of puerperal fever .... 1 2 " phlegmonous abscess, associated with erysipelas . 3 " simple erysipelas . 8 " erysipelas (convalescing) . . . 1 case of endocarditis .... 1 " pleurisy with effusion . . . . . . 1 " " with pericarditis . ... 2 cases of pneumonia . 1 2 " acute articular rheumatism . . ... 1 case of scarlatina . . . . 5 cases of typhoid fever . . . . . • ■ .... 7 " phthisis (in 3 of which a general pyogenic infection was found post mortem ; 2 streptococci) ... 4 120 THE BLOOD. Positive Eesults. Deaths. Becoverieg. 5 cases of sepsis, following phlegmonous abscesses, or pneu- monic infection (4 streptococci, 1 staphylococci) 3 2 9 " puerperal infection ( S streptococci, 1 staphylococci J 3 6 1 case of ulcerative endocarditis (streptococci ) 1 2 cases of mixed infection (streptococci) .1 1 Streptococci are met with frequently in the blood after death from diphtheria, while the Staphylococcus aureus and Loffler's bacillus are seen more rarely. In scarlatinal sepsis streptococci have likewise been found. Of other micro-organisms which may be met with in septic con- ditions the Diplococcus pneumoniae is the most common. It has been found in peritonitis, associated with carcinoma of the uterus, in cases of suppurative oophoritis, following childbirth, in cases of biliary abscess at the time of the chill, etc. Friedlander's bacillus has also been found. In several cases of gonorrhceal septicsemia the gonococcus has been isolated during life. Proteus vulgaris has been found in a few instances. The Bacillus aerogenes capsulatus, which is so frequently seen after death, has also been obtained from the blood of living patients. Quite recently also a newly discovered micro-organism has been isolated from the blood by MacCallum and Hastings, which they term the Micrococcus zymogenes. It is apparently closely related to the pneumococcus and the Streptococcus pyogenes. The Staphylococcus pyogenes aureus occurs in the form of minute spherical bodies, averaging about 0.8 ji in diameter, which readily stain with the basic anilin dyes, as also with Gram's method. They usually occur in clumps, but may also be seen in pairs and in short chains. The organism grows on all culture-media, and in the pres- ence of oxygen gives rise to the formation of an orange-yellow pig- ment. Gelatin is rapidly liquefied ; it coagulates milk and clouds bouillon. The Staphylococcus pyogenes albus and dtreus differ from the aureus by the absence of pigment in the first and by the forma- tion of a lemon-yellow pigment in the second. The StreptoGoocus pyogenes (Plate VII., Fig. 1) occurs in chains of spherical cocci which usually vary from four to twenty in number. The size of the individual organism is somewhat greater than that of the staphylococcus, but may vary even in one and the same chain. It is readily stained with the basic anilin dyes and also with Gram's method. It grows on all culture-media, at the temperature of the room, forming small gray granular colonies on agar and gelatin. As a rule, it does not liquefy gelatin, and it may or may not coagulate milk and cloud bouillon. Several varieties are recognized, viz., Streptococcus brevis, which forms short chains ; Streptococcus longus, which occurs in long chains ; streptococci which render bouillon PLATE VII. FIG. 1, f ■ .J Streptococcus Pyogenes. { Abl3ott,) FIG, 2. ^ Bacillus Aiithracis, highly magni- fied to show Swellings and Concavi- ties at extreniities of the Single Cells. (Abbott.) ooRd' at \ ?Sp ' oo//m -^ '-^^'^ oo 5o8c8® '^ Spi rilla of Relapsing Fever. (v. Jakseh.) t_. SCHMIDT, FEC. Malarial Blood Stained with Chenzinsky-Plehn's Solution. (Personal Observation, i BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD. 121 cloudy, and those which do not ; streptococci which form flocculent, sandy, scaly, or viscous sediments. The Streptococcus oongUymeratus grows, without clouding bouillon, in the form of dense separate particles, scales, or thin membranes at the bottom and sides of the tube, and on shaking the sediment it breaks up into little specks, without producing uniform, diffuse cloudiness. The chains are long and interwoven in conglomerate masses (Welch). LiTEEATUEB. — F. W. White, "Cultures from the Blood in Septicseniia., Pneumonia, Meningitis, and Chronic Diseases," Jour. Exper. Med., vol. iv. p., 425. W. S. Thayer and J. W. Lazear, " Gonorrhceal Septiosemia and Ulcerative Endocarditis," Ibid., p. 81. Petruschky, Zeit. f. Hyg., vol. xvil. p. 59. Sittmann, Deutsch. Arch. f. klin. Med., vol. liii. p. 323. Cannon, Deutsch. Zeit. f. Chir., vol. xxxUi. p. 571. For compara- tively negative results, see Kiihnau, Zeit. f. Hyg., vol. xxv. p. 492. Anthrax. The bacillus of anthrax, as first pointed out by Pollender, Brouell, and Davaine, is frequently met with in the blood, where it should be sought for in doubtful cases by staining with Loffler's method. The number of the organisms present, however, is probably always small. Cover-glass preparations are floated for five to ten minutes on a mixture of 30 c.c. of a concentrated alcoholic solution of methylene-blue and 100 c.c. of a 1 : 10,000 solution of potassium hydrate ; they are then washed for five to ten seconds in a 0.5 per cent, solution of acetic acid, treated with alcohol, dried, and mounted in balsam. Thus stained, the bacilli appear as rods meas- uring from 5 // to 12 /« in length by 1 // in breadth, and usually present a segmented appearance, the extremities being slightly thick- ened. Spores are not found, as the organism multiplies by fission. When present in large numbers it is not even necessary to stain, as the organisms can then be seen without difficulty in fresh specimens (Plate VII., Fig. 2). In doubtful cases, in which a microscopical examination of the blood yields negative results, a few cubic centimeters of the blood may be injected into a mouse or a guinea-pig, in the blood of which the bacilli will soon be found in enormous numbers if the disease is anthrax. LiTEEATUEE. — Polleuder, Casper's Vierteljahrsch. f. gerichtl. n. offentl. Med., 1855, vol. viii. p. 103. Brauell, Virchow's Arohiv, vol. xi. p. 132, and vol. xiv. p. 32. Da- vaine, Compt. rend, de I'acad. d. sci., vol. Ivii. p. 220. Blumer and Young, Johns Hopkins Hosp. Bull., 1885, p. 127. Acute Miliary Tuberculosis. In acute miliary tuberculosis tubercle bacilli have repeatedly been observed in the blood ; but while their presence may be regarded as pathognomonic of the disease, the search for them is most tedious and often in vain. Nevertheless a careful examination of the blood is indicated in doubtful caseSj but the fact should ever be borne in 122 THE BLOOD. mind that only a positive result is of value. According to Lieb- mann, the tubercle bacilli are most numerous in the blood about twenty-four hours after the injection of tuberculin. Working in this manner, he claims to have obtained positive results in fifty- six cases out of one hundred and forty-one. For methods of staining and a description of the tubercle bacillus, the reader is referred to the chapter on Sputum. LiTEKATURE. — Liebmann, Berlin, klin. Woch., 1891, p. 393. Kronig, Deutsch. med. Woch., 1894, vol. v. p. 42. Glanders. In glanders the specific bacillus is constantly present in the blood, and may be demonstrated by staining the dried preparations on a cover-glass for five minutes with a concentrated alcoholic solution of methylene-blue, mixed with an equal volume of a 1 : 10,000 solution of potassium hydrate just before using. From this mixture the specimen is passed for a second or two into a 1 per cent, solu- tion of acetic acid which has been tinged a faint yellow by the addition of a little tropseolin GO solution ; it is then decolorized by washing in water containing 2 drops of concentrated sulphuric acid Fig. 27. Bacillus of glanders. (Abbott.) and 1 drop of a 5 per cent, solution of oxalic acid for each 10 c.c. In specimens thus stained, the bacilli appear as short rods measur- ing from 2 // to 3 ;u in length by 0.3 // to 0.4 (i in breadth, often containing a spore at one end (Fig. 27). LiTEEATUEE.— Duval, Arch. demiSd. exp^r., 1896, p. 361. Influenza. In the sputum of influenza a specific organism has been described by Pfeiffer and Kitasato ; it is said to be constantly present also in the blood of such patients. The organism in question appears in the form of minute rods measuring 0.1 ii in breadth by 0.5 /i in length occurring either singly or in chains of three or four. In suitably prepared specimens, owing to the fact that their poles take up the stain more readily than the middle portion, they convey the impression of diplococci. BACTERIOLOOY AND PARASITOLOGY OF THE BLOOD. 123 Canon advises the following method for demonstrating their pres- ence in the blood : cover-glass preparations that have been allowed to dry at ordinary temperature are placed in absolute alcohol for five minutes and are then stained at a temperature of 37° C. for from three to six hours, with Chenzinsky-Plehn's solution (see page 101). The specimens are washed in water, dried between layers of filter-paper, and mounted in balsam. Stained in this manner, the red corpuscles are colored red, and the leucocytes, as well as the bacilli, blue. As a rule, only from four to twenty are found in one preparation, usually occurring singly, but also in groups. Owing to the fact that they are found in the blood only during the acme of the disease. Canon recommends examination of the sputum for diagnostic purposes, a view with which my own observations are entirely in accord. Some observers indeed deny the occurrence of the organism in the blood altogether (Kiihnau). LiTERATUEE. — Canon, Virohow's Arohiv, vol. oxxxi. p. 401. Klein, Baumgar- ten'a Jahresb., 1893, p. 206. Kiilinaa, Zeit. f. Hyg., vol. xxv. p. 492. Relapsing Fever. Relapsing fever is characterized by the presence in the blood, and here only, of spirilla or spirochsetse which bear the name of their discoverer, Obermeier. In order to search for these organisms no special precautions are necessary. After having carefully cleansed the finger, as described, a drop of blood is mounted on a very thin cover-glass. This is inverted directly upon the slide, when the specimen is ready for examination ; an oil-immersion lens is not required. Attention is drawn to the presence of the organisms by certain disturbances which are noticeable among the red corpuscles, and upon careful examination it will be seen that these are caused by the wriggling movements of the spirilla. The Spirochsetse Obermeieri are long, slender filaments, measuring from 36 fi to 40 ^ in length by 0.3 n to 0.5 [i in breadth, and present from eight to twelve incurvations of equal size with tapering extremities (Plate VII., Fig. 3). These last two characteristics serve to distinguish this species from that described by Ehrenberg, in which the radius of the incurvations is not the same in all, and in which the extremities do not taper. The number of spirilla which may be found in a drop of blood varies, being greater during the access of the fever, when twenty, or even more, may be observed in the field of the microscope. They occur singly or in bunches of from four to twenty, specimens resem- bling those figured in the illustration being frequently seen. In the quiescent stage they are arranged sometimes in the form of rings or of the figure 8. After the crisis they seem to disappear entirely, and their presence during an afebrile period may therefore be regarded as indicating a pseudocrisis. During the afebrile periods small, bright, 124 THE BLOOD. round bodies have been described as occurring in the blood, which according to some are spores, but according to others represent merely debris of the spirilla. Culture-experiments have not been very satisfactory, although Koch observed an increase in their number at a temperature of from 10° to 11° C. That confusion should ever arise in distinguishing the spirilla of relapsing fever from the free flagella observed at times in malarial blood seems to me very improbable. LiTEEATUEE. — Heidenreich. Uutersuch. fiber d. Parasit. d. Euckfallstyphus, Ber- lin, 1877. Moczutkowsky, Deutsch. Arch. f. klin. Med., vol. xxiv. p. 80, and vol. xxx. p. 165. Blisener, Inaug. Diss., Berlin, 1873. Engel, Berlin, klin. Wooh., 1873, p. 409. Malta Fever. In Mediterranean or Malta fever the specific organism, the Micro- coccMS melitensis (Bruce), has been isolated from the blood during life. Diagnosis is greatly facilitated by the fact that a well-pro- nounced agglutination is obtained with the patient's serum. A positive reaction with a dilution of more than 1 : 20, according to Birt and Lamb, may be regarded as proof of the existence of the disease. As a rule, such a result can still be reached with a dilution of from 1 : 600 to 1 : 700. It begins about the fifth day of the disease, and gradually diminishes in intensity during convalescence, but may persist for a year and a half, and even longer. LiTEBATUEE. — C. Birt and G. Iiamb, "Mediterranean Fever," Lancet, Sept. 9, 1899. Wright and Smith, Brit. Med. Jour., April 10, 1897. Musser and Sailer, Phila. Med. Jour., 1898, p. 1408, and 1899, p. 89. E. P. Strong and W. E. Musgrave, " The Occurrence of Malta Fever in Manila," Phila. Med. Jour., 1900, p. 996. Yellow Fever. Wasdin and Geddings, constituting a commission of medical officers of the U. S. Marine-Hospital Service detailed by the U. S. government to investigate the cause of yellow fever, report that Sanarelli's bacillus may be isolated from the blood of the patients during life. They found the organism in twelve cases out of four- teen after the third day of the disease, and also obtained it from the remaining two after death. In other diseases it was not found. A similar commission, consisting of Eeed, Carroll, Agramonte, and Lazear, on the other hand, arrived at negative results. By withdrawing the blood from the veins of nineteen patients they failed to obtain a positive result in every instance. Post-mortem investigations in eleven cases were likewise negative. According to Reed and Carroll, Sanarelli's Bacillus icteroides should be considered a variety of the hog cholera bacillus, and as a secondary invader in yellow fever. Infection occurs through the bite of mosquitoes (Culex fasciatus, BACTERIOLOOY AND PARASITOLOGY OF THE BLOOD. 125 Fabr., and probably other varieties also) which have previously fed on the blood of yellow fever patients. The period after contamina- tion which must elapse before the mosquito is capable of conveying the infection averages twelve days in summer, and eighteen or more days during the winter months! Literature. — " Controversy between G. SanarelU and W. Beed and J. Carroll on the Specific Cause of Yellow Fever," Med. News, 1899, pp. 193, 321, 513, and 737. E. Wasdin and H. D. Geddings, Eeport of Commission of Medical Officers to Investigate the Cause of Yellow Fever, Treasury Dept., U. S. Marine-Hospital Service, 1899. Eeed, Carroll, and Agramonte, Jour. Am. Med. Assoc, 1901, p. 431. Malaria. The discovery in the blood of a specific micro-organism belonging to the class of protozoa, the Plasmodium malarioe of Laveran, and its invariable presence in the different forms of this disease, must be regarded as one of the most important in clinical medicine. This is not the place to state how frequently a diagnosis of malarial fever based upon clinical symptoms alone has proved false, nor how often a tubercular, a syphilitic, or a septic infection has been overlooked and termed malaria. It will suffice to say that errors of this kind, in view of our present knowledge and the ease with which they can be avoided by every physician, should no longer occur. The diagnosis of malaria should in every case be based upon a micro- scopical examination of the blood. The search for the specific organ- ism, it is true, may be very tedious at times, but it will always be crowned with success if the disease in question is malaria. The parasite in question, as I have stated, is a protozoon, and belongs to the class of hsematozoa, representatives of which are found in the blood of various animals, such as the rat, frog, turtle, carp, various birds, etc. Three varieties are known to occur in the blood of man, viz., the parasite of tertian, quartan, and sestivo- autumnal fever. The life-history of these organisms is now well understood, and it is known that in addition to the intra-corporeal cycle of development which takes place in the human body there is yet another, an extra-corporeal cycle, which occurs in certain mos- quitoes of the genus Anopheles. Infection occurs through the bites of such mosquitoes, which themselves have been infected by sucking the blood of malarial patients. This has been abundantly demonstrated by Ross, Manson, . Grassi, and others, and may be regarded as an established fact. Method of Examination. — The necessary amount of blood is obtained best by puncture of a finger or the lobe of the ear, after this has been thoroughly cleansed with soap and water and dried. The first few drops are wiped away. A small drop of blood is then received upon a cover-glass held with a pair of forceps, care being taken that only the tip of the drop is touched, when the specimen is immediately transferred to a slide. Cover- 126 TBE BLOOD. glasses and slides must be absolutely clean, and it is best to keep both in bottles filled with alcohol or a mixture of alcohol and ether. If these precautions are taken and the drop is not too large, the corpuscles will spread out in an even layer between the two glasses and retain their principal features. Pressure should always be avoided. For examination of the specimens an oil-immersion lens is almost indispensable unless the observer has been thoroughly trained in hsematological work. If the specimens cannot be exam- ined at once, it is well to ring them with paraffin. They may then be kept for several hours. But if a longer time must elapse, it is necessary to prepare dried specimens, which are subsequently stained according to one of the following methods : Futcher's Method. — The air-dried films are fixed for one minute in a 0.25 per cent, solution of formalin in 95 per cent, alcohol. But as it is important that this solution should be made up fresh for each examination, it is more convenient to keep a 10 per cent, aqueous solution of formalin on hand, and to add four or five drops of this to 10 c.c. of a 95 per cent, alcohol just before using. The specimens are then rinsed in water, dried between filter-paper, and stained for from ten to fifteen seconds with a carbolated solution of thionin. This is prepared by adding 20 c.c. of a saturated solu- tion of thionin in 50 per cent, alcohol to 100 c.c. of a 2 per cent, solution of carbolic acid. The thionin carbolate thus formed con- stitutes the active staining principle. After washing off the ex- cess of stain the preparations are dried with filter-paper and mounted as usual. Thus prepared, the malarial parasites appear as reddish- violet bodies and are readily seen. The method is of special value in staining the ring-shaped bodies of the sestivo-autumnal infection, which are difficult to see in unstained specimens, and usually do not stain well with eosin and methylene-blue. Staining with Eosinate of Methylene-blue. — This method has already been described (page 99), and, like Futcher's method, furnishes good results. Plehn's Method. — The solution employed has the following com- position : Concentrated aqueous solution of methylene-blue ... 60 c.c. 0.5 per cent, solution of eosin in 70 per cent, alcohol . . 20 c.c. Distilled water ... .... 40 c.c. Aqueous solution of sodium hydrate (20 per cent.) . . 12 drops. The specimens are fixed in absolute alcohol for from three to five minutes. After drying they are stained for from five to six minutes, rinsed in water, dried between filter-paper, and mounted. The red corpuscles are stained red, and the nuclei of the leucocytes and the malarial organisms blue. The Nocht-Romanowsky Method. — This method is employed best BACTERIOLOGY AND PABASITOLOOY OF THE BLOOD. V21 when details of structure are to be studied in the malarial parasite. The cover-glass preparations are fixed by absolute alcohol, and are then immersed, specimen side down, for one to two hours in the staining solution. This should always be prepared freshly from the following stock solutions : 1. A neutral solution of Unna's polychrome methylene-blue, prepared by adding dilute acetic acid (2-3 per cent, solution) to the polychrome methylene-blue (Griibler) until the latter no longer pre- sents an alkaline reaction. As a general rule, 5 drops of a 3 per cent, solution of the acid are sufficient for 1 ounce of the com- mercial liquid dye. The reaction is tested with red litmus-paper, note being taken of the color immediately above the zone which comes in contact with the stain. 2. A 1 per cent, aqueous solution of Griibler's methylene-blue, which should be at least one week old. 3. A 1 per cent, aqueous solution of Griibler's watery eosin. The staining solution is then prepared by adding 4 drops of No. 3, 6 drops of No. 1, and 2 drops of No. 2 to 10 c.c. of dis- tilled water, mixing well. The specimens are fixed in alcohol or by heat, and are immersed in the stain, specimen side down, for one or two hours. They will not overstain in twenty-four hours (Ewing). Staining with Iodine. — The air-dried blood-films are exposed to the vapor of iodine until they assume a pronounced yellow color. To this end, a few grammes of iodine are placed in a small glass dish provided with a well-fitting top. The specimens are left in this dish, arranged on little glass tripods or similar contrivances, blood side down, for ten minutes or longer. They are then mounted in a drop of syrup of laevulose and examined as usual. Special fixation is generally not necessary, but at times specimens are met with in which solution of the haemoglobin takes place in the syrup. In such an event a brief fixation is required, for which purpose Futcher's formalin or absolute alcohol may be employed. With this method the red blood-corpuscles practically present a natural color more or less intensified, and the malarial organisms appear as in fresh blood. I have found this procedure especially serviceable in demonstrating the natural appearance of the parasite at a time when fresh blood was not available. The Parasite. — The following forms of the parasite may be found in the blood : 1. Hyaline Non-pigmentbd Intracellular Bodies. — These apparently represent the earliest stage in the development of the parasite, and are found in all forms of malarial fever ; they are espe- cially abundant during the latter part of the paroxysm or immedi- ately thereafter. At first sight they may be mistaken for vacuoles, but upon closer examination it will be found that they exhibit dis- 128 THE BLOOD. tinct movements of an amoeboid character, and may thus easily be recognized with a little experience. The rapidity with which these changes in the form of the organism occur in the tertian type of ague is most astonishing, and sketches of any one phase can often, indeed, be made only from memory ; in quartan fever the movements are much slower and far less exten- sive. . In the irregular fever of the sestivo-autumnal form amoeboid movements may liltewise be observed, but more commonly the para- site assumes a ring-like appearance, and does not throw out distinct pseudopodia. If these forms are carefuUy observed, however, it will be found that they are not absolutely quiescent, but alternately ex- pand and contract. In tertian fever the organism (Plate VIII.) is pale and indis- tinct, while in quartan fever it is sharply outlined and somewhat refractive (Plate IX., Fig. 2). In the aestivo-autumnal form the organism is usually much smaller than in the tertian type, and the ring-like bodies frequently present at some point in their interior a distinctly shaded aspect which closely resembles the darker por- tion in the centre of a normal corpuscle (Plate IX., Fig. 1). It is thus possible, even at this stage in the development of the para- site, to distinguish between fever of the tertian, quartan, and sestivo- autumnal type. The numbers in which these small, non-pigmented intracellular organisms may at times be met with is most astonishing. In a case of pernicious malarial fever of the algid type, which I had occasion to examine, and in which a history of only one week's illness with- out chills was obtained, normal red corpuscles were indeed only exceptionally found. The case was one of the Bestivo-autumnal form of fever. 2. Pigmented InteacelIjULAh Organisms. — These represent a later stage in the development of the parasite, and, like the non- pigmented intracellular bodies, are met with in all types of malarial fever. Their appearance, however, differs considerably in the vari- ous forms. In tertian fever minute granules of a reddish-brown color appear in the bodies of the organism very soon after the par- oxysm. These gradually increase in number, while the invaded corpuscles proportionately become paler and paler, until finally only an indistinct, shell-like outline can be discerned. In fresh specimens the granules,, which often assume the form of little rods, resembling bacteria, exhibit most active molecular movements, attracting atten- tion at once. The body of the parasite, which during its develop- ment has increased gradually in size, is probably hyaline, and may still be seen to undergo amoeboid movements. These are not nearly so active, however, as in the non-pigmented stage. The move- ments, moreover, cannot be followed so readily, owing to the pres- PLATE VIII. '-Schmidt fecit The Parasite of Tertian Fever. I, Normal Red Corpuscle; 2-4, Non-pigmented Stage of the Organism, showing Amceboid Move- ments; 5-7, Progressive Pigmentation and Growth; 8-11, the Process of Segmentation; 12, Young Forms; r3, Large Extra-cellular Organism ; 14, Mode of Formation of Extra-cellular Body ; 15, Small Fragmented Extra-cellular Organism ; 16, Flagellate Body and Free Flagella. Unstained Specimen. {Personal Observation.) PLATE IX. /L 13 L Schmidt fecit The Parasite of Aestivo- Autun-inal Fever. I, Normal Red Corpuscle; 2-10, Gradual Growth of the Orgauisni ; 11 and 12, Segmenting Bodies; 13, Young Forms ; 14-22, Crescents, Ovoids and Spherical Bodies, with and without Bib; 23, Flagellate Body. Unstained Specimen. (Personal Observation.) % FIG, 2. LSclunidl fecit The Parasite ol' Quartan Fever. I, Normal Red Corpuscle; 2-6, Gradual Growth of the Organism ; 7, Pigmented Extra-cellular Body ; 8, Segmenting Body; m, Young Forms; 10, Vacuolated Extra-cellular Body; i [, Flagellate Form. Un- stained Specimen. (F'ersonal Obser\'ation. ) BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD. 129 ence of the granules. At first sight, these appear to be scattered in small collections throughout the red corpuscle, and the impression may be 'gained that several organisms are present at the same time. Upon closer investigation, however, it will be seen that this is only apparently the case, and that the granules are confined to the bulb- ous extremities of the pseudopodia of a single parasite. Before the end of forty-eight hours the organism has filled out the entire red corpuscle, which at the same time has attained a larger size than normal. The amoeboid movements become less apd less marked, and the pigment-granules, which may still be quite active, tend to collect about the periphery (Plate YIII.). In quartan fever pigmented intracellular bodies likewise appear very soon after the paroxysm. The individual granules, however, are somewhat larger, of more irregular size, and darker in color than those seen in the tertian type (Plate IX., Fig. 2). Instead of exhibiting active molecular movements, moreover, they are almost entirely quiescent, and usually are grouped along the periph- ery of the organism. While amoeboid movements can at first be observed, these become less and less marked, until finally, at the end of from sixty-four to seventy-two hours, they cease. The organism then presents a round or ovoid form, but does not fill the red corpuscle entirely. It is curious to note that in this form of ague the red corpuscles do not become decolorized, but rather darker than normally, and at times specimens may be seen which present a distinctly greenish or brassy appearance. When the parasite has become fully developed the corpuscle is smaller than normally, and, on staining, it may be seen that the organism still is surrounded by a narrow zone of corpuscular protoplasm even when this is not apparent in unstained preparations. The pigmented intracellular bodies which may be found in sestivo- autumnal fever (Plate IX., Fig. 1) can readily be distinguished from those observed in tertian and quartan ague. As in these types, pigment-granules also appear after the paroxysm ; they are never numerous, however, and often only one or two minute dark granules can be detected near the periphery. The organism, even in the later stages of its development, scarcely ever occupies much more than one-third of the corpuscle. Usually the granules exhibit scarcely any movements. As in the quartan type of ague, decolor- ization of the red corpuscles does not occur, and here, as there, a. greenish, brassy appearance often is observed. At times the red corpuscles are shrunken, crenated, or spiculated. At the beginning and during the paroxysm forms are at times seen in which the few pigment-granules that may be present have gathered in the centre of the parasite and formed a solid clump. From the facts that these are observed only during the paroxysm, and that central blocks of pigment are found only during the stage 130 THE BLOOD. of segmentation (see below) in tertian and quartan ague, Thayer and others conclude that these bodies are pre-segmenting forms of the parasite. This belief is strengthened further by the observation that pigment-bearing leucocytes are then also seen, which in the other types of fever likewise are found only at this time. 3. Segmenting Bodies. — In cases of tertian and quartan fever the progress of segmentation may be observed directly under the micro- scope, if specimens of blood are obtained just prior to or during the chill. In tertian fever organisms will then be seen in which the de- struction of the red corpuscles has advanced to a stage in which it is only possible to make out a pale contour of the original host. The parasite itself has assumed gradually a granular appearance, and the pigment-granules, which until then have exhibited pronounced mo- lecular movements, now become quiescent, larger and rounder, and show a distinct tendency to collect in the centre of the body. Here they form a roundish mass in which the individual components can scarcely be made out. While this change in the position of the pig- ment is taking place,, beginning segmentation of the surrounding granular protoplasm will be observed. This at first is most marked at the periphery, from which delicate striae will gradually be seen to extend toward the central mass, dividing up the protoplasm into a number of oval bodies which closely resemble the petals of a flower (Plate VIII.). Still later these bodies, which in reality are the spor- ules of the parasite, will be found scattered in an irregular manner throughout the interior of the organism. The apparent envelope then disappears, and the sporules, which in tertian fever usually number from fifteen to twenty, lie free in the blood. Quite fre- quently, also, a sudden expulsion of the little bodies is observed and the impression gained as though the envelope had been burst asunder. Upon closer inspection, even at the petal stage, it will be seen that almost every sporule presents a tiny dot in its interior, which may at first sight be mistaken for a pigment-granule, but which in all probability is a nucleus. After the expulsion of the sporules these are frequently seen to move about in an active manner, but sooner or later they come to rest. While the progress of segmentation is very frequently observed to proceed in the manner described, this is not invariably the case. It may thus happen that segmentation occurs before the pigment- granules have had time to gather at the centre, or that the parasitic protoplasm breaks up into sporules directly without the intervention of the petal stage. In every case, however, the formation of sporules is associated directly with the occurrence of a paroxysm, and repre- sents the asexual type of reproduction of the parasite. The ultimate fate of the sporules is not definitely known, but it is likely that they in turn invade new corpuscles, cause their destruc- tion, and become segmented, thus giving rise to a new generation. BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD. 131 As the process of segmentation, moreover, coincides in time with the occurrence of the chill, it is apparent that the interval elapsing between two consecutive chills — i. e., the type of the ague — depends upon the rapidity with which the non-pigmented forms arrive at maturity. In (juartan ague the manner in which segmentation takes place differs somewhat from that observed in the tertian form. It will here be observed that the pigment-granules, which have gathered along the periphery of the organism, as the parasite approaches ma- turity become arranged in a stellate manner, and apparently reach the centre through definite protoplasmic channels. Here they finally form a dense clump, and while the protoplasm assumes a finely granular appearance, segmentation proper . begins and proceeds as in the tertian form. In quartan ague, however, the number of segments is smaller, varying between six and twelve; The entire segmenting body, moreover, is smaller than in the tertian form, and the segments are arranged in a more symmetrical manner. Here, indeed, the most perfect rosettes are observed (Plate IX., Fig. 2). In sestivo-autumnal fever segmenting bodies are only exception- ally seen in the peripheral blood, and it appears that the process of reproduction occurs principally in the spleen. The pre-segmenting forms described here undergo segmentation in a manner closely re- sembling that observed in tertian fever. The number of segments, moreover, is about the same, varying, as a rule, between ten and twenty. The segmenting body itself, however, is much smaller than in either the tertian or quartan form, and it is not possible to dis- tinguish any remains of the original host. 4. Crescbntic, Ovoid, and Sphekical Bodies (Plate IX., Fig. 1). — These are observed only in cases of sestivo-autumnal fever when this has persisted for at least one week. At first sight they apparently bear no relation to the other forms which have been de:5cribed, and it has long been a question whether or not these bodies actually represent a stage in the life-history of the common malarial parasites. Grassi and Feletti have applied the name Lawrania malarice to this form. More recent investigations have rendered it probable that they are derived directly from the pig- mented intracellular forms. Specimens may thus be met with in which crescentic bodies are found in the interior of red corpuscles that have lost but little of their original color. Such observations, however, are not common. The typical crescents which are usually seen are highly refractive bodies, somewhat larger than a red cor- puscle, measuring from 7 /i to 9 ;u in length by 2 // in breadth. Their extremities are usually rounded off and joined by a delicate, curved line bridging over their concave border. This is supposed to TiCpresent the remains of the original host. At other times this hood-like appendage is found along the convex border. The little 132 THE BLOOD. pigment-granules and rods, which are always found in the interior of the crescents, are generally collected about the centre of the body, but they are occasionally also seen in one of the horns. While usually quiescent, a migration of some of the granules toward one extremity and back to the central mass may at times be observed. The ovoid and spherical bodies, which are usually much smaller than the crescents, exhibit the same general features, however, and often are provided likewise with a little hood. It is now known that the spherical bodies develop from the ovoids, and these again from the crescents. Like the crescents, the ovoid and spherical forms may be found in the interior of red corpuscles. 5. Extracellular Pigmented Bodies. — In tertian and quar- tan ague some of the pigmented intracellular bodies, instead of undergoing segmentation when they have arrived at maturity, may be seen to leave their hosts and to appear as such in the blood. At the same time they increase considerably in size, and in the tertian form may indeed become as large as a polynuclear leucocyte (Plate VIII.). The pigment-granules, moreover, exhibit an activity in their movements which is most astonishing and never observed under other conditions. The outline of the parasite is then usually irregular and quite indistinct. Upon careful observation it will be seen that in some of these bodies the movements of the granules after a while become less and less marked, and finally cease, while the body of the parasite itself becomes still more irregular in out- line. This appearance is undoubtedly referable to the death of the organism. In others a gradual fragmentation is observed, small particles of the pigmented mother-substance being cut off from the parent-form. It is thus quite common to see the original parasite break up into four or five smaller bodies, in which the movements of the pigment^granules persist for some time. Sooner or later, however, even these cease, the outlines of the bodies become more and more indistinct, and death occurs. In still others the forma- tion of vacuoles may be observed, the pigment-granules 'at the same time becoming quiescent. This process is likewise regarded as one of degeneration. Most interesting, however, is the fact that/ajreZ- laiion may occur in some of these extracellular forms. It will then be observed that the pigment-granules which exhibit a most sur- prising activity tend to collect near the centre of the organism, while at the same time curious undulating movements may be made out along its contours. Suddenly one or more (one to six) extremely slender filaments will be seen to protrude from as many points on the periphery, presenting minute enlargements here and there in their course (Plate VIII.). The length of these filaments, or fla- gella, as they are termed, varies considerably. As a rule, it does not exceed the diameter of from five to eight red corpuscles, but much longer specimens are at times observed, and it appears to me that BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD. 133 in most illustrations they are represented too short. With these flagella the organism exerts most active whipping movements, scat- tering the red corpuscles to the right and left. Attention is, indeed, usually first drawn to the presence of these bodies by the disturb- ance which they cause in the field of vision. Occasionally one of the flagella may be seen to become detached from the body of the parasite and to move rapidly about among the corpuscles in a snake- like manner. In microscopical specimens they gradually come to a rest and often curl into a spiral. That diiSculty should ever arise ^n distinguishing such detached flagella from the spirilla of relapsing fever seems very improbable, as the nature of these formations is shown by the presence or absence of other forms of the malarial organism. Beyond the fact that the flagellate organisms in tertian fever are larger than in the quartan form, no special points of difference exist (Plate IX., Fig. 2). In sestivo-autumnal fever similar changes may be observed. In crescents it is thus not at all uncommon to observe a small hyaline protrusion from the surface of the organism, which later may become detached. This process was formerly regarded as one of regeneration, but it is questionable whether this is actually the case. In other specfmens, again, true fragmentation, or vacuolization, may occur, and flagellate bodies are met with in this type of fever as well as in tertian and quartan ague. The flagellates, as in quartan fever, are smaller than those observed in the tertian form, but other points of difference do not exist (Plate IX., Fig. 1). The significance of the flagellate organisms has until recently not been understood, but we now know that they represent the male element in the sexual reproduction of the malarial parasite, and the beginning of a cycle of development, which takes place outside of the human body, in the bodies of certain mosquitoes. The beginning of this cycle was observed first by MacCallum in the blood of infected crows. He here discovered that when one of the flagella broke loose it almost always sought out another full-grown form of the parasite which had not undergone segmentation, and penetrated this, just as the spermatozoon penetrates the ovum. Sul)sequently he observed the same process in the blood of the human being. The further development of the fertilized forms, however, does not take place in the human blood, but in the bodies of mosquitoes. The fertilized organism then penetrates the stomach- wall of the insect, and here gives rise to the formation of little cysts, in which after about seven days numerous irregular, rounded, ray-like striae appear. After a time the capsules of the cysts burst, and the delicate, thread-like bodies are set free in the body cavity of the mosquito, and shortly after appear in the salivary glands. These bodies apparently represent the young parasites, which result 134 THE BLOOD. from the sexual reproduction of the adult organism. If at this stage of their development the infected mosquito is allowed to bite a human being, malarial infection results, with the appearance in the blood of the hyaline forms already described. From the above description it will be seen that three forms of the malarial parasites may be found in the blood, viz., the parasite of tertian, quartan, and sestivo-autumnal fever, and it has been shown that these forms may readily be distinguished from each other. It should be mentioned, however, that in tertian and quartan fever several groups of the same organism may be present at one time, and as the process of segmentation coincides with the occurrence of a paroxysm, it will readily be seen that the number of paroxysms within a given time depends directly upon the number of groups which may be present in the blood. If a double infection with the tertian parasite has occurred, one group of organisms may thus have just reached the segmenting stage, while the second group has at- tained only a twenty-four hours' growth, the result being that maturity is reached by the two groups on successive days. Quotidian fever is then the result. Should still other groups be present, the clinical picture will accordingly become more complicated. In quartan ague, similarly, double quartan fever will occur if two groups are present, and triple quartan fever if three groups are present at one time. Mixed infections, further, are also possible. In conclusion, it may not be out of place to refer to the presence of pigment-bearing leucocytes in the blood of malarial patients. These are quite constantly met with during the paroxysm, and it is indeed often possible to observe the process of phagocytosis directly under the microscope (see Fig. 15). The forms which are taken up are the central pigment-clumps of organisms that have undergone sporulation, the small, fragmented extracellular forms, the flagellate bodies, and even the segmenting bodies. In every case where pig- ment-bearing leucocytes — which are probably always of the neu- trophilic, polynuclear variety — are observed malarial fever should be suspected and a careful examination made, as a melansemia lias so far been observed only in this disease, in relapsing fever, and in connection with the rare melanotic tumors, in which not only leuco- cytes containing melanin occur in large numbers, but also masses- of this pigment float free in the blood. LiTEEATUEE. — A. Laveran, Nature parasitaire des accidents de Timpaludisme, Description d'un nouveau parasite, Paris, 1881. For a full account of the literature, see the monograph by W. S. Thayer and J. Hewetson, " The Malarial Fevers of Balti- more," Johns Hopkins Hosp. Rep., vol. v. On recent advances in our knowledge concerning the etiology of malarial fever, see W. S. Thayer, Phila. Med. Jour., 1900, p. 1046, where a full account of the literature is given. T. B. Futcher, "A Critical Summary of Recent Literature concerning the Mosquito as an Agent in the Trans- mission of Malaria," Am. Jour. Med. Sci., 1899, p. 318. W. S. MacCallum, "On the Hsematozoon Infection of Birds," Jour. Exper. Med., vol. iii. p. 117. E. L. Opie, "On the HiBmatozoon of Birds, Ibid., p. 79. F. Grohe, Zur Gesch. d. Melanaemie, Vir- chow's Archiv, 1861, vol. xx. p. 306. BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD. 135 Filariasis. Filaria sanguinis hominis (Lewis) : syn., Filaria Wuchereri (da Silva Lima) ; Filaria Bancrofti (Cobbold) ; Filaria Mansoni ; Trichina cystica (Salisbury) ; Trichina sanguinis hominis nocturna (Manson). Several varieties of the parasite (Fig. 28), which belongs to the class of nematodes, have been observed in the blood of man. Among these are the Filaria sanguinis hominis nocturna, Filaria sanguinis hominis diurna, or Filaria sanguinis hominis, var. major, and Filaria sanguinis hominis, var. minor. The female of Filaria noeturna, according to Hanson's description, is "a long, slender, hair-like animal, quite three inches in length, but only one one-hundredth inch in breadth, of an opaline appear- ance, looking as it lies in the tissues like a delicate thread of catgut, animated and wriggling. A narrow alimentary canal runs from the simple club-like head to within a short distance of the tail, the Fig. 28. Filaria sanguinis hominis, showing sheath. (After Lewis.) remainder of the body being almost entirely occupied by the repro- ductive organs. The vagina appears about one twenty-fifth of an inch from the head ; it is very short, and bifurcates into two uterine horns, which, stuffed with embryos in all stages of development, run backward nearly to the tail " (Osier). The male worm is rarely seen, and is much smaller than the female. While the adult parasite has its habitat in the lymphatic channels, the embryos, which are set free in enormous numbers, invade the blood-current, in which they may readily be found at night ; during the day an examination of the blood will usually yield negative results. This periodicity may, however, be reversed by having the patient sleep in the daytime and be about at night. Each embryo has an envelope of its own, which is hyaline in appearance, and within which the young worm, measur- ing 0.34 mm. in length by 0.0075 mm. in breadth, is able to extend and contract itself. In fresh preparations these organisms are readily detected by the disturbance with their movements create among the corpuscles ; they are apparently transparent and homogeneous, but 136 THE BLOOD. after some time, when the worm has come to rest, it will be seen that they are granular and transversely striated. As the mere presence of these parasites usually does not produce symptoms, and as an examination of the blood made in daytime, as already stated, generally yields negative results, attention is drawn to their presence only when symptoms pointing to an occlusion somewhere in the course of the lymphatic channels exist, as evidenced by chyluria (which see), elephantiasis, or lymph scrotum. Infection occurs through the bite of certain mosquitoes, viz., Anopheles, Culex penicillarius, and Culex pipiens. The develop- ment of the different forms seems to take place in different organs of the host. The Filaria perstans is a variety which Manson found in the natives of the western coast of Africa. Its embryos are found in the blood both during the day and at nights. They have no sheath and are actively motile. LiTEEATUEE. — Moslcr and Peiper, Specielle Path. u. Therap., 1894, vol. vi. p. 219, P. Manson, Allbutt's System of Medicine, vol. ii. I. Guit^ras, Med. News, April, 1886. F. P. Henry, Ibid., 1896. E. Opie, Am. Jour. Med. Sci., 1901, vol. cxxii. p. 251. Distomiasis (Bilharziosis). Bilharzia hsematobia (Cobbold) : syn., gynsecophorus (Diesing) ; Distomum hsematobium (Bdharz) ; Schistosoma haematobium (Wein- land) ; Distoma capense (Harley) ; thecosoma (Maguin-Tandon). The Bilharzia hsematobia belongs to the class of trematode pla- todes. According to Bilharz, the greater portion of the Fellah and Coptic population of Egypt is infected. It is abundant also in South Africa, and is now said to occur occasionally in the United States. From Europe no cases have as yet been reported. It may give rise to diatrhcea, haematuria, and ulceration of the mucous surfaces. The male is smaller but thicker than the female, measuring from 12 to 14 mm. in length ; on its abdominal surface a deep groove is found with overlapping edges, which serves for the reception of the female (Fig. 29). While the adult parasite is seen but rarely in the blood, its ova are found frequently. These are slender bodies, measuring 0.12 mm. in length by 0.04 mm. in breadth, and are provided with a dis- tinct, spike-like projection, which issues from one extremity or the side. Infection apparently takes place through unfiltered drinking- water. Literature. — Bilharz, Wien. mcd. Woch.. 185fi, vol.vi. p. 49. Meissner, Schmidt's Jahrbiich., 1882, vol. xxx. p. 193. Efitimeyer, Verhandl. d. Cong. f. inn. Med., ]89a, vol. xi. p. 144. BACTERIOLOGY AND PARASITOLOGY OF TEE BLOOD. 137 Fia. 29. Male and female specimens of the human blood fluke {BUharsia hsematobia), enlarged. X 12. (After Looss.) Anguilluliasis. Of late, Teissier has announced that in a case of intermittent fever numerous embryos of anguillula were found in the blood. They disappeared after expulsion of the parasites from the intestinal tract, and at the same time the fever ceased (for a description of the anguillula, see page 251). LiTEKATUEE. — Teissier, Compt. rend, de I'acad. des sci., vol. cxxi. p. 171. CHAPTER II. THE SECRETIONS OF THE MOUTH. SALIVA. NoEMAL saliva is a mixture of the secretions derived from the submaxillary, sublingual, parotid, and mucous glands of the mouth. It is a colorless, inodorous, tasteless, somewhat stringy and frothy liquid, and serves the purpose of aiding in the acts of mastication, deglutition, and digestion. The quantity secreted in twenty-four hours amounts to about 1500 grammes. Greneral Characteristics. Normal saliva has a specific gravity of from 1.002 to 1.009, cor- responding to the presence of from 4 to 10 grammes of solids. The reaction of the saliva proper is alkaline, the degree of alkalinity corresponding to from 0.006 to 0.048 per cent, of sodium hydrate. Normally an acid saliva is observed only in newly born infants and in sucklings. The reaction of the tongue and the mucous membrane lining the mouth is quite commonly acid early in the morning, owing to the production of lactic acid by some of the bacteria which are constantly present in the mouth. This acid, according to Magittot, corrodes the enamel of the teeth, and may ultimately produce dental caries. Chemistry of the Saliva. In order to give an idea of the general composition of the saliva the following analyses are appended ; the figures correspond to 1000 parts by weight : Water 995.20 994.20 988.10 Ptyalin^ 1.34 1.30 1.30 ^"■l",. \ 1.62 2.20 2.60 Epithelium f Fatty matter . . . . 0.50 Sulpliocyanldes 0.06 0.04 0.09 Alkaline chlorides 0.84 Disodium phosphate . . . 0.94 2.20 3.40 Magnesium and calcium salts . . 0.04 Alkaline carbonates traces. ' These figures are too high, as they refer to the total precipitate obtained with alcohol. 138 SALIVA. 139 In order to demonstrate the presence of the sulphocyanides, it is usually only necessary to heat a few cubic centimeters of the pure saliva, faintly acidified with hydrochloric acid, with a dilute solution of ferric chloride, when a red color will be seen to develop. If necessary, larger quantities, such as 100 c.c, are evaporated to a small volume ; the test is then applied to the concentrated fluid. Of organic matter, ptyalin, a little albumin mixed with mucin, and about 1 gramme of urea pro liter are found. Of all these sub- stances, the ptyalin is especially interesting from a physiological point of view. It may be isolated in a comparatively pure state according to Gautier's method : To a large quantity of saliva alcohol (98 per cent.) is added as long as a flocculent precipitate forms. This is collected upon a small filter and dissolved in a little distilled water. The solution thus obtained is treated with several drops of a solution of mercuric chloride, in order to remove albuminous material, which is filtered off. The excess of mercury is removed by means of hydrogen sulphide, when the remaining liquid is evaporated at a temperature of from 35° to 40° C, and taken up with strong alcohol. The in- soluble residue is dissolved in a little water, filtered, dialyzed in order to remove inorganic salts, and is finally precipitated with strong alcohol, when the ptyalin will separate out in light flakes. Obtained in this manner, ptyalin is a white amorphous substance, soluble in water, dilute alcohol, and glycerin. In neutral or even slightly alkaline solutions, but not in acid solutions, it rapidly transforms boiled starch into dextrin and sugar at a temperature of from 35° to 40° C. This transformation takes place according to the equations : (1) (Ci,HjoOi„)5, + 3HP = 3[(C„H,„0,„)i,.C„H2jOi,]. starch. Erythrodextrin. (2) 3[(C„H,„0,„)„.C,A,0„] + 6H,0 = 9[(Ci,H^O,„)5.C.,H,,0,i]. Erythrodextrin. Achroodextrin (3) 9[(C,jH^O,o)6Ci2H,,Oi,] + 4.5HjO = 54C„H,,0„ = 54C,2H«0„. Achroodextrin. Isomaltose. Maltose. In order to test for ptyalin, a few cubic centimeters of saliva are filtered and added to a solution of starch ; the mixture is placed in the warm chamber for some time, when it is tested with cupric sul- phate or iodine. At" first, starch gives a blue color with iodine ; after the reaction has proceeded further a red or violet-red color is obtained, indicating the presence of erythrodextrin, while no change in color at all results when achroodextrin only is present. The maltose may be recognized by the fact that it turns the plane of polarization more strongly to the right than glucose ; it also reduces Fehling's solution. The test for nitrites, which may likewise be present in the saliva, is conducted in the following manner : about 10 c.c. of saliva are 140 THE SECRETIONS OF THE MOUTH. treated with a few drops of Ilasvai/s reagent and heated to a tempera- ture of 80° C, when in the presence of nitrites a red color will develop. The reagent is prepared as follows : 0.5 gramme of sulph- anilic acid in 150 c.c. of dilute acetic acid is treated with 0.1 gramme of naphtylamin dissolved in 20 c.c. of boiling water. After standing for some time the supernatant fluid is poured ofi" and the blue sediment dissolved in 150 c.c. of dilute acetic acid. The solu- tion is kept in a sealed bottle. Microscopical Examination of the Saliva. If normal saliva is allowed to stand, two layers will be seen to form, viz., an upper clear and a lower cloudy layer, which lattel con- tains certain morphological elements. Among these, salivary cor- puscles, pavement epithelial cells, and micro-organisms are found (Fig. 30). Fig. 30. "^0^?",% •■• Buccal secretion. (Eye-piece III., obj. Eeichert, ^ homogeneous immersion : Abbe B mirror, open condensers.) a, epithelial cells; &, salivary corpuscles; c, fat-drops; d, leuco- cytes ; e, Spirochaeta buccalis ; /, comma-bacillus of mouth ; g, Leptothrix buccalis ; h, i, k, various fungi, (v. Jaksch.) The salivary corpuscles resemble white corpuscles very closely, but differ in their greater size and coarser appearance. The epi- thelial cells are large, irregular, polygonal cells, provided with well- defined nuclei and nucleoli ; they exhibit certain irregularities in size, according to their origin, and belong to the class of pavement or stratified epithelium. Micro-organisms.' — While schizomycetes and moulds are only exceptionally found in the mouth under normal conditions, and are then undoubtedly derived from ingested food, bacteria are always present in large numbers, and it is not surprising that all forms which are found in the air, food, and drink may here be encountered (Plate X., Fig. 1). Some of these, such as the Leptothrix buccalis innominata. Bacillus buccalis maximus, Leptothrix buccalis maxima, ' W. D. Miller, Die Mikroorganismen d. Mundhohle, 1892. PLATE X. Bacteria of the Mouith. (Cornil Babes.; Leptothrix Bueealis. (v. Jakseh.) SALIVA. 141 lodococcus vaginatus, Spirillum sputigenum," and Spirochsete den- tiutn, are always present. Together with other bacteria, they have been found in carious teeth, in abscesses communicating with the mouth and pharynx, and in exudates on the mucous membranes of these parts. In all probability, however, they are non-pathogenic. To this class also belongs the smegma bacillus, which has been en- countered in the saliva, the coating of the tongue, and in the tartar of the teeth of perfectly healthy individuals. In this connection it is interesting to note that, in contradistinction to the bacteria which are only temporarily found in the mouth, the majority of those which are constantly present cannot be cultivated on artificial media. Important from a practical standpoint is the fact that a number of pathogenic micro-organisms may at times be found under normal conditions. The Diplococcus pneumoniae, also known as the pneu- mococcus of Frankel and Weichselbaum, the Diplococcus lanceolatus, the Micrococcus lanceolatus, the Micrococcus septicsemise sputi, and the Micrococcus pneumoniae cruposae (Sternberg), has thus been found in a virulent condition in from 15 to 20 per cent, of healthy indi- viduals, and it is even claimed that in a non-virulent state it is constantly present in the mouth. Streptococci are likewise frequently observed, but usually possess but little virulence or none at all when obtained from the healthy mouth and tested upon animals. Pyogenic staphylococci may also be found at times, but are less common than the streptococci. Most important is the occasional occurrence of the diphtheria bacillus in the mouths of individuals who have not been exposed to contagion. Welch ^ mentions that virulent organisms were found by Park and Beebe in the healthy throats of eight out of three hundred and thirty persons in New York, who gave no history of direct contact with cases of diphtheria. Two of these eight persons later developed the disease. Non-virulent bacilli were found in twenty-four individuals of the same series, and the pseudodiphtheria bacillus in twenty-seven. Other pathogenic bacteria which may be found in normal mouths are the Micrococcus tetragenus, the Bacillus pneumoniae of Fried- lander, the Bacillus crassus sputigenus, and the Bacillus coli com- munis. It is interesting to note that the secretions of the mouth and throat, as most secretions of the body, possess a certain degree of germicidal power. The Staphylococcus aureus, the Streptococcus pyogenes, the Micrococcus tetragenus, the typhoid bacillus, and the cholera spirillum, when present in moderate numbers, are thus killed by the saliva. The diphtheria bacillus, however, is more resistant, and may survive for twenty-four to forty days. It has been found, as a matter of fact, that the organism may be demon- strated in the throats of some individuals who have passed through 1 Dennis' System of Surgery : Surgical Bacteriology. 142 _ THE SECRETIONS OF THE MOUTH. an attack of diphtheria for several weeks after all the clinical symp- toms have disappeared. The Diplococcus pneumonise is even said to grow well in saliva, although it rapidly loses its virulence. By then cultivating it upon pneumonic sputum, however, the virulence of the organism is restored. The individual bacteria will be con- sidered in detail later on. Pathological Alterations. It has been mentioned that about 1500 grammes of saliva are secreted in the twenty-four hours. This quantity is, however, sub- ject to great variation. An increase is thus frequently noted in pregnancy, in various neurotic conditions, in tabes, bulbar paralysis, in inflammatory diseases of the mouth, in dental caries, following the administration of pilocarpin, in poisoning with mercury, acids, and alkalies, etc. The quantity is diminished in all febrile diseases, in diabetes, and often in nephritis. The effect of psychic influences upon the secretion of saliva as well as of other glands is well known, an increase or decrease in the flow being produced under various conditions. In determining whether or not salivation actually exists, the physi- cian should not only be guided by the statements of his patients, but an actual estimation of the amount secreted within a definite period of time should be made. Hysterical individuals not infre- quently complain of "salivation," when a direct estimation will show that the amount is not only not increased, but actually dimin- ished. An acid reaction of the saliva has been noted in various diseases of the intestinal tract, in febrile diseases, and notably in diabetes (Frerichs). According to Strauss and Cohn, however, an alkaline reaction of the saliva is the rule even under pathological conditions. Among the qualitative changes may be mentioned an increase in the amount of urea, which has been repeatedly observed, and especi- ally in nephritis. Urea may be demonstrated as follows : the saliva is extracted with alcohol, the filtrate evaporated, and the residue dissolved in arayl alcohol. This is allowed to evaporate spontaneously, when crystals of urea will separate out, and may then be examined micro- scopically and chemically (see Urine). Bile-pigment and sugar have not been found in the saliva. Of drugs, potassium iodide and potassium bromide rapidly pass into the saliva. Upon this property the indirect examination of the gastric juice for its digestive power — i. e., the presence or absence of free hydrochloric acid — ^by means of the potassium iodide and fibrin packages of Giinzburg, is partly based. In order to test for potassium iodide, strips of filter-paper moist- SPECIAL DISEASES OF THE MOUTH. 143 ened with starch solution are immersed in the saliva, which has been acidified with nitric acid ; in the presence of potassium iodide the starch-paper turns blue. SPECIAL DISEASES OF THE MOUTH. Tuberculosis of the Mouth. — In cases of lupus and the so-called benign form of tuberculosis of the mouth it is rarely possible to demonstrate the presence of tubercle bacilli, even in scrapings taken from the base of the ulcers or in the diseased tissue itself, while in cases of ulcerative stomatitis associated with phthisis in its advanced stages they may be frequently found in large numbers. In some cases, however, their demonstration is by no means easy. In the saliva they are only exceptionally seen. Actinomycosis. — In cases of actinomycosis it is occasionally pos- sible to demonstrate the presence of the specific organism in or about carious teeth. More commonly, however, the patients are not seen until the primary symptoms of the disease have disappeared, when the typical kernels can no longer be found at the original points of entry or have become unrecognizable owing to calcification and retrogressive changes. Usually the -disease has already progressed to the formation of a distinct tumor or abscess, and it may then be necessary to make an exploratory incision, and to examine the scrapings which are brought away. The number of kernels which may be found is at times very small, but a careful examination will probably always lead to their detection if the disease in question is actinomycosis. Catarrhal Stomatitis. — In this affection the quantity of saliva is increased. Microscopically an increased number of epithelial cells and many leucocytes are noted, their number depending upon the intensity of the morbid process. Ulcerative Stomatitis. — In this condition, following mercurial poisoning or scurvy, the same appearance is noted microscopically as in simple stomatitis. In addition there may be necrotic tissue, red blood-corpuscles, and innumerable leucocytes. The reaction of the saliva is intensely alkaline, the color markedly brown, and its odor fetid. Gonorrhoeal Stomatitis. — The number of cases of gonorrhoeal stomatitis that have thus far been recorded is small. The disease, however, has received but little attention, and is probably more common than is generally supposed. In the adult it may be con- tracted through coitus contra naturam, while in the newborn the infection is undoubtedly brought about in the same manner as the corresponding disease of the conjunctiva. In suspected cases the exudate which forms upon the gums, the tongue, and the palate should be examined for the presence of gonococci. In adults the 144 THE SECRETIONS OF THE MOUTH. organism has thus far not always been found ; in the newborn, however, Rosinski has succeeded in demonstrating its presence in all cases examined. Thrush. — Oi'dium albicans (Fig. 31) is most commonly seen in children, but may also occur in adults, and especially in phthisical individuals, and sometimes lines the entire mouth. If in such cases a bit of the membrane is pulled off and examined microscopically, it will be found to consist of epithelial cells, leucocytes, and granular detritus, with a network of branching, band-like formations, which Fig. 31. Oidium albicans, the vegetable parasite of muguet or thrush. (Eeduced from Ch. Robin.) present distinct segments. The contents of the segments are clear, and usually contain two highly refractive granules — the spores, one of which is situated at each pole. These segments diminish in size toward the end of each band, 'their contents at the same time becom- ing slightly granular. TARTAR. In a bit of tartar scraped from the teeth actively moving spiro- chsetse are seen, as well as long, usually segmented bacilli, frequently forming bands which are colored bluish red by a solution of iodo- potassic iodide. Leptothrix buccalis, shorter bacilli (which are not colored by this reagent), micrococci, and a large number of leuco- cytes and epithelial cells which have undergone fatty degeneration, are also found. COATING OF THE TONGUE. A brown coating of the tongue is often observed in severe infec- tious diseases, and consists of remnants of food and incrustated blood. Microscopically, in addition to a large number of epithelial cells, enormous numbers of rnicro-organisms and a large number of dark, cell-like structures, probably derived from desquamated epithelial cells, are found. The white coating of the tongue contains epithelial cells, many micro-organisms, and a few salivary corpuscles. COATING OF THE TONSILS. 145 COATING OF THE TONSILS. Fharyngomycosis Leptothrica. In the props from the crypts of the tonsils in cases of follicular tonsillitis, and also in persons who have had frequent attacks of ton- sillitis, according to Chiari, epithelial cells and long, segmented fungi — the Leptothrix buccalis (Plate X., Fig. 2) — which are col- ored bluish red by a solution of iodo-potassic iodide, are seen. At times patches composed of these fungi extend over a considerable area of the tonsils, so that it may be doubtful whether or not the disease is a beginning diphtheria. A microscopical examination will in such cases settle all doubts. Tonsillitis. In tonsillitis a large number of bacteria have been isolated from the pseudomembranous deposits. Among the more important which are supposed to bear a causative relation to the disease may be mentioned the various streptococci, staphylococci, less commonly the pneumococcus, the diplococcus of Brison, the Bacillus coli communis, the bacillus of Friedlander, the Bacillus septicsemise sputi, and in a few isolated instances the Micrococcus tetragenus. In many cases in which tonsillar deposits were clinically regarded as diphtheritic culture revealed only an abundance of the thrush fungus. Glandular Fever. According to Neumann and Comby, glandular fever generally depends upon infection with a streptococcus. In the cases reported by Lande ' and Froin and by Hirtz ^ bacteriological examination of the throat at the height of the febrile stage revealed the presence of the pneumococcus in a virulent condition. Diphtheria. Recognizing the great importance of an early diagnosis in such a malignant disease as diphtheria, an examination for Loffler's bacillus has become just as important to-day as that for the bacillus of tuber- culosis, and every physician should make himself familiar with the methods employed for its recognition. By means of a sterilized, stout platinum loop, a pair of forceps,, or a cotton swab, a piece of membrane is scraped from the tonsils,, the soft palate, or the pharynx, and at once transferred to a sterilized test-tube closed with a pledget of cotton. A particle of the mem- brane is then spread in as thin and uniform a layer as possible upon a cover-glass. When dry the specimen is fixed by being passed 1 Lande et Froin, Eev. mensuelle des Mai. de I'En&nce, 1901, p. 78. 146 THE SEOBETIOm OF THE MOUTH. through the flame of a Bunsen burner three or four times, wheu it is ready for staining. For this purpose Loffler's alkaline solution of methyleue-blue, which consists of 30 c.c. of a concentrated alco- holic solution of methylene-blue in 100 c.c. of an aqueous solution of potassium hydrate (1 : 10,000), may be advantageously employed, the specimen being stained for from five to ten minutes. It is then rinsed in water, placed on a slide, the excess of water removed with filter-paper, and examined with a one-twelfth oil-immersion lens. A dahlia-methyl-green solution may likewise be employed. This consists of 10 grammes of a 1 per cent, aqueous solution of dahlia- violet and 30 grammes of a 1 per cent, aqueous solution of methyl- green. The specimen is stained for from one to two minutes. If it is desired to employ Gram's method, the specimen is most conveniently stained for three minutes with a freshly prepared con- centrated alcoholic solution of gentian-anilin water. This is pre- pared by adding anilin oil to 10 c.c. of distilled water, drop by drop, thoroughly shaking after the addition of each drop, until the solution becomes opaque. It is then filtered and treated with 10 c.c. of absolute alcohol and 11 c.c. of a concentrated alcoholic solution of gentian-violet. The specimen is decolorized in a solution com- posed of 1 gramme of iodine and 2 grammes of potassium iodide, dissolved in 300 c.c. of water. After remaining in this solution for five minutes the specimen is rinsed in alcohol and the process repeated until the violet color disappears. It is transferred to absolute alco- hol, then to oil of cloves, and mounted in balsam. Cultures should also be made, preferably upon a mixture of blood- serum and bouillon, as recommended by Loffler. This is composed of three parts of blood-serum and one part of bouillon, containing 10 per cent, of peptone, 3 per cent, of grape-sugar, and 0.5 per cent, of sodium chloride, the mixture being solidified in the usual manner. Upon this medium Loffler's bacillus grows so much more rapidly than other organisms which are usually present in the secretions of the mouth and throat, that at the end of twenty-four hours they 'often form the only colonies that attract attention. Should other colonies of similar size be present, these are generally quite dififerent in appearance. In this manner a diagnosis can be made upon the day following inoculation of the tube. In the absence of blood-serum bouillon, alkaline bouillon, nutrient gelatin, nutrient agar, glycerin-agar, and potato may be employed. Coagulated egg-albumin, as pointed out by Booker, and milk are also good soils. The colonies are large, round, elevated, and grayish-white in color, with a centre that is more opaque than the slightly irregular periphery. The surface of the colony is at first moist, but after a day or two assumes a dry appearance. The bacillus (Fig. 32) is non-motile and varies in size and shape, COATING OF THE TONSILS. 147 its average length being from 2.5 /z to 3 //, its breadth from 0.5 /i to 0.8 II. Its morphological characteristics are so peculiar as to render its identification upon cover-slip preparations and in sections of the diphtheritic membrane an easy matter in most cases. Sometimes the organism appears as a straight or slightly curved rod ; but especially characteristic are irregular and often bizarre forms, such as rods with one or both ends terminating in a little knob, and rods broken at intervals, in which short, well-defined, round, oval, or straight segments can be made out. Some forms stain uniformly, others in an irregular manner ; the most common present the appearance of deeply stained granules in faintly stained bacilli. Fig. 32. a Bacillus of diphtheria. (Abbott.) 11. Its morphologji, when cultivated on glycerin agar-agar. b. Its morphology as seen in cultures on LbiBer's blood-serum. Streptococci are also seen, as a rule, and it may be said that the gravity of a case is directly proportionate to the number of strepto- cocci present. It is important to note that diphtheria bacilli may still be found in the throat for weeks after all clinical symptoms have disappeared. Patients should hence be isolated until a bacteriological examination has demonstrated the absence of the organism. LiTEBATUKE. — S. P'lexner, " The Bacteriology and Pathology of Diphtheria," Bull. Johns Hopkins Hosp., 1895, p. 39. W. H. Welch, Am. Jour. Med. Sci., 1894. Heub- ner, Schmidt's Jahrbiicher d. gesammteu Med., 1892, vol. ccxxxvi. p. 270. Klebs, Arch. f. exper. Path., 1875, vol. iv. p. 207. Loffler, Centralbl. f. Bakt. u. Parasit., 1887, vol. ii., p. 105, and 1890, vol. vii., p. 528. C. Frankel, " Die Unterscheidung d. echten u. d. falschen Diphtheriebacillen," Berlin, kiln. Woch., 1897, p. 1087. CHAPTEE III. THE GASTRIC JUICE AND GASTRIC CONTENTS. THE SECRETION OF GASTRIC JUICE. The gastric juice is the result of the glandular activity of the stomach, and is the only secretion of the digestive tract which pre- sents an acid reaction. As is well known, the mucous membrane of the stomach is cov- ered throughout its entire extent by a single layer of cylindrical epithelium, which dips down in places to line the orifices and larger ducts of the numerous tubular glands with which it is beset. Of these, two kinds are described, viz., the fundus and pyloric glands, so named from the location in which they are principally found. In the secretory portion of a fundus gland two sets of cells can be distinguished. The one kind is small, granular, and polyhedral or columnar, bordering upon the narrow lumen of the tube ; these are termed the chief or principal cells (Heidenhain), but are also known as the central or adelomorphous cells. They stain with anilin dyes to only a slight extent. The others, known as parietal, adelomor- phous, or oxyntic cells, are variously situated between the adelomor- phous cells and the membrana propria ; they are most numerous in the necks of the glands. They are larger than the chief cells, oval or angular and finely granular in structure ; they possess a strong affinity for the anilin dyes. The pyloric glands, which are found only in the region of the pylorus, on the other hand, are character- ized by the greater length of their ducts, which are also lined by the cylindrical epithelium of the mucous membrane proper. The secretory portion of these glands is represented by a single layer of short and finely granular, columnar cells, which closely resemble the chief cells of the fundus glands. In addition to these, a few isolated cells, the cells of Nussbaum, are found, which in structure and in their behavior to anilin dyes resemble the parietal cells. Upon chemical examination the gastric juice is found to consist essentially of water, free hydrochloric acid, pepsin, rennet (a milk- curdling ferment), mucus, and certain mineral salts. Of these constituents, the hydrochloric acid is secreted by the parietal cells, pepsin and the milk-curdling ferment by the chief cells of the fundus and the pyloric glands, while the mucus is the product of the cylindrical goblet-cells lining the stomach and the 148 TEST-MEALS. 149 wider portions of its glandular ducts. It should be borne in mind, however, that the ferments mentioned do not exist in the cells as such, but as zymogens, which are transformed into the ferments through the activity of the free hydrochloric acid. According to modern investigations, moreover, the zymogens only are secreted by the cells. Until recently it was supposed that the gastric juice is secreted only upon appropriate stimulation of the nervous mechanism of the stom- ach, either directly or indirectly, and that the stomach in its quiescent state— i. c, when not digesting — is empty. The researches of Schreiber and Martins, however, have rendered the correctness of this view doubtful, as they were able to obtain quantities of gas- tric juice, varying from 1 to 60 c.c, from the non-digesting stom- ach of every normal person examined. I have likewise never failed to obtain a few cubic centimeters under the same conditions. TEST-MEALS. Although the secretion of gastric juice takes place continuously, the amount that can usually be obtained from the non-digesting organ is not sufficient for analytical purposes. It is, therefore, nec- essary to stimulate the glandular apparatus of the stomach to in- creased activity. This may be accomplished with thermic, chemical, electrical, and digestive stimuli, of which the last named are the most convenient and the most effective, furnishing an idea not only of the secretory, but also of the motor and resorptive activity of the organ. The analytical results will, however, depend to a large ex- tent upon the character of the food ingested, starches and fats exert- ing but a slight ' stimulating effect, while proteids cause a copious secretion of gastric juice. The ingestion of fluids at the same time will likewise influence the results obtained, owing to dilution of the gastric juice. The time of the height of digestion, moreover, varies with the kind and quantity of food taken. In order to obtain uniform results it is necessary, therefore, to withdraw the gastric contents at a certain period after the ingestion of a meal of known composition and bulk. Numerous test-meals have been proposed. The following are the most important : t The Test-breakfast of Ewald and Boas. This consists of from 35 to 70 grammes of wheat-bread and of 300 to 400 c.c. of water or weak tea, without sugar. It is best to give this meal to the patient early in the morning, when the stomach is empty — i. e., as a breakfast. The gastric contents are obtained one hour later. 150 THE OASTBIC JUICE AND GASTMIO CONTENTS. The Test-dinner of Riegel. This consists of a plate of soup (400 c.c), a beefsteak (200 grammes), a slice or two of wheat-bread (50 grammes), aud a glass- ful of water (200 c.c). The contents of the stomach are obtained after four hours. The disadvantage of this method lies in the fact that the lumen of the stomach-tube is frequently occluded by large pieces of undigested meat, a source of annoyance which may be guarded against, however, by using finely chopped meat. The Double Test-meal of Salzer. For breakfast the patient receives 30 grammes of lean, cold roast, hashed or cut into strips sufficiently small not to obstruct the stomach-tube ; 250 c.c. of milk ; 60 grammes of rice ; and one soft- boiled egg. Exactly four hours later the second meal is taken, con- sisting of 35 to 70 grammes of stale wheat-bread and 300 to 400 c.c. of water. The gastric contents are withdrawn one hour later. In this manner the gastric juice is not only obtained at the height of digestion, but an idea may at the same time be formed of the motor power of the stomach. Under normal conditions the organ should contain no remnants of the first meal at the time of examination. The Test-breakfast of Boas. This consists of a plateful of oatmeal -soup, prepared by boiling down to 500 c.c. one liter of water to which one tablespoonful of rolled oats has been added. A little salt may be used if desired, but nothing more. The contents of the stomach are obtained one hour later. This test^meal was devised by Boas in order to guard against the introduction from without of lactic acid, which is present in all kinds of bread. The meal is employed in cases of suspected cancer of the stomach in which a quantitative estimation of lactic acid is to be made, the stomach being washed out completely the night before. Still other tfist-meals have been suggested, but they possess no material advantage over those described. THE STOMAOH-TUBE. The stomach-tubes in general use are essentially large Nflaton catheters. They should measure at least from 72 to 75 cm. in length, and be provided with three fenestra, of which one is placed at the end of the tube and two laterally, as near the end as possible. For the purpose of washing out the stomach the tube is connected with a glass funnel by means of ordinary rubber tubing, which can be detached from the stomach-tube proper. There is no advantage In rubber funnels or in having a continuous tube. THE STOMACH-TUBE. 151 It is important that the tubes should be thoroughly cleansed in hot water as soon after use as possible. The advice of Boas, more- over, to have special, marked tubes for tubercular, syphilitic, and carcinomatous patients, should be borne in mind. Patients in whom lavage is to be practised for any length of time should provide their own instruments. Contraindications to the Use of the Tube. Of direct contraindications to the use of the tube, there should be mentioned the existence of the various forms of valvular disease when in a state of imperfect compensation, angina pectoris, arterio- sclerosis of high degree, aneurism of the large arteries, recent hem- orrhages from whatever cause, marked emphysema with intense bronchitis, acute febrile diseases, etc. Fig. 33. Introduction of the Tube. The technique of the introduction of the tube should be as simple as possible ; the exhibition of complicated bottle arrange- ments for the purpose of obtaining the gastric juice Bnly adds to the excitement of a nervous patient, and should be avoided. The patient's clothing and floor of the room should be protected from being soiled by material that may be vomited along the sides of the tube, the dribbling of saliva, etc. For this purpose, Tiirck's rubber bib with pouch may be advantageously employed. " It is so arranged ' as to form a pouch in front, to catch the saliva or stomach contents that may be thrown oif from the mouth or stomach. A detachable tube passes from the bottom of the pouch and is conducted into a basin or any suitable vessel."' Cocainization of the pharynx is not nec- essary, but may be resorted to in hyperses- thetic individuals, a 10 per cent, solution being employed. The tube, held like a pen, is passed to the posterior wall of the pharynx, the patient bending his head forward, and not backward, as is usually advised. The patient is then told to swallow, but this is not necessary. The tube is pushed on until resistance is felt when it meets with the floor of the stomach. The procedure does not occupy ten seconds. At the least sign of cyanosis or of marked ' Manufactured by G. Tiemaun & Co., New York. Boas' bulbed tube. 162 THE GASTRIC JUICE AND GASTRIC CONTENTS. pallor the tube should be withdrawn at once, and the patient ob- served for a day or two before a second attempt is made. If the gastric juice does not flow at once, the patient is instructed to bear down with his abdominal muscles, and, if this is insuificient, to cough a little. Repeated attempts of this kind will usually bring about the desired result, unless the tube has not been introduced far enough or too far ; in the latter case it will double upon itself, so that its end rises above the level of the liquid. Pressing upon the abdomen with the hands is of no effect (Method of Expression). Aspiration must at times be employed. For this purpose, Boas' bulbed tube (Fig. 33) is convenient. The manner in which it is used is the following : the proximal end of the tube, after having been introduced into the stomach, is compressed and the bulb squeezed, when the distal end is clamped and the bulb allowed to expand. When this is repeated several times a partial vacuum is Fig. 34. Arrangement of bottle for aspiration of tlie gastric contents. produced in the tube, which usually causes a flow of gastric juice. In the absence of such an instrument the stomach-tube may be con- nected with a bottle, in which a partial vacuum has been established by aspiration (Fig. 34). Unless the patient is accustomed to the introduction of the tube, however, these more complicated proced- ures should be avoided as much as possible (Method of Aspiration). I have found that in cases in which gastric juice cannot be ob- tained by expression the flow may often be started by suction with the mouth, and I regard this method as preferable to the one just described. With due precautions, viz., holding the tube between the fingers near the mouth of the patient, so as to be informed at once, by the sense of touch, when the stomach contents have reached this point, unpleasant results will be obviated. If only a very small amount of gastric juice is present in the stomach — i. e., when a defi- GENERAL CHARACTERISTICS OF THE GASTRIC JUICE. 153 nite flow cannot be established — it is best to suck lightly with the mouth, to compress the tube firmly, to remove it as rapidly as pos- sible, and empty it into a little dish. A few drops, sufficient to test for free hydrochloric acid, can thus always be obtained, even from the non-digesting organ. Einhorn's bucket-method is of little value, as the amount of gastric juice which can thus be obtained is insufficient for analytical pur- poses. It may be employed, however, in patients who are particu- larly nervous, and who object to the use of the tube, and possibly also when its use is contraindicated. The test for hydrochloric acid can be made, but the information thereby obtained is in itself of comparatively little value. In order to wash out the stomach, the funnel-tube is attached, the funnel filled with lukewarm water or any desired medicated solution, elevated above the head of the patient, and the water allowed to flow. From 500 to 1000 c.c. may be introduced at one time. By suddenly depressing and inverting the funnel over a suitable vessel before all water has left the funnel a siphon arrangement is estab- lished and the stomach emptied. It is well to measure the return- ing water as well as the amount introduced. Should the flow diminish or cease before all the water has been removed, the end of the tube "probably stands above the level of the liquid, and the flow can be started again by pushing the tube on further or by withdrawing it a little, as the case may be. Washing out the stomach soon after the ingestion of a full meal is always very tedious and annoying, if not an impossible procedure, as the fenestra readily become obstructed. Should this occur, the funnel, filled with water, is elevated as high as possible, with a view to overcome the obstruction by hydrostatic pressure ; or, if this proves insufficient, the funnel -tube is detached and the obstruction dislodged by means of air, for which purpose a Politzer bag or the bulb of a Boas tube is very convenient. GENERAL OHARAOTERISTIOS OF THE GASTRIC JUICE. Pure gastric juice is an almost clear, faintly yellowish fluid, of a sour taste and a peculiar, characteristic odor. Its specific gravity varies between 1.002 and 1.003, corresponding to the presence of but 0.5 per cent, of solids. Its reaction, owing to the presence of hydrochloric acid, is acid. Amount. Very little is known of the total quantity of gastric juice that is secreted in the twenty-four hours. The figure given by Beaumont,^ viz., 180 grammes pro die, based upon observations made upon the often-quoted Canadian hunter, Alexis St. Martin, is undoubtedly too ' Beaumont, Experiments and Observations on the G-astric Juice, Boston, 1834. 154 THE GASTRIC JUICE AND GASTRIC CONTENTS low. The amount given by Bidder and Schmidt/ viz., that corrc'^ sponding to about one-tenth of the body-weight, is probably more nearly correct.^ It may be stated a priori, however, that the quantity secreted varies within wide limits, being influenced by numerous factors, and notably by the degree of the appetite and the amount and character of the food taken, especially that of the proteids. The age and sex of the individual, the time of day (notably in its relation to the ingestion of food), the emotions, etc., all influence the glandular activity of the stomach. From the non-digesting organ, as has been pointed out, from 1 to 60 c.c. of gastric juice may be obtained at one time. The amount ■which can be procured during the process of digestion, on the other hand, varies with the amount of liquid ingested, the time of expres- sion, the size and motor power of the stomach, and the degree of transudation ; the process of resorption probably does not play any part, as it has been ascertained that very little water, if any, is absorbed in the stomach. According to Boas, from 20 to 50 c.c. of filtrate can normally be ob- tained exactly one hour after the ingestion of Ewald's test-breakfast.' vVbnormally large quantities of gastric juice are practically found only in cases of so-called hypersecretion, the " Magensaftfluss " of the Germans, which may occur periodically or continuously. For- merly the presence of appreciable quantities of gastric juice in the non-digesting organ was regarded as conclusive evidence of the existence of this condition, but in the light of Schreiber's researches this position can no longer be maintained. The diagnosis should, hence, only be made when in conjunction with the clinical symptoms of hypersecretion from 100 to 1000 c.c. of pure gastric juice can be obtained from the non-digesting organ. To this end, the stomach should be emptied completely by the tube, before retiring, and an examination made on the following morning, no food or liquids being allowed in the meantime. In various pathological conditions abnormally large quantities of liquid may be obtained, which cannot be regarded as gastric juice, how- ever. Attention will be drawn to these conditions at another place. CHEMICAL EXAMINATION OF THE GASTRIC JUICE. Chemical Composition of the Gastric Juice. As has been briefly shown above, gastric juice consists of water, free hydrochloric acid, certain ferments and their zymogens, and mineral salts. Analyses giving the exact chemical composition of pure, un- contaminated gastric juice in man are wanting, owing to the difficulty ' Bidder u. Schmidt, VerdauiinKsaiifte u. d. Stoffwechsel, 1852. ^ Griinewald's figure— i. e.., 1580 grammes — I likewise regard as too low. Acoordiug to my experience, the daily secretion appears to vary between 2000 and 3000 c.c. ' Eiegel, Die Erkrankungen des Magena, Part I. p. 88. CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 155 of excluding the saliva. In patients the subjects of gastric fistula analytical studies have, however, been made, and from the table below, taken from Schmidt, an idea may be formed of the various amounts of solid constituents contained in 1000 parts of gastric juice, uncontaminated by food or the products of digestion, but not free from saliva : Water 994.40 Solids Organic material . . Sodium chloride . . Calcium chloride . • Potassium chloride Ammonium chloride Hydrochloric acid . . Calcium phosphate Magnesium phosphate Iron phosphate 5.60 3.19 1.46. 0.06 0.55 0.20 0.12 The Acidity of the Gastric Juice is Referable to the Presence of Free Hydrochloric Acid. It has been conclusively demonstrated by Schmidt that the acidity of the gastric juice is due to the presence of free hydrochloric acid. P.ffl, 3.0 Fig. 35. 2.5 3.0 1.5 1.85 1.0 0.75 0.5 0.25 t 3fl: 90 loo 10 203040 soeomso Illustrating the curve of acidity after Ewald's test-breakfast. (Rosenheim.) Hydroohlorie acid. Lactic acid. X Beginning of the stage of free hydrochloric acid. P. M. Pro mille. The numbers upon the abscissa indicate the minutes. After accurately determining the amount of chlorine and all basic substances present, it was found that after the latter had been satu- 156 THE GASTRIC JUICE AND GASTRIC CONTENTS. rated a quantity of hydrochloric acid still remained, which in the dog varied between 0.25 and 0.42 per cent., with an average of 0.33 per cent. The amount of free acid was also determined by titration and the same results reached as by gravimetric analysis. While the acidity of pure gastric juice — i. e., gastric juice not contaminated with saliva or food in various stages of digestion — is thus solely due to the presence of free hydrochloric acid, other factors enter into consideration in the examination of the gastric contents during the process of digestion. Acid salts and varying amounts of lactic acid derived from the carbohydrates ingested are Fig. 36. in - _ _ -- 3Q _ - - ' " ~ ""^i ^,u , -- __^ >fc. __ _ ^ _ . __ tf^ -^- - _.- ^^ s " a.u _ _ _.^ ~: ::: : f^::: — :_ - - - / _ _ - ^- _ _ ^ : " 1 K __ ,/ __ " 1-^ . " ? :: : - ^?. :: : -_ - ^____ In ^-_'__ ■^■'* 4 t- __ - ^S Y\\\ rrrrill n - W M 1 iTt' f f MINI \l\ -It _ _ _ ~ ~~ - — " t-,± :::::___ _:: :_:...:.: 30 60 130 150 180 glO 3«) 270 300 Illustrating the curve of acidity after Riegel'a^st-ineal. (Rosenheim.) — ^Hydrochloric acid. Lactic add. X Beginning of^^^Uage of free hydrochloric acid. then also found. At the beginning of d^ipon the acidity, accord- ing to Ewald, is due to a certain extent to the presence of lactic acid.' Hydrochloric acid, it is true, is present at the same time, but is held in combination by albuminous material. Later on, when the albuminous affinities have become saturated, it appears as such, with the result that the formation of lactic acid progressively diminishes, owing to the inhibitory action on the part of the hydro- chloric acid upon the lactic-acid-producing organisms.^ ' Ewald, Klin. d. Verdauungskrankheiten, 1890, vol. i. Ewald u. Boas, Beitr. z. Physiol, u. Path. d. Verdauung. Virchow's Archiv, 1885, vol. ci. p. 355, and 1886, vol. civ. p. 271. See Lactic Acid, p. 183. '' H. Strauas u. F. Bialocnur, " Ueber d. AbhSngigkeit d. Milchsauregahrung v. HCa— Gehalt d. Magensaftes," Zeit. f. klin. Med., vol. xxviii. p. 567. CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 157 The varying degrees of acidity at different periods of digestion, after such test-meals as those of Ewald and Riegel, and the amount of the two acids present, may be seen from the accompanying diagrams (Figs. 35 and 36). Under pathological conditions the amount of free hydrochloric acid, as will be shown, may undergo great variations, diminishing on the one hand to zero, and increasing on the other to 0.5 per cent., or even more. At the same time the amount of lactic acid, which normally is present in very small amounts, and is absent altogether at the height of digestion, may greatly increase. Fatty acids, more- over, which are normally not present in the gastric juice, may then also be observed. It is thus seen that the total acidity of the gas- tric juice, especially in disease, cannot be regarded as indicating the amount of one single acid, unless the absence of other acids and acid salts is insured. Method of determining the Total Acidity of the Gastric Contents. To this end, a known quantity of gastric juice is titrated with a one-tenth normal solution of sodium hydrate, using phenolphthalein as an indicator, when the number of cubic centimeters of the one- tenth normal solution employed, multiplied by the equivalent of 1 c.c. of this solution in terms of hydrochloric acid, will indicate the amount of acid present, from which the percentage-acidity is readily calculated. A normal solution of sodium hydrate is one containing the equiva- lent of its molecular weight in grammes — i. e., 40 grammes — in 1000 c.c. of distilled water; a decinormal solution will, therefore, contain 4 grammes in the same volume of water. This quantity is dissolved in less than 1000 c.c. and the solution brought to the proper strength by titrating it with a solution of oxalic acid of known strength. From the equation ' 2NaOH + CjHjOj = CjNa^O^ + 2H,0, it is seen that two molecules of NaOH (molecular weight 40) com- bine with one molecule of C2H2O4 + 2H2O (molecular weight 126), or 4 parts by weight of the former with 6.3 of the latter. One-tenth gramme of oxalic acid would hence require 15.873 c.c. of the one- tenth normal solution of NaOH for its neutralization, as is apparent from the equation 100 6.3 : 1000 : : 0.1 : a; ; 6.3a: = 100, and a; = 3-3 = 15.673. 158 THE GASTRIC JUICE AND QASTBIC CONTENTS. One-tenth gramme of pure crystallized oxalic acid is dissolved in distilled water, and the solution titrated with the one-tenth normal solution of sodium hydrate, which is to be corrected, using two or three drops of a 1 per cent, alcoholic solution of phenolphthalein as an indicator, until the rose color of the solution has entirely disap- peared ; 15.9 c.c. should bring about this result. As the NaOH solution, however, has been purposely made too strong, less will be required. The amount of water that must then be added in order to bring the solution to its proper strength is determined by the formula Nd (7= — , in which C represents the number of cubic centimeters of n water which must be added to the remaining solution, N the total number of cubic centimeters remaining after one titration, n the number of cubic centimeters consumed in one titration, and d the difference between the number of cubic centimeters theoretically required and that actually used in one titration. The solution hav- ing thus been properly diluted, the correctness of its strength is again tested and a further correction made, if necessary, until abso- lute accuracy has been attained. 1000 c.c. of the one-tenth normal solution containing 4 grammes of NaOH are equivalent to 3.65 grammes of HCl, as is seen from the equation NaOH + HCl = NaCl + Hp 40 36.5 3000 c.c. of the ^ normal solution represent 3.65 grammes of HCl 100 " " " " " " 0.365 gramme " " 10 " " " " " " 0.0365 " " " 1 " " " " " represents 0.00365 " " " Application to the Gastric Juice. — Five or 10 c.c. of the filtered gas- tric juice are titrated with the one-tenth normal solution of sodium hydrate, using two or three drops of a 1 per cent, alcoholic solution of phenolphthalein, as an indicator, until the rose color which appears after the addition of every drop of the sodium hydrate solution no longer disappears on stirring or becomes deeper after the addition of a further drop. The number of cubic centimeters of the one-tenth normal solution employed multiplied by 0.00365 will then indicate the acidity of the 5 or 10 c.c. of gastric juice in terms of HCl, from which the percentage-acidity is calculated. Example. — Ten c.c. of gastric juice required the addition of 6.5 c.c. of the one-tenth normal solution ; 6.5 X 0.00365 (i. e., 0.0237) would hence indicate the acidity of the 10 c.c. of gastric juice in terms of HCl, and 0.0237 X 10 = 0.237, the percentage-acidity. As these figures express the amount of HCl in pure gastric juice obtained only from normal individuals, it has been found more con- venient for clinical purposes merely to indicate the degree of acidity by the number of cubic centimeters of the one-tenth normal solution CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 159 employed. In the above example, in which 6.5 c.c. were used, the percentage acidity would thus be indicated by the figure 65 — i. e., the number of cubic centimeters of the one-tenth solution necessary to neutralize 100 c.c. of gastric juice. Under normal conditions figures varying from 40 to 60 are usually obtained one hour after the ingestion of Ewald's test-breakfast, while in pathological conditions greater variations are observed. In acute and chronic inflammatory conditions of the stomach, as well as in some of the neuroses, the acidity of the gastric contents is below normal. Higher figures are met with in cases of ulcer, in some cases of dilatation, and are especially frequent in some of the neuroses, in which a degree of acidity corresponding to 90 or even more is not infrequently observed. Increased acidity, usually asso- ciated with hypersecretion of gastric juice, is met with in the so- called hyperseeretio acida et continua of Reichmann.' It has been pointed out that the reaction of normal gastric juice is always acid, owing to the presence of free hydrochloric acid, and the same may be said to hold good for the gastric contents in general, obtained from a normal individual. Pathologically an acid reaction is also the rule, as in those cases in which hydrochloric acid is absent fatty acids and lactic acid usually make their appearance. It is, therefore, not surprising that an alkaline, neutral, or amphoteric reaction is but rarely, or at least not commonly, observed in the gastric contents artificially obtained, and practically seen only in the so-called mucous form of chronic gastritis, or in those rare cases of anadeny, in which a complete destruction of the gastric glands has taken place. In vomited material, on the other hand, such observations are common, owing to the presence of large amounts of saliva. The vomited material in cases of so-called vomitus matutinus, which is usually referable to a chronic catarrhal condition of the pharynx, generally presents an alkaline reaction, owing to the fact that the fluid brought up is largely unchanged saliva. Source of the Hydrochloric Acid. That the hydrochloric acid is not directly derived from the chlo- rides ingested is shown by the fact that it is secreted by starving animals. The same point is also proved by the observations of Schreiber, which go to show that the secretion of the acid is con- tinuous, not to mention the well-known fact that even after the ingestion of material free from chlorine an acid gastric juice is secreted. It is apparent, then, that the chlorides of the blood must furnish the necessary chlorine, and as the pyloric glands, which con- 'Eeichmann. Berlin, klin. Woch., 1882, Vol. xix. p. 606; 1884, vol. xxi. p. 768; 1887, vol. xxiv. p. 12. 160 THE GASTRIC JUICE AND GASTRIC CONTENTS. tain no parietal cells, furnish an alkaline, and the fundus glands, which do contain parietal cells, an acid secretion, it is thought that these parietal cells are in some manner concerned in the production of the hydrochloric acid. The exact manner in which this takes place has not been definitely ascertained, but it is not improbable that the acid results from a " Masseneinwirkung " on the part of the carbonic acid, which is present in large quantities in the blood as such, upon the sodium chloride, and that owing to a specific action on the part of the parietal cells the hydrochloric acid is secreted into the ducts of the glands of the stomach, while the sodium carbonate which is formed at the same time returns to the blood. Two factors are thus necessary in order that a normal amount of hydrochloric acid should be secreted — i. e., a normal condition of the blood and a normal condition of the cells. Whenever the integrity of either of these factors becomes impaired, it is clear that an abnormal secretion of hydrochloric acid or none at all will result. The nervous system, furthermore, must be taken into con- sideration as a third factor, as normal innervation is the sine qua non for the normal activity of any organ. The secretion of the acid is impaired whenever the nutrition of the cells of the stomach suffers, whether this be the result of inflammatory lesions, new growths, or hypersemic conditions of the stomach, the effect of renal, hepatic, or pulmonary diseases, etc., or in consequence of central or peripheral nerve influences. In the secondary dyspepsias, then, the result of renal, hepatic, cardiac, or hsemic diseases, etc., an examination of the gastric juice for free hydrochloric acid is of comparatively little value from a diagnostic standpoint, although it may suggest valuable points for the dietetic treatment of such patients. Significance of Free Hydrochloric Acid. Formerly it was believed that the principal function of the stomach was a digestive one, and that in the stomach, owing to the action of hydrochloric acid and pepsin, albumins were to a large extent transformed into peptones and albumoses. As pepsin is active only in the presence of a free acid, it was thought, moreover, that the power of the hydrochloric acid to render pepsin physiologically active constituted its entire field of usefulness. It had been noted over one hundred years ago, however, by the Abb6 Spallanzani, that pieces of meat immersed in gastric juice resist the process of putrefaction for days. When it was shown, later on, that the free mineral acids are powerful antiseptics, and that the stomach secretes an amount of free hydrochloric acid sufficient to prevent the development of most of the putrefactive CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 161 organisms, the time had come to doubt the correctness of the view previously held. Numerous experiments have been made in order to test the anti- septio and germicidal power of the gastric juice. Among the more important results achieved the following may be mentioned : the comma bacillus of cholera Asiatica is destroyed by normal acid gastric juice, while infection results when this has previously been neutralized. The same holds good for numerous other pathogenic organisms which are of special interest to the clinician. Among these may be mentioned the various species of streptococcus, Staphylo- coccus pyogenes aureus, the bacillus of anthrax, etc. ' Unfortunately, however, not all species of pathogenic organisms are destroyed by the acid of the gastric juice, and the spores, moreover, of some of those that are destroyed are possessed of a considerable degree of resist- ance. This is especially true of the tubercle bacillus and in many cases of tha spores of the anthrax bacillus. Those bacteria also which cause lactic acid and butyric acid fer- mentation resist the antifermentative power of the gastric juice to a certain extent, as may be concluded from the fact that they are always present in the intestines. At the beginning of the process of gastric digestion, when the hydrochloric acid secreted is immediately taken up by the albuminous bodies present, traces of lactic acid can usually be demonstrated in the gastric contents if carbohydrates have been ingested. Later on, when free hydrochloric acid appears, lactic acid fermentation ceases. This observation is in accord with the fact that the action of the lactic acid producers is prevented by the presence of 0.7 pro mille of free hydrochloric acid. From what has been said it may be argued that as the principal function of the stomach consists in the furnishing of an antiseptic and germicidal fluid, under suitable conditions life could go on in the absence of the stomach. That this is possible has been demon- strated by Czerny, who succeeded in removing almost the entire organ from a dog. Five or six years later the same animal was killed in Ludwig's laboratory, and it was found at the autopsy that "near the cardia a small portion of the stomach had remained, surrounding a globular cavity filled with food." This dog then had lived for almost six years practically without a stomach, had gained m weight, and was to all intents and purposes as healthy an animal as one provided with an entire organ. In the human being similar observations have been made on subjects of carcinoma of the stomach. It is thus very probable that the stomach, so far as the process of digestion is concerned, is not necessary for the maintenance of life. LiTBEATOEE. — Spallanzani, Experiences sur la digestion de I'homme et de diflgr- eutes espSoes d'animaux, Geneve, 1784. Bunge, Lehrbuch d. physiol. Chem., 1889, p. 44. Mester, "Ueber Magensaft u. Danufaulniss," Zeit. f. Win. Med., vol. xxiv. p.. 441. Schmitz, " Zur Kenntniss d. Darmf aulniss." Zeit. f. physiol. Chem., vol. xvii. p. 401 ; " Die Beziehung d. Salzsaure d. Magensaftes z. Darmf aulniss," Ibid., vol. xix. 11 162 THE GASTRIC JUICE AND GASTRIC CONTENTS. p. 401. C. E. Simon, " On Indicanuria," Am. Jout. Med. Sci., 1895, vol. ex. p. 48. Czerny, Beitrage z. operatlven Chirurgie, Stuttgart, 1878, p. 141. Ludwig u. Ogata, "Ueber d. Verdauung nach d. Ausschaltung d. Magens," Du Bois' Archiv, 1883, p. 89. J. Carvallo u. V. Pachon, " Untersuchungen liber d. Verdauung bei einem Hunde ohne Magen," Arcli. der Physiol., 1894, p. lOd. The Amount of Free Hydrochloric Acid. Pure gastric juice, according to Ewald/ Szabo/ and Boas,' con- tains from 2 to 3 pro mille of free hydrochloric acid. In the digesting organ such amounts are met with only at the height of digestion, and after all albuminous and basic affinities have been saturated. The time at which free hydrochloric acid can be demonstrated in the gastric contents after the ingestion of a meal will, hence, vary with the character of the food and its amount. When but little work is to be accomplished free hydrochloric acid is found much sooner than otherwise. After Ewald's test-breakfast, for example, it appears after thirty -five minutes ; the point of maxi- mum acidity is reached after from fifty to sixty minutes, and corre- sponds to the presence of 1.7 pro mille. Following Riegel's meal, on the other hand, the free acid appears after one hundred and thirty- five minutes, and reaches its highest point (corresponding to 2.7 pro mille) in from one hundred and .eighty to two hundred and ten minutes (Figs. 35 and 36). Clinically it is necessary to distinguish between euchlorhydria, or the secretion of a normal amount of free hydrochloric acid (0.1 to 0.2 per cent.), hypochlorhydria, or the secretion of a deficient amount (less than 0.1 per cent.), hyperchlorhydria, in which more than 0.2 per cent, is found, and, finally, anachlorhydria, in which no hydrochloric acid at all is secreted. Euchlorhydria. — Euchlorhydria, when associated with clinical symptoms pointing to gastric derangement, is most commonly ob- served in nervous dyspepsia. A chronic gastritis can always be excluded in the presence of a normal amount of the free acid, thus constituting a most important point in the differential diagnosis between the two conditions. A normal secretion of free hydro- chloric acid is, furthermore, observed in some cases of atony or hypatony of the muscular walls of the stomach. Hypochlorhydria. — Hypochlorhydria is associated with all those diseases in which the secretory elements have been more or less damaged, as in subacute and chronic gastritis, in some cases of ulcer of the stomach or the duodenum, in incipient carcinoma, dilatation, and atony. Anachlorhydria. — Not many years ago it was thought that the absence of free hydrochloric acid from the gastric contents was pathognomonic of carcinoma of, the stomach. This view was soon abandoned, however, as it was shown that cases of carcinoma occur ' Loc. cit. 2 D. SzabA, Zeit. f. physiol. Chem., 1877, vol. i. p. 155. ' Loo. cit. See also A. Sohiile, Zeit. f. klin. Med., 1896, vols, xxviii. and xxix. CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 163 in whicli hydrochloric acid is not only present, but present in ex- cessive amounts. This is true especially of those cases in which the malignant growth has started upon the base of an old ulcer. It was, furthermore, shown that anachlorhydria exists in almost all cases of advanced chronic gastritis, and is a very common occur- rence in neurasthenic and hysterical individuals, constituting the so-called hysterical anacidity. Hyperchlorhydria. — The existence of hyperchlorhydria is gen- erally indicative of a gastric neurosis, and is thus frequently met with in its simplest form in certain neurasthenic individuals. Asso- ciated with a continuous hypersecretion of gastric juice it constitutes the neurosis that has been described under the term hypersecretio aoida et aontinua. Hyperchlorhydria is also of frequent occurrence in cases of gastric ulcer, and may even occur in carcinoma, notably in those cases in which, as stated above, the new growth has started from an old ulcer. Test for Free Acids. Following a physical examination of the gastric contents, and, if acid, a determination of the total acidity, the next step will be to determine whgther or not the acid reaction is referable to the pres- ence of a free acid, of combined acids, or of acid salts. The Congo-red Test.^ — Congo-red is a carmin-colored powder, while its solutions are of a peach- or brownish-red color, which changes to azure blue upon the addition of a free acid, but remains unaffected in the presence of an acid salt. Congo-red may be em- ployed in solution or in the form of a test-paper. The latter, how- ever, is less delicate than the solution, and indicates only the pres- ence of 0.01 per cent, of hydrochloric acid, while a positive reaction can still be obtained with the aqueous solution in the presence of 0.0009 per cent. The solution should be moderately dilute. The test-paper is prepared by soaking filter-paper, free from ash, in this solution, drying, and cutting it into suitable strips. In order to test for the presence of a free acid, it is only necessary to immerse a strip of the test-paper in the filtered gastric juice, or to add a drop or two of the solution to a small amount of the juice, when in the presence of a free acid a blue color will develop, which varies from a sky-blue to a deep azure according to the amount present. A negative result will exclude at once the possibility of peptic activity, as pepsin acts only in solutions containing a free acid. If the result of the test is positive, the nature of the free acid must still be ascertained, and it is, therefore, necessary to test for free hydrochloric acid, or in its absence for lactic acid and certain fatty acids. ' Eiegel, Deutsch. med. Woch., 1886, No. 35 ; and Boas, Diagnostik n. Therapie d. Magenkran kheiten. 164 THE GASTRIC JUICE AND OASTBIC CONTENTS. Tests for Free Hydrochloric Acid. The various reagents which may be employed are given below, and are arranged according to their degree of delicacy, viz.: 1. Dimethyl-amido-azo-benzol 0.02 pro mille 2. Phlorogliicin-vanillin 0.05 " 3. Eesorciii 0.05 " 4. Tropsolin 00 0.30 5. Mohr's reagent 1.00 " The Dimethyl-amido-azo-benzol Test.' — This test is known also as Topfer's test, and is destined to replace the older phloroglucin- vanillin and resorcin tests in the clinical laboratory. The delicacy of the reagent is such that the natural yellow color of the indicator is changed to a reddish tinge upon the addition of but one drop of a one-tenth normal solution of hydrochloric acid in 5 c.c. of dis- tilled water. Its superior delicacy, as compared with the phloro- glucin-vanillin and resorcin tests, is apparent from the fact that 6 c.c. of a 0.5 per cent, solution of egg-albumin, to which six drops of a one- tenth normal solution of hydrochloric acid have been added, still give a positive reaction with dimethyl-amido-azo-benzol, while the phloroglucin-vanillin and resorcin reactions are negative. Organic acids yield a red color only when present in amounts exceeding 0.5 per cent.; but even then a negative reaction is ob- tained, if, as in the stomach, small quantities of albumins, peptones, or mucin are present at the same time. A positive reaction is then obtained only when the organic acids are present in amounts far exceeding 0.5 per cent. Loosely combined hydrochloric acid and acid salts do not produce this change in color. For practical purposes a 0.5 per cent, alcoholic solution is em- ployed. One or two drops of this are. added to a trace of the gastric contents, which need not be filtered : in the presence of free hydro- chloric acid a beautiful cherry-red color develops, which varies in intensity according to the amount of free acid present. A test-paper, prepared by soaking strips of filter-paper in the 0.5 per cent, solu- tion and allowing them to dry, may also be employed. With gastric juice containing no free hydrochloric acid, as with distilled water, a yellow color results, the fluid at the same time becoming cloudy and beautifully fluorescent. I have personally used Topfer's test during the' last seven years, and prefer it to all others. The Phloroglucin-vanillin Test.^ — The solution employed con- tains 2 grammes of phloroglucin and 1 gramme of vanillin, dissolved in 30 C.C. of absolute alcohol : a yellow color results, which gradu- ' Topfer, Zeit. f. physiol. Chem,, vol. xlx. Hari, Arch. f. Verdauungskrank., vol.ii. pp. 182 and 332. 2 Giinzburg, Centralbl. f. klin. Med., 1887, vol. viii. No. 40. CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 165 ally turns a dark golden red, changing to brown when exposed to light. The solution should therefore be kept in a dark-colored bottle. Lenhartz suggests the use of separate solutions of phloro- glucin and vanillin, one or two drops of each being employed in the test. Boas recommends a solution of the phloroglucin and vanillin, in the proportions indicated, in 100 grammes of 80 per cent, alcohol, and claims that the reagent is then still more sensitive and more stable. If a few drops of gastric juice, or even of the unfiltered gastric contents, containing 0.05 per cent, or more of free hydro- chloric acid, are treated with the same number of drops of the reagent, no change in color results, but upon the application of gentle heat — boiling and rapid evaporation are to be avoided — a, rose-tint or exceedingly fine rose-colored lines develop, which are characteristic of the presence of the free acid. For practical purposes it is best to carry on this slow evaporation on a thin porcelain butter-dish, the porcelain cover of a crucible, or in a small evaporating-dish of the same material. The color obtained in the presence of free hydrochloric acid is a rose color in every in- stance, and varies in intensity with the amount of acid present. A brown, brownish-yellow, or brownish-red color always indicates that excessive heat has been applied or that free hydrochloric acid is absent. Organic acids do not produce the reaction, nor is it interfered with by their presence, or that of albumins, peptones, or acid salts. A phloroglucin-vanillin test-paper, prepared by soaking strips of filter-paper, free from ash, in the solution and drying them, may also be employed. If a strip of this is moistened with a drop of gastric juice and gently heated in a porcelain dish, the rose color will develop in the presence of free hydrochloric acid, and does not disappear upon the addition of ether. The Resorcin Test.^ — The solution consists of 5 grammes of resublimed resorcin and 3 grammes of cane-sugar, dissolved in 100 grammes of 94 per cent, alcohol. It is equally as delicate as the phloroglucin-vanillin solution and has the advantage of greater stability. Five or six drops of gastric juice are treated with three to five drops of the reagent and slowly evaporated to dryness over a small flame, when a beautiful rose- or vermilion-red mirror will be obtained, which gradually fades on cooling. If the reagent is employed in the form of a test-paper, a violet color at first develops, which upon the application of heat turns brick red and does not disappear on treatment with ether. The presence of acid salts, organic acids, albumins, or peptones does not interfere with the reaction. 1 Boas, Centralbl. f. klin. Med., 1888, vol. ix. No. 45. 166 THE GASTRIC JUICE AND GASTRIC CONTENTS. The Tropseolin Test.' — Tropseolin 00, when employed according to the method suggested by Boas, is a very reliable reagent, indi- cating the presence of 0.2 to 0.3 per cent, of free hydrochloric acid. Three or four drops of a saturated alcoholic solution of tropseolin 00, which has a brownish-yellow color, are placed in a small porcelain dish or cover, and allowed to spread over the surface. A like amount of gastric juice is then added and likewise allowed to flow over the surface of the dish ; upon the application of gentle heat beautiful lilac or blue stripes appear, which are said to be absolutely character- istic of free hydrochloric acid. A tropseolin test-paper may also be prepared by soaking filter- paper, free from ash, in the alcoholic solution, and then drying and cutting it into strips. A fev/ drops of gastric juice containing free hydrochloric acid produce a more or less pronounced brown color upon this paper, which turns lilac or blue upon the application of gentle heat. Organic acids, when present in large amounts, likewise produce a brown color, but this disappears on heating, and a lilac or blue color does not result. For ordinary purposes this test is sufficient, and recourse need only be had to the more delicate reagents when a negative or a doubtful result is obtained. Mohr's Test, as modified by Ewald.^ — Two c.c. of a 10 per cent, solution of potassium sulphocyanide are treated with 0.5 c.c. of a neutral solution of ferric acetate, and diluted to 10 c.c. with distilled water, a ruby-red solution resulting. Of this, a few drops are placed in a porcelain dish, when a drop or two of the filtered gastric con- tents are allowed to come into contact with the reagent. In the presence of free hydrochloric acid a light- violet color develops at the point of contact between the two fluids, and turns a, deep mahogany- brown upon mixing. The test is not interfered with by the presence of acid salts or peptones, but is not so sensitive as those already described. The Benzopurpurin Test.^ — Benzopurpurin 6B has been highly recommended by v. Jaksch as a very sensitive test for hydrochloric acid. It is best used in the form of a test-paper, prepared by soak- ing strips of filter-paper, free from mineral ash, in a concentrated watery solution of the reagent and allowing them to dry. In the presence of more than 0.4 gramme of hydrochloric acid in 100 c.c. of gastric juice the color of the test-paper immedi- ately turns a deep blackish-blue. Should a brownish-black color develop, this is likely due to the presence of organic acids, or, a mixt- ure of these and hydrochloric acid. If the color is caused by or- 1 Ewald, Klinik d. Verdauungskrank., Berlin, 1888, vol. ii. ; and Boas, Deutsch. mecL Woch., 1877, vol. xiii. p. 852. 2 Ewald u. Boas, Virchow's Arohlv, vol. cl. p. 325 ; vol. civ. p. 271. ' V. Jaksch, Klinische Diagnostik, 1896, p. 177. CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 167 ganic acids only, it will disappear on washing the strip with a little neutral ether, the original color of the test-paper being thus restored ; but if due to a mixture of the two, the reaction is less marked, and does not disappear. According to Hellstrom,' 0.39 milligramme of hydrochloric acid, dissolved in 6 c.c. of water, can be recognized by the addition of only 5 milligrammes of benzopurpurin. Acid salts, peptones, and serum-albumin do not seriously inter- fere with the reaction. V. Jaksch claims that the benzopurpurin test-paper is more sensi- tive than the Congo-red paper. The Combined Hydrochloric Acid. It has been stated (see page 155) that the total acidity of the gastric juice can only be referred to hydrochloric acid when organic acids and acid salts are absent. But at the same time the free acid is titrated together with the loosely combined acid. The presence of free hydrochloric acid in normal amounts implies, of course, the existence of peptic activity, and indicates that all albuminous affinities have been saturated. In the absence of free hydrochloric acid, however, it is important to know whether or not hydrochloric acid is secreted at all — i. e., whether peptic digestion is at a standstill or whether an amount is secreted that is sufficient to saturate only certain albu- minous affinities without appearing in the free state. In the treat- ment of the various forms of gastric disease, more especially those associated with an absence of free hydrochloric acid, accurate knowl- edge in this respect is important. If no hydrochloric acid at all is secreted, the stomach can only 'be regarded as a storehouse, as it were, and proteids must be ordered in such a form that they may be subjected to the process of pancreatic digestion with as little delay as possible, the nutrition of the body being aided, if necessary, by a suitable administration of predigested food. If, on the other hand, an amount of hydrochloric acid is secreted which is sufficient to saturate the albuminous affinities of an ordinary meal, or at least of moderate amounts of proteids, the dietetic directions need not be so stringent. While in the former case the absence of loosely com- bined hydrochloric acid usually indicates complete destruction of the glandular elements of the stomach — in other words, an irrepar- able condition — a fair prognosis may be given when the amount of acid secreted is sufficient for the saturation of the albuminous affinities of an ordinary meal. The following table ^ shows the amount of hydrochloric acid necessary to saturate the affinities of known quantities of various articles of food, the figures given having reference to 100 c.c. or 100 grammes : ' Cited by v. Jaksch. ' Taken, in part from personal observations, and in part from Ehrlich, Dissert., Erlangen, 1893. 168 THE GASTRIC JUICE AND OASTBIO CONTENTS. Milk Beef (boiled) . Mutton (boiled) . . . Veal (boiled) . . Pork (boiled) . . Sweetbread (boiled) . Calves' brains (boiled) Ham (raw) Ham (boiled) . . . . Flounder . . . . Liver sausage . Cervelat sausage Mettwurst . . . Bologna sausage . . • Blood sausage . . . Potato (mashed) Rice (milk) . . . . Corn . . . Graham bread . Pumpernickel . Wheat bread . . Eye bread Swiss clieese Fromage de Brie Edam cheese Roquefort cheese Beer (German) . 0.32-0.56 gramme of pure HOI. 1.95-2.0 grammes " " 1.9 " " " 2 2 *' " " 1.. 5-1^6 " " " 0.9-^0.95 gramme " " 0.56-0.65 " " 1.9 grammes " 1.3-1.8 " " " 1.41 " " " 0.8-6.9 gramme " " 1.1 grammes '' " 1.0 gramme " " 1.49 grammes " " 0.3 gramme " " 0.48 " " " 1.22 grammes " " 0.27 gramme " " 0.3 " " " QfJ it It it 0.3-0.'5 " " 0.3-0.5 " " 2.6-2.7 grammes " " 1.3 " " " 1.4 21 " '' '' 0.07-0.15 gramme " " Quantitative Estimation of the Hydrochloric Acid of the Gastric Juice. Tbpfer's Method.' — The free and combined hydrochloric acid is most conveniently estimated according to Topfer's method, which is both simple and sufficiently accurate for clinical purposes. In this method the total acidity (a) of a given amount of gastric"^ juice — i. e., the acidity referable to the presence of free hydrochloric acid, combined hydrochloric acid, acid salts, and any organic acids that may be present — is first determined (lactic acid and the fatty acids, if present, need not be removed), using phenolphthalein as an indicator. This is followed by a determination of the acidity refer- able to free acids and acid salts in the same amount of gastric juice (6), using alizarin (alizarin monosulphonate of sodium) as an indi- cator. As this does not react with loosely combined hydrochloric acid, the diiferencc between a and 6 will indicate the amount of the latter. The free hydrochloric acid (c) finally is estimated with dimethyl-amido-azo-benzol as an indicator, the difference between a and 6-f-e giving the acidity referable to organic acids and acid sattST^ The solutions required are the following : 1. A decinormal solution of sodium hydrate. 2. A 1 per cent, alcoholic solution of phenolphthalein. 3. A 1 per cent, aqueous solution of alizarin. 4. A 0.5 per cent, alcoholic solution of dimethyl-amido-azo-benzol. Three separate portions of 5 or 10 c.c. of filtered ga.stric juice are measured into three small beakers or porcelain dishes. To the ' Loo. cit. CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 169 first portion 1 or 2 drops of phenolphthalein are added, when it is titrated with the one-tenth normal solution of sodium hydrate. It is necessary, however, to titrate to the point of a deep red, and not to the rose hue which first appears. It will be seen that upon the addition of the first few drops of the one-tenth normal solution the red color, which first appears, disappears on stirring. Upon further titration a point is reached when this no longer occurs, and the color of the entire solution suddenly turns to a rose. This, however, is not the end-reaction that is desired. If the titration is continued, it will be observed that a dark-red cloud forms in the light rose-colored solution, which disappears on stirring ; finally, a point is reached when an additional drop no longer intensifies the color of the solution. This point is the end-reaction which must be reached. To the second portion 3 or 4 drops of the alizarin solution are added, when it also is titrated with the one-tenth normal solution of sodium hydrate until a pure violet color is obtained. As practice is required in order to determine this point with accuracy, Topfer advises to make previously the following simple tests : 1. To 5 c.c. of distilled water add 2 or 3 drops of alizarin solu- tion, when a yellow color will result. 2. To 5 c.c. of a 1 per cent, solution of disodium phosphate add the same number of drops, when a red or slightly violet color will be obtained. 3. Five c.c. of a 1 per cent, solution of sodium carbonate, treated with 2 or 3 drops of the alizarin solution, will strike a pure violet ; this is the color to which the titration must be carried. In the third portion of the gastric juice the free hydrochloric acid is titrated, after the addition of 3 or 4 drops of the dimethyl-amido- azo-benzol, until the last trace of red — in the presence of free hydro- chloric acid — has disappeared. A yellow color resulting upon the addition of the indicator demonstrates the absence of the free acid," as has been shown on page 164. The results are then calculated as in the following example : Ten c.c. of gastric juice, using phenolphthalein as ■ an indicator, required 10 c.c. of the one-tenth normal solution in order to bring about the end-reaction, while a like amount titrated in the same manner with alizarin required 7 c.c. in order to bring about the same result. The difference between 10 and 7 — /. e., 3 — would thus indicate the number of cubic centimeters necessary to neutralize the amount of hydrochloric acid in combination with albuminous material. As 1 c.c. of the one-tenth normal solution represents 0.00365 gramme of hydrochloric acid, the amount of acid thus held will be equivalent to 0.00365 X 3 = 0.01095 gramme of hydrochloric acid — i. e., 0.1095 per cent. In the estimation of the free hydrochloric acid, 2.3 c.c. of the one- tenth normal solution were required, using dimethyl-amido-azo-ben- 170 THE GASTRIC JUICE AND GASTRIC CONTENTS. zol as an indicator ; this would correspond to 0.00365 X 3.2 — i. e., 0.1168 per cent. The value of the total acidity in terms of hydro- chloric acid is 10 X 0.00365 ^0.0365 gramme for every 10 c.c. of gastric juice, or 0.365 per cent. By deducting the amount of the free and combined hydrochloric acid, viz., 0.1095 + 0.1168 = 0.2263, from this, it is found that the acidity of the gastric juice referable to organic acids and acid salts amounts to 0.1387 per cent., so that the results can be tabulated as follows : Free hydrochloric acid 0.1168 per cent. Combined hydrochloric acid 0.1095 " Organic acids and acid salts 0.1387 " Total acidity 0.3650 per cent. The Method of Martius and Liittke (modified).' — ^This method is equally exact, but requires a greater expenditure of time. It is based upon the fact that upon incineration" of the gastric juice the free hydrochloric acid and that loosely combined with albu- minous material escape, while the chlorine in combination with inorganic bases remains in the mineral ash unless a very intense heat is applied for some time. By subtracting the amount of chlorine present in the latter form from the total amount, the quantity in combination with albuminous material and that occurring as free acid will be found. The total acidity of the gastric juice is then determined, and that referable to the presence of the free and com- bined hydrochloric acid subtracted, the difference giving the amount of organic acids present. By determining the acidity due to the pres- ence of free hydrochloric acid according to Topfer's method, and deducting the amount found from that referable to the presence of free and combined hydrochloric acid, the amount of the latter is obtained. Reagents required : 1. A solution of silver nitrate in nitric acid of such strength that 1 c.c. shall represent 0.00365 gramme of hydrochloric acid. 2. Liquor ferri sulphurati oxydati. 3. A decinormal solution of ammonium sulphocyanide. 4. A one-tenth normal solution of sodium hydrate. 5. A 1 per cent, alcoholic solution of phenolphthalein. 6. A 0.5 per cent, alcoholic solution of dimethyl-amido-azo-benzol. Preparation of the solutions : 1. The silver nitrate solution. As a solution is required of such strength that 1 c.c. shall be equivalent to 0.00365 gramme of hydro- chloric acid, the amount of silver nitrate that must be dissolved in 1000 c.c. of water is ascertained in the following mariner : since 169.66 (molecular weight) parts by weight of silver nitrate combine with 36.5 parts of hydrochloric acid (molecular weight), the amount ' F. Martius u. L. Liittke, Die Magensaure des Menschen, Stuttgart, 1892. CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 171 of silver nitrate required for each cubic centimeter is found from the equation 169.66 : 36.5 : : x : 0.00365 ; 36.5 1 = 0.6192590 ; x = 0.0169. In 1 CO. of the silver solution 0.0169 gramme of silver nitrate must thus be present, or 16.9 grammes in the liter. This quantity, or roughly 17 grammes, is weighed off and dissolved in 900 c.c. of a 25 per cent, solution of nitric acid ; as the acid must be present in excess, the solution is purposely made too strong. To this solution 50 c.c. of the liquor ferri sulphurati oxydati are added. The solu- tion is then brought to the proper strength by titration of a known number of cubic centimeters of a one-tenth normal solution of hydrochloric acid and correcting as usual. 2. The ammonium sulphocyanide solution. A normal solution of ammonium sulphocyanide contains 75.98 grammes (molecular weight) per liter, and a decinormal solution 7.598 grammes. This quantity, or roughly 8 grammes, is dissolved in about 900 c.c. of water and the solution brought to the proper strength by titrating a known number of cubic centimeters of the silver nitrate solution, when each cubic centimeter should correspond to 1 c.c. of the silver solution — i. e., to 0.00365 gramme of hydrochloric acid. It is corrected as described elsewhere. Method. — 1. To determine the total amount of chlorine present : 10 c.c. of filtered gastric juice — ^JVIartius and Liittke make use of the unfiltered gastric contents — are measured into a small ilask bearing a 100 c.c. mark, and treated with an excess of the one-tenth normal solution of silver nitrate. Experience has shown that 20 c.c. are sufficient. The mixture is agitated and allowed to stand for ten minutes. Distilled water is then added to the 100 c.c. mark; the mixture is agitated once more and filtered through a dry filter into a dry beaker. Fifty c.c. of the filtrate are titrated with the one- tenth normal solution of ammonium sulphocyanide until the blood- red color which appears upon the addition of every drop — due to the formation of ferric sulphocyanide — no longer disappears on stirring. By multiplying the number of cubic centimeters of the ammonium sulphocyanide solution used by 2 (the number of cubic centimeters that would have been necessary for the precipitation of the excess of silver in 100 c.c.) and deducting the result from the number of cubic centimeters of the one-tenth normal solution of silver nitrate employed, viz., 20, the number of cubic centimeters of the latter solution is found which was necessary to precipitate the chlorine in 10 c.c. of the gastric juice. As 1 c.c. of the solu- tion represents 0.0036 gramme of hydrochloric acid, it is only nec- essary to multiply this figure by the number of cubic centimeters used in precipitation of the chlorine. The resulting value. T, expresses the total amount of chlorine present. 172 THE OASTBIC J VICE AND GASTRIC CONTENTS. As a general rule, it is not necessary to decolorize the gastric juice. It' desired, however, 5 to 15 drops of a 5 per cent, solution of potassium permanganate may be added to the 10 c.c. employed, after the mixture has stood for ten minutes. 2. Determination of the amount of chlorine in combination with inorganic bases, F. Ten c.c. of the filtered gastric juice are carefully evaporated to dryness in a platinum crucible, on a water- bath or upon a plate of asbestos, in order to avoid sputtering (as the heat applied in the process of incineration is not very intense, a porcelain crucible may also be employed). The residue is then care- fully incinerated over an open flame, the process being carried only to the point when the organic ash no longer burns with a luminous flame. Intense heat should be avoided, as the. chlorides are volati- lized upon the application of red heat. On cooling, the ash is moistened with a few drops of distilled water and mixed with a stirring-rod, when the residue is extracted in separate portions with 100 c.c. of hot distilled water and filtered. This amount is usually sufficient to dissolve all the chlorides present. If any doubt should exist, however, it is only necessary to add a drop of the silver solu- tion to a few drops of the last portion of the filtrate : the formation of a cloud, referable to silver chloride, will necessitate still further washing. The whole filtrate is then treated with 10 c.c. of the one- tenth normal solution of silver nitrate, and the amount consumed in the precipitation of the chlorides determined by titration with the one-tenth normal solution of ammonium sulphocyanide, as de- scribed above. The hydrochloric acid present in combination with inorganic bases is thus determined. The difference between the amount of hydrochloric acid in combination with inorganic bases and the total amount of chlorine in terms of hydrochloric acid will then indicate the amounts of the free and of the combined hydro- chloric acid, which are termed L and C, respectively ; hence T-~F=L+C. 3. The total acidity in terms of hydrochloric acid is further de- termined according to the method given elsewhere (see page 157) and indicated by the letter A. The difference between the total acid- ity and the amount of free and combined hydrochloric acid will represent the amount of organic acids and acid salts, 0; hence = A-{L+C). The free hydrochloric acid finally is determined according to the method of Topfer. The difference between the value thus found and that expressing the amount of free and combined hydro- chloric acid will indicate the amount of the latter; hence (L -\- C) — L=C. Leo's Method.^ — This method is based upon the observation that calcium carbonate combines with free and combined hydrochloric acid 1 Leo, Centralbl. f. d. med. Wise., 1889, vol. xxvii. p. 481. CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 173 at ordinary temperatures to form neutral calcium chloride, while the acid phosphates are not affected. It is thus clear that by determin- ing the total acidity of the gastric juice, and deducting from this the acidity referable to acid salts, the amount of the physiologically active hydrochloric acid — i. e., of the free and combined hydrochloric acid — is obtained. As it has been shown that in the presence of calcium chloride (formed, as indicated above, upon the addition of calcium carbonate), owing to the formation of calcium monophosphate — CaHPO^, twice the quantity of sodium hydrate is taken up, it is necessary to make the first titration also after the addition of an excess of calcium chloride. Reagents required : 1. A one-tenth normal solution of sodium hydrate. 2. A 1 per cent, alcoholic solution of phenolphthalein. 3. A concentrated solution of calcium chloride. 4. Chemically pure calcium carbonate. The purity of the salt may be tested by stirring a small piece with water ; the solution should not color red litmus-paper blue. A solution of the salt in dilute hydrochloric acid should not yield a precipitate when treated with sulphuric acid. Method. — Organic acids that may be present are iirst removed by shaking with ether, 50 to 100 c.c. being required for each 10 c.c. of gastric juice. The total acidity of the gastric juice is then de- termined in 10 c.c. of the filtered liquid after the addition of 5 c.c. of the concentrated solution of calcium chloride, the result being termed A. The acidity referable to the presence of acid phosphates is deter- mined as follows : 15 c.c. of filtered gastric juice are treated with a pinch of dry and chemically pure calcium carbonate ; the mixture is thoroughly stirred, and passed at once through a dry filter. Ten c.c. of the filtrate, from which the carbon dioxide is expelled by means of a current of air, are then treated with 5 c.c. of the calcium chloride solution and titrated as above, the resulting value being termed P. A — P is hence equivalent to L -\- C. The value of C can then be ascertained by determining the acidity referable to free hydrochloric acid according to Topfer's method, and deducting the value found from L -\- G. This method is sufficiently accurate for practical purposes, and has the advantage of not requiring the expenditure of much time. The Ferments of the Gastric Juice and their Zymogens. Pepsin and Pepsinogen. — According to our present knowledge, the zymogen of pepsin, viz., pepsinogen or propepsin, and not pepsin itself, is secreted by the chief cells of the fundus glands. This view 174 THE QASTRIG JUICE AND OASTRIQ CONTENTS. is based upon the observation that an aqueous extract of the mucous membrane of the stomach of a fasting animal recently killed does not lose its digestive power for a considerable length of time when treated with a 1 per cent, solution of sodium carbonate at a tempe- rature of from 38° to 40° C, whereas pepsin itself is thus rapidly destroyed. It is natural then to conclude that the glands of the stomach do not contain pepsin, but some other substance during the process of fasting, which is capable of resisting the action of sodium carbonate, and which can be transformed into pepsin by the addition of hydrochloric acid. This substance has been termed pepsinogen or propepsin. As a rule, pepsin is obtained only from the mucous membrane of the digesting organ, wMle at other times the physio- logically inactive zymogen is found. As the zymogen, moreover, is probably always present together with pepsin in the gastric juice obtained from healthy individuals during the process of digestion, it is not clear whether the transformation of the zymogen into its fer- ment takes place in the body of the cell or after secretion. There is evidence to show, however, that the latter view is correct.^ This is not the place to enter into a detailed consideration of the various properties of pepsin, and it will suffice to say that the activity of the ferment is destroyed by even very dilute solutions of the alkaline carbonates. The same result is reached by exposing a watery solution of pepsin to a temperature of 70° C, while in a dry state a temperature of 100° C. will not destroy its activity ; this is shown by the fact that a specimen of pepsin thus treated is, on cooling, still capable of digesting albumins in the presence of hydrochloric acid. While pepsin is capable of digesting albumins in the presence of other acids, viz., phosphoric, sulphuric, oxalic, acetic, lactic, and salicylic acids, the solutions must be stronger than in the case of hydrochloric acid. With lactic acid, for example, a satisfactory result is reached only with a concentration of from 12 to 18 pro mille, while of hydrochloric acid 2 to 4 pro mille are sufficient. Larger or smaller amounts do not act so promptly. Very important from a practical standpoint is the fact that but small quantities of pepsin are required to digest large amounts of albumin. Petit ^ thus claims that a pepsin preparation from his laboratory was capable of dissolving 500,000 times its weight of fibrin in seven hours. This property possessed by pepsin, of doing an amount of work that is widely out of proportion to the amount of ferment present, is common to all ferments, and is dependent upon the fact that the ferment itself undergoes no change during the process. Figures expressing the exact quantity of pepsin or of its zymogen produced in the twenty -four hours are lacking, and inferences can ' C. E. Simon, Physiological Chemiatry, Lea Bros. & Co., 1901. 'Petit, " Etude sur les ferments digestifs," Jour. de. Th6rap,, 1880. CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 175 hence only be drawn as to the physiological activity of the ferment from the rapidity with which given amounts of albuminous material are digested. This, however, depends to a large extent upon the nature and concentration of the free acid present. Under normal conditions 25 c.c. of gastric juice will dissolve 0.05 to 0.06 gramme of serum-albumin in one hour, the same amount of coagulated egg- albumin in three hours, and a like amount of fibrin in one hour and a half. As abnormalities in the circulation and innervation of the stomach apparently do not influence the production of pepsin, or rather of its zymogen, a diminution in the degree of peptic activity, or its total absence, may be referred directly to disease of the stomach itself, viz., its glandular apparatus. The determination of the presence or absence and relative amount of pepsin in the gastric juice, hence, furnishes more useful information than the recognition of the presence or absence of free hydrochloric acid. As pepsin is formed from pepsinogen through the agency of a free acid, its presence, in the absence of organic acids in notable quan- tities, indicates at once the presence of hydrochloric acid. It may be said, vioe versa, that if free hydrochloric acid is present in the gastric juice, and the latter digests albumins, pepsin also will be found. Should the zymogen alone be present, digestion will take place only upon the addition of an acid, while an absence of diges- tion upon the addition of hydrochloric acid indicates the absence of both pepsin and its zymogen. At times, though rarely, a " gastric juice " is met with which is capable of digesting albumin in the absence of hydrochloric acid, owing to the presence of pancreatic juice — a point which is important, both from a diagnostic and a prognostic point of view. In the differential diagnosis of a chronic gastritis and a neurosis, or a dyspeptic condition referable to hypersemia of the gastric mucous membrane, the demonstration of the presence of the zymogen in the absence of hydrochloric acid may, at times, be very important, bear- ing in mind the fact that circulatory and nervous disturbances apparently do not influence the production of pepsinogen. An entire absence of the latter would, of course, warrant the diagnosis of complete anadeny of the stomach. Tests for Pepsin and Pepsinogen. — Test for the Enzyme. — If the presence of free hydrochloric acid has previously been ascertained, 25 c.c. of filtered gastric juice are set aside and kept at a tempera- ture of from 37" to 40° C., a bit of coagulated egg-albumin, fibrin, or serum-albumin being added. In order to permit of a comparison of results, the same amounts should always be taken ; 0.05 to 0.06 gramme of egg-albumin, as has been shown, ought, under physiolog- ical conditions, to be digested in three hours. Test for the Zymogen. — Should hydrochloric acid be absent, the 176 THE GASTRIC JUICE AND GASTRIC CONTENTS. test is made in the same manner, after the addition of from 3 to 5 drops of the officinal solution of hydrochloric acid to 25 c.c. of the filtrate. Under such conditions usually pepsinogen alone is found. Quantitative Estimation. — Of Pepsin. — Accurate methods for the quantitative estimation of pepsin are unknown, and relative values only can be obtained. Most convenient is the method suggested by Hammerschlag.^ Three Esbach's tubes (albuminimeters) are em- ployed. Tube A is filled to the mark U with a mixture of 10 c.c. of a 1 per cent, solution of serum-albumin in 0.4 per cent, of hydro- chloric acid and 5 c.c. of filtered gastric juice. The second tube, B, which is the standard, is likewise filled to the mark U, but 0.5 gramme of pepsin is added to the serum solution, instead of the gastric juice. The third tube, C, contains merely a mixture of the serum solution and 5 c.c. of water. After having been kept in the thermostat for one hour, at a temperature of 37° C, Esbach's reagent is added to each tube to the mark R. After standing for twenty-four hours the amount of precipitated albumin is read oif, and the difference between that in tube A and tube C compared with that in tube B. Of Pepsinogen. — In order to estimate the amount of pepsinogen ' the method of Boas may be employed. To this end, the gastric juice is diluted with distilled water in varying proportions, such as 1 : 5, 1 : 10, 1 : 20, etc. A known quantity of coagulated albumin is added to each specimen, as also 1 or 2 drops of an officinal solution of hydrochloric acid, for each 10 c.c. employed. These tubes are kept at a temperature of from 37° to 40° C., when the degree of dilution is noted at which the bit of egg-albumin is still dissolved. The greater the degree of dilution at which digestion still takes place, the greater the amount of pepsin or of its zymogen present. If it is desired to exclude definitely the presence of pepsin and pepsinogen in the stomach, the method of Jaworski should be em- ployed. To this end, about 200 c.c. of a decinormal solution of hydrochloric acid are poured into the stomach through a tube and aspirated after one-half hour. If the fluid removed contains no pepsin, the absence of both the enzyme and its zymogen may be inferred. The Milk-curdling Ferment and its Zymogen, viz., Chymosin and Chymosinogen. — A great deal of what has been said above regarding pepsin and its zymogen also holds good for chymosin and its pro-enzyme. The pro-enzyme thus also appears to be formed by the cell, as a neutral aqueous extract of the mucous membrane of the stomach does not, as a rule, contain the ferment, but the zymogen, the ferment resulting only when the latter is treated with a free acid. It differs from pepsin in that it can exert its physiological activity 1 HammerscMag, Wien. med. Prosse, 1894, vol. xxxv. p. 1654, CHEMICAL EXAMINATION OF THE GASTRIC JUICE. Ill in feebly acid, neutral, and even feebly alkaline solutions. Exposure of an active solution of chymosin to gastric juice containing 3 pro mille of free hydrochloric acid, moreover, at a temperature of from 37° to 40° C, leads to its destruction. Its specific action is exerted upon milk, or lime-containing solu- tions of casein, which are coagulated in neutral or feebly alkaline solutions. In this connection it is important to note that the addition of a few cubic centimeters of a solution of calcium chloride, or any other soluble lime salt, results in a transformation of the zymogen into the physiologically active ferment, and that hydrochloric acid, while it normally causes such transformation, is not absolutely necessary in the presence of calcium chloride. Under physiological conditions chymosin and its zymogen are always present in the gastric juice. In disease the inferences that may be drawn from a quantitative estimation of the ferment and its zymogen have been well formulated by Boas/ to whom we are espe- cially indebted for a great deal of valuable information in this con- nection : 1. ISTotwithstanding the absence of free hydrochloric acid, chymo- sin may be present, although in minimal traces — i. e., demonstrable with a dilutiofl of from 1 : 10 to 1 : 20 (see method on page 178). 2. In the absence of free hydrochloric acid the zymogen may still be present in normal amounts — i. e., demonstrable with a dilution of from 1 : 100 to 1 : 150. The presence of the zymogen, especially when repeatedly observed, probably always permits of the conclusion that we are not dealing with an organic disease of the stomach, but with a neurosis or a hypersemic condition of the mucous membrane referable to disease of other organs. 3. The zymogen may occur in moderately diminished amount, 50 per cent, only being present. This is usually owing to the existence of a gastritis which has not reached its highest degree of severity. The nearer the amount of zymogen approaches the normal, the greater will be the probability of an ultimate recovery under suit- able treatment. 4. The amount of the zymogen is greatly diminished (dilutions ofl :10to 1 :25 yielding a negative result) or may be absent alto- gether. In cases of this kind a severe and usually incurable gas- tritis exists, either primary or occurring secondarily to carcinoma, amyloid degeneration, etc. 5. In conditions 1, 2, and 3, the re-establishment of the secretion of hydrochloric acid may be attempted with some prospect of success by means of stimulating remedies. These conclusions are based upon the employment of Ewald's 1 Boas, Centralbl. f. d. med. Wiss., 1887, vol. xxv. p. 417 ; and Zeit. f. klin. Med., 1888, vol. xiv. p. 240. See also J. Friedenwald, Med. News, 1895. 12 178 THE GASTRIC JUICE AND OASTBIC CONTENTS. test-breakfast, and cannot be applied to observations made after other test-meals, without previous studies in this direction. Testing for the presence of chymosin and its zymogen, moreover, is of decided value in cases in which alkaline material is vomited, and where we may be called upon to decide whether this contains constituents of the gastric juice or not. Tests for Chymosin and Chymosinogen. — Test for the Enzyme. — Five to 10 c.c. of milk are treated with from 3 to 5 drops of the filtered gastric juice and kept at a temperature of from 37° to 40° C. for ten to fifteen minutes. If coagulation occurs during this time, it may definitely be concluded that the enzyme is present. Test for the Zjrmogen. — The milk is treated with 10 c.c. of the filtered and feebly alkalinized gastric juice and with 2 or 3 c.c. of a 1 per cent, solution of calcium chloride. The mixture is kept at a temperature of from 37° to 40° C, when in the presence of the zymogen the formation of a thick cake of casein will be observed to occur within a few minutes. Quantitative Estimation. — Of the Enzyme. — This is based upon the fact that on gradually diluting a specimen of gastric juice a point finally is reached at which a chymosin reaction can no longer be obtained, the value being, of course, a relative one. Under phys- iological conditions a positive reaction can still be observed with a degree of dilution varying between 1 : 30 and 1 : 40. The gastric juice is neutralized with a very dilute solution of sodium hydrate. Tubes are then prepared containing from 5 to 10 c.c. of the gastric juice, diluted in the proportion of 1 : 10, 1 : 20, 1 : 30, etc., to which an equal amount of neutral or amphoteric milk is added. The tubes, properly labelled, are kept at a temperature of from 37° to 40° C, and the degree of dilution noted at which coagulation still occurs. Of the Zymogen. — The gastric juice is rendered feebly alkaline and tubes are prepared containing equal amounts of milk and gastric juice, the latter variously diluted, as above directed ; the examination is then carried on in the same manner. Normally a positive reaction is obtained with a dilution varying between 1 : 150 and 1 : 100. Allowance must, of course, be made for the amount of fluid which is added during the process of neutralization. The Products of Gastric Digestion. Digestion of the Native Albumins. — The first step in the proc- ess of albuminous digestion in the stomach is one of swelling, which may be observed when a flake of fibrin, for example, is placed in gastric juice and the temperature maintained between 37° and 40° C. Very soon simple solution takes place, which is followed by the process of " denaturization," as Neumeister terms it, in which the native albumins are transformed into acid albumins or CHEMICAL EXAMINATION OF THE OASTBIC JUICE. 179 syntonins, owing to the continued activity of the hydrochloric acid and pepsin. The pepsin, however, acts only as an adjuvant to the acid, and hydrochloric acid alone is capable of effecting the same result. But while in the absence of pepsin more concentrated solu- tions of the acid and a higher temperature are required, the tem- perature of the body and the amount of hydrochloric acid secreted by the stomach are sufficient when pepsin is present. Pepsin, in the absence of free hydrochloric acid, is perfectly inert. The " denaturization " of the native albumins is followed by a splitting up of the albuminous molecule and a process of hydration, the so-called primary albumoses being the first products thus formed. During the further process of digestion the deutero-albumoses then result, and from these the peptones, to which, in contradistinction to the peptones formed during the process of pancreatic digestion, the term amphopeptone has been applied by Kiihne. Digestion of the Proteids. — The digestion of casein, which belongs to the class of nucleo-albumins, differs from the process described. The casein of the milk is present in solution as a neutral calcium salt, and as it has the character of a polybasic acid, calcium chloride and the corresponding acid casein salt will result in the presence of the hydrochloric acid of the stomach ; still later, when more hydrochloric acid has been secreted, insoluble casein, as such, will be found. While the acid is thus capable of causing the pre- cipitation of casein, it has also been shown that the same result may be reached in the absence of hydrochloric acid. According to Hammarsten, this is brought about in consequence of the hydrolytic action on ths part of the chymosin, the calcium salt of paracasein (cheese), and a small amount of an albumose-like posset-albumin being formed. This latter process is now supposed to take place in the stomach after the hydrochloric acid has previously transformed the neutral into the acid casein salt. When this stage is reached the paracasein is decomposed into an albumin and an insoluble nuclein. The albumin is then further digested as described ; a hetero-albumose, however, does not result. The remaining proteids, such as haemoglo- bin, glucosides, etc., are similarly acted upon by the gastric juice, and are first split up into the corresponding albumins and their pairlings. The albuminous radicles are then digested, as described. Digfestion of the Albuminoids. — Of the albuminoids, only col- lagen and elastin undergo digestion in the stomach, gelatoses and elastoses being formed during the process, while keratin passes off undigested. Hetero-albumoses, however, are formed from neither collagen nor elastin, but merely proto-albumoses, which in turn ai-e transformed into deutero-albumoses, and these into peptone. Digfestion of the Carbohydrates. — The secretion of the stomach itself is not capable of digesting carbohydrates. There appears to be no doubt, however, that a transformation of starches into sugar 180 THE GASTRIC JUIGE AND GASTRIC CONTENTS. takes place during the earlier stages of digestion. This is owing to the continued action of the ptyalin of the saliva (see page 139) in the stomach, which proceeds until the amount of hydrochloric acid secreted reaches 0.01 per cent, or more, it being remembered that the transformation of starches into sugar takes place best in a neutral or feebly alkaline medium. The question whether or not a diastatic ferment occurs in the mucus secreted by the stomach itself is unimportant, as cases have but rarely been observed in which there was an absence of ptyalin from the saliva. As indicated in the chapter on the Saliva, a number of intermediary products are formed in the transformation of starch into sugar, of which an idea may be had from the accompanying ta,ble : Starch. .1 Amidulin. I I Erythrodextrin. Maltose. I ^ I Achroodextrin a Maltose. I I Achroodextrin /3 Maltose. Achroodextrin y (maltodextrin) Maltose. Maltose. Maltose. In the mouth this transformation is effected very rapidly in the case of certain starches, such as corn-starch and rye-starch, and it is possible to demonstrate the presence of sugar after from two to six minutes. Potato-starch, on the other hand, requires a much longer time, viz., from two to four hours. This difference is entirely de- pendent upon the varying degree of resistance offered to the action of the saliva by the enclosing envelope of cellulose, as is apparent from the fact that a paste made from potatoes is digested just as rapidly as one made from rye. For practical purposes, the digestion of carbohydrates in the stomach may be disregarded as insignificant. Fats are not digested in the stomach. From the above considerations it is apparent that under physio- logical conditions a mixture of various products is met with in the stomach at the height of digestion, and it might be expected that from a preponderance of the one over the other definite and valuable conclusions as to the digestive power of the organ could be reached. While this is true in a certain sense, the quantitative methods of analysis that would have to be employed in order to obtain definite data are as yet too complicated for the purposes of the clinician, and CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 181 from the simple qualitative tests not much information can be de- rived. The recognition of the presence of peptones would thus merely indicate the presence of hydrochloric acid and pepsin in a general way, as peptones may be formed in the absence of hydro- chloric acid and in the presence of organic acids, which may be found in pathological conditions. A portion of the albumin of milk, eggs, meat, etc., is, moreover, already peptonized during the process of boiling. It is not surprising then that peptones may be demonstrated in practically every specimen of gastric contents. A large amount of syntonin and primary albumoses in the presence of a feeble peptone-reaction must, of course, be regarded as abnormal, pointing to a defective secretion of either hydrochloric acid or the enzymes, or of both. The same may be said to hold good when a pronounced peptone-reaction disappears upon the removal of syntonin and the primary albumoses. So far as the examination for the products of carbohydrate diges- tion is concerned, it may be stated, as a general rule, that in the presence of a normal amount of hydrochloric acid erythrodextrin can usually be demonstrated toward the end of gastric digestion, while achroodextrin is nearly always obtained at that time when free hydro- chloric acid is absent, so that the tests for the presence of these two bodies may be regarded as roughly indicating the presence or absence of free hydrochloric acid. Boas draws attention to the fact, however, that ptyalin may, at times, though rarely, be absent, when conclusions drawn from these tests as to the presence of hydrochloric acid would be erroneous. The tests for sugar in the gastric juice do not furnish any infor- mation of practical value. Analysis of the Products of Albuminous Digestion. In order to separate the various bodies referred to from each other the following procedure may be employed : The filtered gastric contents are carefully neutralized with a dilute solution of sodium hydrate, using litmus-paper to determine the re- action ; a small drop of the mixture is placed upon the paper from time to time during the addition of the sodium hydrate until no change in color is produced either on the red or the blue paper. If syntonin is present, it will be precipitated, and can be collected on a small filter. Upon the addition of an excess of dilute acid or an alkali this precipitate will again be dissolved. The filtrate is feebly acidified by the addition of a few drops of a very dilute solution of acetic acid, treated with an equal volume of a saturated solution of common salt, and brought to the boiling-point. Any native albumin that may be present in solution is thus coagulated and can be filtered ofi" on cooling. In the filtrate the albumoses and peptones remain. The presence of the former may be demonstrated by adding a few 182 THE OASTRIC JUICE AND GASTRIC CONTENTS. drops of nitric acid to a specimen, when a precipitate will form which dissolves upon the application of heat, and reappears on cooling; if necessary, the specimen may be diluted. Should the deutero-albumoses of vitellin or myosin be present, however, this test yields a negative result, and a precipitate only occurs when the solution, acidified with nitric or acetic acid, is com- pletely saturated with sodium chloride. The presence of primary albumoses may be established by adding pieces of rock-salt to the neutral solution, when a precipitate occurs. The albumoses may roughly be separated from the peptones by satu- rating the acidified filtrate just obtained with pulverized ammonium sulphate, whereby the albumoses are precipitated almost entirely. A small portion of deutero-albumoses, however, which resulted from the proto-albumoses, remains in solution and passes into the filtrate, which also contains all of the amphopeptone. In the filtrate this may be demonstrated as follows : a concentrated solution of sodium hydrate is added until all the ammonium sulphate has been trans- formed into sodium sulphate, and a slight excess of the hydrate is present ; care should be had, however, that the temperature does not rise too high, by unmersion in cold water. The sodium sulphate, which separates out during this process, is allowed to settle. A 2 per cent, solution of cupric sulphate is then carefully added drop by drop, to a specimen of the supernatant fluid, when in the pres- ence of peptones a rose to a purplish-red color will develop. To obtain the peptones, the filtrate is diluted with an equal volume of water, neutralized, and then treated with a solution of tannic acid, care being taken to avoid an excess, as otherwise the peptone precipi- tate is partly dissolved.^ Tests for the Products of Carbohydrate Digestion. Starch may be recognized by the fact that it strikes a blue color with a solution of iodo-potassic iodide, while the same solution gives a violet or mahogany brown with erythrodextrin. To this end, it is only necessary to add a drop or two of Lugol's solution to a few cubic centimeters of the filtered gastric juice. The presence of achroodextrin may be inferred if no change in color occurs upon the addition of the reagent. Maltose and dextrose, which both react with Fehling's solution and undergo fermentation, differ from each other in the fact that the former does not reduce Bdrfoed's reagent on boiling. This is prepared by adding 1 per cent, of acetic acid to a 0.5 to 4 per cent, solution of cupric acetate. The rotatory power of maltose is about three times as strong as that of dextrose ; («) D = 150.4, as com- pared with 52.5. 1 For a more detailed account of the chemistry of digestion and the analysis of the resulting products, see C. E. Simon, Physiological Chemistry, Lea Bros. & Co., 1901. CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 183 Lactic Acid. Mode of Formation and Clinical Significance. — It was for- merly thought that the acidity of the gastric juice was referable to the presence of lactic acid, as this can always be demonstrated in the beginning of the process of digestion. The hydrochloric acid was then supposed to result from the action of the lactic acid upon the chlorides of the food. That this view is erroneous C. Schmidt ' succeeded in demonstrating beyond a doubt, as has been shown on page 155. An explanation of the presence of lactic acid suggested itself when Miller found that in the mouth various bacteria normally occur which are capable of forming lactic acid from sugar, and that from the gastric contents a number of bacteria can be isolated which are capable of causing acid fermentation in sugar-containing media. There would, hence, be nothing surprising in the constant occur- rence of lactic acid, as the two principal factors necessary for its formation are always present after the ingestion of an ordinary meal, viz., carbohydrates and bacteria capable of causing lactic acid fermentation. The absence of the lactic acid during the later stages of digestion was, furthermore, explained by the fact that lactic acid fermentation ceases in the presence of from 0.7 to 1.6 pro mille of hydrochloric acid — i. e., in the presence of amounts of hydrochloric acid which are found in the normal gastric juice. The normal occurrence of lactic acid in the stomach was, until recently, regarded as an established fact. But at this stage Martins and Liittke, employing the method already described, found " that the accurately determined curve of acidity referable to hydrochloric acid coincided in all respects, even at the beginning of the process of digestion, with the curve referable to the total acidity," so that lactic acid as a physiological constituent could not have been present. Recent researches of Boas,^ moreover, appear to prove beyond a doubt that in physiological conditions no appreciable amounts of lactic acid are formed during the process of digestion, and that the lactic acid found after an ordinary meal has been introduced into the stomach as such. That lactic acid is actually present in the various kinds of bread has definitely been proved, and it is, hence, not permissible to make use of any test-meal containing lactic acid when the question as to its formation in the stomach is to be con- sidered. For these reasons Boas suggests the use of simple oatmeal- soup to which salt only has been added. For practical purposes this is probably not always necessary, as the small amount of lactic acid found after Ewald's test-breakfast may usually be disregarded ; an increased amount can be referred directly to pathological con- ditions. ^ Loc. cit. ^ J. Boas, " Ueber d. Vorkommen v. Milchsaure im Gesunden u. Kranken Magen," Zeit. f. klin. Med., 1894, vol. xxv. p. 285. 184 THE GASTRIC JUICE AND GASTRIC CONTENTS. The fact that the lactic acid disappears, or is at least no longer demonstrable, at the height of digestion, Boas refers to a resorption or a carrying-oif of the acid introduced, on the one hand, or to an interference of the hydrochloric acid with the delicacy of the reagent usually employed — i. e., Uffelmann's reagent — on the other. Patho- logically the same rule may be said to hold good, as Boas was un- able to demonstrate its presence after the exhibition of his test-meal in the most diverse diseases of the stomach, viz., chronic gastritis, atony and dilatation referable to myasthenia, or pyloric stenosis following ulcer, etc. Mere traces, which were occasionally observed, ^re of no significance, and possibly referable to lactic acid fermenta- tion having taken place in the mouth. In all the cases examined, moreover, no organic acids could be demonstrated by the method of Hehner-Seemann (see page 192). It is apparent then that notwithstanding stagnation of the gastric contents and the absence of free hydrochloric acid in normal amounts, lactic acid is not necessarily formed in the stomach, even in the presence of carbohydrates. In only one disease of the stomach was lactic acid found in notable quantities, viz., in carcinoma. This ob- servation is in accord with the fact that Uffelmann's test here yields a marked reaction — i. e., a deep-lemon or a canary-yellow color — even upon the addition of but a few drops of the gastric juice, while in the benign affections only a pale-yellow, brownish, or grayish color is obtained. Boas' test-meal should be given the evening before the examina- tion, the stomach having previously been washed free from all remnants of food ; the remaining contents are obtained the next morning. In an analysis of fourteen cases of carcinoma Boas was able to demonstrate the presence of lactic acid in amounts varying between 1.22 and 3.82 pro mille in all cases but one, while in other diseases after the ingestion of Ewald's test-breakfast only 0.1 to 0.3 pro mille could be obtained. Unfortunately, recent investigations have shown that notable amounts of lactic acid may also be found in gastric anadeny, and in cases of dilatation referable to benign causes. Such cases, however, are rare, and it may safely be stated that the presence of large amounts of lactic acid will almost invariably justify the diagnosis of carcinoma of tlie stomach.' That stagnation of the gastric contents and the absence of free hydrochloric acid alone are not capable of causing the formation of lactic acid has been seen, and it is, hence, difficult to explain why in carcinoma practically only lactic acid fermentation should occur. ' J.H. de Jong, " Der Nachweis d. Milclisaure u. ihre klinischo Bedciitung," Arch, f. VerdauuiiKskrank., vol. ii. p. .53. .T. Friedenwald, ■' The Significance of the Presence of Lactic Acid in the Stomach." N. Y. Med. Jour., ISO.'i. Eosenhaim u. Eichter, " Ueber Milchsiiurebildung im Magen," Zeit. f. klin. Med., vol. xxviii. p. 505. CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 185 Whether the malignant growth itself must be regarded as one of the principal factors in this connection, as Boas suggests, must still re- main an open question. Owing to the interest which attaches to this subject, it may not be out of place to refer briefly to the following observation of Koch : In a case in which ulcer of the stomach existed, the hydrochloric acid suddenly disappeared and gave place to lactic acid, which then steadily increased in amount from week to week. A tumor could not be demonstrated on physical examination. Soon after, the patient died, and at the autopsy a carcinoma of the stomach was found upon the base of the pyloric ulcer. An exploratory operation should hence be made whenever notable amounts of lactic add can repeatedly be de- monsirated in the stomach contents after the ingestion of Boas' test-meal. Negative results, however, do not exclude the existence of carcinoma. The formation of lactic acid from starch may be represented by the following equations : (1) 2C6Hi„05 + H,0 = CijHjjOii (milk-sugar). (2) CijFIj^Oii + H,0 = 2C6H1A (glucose). (3) 2C6H12O6 = 4C3H6O3 (lactic acid). It should, finally, be mentioned that only that form of lactic acid which results from fermentative processes is of interest in this con- nection, and not the sarcolactic acid contained in meat. Tests for Lactic Acid. — For the reasons indicated. Boas' test- meal (see page 150) should be employed whenever it is desired to test for lactic acid in the gastric contents. If the case under examina- tion shows well-marked symptoms of stagnation, the stomach should be washed out completely in the evening, the soup then given, and the gastric contents procured the next morning, before any food or liquid is taken. Otherwise the test-meal may be given in the morn- ing on an empty stomach, without previous lavage, and the contents examined one hour later. Uffelmann's Test.' — Heretofore Uffelmann's reagent was quite com- monly employed in testing for lactic acid, but everyone who has had occasion to make frequent use of this I'eagent in clinical work must have been struck with the uncertainty of the results so often obtained. In a large majority of the cases thus examined, particu- larly if Ewald's test-breakfast is employed, a characteristic reaction — i. e., the occurrence of a lemon or canary-yellow color — is not seen, notwithstanding the presence of lactic acid, but a pale-yellow, brownish, grayish-white, or even gray color is obtained instead, often leaving in doubt whether lactic acid is present or not. Aside from doubtful results, the value of the test is greatly diminished by the • Uffelmann, Deutsch. Arch. f. klin. Med., 1880, vol. xxyi.; and Zeit. f. klin. Med., vol. viii. p. 392. 186 THE GASTRIC JUICE AND GASTRIC CONTENTS. fact that glucose, acid phosphates, butyric acid, and alcohol give the same reactiou, and that in the presence of such amounts of hydro- chloric acid as are found at the height of normal digestion lactic acid is not indicated by the reagent. All these difficulties have long been appreciated, and in order to obviate at least some of them it was proposed to apply the test to an aqueous solution' of the ethereal extract of the gastric contents : To this end, 5 or 10 c.c. of the filtered gastric juice ai:e extracted by shaking with from 50 to 100 c.c. of neutral sulphuric ether in a stoppered separating-funnel for about twenty or thirty minutes ; the ethereal extract is then evaporated on a water-bath or the ether distilled off (no flame). The residue is taken up with from 5 to 10 c.c. of distilled water, and tested as follows : three drops of a saturated aqueous solution of ferric chloride are mixed with three drops of a concentrated solution of pure carbolic acid and diluted with water until an amethyst-blue color is obtained ; to this solution a portion of the ethereal extract is added, when in the presence of only 0.1 per cent, of lactic acid a lemon or canary-yellow color is obtained. Kelling's Method.' — Five or 10 c.c. of gastric juice are diluted with from ten to twenty volumes of water and treated with one or two drops of a 5 per cent, aqueous solution of ferric chloride. In the presence of lactic acid a distinct greenish-yellow color is seen if the tube is held to the light. This test is more reliable than that of Uffelmann, as a positive reaction is obtained only in the presence of lactic acid. Strauss' Method.^ — Instead of evaporating the ether as in the above method, the ethereal extract may be directly examined by shaking with a freshly prepared solution of ferric chloride, as sug- gested by Fleischer. Making use of this principle, Strauss has constructed an apparatus (Fig. 37) which may be found very con- venient, and which permits of roughly determining the amount of lactic acid present. The instrument is essentially a separating- funnel of 30 c.c. capacity, bearing two marks, of which the one corresponds to 5 c.c, the other to 26 c.c. The apparatus is filled with gastric juice to the mark 5, when ether is added to the 25 c.c. line. After shaking thoroughly, the separated liquids are allowed to escape by opening the stopcock until the 5 c.c. mark is reached. Distilled water is then added to the 25 mark, and the mixture treated with two drops of the officinal tincture of ferric chloride, diluted in the proportion of 1 : 10. Upon shaking, the water will assume an intensely green color if more than 1 pro millo of lactic acid is pres- ent, while a pale green is obtained in the presence of from 0.5 to 1 1 O. Kelling, " Ehodan im Mageninhalt ; Zugleich ein Beitrag z. Uffelmann 'schen Milchsaurereagens," Zeit. f. physiol. Chem., vol. xviii. 2 H. Strauss, " Ueber eine Modiflkation d, Uffelmann'soheu Eeaktion," Berlin, klin. Woch., 1895, No. 37. CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 187 pro mille. The tincture of iron should be kept in a dark-colored dropping-bottle of about 50 c.c. capacity. It will be observed that only large amounts of lactic acid, which alone are of importance from a diagnostic point of view, are indi- cated by the apparatus. Small amounts, as those introduced with Ewald's test-breakfast, or referable to lactic acid fermentation in the mouth, are not indicated, so Fig. 37. that confusion as to the presence or absence of the acid can never arise. Boas' Method.' — In doubtful cases the follow- ing method should be employed, as with it, and following the exhibition of Boas' test-meal, all possible errors can be avoided. T.he stomach must, however, be washed perfectly clean before the test- m^cd is introduced. It is my belief that some of the positive results which have been obtained in other diseases than carcinoma are referable to neglect in this particular point. Aldehyde is not infrequently found in the stomach contents when sarcinse are present in large numbers, and may be mistaken for lactic acid, as I discovered to my regret not long ago. Principle of the Method. — When a solution of lactic acid is treated with a strong oxidizing agent and heated, the lactic acid is decomposed into acetic aldehyde and formic acid, according to the equation CHs-CH(OH)— CO.OH = CH3.CHO + H.CO.OH. Lactic acid. Acetic aldehyde. Formic acid. Practically, then, the test for lactic acid resolves „ ■j. i_p • i . ; /■ i- 1111 1 • 1 Straufss' apparatus for itseli into a test tor acetic aldehyde, which can the approximative Ti 1 . 1 1 J !_■ •,! • estimation of lactic readily be recognized by testing with various re- acid, agents, such as an alkaline solution of iodo-potassic iodide, Nessler's reagent, and others. Nessler's reagent is prepared as follows : 2 grammes of potassium iodide are dissolved in 50 c.c. of water and treated with mercuric iodide while heating, until some of the latter remains undissolved. Upon cooling, the solution is diluted with 20 c.c. of water. Two parts of this solution are then treated with 3 parts of a concentrated solution of potassium hydrate ; any precipitate that may have formed is filtered off, and the reagent kept in a well-stoppered bottle. When aldehyde is added to such a solution a yellowish-red or red precipitate results, the exact color depending upon the amount of aldehyde present. One part of the ' Boas, Deutsoh. med. Woch., 1893, No. 39 ; and Miiuch. med. Woch., 1893, No. 43. 188 THE QASTBIG JUICE AND GASTRIC CONTENTS. aldehyde may still be recognized when diluted with 40,000 parts of water. With an alkaline solution of iodo-potassic iodide, aldehyde in a dilution of 1 : 20,000 will still produce a cloudiness, referable to the formation of iodoform, which is readily recognized by its character- istic odor (Lieben's test for acetone). Method. — The filtered gastric juice is tested for the presence of free acids with Congo-red (see page 163). If present, from 10 to 20 c.c. are evaporated to a syrup on a water-bath, after the addition of an excess of barium carbonate, while the latter is unnecessary in the absence of free acids. The syrup is treated with a few drops of phosphoric acid, and the carbon dioxide removed by bringing it to the boiling-point once only, when- it is allowed to cool and extracted with 100 c.c. of neutral sulphuric ether (free from alcohol), by shaking for half an hour. The layer of ether is poured off after half an hour, the ether is evaporated (no flame), the residue taken up with 45 c.c. of water, shaken and filtered, and finally treated with 5 c.c. of sulphuric acid and a pinch of manganese dioxide in an Erlenmeyer flask. This is closed with a perforated stopper carrying a glass tube bent at an obtuse angle, the longer limb of which passes into a narrow glass cylinder containing from 5 to 10 c.c. of Nessler's reagent or a like quantity of an alkaline solution of iodo-potassic iodide. If heat is now carefully applied, the aldehyde, formed by the oxidation of the lactic acid with manganese dioxide and sulphuric acid, passes over when the boiling-point is reached, and causes the precipitation of yellowish-red aldehyde of mercury in the tube containing the Nessler's reagent, or of iodoform if the alkaline solution of iodine is employed. Quantitative Estimation of Lactic Acid according to Boas' Method.' — The principle already set forth also applies to the quanti- tative estimation of lactic acid. Solutions required : 1. A one-tenth normal solution of iodine. 2. A one-tenth normal solution of sodium thiosulphate. 3. Hydrochloric acid (sp. gr. 1.018). 4. A potassium hydrate solution (56 : 1000). 5. Starch solution. Preparation of these solutions : 1. A normal solution of iodine should contain 126.53 (molecular weight of iodine) grammes of iodine in the liter, and a one-tenth normal solution, hence 12.6 grammes. In order to dissolve the iodine 25 grammes of potassium iodide are dissolved in about 200 c.c. of distilled water, when the 12.6 grammes of resublimed iodine are added. This solution is then diluted with distilled water to the 1000 c.c. mark, and requires no further correction. Loc. oit., p. 187. CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 189 2. The one-tenth normal solution of sodium thiosulphate is pre- pared as described in the chapter on Acetone (see Urine). When treated with 1 gramme of ammonium carbonate pro liter it will retain its titre almost indefinitely. 3. Preparation of the starch solution : 5 grammes of starch are dissolved in 900 c.c. of water by heating, when 10 grammes of zinc chloride in 100 c.c. of water are added. Method. — Ten to 20 c.c. of the filtered gastric juice are first treated as indicated above, viz., evaporated to a syrup after the addition of bariuna carbonate if free acids are present. A few drops of phosphoric acid are added, the carbon dioxide driven off by boiling, and the residue extracted, on cooling, with 100 c.c. of ether free from alcohol ; the ether is evaporated after separation, the residue taken up with 45 c.c. of distilled water, and treated with manganese dioxide and sulphuric acid. The flask is closed by a doubly perforated stopper ; through one aperture a bent tube passes to the distilling-apparatus, and a straight tube provided with a piece of rubber tubing, clamped off, through the other. The latter should dip well down into the liquid, and serves for passing a current of air through the solution when the distillation is completed. The mixt- ure is distilled until about four-fifths of the contents have passed over, excessive'heat being carefully avoided, as otherwise the aldehyde will be decomposed, according to the equations : (1) CH3 - CH(OH) - CO.OH = CH3.CHO + HCOOH. Lactic acid. Aldeliyde. Formic acid. (2) CH3.CHO + HCOOH + 20 = CH3.COOH + CO2 + HjO. Aldehyde. Formic acid. Acetic acid. To the distillate, which is best received in a high Erlenmeyer flask, well stoppered, 20 c.c. of the one-tenth normal solution of iodine are added, mixed with 20 c.c. of the 5.6 per cent, solution of potassium hydrate. The mixture is shaken thoroughly and allowed to stand for a few minutes. In order to liberate the iodine not used in the reaction, 20 c.c. of hydrochloric acid are added, and the ex- cess of iodine determined by titration with the one-tenth normal solu- tion of sodium thiosulphate. The titration is carried almost to the point of decolorization, when a little starch solution is added ; the mixture is then titrated until the blue color has disappeared. The number of cubic centimeters of the one-tenth normal solution em- ployed, viz., 20, minus the number of cubic centimeters of the one- tenth normal solution of sodium thiosulphate, will then indicate the number of cubic centimeters of the former required for the formation of iodoform, viz., the amount of lactic acid present in 10 or 20 c.c. of gastric juice, as the case may be. As 1 c.c. of the one-tenth normal solution of iodine has been found to indicate the presence of 190 THE GASTRIC JUICE AND OASTBIC CONTENTS. 0.003388 gramme of lactic acid, it is only necessary to multiply the number of cubic centimeters used by this figure, and the result by 10, in order to obtain the percentage. The method described is reliable and suiSciently accurate for clini- cal purposes. At the same time it may be said that no more time is required than in the ordinary quantitative estimation of sugar by means of Fehling's method, or of hydrochloric acid according to the method of Martins and Liittke. Boas' Eapid Method. — This method is less accurate than the preceding one, but may be advantageously employed in the absence of the various reagents necessary with the former. Ten c.c. of filtered gastric juice are treated with a few drops of dilute sulphuric acid, and the albumin present removed by heat. The filtrate is evapo- rated to a syrup on a water-bath, water added to the original amount, and this again evaporated to a small volume, fatty acids being thereby removed. The lactic acid remaining is now extracted with ether (200 c.c. for. every 10 c.c. of gastric juice) ; the ether is evaporated, the residue taken np with water and titrated with a one-tenth normal solution of sodium hydrate, using phenolphthalein as an indicator. As 40 parts by weight of sodium hydrate (molecu- lar weight) combine with 90 parts by weight of lactic acid (molecu- lar weight), and as 1 c.c. of the one-tenth normal solution of sodium hydrate contains 0.004 gramme of sodium hydrate, the corresponding amount of lactic acid is found from the equation : 40 : 90 : : 0.004 ■.x;AOx = 0.360 ; x = 0.009. The value of 1 c.c. of the one-tenth normal solution in terms of lactic acid is thus 0.009. By multiplying the number of cubic centimeters used by this figure, the amount of lactic acid present in 10 c.c. of gastric juice is ascertained. The result multiplied by 10 indicates the percentage. The Fatty Acids. Mode of Formation and Clinical Significance. — Unless much milk or carbohydrates have been ingested, fatty acids do not occur in the gastric contents under physiological conditions, and it would appear from the researches of Boas ^ that their formation is intimately associated with that of lactic acid. After the exhibition of his test- meal (see page 150) he was unable to demonstrate their presence either in health or in various diseases of the stomach, such as chronic gastritis, atony or dilatation referable to benign causes, etc. In carcinoma, however, fatty acids, just as lactic acid, were quite constantly found. That butyric acid can be derived from lactic acid has been demon- strated by Fliigge, the reaction taking place according to the equation 2C,HA = C.HbO, + 2C0„ + 4H. ' Loo. cit. CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 191 This observation is probably explained by the fact that most of the organisms causing butyric acid fermentation are anaerobic, while the Bacillus acidi lactici and the Oidium lactis eagerly absorb oxygen. Acetic acid fermentation, on the other hand, presupposes the pres- ence of alcohol, whether this is introduced into the stomach as such or whether it results from the action of yeast (Saccharomyces cere- visiffi) upon sugar. The transformation of alcohol into acetic acid is represented by the equation C2H5OH + 20 = C^HA + HA while the formation of alcohol during the process of fermentation from glucose is shown below : C^HiA + 2H,0 = 2C,H60 + 2H,0 + 200,. It is, hence, necessary, whenever acetic acid is met with in the gastric contents, to exclude the presence of alcohol, as only then is it permissible to refer its presence to stagnation and advanced decomposition of carbohydrates. If the examination is confined to an analysis of the gastric contents obtained otherwise than after the exhibition of Boas' or Ewald's test-meal, the diagnosis of pyloric stenosis, with dilatation is probably always justifiable in the presence of notable quantities of butyric acid and acetic acid, while the same after a previous washing-out of the stomach and the exhibition of Boas' test-meal would suggest carcinoma as the cause of the stenosis. That butyric acid may occur in the gastric contents when butter or fats in general have been ingested is, of course, not surprising, and its presence then should be looked upon as a physiological occur- rence. At the same time it should not be forgotten that butyric acid, just as lactic acid, may possibly have been formed in the mouth, and conclusions should, hence, only be drawn when such sources of error can be definitely excluded, and the amount found exceeds mere traces. In conclusion, it may be said that in disease butyric acid is far more frequently encountered in the gastric contents than acetic acid, but the significance of the two, if alcoholism can be excluded, is the same. Tests for Butyric Acid. — 1. Butyric acid can usually be recog- nized by its odor alone, which is that of rancid butter. Often, how- ever, it will be necessary to resort to more definite tests, such as the following : 2. Ten c.c. of filtered gastric juice are extracted with 50 c.c. of ether. The ether is evaporated and the residue taken up with a few cubic centimeters of water. If a trace of calcium chloride in sub- stance is now added, the butyric acid will separate out in the form of oil-droplets, the nature of which is readily recognized by the pungent 192 THE OASTRIC JUICE AND OASTBIO CONTENTS. odor. If, instead of adding calcium chloride, a slight excess of baryta-water is used, strongly refractive rhombic plates or granular, wart-like masses of barium butyrate are obtained upon evaporation. 3. Butyric acid may also be recognized by the peculiar odor of pineapple which develops when the dry residue of the ethereal solution is treated with a little sulphuric acid and alcohol. The reaction is due to the formation of butyl ethylate (Pineapple test). Tests for Acetic Acid. — 1. Like butyric acid, acetic acid can usually be recognized by its odor. 2. Ten c.c. of filtered gastric juice are extracted with ether. The ether is evaporated, the residue dissolved in a few drops of water, and accurately neutralized with a dilute solution of sodium hydrate, sodium acetate being formed. If to this a drop or two of a very dilute solution of ferric chloride is added, a dark-red color results in the presence of acetic acid. With silver nitrate a precipitate is obtained which is soluble in hot water. Quantitative Estimation of the Fatty Acids. — Method of Cahn- Mehring, modified by McNaught.' — The total acidity is determined in 10 c.c. of filtered gastric juice. Another 10 c.c. are evaporated to a syrup, diluted with water, and similarly titrated. The difference between the two results will indicate the amount of fatty acids present. Quantitative Estimation of the Organic Acids. — ^Method of Hehner-Seemann.^ — This method is based upon the observation that if a certain amount of a one-tenth normal solution of sodium hydrate is added to organic acids and the mixture is evaporated and incinerated, the organic acids are decomposed, with the liberation of carbon dioxide, while their alkali is left behind in the form of a carbonate ; this is then determined by titration with a one-tenth normal solution of hydrochloric acid. The amount of physiologi- cally active hydrochloric acid can be estimated at the same time by deducting from the total acidity the acidity referable to organic acids. Method. — Ten or 20 c.c. of filtered gastric juice are titrated with a one-tenth normal solution of sodium hydrate, evaporated to dry- ness, and incinerated, the application of heat being discontinued as soon as the ash has ceased to burn with a luminous flame. The residue is taken up with water and titrated with a one-tenth normal solution of hydrochloric acid. This is prepared by diluting 146 grammes of the concentrated acid (sp. gr. 1.14) with distilled water to about 900 c.c, when the solution is brought to the proper strength by comparing it with a one-tenth normal solution of sodium hydrate, according to directions given elsewhere. The number of cubic cen- timeters of the one-tenth normal solution of hydrochloric acid ' CSted by Boas, Diagnostik u. Therapie d. Magenkrankheiten, 2d ed., 1891, p. 140. ' Seemann, "Ueber d. Vorhandenaein freier Salzsaure im Magen," Zeit. f. klin, Med., vol. V. p. 272. CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 193 employed multiplied by 0.00365 will indicate the amount of fatty acids in the 10 c.c. of gastric juice, in terms of hydrochloric acid ; the percentage is ascertained by multiplying by 10 or 5, as the case may be. By deducting the number of cubic centimeters employed from that of the one-tenth normal solution of sodium hydrate, first used, the number of cubic centimeters of the latter required for the neutralization of the physiologically active hydrochloric acid is ascertained, and the amount determined by multiplying by 0.00365. The stomach always contains a certain quantity of gases which have partly been swallowed and partly have passed into the stomach from the duodenum. As fermentative processes in health occur only when carbohydrates or fats have been ingested, and then only to a slight degree, nitrogen, oxygen, and carbon dioxide are the only gases found during the process of albuminous digestion. As the oxygen swallowed is, moreover, largely absorbed by the blood, and two volumes of carbon dioxide are returned for one volume of oxy- gen, the presence of large amounts of the former and small amounts of the latter is readily explained. In an analysis of the gases con- tained in the stomach of a dog which had been fed on meat, Planer found the following proportions : Carbon dioxide . . . 25.2 vol. per cent. Oxygen 6.1 " Nitrogen 68.7 " " With a strict vegetable diet, on the other hand, hydrogen may also be found (Planer) : Man. Dog. Carbon dioxide .... 20.79 33.83 32.9 vol. per cent. Oxygen 0.37 0.8 " Nitrogen 72.50 38.22 66.3 " Hydrogen 6.71 27.58 The presence of hydrogen is readily understood, if it is remembered that during the process of butyric acid fermentation hydrogen and carbon dioxide are formed. Lactic acid or acetic acid fermentation does not give rise to the formation of gases. Marsh gas, CHj, a product of the fermentation of cellulose, may also be found in pathological conditions, and is formed according to the equation (QHi„05)„ + (H,0)„ = 3(C0,), + 3(CH,)„. It is yet an open question whether marsh gas is formed in the stomach or passes into the stomach from the small intestine. 13 194 THE GASTRIC JUICE AND GASTRIC CONTENTS. Such observations must, however, be regarded as rarities. In one case of this kind, examined by Ewald and Ruppstein,' in which alcohol, acetic acid, lactic acid, and butyric acid were found in the vomited material, au analysis of the gases gave the following result : Carbon dioxide 20.6 vol. per cent. Oxygen 6.5 " " Nitrogen 41.4 " " Hydrogen .... 20.6 " Marsh gas 10.8 " Traces of defiant gas and of hydrogen sulphide were also found. It is curious to note ■ that in this case the patient, who, according to his own statement, had a " vinegar-factory in his stomach on one day and gas-works on another day," was occasionally able to light the eructated gas at the end of a cigar-holder, where it burnt with a faintly luminous flame. McNaught has reported a similar case, in which the analysis furnished the following results : carbon dioxide, 56 per cent.; hydrogen, 28 per cent.; marsh gas, 6.8 per cent.; atmospheric air, 9.2 per cent.^ Ammonia and hydrogen sulphide are also at times met with ; their presence is always due to albuminous putrefaction. Boas ^ found that hydrogen sulphide is quite commonly present in cases of dilatation referable to benign causes, while it is almost always absent in carcinoma. He adds that it is never found when lactic acid is present. In acute gastritis it may be observed tem- porarily. In a number of cases of carcinoma I have never found hydrogen sulphide. In one case reported by Strauss the Bacillus coli communis was apparently concerned in its production. To obtain a knowledge of the gases formed in the stomach during the process of digestion it is only necessary to fill an ordinary Doremus ureometer, or an Einhorn saccharimeter, with the unfiltered gastric contents, and to keep it at a temperature of from 37° to 40° C, when the evolution of gas can be followed closely and the necessary tests made. The presence of carbon dioxide is readily recognized by passing a small amount of sodium hydrate, in concen- trated solution or in substance, into the tube, after the evolution has entirely ceased, when the fluid will rise. If other gases are present at the same time, they will remain after the carbon dioxide has been absorbed. Hydrogen sulphide is readily recognized by its odor and by the fact that it will color a piece of filter-paper, moistened 1 Ewald, Arch. f. Anat. u. Physiol., 1874, p. 217. ' Kuhn, "TJeber Hefegahrung vind Bildung brennbarer Gase im mensohlichen Magen," Zeit. f. klin. Med., vol. xxi. ; and Deutsch. med. Wooh., 1892, No. 49, and 1893, No. 15. ' Boas, " Ueber Sohwefelwasserstoff bildung in Magenkrankhciten," Centralbl. f. inn. Med., 1895, No. 3; Dentsch. med. Wooh., 1892, No. 49. Zawadzki, "Sohwefel- wasserstoff im erweiterten Magen," Centralbl. f. inn. Med., 1894, No. 50. Dauber, "Sohwefelwasserstoff im Magen," Arch. f. Verdanungskrank., vol. iv. p. 4. CHEMICAL EXAMINATION OF THE OASTBIC JUICE. 195 with a few drops of sodium hydrate and lead acetate a more or less pronounced brown or black. The test is conveniently made by filling a test-tube about half-full with the gastric contents and clos- ing it with a cork stopper to which a strip of lead-paper, prepared as indicated, is fastened. The eructation of gas formed in the stomach should not be con- founded with the so-called eructatio nervosa, in which no gas is either eructated, or air simply enters the oesophagus and is expelled again with a loud, explosive noise. This may frequently be observed in neurasthenic and hysterical individuals, and is to a greater or less degree under the control of the will. It is hardly likely, however, that the physician will be called upon in the laboratory to differen- tiate between this form and that of true ructus, caused by fermenta- tive processes taking place in the stomach. The gases brought up in the former condition are without odor or taste, and thus differ from those found in true dyspepsia. Acetone. The presence of acetone in the gastric contents in pathological conditions has repeatedly been observed, especially by v. Jaksch and Lorenz,^ and it is curious to note that the latter was at times able to demonstrate larger quantities of the substance in the gastric con- tents than in the urine. In the chapter on Acetonuria the relation existing between diges- tive diseases and the elimination of acetone will be dealt with more fully, but it may here be mentioned that in the j)>'imary diseases of the gastro-intestinal tract acetone is met with quite constantly in the gastric contents, while it is observed but rarely in the secondary forms, and never is seen in the gastric neuroses. This statement, however, is denied by Sovelieff, who claims to have found traces of acetone in only one case of nervous dyspepsia, while negative results were obtained in all other diseases of the stomach. I have re- peatedly been able to demonstrate the presence of acetone in cases of carcinoma, and never have found it in neurotic conditions. In order to test for acetone, the gastric contents are distilled after the previous addition of a small amount of phosphoric acid (1 : 1000), when the tests of Reynolds and Gunning (see Urine) are applied to the distillate. If both reactions furnish a positive result, the presence of acetone may be regarded as demonstrated. Den- nigfes' test may also be employed, and can be applied to the filtered contents directly (see Urine). Ftomams and Toxalbumins. Remembering that ptomains and toxalbumins have been obtained directly from tainted meat, sausage, fish, clams, crabs, cheese, etc., it 1 Lorenz, Zeit. f. kliu. Med., 1891, vol. xix. p. 19. 196 THE GASTRIC JUICE AND GASTRIC CONTENTS. is to be expected that these bodies may be met with in the gastric contents also. At the same time it may be mentioned that the stomach appears to possess the power of eliminating from the system poisons of this nature which are circulating in the blood. This is shown by the observations of Alt, who found that the water with which the stomach of an animal had been irrigated, after the sub- cutaneous injection of the poison of Pelias berus and Echidna arictans, or the direct bite of the snake, produced identical symp- toms of poisoning when injected into another animal. It is inter- esting to note that with lavage of the stomach the poisoned animal recovered. Similar observations have been made in cholera Asiatica. Certain vegetable alkaloids, such as morphin, are also known to be eliminated to a large extent by the stomach. Of the nature of the ptomai'ns and toxalbumins which may occur in the stomach, very little is known.* Vomited Material. Food-material. — The vomiting of large amounts of totally undi- gested meat two or three hours after its ingestion is met with only in conditions associated with an entire absence of digestive juices Pig." 38. ■'•:l-/>^°%w)-... Collective view of vomited matter. (Eye-pieoe III., objective 8 A, Eeiehert.) a, muscle- flbres ; 6, white blood-corpuscles ; c, c', squamous epithelium ; c'', columnar epithelium ; a, starch-grains, mostly changed by the action of the digestive juices ; e, fat-globules ;/, sarcmae ventriculi ; g, yeast- fungi ;7(, forms resembling the comma-bacillus found oy the author pice in the vomit of intestinal obstruction ; i, various micro-organisms, such as bacilli and micro- cocci ; k, fat-needles, between them connective-tissue derived from the food ; I, vegetaole cells. (V. Jaksch.) from the stomach — i. e., in cases of atrophic cirrhosis of the stomach (anadeny of Ewald). This condition is not to be confounded with 1 Brieger, Untersuohnng'en fiber Ptomaine, Hirschwald, Berlin, 1886. CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 197 the regurgitation of undigested food, mixed with mucus and saliva, which is seen in cases of stricture of the oesophagus or of the car- diac oriiice of the stomach. While at the outset of the latter disease the regurgitation of food occurs immediately, or at least very soon, after a meal, it may take place between meals in the later stages of the disease when dilatation has occurred. The recognition of the origin of the material brought up may then be exceedingly difficult. In such cases an examination should be made for biliary coloring-matter, which, if present, will, of course, immediately exclude the oesophagus as the source of the material ejected. Unfortunately, however, the reverse does not hold good. Small amounts of undigested meat are of no significance. The vomiting of well-digested food is observed in some of the neuroses of the stomach, and also in certain cases of acute and subacute gas- tritis, ulcer of the stomach, and chronic gastritis in its early stages. The vomiting referable to cerebral and spinal diseases also belongs to this category. In this connection it is very important to inquire into the existence of nausea previous to the vomiting, for, as is well known, considerable amounts of saliva and mucus may be swal- lowed if much nausea has existed, the result being that the process of digestion is arrested before the occurrence of vomiting. In such an event it would be erroneous to conclude that, because the mate- rial ingested has not reached that stage of digestion which would be expected at the time of the vomiting, the stomach is incapable of properly performing its functions. Mucus. — The constant presence of large amounts of mucus in the gastric contents obtained with the stomach-tube is almost pathognomonic of the mucous form of gastritis, while its presence in vomited matter may be referable to pre-existing nausea. In cases of pharyngitis moderate amounts of mucus are frequently found. The vomiting of pure mucus, according to Boas, is always pathognomonic of the absence of dilatation of the stomach, a statement founded on reason, as it is altogether unlikely that no particles of food should be brought up at the same time. Under the term gastrosuocorrhoea mucosa Dauber ' has described a condition in which large amounts of mucus are secreted by the noa-digesting organ, in the absence of symptoms pointing to a gastritis. I have observed a similar case occurring in a neuras- thenic patient, in which enormous quantities of mucus could at times be obtained from the fasting organ, but never during the process of digestion. A mild degree of hyperchlorhydria existed at the same time, as well as enteritis mucosa and rhinitis mucosa. The motor power was practically normal. Mucus is readily recognized on simple inspection by its glossy ^ Dauber, " Ueber kontinuirliche Magen-Schleimsecretion,'' Arch. f. Verdauungs- krank., vol. ii. p. 167. 198 THE OASTBIG JUICE AND GASTRIC CONTENTS. appearance. Chemically, it is distinguished by its behavior toward acetic acid (see Urine). Saliva. — The vomiting of pure saliva in the morning upon rising is a fairly common symptoitn of chronic pharyngitis, which in turn frequently carries in its train a chronic gastritis ; it constitutes the so-called vomitus matutinus. Saliva, like mucus, is, of course, always present in the gastric contents in small amounts. Larger amounts are usually referable to an increased secretion owing to the existence of nausea. Chemically, saliva is best recognized by test- ing for the presence of the sulphocyanides (see Saliva, page 138). Bile. — Bile is rarely observed in the gastric contents brought up by the stomach-tube, but is frequently seen in vomited matter, of which it may be said to be a constant constituent whenever the vomiting has been very intense or frequently repeated. Its presence in the former case should always excite suspicion of the existence of stenosis of the descending or horizontal portion of the duodenum or the beginning of the jejunum. This diagnosis becomes the more probable the more constant its presence. Pancreatic Juice. — Mixed with the bile there is probably always present some pancreatic juice, and it has even been suggested that the constant absence of this constituent, in the presence of bile, is strongly suggestive of pancreatic disease or of obstruction of the pancreatic duct (the ductus Wirsungianus). Blood. — The presence of unaltered blood in the gastric contents is usually recognized without difficulty. As marked changes in color, varying from a deep red to a coffee or chocolate brown, may occur, however, when free acids are present, it is at times necessary to resort to a more detailed examination. In order to recognize mere traces when the macroscopical and even the microscopical examination do not point to the presence of blood, the method of Miiller and Weber or that of Donogany should be employed. Kuttner claims that he was thus able to demonstrate the presence of blood in nu- merous cases of chlorosis in which other tests furnished negative results. I have been less successful in the disease in question, but admit that in cases of carcinoma and ulcer of the stomach it is with this method often possible to find traces of blood which would other- wise have remained unnoticed. Method of Miiller and Weber. — The gastric contents are treated with a few cubic centimeters of strong acetic acid and extracted with ether. Should the ether not separate in a clear layer after a few minutes, a few drops of alcohol are added. If the ether then remains colorless, no blood-pigment is present, while a brownish- red color indicates the presence of acetate of htematin. As a similar but yellowish-brown and much less intense discoloration of the ether may be produced by other pigments, such as biliary coloring- matter, it is well, in doubtful cases, to test the ethereal extract with CUKMWAL EXAMINATION OF THE GASTRIC JUICE. 199 tincture of guaiacum. A positive result indicates the presence of blood coloring-matter. The same may be said if, upon spectroscopic examination of the ethereal extract, an absorption-band is discov- ered at the junction of the red and yellow. Donogany's Method. — A small amount of the suspected material is extracted with a 20 per cent, solution of sodium hydrate and filtered. A drop of the filtrate is then mixed on a slide with a drop of pyridin and covered with a cover-glass, when, in the presence of blood, orange-red crystals of hamochromogen will separate out on standing for a few hours. . On spectroscopic examination these crystals will show the characteristic band of absorption between the yellow and the green. Hemorrhage from the stomach, hcematemesis, may be observed in the most diverse conditions. It is either dependent upon a primary disease of the organ, such as ulcer and carcinoma, or it occurs sec- ondarily to disease of other organs, leading to a hypersemic condi- tion of the gastric mucosa, such as the various forms of cardiac, renal, and hepatic disease, in connection with menstrual abnormali- ties, etc. In melsena, purpura hsemorrhagica, pernicious antemia, etc., the cause of the hemorrhage cannot always be determined. It appears to be certain, however, that nervous influences may also take part in the causation of gastric hemorrhage. Pus. — Thei occurrence of pus in vomited matter, referable to disease of the stomach itself, is uncommon. It is seen practically only in cases of phlegmonous and diphtheritic gastritis, and, as Strauss ' has pointed out, in carcinoma affecting the smaller curva- ture and the region of the fundus. In such cases it is not uncom- mon to obtain as much as one-half to two tablespoonfuls of a mucopurulent fluid from the non-digesting organ. As the motor function in this form of carcinoma is often unimpaired, the symptom may be of considerable value in diagnosis. The presence of larger quantities usually indicates perforation into the stomach of an accumulation of pus from a neighboring organ. An abscess of the liver, a suppurative pancreatitis, an abscess of the colon, or a sub- phrenic abscess may thus prove to be its primary source. When present in considerable amount pus is, of course, readily detected with the naked eye ; if any doubt should arise, a microscopical examination will determine the question. Stercoraceous Material. — ^Very important from a clinical stand- point is the vomiting of stercoraceous matter which is notably observed in cases of ileus. Usually this is recognized without diffi- culty by its odor, which is referable to the presence of skatol. If any doubt should arise, it is only necessary to distil the vomited matter after the addition of a little phosphoric acid, and to test for the presence of phenol, indol, and skatol in the distillate, as ' H. Strauss, " Ueber Eiter im Magen," Berlin, klin. Wocli., 1899, p. 870. 200 THE GASTRIC JUICE AND GASTRIC CONTENTS. described in the chapter on Feces (see page 216). When chiefly derived from the small intestine, the vomited matter, according to V. Jaksch, will contain bile-acids and bile-pigment together with an abundance of fat, which may be detected by chemical or microscop- ical examination. The reaction is usually alkaline or feebly acid. I have had occasion to examine the vomited matter of a patient in whom an almost complete obstruction existed immediately above the ileo-csecal valve ; the color of the material was a golden yellow, the reaction neutral ; no bile-pigment or biliary acids were found, while hydrobilirubin was present. Parasites. — Of parasites, ascarides, segments of tsenise, trichinae, Anchylostoma duodenale, and Oxyuris vermicularis are, at times, encountered. The Trichomonas vaginalis has also been seen in one case of carcinoma of the oesophagus.' For a description of these parasites see the chapter on the Feces. The Odor. — The odor of normal gastric juice is peculiar, suggesting the presence of some acid, which can be sharply dis- tinguished from the odor referable to acetic acid or butyric acid. If blood is present in large amount, the vomited matter emits an odor which is so characteristic as never to be mistaken. A feculent odor is met with in cases of enterostenosis or in the presence of an abnormal communication between the stomach and the small or large intestine. A putrid odor may be observed in cases of ulcera- tive carcinoma, pyloric stenosis referable to ulcer, simple carcinoma of the stomach, muscular hypertrophy of the pylorus, stenosis due to inflammatory adhesions, etc. In cases of phosphorus poisoning the vomited matter emits an odor of garlic ; the odor observed in uraemic conditions is referable to ammonia ; a carbolic acid odor is met with in cases of poisoning with this substance. MICROSCOPICAL EXAMINATION OF THE GASTRIC CONTENTS. In the gastric juice obtained from the non-digesting stomach the various morphological constituents of mucus and saliva, which have been described elsewhere, are found. Microscopical particles of food, such as elastic tissue-fibres, starch-granules, fat-droplets, fatty acid crystals, vegetable- and muscle-fibres, are, furthermore, quite constantly seen. Leucocytes and isolated nuclei also are observed ; the latter are set free by the action of the gastric juice upon the mucous corpuscles and epithelial cells. If gastric juice is allowed to stand, small tapioca-like bodies will collect at the bottom of the vessel, which upon microscopical exami- nation will be seen to contain numerous snail-shell -like formations, ' G. Striibe, " Triohomonaa hominis bei Carcinoma ventriculi," Berlin, klin. Woch., 1898, p. 708. PLATE XI. \ ,; ' # 3, 584, 616. Hogg, Brit. Med. Jour., 1888, p. 121. Kartulis, Centralbl. f. Bakt. n. Parasit., vol. 1. p. 65. ' K. A. Eudolphi, Arch. f. Zool. u. Zoot., 1803, vol. iii. Pt. 2, p. 1. Idem, Entozoorum o. vermium intestinal, historia naturalis, Amstelaedami, ii. 2. 248 THE FECES. Ascaris maritima, Leuckart, also belongs to this class. It has been observed in only one case — in Greenland. Oxyuris vermicularis, Bremser •.'syn., Ascaris vermicularis (Linn6); Ascaris grsecorum (Pallas) (Fig. 60). The male is 4 mm., the female 10 mm. long. At the head Fig. 59. Fig. 60. Ascaris mystax. (v. Jaksch.) u, male ; b, female ; c, head ; d, egg. Oxyuria vermicularis. (v. Jaksch.) a, head ; 6, male ; c, female ; d, eggs. three lip-like projections with lateral cuticular thickenings may be The tail of the male is provided with six pairs of papillae, and seen. Fig. 61. Anchylostoraum duodenale. (v. Jaksch.) a, male, natural size ; b, female, natural size ; c, male, magnified ; d, female, magnified ; e, head (eye-piece II., objective C, Zeiss) ; /, eggs. the female with two uteri. The eggs are 0.05 by 0.02 to 0.03mm. in size, and covered with a membrane showing a double or triple contour ; in the interior, which is coarsely granular, the embryos are contained. PATHOLOGY OF THE FECES. 249 Fig. 62. The female worm lives in the caecum, but after impregnation travels downward to the rectum. Here it causes most annoying symptoms, which are especially distressing at night, when the organ- ism emerges from the anus. In doubtful cases of pruritus ani aut vulvae an examination of the feces should be made for this parasite. The ova themselves do not occur in the feces.' Anchylostomum duodenale (Dubini) : s'yn., Anchylostoma duode- nale (Dubini) ; Strongylus quadridentatus (v. Siebold) ; Dochmius anchylostomum (Molin) ; Sclerastoma duodenale (Cobbold) ; Stron- gylus duodenalis (Schneider) ; Dochmius duodenalis (LeucJiart) ; Uncinaria duodenalis (Roilliet) (Fig. 61). This organism belongs to the family Strongyhides, and is one of the most dangerous parasites met with in the human being. It has been found in Italy, Germany, Switzerland, Belgium, Egypt, and in the West Indies (Jamaica). Within recent years several cases have also been reported in the United States. From a pathological standpoint the parasite is of spe- cial interest, as its presence gives rise to severe and often fatal anaemia. Griesiuger was the first to point out that the so-called Egyptian chlorosis is produced by this organism. In every case of severe anaemia, particularly when occurring in patients who have been working in mines, tunnels and brick- yards, the feces should be carefully exam- ined for the ova of this parasite. The worm itself is rarely found. Its habitat is in the jejunum. Infection takes place through contaminated drinking-water, or possibly by direct transference of the em- bryos with dirty hands.^ The male is 6 to 11.5 mm. long, the female 10 to 18 mm. The head, which tapers somewhat, is turned toward the back; the mouth capsule is hollowed out and sur- rounded by four teeth ; the tail of the male forms a three-lobed bursa, while that of the female tapers conically ; the genital opening is behind the middle of the body. Its eggs have an oval form and a smooth surface, measuring from 0.05 to 0.06 by 0.03 to 0.04 mm. In their interior two or three segment- ing bodies are found, which rapidly develop outside of the human body, so that after twenty-four to forty-eight hours embryos may be ' Lutz, loo. oit. ' Leichtenstern, Centralbl. f. klin.'Med., 1885, vol. vi. p. 195; Deutsch. med. Woch., 1885, vol. xl. ; 1886; vol. xii. : 1887, vol. xiii. Lutz, Volkmann's Sammlung, 1885, Nos. 2.'i5 and 256. American cases : W. L. Blickhahn, Med. News, 1893, p. 662. F. S. MoUau, ■ Buffalo Med. and Surg. Jour., 1895, p. 573. Trichocephalus dispar. (V. JAKSCH.) a, male, slightly magnified ; ft, female, slightly magnified; c, eggs (eye-piece 11., objective A, Keiehert). bjective 8 250 THE FECES. found in the same feces in which the eggs were observed, or fiilly developed, ova may be found after allowing the feces to stand for only a few hours. When allowed to dry, the young parasites become encysted, but after remaining so for from one to two weeks they are capable of infection. A second host for its cycle of development is, according to Leichtenstern, not necessary. Trichocephalus hominis, Schwank : syn., Trichocephalus dispar (Rudolphi) ; mastigodes (Zeder) ; trichuris (Biittner). This parasite, which belongs to the family Triohotrachdides, is formed like a whip, Fig. 63. Trichina spiralis in muscle. the last-end being the head-end, while the tail-end is very much thicker. The male measures 46 mm. and the female 50 mm. in length. The eggs are brownish in color, measuring 0.05 by 0.06 mm. in size, and present a doubly contoured shell, with a de- pression at each end, closed by a lid. The contents are coarsely granular. The organism is said to be the most widely distributed PATHOLOGY OF THE FECES. 251 Fig. 64. intestinal parasite, occurring in Europe, North America, Asia, Africa, and Australia. Its habitat is in the csecum. The living worm is only rarely found in the feces.' Trichina spiralis (Owen) (Fig. 63) is rarely found in the feces. The male measures 1.5 mm. in length, and is provided with four papillae between the conical lips. The female is 3 mm. long. The uterus is situated nearer the head than the ovary, which opens into it. Fertilization occurs in the intestinal canal. The eggs develop into em- byros in the uterus, emerge from this, and penetrate the intestinal walls, whence they are carried by the blood-current to the muscles. Trichinosis is far less common in the United States than in Europe.^ The diagnosis of sporadic cases has been greatly facilitated by the dis- covery of Brown that eosinophilia, often of high grade, is practically of constant, occurrence during the acute stage of the disease (see page 90). Angnillula intestinalis is 2.25 mm. long and 0.04 mm. broad ; its mouth is three-cornered and bounded by three lips. The genital aperture is located between the middle and posterior third of the body. Its eggs are similar to those of Anchy- lostomutn duodenale, with which the angnillula is not infrequently asso- ciated ; but they are longer and more elliptical, with tapering poles ; they are never found in the feces unless active catharsis is established. Other- wise the embryos only are found, as the development of the ova occurs with great rapidity. When sexually mature, the parasite is called Angnillula stercoralis ; this again gives rise to embryos, which may in turn enter the intestinal canal. The Anguillula ster- coralis (Fig. 64) has a rounded body, which presents an indistinct cross-striation. Its head is like the top of a cane, and is provided with two lateral jaws, each of which is armed with two teeth. The male measures 0.08 mm., the female 1.22 mm. in length. The patho- 'Emi, Berlin, klin. Woch., 1886, vol. xxiii. p. 614. ^ Leuckart, loc. cit., Anguillula stercoralis. (Bizzozeeo.) 252 THE FECES. logical significance of this parasite has not been definitely ascer- tained, but from its resemblance to Anchylostomum duodenale it has become important from a diagnostic point of view. Some observers regard the parasite as harmless, while others, and notably Davaine, associate its presence with ansemia.^ Insecta. — As the larvae of the various insects met with in the feces have been very little studied, they will not be considered at this place; they are apparently of no clinical importance. Vegetable Parasites. — Among the pathogenic vegetable parasites the bacillus of cholera, of typhoid fever, and of tuberculosis, as well as the bacilli of Booker, the Bacillus coli communis, the Bacillus pyo- cyaneus, the Bacillus lactis aerogenes, the bacillus of Shiga, and the Proteus vulgaris, deserve especial consideration. The Comma-bacillus. — As early as 1848 certain "vibrios" were observed in abundance in the stools of cholera patients by Yirchow, and in 1849 by Pouchet, Britton, and Swayne, no importance, how- ever, being attached to their presence at the time. The first accurate and detailed studies of the organism found in cholera stools were made in 1883 by the members of the French and German commissions sent to Egypt to investigate the nature of the dreaded disease. The results of their work were first pub- lished by Koch in his report to the Berlin Sanitary Office in 1883, and in 1884 by Strauss, Roux, Nocard, and Thuillier. The clinical recognition of cholera Asiatica has now become a simple matter since Pfeiffer has demonstrated that the blood-serum of cholera patients possesses the property of causing arrest of motility and agglutination of the specific bacilli. Ordinary bouillon- cultures, however, can usually not be employed, as particles of the film when broken up may easily be mistaken for agglutinated bacilli. It is best in every case to make use of agar-cultures sixteen to twenty-four hours old, and to prepare emulsions in bouillon or normal salt solution as occasion requires. The emul- sion, moreover, should always be examined microscopically before use, so as to insure the absence of any conglomerations of bacilli. The blood is then diluted in the proportion of 1 : 10 or 1 : 15. If the test-tube method is employed, the tubes should be kept in the incubator (37° C.) for only one or two hours. Upon the slide the reaction is obtained in from five to twenty minutes. If no distinct agglutination is observed at the end of one hour, the diagnosis of cholera is rendered improbable. Dried blood retains its agglutinating properties for a considerable length of time, and may also be used for examination. The comma-bacillus is a slightly arched or half-moon-shaped little rod, and is somewhat shorter than the tubercle bacillus (Plate ' Grassi, Centralbl. f. Bakt. u. Parasit., 1887, vol. ii. p. 413. Leiohtenstern, Deutsch. med. Woch., 1898, p. 118. Perroncito, Arch. p. 1. sci. med., 1881, No. 2. Compt. rend, de I'Aoad. des Sci., 1882, No. 1. Teissier, Ibid., vol. cxxi. p. 171. PLATE XI ^/'\y\ A>^ ^^'.k'l S| )i I'i I i Li 111 of Asiatic Cholera. Impression Cover-slip from a Colony Thirty-four Hours Old. (Abbott.) Bacillus of Finkler smcl Prior. (Cornil and Babes.) Bacillus of Typhoid Fever from a Culture Twenty-four Hours Old, on Agar-agar. (Abbott.) PATHOLOGY OF THE FECES. 253 XII., Fig. 1). Occasionally two are placed end to end with their convexities in opposite directions, thus presenting the appearance of the letter S. They are provided with flagella. Koch detected these bacilli in the intestinal contents and feces, but rarely in the vomited matter, in Asiatic cholera only. In the stools they at times occur in such numbers as to constitute pure cultures. In plate- cultures kept at a temperature of 22° C. white colonies with serrated borders may be observed after twenty-four hours. The color of such a colony is slightly yellow or rose red, its central portion gradu- ally assuming a deeper tint, and finally becoming liquefied. Upon agar-plates the bacilli form a grayish-yellow, irregular, slimy coat- ing, but do not liquefy the culture-medium. In stab-cultures, after twenty-four hours, a whitish color may be observed along the line of the stab ; around this there is formed a funnel-shaped depression, which gradually increases in size and apparently contains a bubble of gas. The upper portion of the culture-medium at the same time becomes liquefied, while the lower portion remains solid for days. In a suspended drop spirochsetse-like spirals are observed at the margins, which often present as many as twenty distinct arches.^ Closely related to Koch's comma-bacillus, and possibly bearing to cholera nostras the same relation that the former bears to cholera Asiatica, is the badUus of Finkler and Prior, discovered in 1884 and 1885 (Plate XII., Fig. 2). This is, however, readily distinguished from the former by the following characteristics : it is larger and thicker than the comma-bacillus ; the colonies on gelatin plate- cultures show equally round and sharp-edged forms, which present a granular appearance under a low or medium power, and are usu- ally of a brown color. The organism liquefies gelatin very rapidly, a penetrating, excessively fetid odor being developed at the same time. In stab-cultures the bacillus of cholera Asiatica forms a funnel-shaped depression, while the bacillus of Finkler and Prior forms a stocking-like depression.^ In this connection the green haciUus of Le Sage, discovered in certain forms of infantile diarrhoea, must briefiy be referred to, the stools, as has been mentioned, being of a grass-green color. The production of this pigment in cultures is one of the characteristics of the organism ; when injected into the intestines of animals it is said to produce diarrhoea and a catarrhal inflammation of the mucous membrane. Booker ' has described nine different bacilli, as occurring in cases of ' E. Koch, Berlin, klin. Woch., 1884, vol. xxi. pp. 477,493, 509. ' Finkler, Deutsch. med. Wooh., Tageblatt der Naturforscherversammlung, 1884, vol. ^- p. 36, and 1885, p. 438. Finkler u. Prior, Erganzungshefte z. Centralbl. f. allg. Gesundheitspflege, 1885, vol. i. ' W. D. Booker, " A Bacteriological and Anatomical Study of the Summer Diarrhoeas of Infants." Johns Hopkins Hosp. Eep., vol. vi. 254 THE FECES. infantile diarrhoea. Seven of these closely resemble the Bacillus coli communis. Bacillus " A " is a bacillus with rounded ends, measur- ing from 3 /i to 4 /i in length by 0.7 n in breadth. It is motile and liquefying. Colonies on agar and potato present a dirty-brown color. The typhoid bacillus, discovered by Eberth' in 1880 in the ab- dominal organs of patients dead with typhoid fever, is unfortunately not so readily recognized in the feces as the organisms just described. This is owing to the intimate relation which apparently exists be- tween the bacillus in question and the Bacillus coli communis, with which it has many properties in common. A few years ago Eisner suggested a method which, it was hoped, would effectually overcome this difficulty, and in the hands of numerous observers good results were obtained. Widal's agglutination test, however, which was almost simultaneously introduced, diverted attention from the study of the feces, and Eisner's work has practically been forgotten. In the meantime Widal's test has been carefully investigated, and although the reaction must unquestionably be considered as a specific reaction of typhoid fever, its value in diagnosis is neverthe- less limited (see page 100). As a consequence, further attempts have been made to discover a method which will enable the general practitioner to establish definitely the diagnosis of typhoid fever at an early stage of the disease. Whether or not Eisner's method (v. i.) has been deservedly abandoned, further investigations will show. At the present time another procedure, which was suggested by Piorkowski, is attracting widespread attention, as it is claimed that with this method the diagnosis can be made within twenty -four hours. PiOEKOWSKi's Method.^ — The necessary culture-medium is pre- pared as follows : normal urine of a specific gravity of about 1.020 is allowed to stand until the reaction has become alkaline ; it is then treated with 0.5 per cent, of peptone and 3.3 per cent, of gelatin, boiled for one hour, and filtered immediately into test-tubes with- out any further application of heat. The test-tubes are closed with cotton, sterilized for fifteen minutes in a steam sterilizer at 100° C, and resterilized after twenty-four hours for ten minutes. To examine the feces, one tube is inoculated with 2 oesen of the fecal matter, which should be as fresh as possible. From this tube 4 oesen are transferred to a second tube, and a third is inoculated with from 6 to 8 oeesen from the one preceding. Plates are finally prepared and kept at a temperature of 22° C, as the presence of so small an amount of gelatin does not permit of exposure to higher temperatures. After sixteen to twenty-four hours an examination is 1 Eberth, Virchow's Arohiv, 1881, vol. Ixxxiii. p. 486. 2 Piorkowski, " Ein einfaches Verfahren z. Sicherstellung d, Typhusdiagnose, ' Berlin, klin. Woch., 1899, p. 145. PATHOLOGY OF THE FECES. 255 made with a low power. At the expiration of this time the colonies of the colon bacillus appear as round, yellowish-brown, and finely granular specks, with well-defined borders, while the typhoid colo- nies show a peculiar flagellate appearance, from two to four fine colorless radicles usually starting from a light, highly refractive central focus. After forty-eight, hours the radicles have greatly extended, and after forty-eight to fifty-six hours the colonies are perfectly developed and present a picture which strongly suggests the appearance of radishes, minute interweaving branches being given off in every direction, while no difference can be observed at this time between typhoid and colon bacilli which have been grown for control in 10 per cent, normal or bouillon-gelatin. Piorkowski claims that he has thus been able to demonstrate the presence of typhoid bacilli in infected drinking-water, and in the feces of typhoid fever patients at a time when a positive result could not yet be obtained with Widal's test. Recent reports bear out the claims of Piorkowski, and the method can hence be recommended in doubtful cases.* Elsner's Method.^ — The culture-medium is prepared as follows : an aqueous extract of potato (500 grammes to the liter) is treated with 10 per cent, of gelatin and boiled. The solution is then treated with 2.4 to 3.2 c.c. of a one-tenth normal solution of sodium hydrate, in order to secure the necessary degree of acidity, and then filtered and sterilized. When needed, a portion is placed in an Erlenmeyer flask and treated with 1 per cent, of potassium iodide. The mixture is inocu- lated with fecal material and the necessary plates prepared. Upon this medium only a few species of bacteria will grow, principally the Bacillus coli and the typhoid bacillus. After twenty-four hours the Bacillus coli colonies are already mature, while the typhoid colonies can scarcely be made out with a low power. After forty-eight hours, however, they appear as small, highly refractive, extremely fine, granular colonies, closely resembling drops of water, which can be readily distinguished from the large, much more granular, brownish colonies of the Bacterium coli. This difference is brought out par- ticularly well if diluted plates have been prepared. Brieger,' who carefully repeated the experiments of Eisner, states that typhoid bacilli are found in abundance in the stools so long as fever exists, but with approaching convalescence they diminish in number and ultimately disappear. If, notwithstanding the absence of fever, bacilli are found in notable numbers during convalescence, a relapse may be anticipated. In pure cultures the typhoid bacilli present the following features : ' A. Schiitze, "Ueber d. Nachweis v. Typhusbacillen in den Faeces," Zeit. f. klin. Med., vol. xxxviii. p. 39. '^ Eisner, Zeit. f. Hyg. u. Infektionskrank., 1895, vol. xxi. p. 25. ' Brieger, Deutsch. med. Woch., 1895, vol. xxi. p. 835. 266 THE FECES. they occur in the form of rods of almost one-third the size of a red blood-corpuscle, or in threads composed of several rods joined end to end (Plate XII., Fig. 3). Their ends are rounded ; their length is equivalent to about three times their breadth. They are actively motile and provided with polar as well as lateral flagella. They grow very readily on bouillon-peptone gelatin, and after twenty-four hours colonies begin to appear. When slightly magnified these present a faintly yellowish color ; microscopically they are barely visible. When kept at a temperature of 37° C. the formation of spores may be observed, especially when the organism is grown on media colored with phloxin-red or benzopurpurin. Gelatin is not liquefied ; the growth is white and fine, both along the line of the stab and on the surface. Cultivation in glucose-bouillon, orglueose- agar, in fermentation-tubes, does not give rise to the formation of gas, but after twenty-four hours the entire fluid becomes turbid. Milk is rendered feebly acid, but is not coagulated. No indol reac- tion is obtained when the organism is grown on peptone-containing media. On potato a very faint, whitish, almost invisible growth takes place. Absolute identification is possible by means of Pfeifi«r's agglutination-test (see Widal's reaction). Tubercle bacilli, when present in the feces, are indicative of intestinal tuberculosis, providing they are observed upon repeated examination and there are clinical symptoms pointing to the bowels as the seat of the disease ; otherwise they may be referable to swallowed sputa. They may be demonstrated as described in the chapter on Sputum. The Bacillus coli communis,' while constantly present in normal feces, is described at this place, as modern investigations have shown that it may at times develop pathogenic properties. It has been found in the pus in eases of purulent perforating peritonitis, angio- cholitis, pyelonephritis, etc., and, as indicated elsewhere, at times forms the nucleus of gall-stones. It occurs in the form of delicate or coarse rods, measuring about 0.4 /i in length, which manifest a certain degree of motility, due to the presence of one or two polar flagella. The organism is stained by the usual anilin dyes, and is decolorized by Gram's method. The colonies upon gelatin closely resemble those of the bacillus of typhoid fever, forming small whitish specks in the gelatin, and delicate films with serrated borders upon the same medium, which, moreover, is not liquefied. They also grow upon potato. As in the case of the cholera bacillus, the nitroso- indol reaction can be obtained when the organism is grown upon peptone-containing media. In solutions of glucose active fermentation takes place. The Bacterium lactis agrogenes (Escherich) closely resembles the organism just described, and may also at times develop pathogenic properties. It was recently found in a case of pneumaturia and in • Flugge, Die Microorganismen. PATHOLOGY OF THE FECES. 257 one of idiopathic bacteriuria. It is seen quite constantly . in the stools of sucklings, but may also be met with in those of adults. It occurs in the form of rather stout rods, which frequently lie in pairs, resembling diplococci. The organism is non-motile. Like the Bacillus coli communis, it is decolorized by Gram 's method. In plate-cultures it forms a dense white film ; in stab-cultures a chain of white colonies resembling beads is i.een. In the latter, moreover, if the stab is closed, bubbles of gas will be seen to form, which rapidly increase in number and size. Milk is coagulated in large lumps in twenty-four hours ; at the same time, the formation of gas is much more intense than in the case of the Bacillus coli communis. The Bacillus pyocyaneus has within recent years been isolated from the stools of dysenteric patients, and has been proved the cause of several epidemics. The organism in question is a small motile bacil- lus measuring from 1 ^ to 2 ^ in length by 0.3 /i to 0.5 /jt in breadth. It sometimes occurs in short chains, but is usually single. It is stained with the common anilin dyes, and is decolorized with Gram's method. It grows on the usual culture-media, and liquefies gelatin. In 2 per cent, glucose-bouillon no fermentation takes place. Litmus- milk is curdled in about forty-eight hours. Some varieties produce indol. Most" characteristic is the production of certain pigments, viz., pyocyanin and a fluorescent bluish-green pigment which is common to almost all varieties.^ Bacillus acidophilus, Moro.^ This organism has recently been described by Moro as occurring in the stools of breast-fed infants, in which it normally prevails over all other forms ; under pathological conditions, on the other hand, as also in the stools of children, which have been fed with cows' milk their number is found dimin- ished, while the members of the coli-group enter into the foreground. Beyond the stools, the bacillus has been found in the outer portion of the secretory duct of the human mammary gland, in the milk, and the skin of the nipple and its immediate surroundings. It is apparently not pathogenic. The organism occurs in the f jrm of slight rods measuring 1.5 // to 2 /i in length, by 0.6 ;u to 0.9 /i in breadth. It is non-motile. It is not decolorized by Gram's method, but loses this property after from thirty-six hours to nine days. The best growths are obtained on beer wort bouillon and common bouillon when acidified with a mineral acid ; the acidity of 10 c. c. of the medium may correspond to 10 c.c. of a decinormal solution of potassium hydrate. The optimum temperature is 37° C; between 20° C. and 22° C. no ' A. J. Lartigau, " A Contribution to the Study of the Pathogenesis of the Bacillus. Pyocyaneus," etc., Jour. Exper. Med., 1898, No. 6. ''Moro, "Ein Beitrag zur Kenntniss der normalen Darmbacterieu des Sanglings,'" Jahrbuch f. Einderheilk., vol. lii. Also: "Ueber die nach Gram farbbaren Bacillen d. Sauglingstuhles," Wien. klin. Woch., 1900, No. 5. 17 258 THE FECES. growth? occurs. On the various agar-slants imperfect development takes place ; on potato the organism does not grow. It is an active acid-producer, but does not give rise to the formation of gas ; with Escherich's stain it is colored blue. Escherich's Stain. — This stain is now extensively used by psediat- rists in order to ascertain any deviations from the normal in the flora of the feces. Under strictly normal conditions the bacilli which are found in the stools of breast-fed children are thus nearly all colored blue (see above), while red bacilli are but little numerous. In the case of infants, on the other hand, which are fed exclusively on cows' milk the red bacilli predominate, while in mixed feeding the blue enter into the foreground in about the proportion in which breast- milk is employed. The red bacilli belong to thfe coli-group. These further predominate, or may be found exclusively, if for any reason intestinal digestion is impaired. Staphylococci, streptococci, etc., when simultaneously present, are in either event stained blue. In staphylococcus enteritis the blue bacilli which normally exist in the stools of breast-fed infants are almost entirely replaced by staphylo- cocci. At the beginning of the enteritis they are not numerous, but they increase during the progress of the disease, and finally disappear when the child recovers. In staining, the following solutions are employed : 1. An aqueous solution of gentian- violet (5 : 200). This is boiled for one-half hour and is then filtered ; it keeps for a long time. 2. A mixture containing 11 parts of absolute alcohol and 3 parts of oil of anilin. (1) and (2) are mixed in the proportion of 8.5 : 1.5 ; the resulting solution keeps for from two to three weeks, but not longer. 3. A solution of iodo-potassic iodide containing 1 part of iodine and 2 parts of potassium iodide in 60 parts of water. 4. A mixture of equal parts of oil of anilin and Xylol. 5. A concentrated alcoholic solution of fuchsin, diluted with an equal volume of absolute alcohol. A bit of the stool is spread upon a slide in as thin a layer, as possible. After drying in the air the specimen is fixed by passing through the flame of a Bunsen burner. It is then stained for a few seconds with the mixture of (1) and (2), blotted, placed in the iodine solution for a few seconds, blotted again, decolorized with (4) until a notable extraction of color no longer occurs. It is washed with xylol, dried, and finally stained for a few seconds with the fuchsin solution, washed with water, blotted, and is then ready for examination.' Proteus vulgaris, Hauser. This organism, while usually regarded as non-pathogenic, should be numbered among the bacteria which may at times develop pathogenic properties. Baginsky and Booker ' Moro, loc. cit. PATHOLOGY OF THE FECES. 259 have frequently found it in the stools in cases of infantile summer diarrhoea. Escherich observed it at times in the meconium. Brudzinski examined the dyspeptic and fetid stools of a number of artificially fed infants in Escherich's clinic, and in all the cases found the proteus. Others have encountered it in inflammatory conditions of exposed surfaces, in appendicitis, in perforative peri- tonitis, and even in closed abscesses, either alone or in association with other bacteria (Welch). A mixed infection with the proteus and Loffler's bacillus has also been observed. The organism forms little rods, measuring about 0.6 // in diameter, while their length is variable ; at times a more roundish form is observed ; at others little rods measuring from 1.25 // to 3.75 fi in length, or even long threads. They are readily stained, but are easily decolorized by alcohol or Gram's method. Most characteristic is their growth upon nutrient gelatin. At the temperature of the room little depressions will be observed after six to eight hours, which are surrounded by a narrow zone of bacilli from which a thin, wide film, provided with irregular projections, extends over the culture-medium. From this film islets become separated, which slowly extend over the gelatin and cause its liquefaction. The organism is motile. It decomposes urea and causes albuminous putrefaction. The nitroso-indol reaction is readily obtained in bouillon-cultures. "^ In boiled milk the organ- ism grows well, while in fresh milk it develops only irregularly, and in acid milk no growth takes place at all. Bacillus dyseEteriae, Shiga. This organism is now regarded as the specific cause of one form of dysentery which prevails in the tropics. It was discovered by Shiga in Japan (1897), and has since been encountered, by Flexner and Strong more especially, in the acute form of the disease which prevails in the Philippine Islands and in Porto Rico. In some of the cases amcebse also were found, but they are rarely numerous. The stools at first contain a small amount of mucus ; this rapidly increases and soon becomes blood- streaked. Generally within forty-eight hours, or a shorter time, the stools consist of nothing but reddish, bloody mucus, and on micro- scopical examination red blood-corpuscles, leucocytes, and epithelial cells are found. The bacillus in question Shiga describes as a short rod with rounded ends, much resembling the bacillus of typhoid fever, or the greater portion of the coli-group. It is possessed of moderate motility, but flagella have not as yet been demonstrated ; neither has spore-formation been observed. The organism decolorizes by Gram's stain. Upon gelatin plates at room temperature there appear, after a few- days, small round dots, which, magnified under low powers, are slightly yellow and finely granular. After a few days they increase ' Fliigge, loc. cit. 260 THE FECES. in size ; the middle portion of the colonies then appears darker under a low power, while the outer zone appears brighter and more seed-like. The superficial and deeper colonies show no marked variation. In stab-cultures of gelatin a whitish strand forms the whole length of the stab. The gelatin is not liquefied. After twenty-four hours in the incubator single colonies upon slanted agar appear moist, bluish, and partially translucent. After two days they present a combination of a middle dark and a periph- eral bright, sharply defined zone. The growth on glycerin-agar is slightly more abundant than on ordinary agar. The organism grows on blood-serum, without lique- fying it. In the stab-cultures of glucose-agar there is formed along the whole line of the puncture a thick gray-white strand without the development of gas. Upon potato after twenty-four hours in the incubator there is hardly any perceptible growth, only the surface appears slightly shiny. After two days this changes to a yellow brown. In the course of a week the growth is heavier and of a deeper brown color. Bouillon cultures show after a day in the incubator a somewhat intense cloudiness, with a moderate precipitate. No pellicle is formed on the surface. No indol reaction is present. Litmus-milk after twenty -four hours appears reddish; otherwise, however, it undergoes no change. The milk never coagulates. The bacillus is pathogenic for mice, rabbits, and guinea-pigs. It is agglutinated by the patient's blood-serum, and it is interesting to note that this reaction is obtained only with cases definitely known to have been infected with the micro-organism in question. In several cases of amcd)ia dysentery, which were examined in this direction at the Johns Hopkins Hospital, the blood-serum failed to produce the reaction with the bacillus obtained at Manila. As Flexner states, these results tend to emphasize the distinction of types of dysentery occurring in the tropics.' CHEMISTRY OF THE FECES. According to Hoppe-Seyler, mucin is a constant constituent of the feces, both under physiological and pathological conditions. Nor- mally, however, it is never possible to recognize its presence either with the naked eye or with the microscope. In order to demonstrate the presence of mucin in the feces they are digested with water and treated with an equal volume of milk of lime ; the mixture is allowed to stand for several hours, when it is filtered and the filtrate 1 K. Shiga, Centralbl. f. Bakt., Parasit. u. Infeotionakrankh., 1898, vol. xxiv. E..P- Strong and Musgrave, " Preliminary Note regarding the .ffltiology of the Dysenteries of Manila," Eeport of the Surgeon-General of the Army, Washington, 1900, p. 251. S. Flexner, " On the Etiology of Tropical Dysentery," Bull. .Johns Hopkins Hosp., 1900, p. 231. CHEMISTRY OF THE FECES. 261 tested with acetic acid. In the presence of mucin a cloud develops upon addition of the acid. Albumin is demonstrated in the feces by treating them repeatedly with water slightly acidified with acetic acid. The filtrate is then examined for albumin according to methods given elsewhere (see Urine). Under normal conditions these reactions prove negative. Pathologically, serum-albumin has been observed in cases of typhoid fever and chlorosis. Peptones (albumoses) are normally absent from the feces. They have been observed in typhoid fever, dysentery, tubercular ulcera- tion, purulent peritonitis with perforation into the gut, atrophic cirrhosis, and carcinoma of the liver. Acholic stools are also usually rich in peptones. The peptones are demonstrated in the following manner : the feces are digested with water, so as to form a thin mush ; they are then boiled, filtered while hot, and the filtrate examined for albumin, so as to be sure that all of this has been removed. The mucin is removed by treating with lead acetate, when the filtrate is examined for peptones as described in the chapter on Urine (which see). Of the carbohydrates, starch, glucose, and certain gums may be found, ^n order to demonstrate these the feces are boiled with water, filtered, and evaporated to a small volume. This solution may now be tested with phenylhydrazin or Trammer's reagent for glucose (see Urine), and with a solution of iodo-potassic iodide for starch (see Saliva, page 139). The residue is extracted with alcohol and ether, as described under the heading of fatty acids, and, then with water. The filtrate of the aqueous extract is con- centrated, boiled with dilute sulphuric acid, and then over-saturated with sodium hydrate. This mixture is treated with cupric sulphate and boiled, in order to test for dextrin and gums. Bik-pigment, which is normally absent from the feces, occurs in large amounts in catarrhal conditions of the small intestine, and may be demonstrated by Gmelin's method, viz., a drop of the filtered liquid, or a particle of highly colored fecal matter, is brought into contact with a drop of fuming nitric acid, when the yellow color will be seen to pass through the various shades of the spectrum, the green shade being the most characteristic. At times, however, it is not possible to obtain a positive reaction in this manner, although bile-pigment is present. In such cases the examination should be conducted under the microscope, and attention directed to bile-stained epithelial cells, leucocytes, particles of mucus, and crystals. Whenever there is increased intestinal putrefaction the fatty acids, phenol, indol, and skatol will, of course, be found in increased amounts.' ' A. E. Austin, "The Chemical Examination of the Feces for Clinical Purposes," Phila. Med. Jour., 1900, p. 551. 262 THE FECES. Ptomains. — Of ptomains, only two have been isolated from the feces, under pathological conditions, viz., putrescin and cadaverin. They have been found in Asiatic cholera, in cholerina, dysentery, and in connection with cystinuria. In cholera and cystinuria their amount may be quite large. Baumann and v. Udranszky thus obtained 0.5 gramme of the benzoylated compounds from the col- lected feces of twenty-four hours. In cholera the cadaverin seems to predominate, while in cystinuria more putrescin is found.' To isolate the diamins in question, the feces are digested with alcohol which has been acidified with sulphuric acid. The alcoholic extract is evaporated, the residue dissolved in water, and further benzoylated, as described in the section on Urine. THE FECES IN VARIOUS DISEASES OF THE INTESTINAL TRACT. Acute Intestinal Catarrh. — This condition follows the ingestion of excessive quantities of normal food, of tainted food (meat, fish, cheese, etc.), beer, and of certain poisons, such as acids, alkalies, arsenic, corrosive sublimate, etc., when taken in toxic quantities. It is also observed as the result of a general infection, as in summer diarrhoea, cholera nostras, typhoid fever, and severe malaria, and is associated with disturbed circulatory conditions, producing a passive hypersemia of the gastro-intestinal mucosa, as in diseases of the liver and portal system, in chronic heart and lung diseases, etc. How far these circulatory disturbances may be considered as primary causes remains to be seen. Possibly they merely act as predisposing causes of certain chemical processes taking place in the intestinal contents. The stools are usually increased in number in proportion to the degree in which the large intestine is affected. Two or three, or ten or more, stools may be passed within the twenty-four hours. In consistence they are mushy or even watery, containing in some cases 90 or 95 per cent. Their color is usually light yellow, but may, at times, be green. Microscopically, remnants of food may be found in large quantities, as also numerous bacteria, triple phosphates, isolated pus-corpuscles, and desquamated cylindrical epithelial cells. A duodenal catarrh can only be diagnosed when icterus exists at the same time. In catarrh of the j^unum and ileum, when the large intestine is not affected, tlae stools are firm, formed, and speckled with small hyaline particles of mucus, which are visible only with the micro- scope. Usually, however, the large intestine also is affected, when the stools are loose and contain undigested particles of food, the latter indicating abnormal conditions in the small intestine. Bile- ' C. E. Simon, "Cystinuria and its Relation to Diaminuria," Am. Jour. Med. Sci., Jan., 1900. THE FECES IN DISEASES OF THE INTESTINAL TRACT. 263 pigment is also met with, as the contents of the small intestine only give Gmelin's reaction. Catarrh of the large intestine probably always exists whenever diarrhoea occurs. When the colon is extensively affected mneus appears in larger masses than otherwise ; and if the catarrh is very low down the feces may be formed, but are covered with mucus. Chronic Intestinal Catarrh. — This may follow an acute attack, and may also occur after dysentery, severe malaria, typhoid fever, etc. Diarrhoea usually alternates with constipation. It is not very Fig. 63. Rectal discharge from a case of enteritis membranosa. common in adults, while in children it is quite frequently observed. Macroscopically and microscopically it presents the same picture as in the acute form. Enteritis memhranosa is a form of chronic intestinal catarrh which is essentially characterized by the evacuation of cylindrical masses of mucus, as described on page 226 (Fig. 65). Cholera Nostras. — This is an infectious disease affecting both stomach and intestines, and is probably dependent upon the pres- ence of the bacillus of Finkler and Prior. The stools are first feculent, but soon become colorless and more and more watery, until they ultimately resemble the so-called rice- water stools of cholera Asiatica, and contain much serum-albumin and mucin. Summer Diarrhoea of Infants. — In this disease six or seven stools are passed daily, which are more liquid than normally, of a fetid odor, and contain flakes of casein. They are often green when passed, or may assume that color on standing. Mucus is present, and when the colon is especially affected may occur in sago-like par- ticles. Pus-corpuscles, epithelial cells, and small amounts of blood may be present in severe forms. Booker, in his classical work on the summer diarrhoea of infants, arrives at the conclusion that the disease should not be attrib- 264 THE FECES. uted to the presence of any particular micro-organism, but that the "affection is the result of the activity of a number of varie- ties of bacteria, some of which belong to well-known species and are of ordinary occurrence and wide distribution, the most impor- tant being the streptococcus and Proteus vulgaris." He also found tha± in the colon the Bacillus lactis aerogenes occurs in greater num- ber than in the normal intestine, and that it may even predominate over the Bacillus eoli communis. Among other forms of bacteria which occur frequently and in great abundance are small, short, faintly staining bacilli ; long, very slender bacilli ; large bacilli with pointed ends, and small, faintly staining spirilla. Dysentery. — This is an infectious disease, and may be caused by several varieties of bacteria, such as the bacillus of Shiga, the Bacillus pyocyaneus, and others. The stools during the first few days are irregular. A moderate diarrhoea then sets in ; the stools are thin, but still feculent, and number five or six per diem. After several days the diarrhoea increases and the stools assume a definite char- acter, numbering from ten to twenty or even fifty or sixty in the twenty-four hours. At the same time they become scanty in amount, usually not exceeding 10 or 15 grammes at a time. They are now sero-sanguineous in character, and in them may be found pieces of necrotic tissue. Microscopically, blood-corpuscles, particles of mucus, pus-corpuscles, and numerous bacteria are seen. According to the preponderance of blood, pus, mucus, etc., the stools are termed sanguineous, sero-sanguineous, putrid, mucoid, etc. Shreds of mucus, resembling frogs' eggs or kernels of tapioca, which are, in all proba- bility, casts of follicles, are also found. Typical dysenteric stools do not, as a rule, emit a marked odor, but those of the gangrenous form are very offensive. Amoebic Dysentery. — This form of dysentery is especially inter- esting, not so much on account of its prevalence, however, as from the importance attaching to an early diagnosis, since successful treatment is altogether dependent thereupon, and differs materially from that employed in other forms. The number of stools may vary within very wide limits — ^from six to twenty or even thirty in the twenty-four hours. They may be wholly mucoid, streaked here and there with pus, and presenting a few grayish threads. Others seem to be made up of a greenish, pultaceous mass, in which at times large greenish, irregular sloughs are observed. Such stools are usually slight in amount. Occasion- ally large brownish liquid evacuations are seen, in which small grayish-white masses occur, imbedded in blood-stained mucus. Such stools contain the diagnostic amoebae most abundantly. For a satisfactory examination the bed-pan should be well warmed and brought to the laboratory iinmerJiatdy for examination. If this is impractical, some of the material may be deposited in a suitable MECONIUM. 265 receptacle, and the small, grayish-white masses placed upon a warmed slide, if a warm stage is not at hand. One preparation after another must now be carefully looked over for actively mov- ing amoebse, or for amoeba-like bodies which exhibit definite move- ments (for a description of these parasites see page 233). In addition to the amoebse, other animal parasites may also be met with, such as the Trichomonas intestinalis, which is at times present in very large numbers. Red blood-corpuscles in greater or less abundance, numerous pus- corpuscles, more or less degenerated cylindrical epithelial cells, Charcot-Leyden crystals, bacteria of all kinds, and even large pieces of necrotic tissue may be found. Cholera Asiatica. — In this disease the stools are very numerous, being at first feculent, but soon becoming rice-water-like. As large a quantity as 200 grammes may be passed at each evacuation. The stools are colorless, almost odorless, watery, and on standing a finely granular, grayish-white sediment may be seen to form at the bottom. The reaction is neutral or alkaline. They contain only 0.5 per cent, of solids, a little serum-albumin, and a large amount of sodium chloride. In severe cases blood is present in variable amount. Microscopically, epithelial cells, triple phosphate crystals, and numer- ous micro-organisms are found. Of the latter, the comma-bacillus is, of course, the most important (see page 252). Typhoid Fever. — Typhoid . stools are usually described as re- sembling pea-soup both in consistence and color. Their odor is generally highly offensive and characteristic. They contain a large amount of biliary coloring-matter and have almost always an alka- line reaction. Microscopically, many bile-stained epithelial cells, some leucocytes, many triple phosphate crystals, and an enormous number of micro-organisms, especially the Clostridium butyricum of Nothnagel and Eberth's bacillus, are found. Later on, they may assume the appearance of ulcerative stools and become almost black, owing to the presence of blood. MECONIUM. By meconium are meant those masses which are first excreted from the bowel after birth. It is a thick, tenacious, greenish-brown mate- rial, which has accumulated during the intra-uterine life of the infant. Microscopically, a few cylindrical epithelial cells, a few fat-droplets, numerous cholesterin-crystals, bilirubin-crystals, and lanugo-hairs are found. Micro-organisms are absent, but soon after suckling has com- menced they appear in abundance. The most important of those which are then constantly present are the Bacillus lactis aerogenes, which predominates in the small intestine, and the Bacillus coli 266 ■ THE FECES. communis, which is found more particularly in the large intestine. Both have already been described (see page 256). In addition to these, the Proteus vulgaris, Streptococcus coli brevis, Micrococcus ovalis, tetragencoccus, Torula cerevisise, Torula rubra, and a few less important micro-organisms have been found. Chemically, meconium contains bilirubin in considerable amount (recognizable by Gmelin's reaction), biliary acids, fatty acids, chlo- rides, sulphates, phosphates of the alkalies and their earths. It does not contain urobilin, glycogen, peptones, lactic acid, tyrosin, or leucin. An idea may be formed of its composition from the following analysis of Zweifel :' Water 79.8-80.5 per cent. Solids 19.5-20.2 " Mineral matter 0.978 " Cholesterin 0.797 " Fats 0.772 " ' C. E. Simon, Physiological Chemistry, Lea Bros. & Co., Phila., 1901. CHAPTEK V. THE NASAL SECRETION. In the nasal secretion, which normally is small in amount, trans- parent, colorless, odorless, tenacious, and of a slightly saline taste, pavement-epithelial cells in large numbers, ciliated epithelial cells, as well as some leucocytes and an enormous number of micro-organisms, are found (Fig. 66). Its reaction is alkaline. Fig. 66. Bpithelial cells and mucous corpuscles found in the nasal secretion. In acute* coryza the amount is diminished at first, but soon a very copious secretion occurs, which contains numerous epithelial cells and micro-organisms. When complicated with an ulcerative condi- tion pus is observed in considerable amount. Occasionally, as in cases of traumatism, cerebral tumors, etc., cerebrospinal fluid is discharged through the nose, aud may be recognized by the fact that it is free from albumin and contains a substance which reduces Fehling's solution. Of pathogenic organisms, the tubercle bacillus and the bacillus of glanders may occur in ulcerative diseases of the nose, their presence indicating the existence of the corresponding affection. In ozsena a large diplococcus has been described by Lowenberg, which is said to be characteristic of the disease. Oidium albicans has been observed in rare cases. The Meningococcus intracellularis of Weichselbaum, which is now quite generally regarded as the cause of epidemic cerebrospinal meningitis, has also been demonstrated in the nasal secretion of healthy individuals. This fact helps to explain the origin of those cases of meningitis which develop after injuries to the skull. 267 268 THE NASAL SECRETION. Ascarides and other entozoa have also been found. Charcot- Leyden crystals (see page 291) have been observed in the nasal secretion in cases of bronchial asthma and in connection with nasal polypi. Their presence is usually accompanied by the simul- taneous occurrence of eosinophilic leucocytes. Literature. — Iteimann, Baumgarteu's Jahresber., 1888, vol. iil. p. 417. Loweuberg, Deutsch. med. Woch., 1885, vol. xi. p. 6, and 1886,, vol. xil. p. 446. Tost, Ibid., p. 161. Gerber u. Podack, Deutsch. Arch. f. klin. Med., 1895, vol. liv. p. 262. Leyden, Deatsoh. med. Woch., 1891, vol. xvii. p. 1085. Sticker, Zeit. f. klin. Med., 1888, vol. xiv. p. 81. Nothnagel, Wien. med. Blatter, 1888, Nos. 6, 7, 8. CHAPTER VI. THE SPUTUM. GENERAL TECHNIQUE. The sputum should be collected in receptacles so constructed as to permit of their complete and easy disinfection. The paper spit- cups (Fig. 67) which have been introduced within late years are admirably adapted to this purpose, as they may be destroyed imme- diately after use. Fig. 67. Sanitary spit-cups. When working with sputa which are hnown or suspected to he of tubercular origin, the greatest care should be exercised to keep the expec- toration from drying and becoming disseminated in the air. Negligence in this respect may result in the most serious consequences. The macroscopical examination of sputa is most conveniently carried out by placing small portions of the material upon a plate of ordinary window-glass, of suitable size, which has been painted black upon its lower surface, and covering the same with a second, smaller plate. If it is desired to examine individual constituents which have been discovered in this manner, the upper plate is slid off until the particle in question is uncovered, when it may be removed to a microscopical slide and examined under a higher power. It is also very convenient to have a portion of the laboratory table painted black, when unstained plates of glass may be utilized. If these measure about 15 by 15 cm. and 10 by 10 cm., respectively, fairly large quantities of sputum may be examined in situ with a low power. 269 270 THE SPUTUM. GENERAL CHARACTERISTICS OF SPUTA. Amount. — ^The amount of sputum expectorated in the twenty- four hours varies within wide limits, depending largely upon the nature of the disease. Thus, only a few cubic centimeters may be eliminated, or the amount may reach 600 to 1000 c.c, and even more. Very large quantities are expectorated in cases of pulmonary hemorrhage and oedema of the lungs, also following the perforation of accumulations of pus from the thoracic or abdominal cavities into the respiratory passages ; furthermore, in cases in which large vomicae of tubercular or gangrenous origin exist, and finally in cases of abscess of the lung, bronchiectasis, and even in simple bronchial blennorrhcea. In incipient phthisis, acute bronchitis, and in the first and second stages of pneumonia, on the other hand, the amount is usually small. In private practice, as well as in hospital work, an idea should always be formed of the amount expectorated in the twenty-four hours, especially in cases in which this is abundant. It is apparent that a copious and long-continued expectoration cannot continue without exerting very detrimental effects upon the patient's general nutrition ; in cases of pulmonary phthisis, for example, Renk has shown that 3.8 per cent, of all nitrogen eliminated in such cases is removed in this manner. Lenz in his recent experiments found even 5 per cent. Consistence. — The consistence of the sputum corresponds, in a general way at least, to its amount, and may vary from a liquid to a highly tenacious state. The cause of the tenacity of the sputum is but imperfectly understood. The mucin present does not appear to be the most important factor, as it has been observed to occur in diminished amount in pneumonic sputa, which are noted for their high degree of tenacity. Kossel ^ has suggested that the phenomenon may be due to the presence of nucleins or nuclein derivatives, while others again refer it to the presence of abnormal albuminous bodies of unknown character. However this may be, sputa are not infrequently seen where it is possible to invert the cup without losing a drop of its contents. This is observed especially in cases of acute croupous pneumonia up to the time of the crisis, pro- viding that a catarrh of the bronchi does not exist at the same time. It is noted, furthermore, immediately after an attack of acute bronchial asthma, and also in the initial stage of acute bronchitis. In cases of oedema of the lungs, on the other hand, the sputa are liquid and present the general characteristics of blood-serum, being covered, like all albuminous liquids when brought into contact with the air, by a frothy surface-layer. The sputa observed in cases of acute pulmonary gangrene, pulmonary abscess, putrid bronchitis, and ' Kossel, Zeit. f. klin. Med., 1888, vol. xiii. p. 152. GENERAL CHARACTERISTICS OF SPUTA. 271 following perforation into the lungs of an empyema or an accumula- tion of pus situated beneath the diaphragm, are fluid and consist of pure pus. Color. — The color of the sputa may vary greatly. They may be perfectly clear and transparent, gray, yellow, green, red, brown, and even black. Purely mucoid expectoration is almost transparent and colorless, as is also the sputum of pulmonary oedema when not mixed with blood or pus. The larger the number of leucocytes the more opaque does the sputum become, assuming at first a white, then a yellow, and finally a greenish color, the two latter colors being usually indicative of the presence of pus. Green sputa, however, may also be observed when bile-pigment has become admixed with the sputa, as in cases of perfo- ration of a liver-abscess into the lung. Green sputa may also be observed in cases of jaundice, and especially in pneumonia when accompanied by icterus. In cases of amoebic liver-abscess with perforation into the lung the sputa present a color resembling anchovy sauce, which is very characteristic. In one case I recog- nized the nature of the disease by simple inspection of the sputa.^ The inhalation of particles of carbon gives the sputum a grayish or even a black color ; the same or an ochre-yellow or red color is observed in cases of siderosis. A red color is usually indicative of the presence of blood, the in- tensity of the shade depending upon the character of the disease. It is seen especially after the formation of cavities, in caseous pneu- monia, in incipient phthisis, heart-disease, etc. In general, it may be said that a clear, bright-red color indicates an arterial, a dark- red or bluish-red a venous origin of the hemorrhage. The exact shade will depend upon the length of time that the blood, no matter what its origin may be, has remained in the lungs. In pulmonary gangrene a dirty brownish-red color is observed, owing to the pres- ence of methsemoglobin, and, to some extent also, of hsematin. Quite characteristic is a chocolate color, which is observed when- a croupous pneumonia terminates in necrosis and gangrene. Equally characteristic is the rusty and prune-colored expectoration seen in cases of pneumonia. Occasionally a breadcrust-brown color is observed in cases of gangrene and abscess of the lung, which is quite characteristic, the color being due to the presence of hsematoidin or bilirubin. Rust-colored punctate or striped sputa, moreover, are said to be diagnostic of brown induration of the lung. Odor.. — Most sputa are odorless. Under certain conditions, how- ever, there may be a very marked odor. In cases of pulmonary gangrene or putrid bronchitis the odor is of a kind never to be for- gotten, the stench, indeed, being frightful. A somewhat similar, 1 See Johns Hopkins Hosp. Bull., November, 1890. 272 THE SPUTUM. slightly sweetish odor is observed in certain cases in which putre- factive organisms have entered the lungs, and there exert their action upon the accumulated sputa, in the absence of gangrene, as in cases of bronchiectasis, perforating empyema, and where ulcerative proc- esses are taking place in the lungs, whether these be of tubercular origin or not. An odor like that of old cheese is occasioDally observed in cases of perforating empyema ; under such conditions tyrosin is usually found. This body, however, has nothing to do with the odor of the sputa ; both factors are merely indicative of certain putrefactive changes going on in the lungs. According to Leyden, the occurrence of tyrosin in sputa is usually indicative of the perforation of an old accumulation of pus into the lungs. Specific Gravity. — The specific gravity of sputa varies within wide limits ; mucous sputa have a specific gravity of 1.004 to 1.008, purulent sputa one of 1.015 to 1.026, and serous sputa one of 1.037 or more. Configuration of Sputa. — As a general rule, the following forms of sputa, which may be termed pure sputa, present a homogeneous appearance : Mutoid sputa, 1 Purulent sputa, I -q- Serous sputa, \ Homogeneous sputa, Sanguineous sputa, J with one exception, perhaps — the typically rusty sputa of croupous pneumonia ; while mixtures of any two or three of these may be classed as heterogeneous sputa : Mucopurulent sputa, Mucoserous sputa, Serosanguineous sputa, Sanguino-mucopurulent sputa. Heterogeneous sputa. The so-called sputum crudum of the first stage of acute bronchitis may be regarded as an example of a purely mucoid sputum. A purely purulent sputum is usually indicative of the perforation of an empyema or any other accumulation of pus into the lungs or bronchi, of pulmonary abscess, or of bronchial blennorrhoea. A purely serous sputum is found in cases of pulmonary oedema, and a purely hemor- rhagic sputum in cases of severe pulmonary hemorrhage. Of the heterogeneous sputa, the most important are the so-called nummular sputa of the second and third stages of phthisis. These are characterized by the fact that when thrown or expectorated into water they sink to the bottom, and there form coin-like disks, from which property they have received their name. Such sputa are mucopurulent in character, and contain a focus of almost pure pus imbedded in a more or less homogeneous mass of mucus. Quite different from these are the so-called sputa globosa of the ancients, which consist of fairly dense, roundish, grayish-white masses ; they MICROSCOPICAL CONSTITUENTS OF SPUTUM. 273 are secreted in old cavities which have become lined with a granu- lation-membrane. Very important is the presence of small, cheesy particles, which are occasionally found at the bottom of the spit-cup. They vary in size from that of a millet-seed to that of a pea, and are observed espe- cially in the second and third stages of phthisis. Usually they con- tain tubercle bacilli in large numbers, and frequently also elastic tissue. Not to be confounded with these, are certain small, caseous masses which are at times expectorated by perfectly normal indi- viduals, and also by patients suffering from acute tonsillitis, ozsena, etc., and which probably come from the tonsils or mucous cysts. Formerly they were regarded as tubercles, and in hypochondriac individuals their expectoration may cause a great deal of anxiety. They are quite readily distinguished from the true caseous masses expectorated by phthisical individuals by the following character- istics : as a rule, they are expectorated unaccompanied by pus or even by mucus ; rubbed between the fingers they emit an extremely offensive odor, which is referable to the presence of fatty acids ; an examination for tubercle bacilli, moreover, will prove entirely nega- tive. Quite characteristic, furthermore, is the peculiar, finely floc- culent, granular appearance of the sputa seen after perforation of an empyema Into the lungs through a small aperture, which is not followed by pneumothorax. Occasionally, as in putrid bronchitis, and gangrene of the lungs, and also in chronic bronchitis, ultimately leading to the formation of bronchiectatic cavities, an exquisite sedimentation is observed. Such sputa when collected in a conical glass present three distinct zones : the one at the bottom contains the cellular elements of the sputum, the second the pus-serum ; and the third or superficial layer consists of mucus and contains many air-bubbles. MICROSCOPICAL CONSTITUENTS OF SPUTUM. Elastic Tissue. — Of macroscopical constituents which may be observed in sputa, there may be mentioned, first of all, the occur- rence of threads of elastic tissue and pulmonary parenchyma, which are seen in cases of phthisis, pulmonary abscess, and gangrene. As their ultimate recognition, however, largely depends upon a micro- scopical examination, this subject will be considered later on. Fibrinous Casts. — Fibrinous casts are observed especially in cases of croupous pneumonia (Fig. 68), immediately before or after resolution has taken place. They are seen also in cases of so-called fibrinous bronchitis (Fig. 69), and in diphtheria, when the membrane has extended into the finest ramifications of the bronchi. These casts may vary in size from 12 cm. in length by several millimeters in thickness to small fragments which measure only from 0.5 to 3 18 274 THE SPUTUM. Fig. 68. Fibrinous coagulum from a case of croupous pneumonia, (Bizzozeeo.) Fig. 69. Fibrinous coagulum from a case of plastic bronchitis, (v. Jaksoh.) cm. in length. The fibrinous casts observed in cases of pneumonia, MICROSCOPICAL CONSTITUENTS OF SPUTUM. 275 usually from the third to the seventh day, are of the latter size or even smaller, being derived from the ultimate twigs of the finest bronchioles. Those found in the rather rare disease, fibrinous bron- chitis, stand between these two in size, being casts of the smaller and medium-sized bronchi. Attention is usually attracted to the presence of such casts by their white color ; often, however, they are yellow- ish brown or reddish yellow, owing to the presence of blood-coloring matter which has become deposited upon the easts ; at other times they are enveloped in mucus, when their recognition may become quite difficult. Such casts, when examined carefully, will be seen to branch dichotomously, and to contain a cavity in their larger portion, while the finer branches appear to be solid. Microscopically, they may be shown_to consist of a large number of fibres, which are arranged longitudinally or in a net-like manner, and contain blood- corpuscles and epithelial cells in their meshes. When treated with Weigert's fibrin-stain they are beautifully resolved. Charcot-Leyden crystals have at times been observed in these formations. Whenever it is desired to examine sputa for casts it is best to pick out particles that look promising, upon a dark or light surface, and then to shake them out in water. For such purposes Kronig's sputum-plate can be recommended. Curschmann's Spirals.^ — Quite distinct from the formations just described are the so-called spirals of Curschmann, which are observed especially in cases of true bronchial asthma, but occur also in chronic bronchitis, and even in croupous pneumonia. Upon careful examination they will be seen to consist of thick, yellowish- white masses, which exhibit a spirally twisted appearance, and are characterized, moreover, by their more solid consistence and light color. On microscopical examination they are seen to be composed of a spirally twisted network of extremely delicate fibrils, containing epithelial cells and numerous leucocytes ; the latter are almost all of the eosinophilic variety.^ Usually, but not invariably, Charcot- Leyden crystals also are seen.' The spirally twisted mass is found to be wound around a central, very light and clear thread, which usually has a zigzag course (Fig. 70). Other formations, probably mere varieties of those just described, have also been observed, in which the central thread is absent or in which the spiral arrangement is deficient. The spiral form, how- ever, with the central thread, must be considered as the most char- acteristic. Their length and breadth may vary a great deal, but rarely exceed 1 to 1.5 cm. Their occurrence seems always to indi- cate a desquamative catarrh of the bronchi and alveoli, but practi- ^ Leyden, Virchow's Archiv, 1872, toI. liv. p. 328. Curschmann, Deutsch. Arch. f. Win. Med., 1883, vol. xxxii. p. 1, and vol. xxxvi. p. 578. v. Jaksch, Centralbl. f. kUn. Med., 1883, vol iv. p. 497. ^ Schmidt, Zeit. f. klin. Med., 1892, vol. xx. p. 92. v. Noorden, Ibid., p. 98. ' Leyden, loo. cit. 276 THE SPUTUM. cally nothing is known concerning their formation. If in a given case the diagnosis rests between true bronchial and what may be termed reflex asthma, the presence of these formations points to the existence of the former disease. Chemically, the spirally wound Fig. 70. A Curschmann spiral from a case of true bronchial asthma. mass seems to consist of a mucinous substance, while the central thread is possibly of fibrinous origin. Charcot-Leyden crystals (Fig. 71), which are usually absent at the beginning of an attack of asthma, at which time only the spirals are observed, may be seen to develop from the spirals when these Fig. 71. Fig. 72. \^ % ^/ fh^. c& 50 / « ^"^ / "^y^ 9s. Charcot-Leyden crystals. (Scheube.) Wall of a hydatid cast, showing the laminated structure; not mag- nified. (Davaine.) are kept for several days. They will be considered later on in studying the chemistry of the sputum. Echinococcus Membranes. — Echinococcus membranes come from a perforating cyst of the liver, kidney, or lung. They consti- tute rather thick, and at the same time tough, pieces of membrane (Fig. 72) ; occasionally entire sacs are seen, of the color of white porcelain, in sections of which it is possible to make out a fibrillated structure. The disease is rare in this country. PLATE Xlir. ■■9 ■Si. "Viy ,:,i.m .-.■.iT^ ,■'**&&■ '■^!!ii»r ? f •. >' m IfUl .,^. ^^^'^, ■'ff. -# Sputum from a case of Bronchial Asthma, showing large num- bers of Eosinophilic Leucocytes and Free Granules. It will be noted that the leucocytes are all mononuclear. (Eye-ptece r, objective i-8, Bausch and I.omb.) MIOBOSGOPIOAL EXAMINATION. 277 Concretions. — Still rarer is the expectoration of concretions which have formed in dilated portions of the bronchi or in tubercular cavities, or of calcified bronchial glands that have found their way into the lungs. Curious examples of the occurrence of such con- cretions have been reported. Andral thus cites a case of phthisis in which within eight months as many as 200 stones were expec- torated, and Portal mentions a case in which 500 were thus expelled.' Foreign Bodies. — Foreign bodies which have accidentally entered the air-passages and have remained there for a long time may also be found in the sputum. Heyfelder mentions a case in which a man coughed up a wooden cigar-holder with pus and blood after eleven and a half years. MICROSCOPICAL EXAMINATION. Under this heading it is necessary to consider leucocytes, red blood-corpuscles, epithelial cells, elastic fibres, corpora amylacea, parasites, and crystals. Leucocytes. — Leucocytes, usually polynuclear in character, are found in every sputum in considerable numbers, imbedded in a homogeneous, more or less tenacious material. At times they appear very granular, containing fat-droplets, or granules of pigment, such as carbon or hematoidin. Their number varies considerably, being naturally greatest in cases of perforating abscess, empyema, putrid bronchitis, etc. While the leucocytes which usually are found in the sputum are of the neutrophilic variety, eosinophiles may also be observed, and especially in asthmatic sputa, in which they often predominate. Free eosinophilic granules are then also seen, and I have repeatedly observed specimens in which the spirals (see above) were literally covered with these granules (Plate XIII.). The presence of eosino- philic leucocytes is, however, not characteristic of the sputa of bronchial asthma, as they may be met with in other diseases as well. Teichmiiller has pointed out that they are present in a large percentage of tubercular cases, and may be found months before tubercle bacilli can be demonstrated. He regards their occurrence as evidence of a defensive struggle on the part of the body, which is most evident in fairly strong individuals. In recovery a gradual increase in their number is always noticeable, and a diminution, Teichmiiller thinks, is indicative of a relapse, or, if the diminution occurs rapidly, of florid consumption. These statements, however, lack confirmation and are probably too dogmatic. The same observer has also described an "eosinophilic" bronchitis, which differs from other forms of the disease in the abundance of eosinophilic cells which are encountered. The sputum in such cases is described ' L. W. Atlee, " Bronchial Concretions," Am. Jour. Med. Sci., 1901, vol. cxxii. p. 49. 278 THE SPUTUM. as transparent, mucoid, and loose, with yellow purulent admixtures. It is said to be markedly different from the tough, thick sputa of bronchial asthma. Typical spirals are absent, but rudimentary forms may be encountered. Charcot-Leyden crystals are present.' Basophilic leucocytes have also been observed in the sputa. Red Blood-corpuscles. — The presence of red blood-corpuscles in small numbers does not, by any means, indicate serious pulmonary or cardiac disease, as they may be found in almost any sputum, and especially in that of individuals who smoke much or live in a smoky atmosphere ; they are, without doubt, derived from the catarrhally inflamed bronchial or tracheal mucosa. Whenever they occur in large numbers, however, their presence becomes important. They may be observed in acute bronchitis, pneumonia, oedema of the lungs, bronchiectasis, abscess, gangrene — in fact, in all pulmonary- diseases. Their occurrence is most important in phthisis, and is, in fact, one of the most constant symptoms of the disease. The form of the red corpuscles will depend upon the length of time they have remained in the lungs, and all gradations from the typical red corpuscle to its shadow, or even fragments, may thus be observed. In pneumonia the microscopical examination may at times be disappointing, the appearance of the sputum suggesting that red corpuscles in large numbers are present, while, as a matter of fact, they are almost all destroyed, the color being due to altered pigment. It may even be necessary at times to depend upon chemi- cal methods to clear up any doubt as to the source of the color of the sputum. It should always be remembered that the presence of blood-pigment is not always indicated by a red color, but that it may also assume a golden-yellow or even a greenish tinge, owing to cer- tain chemical changes which have taken place. The golden-yellow and the grass-green sputa observed in cases of pneumonia during convalescence belong to this class. To demonstrate the presence of traces of blood in the sputum, Donogany's method, or that of Miiller and Weber, may be con- veniently employed. With the former method the sputum is first boiled with a 20 per cent, solution of sodium hydrate (see page 199). Epithelial Cells. — Epithelial cells may also be observed in the sputum. While a great deal of information might be expected from their presence from a diagnostic point of view, as accurately indi- cating the parts of the respiratory tract attacked by disease, the data obtained are practically of little value. Cylindrical epithelial cells, providing they do not come from the nose, indicate in a general way an inflammatory condition of the ' Tcichmiiller, "Die eosinophile Bronchitis," Deutsch. Arch. f. klin. Med., vol. Ixiii. Hefte 5, 6. See, also, K. Sohonbrod, Ueber don gegenwartigen Stand der Beurthei- lung der eosinophilen Zellen im Blute und im Sputum, Inaug. Diss., Erlangen, 1895. A. Hein, Ueber das Vorliommeii eosinophiler Zellen im Sputum, Inaug. Diss., Erlan- gen, 1894. MICROSCOPICAL EXAMINATION. 279 lower laiynx, trachea, or bronchi. They are not of much impor- tance, however, as their form is usually so much altered that it is often difficult to recognize them ; they may thus become polyhedral, cuboidal, or even round, and can then hardly be distinguished from leucocytes. Actively moving cilia can be found only in perfectly fresh sputa, immediately after being expectorated, if ciliated epi- thelial cells can be definitely recognized in a sputum, it may be in- ferred that we are dealing with a pathological condition of an acute nature, providing, of course, they did not come from the nose. Formerly much importance was attached to the so-called alveolar epithelial cells (Fig. 73) as an aid in diagnosis. Buhl thus imagined these, particularly when undergoing fatty or myelin degeneration, to be absolutely pathognomonic of pulmonary disease, and especially of that form of pneumonia which has been termed essential idio- pathic desquamative pneumonia. Bizzozero, however, as well as Fig. 73. Epithelium, leucocytes, and crystals of the sputum. (Eye-piece III., ohjective 8 A, Eeich- ert.) o, a', a", alveolar epithelium ; 6, myelin forms ; c, ciliated epithelium ; d, crystals of calcium carbonate : e, hsematoidin crystals and masses ; /,/,/, white blood-corpuscles ; g, red blood-corpuscles ; ft, squamous epithelium, (v. Jaksch.) others, has shown that these cells not only occur in almost every known pulmonary disease, but that they are present also in the so- called "normal" expectoration which at times is obtained upon making a very forcible expiration. Bizzozero ' describes these cells as round, oval, or polygonal bodies, varying in size from 20 (i to 50 [i. They may contain one, two, or three oval nuclei, which are rather small and provided with nucleoli. Usually the latter are hidden beneath numerous granules. Some of these granules are albuminous, but most of them are either pigment- granules, fatty granules, or myelin granules. The myelin granules were first discovered by Virchow ^ in 1854, and termed myelin gran- ules on account of their resemblance to mashed nerve-matter. They are distinguished from the other forms by their clear, pale, color- ' Bizzozero, Microscopie clinique, 2d ed. Franfaise, Paris, 1885. ' Virchow, Virchow's ArcMv, 1854, vol. vi. p. 562. 280 THE SPUTUM. less appearance, and the fact that at times fine concentric striations can be detected. These forms may be round, but more often they are irregular. At times fatty, myelin, and pigment-granules may be seen in one and the same cell. Possibly they are derived from the pulmonary alveoli, but this is still an open question. Chemi- cally, the myelin droplets have been shown to contain a considerable amount of protagon, besides traces of lecithin and cholesterin.' Liver-cells may at times be observed in the sputa in cases of liver- abscess, and are easily recognized by their characteristic form. Elastic Tissue. — Much more important from a clinical stand- point are the elastic fibres and shreds of elastic tissue which may be found in sputa. They vary much in length and breadth, and are provided with a double, undulating contour ; they are usually curled at their ends. Very often they exhibit an alveolar arrange- ment (Fig. 74), which at once determines their origin. Fig. 74. Elastic fibres in tlie sputum. (Eye-piece III., objective 8 A, Eeichert.) (v. Jaesch.) Whenever present, elastic tissue is an absolute indication that a destructive process is going on in the lungs. It is found in cases of abscess of the lungs, bronchiectasis, occasionally in pneumonia, and, most important of all, in phthisis. In gangrene of the lung elastic tissue is usually not found ; this is probably owing to its destruction by a ferment, as suggested by Traube. In every case it is necessary to determine whether the elastic tissue may not be owing to the presence of animal food in the sputum, and it may, hence, be stated as a rule that it can only be regarded as absolutely^ characteristic when showing the alveolar arrangement. In order to demonstrate the presence of elastic tissue in the 1 A. Schmidt, "Ueber Herkunft u. ohetn. Natur d. Myellnformen d. Sputums," Berlin, klin. Wooh., 1898, p. 73. See, also, Zoja, Maly'a Jahresberiohte, vol. xxiv. p. 694. MICROSCOPICAL EXAMINATION. 281 sputum, it is necessary to examine large quantities with a moder- ately low power, and best after the addition of a strong solution of sodium hydrate. The sputum may also be boiled with a 10 per cent, solution of the reagent, an equal volume being added ; after dilution with four times its volume of water it is allowed to settle for twenty-four hours. The centrifugal machine will here be found of great assistance. The following method, in use at the Johns Hopkins Hospital, is most convenient : a small amount of the thick, purulent portion of the sputum is pressed out into a thin layer between two pieces of plain window-glass, 15 by 15 cm. and 10 by 10 cm. The particles of elastic tissue appear on a black background as grayish-yellow spots, and can be examined in situ under a low power. Or, the upper piece of glass is slid off tUl the piece of tissue is uncovered, when it is picked out and examined on a slide, first with a low and then with a higher power. At first there will be some difficulty in distinguishing with the naked eye between elastic fibres and particles of bread, or milk globules, or collections of epithelium and debris, but with practice such mistakes are rarely made, and the microscope always reveals the difference. To stain elastic tissue, Michaelis suggests the following method : suspected bits of sputum are spread upon a slide in a thin layer, dried, and then placed for one-half hour in a jar containing Weigert's solu- tion. The specimen is then washed with water, decolorized in acid alcohol (containing 3 per cent, of hydrochloric acid), dried, covered with a thin layer of oil of cedar, and examined without a cover-glass with a low power ; the elastic fibres are stained a dark violet. Wdgerfs Elastie Tissue Stain. — This is prepared as follows : 200 c.c. of an aqueous solution of fuchsin and resorcin, containing 1 and 2 per cent, of the ingredients, respectively, are boiled in a porcelain dish. When the boiling-point is reached 25 c.c. of liquor ferri sesquichloridi (Ph. G. III.) are added. "While stirring the solution is boiled for from two to five minutes longer. It is then allowed to cool ; the precipitate is collected on a filter, dried, and boiled in 200 c.c. of 94 per cent, alcohol while stirring. On cool- ing, alcohol is added to the 200 c.c. mark, when the solution is treated with 4 c.c. of hydrochloric acid, and is ready for use. Animal Parasites. Portions of echinococcus cysts, viz., pieces of membrane (Fig. 73) and booklets (Fig. 75), are occasionally seen when the parasite has lodged in the lungs or in the neighboring organs. The disease, however, is exceedingly rare in this country. The adult parasite (Fig. 76), Taenia echinococcus (v. Siebold), is found in the intestinal canal of the dog, the dingo, the jackal, the 282 THE SPUTUM. wolf, etc. The larval form, Eohinococeus polymorphus, develops in cattle, sheep, and swine, and is also found in man. The parasite, in fact, is the most dangerous animal parasite which is encountered in the human being. In America it is at present not common. Fig. 75. <3s=- HooWets from Tsenia echlnocoocus. X 350. If the eggs of the parasite are introduced into the digestive tract of man, the embryos may make their way into the lungs, liver, or other organs, and there give rise to the formation of cysts, which are often of enormous size. The body of the adult animal is from Human eohinococeus. (From Pinlayson, after Davaine.) A, a grouj) of echinooocci, still adiiering to the germinal memhrane by their pedicles. X 40. J5, an echinococcuB with head invaginated in the body, X 107. C, the same compressed, showing suckers and hooks of the retracted head. D, eohinococeus with head protruded. E, crown of hooks, showing the two circles. X 350. 4 to 5 mm. long, with only 3 or 4 segments, the largest of which may measure 0.6 mm. in length by 2 mm. in breadth. On the head there are from 28 to 50 booklets (see Fig. 76).' Trichomonades have at times been observed in cases of gangrene 1 Hydatid disease in man : Neisser, Die Echinococcen-Krankheit, 1877, Berlin. Davaine, Traits des Entozoairea et des Maladies vermineuses, Paris, 1877, 2d ed. MICROSCOPICAL EXAMINATION. 283 of the lung, and in the pus removed post mortem from lung-cavities. They are identical with the Trichomonas vaginalis of Donn6. Most important is the presence of the AmoBba coli, as the diag- nosis of hepatic abscess with perforation into the lung may be made in every instance in which this organism is encountered in the sputa (see Feces).' A form of pulmonary disease closely simulating phthisis is very common iu Japan, and has been shown to be referable to the pres- ence of a parasite in the lungs, the Distoma pulmonale, Balz : syn., Distoma Westermanni (Kerbert), Distoma Ringeri (Cobbold). The worm and its ova are found in the sputum. " The parasite is 8 to 10 mm. long, 5 to 6 mm. wide, of a club shape, rounded very markedly in front, less rounded posteriorly. The color during life is almost like that of earth-worms. The two sucking disks are nearly equal in size. The ova are brown, with a thin shell, lidded, 0.1 mm. long and 0.05 mm. wide." (Huber.) In this country the patasite has been found in the cat and in the dog ; in the human being one case at least, occurring in a Japanese student, has been reported.^ It is interesting to note that many Charcot-Leyden cryste,ls are at the same time found in the sputum.^ Mansoa found the ova of a species of Distoma hcematobium in the bloody expectoration of a Chinaman who had lived for some time on the island of Formosa. Vegetable Parasites. Pathogenic Organisms. — The Tubercle Bacillus. — The most im- portant vegetable parasite met with in the sputa is the baeiUus of tuberculosis. The history of the discovery of this organism, and the theories which were held before its pathogenic importance was estab- lished, cannot be considered here. Suffice it to say that the study of bacteriology has given no other discovery of equal importance from a clinical point of view. How primitive and wholly inadequate were the means formerly employed in making the diagnosis of this, the most formidable disease of modern times ! The presence or absence of elastic tissue in the sputa was practically all that physicians had to guide them beyond the history of the patient and the results of a physical examination. The demonstration of elastic tissue, however, as has been pointed out, merely indicates the existence of a destructive process in the lungs. Under such condi- tions it was of necessity impossible to diagnose tubercular disease in its incipiency. It is true that cases are occasionally observed in which tubercle bacilli are never present in the sputa, and are only discovered post mortem. Such cases, however, are extremely rare, ' C. E. Simon, Johns Hopkins Hosp. Bull., Nov., 1890. ' C. W. Stiles, "Distoma Westermanni," Johns Hopkins Hosp. Bull., 1894, p. 57. ' Braun, Die thierischen Parasiten, etc., Stuber, Wiirzburg, 1895. 284 THE SPUTUM. and do not in the least detract from the importance which attaches to careful and repeated examinations of the sputa in all doubtful cases. , From a macroscopical examination it is impossible to decide whether or not a particular sputum is of tubercular origin. At times a sputum may have a suspicious appearance, but it is never possible to speak with certainty from simple inspection, as a mucoid sputum may contain tubercle bacilli in large numbers, while a muco-purulent sputum may be entirely free from them, and vice versa. Reliance should, hence, only be placed upon a careful microscopical examina- tion. When found, their presence is, of course, pathognomonic. A negative result, however, does not exclude the existence of tuber- cular disease. The possibility that they may be altogether absent from the sputum has been mentioned. In some instances they may be present at times and absent at others. In all cases in which the existence of phthisis is suspected, it is imperative to make use of every device which may aid in its detection. In this connection, I wish to insist upon the method of "growing the bacilli," as it were, in the warm chamber for from twenty-four to forty-eight hours, and then re-examining the sputa in doubtful cases, as Nuttall ' demonstrated beyond a doubt that the tubercle bacillus will multiply in the sputum itself at a certain temperature. The value of this observation is obvious, and I have repeatedly been able to demonstrate their presence in this manner when it was impossible to detect them in the fresh sputum. The centrifugal machine in such cases is also useful and yields valuable results, the probabilities of finding the bacilli when present in small number being very much increased. If but few bacilli are present, the following procedure may also be employed : about 100 c.c. of sputum are boiled with double the amount of water, to which from six to eight drops of a 10 per cent, solution of sodium hydrate have been added, until a homogeneous solution has been obtained, water being added from time to time to allow for evaporation. The mixture is then centrifugated or set aside for twenty-four to forty-eight hours and examined for tubercle bacilli and elastic tissue. In the examination of tubercular sputa the fine caseous particles previously described (page 273) should be carefully sought for, as they contain the largest number of bacilli. In their absence reliance should be placed upon the examination of a large number of prepa- rations. If, notwithstanding the fact that all due precautions have been taken, no bacilli can be demonstrated in the sputum, and the clinical history and the physical signs are indefinite or negative, the proba- bilities are that we are dealing with a benign process. From an ' Nuttall, Johns Hopkins Hosp. Bull., 1891. MICROSCOPICAL EXAMINATION. 285 examination of the sputa alone in such cases it is utterly impossible to reach a definite conclusion. When the amount of sputum, more- over, is small and coatains but little pus, the absence of tubercle bacilli in doubtful cases is less suggestive of the absence of tuber- cular disease than in cases in which the sputum is more abundant and mucopurulent. Only two bacilli are likely to be mistaken for the tubercle ba- cillus, viz., the bacillus of leprosy and the smegma bacillus. All three are characterized by the difficulty with which they take up basic dyes, and the great tenacity with which these are retained when once stained, even upon treatment with mineral acids. This peculiarity has been quite generally referred to the presence of fat in the bacilli, but it appears from more recent researches that the chitin or chitinous substances in the bodies of the tubercle bacilli are at least primarily concerned in the reaction (Helbing '). Sata,^ moreover, has shown that other bacteria, such as the anthrax bacillus, the bacillus of glanders, the Staphylococcus aureus, etc., give a fat reaction which is as intense as that of the tubercle bacillus, while these organisms are not in the least resistant to the action of acids when stained. That confusion should arise in the differentiation between the tubercle bacillus and the baoUlus of leprosy is very unlikely. More important is the smegma baaiUus, which is now known to occur at times upon the tonsils, the tongue, and in the tartar of the teeth of perfectly healthy individuals. In sputum coming from the lungs it has been observed by Pappenheim,^ Frankel,* and others. To Pappenheim we are indebted for a method by which we are enabled to differentiate such cases from tuberculosis. This is essentially based upon the greater ease and rapidity with which the smegma bacillus is decolorized by means of fluorescein-alcohol, as compared with the tubercle bacillus. As the other methods which have hitherto been in use in the clinical laboratory do not permit of differentiation between the two organisms, I have given Pappenheim's method the first place, but have retained the others also. They may be emp- ployed as heretofore, unless special reasons exist for eliminating the smegma bacillus, the occurrence of which in the sputum must after all be regarded as a medical curiosity. In the examination of urinary deposits, however, in which the smegma bacillus is far more commonly seen, these older methods are not applicable (see Urine). Methods of Staining the Tubercle Bacillus. — 1. Fappen- ' C. Helbing, " Erklarnngsversuch f. d. specifische Farbbarkeit d. Tuberkelbacillen," Deutsch. med. Wooh., 1900, V. B. p. 133. ^Sata, "Ueber d. Fettbildung durch verschiedene Batterien," etc., Centralbl. f. allg. Path. u. path. Anat., 1900, Nos. 3, 4. ' A. Pappenheim, "Befund v. Smegmabacillen im mensehlichen Lungenauswurf." Berlin, klin. Woch., 1898, No. 37. *A. Prankel, "Einige Bemerkungen liber d. Vorkommen v. Smegmabacillen im Sputum," Ibid., 1898, p. 880. 286 THE SPUTUM. heim's Method} — A drop of the sputum — or, if the cheesy particles described above, are present, one of these — is spread in a thin layer between two cover-glasses. These are then»drawa apart, dried in the air, and fixed by being passed three times through the flame of a Bunsen burner or an alcohol lamp. Larger -quantities of the sputum may also be employed, and are spread upon slides and examined in the same manner, a drop of immersion oil being placed directly upon the dried and stained preparation. The speci- mens are covered with a few drops of carbol-fuchsin solution and heated to the boiling-point. The solution is composed of 1 part of fuchsin, 100 parts of a 5 per cent, solution of carbolic acid and 10 parts of absolute alcohol. The excess of the staining fluid is drained off, when the preparations are immersed from three to five times in Pappenheim's solution, care being taken to let the fluid drain off slowly after each immersion. The reagent consists of 1 part of corallin (rosolic acid) in 100 parts of absolute alcohol, to which methylene-blue is added to saturation. This mixture is further treated with 20 parts of glycerin, and is then ready for use. The specimens are finally washed in water, dried between filter-paper, and mounted in balsam or oil of cedar. A ^ oil immersion lens is very convenient, but not a necessity, as the organisms are seen quite readily with lower powers, such as Zeiss' DD, Leitz' 7, or Bausch and Lomb's \ or ^, with a correspondingly high eye-piece. 2. Gabett's Method. — The dried preparations are floated for two minutes upon the carbol-fuchsin solution described above, and are immediately transferred, without washing, to a solution composed of 2 parts of methylene-blue in 100 parts of a 25 per cent, solu- tion of sulphuric acid, in which they remain one minute. They are then washed in water and mounted. This method of staining is very convenient, and is the one most generally employed. The smegma bacillus, however, is also stained.^ 3. The Weigert-Ehrlich Method. — Dried specimens are prepared, and stained for twenty-four hours with a solution of fuchsin in anilin-water, by floating upon the surface. The staining fluid is prepared as follows : A small test-tube full of water is shaken with about twenty drops of pure anilin oil (1 : 20), and after standing for a few minutes fil- tered through a moistened filter. To this solution a few drops of a concentrated alcoholic solution of fuchsin or of methyl-violet are added until the mixture becomes slightly cloudy — i. e., until a metal- lic lustre is noted on the surface. After twenty-four hours the preparations are washed with water in order to remove an excess of staining fluid. They are then immersed for several seconds in a ' Pappenheim, loc. cit. ' Frankel, Berlin, klin. Woch., 1884, vol. xxi. p. 195 ; and Deutsch. med. Woch., 1887, vol, xvii. p, 552, PLATE XIV. ** :^y -'i I i / !iS'-- mfi V"'^ Tuberculous Sputum Stained by Gabbett's Method. The Turberele Bacilli are seen as Red Rods, all else is Stained Blue. (Abbott.) L bCHMIOT, FEC. The Diploeoecus Pneui-nonise, Stained with Methylene Blue and Fuchsin as a Counterslain. Taken from the Sputum of a Case of Acute Croupous Pneumonia. FIG. S. f^l i:>JS Heart-Disease Cells, showing Alveolar Epithelial Cells, Loaded Down with Granules MICROSCOPICAL EXAMINATION. 287 dilute solution of nitric or hydrochloric acid (1 : 6, 1 : 3, or 1 : 2), and washed again with water or with absolute alcohol. At this time the specimens should have a faintly red or violet color. They are then dried between layers of filter-paper or in the air, and mounted as usual. If it is desired to use a counter-stain, Bismarck-brown, vesuvin, or methylene-blue in watery solutions may be used for the purpose. Into such a solution the specimen is placed after treatment with nitric acid and washing in water. It remains for about two min- utes, and is then washed, dried, and mounted as above. 4. Ziehl-Neeken's Method. — A mixture of 90 parts of a 5 per cent, solution of carbolic acid and 10 parts of a concentrated alcoholic solution of fuchsin is used. The procedure is the same as that described under the Weigert-Ehrlich method. With both methods, however, it is unnecessary to stain the preparation for twenty-four hours, unless special accuracy is required, and, as a rule, it is suffi- cient to place a few drops of the staining fluid upon the cover-glass and to boil this for a few seconds over the free flame, when the specimen is further treated as described. In this manner excellent results may be obtained in a few minutes. Stained according to one of these methods, the bacilli appear as rods measuring about 3 ^ to 4 // in length by 0.3 /j. to 0.5 // in breadth (Plate XIV., Fig. 1). Usually they are not swollen at their extremities, but simply rounded ofi". They occur as homo- geneous rods or may present within their stained bodies small round or ovoid granules, placed end to end, which do not stain. They may also have a straight or a curved form, or the bacillus may appear to be doubled upon itself in the form of the letter S. The small hyaline bodies in the bacilli have been regarded as spores. The number of bacilli which may be found in a sputum varies greatly, and while in general it may be said that it is in direct ratio to the intensity of the disease, and may thus be considered as of some prognostic value, too much reliance should not be placed upon this statement, as in acute miliary tuberculosis, and in cases that have gone to the formation of cavities, the number may be very small or they may be altogether absent. In an incipient case, on the other hand, in a little mucoid sputum the number may be very large. Of the variations in number- and form of the tubercle bacilli during treatment with Koch's tuberculin it is unnecessary to speak at this place, as the prognostic significance attaching to such varia- tions is questionable. The Diplococcus Pneumonise. — In doubtful cases the sputum may be examined for the Diplococcus pneumonise, and it may be accepted at the present time that its presence in a given case, providing that the cUnical history and the physical signs point to a pneumonia, renders the diagnosis of acute croupous pneumonia very probable. Method. — Cover-glass specimens, prepared as indicated above, 288 THE SPUTUM. are placed for one or two minutes in a 1 per cent, solution of acetic acid ; they are then removed, the excess of acetic acid is drawn oflF by means of a pipette, when they are allowed to dry in the air ; they are subsequently placed for several seconds in saturated aniHn-water and gentian-violet solution, washed in water, and examined. Rod- shaped diplococci (Plate XIV., Fig. 2), surrounded by a capsule, which latter is considered the characteristic feature of this organ- ism, will be seen in cases of acute croupous pneumonia;' The bacillus of influenza has already been considered in Chapter I. (page 122). In the sputum it is frequently associated with pyo- genic cocci and pneumococci. In whooping-cough protozoa have been observed by Deichler and Kurloff; their observations have not been confirmed, however, and other observers attribute the disease to the presence of bacteria. Among these may be nientioned Affanasiew, Ritter, Czaplewski, Hensel, Koplik, and others. All these investigators claim to have isolated from the sputum of whooping-cough a micro-organism, which they regard as the cause of the disease. Whether or not Aifanasiew's bacillus is identical with Ritter's diplococcus and with the pole-bacillus of Czaplewski, Hensel, and Koplik^ is, however, not clear. Koplik's organism is extremely minute, measuring from 0.8 /i to 1.7 /^ in length by 0.3 /i to 0.4 ji in breadth. When stained with LoiHer's blue it has a finely punctate appearance,, like the diphtheria bacillus. In pure culture it is not decolorized by Gram's method. It is anaerobic as well as aerobic, and is apparently not motile. To isolate it from the sputum, it is best to obtain some of the grayish-white pellets which are expectorated during the con- vulsive stage. In these, small particles will be seen, resembling scales of dandruif. Such particles are isolated and planted first on hydrocele fluid, in order to obtain the crude culture. Later the organism may be grown in bouillon, on agar, gelatin, etc. On Loffler's serum a whitish growth is obtained which closely simulates that of the diphtheria bacillus. The organism is pathogenic for mice, particularly after intraperitoneal inoculation, but it does not produce whooping-cough in the lower animals. The Smegma Bacillus. — In a few isolated cases the smegma bacil- lus has been encountered in the sputum, and, as I have already stated, the same organism may normally be present in the saliva, the coating of the tongue, the tartar of the teeth, etc. Like the tubercle bacillus, it resists the decolorizing action of acids when once stained, and may hence be confounded with it unless special precautions are observed (page 285). iFrankel, Zeit. f. klin. Med., 1886, vol. ii. p. 437. Weichselbaum, Wien. nied. Wooh., 1886, vol. xxxix. pp. 1301, 1339, 1367. 2 E. Czaplewski u. E. Hensel, "Bacteriol. Untersuchungen bel Keuchhusten," Deutsch. med. Woch., 1897, p. 586. H. Koplik, " The Bacteriology of Pertussis," Johns Hopkins Hosp. Bull., 1898, p. 79. MIOBOSCOPICAL EXAMINATION. 289 Rabinowitch ^ recently succeeded in cultivating from the spu- tum of a case of pulmonary gangrene an organism which is either identical with the smegma bacillus or closely allied to it; she gives the following account of its cultural characteristics : on glycerin-agar, after twenty -four to forty-eight hours the organism forms grayish-white, lustrous colonies of the size of the head of a pin, which gradually coalesce to a whitish, cream-like coating. On further growth the lustre disappears, the surface appears dry, the coating becomes wrinkled and assumes a yellowish color. Still later, when kept at the temperature of the room it turns to a deep orange. The organism is non-motile. It occurs in the form of little rods, which in older cultures manifest a tendency to the formation of long threads. In gelatin stab-cultures small colonies appear along the line of the puncture, which are separated from each other. On the surface a thickish, white, lustrous coating develops, which gradu- ally turns orange. The gelatin is not liquefied. On potato the cultures form a moist, gray coating after two or three days. Bouil- lon remains clear, but on the surface a wrinkled membrane appears ; at the same time a disagreeable odor develops, and a marked indol reaction is then obtained. "When injected as such the organism was not pathogenic for guinea-pigs, while inoculation together with ster- ile butter produced changes identical with those obtained by the same observer in the case of an acid-resisting bacillus which has repeatedly been found in butter. Unlike Pappenheim's organism, the bacillus which was isolated by Rabinowitch was not decolorized by Pappenheim's method. Nevertheless, she regards the two as identical, and looks upon similar acid-resisting bacilli which have been obtained from butter, manure, and various grasses, as closely related organisms. Actinomycosis of the lungs may at times be diagnosed from the presence of the characteristic granules and thread-like formations in the sputum. In America the disease is very rare. The organism in question (Fig. 77) probably belongs to the species cladothrix, occupying a unique position among the patho- genic bacteria. Infection in man and animals (cattle and pigs) possibly occurs through ears of barley or rye, a supposition with which the observation that the disease frequently begins in the autumnal months accords. In the pus derived from ulcerating actinomycotic tumors, in the sputum in cases of pulmonary actinomycosis, and also in the feces when the disease has attacked the intestines, yellow granules will be observed, measuring from 0.5 to 2 mm. in diameter. If such a granule is examined microscopically, slight pressure being applied to the cover-glass, it will be seen to consist of numerous ' L. Rabinowitch, " Befund v. saurefesten tuberkeibacillenahnlichen Bakterien beii Lungeugangran," Deutsch. med. Woch., 1900, No. 16. 19 290 THE SPUTUM. threads wbich radiate from a centre in a fan-like manner and present club-shaped extremities. The organism may be demonstrated in the following manner: dried cover-glass preparations are stained for five to ten minutes with a saturated anilin-water and gentian-violet mixture (see page 146), when they are rinsed in normal salt-solution, dried between filter-paper, and transferred for two or three minutes to a solution of iodo-potassic iodide (1 : 100 or 1 : 50). They are then again dried between layers of filter-paper, decolorized in xylol-anilin oil Fig. 77. Actinomyces. (Musser.) (1 : 2), washed in xylol, and mounted in balsaln. The mycehum assumes a dark-blue color.^ Non-pathogenic Organisms. — Of the non-pathogenic micro- organisms which may be observed in sputa little is known. Oidium albicans may be seen in children, and is usually derived from the mouth. Of other fungi which are occasionally observed, there may be mentioned the Aspergillus fumigatus and Mucor corymbifer. Sac- charomyces has been seen in pus from pulmonary abscesses. Sar- cina pulmonalis has been found at times, and especially in the so-called mycotic bronchial props occurring in putrid bronchitis. They are usually smaller than the Sarcinse ventriculi, but larger than those observed in the urine; they present the characteristic form of the latter. Various other bacilli and micrococci, in addi- tion to those mentioned, are also found in the sputa in large num- bers, but have not been closely studied, excepting the pus-organisms, which may almost always be demonstrated. Crystals. — Of crystals which may occur in sputa, it will be neces- sary to consider briefly the crystals of Charcot-Leyden, hsematoidin, 1 E. Paltauf, Sitzungsber. d. K. K. Gesellsch. d. Aerzte Wien, 1886. MICROSCOPICAL EXAMINATION. 291 cholesterin, margarin, tyrosin, calcium oxalate, and triple phos- phates. Charcot-Leyden Crystals.^ — These crystals were discovered in the sputa of patients suffering from bronchial asthma, and were supposed to stand in a causative relation to the disease. This view, however, has been disproved, and it is now known that they may occur in other diseases as well. But while their presence is almost constant in true bronchial asthma at a time when Curschmann's spirals can also be demonstrated in the sputa, they are only exceptionally met with in other diseases, such as acute and chronic bronchitis, phthisis, etc. They were formerly regarded as identical with Bottoher's sperma crystals, but modern research has shown that this is not the case. They are straight hexagonal double pyramids, and appear under the microscope as flattened needles of variable size (Fig. 71). Some attain a length of from 40 // to 60 /i, while others are scarcely visible even with a comparatively high power of the microscope. They show a feeble, positive double refraction, and have but one optical axis, while the sperma crystals are biaxial and strongly double refracting. Their behavior to solvents is essentially the same as that of the sperma crystals, but they differ from these in their insolubility in formol. They are colored yellow with Florence's reagent, while the sperma crystals are stained a bluish black. Very curiously the appearance of Charcot-Leyden crystals is closely asso- ciated with the presence of eosinophilic leucocytes, and they have hence not inaptly been termed leuoocytio crystals. In true bron- chial asthma it is not uncommon to find microscopical preparations of the sputum literally studded with eosinophilic leucocytes and free granules. Outside the sputum they are also found in the blood in myelogenous leukaemia, and in the stools in association with animal parasites. They readily form in both normal and abnormal red bone-marrow, and excellent specimens may be obtained for purposes of demonstration if a piece of a rib is allowed to remain exposed to the air for a few days. The marrow then usually contains large numbers. The crystals also form in decomposing viscera in general, and at times form a complete covering of old anatomical prepara- tions. Their occurrence may indeed be regarded as evidence of retrogressive changes in the cellular elements of any organ. Of the relation which they bear to the eosinophilic leucocytes, with which they are so constantly associated, nothing whatever is known. Haematoidin crystals may be observed in the sputa following ex- travasations of blood into the lung. They frequently occur in the form of ruby-red columns or needles (Plate I., Fig. 2) ; amorphous granules, however, are also seen, enclosed in the bodies of leucocytes, ^ Leyden, Virchow's Arohlv, 1872, vol. liv. p. 324. Schreiner, Liebig's Annal., 1878, vol. cxciv. p. 68. Cohn, Centralbl. f. allg. Path. u. path. Anat, vol. x. p. 940. Brown, Phila. Med. Jour., 1898, p. 1076. 292 THE SPUTUM. in which case they are probably always indicative of a previous hemorrhage, while the needles are generally observed when an ab- scess or empyema has perforated into the lungs. The substance is derived from blood-pigment, and is now known to be identical with bilirubin. Cholesterin crystals are at times seen in the sputa in cases of phthisis, pulmonary abscess, and, in general, whenever old accumula- tions of pus have entered the lung from a neighboring organ. They are readily recognized by their characteristic form and chemical properties (see Feces, page 218). Fatty acid crystals are frequently observed in cases of putrid bron- chitis and gangrene of the lung, and also in cases of bronchiectasis and phthisis. They occur in the form of single needles or groups of needles, which are long and pointed. They are easily soluble in ether and hot alcohol ; insoluble in water and acids. Chemically, they are probably composed of the higher fatty acids, such as pal- mitic and stearic acids. Tyrosin crystals have been observed in cases of putrid bronchitis, perforating empyema, etc. Leucin is likewise probably always pres- ent, occurring in the form of highly refractive globules. For the recognition of these bodies, particularly of tyrosin, a chemical examination should always be made, as crystals of the soaps of fatty acids have frequently been mistaken for those of tyrosin (see Urine). Oxalate of calcium crystals are rarely seen. Fiirbringer observed them in large numbers in a case of diabetes, and linger found them in a case of asthma. They are readily recognized by their envelope- form, but they occur also in amorphous masses. They are soluble in mineral acids ; insoluble in water, alkalies, organic acids, alcohol, and ether. Triple phosphate crystals also are rarely seen, but may occur in cases of perforating abscesses, etc. They are recognized by their coffin-lid shape and the readiness with which they dissolve in acetic acid. CHEMISTRY OF THE SPUTUM. In addition to the substances described, sputum contains certain albumins, volatile fatty acids, glycogen, ferments, and various inor- ganic salts. Among the albumins which have been observed in sputa may be mentioned serum-albumin, and especially mucin, which is often pres- ent in large amounts. In pneumonic and purulent sputa albumoses also have been found. In order to demonstrate the presence of serum-albumin the sputa are treated with dilute acetic acid, when the filtrate is tested' with potassium ferrocyanide, as described in the chapter on Urine. THE SPUTA IN VARIOUS DISEASES 293 Serum-albumin is, of course, found in notable quantities in cases of oedema of the lungs. The volatile fatty acids contained in sputa may be obtained by diluting with water, acidifying with phosphoric acid, and distilling, when the distillate is further examined as described in the chapter on Feces. Acetic, butyric, propionic, and capronic acid has been found. The fats and fixed fatty acids are extracted from the residue with ether, and shaken with a solution of sodium carbonate in order to transform them into their sodium salts, when the ether is decanted and evaporated, leaving the soaps behind. Glycogen has repeatedly been demonstrated in sputa, and may be detected by Ehrlich's method (see page 52). The sputa of gangrene of the lung and putrid bronchitis have been shown to contain a ferment resembling trypsin. In order to test for this ferment, the sputa are extracted with glycerin ; the examination is then continued as described in the chapter on the Examination of Cystic Contents. The myelin granules, as I have already indicated, consist largely of protagon, lecithin, and cholesterin. The following are the inorganic salts which may be demonstrated in the sputum : sodium and magnesium chloride, phosphates of the alkalies and the alkaline earths (viz., calcium and magnesium), cal- cium and sodium sulphate and carbonate, phosphate of iron, and silicates. THE SPUTA IN VARIOUS DISEASES. Acute Bronchitis. — In the beginning of the disease the expecto- ration is small in amount, transparent, and contains very few cellular elements, constituting the so-called sputum crudum of the ancients. Microscopically, there is evidence of the existence of a desquamative process extending toward the pulmonary alveoli to a greater or less extent, and especially implicating the bronchi and trachea. Epithelial cells of various forms are found, and are probably derived from cells which were originally ciliated. Ciliated cells may occasionally be observed in perfectly fresh specimens, but are usually absent. Leu- cocytes in small numbers and alveolar cells are also seen. The presence of a few red blood-corpuscles is a common occurrence, and is probably due to rupture of a capillary blood-vessel. Later on, the sputa become more abundant, opaque, and assume a yellow color tending to green, owing to an increase in the number of leucocytes, while the other cellular elements diminish in number. Chronic Bronchitis. — The amount and consistence of the sputum in this condition vary greatly ; it is most abundant in cases of so- called bronchorrhoea, in which whole mouthfuls may be expectorated at a time. The color is usually a yellowish green, owing to the 294 THE SPUTUM. presence of numerous pus-corpuscles in various stages of degenera- tion. Microscopically, enormous numbers of micro-organisms are found, especially in cases in which the sputa have remained for some time in the bronchi. In addition, red corpuscles and epithelial cells are found ; the latter, however, are not so abundant as in the iirst stage of an acute bronchitis. A few alveolar epithelial cells in a state of fatty and myelin degeneration will also be seen. Putrid Bronchitis and Pulmonary Gangrene. — The sputa of putrid bronchitis and pulmonary gangrene resemble each other so closely that it is only possible to distinguish between the two by the presence of d6bris of pulmonary parenchyma in the latter disease. In pulmonary gangrene an exquisite sedimentation is also quite com- monly observed when the sputum is placed in a conical glass : The bottom layer is then of a greenish-yellow or brownish color, and contains a large amount of pus and greenish or brownish masses which vary in size from that of a millet-seed to that of a bean. Fragments of lung-tissue are also quite frequently seen. Micro- scopically, more or less degenerated leucocjtes, crystals of ammonio^ magnesium phosphate, and perhaps also of tyrosin and leucin, as well as hsematoidin, are found. The greenish or brownish material referred to contains amorphous masses of pigment, probably derived from haemoglobin, at times elastic tissue, fatty acid crystals, fat- droplets, and innumerable micro-organisms. Among these, the Leptothrix pulmonalis is quite conspicuous, and maiy be recognized by the violet or bluish color which it assumes when treated with Lugol's solution. Most important in the differential diagnosis between pul- monary gangrene and putrid bronchitis is the occurrence of elastic fibres arranged in an alveolar manner. The middle layer is whitish, transparent, and contains flakes of mucus in suspension. The super- ficial layer is frothy and of a dirty greenish-yellow color, the entire sputum emitting an odor never to be forgotten. Fibrinous Bronchitis. — The sputum presents all the character- istics of an ordinary chronic bronchitis ; but in addition well-defined fibrinous casts may be seen, which have been described (see page 273). Bronchial Asthma. — In this affection, and especially at the com- mencement of an attack, the expectoration is scanty, frothy, and grayish, or at times rose-colored owing to admixture of blood. Most characteristic are plug-like masses of a greenish-yellow or grayish color, containing spirals of Curschmann, Charcot-Leyden crystals, and a large number of eosinophilic and some basophilic leucocytes (see page 275). Pulmonary Abscess. — The sputum as long as it is fresh does not emit a fetid odor, and thus differs from that observed in cases of gangrene of the lung. It consists almost entirely of pus ; elastic fibres are present in abundance, as also a brownish or yellow pig-' ment-hsematoidin. Fragments of lung-tissue enclosed in a mass of THE SPUTA IN VARIOUS DISEASES. 295 pus have at times been observed, together with fatty acid and choles- terin crystals. Abscess of the Liver with Perforation into the Lung. — The sputa are of a reddish-yellow or reddish-brown color, viscid, muco- purulent, and are frequently discharged in large amounts. Micro- scopically, pus-corpuscles, red blood-corpuscles, pigmented alveolar cells (often undergoing fatty degeneration), as well as elastic tissue and granular detritus, are found. The presence of actively moving amoebse is, of course, most important from a diagnostic point of view, and is absolutely pathognomonic. Liver-cells, pieces of echinococcus- membranes, and booklets may be observed in other cases. Pneumonia. — During the first and third stages a simple catarrhal sputum is observed which does not present any special characteristics. During the second stage, however — i. e., that of hepatization — the sputum is usually quite characteristic. Its color is then reddish- brown — the classical rust-colored expectoration. The sputum at the same time is generally so tenacious that the spit-cup may actually be inverted without losing a drop of its contents. Microscopically, the following elements may be found : red corpuscles (to the presence of these the reddish color is in part due) ; at times, however, only a small number is observed, when the color is referable to haemoglo- bin which has been dissolved out from the corpuscles. Leucocytes are always present in considerable numbers. Fibrinous casts of the finer bronchioles may also be seen, and may, in fact, be visible with the naked eye. Alveolar epithelial cells, often loaded with granules of pigment, fat, and myelin, as well as others derived from the larger bronchi and the trachea, are seen. Should abscess of the lung or gangrene complicate the case, the elements described above under these headings will be found in addition, the presence of elastic tissue being, of course, the most important. Note may be taken at the same time of the occurrence of pneumococci, bearing in mind, however, that their presence is not absolutely pathognomonic. In doubtful cases, as indicated, their presence may be regarded as pointing to croupous pneumonia, providing that the clinical history and the physical signs are in accord. Phthisis Pulmonalis. — The appearance of the sputum in phthisis offers nothing that is characteristic, and is dependent upon the stage of the disease, its extent, the existence of complications, etc. In a general way it may be said that the sputa in incipient cases are usually small in amount, of a grayish-yellow color, and tenacious, the amount increasing gradually as the disease progresses, the largest quantities at this stage being expectorated in the morning upon rising. When well advanced, nummular sputa are seen. The macroscopical examination of the sputa of tubercular patients offers no characteristic features, the elements found being practically 296 THE SPUTUM. the same as those observed in cases of simple chronic bronchitis, with one exception — i. e., the occasional admixture of blood, which is usually visible with the naked eye, but may vary greatly in amount. On the one hand, small specks or streaks of blood may thus be observed ; while, on the other hand, the sputa may consist almost entirely of blood. The color of the sputum, of course, is influenced largely by the amount of blood present and the length of time that it has remained in the lungs, and varies from a bright red to a dirty brown. In cases in which a considerable hemorrhage has taken place it is necessary to exclude every other source before attributing the hemorrhage to a pulmonary origin, and in case of rupture of an aneurism, or long-continued hypersemic conditions of the lungs, so frequently observed in cases of heart-disease, in hemor- rhage of gastric origin, and in hemorrhage from the mouth or pharynx, it may at times be difficult to determine the source of the blood. The diagnosis of phthisis is thus altogether dependent upon a microscopical examination, and, above all, upon the demonstration of tubercle bacilli and elastic tissue, which have both been considered in detail. In addition, leucocytes, alveolar epithelial cells, hsema- toidin-crystals and granules are met with, which latter may be present in large numbers if a hemorrhage has occurred some time before. If the process has gone on to the formation of cavities, various constituents are also observed which are found when putre- factive processes take place in the lung. (Edema of the Lungs. — The sputa are abundant, thin, liquid, and frothy, the color of the foam varying from white to a dirty reddish-brown. Chemically, such sputa consist almost entirely of transuded serum, and are hence particularly rich in serum-albumin. Microscopically, only a small number of leucocytes and a variable number of red blood-corpuscles are found, the number of the latter, however, being scarcely large enough to account for the red color, which V. Jaksch ascribes to the presence of methsemoglobin. Heart-disease. — The sputa observed in chronic bronchitis the result of chronic heart-disease are characterized by the presence of so-called " heart-disease cells " — i. e., alveolar epithelial cells con- taining numerous hsematoidin-granules (Plate XIV., Fig. 3). If, in consequence of the existence of chronic hear1>disease hemorrhagic infarcts have occurred in the lungs, the patient may at times expec- torate numerous masses of a markedly red color, while later on — *'. e., after several days — they assume a brownish-red appearance, the sputum then presenting the characteristics noted some time after a hemorrhage. The Pneumoconioses. — Among the pneumoconioses, anthracosis, siderosis, chalicosis, and stycosis may be briefly considered. These conditions are interesting not only from a physiological, but also from a pathological standpoint. THE SPUTA IN VARIOUS DISEASES. 297 Anthracosis. — To some extent particles of carbon may be found in the sputum of almost every individual, and especially in smokers. The expectoration in such cases is of a pearl-gray color, and is brought up in larger or smaller masses, especially in the morning upon rising. Larger amounts are noted in miners and in those who are brought into close contact with coal-dust. Microscopically, particles of carbon and epithelial cells, especially of the alveolar type, as well as leucocytes loaded with the pigment, are seen. Siderosis. — In siderosis the sputum presents a brownish-black color and contains cells enclosing particles of ferric oxide. These may be readily recognized by treating the preparation with a drop of ammonium sulphide or potassium ferrocyanide solution in the presence of hydrochloric acid, when a black color on the one hand or a blue color on the other is obtained in the presence of iron. Ohalioosis. — In chalicosis silicates are found in the sputa.^ Stycosis. — This condition was first described by A. Robin in a man> aged seventy, who from his seventeenth year suffered from cough and frequent attacks of diarrhoea, and whose condition at various times had been diagnosed as phthisis pulmonalis et intes- tinarum, although tubercle bacilli could not be demonstrated. The patient died from acute pericarditis complicating an attack of acute mono-articular rheumatism. Post mortem the lungs were found perfectly normal ; the bronchial and anterior mediastinal glands, as well as the mesenteric glands, however, were completely solidified and composed almost wholly of calcium sulphate. The man, it was then found, had been working in plaster of Paris all his life, and the symptoms observed — viz., cough, expectoration, and diarrhoea — Robin is inclined to attribute to the pressure of the solidified glands upon the bronchi and intestines. ' Betts, "Chalicosis Pulmonum," Jour. Am. Med. Assoc, 1900, No. 2. CHAPTER VII. THE URINE. GENERAL CONSIDERATIONS. This is not the place to enter into a discussion of the various hypotheses which have been advanced to explain the manner in which waste-material is removed from the body through the kidneys. It will suffice to state that while the water and mineral constituents of the urine in part at least undoubtedly pass into the uriniferous tubules by a process of transudation, a selective glandular activity of the cells lining the convoluted tubules and the loop of Henle appears to be necessary for the elimination of the most important organic constituents. As the physical characteristics of the urine, as well as its chemi- cal composition, are influenced not only by the age and sex of the individual, but also by the character of the food ingested, the proc- ess of digestion, exercise, climate, temperature, race, etc., it is apparent that a quantitative analysis of any one urine, or even average figures, can give only an approximate idea of its composi- tion. The reader is accordingly referred for information to the special paragraphs concerning the variations in the individual con- stituents observed in health. It is important, however, to note that, notwithstanding the fairly wide variations here observed, the composition of the blood, as pointed out in a previous chapter, remains quite constant, showing the perfect manner in which the nervous system through the kidneys guards against an undue accu- mulation of what may be termed normal waste-products in the blood, and in virtue of which abnormal substances are also imme- diately eliminated. Moreover, as will be pointed out later on, a perfect mechanism appears to exist which prevents an undue accu- mulation of material in the blood that can hardly be regarded as waste. The presence of an amount of sugar in the blood exceeding 6 pro mille, for example, appears to be invariably followed by gluco- suria, and the introduction of excessive quantities of sodium chloride similarly and almost immediately leads to an elimination of the excess. 298 GENERAL CHARACTERISTICS OF THE URINE. 299 GENERAL CHARACTERISTICS OF THE URINE. General Appearance. Normal urine, just voided at an ordinary temperature, is either perfectly clear or but faintly cloudy, owing to the fact that the acid and normal salts present are all soluble in water. It may be stated, as a general rule, that whenever a urine freshly passed presents a distinct cloudiness some abnormality exists. When allowed to stand for a time a light cloud develops, which gradually settles to the bottom, constituting the so-called nubecula of the ancients. Examined under the microscope this is found to contain a few round, granular cells, somewhat larger than normal leucocytes, the so-called mucous corpuscles, and a few pave- ment-epithelial cells, derived from the bladder or genital organs. Chemically the nubecula probably consists of traces of mucus. When kept for twenty-four hours at an ordinary temperature crystals of uric acid are frequently observed in addition to the above elements, usually presenting the so-called whetstone-form. If, however, the temperature at which the urine is kept approaches the freezing-point, the entire volume becomes cloudy, owing to pre- cipitation of acid urates, as these are much less soluble in cold than in warm water ; on standing they gradually settle to the bottom of the vessel, and form what is known as a sediment, while the super- natant fluid again becomes clear. If kept still longer exposed to the air, at the temperature of the room, the entire volume of urine again becomes cloudy, owing to a diminution of its normal acidity, the result being a precipitation of ammonio-magnesium phosphate, calcium phosphate, and still later, when the urine has become alkaline, of ammonium urate. Gradually a heavy sediment, containing these salts in addition to the constituents of the primitive nubecula, forms at the bottom of the vessel ; the supernatant fluid, however, remains cloudy. On microscopical examination it will be seen that this cloudiness is due to the presence of enormous numbers of bacteria. The changes which take place in a normal urine when allowed to stand at ordinary temperature may be tabulated as follows : (1) Urine clear, no sediment — reaction acid. (2) Urine slightly cloudy, owing to development of the nubecula — ^reaction acid. vr u 1 f Mucous corpuscles, Nubecula | Pavement-epithelial cells. (3) Urine clear, the nubecula has settled — reaction acid. r Mucous corpuscles, „ ,. , I Epithelial cells. Sediment .i Uric acid crystals, I A few bacteria. 300 THE URINE. (4) Urine cloudy, owing to the precipitation of phosphates — reaction faintly acid. (5) Urine cloudy, owing to the presence of bacteria — reaction alkaline. Sediment Bacteria, Mucous corpuscles, Epithelial cells. Triple phosphates, Tri-calcium phosphate, Ammonium urate. Color. The color of normal urine may vary from a very light yellow to a brownish red, the particular shade depending essentially upon the specific gravity, becoming lighter with a diminishing, and darker with an increasing density. Pathologically the same rule holds good, except in diabetes, in which a very high specific gravity is gen- erally associated with a very light color. The reaction of the urine also exerts a marked influence upon its color, an acid urine being more highly colored than an alkaline urine, which can be readily demonstrated by allowing a specimen of acid urine to become alka- line, and by treating an alkaline urine with dilute hydrochloric or acetic acid. At the same time it may be said that every urine darkens slightly on standing, the reaction remaining acid. The various shades observed in normal urines may be grouped under the following headings : 1. Pale urines vary from a faint yellow to a straw color. 2. Normally colored urines are of a golden or an amber yellow. 3. Highly colored urines present a reddish-yellow to a red color. 4. Dark urines vary between brownish red and reddish brown. As these shades may occur in both normal and pathological urines, definite conclusions cannot, as a rule, be drawn from mere inspection. A very pale urine indicates simply an excess of water, which may be normal, but may also occur in such diseases as chronic interstitial nephritis, diabetes mellitus, diabetes insipidus, hysteria, and the various ansemias ; it is further seen during convalescence from acute febrile diseases, while a highly colored urine, though also occurring in health, may indicate the existence of a febrile process. It may be stated, as a general rule, that a pale urine always excludes the existence of a febrile disease of any severity, and that the continued secretion of a very pale urine is usually associated with a certain degree of antemia. The normal color of the urine is probably owing to the presence of several pigments, which are most likely closely related to each other and to hsematin. In addition to these colors others may be observed at times which are either pathological or accidental — i. e., due to the presence of cer- GENERAL CHARACTERISTICS OF THE URINE. 301 tain drugs. The former are, on the whole, of greater importance to the physician than those mentioned above, as more definite conclu- sions can be drawn from their presence. Most important among such pathological pigments are those due : 1. To the presence of blood-coloring matter. The color in such cases may vary from a bright carmin to a jet black, the exact shade depending upon the quantity of blood-coloring matter present, upon changes that the blood may have undergone either before or after being passed, and also upon the presence of the pigment in solution or contained in red corpuscles. 2. Those due to the presence of biliary coloring matter. The color here varies from a greenish yellow to a greenish brown. 3. A milky-colored urine is observed in cases of chyluria. Among the accidental abnormalities in color, on the other hand, are those due to the presence of substances like carbolic acid and its congeners, santonin, etc. As the recognition of the causes of such alterations, normal, pathological, and accidental, largely depends upon a more detailed study of the individual pigments, this subject will be dealt with more fully further on (see Pigments and Chromogens). Odor. The odor of the urine is usually of little significance. Normally it resembles that of bouillon, and in some cases that of oysters ; it is probably due to the presence of several volatile acids. The odor of urines undergoing decomposition is characteristic and has been termed " the urinous odor of urine," an ill-chosen term, as this odor is always indicative of an abnormal condition. The ingestion of asparagus, onions, oil of turpentine, etc., pro- duces a characteristic odor which is of no significance. Consistence. Urine, while normally fluid and but slightly viscid, may in dis- ease acquire a marked degree of viscidity, which becomes especially apparent upon attempting its filtration ; the liquid passes through the paper with more and more difficulty, and finally clogs its pores altogether. Quantity. The quantity of the urine is normally subject to great variations, the amount eliminated in the twenty-four hours being influenced by that of the fluid ingested, the nature and quantity of the food, the process of digestion, the blood -pressure, the surrounding tempera- ture, sleep, exercise, body-weight, sex, age, etc. It is easy to understand, then, why figures given by different 302 THE URINE. observers in diiferent countries should vary considerably. Salkow- ski, in Germany, thus gives 1500 to 1700 c.c. as the normal amount; v. Jaksch, in Austria, 1500 to 2000 c.c. ; Landois and Sterling, in England, 1000 to 1500 c.c. ; Gautier, in France, 1250 to 1300 c.c. In the United States I have found an average secre- tion of from 1000 to 1200 c.c. in the adult male, and 900 to 1000 c.c. in the adult female. It is thus seen that the secretion of urine is greatest in Germany and Austria, where the body-weight and ingestion of liquids are greater than in England, France, arid the United States. Children pass less, but relatively more (considering their body- weight) urine than adults. Women pass somewhat less than men. During the summer months, when a larger proportion of water is eliminated through the skin and lungs than in cold weather, less urine is voided. The same occurs during repose, more urine being passed during active exercise, and hence less during the night than during the day. The amount of urine secreted in the different hours of the day varies greatly, reaching its maximum a few hours after meals. It decreases toward night, and reaches its lowest point in the first hours of the night, after vvhich it begins to rise rapidly until 2 or 3 o'clock in the morning. The ingestion of large amounts of liquid, of course, increases the daily amount considerably, and 3000 c.c. may be passed under such conditions by an individual in good health, while it may decrease to 800 or 900 c.c. when but little liquid is taken. After the ingestion of much solid food the secretion of urine is temporarily diminished. Water containing no salts possesses distinctly diuretic properties, as do also beer, wine, coffee, tea, etc. The most important medicinal diuretics are digitalis, squill, broom, spirit of nitrous ether, juniper, urea, etc. Pathologically the amount of urine varies within wide limits. In a given case, moreover, it may be exceedingly difficult to determine whether or not the secretion is within physiological limits. As a general rule, whenever less than 500 c.c. or more than 3000 c.c. are passed some abnormal condition exists, providing all other causes which might lead to the secretion of such an amount can be eliriii- nated. Clinically we speak of polyuria and oliguria. Polyuria. — Polyuria is observed in many diseases, and is present under such varied conditions that a classification is only warrant- able upon a hypothetical basis, especially as the causative factors concerned in its production are mostly unknown. As polyuria is almost invariably associated with diabetes mel- GENERAL CHARACTERISTICS OF THE URINE. 303 litus, its presence in any case should always excite suspicion and lead to a proper examination. The quantity of fluid eliminated in diabetes is usually dependent upon the amount ingested. The ex ere-, tion of a proportionately large amount of fluid, however, does not necessarily follow the ingestion directly, and retention of a large amount may occur ; it has been shown, as a matter of fact, that the diabetic patient excretes liquids with greater difliculty than the healthy subject. At the same time it should be borne in mind that the polyuria in diabetes is not necessarily continuous, and that periods during which a normal or even a subnormal amount of urine is observed may alternate with true polyuria. From 2 to 26 or even 50 liters may be passed within twenty-four hours. Intercurrent dis- eases of a febrile character may modify the quantity very materially and cause the elimination of a normal or subnormal amount. The cause of the polyuria in diabetes mellitus is unknown. The ingestion of large amounts of liquids, of course, leads to a cor- respondingly large elimination, and the existing polydipsia could, hence, be made responsible for the polyuria; the latter would thus be the result of an increased stimulation of the thirst-centre, pos- sibly owing to the presence of some abnormal constituent of the blood. The^ polydipsia, however, may also be the result of a pri- mary polyuria. The polyuria associated with the resorption of large pericardial, pleural, ascitic, and subcutaneous effusions is more readily under- stood, although the primum mobile may be unknown ; it depends in such cases entirely upon the presence of excessive quantities of fluid in the bloodvessels. A form of polyuria which has been termed " epicritic polyuria " is frequently observed during convalescence from acute febrile dis- eases, and is of prognostic importance. Its occurrence in a given case is regarded by many as a good omen, especially in typhoid fever ; still it must not be forgotten that a polyuria may occur after subsidence of the fever, and be followed by a considerable degree of oliguria, and in some cases may precede death. A polyuria of this kind probably always indicates the elimination of waste- products which had accumulated in the blood during the course of the disease, but it may, at the same time, be due to the presence of retained water. Second in constancy is the polyuria associated with granular atrophy of the kidneys, constituting one of the most important symp- toms of the disease. Cases have been reported in which as much as 10,000 c.c. of urine were secreted in the twenty-four hours ; 2000 to 4000 c.c. represent the usual amount in such cases. Polydipsia commonly exists at the same time, and the explanation of the poly- uria again becomes a very difficult matter. That generally given is based upon the following considerations : 304 THE URINE. In granular atrophy of the kidneys large tracts of renal paren- chyma are destroyed, the result being a diminution in the area of glandular material, which in itself would lead to a diminished secre- tion of urine. The coexisting cardiac hypertrophy, however, by raising the blood-pressure in the kidneys, is supposed to counter- balance the renal deficiency and even to lead to an increase in the amount of urine. There appears to be some doubt as to the cor- rectness of such an explanation, however, as the existence of hyper- trophy of the left ventricle in the absence of glandular disease of the kidneys by no means leads to a degree of polyuria comparable to that observed in this disease. It is possible that while cardiac hypertrophy in itself may be one of the causative factors, still another may be a vicarious action of the sound glandular elements. If such be the correct explanation, the coexisting polydipsia is merely secondary. This, however, can only be regarded as an hypothesis, and the diminished renal secretion associated with a gradually developing cardiac dilatation should not be upheld as • an absolute proof of its correctness. Very curiously, polyuria may occur also in association with mul- tiple myelomata of the bones and the presence of Bence Jones' albumin in the urine. In one of the cases reported by Hamburger,^ and which I had occasion to study in greater detail from a chemical point of view, 3500 c.c. were voided in the twenty-four hours. The symptom, however, is not constant. Polyuria, furthermore, has been observed in the most diverse diseases of the nervous system, both functional and organic. It is frequently observed both as a transitory and a permanent symptom in cases of hysteria. Large quantities of a very pale urine are secreted after the occurrence of severe hysterical seizures, but the same may be observed throughout the course of the disease. A similar condition is frequently seen in neurasthenia, migraine, chorea, and epilepsy. Generally speaking, it may be said that a paroxysmal polyuria in nervous diseases is associated with functional derangement, while a continuous polyuria appears to be connected rather with true organic changes. It has been observed in certain cases of tabes, cerebrospinal and spinal meningitis, during the first stage of general paresis, in association with tumors involving the medulla, the cere- bellum, and the spinal cord, in injuries affecting the central nervous system, in Basedow's disease, etc. Cases of idiopathic diabetes insipidus also should probably be classified under this heading. Enormous quantities of urine may be secreted in this disease, which are equalled only by cases of diabetes mellitus, and may at times reach 43 liters per diem. 1 L. p. Hamburger, " Two Examples of Bence Jones Albuminosuria associated with Multiple Myeloma," Johns Hopkins Hosp. Bull., Feb., 1901. GENERAL CHARACTERISTICS OF THE URINE. 305 Oliguria. — Oliguria is, on the whole, more frequent than polyuria, and is met with in almost all conditions associated with a lowered blood-pressure. First in order stand those cases of cardiac disease in which compensation has failed, whether the cardiac weakness is primary or occurring secondarily to other diseases — i. e., pulmonary, hepatic, and renal. The oliguria observed in the so-called continued fevers, notably typhoid fever, is probably also referable to cardiac weakness. It should be remembered, however, that a larger proportion of water is eliminated through the skin and lungs than normally, and that a retention of fluids also undoubtedly occurs which is not due to cardiac weakness ; still other factors may be concerned in its production. The oliguria occurring in acute nephritis and in chronic paren- chymatous nephritis in all probability depends largely upon mechani- cal causes, the increased intra-canalicular resistance in the form of desquamated epithelium and tube-casts, as well as the pressure of the exudate upon the bloodvessels obstructing the passage of urine, while the functional activity of the diseased glandular elements is at the same time lowered. Upon mechanical causes, also, depend all those cases of oliguria which are associated with the presence of a stone or tumor pressing upon a portion of the urinary tract. Oliguria may occur as a nervous manifestation in connection with puerperal eclampsia, lead colic, hysteria, psychic depression, preced- ing and during epileptic seizures, etc. Whenever there is a diminu- tion in the amount of bodily fluids oliguria is also observed ; this is particularly marked in cholera and following severe hemorrhage. Obstruction to the flow of blood in the vena cava or liver, lead- ing to an increase of venous pressure and a decrease of arterial pressure in the kidneys, likewise results in oliguria, as is seen in atrophic hepatic cirrhosis, acute yellow atrophy, thrombosis of the vena cava and the renal vein, or in cases in which pressure is exerted upon these by tumors, ascitic fluid, etc. In any case the oliguria may go on to complete anuria, which condition not infrequently precedes death.. Anuria may, however, also occur independently of a pre-existing oliguria, as in hysteria. Specific Gravity. The specific gravity of normal urine varies between 1.015 and 1.025, corresponding to 1200 to 1500 c.c, viz., the normal amount of urine voided in twenty-four hours. Pathologically, a specific gravity of 1.002 on the one hand and 1.060 on the other may occur, depending upon the amount of solids and fluids present, increasing as the solids increase, the amount of urine remaining the same, and decreasing as the amount of fluid increases, the solids remaining the 20 306 THE URINE. same. The specific gravity is thus an index in a general way of the metabolic processes taking place in the body. The necessity of determining the specific gravity of the total amount of urine voided in a given case, and not that of an individual specimen passed during the twenty-four hours, becomes apparent upon considering the variations which may occur in the quantity of solids and liquids ingested during the day. The ingestion of large amounts of water or beer would, of course, result in the passage of a correspondingly large quantity of urine within the next few hours, containing but a small amouiit of solids, and hence presenting a low specific gravity. From such an observation it would be erroneous to infer a diminished excretion of solids for the day, as succeeding specimens would in all probability be passed presenting a higher specific gravity. An observation made upon a specimen taken from the collected urine of the twenty-four hours, moreover, can only then convey a correct idea if the total quantity is within the normal limits. If this should not be the case, the volume of the urine passed must first be reduced to the normal and the specific gravity then taken. Supposing a known quantity of common salt to be dissolved in 1000 c.c. of water, so that the resulting specific gravity is 1.24 ; by doubling the amount of salt and water the specific gravity would still remain the same, while the amount of salt would actually be twice as large as at first. In order to obtain the specific gravity indicating the actual amount of solids present it would be necessary to concentrate the fluid to 1000 c.c. The specific gravity being inversely proportionate to the amount of fluid secreted, the necessary correction is made according to the following formula : Sp.gr. = ^, in which Sp. gr. indicates the specific gravity to be determined, q the amount of urine actually passed, d the specific gravity observed, and iVthe normal amount of urine — i. e., 1200 c.c. Example. — A patient has passed 3000 c.c. of* urine in the twenty- four hours with a specific gravity of 1.017 ; this is corrected accord- ing to the above formula : Sp.gr.=300iXJZ_ 1.042. ^ ^ 1200 From the specific gravity the amount of solids can be calculated with sufficient accuracy for clinical purposes by multiplying the last two decimal points by 2, the number obtained indicating the amount of solids in 1000 c.c. of urine. To illustrate the necessity of either indicating the total amount of urine passed within the twenty-four hours, and of taking the specific GENERAL CHARACTERISTICS OF THE URINE. 307 gravity from this collected urine, or of correcting the specific gravity as shown above, the following case may be supposed : A " specimen " of urine is taken, presenting a specific gravity of 1.002 ; by multiplying the 2 by 2, the person would be supposed to pass 4 grammes of solids in every 1000 c.c. of urine. Had the specific gravity been observed in the total amount of urine passed in the same twenty-four hours, it would have been found to be 1.012, the man having passed 3000 c.c. of urine ; by multiplying 12 by 2, 24 grammes of solids would have represented the amount in every 1000 c.c. — i. e., 24 X 3 = 72 grammes in toto. The same result would have been reached by correcting the specific gravity of 1.012 for the normal amount of urine. The first calculation then would have indicated a considerable deficit as compared with the second. The following rules for practice may thus be stated : 1. Whenever the specific gravity only is to be indicated in a uri- nary report it should always be the corrected one ; if this is not done, the amount of urine should be stated in every case. 2. The specific gravity should always be taken from a specimen of the collected urine of the twenty-four hours, and never from a specimen ad libitum. From the rule, that the specific gravity of a urine is inversely proportionate to the amount of fluid eliminated, it must follow that whatever causes produce oliguria will also produce a high specific gravity, while all those causes which produce a polyuria will similarly produce a low specific gravity, with the following exceptions : 1. A diminished amount of urine with a lowered specific gravity occurs in many chronic diseases and toward the fatal termination of acute diseases, indicating a defective elimination of solids. 2. The same may be observed in certain cases of oedema. 3. Following copious diarrhoea, vomiting, and sweating. 4. A high specific gravity is associated with polyuria in diabetes mellitus. Unfortunately the determination of the specific gravity and the solids contained in urines does not furnish as valuable information in many cases as would be expected d, priori. This is largely owing to the fact that the organic constituents of the urine have a lower specific gravity than the inorganic salts, and especially the chlorides, which are usually present in considerable amount. It thus not in- frequently happens that the nitrogenous constituents are considerably increased, while the specific gravity is relatively low, owing to the absence or a diminution in the amount of chlorides. In other words, while the specific gravity may be regarded as a fair index of the total amount of solids excreted, its increase or decrease furnishes no information as to the nature of the constituents causing such a change. 308 THE URINE. Fig. 78. Determination of the Specific Gravity. — The specific gravity of the urine is most conveniently determined by means of a hydrometer indicating degrees varying from 1.002 to 1.040. Such instruments, constructed especially for the examina- tion of urine, are termed urinometers (Fig. 78). A good instrument should have a stem upon which the individual divisions are at least 1.5 mm. apart, and each division should correspond to 0.5 degree. Urinometers may also be purchased which are provided with a thermometer, a matter of great convenience. Every instrument should be carefully tested by comparison with a standard hydrom- eter. In order to determine the specific gravity in a given case a cylindrical ves- sel is nearly filled with urine and the urinometer slowly introduced, the read- ing being taken at the lower meniscus as soon as the instrument has come to rest. Precautions : 1 . The urinometer must be given ample room, and the read- ing should never be taken when the in- strument touches the sides of the ves- sel, as owing to capillary attraction it is otherwise raised, causing the reading to be too high. 2. The instrument must be perfectly dry and clean before being used, and should never be allowed to "drop" into the urine, as otherwise the weight of the instrument is increased by adhering drops of water, and the reading is too low. 3. Any foam upon the surface of the urine should first be removed by means of a piece of filter-paper, as it interferes with the accuracy of the reading; bubbles of air adhering to the instrument, and thereby elevating it, should be carefully removed with a feather. 4. The specific gravity should always be determined in specimens taken from the twenty-four-hour urine, and corrected according to the formula given above. 5. If the quantity of urine is too small to determine its specific gravity with a urinometer, the following method may be advan- tageously employed : Urinometer. (W. Simon.) GENERAL CHARACTERISTICS OF THE URINE. 309 About 50 c.c. of urine are measured into a small bottle pro- vided with a ground-glass stopper, or into a pyknometer like the one pictured in Fig. 79, and accurately weighed. The weight of the Ftg. 79. The pyknometer. urine divid^ by its volume gives the specific gravity, which must, however, be corrected for the temperature of the urine. If accuracy is required, such corrections should be made in every case, as the specific gravity increases or decreases by one degree for every three degrees C. above or below the point for which the instrument is reg- istered, viz., 15° C. According to Bouchardat and Mercier, this method is not strictly accurate, and the following table has been constructed by which the proper corrections can be readily made : Temperar Normal Sugar Tempera- Normal Sugar ture. urine. urine. ture. urine. urine. 0° 0.9 1.3 18° 0.3 0.6 1 0.9 1.3 19 0.5 0.8 2 0.9 1.3 20 0.9 1.0 3 0.9 1.3 21 0.9 1.2 4 0.9 1.3 22 1.1 1.4 5 0.9 1.3 23 1.3 1.6 6 0.8 1.2 24 1.5 1.9 7 0.8 1.1" 25 1.7 2.2 8 0.7 1.0 26 2.0 2.5 9 0.6 0.9 27 2.3 2.8 10 0.5 0.8 28 2.5 3.1 11 0.4 0.7 29 2.7 3.4 12 0.3 0.6 30 3.0 3.7 13 0.2 0.4 31 3.3 4.0 14 0.1 0.2 32 3.6 4.3 15 0.0 0.0 33 3.9 4.7 16 0.1 0.2 34 4.2 5.1 17 0.2 0.4 35 4.6 5.0 Emample. — Supposing the specific gravity to have been 1.030, at temperature of 20° C, it would be necessary to add 0.9 to 1.030, 310 THE UBINE. making this 1.0309 ; at a temperature of 10° C, it would similarly be necessary to subtract 0.5. Determination of the Solid Constituents. — As indicated above, the amount of solids can be calculated with a degree of accuracy sufficient for clinical purposes by multiplying the last two figures of the specific gravity by 2 ; the number obtained indicates the amount of solids in every 1000 cc. of urine. If greater accuracy is re- quired, the following method may be employed : Five cc. of urine, accurately measured, are placed in a watch- crystal containing a little dry sand (sand and crystal having been previously weighed) ; this is placed over a dish containing concen- trated sulphuric acid, and under the receiver of an air pump which has been made perfectly air-tight by thoroughly lubricating the ground-glass edge of the bell with mutton tallow and applying the bell with a slightly grinding movement to the ground-glass plate. The receiver is now exhausted and the urine allowed to remain in the vacuum for twenty-four hours, when the bell is again exhausted and left for twenty -four hours longer ; at the end of this time the crystal is weighed, the difference between the two weights obtained indicating the amount of solids in 5 cc of urine, from which the percentage and total amount are readily calculated. The slight loss of ammonia which results when this method is employed scarcely affects the accuracy of the result. REACTION. The reaction of the twenty-four-hour urine is, as a rule, acid ; individual specimens, passed in the course of the same twenty-four hours, may be either alkaline, acid, or amphoteric. When a mixture of different acids is brought into contact with a mixture of alkalies, the acids combine with the alkalies according to the degree of affinity which exists between them and the amount present of each. Upon the excess of acids over alkalies, and vke versa, depends the resulting reaction. If the alkalies are not suf- ficient in amount to saturate the acids, an acid reaction will result, while an insufficient amount of acid will give rise to an alkaline reaction. The same principle holds good for the acids and alka- lies giving rise to the salts present in the urine. As here the alkaline substances are not present in sufficient amount to saturate the acids, which can readily be seen from the following table, the acid reaction of normal urine is explained : HCl SO, PjOs K Na NH, Ca Mg 10.1265 6.3811 2.3157 1.3315 3.0334 0.9827 2.5830 1.5194 5.4780 5.4780 0.5977 0.8087 0.0405 0.0233 0.0880 0.0843 The figures in the first column indicate the average daily amount REACTION. 311 of the inorganic acids and alkalies present in the urine of twenty- four hours, and the figures in the second column their equivalents in terms of sodium, that of phosphoric acid having been estimated as diacid sodium phosphate. From this it is seen that the acid equivalents, 8.6963, exceed the alkaline equivalents, 7.9137, by 0.7816 gramme of sodium. There are present then in the urine, in addition to the . normal salts of the monobasic acids, acid salts and especially diacid sodium phosphate, NaHjPO^. To the latter the acidity of the urine is due. If, on the other hand, the alkalies exceed the acids in amount, an alkaline urine will result, which may occur physiologically under various conditions.^ The so-called amphoteric reaction will be observed when the diacid and neutral sodium phosphates, NaHjPO^ and Na2HPO^, are present in a certain definite proportion ; the urine then changes the color of red litmus paper to blue, and viae versa.^ A neutral urine is never observed under normal conditions. The presence of a free acid, moreover, is not possible, as it would imme- diately combine with ammonia, which is constantly being set free in th^' tissues of the body as ammonium lactate, and is normally trans- formed into urea.^ The question now arises. How does the acidity of the urine re- sult ? and What are the ultimate factors that will produce an alka- line and an amphoteric reaction ? These are problems which as yet await a final answer. Our present ideas, however, may be formulated as follows : In the me- tabolism of the body -tissues acids are constantly produced ; chief among these is sulphuric acid, which results from albuminous decom- position, and hydrochloric acid, which at a certain period of digestion i=3 reabsorbed from the stomach. As the alkalinity of the blood is due to neutral sodium phosphate and sodium carbonate, these salts are attacked by the free acids as soon as they enter the blood, the result being the formation of acid salts, and, as the latter diffuse more readily through an animal membrane than alkaline salts, the secretion of an acid urine from the alkaline blood is in part ex- plained. Nevertheless it is impossible to exclude a certain specific action on the part of the glandular elements of the kidneys, as otherwise the secretion of all glands, supposing this to depend upon a process of filtration or diffusion only, would necessarily be acid. As the alkalinity of the blood increases the acidity of the urine decreases, until finally an alkaline urine results. The degree of the alkalinity of the blood, however, depends essentially upon the nature of the food and the secretion of the gastric juice, viz., the hydro- 'Brucke, Maly's Jahresber., 1887, vol. xvii. p. 189. Liebig, Annal. d. Chem. u. Pharmakol., 1844, vol. 1. p. 61. 'Heintz. Jour. f. prakt. Chem., 1872, vol. vi. p. 274. 'F. Walters, Arch. f. exper. Path. u. Pharmakol., 1877, vol. vii. p. 148. 312 THE URINE. chloric acid. The ingestion of vegetable food, rich in salts of or- ganic acids, which become oxidized in the body to the carbonates of the alkalies, will result in the passage of an alkaline urine, for the alkalies thus formed when absorbed into the blood are more than suificient to neutralize completely all the acids present, and the elimi- nation of neutral sodium phosphate alone takes place. In the case of animal food the reverse holds good. The alkaline carbonates here formed are not sufficient to neutralize the excess of acids, and diacid phosphate of sodium is hence eliminated in large quantity.^ An amphoteric urine results whenever the elimination of neutral and acid sodium phosphate is the same ; such an occurrence is, therefore, more or less accidental. As the alkalinity of the blood is increased during the secretion of the acid gastric juice, it may frequently happen, especially follow- ing the ingestion of a • large amount of food, that an alkaline urine is voided. If this does not take place, the acidity of the urine is at least diminished, but increases again during the process of resorp- tion of hydrochloric acid and peptones. The statement so generally found in text-books, that the urine secreted after a meal is alka- line, is not strictly correct ; in a series of observations which I made dn this direction an alkaline urine was observed in only 20 per cent, of the cases examined.^ It may thus be stated that an alkaline urine will result under physiological conditions whenever the alkaline salts present in the food are sufficient to neutralize all the acids formed, as occurs in the case of a vegetable diet, and, furthermore, whenever the period of gastric secretion is lengthened. If an acid urine is allowed to stand exposed to the air for a cer- tain length of time, its degree of acidity gradually diminishes, and the reaction finally becomes alkaline. At the same time the urine becomes cloudy and deposits a sediment, which consists of ammbnio- magnesium phosphate, MgNH^PO^-l-eH^O, neutral calcium phos- phate, C&^(70^2> ^^^ still later contains ammonium urate, C,H2- (NH4)2N403, in addition to the constituents of the primitive nubecula — i. c, a few mucous corpuscles and pavement epithelial cells. The entire volume of urine, moreover, remains cloudy, owing to the presence of innumerable bacteria. The odor becomes extremely disa- greeable, and distinctly "urinous." In short, "ammoniacal decom- position " has occurred. This has been shown to depend upon the action of certain bacteria, notably the Micrococcus urese and the Bac- terium urese, which are present in the air.' These organisms cause decomposition of the urea found in every urine, with the forma- tion of ammonium carbonate, according to the following equations : •E. Salkowski u. J. Munk, Virchow's Arcliv, 1877, vol. Ixxvi. p. 500. ''Quincke, Zeit. f. klin. Med., vol. vli. " W. Leube, "Ueber die ammoniakalisohe Harngahrung," Vircbow's Arohiv, 1886, vol. c. p. 555. REACTION. 313 C0(NH2)j + 2HjO = (NHJ.COs (NHJjCO, = 2NH3 + H,0 + COj. It is not the bacterium, however, which directly produces the re- sult, but a bacterial product, and in this case an enzyme. An alkaline urine, the alkalinity of which is not due to ammo- niacal fermentation, however, but to other causes, as indicated above, may, of course, undergo the same change as an acid urine ; but it is necessary to distinguish sharply between these two varieties of alkaline urines, as the recognition of the cause of the alkalinity is very often most important in diagnosis. The distinction is readily made by fastening a piece of sensitive red litmus-paper in the cork of the bottle containing the urine. If the alkalinity of the urine is due to the presence of ammonia, the litmus-paper will turn blue, but soon changes to red when exposed to the ait ; while a urine, the alkalinity of which is due to the presence of fixed alkalies, will turn red litmus-paper blue only when immersed in the urine, the change in color at the same time persisting. As ammoniacal decomposition can also occur within the urinary passages, it is important, whenever an alkaline reaction due to the presence of ammonia is observed, to test the urine at once upon being voided, or, still better, to procure a portion with a catheter. Such urines are frequently seen in cases of cystitis the result of paralysis, urethral stricture, gonorrhoea, etc. An intensely acid reaction is observed in almost all concentrated urines, especially in fevers, in certain diseases of the stomach asso- ciated with a diminished or suspended secretion of hydrochloric acid, in gout, lithiasis, acute articular rheumatism, chronic Bright' s dis- ease, diabetes, leuksemia, scurvy, etc. Whenever a very acid urine is secreted for a considerable length of time the possibility of renal irritation and the formation of concretions should be borne in mind. An alkaline urine, the alkalinity of which is not owing to the presence of ammonia, but to a fixed alkali, is observed in certain cases of debility, especially in the various forms of ansemia, follow- ing the resorption of alkaline transudates, the transfusion of blood, frequent vomiting, a prolonged cold bath, etc. It may also be due to the ingestion of certain drugs, viz., salts of the organic acids and alkaline carbonates, the former being transformed into the latter, as has been mentioned. An increase in the degree of acidity may similarly take place after the ingestion of mineral acids. Of interest is the observation of Pick ' that in twenty-four to forty-eight hours after the crisis in pneumonia the urine shows a marked decrease in its acidity, becoming neutral or even alkaline. ' F. Pick, "The Urine in Pneumonia," Miinch. med. Woch., 1898, No. 17. 314 THE URINE. This phenomenon, which was observed in thirty-one out of thirty- eight cases, persists for a day or a day and a half, and then the acidity returns. In all likelihood the change is due to absorption of the large amounts of sodium which are present in the exudate. An increase in the acidity of the urine upon standing has repeat- edly been observed, and is probably due to the formation of new acids from pre-existing acid-yielding substances, such as certain carbohydrates, alcohol, etc., which have undergone fermentation. This phenomenon is frequently observed in diabetic patients. A decrease in the acidity of normal urine upon standing, however, is the rule, owing to a gradual decomposition of sodium urate by the acid sodium phosphate, acid sodium urate, and, later on, uric acid resulting, which are thrown down as a sediment in consequence of the diminished acidity of the urine, and which, hence, no longer influence its reaction. This is shown in the equations : (1) NaHjPO, + CsHjNajNA = Na^HPOi + CsHjNaNA (2) NaHjPOi + C5H3NaNA = Na^HFOi + CsH.NA- Determination of the Acidity of the Urine. — The old method of titrating a certain amount of urine with a decinormal solution of sodium hydrate has been abandoned and replaced by that of Freund. This is essentially based upon the observation that the acid reaction of the urine is referable exclusively to diacid phosphates. Freimd's Method.' — In 50 c.c. of urine the total amount of phos- phoric acid is estimated as described on page 331. The result is termed T. In a second portion of 50 c.c. the monacid phosphates, 31, are then precipitated with a normal solution of barium chloride — i.e., one containing 122 grammes of the crystallized salt in 1000 c.c. of water — 10 c.c. being added for every 100 ragrms. of the total amount of phosphoric acid found. After the addition of the barium the mixture is diluted to 100 c.c, filtered, and the phosphoric acid estimated in 50 c.c. of the filtrate. The result obtained is termed -D. Owing to the fact that not only are the monophosphates pre- cipitated on the addition of the barium chloride, but also a small amount of normal phosphates, and that a small amount of diacid phosphate is formed at the same time and passes into solution, an error is incurred. Tiiis, however, remains constant, and amounts to 3 per cent, in favor of the diacid phosphates. As the total amount of phosphoric acid is subject to fairly wide variations, even in health, it is best to calculate the relative propor- tion of r to D for 1 00 c.c. of urine, and then to determine the abso- lute degree of acidity for the twenty-four hours. Figures are thus obtained which are directly comparable with one another. 1 E. Freund, Centralbl. f. d. med. Wiss., 1892, p. 689. CHEMISTRY OF THE URINE. 315 Example. — Supposing that T amounted to 0.386 gramme for 100 c.c. of urine, and D to 0.338 gramme. Three per cent, of D would have to be deducted for reasons just given, making the true value of D 0.3279. The relative proportion of T to Z> would then be 84.9, as determined according to the equation 0.386 : 0.3279 :: 100 : r ; and a; = 84.9. Supposing, further, that the total amount of urine was 2000 c.c, the total acidity of the twenty-four hours would correspond to 1698, according to the equation 100 : 84.9 : : 2000 : x; and x = 1698, and the total acidity per hour to , i. e., 70.7. The results obtained can also be expressed in terms of hydrochloric acid, 100 mgrms. of the diacid phosphates corresponding to 102.8 mgrms. of hydrochloric acid. This mode of indicating the total acidity of the urine would actually be the better. If the urine should be alkaline and cloudy, the sediment is first dissolved by carefully adding a one-tenth or one-fourth normal solu- tion of hydrochloric acid, the amount added being then deducted from the total acidity. Should negative values be found, these could be expressed* in terms of sodium hydrate.^ With this method a complete revision of all the work previously done will be necessary. The results given above have reference only to the old method of titration with a one-tenth normal solution of sodium hydrate. CHEMISTRY OF THE URINE. General Chemical Composition of the Urine. — A general idea of the chemical composition of the urine and the quantitative varia- tions of the individual components may be formed from the follow- ing table, which I hq,ve constructed from analyses made in my labor- atory. The individuals from which the urines were obtained were adults, and their general mode of life, as regards diet, exercise, etc., was that of the average American city-dweller. In addition, the following substances may be encountered under pathological con- ditions : serum-albumin, serum-globulin, albumoses, mucin (nucleo- albumin), glucose, lactose, inosit, dextrin, biliary constituents, viz., bile-acids and bile-pigments, blood-pigments, melanin, leucin, tyro- sin, oxybutyric acid, allantoin, fat, lecithin, cholesterin, acetone, alcohol, Baumstark's substance, urocaninic acid, cystin, hydrogen sulphide, and still others. ' The urine is carefully guarded against ammoniacal decomposition by the addition, to the first portion voided, of from 20 to 25 c.c. of a solution of 10 grammes of oil of peppermint to 100 c.c. of alcohol ; or, a few cubic centimeters of chloroform are added, which answer the same purpose. 316 THE URINE. Analysis of Urine. Water 1 200 -1700 grammes. Solids 60.0 " Inorganic solids 25.0 -26.0 " Sulphuric acid (H^SO,) 2.0-2.5 Phosphoric acid (P-O.) 2.5-3.5 " Chlorine (NaCl) 10.0 -15.0 " Potassium (KjO) 3.3 " Calcium (CaO) 0.2-0.4 " Magnesium (MgO) 0.5 " Ammonia {NH3) 0.7 " Fluorides, nitrates, etc 0.2 " Organic solids 20.0 -35.0 " Urea 20.0 -30.0 « Uric acid 0.2-1.0 " Xanthin bases 1 .0 " Kreatinin 0.05- 0.08 " Oxalic acid 0.05 " Conjugate sulphates 0.12- 0.25 Hippurio acid 0.65- 0.7 " Volatile fatty acid 0.05 " Other organic solids 2.S " Quantitative Estimation of the Mineral Ash of the Urine. — In order to estimate the amount of mineral ash in the urine the following method may be employed : Fifty c.e. of urine are evaporated to dryness in a weighed porce- lain dish, at a temperature of 100° C, and then heated, while Fig. 80. I tm M Desiccator. (W. Simon. covered, over the free flame until gases cease to be evolved, care being taken not to heat too strongly in order to avoid sputtering. The residue is taken up with distilled boiling water, and, after standing, filtered through a Schleicher and Schiill's filter, the weight of the ash of which is known. The dish and the contents of the filter are well washed with hot water. Filtrate and washings are set aside and the dish and filter dried in the oven at 115° C. The CHEMISTRY OF THE URINE. 317 filter is now placed in the dish and slowly incinerated. So soon as the ash has turned white the filtrate and washings are placed in the same dish, evaporated at 100° C, and then carefuUy heated over the free flame. Upon cooling in the desiccator (Fig. 80) the dish with its contents is weighed, the difference between its present and previous weight indicating the quantity of ash contained in 50 c.c. of urine. Precautions : 1. Care should be taken to allow the dish to be- come faintly red only for a moment, as some of the chlorine is otherwise volatilized. Some phosphoric acid may also escape, and too strong a heat, moreover, may cause the transformation of sul- phates into sulphides, the organic material present acting as a reducing agent. 2. If the organic ash is not completely incinerated, it is best to allow the dish to cool and then to moisten the ash with a few drops of dilute sulphuric acid, when the heating is continued. The Chlorides. The chlorides which are excreted in the urine are derived from the food. As they are thus present in a much larger amount than all other inorganic salts combined, and in quantity more than suf- ficient to supply the needs of the body-economy, the relatively large amount of chlorides found in the urine under physiological condi- tions, as compared with the other inorganic constituents, is readily explained. Of the alkalies in the urine, sodium in combination with chlorine exists in greatest amount, and for clinical purposes it is most con- venient to calculate the total quantity of chlorides found in terms of sodium chloride ; a small proportion also occurs combined with potassium, ammonium, calcium, and magnesium. Prom 11 to 15 grammes of sodium chloride, representing the total quantity of chlorine, are normally eliminated in the twenty- four hours, the amount depending, of course, directly upon that contained in the food ingested. If the amount of nourishment is diminished, a decrease in the elimination of the chlorides is observed. If this is carried to the point of starvation, the chlorides disappear almost entirely from the urine, the traces remaining being derived from the body-fluids. The lattei* retain tenaciously a certain amount, which differs but slightly from that normally present. If at this stage food containing sodium chloride is again taken, a portion will be retained in the body until the original equilibrium is restored. A similar retention may be observed for a few days following the ingestion of large quantities of water, which causes an increased elimination of chlorides. This tenacity on the part of the body in retaining sodium chloride 318 THE URINE. is strikingly seen when the potassium salt is substituted for the sodium salt ; in this case the amount of the sodium in the serum of the blood will be found to vary very slightly. It has also been shown that the excretion of sodium chloride can be increased very materially by the ingestion of potassium salts, notably the neutral potassium phosphate (KgHPO^). This is supposed to decompose the sodium chloride present in the serum, resulting in the formation of potassium chloride and neutral sodium phosphate, which are both eliminated as foreign material ; a point is finally reached, however, when the sodium chloride ceases to be excreted. This provision of the economy, in virtue of which an increase in the elimination of the salt is followed by its retention, and a pre- vious retention by an increased elimination, is supposed to be refer- able to the albuminous metabolism taking place in the body. It may be stated, as a general rule, that any increase in the amount of circulating albumin will be followed by an increased elimination of chlorides, these having been previously retained by the albuminous bodies in consequence of the great af&nity which exists between them. At the same time the elimination of the chlorides is influ- enced by the quantity of urine excreted, increasing and decreasing with its volume. Pathologically the excretion of the chlorides may vary within wide limits, diminishing on the one hand to zero and increasing on the other to 50 grammes or more in the twenty-four hours. A marked diminution, which in some cases may go on to a total absence, was formerly thought to be pathognomonic of acute croup- ous pneumonia.^ More modern investigations, however, have shown that such a condition occurs to a greater or less degree in most acute febrile diseases, such as scarlatina, roseola, variola, typhus and typhoid fevers, recurrens, and acute yellow atrophy. The explanation of this phenomenon must be sought for, first, in a diminished ingestion of chlorides ; secondly, in a retention of these in the blood, which probably is associated with an increase in the amount of the circulating albumin ; thirdly, in a diminished renal secretion of water ; fourthly, in a possible elimination of a portion of the chlorides through other channels, as in cases of severe diar- rhoea, the formation of serous exudates, etc.^ Intermittent fever appears to be an exception to this rule ; usually it is true the chlorides are diminished, but not to the extent seen in the other diseases mentioned. They have, moreover, been found to increase during and sometimes immediately after a paroxysm, this increase being, of course, followed by a corresponding diminution. The chlorides are diminished in all acute and chronic renal dis- 1 Eettenbaoher, Wion. med. Zeit., 1850, p. 373. Heller, Heller's Arcliiv, 1844, vol. i. p. 23. ' Salkowskl u. Leube, Lehre voni Haru, 1882, p. 174. CHEMISTRY OF THE URINE. 319 eases associated with albuminuria, owing to some extent at least to a diminished secretion of water.' In all cases of carcinoma of the stomach, and in chronic hyper- secretion associated with dilatation, a decrease is also observed, which in certain cases of hypersecretion and hyperacidity, the result of gas- tric ulcer, may go on to a total absence.^ In anaemic conditions the chlorides are likewise diminished, as also in rickets. In melancholia and idiocy a striking decrease is observed ; in dementia, chorea, and pseudohypertrophic paralysis this is less marked. A total absence has been noted in pemphigus foliaceus, and a considerable diminution in the beginning of impet- igo, as also in chronic lead poisoning. The chlorides are found in increased amount, on the other hand, in all conditions in which retention has previously occurred, chief among these being the acute febrile diseases and cases in which a re- sorption of exudates and transudates, associated with an increased diuresis, is taking place. A marked increase has also been noted in some cases of diabetes insipidus, in which 29 grammes have been eliminated in the twenty-four hours.* A similar increase may occur in prurigo, in which, in one instance, 29.6 grammes were passed in twenty-four hours.* In cases of general paresis, during the first stage, an increased elimination goes hand in hand with an in- creased ingestion of food. In epilepsy the polyuria following the attacks is associated with an increase in the chlorides. Of drugs, certain diuretics, and some of the potassium salts, as has been mentioned, produce an increase : the chlorine contained in chloroform, whether administered internally or as an anaesthetic, is in part excreted in the form of a chloride. Salicylic acid, on the other hand, is said to cause a temporary diminution. It is of practical importance to note that in acute febrile diseases the diminution in the chlorides appears to vary with the intensity of the disease, a decrease to 0.05 gramme pro die justifying the con- clusion that the case under observation is of extreme gravity. It may at times also indicate the previous occurrence of severe diarrhoea or the formation of exudates of considerable extent. A continued increase, on the other hand, should lead to the conclusion that the patient's condition is improving. The elimination of the chlorides also furnishes a fair index to the digestive powers of the patient. This rule also holds good for most chronic diseases. All other causes which might lead to an increase or decrease being eliminated, an excretion of from 10 to 15 grammes indicates a fair condition of the appetite and a normal digestive power, a decrease being associated with the reverse. ' Eohmann, Zeit. f. klin. Med., 1886, vol. i. p. 513. 2 Gluzinski, Berlin, med. Woch., 1887, vol. xxiv. p. 983. ^ Oppenheim, Zeit. f. klin. Med., vol. vl. * V. Brueff, Wien. med. Woch., 1871, p. 552. 320 THE URINE. An increased elimination of chlorides occurring in cases of oedema) and associated with the existence of serous exudates, is always of good prognostic omen, pointing to a resorption of the fluid. A continued elimination of more than 1 5 to 20 grammes, all other causes being excluded, may be considered as pathognomonic of dia- betes insipidus. Test for Chlorides in the Urine. — The recognition of the chlo- rides in the urine is based upon the fact that the addition of a solu- tion of silver nitrate causes their precipitation, the reaction taking place according to the equation AgNOs + NaCl = AgCl + NaNO,. The silver chloride thus formed is insoluble in nitric acid. The test is made in the following manner : after having removed any albumin that may be present, according to methods given else- where (see Albumin), a few cubic centimeters of urine are acidified in a test-tube with about 10 drops of pure nitric acid, and treated with a few cubic centimeters of silver nitrate solution (1 ; 20). The occurrence of a white precipitate indicates the presence of chlorides. An idea may be formed at the same time of the quantity present ; the occurrence of a heavy, caseous precipitate points to a large amount. Quantitative Estimation of the Chlorides by the Method of Salkowski-Volhard.^ — ^When a solution of silver nitrate acidified with nitric acid is treated with a solution of potassium sulphocyanide or ammonium sulphocyanide, in the presence of a ferric salt, the potassium sulphocyanide first causes the precipitation of white silver sulphocyanide, which, like silver chloride, is insoluble in nitric acid : AgNOs + KSCN = AgSCN + KNO3. As soon as every trace ■ of silver is precipitated, it combines with the ferric salt to form ferric sulphocyanide, which is of a blood-red color : 6KSCN + Fe2(S04)3 = Fe2(SCN)e + SK^SOi. If the potassium sulphocyanide solution is of known strength, it is possible to estimate accurately the amount of silver present in the solution, the ferric salt serving as an indicator of the end of the re- action between the silver and the potassium sulphocyanide. Application to the urine : to urine which has been acidified with nitric acid an excess of a silver solution of known strength is added, and the silver not used in the precipitation of the chlorides then esti- mated as indicated above. The difference between the quantity thus found and the total amount used will be that consumed in the pre- 1 E. Salkowskl, Zeit. f. physiol. Chem., vol. i. p. 16, and vol. il. p. 397. CHEMISTRY OF THE URINE. 321 cipitation of the clilorides, from which, knowing the strength of the silver solution, its equivalent in terms of sodium chloride is readily determined. Reagents required : 1. A solution of silver nitrate of such strength that each cubic centimeter shall correspond to 0.01 gramme of sodium chloride. 2. A solution of potassium sulphocyanide of such strength that 25 c.c. shall correspond to 10 c.c. of the silver nitrate solution. 3. A solution of a ferric salt, such as ammonio-ferric alum, satu- rated at ordinary temperature. 4. Nitric acid (specific gravity 1.2). Preparation of these solutions : 1. As pointed out, the silver nitrate solution is made of such strength that each cubic centimeter shall correspond to 0.01 gramme of sodium chloride ; in other words, a standard solution is employed. The silver nitrate must be pure, and it is best to use the crystal- lized salt, and not the sticks wrapped in paper, which always contain reduced silver. In order to test the purity of the salt, about 1 gramme is dissolved in distilled water, heated to the boiling-point, the silver precipitated by dilute hydrochloric acid and filtered off. When evaporated in a platinum crucible the filtrate should leave either no residue at all or only a very faint one ; otherwise it is necessary to recrystallize the salt until the desired degree of purity is reached. The determination of the quantity to be dissolved in 1000 c.c. of water is based upon the fact that 1 molecule of silver nitrate (molecular weight 170) combines with 1 molecule of sodium chloride (molecular weight 58.5) to form silver chloride and sodium nitrate. As the solution of silver nitrate shall be of such strength that 1 c.c. corresponds to 0.01 gramme of sodium chloride, or 1000 c.c. to 10 grammes, the quantity to be dissolved- in 1000 c.c. is found according to the following equation : 58.5 : 170 : : 10 : X, 58.5 x = 1700, x = 29.059. Theoretically, then, this quantity should be dissolved in 1000 c.c, of water. It is better, however, to dissolve it in a quantity some- what less than 1000 c.c, such as 900 or 950 c.c, as the silver salt contains water of crystallization and the weighed-off quantity would not represent the exact amount required, but less, the correcting of a solution which is too strong being a much simpler matter than that of a solution which is too weak. To make this correction, or, in other words, to bring the solution to its proper strength, 0.16 gramme of sodium chloride which has previously been dried carefully by heating in a platinum crucible, is accurately weighed off, dissolved in a little distilled water, and further diluted to about 100 c.c. To this solution a few drops of a solution 322 THE URINE. of potassium chromate are added, when the mixture is titrated witn the silver solution. The silver nitrate will first precipitate the sodium chloride, and thea combine with the potassium chromate, forming red silver chromate, according to the equation 2AgN03 f KjCrOi = AgjCrO^ + 2KNO3. The slightest orange tinge remaining after stirring indicates the end of the reaction. Were the solution of the silver nitrate of the proper strength, exactly 1 5 c.c. should have been used, as each cubic centimeter shall represent 0.01 gramme of sodium chloride. As a matter of fact, less will in all probability be needed, the solution having been purposely made too strong. Its correction then be- comes a simple matter, as it is merely necessary to determine the degree of dilution required. Supposing that 29.059 grammes of silver nitrate were dissolved in 900 c.c. of water, and that 14.5 c.c. instead of 15 c.c. had been required to precipitate the 0.15 gramme of sodium chloride, it is evident that each 14.5 c.c. of the remaining solution must be diluted with 0.5 c.c. of water. It is, hence, only necessary to divide the number of cubic centimeters of the silver nitrate solution remaining by 14.5 ; the result multiplied by 0.5 represents the amount of water which must be added in order to bring the solution to the required strength. Hence the rule for the correction of a solution which has been found too strong : n in which C represents the number of cubic centimeters of water which must be added to the solution remaining ; iVthe total number of cubic centimeters remaining after titration ; n the number of cubic centimeters consumed in one titration ; and d the difference between the number of cubic centimeters theoretically required and that actually used in one titration. In the example given the equation would then read : „_ 936.5 X 0-5 _ 32 09 14.5 32.29 c.c. of distilled water are added to the remaining 936.5 c.c, when the strength of the solution is tested by a second titration. If the solution is found too weak, it is best to make it too strong, and then to correct as described. 2. Preparation of the potassium sulphocyanide solution : from the equation AgNO., + KSCN = AgSCN + KNO3, it is seen that 1 molecule of silver' nitrate (molecular weight 170) combines with 1 molecule of potassium sulphocyanide (molecular weight 97). The CHEMISTRY OF THE URINE. 323 quantity of the latter to be dissolved in 1000 c.c. of water is then found from the following equation : 170 : 97 : : 11.6236 : a: ; 170 a; = 11.6236 X 97 ; x = 6.6. As potassium sulphocyanide is extremely hygroscopic, a solution is made which is too strong, by dissolving about 10 grammes of the salt in 900 c.c. of distilled water. In order to bring this solution to its proper strength, 10 c.c. of the silver solution are diluted to 100 c.c; 4 c.c. of nitric acid (specific gravity 1.2) and 5 c.c. of the am- monio-ferric alum solution are added, when the mixture is titrated with the potassium sulphocyanide solution ; the end-reaction is recognized by the production of a slightly reddish color, which per- sists on stirring. The sulphocyanide solution having been purposely made too strong, it will be found that less than 25 c.c. are needed to precipitate all the silver present. The quantity of water necessary for dilution is ascertained, as above, according to the formula C=^. n 3. The solution of. ammonio-ferric alum is a solution saturated at ordinary temperatures, care being taken to insure the absence of chlorides inC I HS-^ I COOH COOH. Cystin. Cystein. and I have pointed out elsewhere that cystein may be derived from phenyl-alanin, which latter occurs as a normal decomposition-prod- uct of the proteid molecule. Since putrescin, moreover, may be obtained from ornithin, and this from arginin, which in turn is formed during decomposition of the protamin radicle of the albu- minous molecule, we can readily imagine that both cystin and diamins will result if for any reason the oxidation-processes of the body are seriously impaired. The relation between phenyl-alanin — phenyl- a-amido-propionic acid — and cystein is represented by the formulae: CH3 — CH(NHj)— COOH CHs — C(NH2)HS — COOH. Phenyl-alanin. Cystein. Cystin crystallizes in hexagonal plates which are quite characteristic, and not liljely to be confounded with other crystalline elements that may be present in urinary sediments. If doubt should arise, their solubility in ammonia and hydrochloric acid, and their insolubility in acetic acid, water, alcohol, and ether, will lead to their identifi- cation. The quantitative estimation of cystin is rather unsatisfactory, as no method is known which yields reliable results. On the whole, it is perhaps best to determine the neutral sulphur, and to refer the increase beyond its normal value to the presence of cystin. Quantitative Estimation of the Neutral Sulphur in the Urine. — In 100 c.c. of urine the oxidized sulphur, viz., the mineral and the' CHEMISTRY OF THE URINE. 343 conjugate sulphates, are estimated as described on page 338. In the second portion the total sulphur then is determined, the differ- ence indicating the amount of the neutral sulphur. To determine the total amount of sulphur, 100 c.c. of urine are treated with 12 grammes of a mixture of sodium and potassium carbonate (11 : 14), and evaporated to dryness in a nickel crucible. The residue is fused thoroughly, allowed to cool, and extracted with hot water. The carbonaceous residue is filtered off and the filtrate and washings are treated with a few crystals of potassium permanganate. After heating for about fifteen minutes (more potassium permanga- nate should be added if during this time the solution becomes de- colorized, when heat' is again applied for fifteen minutes), concentrated hydrochloric acid is added until the reaction is distinctly acid. This solution is then brought to the boiling-point and treated with about 20 c.c. of a saturated solution of barium chloride. The barium sulphate thus formed is then collected and weighed as described on page 339. The difference between this result and the first indicates the amount of neutral sulphur. lilTBEATUEE. — E. Salkowski, Virchow's Arohiv, vol. Ixvi. p. 313, and vol. cxxxvii. p. 381. Goldmann u. Baumanu, " Zur Kenutniss der Schwefelhaltigen Verblndungen des Harns," Zeit. f. physiol. Chem., vol. xii. p. 254. E. Salkowski, Virchow's Archiv, vol. Iviii. p. 461. J. Munk, Ibid., vol. Ixix. p. 354; and Deatsch. med. Woch., 1877, No. 46. 0. Schmiedeberg, " Ueber das Vorkommen von Unterscbwefliger Saure im Harn," Arch. d. Heilk., vol. viil. p. 425. A. Strumpell, Ibid., vol. xvii. p. 390. J. Abel, "Ueber das Vorkommen von Ethylsiilfid im Hundeharn," etc., Zeit. f. physiol. Chem., vol. XX. p. 253. (See also Cystinuria and Hydrothionuria.) Urea. Urea is by far the most important nitrogenous constituent of the urine, and normally represents from 85 to 86 per cent, of the total amount of nitrogen which is eliminated by the kidneys. Chemically, it may be regarded as carbamide — i. e., as the amide of carbonic acid — and is represented by the formula C0< It is thus a comparatively simple substance, and the question natu- rally arises : In what relation does urea stand to the highly complex albuminous molecule from which it is derived ? Numerous hypoth- eses have been offered to explain this problem, but, although we are in possession of a number of very suggestive data, a final answer to the question cannot be given at the present time. In all likelihood, however, the urea may originate from the albumins in different ways. During the hydrolytic decomposition of the albumins by acids and alkalies bodies are constantly formed which belong to the class of amido-acids, and these bodies Schultzen and Nencki have accord- ingly regarded as intermediary products in the formation of urea 344 THE URINE. within the tissues also. The most important members of this group are, leucin, tyrosin, glycocoll, asparaginic acid, and gluta- minic acid. They are represented by the formulae : CHjC — Glycocoll, or amido-acetic acid. \COOH CH,, ^CH.CH^.CH.NHj.COOH — Leucin, or amido-capronic acid. /0H(1) ^CH2.CH(NHj).COOH(4) — Tyrosin, or para - oxy - phenyl - amido - propionic acid. C00H.CH2.CH(NHj). COOH — Asparaginic acid, or amido-succinic acid. COOH.CH (NH,). CH2.CHj.COOH— Glntaminic acid, or amido-glutaric acid. When introduced into the mammalian organism from without, the nitrogen of these bodies appears in the urine, to a large extent at least, as urea. An analogous formation from the tissue-albumins was hence also supposed to occur, but nothing is known of the manner of their transformation in the body into urea. Different possibilities suggest themselves. It is thus conceivable that cyanic acid — CONH — may be produced as an intermediary product, and that urea then results through the interaction of two molecules of the substance, in statu nascendi, according to the equation /NH, CONH + CONH + H,0 = C0< " + COj ^NH^ On the other hand, a transformation of the amido-acids into the ammonium salts of the fatty acids .standing next in order in the downward scale may also be imagined. Ammonium carbonate would then result, which, through loss of water, could give rise to urea. In the case of glycocoll this transformation could be repre- sented by the following equations : (1) (2) rHj.NHj.COOH + 20 = NH^.COOH + CO, Amido-acetic acid. Ammonium formate. 2NH,.C00H Ammonium formate. + 20 = ( NHJ 2CO, + HjO + COj Ammonium carbonate. {mi,)fio. /NH, = C0< + 2Hj0 (3) According to Drechsel, further, the amido-acids are transformed into carbonic acid, two molecules of which then unite to form urea, carbon dioxide, and water : /NHj /NHj /NH™ C0< + C0< = C0< ' H- CO, + HjO \0H \0H \NHa CHEMISTRY OF THE URINE. 345 The hypothesis of Schultzen and Nencki regarding the origin of urea from amido-acids is supported by the fact that these substances, when introduced into the mammalian organism from without, are largely transformed into urea during their passage through the body. It is known, moreover, that in certain diseases, such as acute yel- low atrophy, the urea may disappear from the urine almost entirely, its place being taken by leucin and tyrosin. In other conditions, however, in which the formation of urea is even more seriously impaired, leucin and tyrosin do not appear in the urine, and there is a growing tendency among physiologists at the present time to abandon the older view of Schultzen and Nencki, and to explain the apparently vicarious elimination of the amido-acids in acute yellow atrophy upon a different basis. Leucin and tyrosin are normally scarcely ever encountered in the mammalian organism, and the opinion now prevails that the greater portion of the nitro- gen which is to be eliminated from the body leaves the tissues as the ammonium salt of paralactic acid. In the liver this is trans- formed into ammonium carbonate, from which urea then results synthetically, with the intermediary formation of ammonium car- bamate. This transition may be represented by the equations : (NH,),C03 = = C0< Ammonium carbonate. Ammonium carbamate. (2) C0< =C0< +HjO Ammonium Urea, carbamate. This hypothesis has much in its favor. We thus find that after extirpation of the liver in geese the uric acid, which in birds plays the same part as the urea in mammals, disappears, and is largely replaced by ammonium lactate. In diseases of the liver, moreover, in which an extensive destruction of the parenchyma is taking place, as in some cases of acute yellow atrophy, in phosphorus poisoning, etc., the elimination of urea is diminished, and in its place a cor- responding amount of ammonia in combination with lactic acid is found. In dogs in which the liver has been in part excluded from the general circulation by the establishment of an Eck-fistula, and in which the hepatic artery has at the same time been ligated, the elimination of urea is much diminished, while that of ammonia increases rapidly so soon as the first symptoms of illness appear in the animals. In such cases, owing to the incomplete isolation of the organ, ammonium carbamate appears in the urine, instead of ammonium lactate. From these observations it is apparent also that the synthesis of urea takes place in the liver. This is further 346 THE URINE. proved by the fact that oh transfusion of isolated livers of dogs with blood to which ammonium carbonate or ammonium lactate has been added, urea is formed as a result. In other organs of the body this synthesis apparently does not occur, but there is evidence to show that at least a small amount of urea originates elsewhere within the body through processes of hydrolysis. This amount, however, is unquestionably slight. That a fraction, moreover, is formed from uric acid, and in the last instance from the xanthin- bases through processes of oxidation, can scarcely be doubted, but this transformation apparently also takes place in the liver.* Before going on to a consideration of the quantitative excretion of urea in health and disease it will be well to form an idea of its ultimate sources. To this end, the theory of Voit^ should be recalled, according to which, albuminous material exists in the body in two different forms — i. e., as organized albumin, which is built up in the form of the tissues of the body, and as unorganized albu- min or circulating albumin, which must be regarded in a manner as a reserve, to be used in tissue-repair or to be broken down if not used, and to be replaced by the proteids ingested with the next meal. It may hence be said that, as in the case of the mineral constituents of the urine, the urea is referable on the one hand to the proteids of the food, and on the other to the proteids of the body-tissues. It is clear then that elimination of urea will continue during starvation. It has been stated that 84 to 86.6 per cent, of all the nitrogen eliminated in the urine is in the form of urea, the remaining 13.4 per cent, being excreted as uric acid, hippuric acid, kreatinin, xanthin-bases, etc. It might hence be supposed that an accurate idea of the degree of tissue-destruction could be formed from a quantitative estimation of urea. This, however, is not the case, and especially in pathological conditions, as the quantitative relations existing between the excretion of urea and the remaining nitrogenous constituents are subject to wide variation. In acute yellow atrophy, for example, as pointed out above, urea may disappear entirely from the urine, the nitrogen being eliminated in the form of other com- pounds. Whenever it becomes desirable then to gain an accurate insight into the degree of proteid-destruction or proteid-assimilation — in other words, into the nitrogenous metabolism — taking place in the body, it is necessary to resort to a quantitative determination of 'Tlie origin of urea: O. Schultzen u. M. Nenoki. Zeit. f. Biol., 1872, vol. viii. p. 124. E. Salkowski, Zeit. f. physiol. Chem., 1879, vol. iv. p. 100. v. Knieriem, Zeit. f. Biol., 1874, vol. X. p. 279. E. Salkowski, Zeit. f. physiol. Cheiii., 1877, vol. i. p. 38. Hoppe- Seyler, Physiol. Chem., 1881, p. 810. Drochsel, Jour. f. prakt. Chem., vol. xv. p. 417, vol. xvi. pp. 169 and 180, and vol. xxii. p. 476. M. Hahn, V. Massen, M. Nencki, and J. Pawlow, " Lii fistula d'Eck." etc., Arch. d. Sci. biol. de St. Petersburg, 1892, vol. i. Seat of formation ; W. v. Schroder, Arch. f. exper. Path. u. I'harmakol., 1882, vol. XV. p. 364. W. Salomon, Virchow's Archiv, 1H84, vol. xovii. p. 149. Minkowski, " Ueber d. Einfluss d. Lelierextirpation auf d. Stoifwpchsel, " Arch. f. exper. Path, u, Pharmakol., 1888, vol. xxi. p. 41, and 1893, vol, x.\xi. p. 214. '' C. Voit, Physiol, d. allg. StotTwechsels u, d. Erniihrung. Herman's Handbuch d. Phvsiol., 1881, vol. vi. I. p. 301. CHEMISTRY OF THE VBINE. 347 the total amount of nitrogen excreted by the kidneys ; the quantity found is then conveniently expressed in terms of urea. At the same time it is customary to express the amount of proteid tissue which is destroyed, as muscle-tissue, as this serves as a fair type of body-tissue in general. As 100 grammes of lean muscle-tissue contain about 3.4 grammes of nitrogen, corresponding to 7.286 grammes of urea, 1, gramme of the latter is equivalent to 13.72 grammes of muscle-tissue. It is, hence, only necessary to multiply the quantity of urea eliminated in the twenty-four hours, corresponding to the total amount of nitrogen found, by 13.72, in order to obtain an idea of the extent of albu- minous destruction taking place in the body. If accurate results are desired, it becomes necessary to determine also the amount of nitrogen eliminated in the feces, a know^ledge of the quantity in the food ingested being, of course, presupposed. With all these data given, the nitrogenous metabolism of the body can be accurately controlled. Example. — A patient eliminates 50 grammes of urea in twenty- four hours ; these 50 grammes correspond to 50 X 13.72 — i. e., 686 grammes of lean muscle-tissue ; on the other hand, he ingests an amount of nitrogenous material corresponding to only 10 grammes of urea, equivalent to 10 X 13.72 — i. e., 137.2 grammes of muscle- tissue. The difference between the amount ingested and that ex- creted in this case — i. e., 548.8 grammes — must be referable to the destruction of organized albumin. When the amount of nitrogen eliminated is equivalent to that in- gested, nitrogenous equilibrium is said to exist. A healthy person is approximately in this condition. It has been pointed out that during starvation urea is still elimi- nated from the body, although in diminished amount. The question now arises, Whq,t happens if at this time an amount of nitrogenous food is given which corresponds exactly in amount to that elimi- nated? Under such conditions an increased elimination of nitrogen takes place, all of the nitrogen ingested, in addition to that resulting from a breaking down of body-tissues, being excreted. The amount of nitrogen referable to the latter source, however, is somewhat less than that eliminated in the total absence of food. Unless starvation has been pushed too far, the body accommodates itself to the amount of food thus given and nitrogenous equilibrium is restored. If more food is allowed, an increased elimination results, which again leads to a condition of nitrogenous equilibrium, different levels, so to speak, being possible. This is well illustrated by comparing the condition of the poorly nourished North German laboring population with that of the well-fed merchants, the excretion of urea in the former amounting to 17.5 to 33.5 grammes, and in the latter to 30 or even 40 grammes. 348 THE URINE. It is apparent, then, that the elimination of urea, and of nitrogen in general, is subject to great variation, depending upon the amount ingested and that resulting from tissue-destruction, which in turn is influenced largely by the body-weight. A statement in figures expressing the daily elimination of urea and of nitrogen would, hence, be of very little value, especially in pathological conditions, in which the amount of nitrogen ingested is frequently very small. The elimination of nitrogen should hence always be compared with the amount ingested, for which purpose the tables of Konig' will be found most convenient. At the same time it must be remem- bered that not all the nitrogen taken into the body as food under- goes resorption, and that a variable amount, which in disease may be considerable, is eliminated with the feces, so that in accurate work this nitrogen also must be taken into account. In order to obviate the tedious estimation of nitrogen in the feces, it has been proposed to determine the standard amount of urea which should appear in the urine of a healthy person under different forms of diet. Such experiments, of course, presuppose the control-person to be in a condition of nitrogenous equilibrium, which, from what has been said above, is readily accomplished, as the human body adapts itself with ease to different forms of diet. In private practice, however, such a procedure would be difficult, but here approximate results can be obtained from a parallel estimation of the chlorides. In health the elimination of the chlorides may be placed at about one- half of the urea. Whenever the nitrogen resulting from tissue- destruction is in excess of that referable to the proteids ingested, this relation between the excretion of chlorides and urea will be disturbed, as the tissues of the body contain very little sodium chloride. When- ever the amount of urea is in excess of the normal amount of chlo- rides, as indicated above, an increased tissue-destruction may be in- ferred, and vice versa. If, on the other hand, the chlorides are present in diminished amount, the conclusion may be drawn that a retention of albumins is taking place in the body ; this is observed frequently during convalescence from acute febrile diseases. An increase in the a/mount of urea, and, as a matter of fact, of all the nitrogenous constituents, is observed especially in the acute febrile diseases, notwithstanding the diminished ingestion of nitrog- enous material, and is due to the greatly increased tissue-destruc- tion.^ An excretion of 50 grammes or more is here frequently observed. Formerly it was thought that the fever itself was re- sponsible for this increased elimination. But this view became untenable when it was shown that the excretion of urea in the beginning of a febrile attack is not proportionate to the height 1 J. Konig, Chemie d. menschlichen Nahrungs u. Genussmittel, Berlin, 1893. 2 Vogel, Zeit. f. rationclle Med., N. F., vol. iv. p. 362. Huppert, Arch. d. Heilk., vol. vii. p. 1. L6bisch,Wlen. med. Presse, 1889, vol. xxxix. p. 1521. Huppert u. Eiesellt, Arch. d. Heilk., vol. x. p. 329. Bauer u. Kunstle, Doutsch. Arch. f. klin. Med., vol. xxiv. p. 53. CHEMISTRY OF THE VRINE. 349 of the temperature, reaching its highest point only when the fever has been continuous for several days. Still larger amounts, more- over, may be eliminated when the fever is abating. Similar obser- vations have since been made. An increased elimination of nitrogen may also be noted in almost every case of ague preceding the onset of the fever. The latter, therefore, cannot be the only factor which causes the increased excretion of urea, and it has been suggested that the cells of the body have lost the power of taking up nitrogen. The question, however, whether this is dependent upon the increase in temperature or the action of certain toxic substances circulating in the blood, or upon both, still remains unanswered. The large increase in the elimination of nitrogen in febrile dis- eases is especially striking in those which end by crisis. This is notably the case in pneumonia, in which it may persist for two or three days after the occurrence of the crisis. The assumption of an underlying insufficiency on the part of the cells furnishes a very sat- isfactory explanation for the continued increased elimination of urea. An increase beyond the amount eliminated during the febrile stage is possibly owing to a retention analogous to that of the mineral constituents of the urine. Apparently, the only exception to the rule that the amount of urea is increased in acute febrile diseases, is acute yellow atro- phy, in which the excretion of urea is not only greatly diminished, but may cease altogether, its place being taken by other nitrogenous bodies, such as ammonium lactate, leucin, and tyrosin. Among afebrile diseases in which an increased elimination of urea has been noted, may be mentioned the ordinary forms of diabetes mellitus, in which the highest figures have been obtained, viz., 150 grammes or more pro die. This is, in all probability, explained, in part at least, by the ingestion of excessive amounts of proteid food by such patients, but carefully conducted experiments seem to show that a not inconsiderable portion of the urea is directly referable to increased tissue-destruction. The cases described by Hirschfeld,' however, which will be considered later on, form an exception to this rule. An increase is observed also in dyspnoeic conditions, and particu- larly in pneumonia, in which it is most marked on the day following the greatest difficulty in breathing. These observations, however, are not free from objections, as an increase has also been noted in conditions of apncea. A moderate increase has been found in cases of pernicious anamia, in severe cases of leukaemia, scurvy, minor chorea, and paralysis agitans. Observations made in cases of hystero-epilepsy have given rise to conflicting results. It is claimed, on the one hand, that the 'F. Hirschfeld, "XJeber eine neue klin. Form d. Diabetes," Zelt. f. klin. Med., vol. xix. pp. 294 and 325. 350 THE UBINK excretion of urea is diminished following convulsive seizures of a hystero-epileptic nature, in contradistinction to an increased elimina- tion following true epileptic attacks. In cases of functional albuminuria associated with an increased elimination of uric acid or oxalic acid, or of both, as well as in numerous cases of gastro-intestinal disease, I have observed an in- creased elimination of urea, and believe that in the treatment of these diseases a systematic study of the excretion of nitrogen is of funda- mental importance. Of drugs, an increased elimination is produced by coffee, caffein, morphin, codein, ammonium chloride, sodium and potassium chlo- rides, lithium carbonate, following the ingestion of large amounts of water, etc. The data concerning the action of quinin, salicylic acid, cold baths, etc., are conflicting. A large increase has been observed in cases of phosphorus poisoning. Electricity also appears to exert a marked influence upon the excretion of urea, producing an increased elimination. The diminished elimination of urea observed in certain diseases of the liver,' notably in acute yellow atrophy, carcinoma, cirrhosis, and even in Weyl's disease, is of especial interest, and is in perfect accord with the theory that the liver is the main seat of its pro- duction. As has been stated, urea may disappear altogether from the urine in acute yellow atrophy and also in Weyl's disease, notwithstanding the frequently not inconsiderable degree of fever. In cirrhosis, hypersemia of the portal system has been thought to cause the dimi- nution, which may be increased further in some cases by the occur- rence of ascites. In short, the factors which may be regarded as causing a diminished elimination of urea in hepatic diseases may be summarized under the following headings : 1. Destruction of hepatic parenchyma. 2. Diminished velocity of the flow of blood through the liver. 3. Insufficient excretion of bile and coincident digestive disturb- ances. Whenever there is disease affecting that portion of the renal parenchyma which is concerned especially in the elimination of urea, a diminished amount will, of course, be met with, and carefully con- ducted observations upon the excretion of the various urinary constituents are here of considerable value from a diagnostic as well as a therapeutic standpoint. As the glomeruli of the kid- neys are mainly concerned in the elimination of water and salts from the blood, and as the striated epithelium of the convoluted tubules appears to provide for the excretion of urea, the elimination ' Hallerworden, Arch. f. exper. Path. u. Pharmakol., vol. xii. Weintraiid, Ibid., vol. xxxi. Stadelmann, Deutsch. Arch. f. kliu. Med., vol. xxxiii. Fawitzki, Ibid., vol. xlv. Frankel, Berlin, kliii. Woch., 1878 and 1892. v. Noorden, Lehrbuch d. Path, d. Stoffwechsels, p. 287. , CHEMISTRY OF THE URINE. 351 of a fair amount of the latter with a diminished elimination of salts, the phosphates being of especial interest, as they are derived to a large extent from albuminous material, would point more particu- larly to glomerular disease. On the other hand, a fair excretion of phosphates and a diminished excretion of urea would be indicative of tubular disease. Whenever glomeruli and tubuli contorti are equally diseased an insufficient elimination of both phosphates and urea will be observed. While, as a rule, the excretion of urea is greatly increased in diabetes mellitus, certain cases, which have been elaborately described by Hirschfeld,' must be excepted. His researches have established beyond a doubt that the resorption of nitrogenous material from. the intestines may be very much below normal, and with it the elimina- tion of urea. Upon these grounds he has advocated the recognition of a distinct form of diabetes, which is characterized by a com- paratively rapid course, the occurrence of colicky abdominal pains before or at the onset of the diabetic symptoms proper, the existence of pancreatic lesions in a certain proportion of the cases, a more moderate degree of polyuria, etc. In mental diseases a diminished excretion of urea has been ob- served in melancholia and in the more advanced stages of general paresis, while an increase is associated with the increased ingestion of food during the first stage of profound dementia. Following epileptic, cataleptic, and hysterical seizures, as well as in pseudohypertrophic paralysis, a decrease has been noted by some observers. The diminished excretion observed in Addison's disease has also been regarded as of nervous origin. All forms of chronic, non-progressive ansemia are associated with a decrease, as are also osteomalacia, impetigo, lepra, chronic rheu- matism, etc. In chronic lead poisoning the elimination of urea may be greatly diminished. Little is known of the influence of drugs in bringing about a diminished excretion of urea. In conclusion, the relation existing between phosphatic excretion and that of nitrogen should be especially noted, for a consideration of which, see page 329. Properties of Urea. — Urea crystallizes in two forms, viz., in long, fine, white needles if rapidly formed, or in long, colorless, quadratic rhombic prisms when allowed to crystallize gradually from its solutions. At 100° C it begins to show signs of decomposition ; at 130° to 132° C. it melts ; and when heated still further it is decomposed into cyanic acid and ammonia, of which the former is immediately trans- formed into its polymeric compound, cyanuric acid. The reaction which takes place is represented by the equations : ^ Loc. cit. 352 THE UB.INE. (2) 3C0NH =C303NsH3. (1) C0< =CONH + NH„ Biuret is formed as an intermediary product during this decom- position, 2 molecules of urea yielding 1 molecule of ammonia and 1 molecule of biuret, as represented in the equation ^1^ = NH + NH,. As this substance, obtained on dissolving the residue remaining after all the ammonia has been driven off by careful heating, yields a beautiful reddish-violet color when a drop or two of a very dilute solution of cupric sulphate is added to its solution alkalinized with sodium hydrate, this reaction may be employed as a test in the detection of urea (Biuret test). Urea is readily soluble in water, fairly so in alcohol, and insoluble in anhydrous ether and benzol. The aqueous solution of urea is neutral in reaction, but this substance combines with acids, bases, and salts to form molecular compounds. Of special interest are the compounds of urea with nitric acid, oxalic acid, and mercuric nitrate. Urea nitrate, CON2H^.HN03, crystallizes in two different forms : in thin rhombic or six-sided colorless plates, which are frequently Fig. 83. Urea nitrate crystals. (Kebkenbtjrg, after Kuhne.) observed arranged like shingles one on top of the other when rapidly formed (Fig. 83), while larger and thicker rhombic columns or plates are obtained if the process of crystallization is allowed to proceed more slowly. Urea nitrate is readily soluble in distilled water, while CHEMISTRY OF THE URINE. 363 in alcohol and in water containing nitric acid it dissolves with difficulty. Upon heating, it evaporates without leaving a residue. Urea oxalate, CON2H4.C2H2O4, crystallizes in rhombic or six-sided prisms or plates (Fig. 84), which are less soluble in water than the nitrate ; in alcohol and in water containing oxalic acid it is only imperfectly soluble. Fig. 84. Urea oxalate crystals. (Keukenbekg, after KtiHNE.) With mercuric nitrate urea forms three different compounds, accord- ing to the concentration of the two solutions, viz., (CON2H4)Hg2(N03)^ (CON2H,).Hg3(N03)„ and (CON,H,)2.Hg(N03)2 + 3HgO. The lat- ter compound is of special importance, as Liebig's quantitative esti- mation of urea was based upon its formation. It results when a 2 per cent, solution of urea is treated with a dilute solution of mer- curic nitrate, the reaction taking place according to the equation 2CON,H4 + 4Hg (NOs), + 3H,0 = [2(CON,H,),Hg(N03), + 3HgO] + 6HNO3. Very important is the behavior of urea when treated with a solu- tion of sodium hypochlorite or hypobromite, the most usual method of estimating urea being based upon this reaction, which may be represented by the equation CONjHj + 3NaOBr = 3NaBr + 2N+CO2 + 2H2O. In the chapter on Reaction it was pointed out that urine gradually undergoes ammoniacal decomposition when exposed to the air, and that this process is due to the action of a non-organized ferment ; the ammonia is liberated according to the equation TTFT Co/ ' + H20 = 2NH3 + C02. This decomposition may also be effected by heating a watery solu- tion of urea in a sealed tube to 100° C 23 354 THE VBINE. Separation of Urea from the Urine. — Fifty to 100 c.c. of urine are evaporated to a syrupy consistence upon a water-bath, and extracted with 100 to 150 c.c. of strong alcohol, by rubbing up the residue, while still hot, with the alcohol. Upon cooling, the mixture is filtered, the alcohol evaporated, and the residue treated with pure cold nitric acid. Urea nitrate then separates out either immediately or on standing. After twenty-four hours the crystalline mass is collected on a muslin filter, well strained, and freed from liquid by placing it upon plates of clay. The material is then dissolved in hot water, and the solution, if strongly colored, gently warmed with animal charcoal and filtered. This solution is neutralized with barium carbonate, and rendered alkaline with barium hydrate. The urea nitrate is thus decomposed, barium nitrate and urea being formed : 2CON2Hj.HN03 +BaC03 = 2CON2H4+ Ba(N03)j + HjO. The barium is now removed by passing a stream of carbon dioxide through the solution and filtering off the precipitate. The filtrate is evaporated until any barium nitrate still remaining crystallizes out. This is removed by decantation, when upon further evapora- tion the urea crystallizes out, and may be dried between layers of filter-paper and recrystallized from 95 to 98 per cent, alcohol. The crystals thus formed may now be subjected to further tests. To this end, a few drops of an aqueous solution are added to a few cubic centimeters of a sodium hypobromite solution, when in the presence of urea bubble* of gas will be given off. With a solution of sodium hypochlorite the same result may be obtained, but in this case the evolution of gas takes place only upon the application of heat. The formation of biuret may also be demonstrated by carefully melting a few of the crystals in a test-tube, dissolving the residue when cool in a little water, and alkalinizing the solution with a little sodium hydrate ; upon the addition of a dilute solution of cupric sulphate a beautiful reddish-violet color will develop, owing to the presence of biuret. The addition of oxalic or nitric acid to a solution of urea will give rise to the formation of urea nitrate and oxalate, as described above. This latter test may very conveniently be made under the micro- scope. A drop of the concentrated solution is placed upon a slide, covered, and a drop of pure nitric acid added from the side. Crystals of urea nitrate will then be seen to separate out, and may be recog- nized by their characteristic shingle-like arrangement (see Fig. 83). When a urine is very rich in urea the mere addition of nitric acid will cause a more or less abundant precipitation of urea nitrate, and with this simple test an idea may even be formed of the amount present. An appearance of hoar-frost is thus noted when not less CHEMISTS Y OF THE URINE. 355 than 25 grammes are present in the liter, while the formation of spangles of urea nitrate requires the presence of at least 45 grammes, and an abundant sediment occurs when 50 grammes or more are present. Quantitative Estimation of Urea. — Hypobromite Method. — The method most commonly used in the clinical laboratory is the one based upon the decomposition of urea into carbon dioxide and nitro- gen in the presence of sodium hypobromite. The reaction takes place according to the equation CONjHi + SNaOBr = NaBr + CO^ + 2HjO + 2N. The carbon dioxide thus formed is absorbed by an excess of sodium hydrate added to the hypobromite solution, while the nitrogen is set free, and can be collected and measured ; the determination of the corresponding amount of urea is then a simple matter. The only solution that is necessary is one of sodium hypobromite containing an excess of sodium hydrate. A 30 per cent, solution of the latter should be kept on hand and the sodium hypobromite solution prepared when required. To this end, 70 c.c. of the sodium hydrate solj^tion are diluted with 180 c.c. of water and treated with 5 c.c. of bromine in a bottle provided with a ground-glass stopper, the mixture being thoroughly shaken until every trace of free bromine has disappeared. The sodium hypobromite solution, if kept in a perfectly dark and cool place, may be preserved for a week or two. The reaction which takes place between the sodium hydrate and the bromine may be represented by the equation 2NaOH + 2Br = NaBr + NaOBr + HjO. Various forms of apparatus, termed ureometers, have been sug- gested for the estimation of urea by this method. One which I have found very satisfactory is represented in Fig. 85. It consists essentially of a burette, C, with an ascending rubber tube attached to the reservoir B, which can be raised or lowered as required for the purpose of equalizing the pressure after collection of the gas. A descending tube leads to a wide-mouthed bottle. A, which con- tains the hypobromite solution. This is closed by a tightly fitting rubber stopper, to which a loop of platinum wire is attached carry- ing a little bucket made of glass or porcelain ; this can be swung from its support by inclining the bottle. Method. — The rubber stopper is removed from the bottle A, and water poured into B until the system BCA is filled to such an ex- tent that the water-level is visible in B above the point where the rubber tube is attached. About 25 to 30 c.c. of the hypobromite solution are placed in the bottle A, and 2 c.c. of urine in the bucket ; this is then attached to the wire loop. The stopper is now 356 THE URINE. Fig. 85. carefully adjusted and the water in B and C brought to the same level, when the first reading is taken. A is then inclined until the bucket drops into the liquid- below. The nitrogen which is liber- ated collects in the burette C ; as a consequence the water falls in C and rises in B. After twenty to thirty minutes the pressure in G is equalized by lowering B until the water in both tubes is at the same level. The sec- ond reading is then taken, the difference between the two indi- cating the volume of nitrogen liberated from 2 c.c. of urine at the temperature of the water in CB, which, as well as the baro- metric pressure, should be pre- viously noted. As the volume of gases is greatly influenced by the tem- perature, the barometric pressure, and the tension of the aqueous vapor, it becomes necessary, in order that the results reached shall be comparable with those obtained by other observers, to reduce the volume of nitrogen actually noted to a certain stand- ard. This has been placed at 0° C. and 760 mercury millimeters pressure, in the absence of moist- ure. This correction is made ac- cording to the following formula : The author's ureometer. F = v.{B — T ) in , ^^ which V represents the corrected 760.(1 + 0.00366.<)' ^ volume of the gas in terms of c.c, v the volume actually ob- served, B the barometric pressure in Hgmm., jPthe tension of the aqueous vapor at the temperature noted, t. The volume of nitrogen observed being thus corrected, the calculation of the corresponding amount of urea is based upon the following considerations : from the formula CON^H^ it is apparent that 2 atoms of nitrogen are contained in 1 molecule of urea ; in other words, that 28 parts by weight of nitrogen correspond to 60 parts by weight of urea. The equivalent of 1 gramme pf urea is then found according to the equation : 60 : 28 : : 1 : a;; and x = 0.46666. The volume corre- sponding to 0.4666 gramme of dry nitrogen at 0° C. and 760 CHEMISTRY OF THE URINE. 357 Hgmm. pressure is 372.7 c.c. It has been found, however, that only 354.3 c.c. of nitrogen are evolved from 1 gramme of urea at best when the hypobromite method is employed. Knowing that 354.3 c.c. of nitrogen correspond to 1 gramme of urea, the amount of urea to which the volume of nitrogen actually observed is refer- able would then be found according to the equation 1 : 354.3 •.-.x-.y; and x = ^ , in which y denotes the number of 354. o cubic centimeters of nitrogen evolved from 2 c.c. of urine, and x the corresponding amount of urea. In order to ascertain the percentage- amount of urea it is only necessary to multiply the figure just obtained by 50. Precautions : 1. The urine must be free from albumin. 2. It should con- tain only about 1 per cent, of urea — i. e., not more than 0.025 gramme in 2 c.c. Whenever a greater amount is noted, therefore, the urine is diluted to the proper degree, due allowance being made in the calculation. In ordinary clinical work the barometric pressure, as well as the tension of the aqueous vapor, may be ignored, and in the accompany- ing tables the corresponding amount of urea may be directly read off at the temperatures 5°, 10°, 15°, 20°, 25°, and 30° C, Of other forms of apparatus, the ureometers devised by Doremus, Green, Marshall, Hiiffner, and Squibb may be mentioned. The latest modification of Dore- mus' apparatus is certainly most convenient, and can be highly rec- ommended. Its general construction is seen in Fig. 86. A small amount of urine is poured into B while the stopcock (C) is closed. This is then opened for a moment and again closed, so as to fill its lumen. The tube A is washed out with water and filled with the hypobromite solution. The tube B is filled with urine, and 1 c.c. (or less, if the urine is concentrated) is allowed to mix with the hypo- bromite solution in A. After all bubbles of gas have disappeared the reading is taken. The degrees marked upon the tube indicate Doremus' ureometer. 358 THE URINE. Ukea. Table for a Temperature of 5° C. 1 ^ A 1^ A 1^ A i^ iV A 1 1.32 1.45 1.58 1.71 1.85 198 2.11 2.24 2.37 2.51 2 2.64 2.77 2.90 3.03 3,17 3.30 3.43 3.56 3,69 3.83 3 3.96 4.09 4.22 4.36 4.49 4.62 4.75 4.88 6,02 6.15 4 5,28 6,41 6.54 5.68 6.S1 5,94 6.07 6.20 6.34 6.47 5 6.60 73 6.87 7.00 7.13 7,26 7.39 7.53 7.66 7.79 6 7.92 8,05 8.19 8.32 8.45 8.53 8.71 8.85 8.98 r.u 7 9.24 9.38 9.51 9.64 9.77 9.90 10.04 10.17 10.30 10.43 8 10.56 10.70 10.83 10.96 11.09 11,22 11.36 11.49 11.62 11.75 9 11,89 12.02 12.15 12,28 12.41 12,55 12.68 12.81 12.94 13.07 10 13.21 13.34 13,47 13.60 13.73 13.87 14.00 14.13 14.26 14.39 11 14.63 14.66 14.79 14.92 16.06 15.19 16.32 15.45 15.1)8 15.72 12 15.85 15.98 16.11 16.24 16.38 16.51 16.64 16.77 16.90 17.04 IS 17.17 17,30 .17.43 17.57 17.70 17.83 17.96 18,09 18,23 18.36 U 18.49 18.62 18.75 18.89 19.02 19.15 19.28 19.41 19..56 19.08 15 19.81 19.94 20,08 20.21 20.34 20.47 20.60 20.74 20.87 21.00 16 21.13 21,26 31,40 21.53 21.66 21.79 21.92 23.06 22,19 22.32 17 22.45 23.69 22,72 22.85 22.98 23.11 23.25 23,38 23.51 23.64 18 23.77 23.91 24,04 24.17 24.30 24.43 24.67 24.70 24.83 24.96 19 25.10 25.23 25.36 26.49 25.62 25.76 25.89 26.02 26,15 26,28 20 26.42 26.55 26.63 26.81 26,94 27.08 27.21 27.34 27.47 27.60 21 27.74 27.87 28.00 2813 28.27 28.40 28.55 28.66 28.79 28.93 22 29.06 29.19 29.82 29.45 29.59 29.72 29.85 29.98 30.11 30 25 23 30.38 30.61 30,64 30.78 30.91 31.04 31.17 31.80 31,44 31„')7 24 31.70 31,83 31.96 32.10 32.23 32:36 32,49 ,'!2.62 32,76 32.89 25 33.02 33.15 33.29 33.42 33.55 33.68 33.81 33.95 34.08 34.21 26 34.34 34.47 34.61 34.74 34.87 35.00 35,13 35.27 35.40 35.53 27 85.66 35.80 35.93 36.06 36.19 36.32 36.46 36 59 36.72 36.85 28 36.98 37.12 37.25 37.38 37.51 37.64 37.78 37.91 38,04 38.17 29 38.81 88.44 38 57 38.70 38.83 38.97 39.10 39.28 39.36 30.49 30 39.63 89.76 39.89 40.02 40.15 40.29 40.42 40,65 40.68 40,81 Urea. Table for a Temperature of 10° C. 1 1^ -h A A A 1^ iV A A 1 1.31 1.43 1.56 1.69 1.82 1.95 2.08 2.21 2,34 2.47 2 2,60 2.73 2.86 2.99 3.12 3.26 3.33 3.61 3.64 3.77 3 3,90 4,03 4.16 4.29 4,42 4.55 4.68 4.81 4.94 6,07 4 5.20 5.33 6.46 5.59 6,72 5.85 5.98 6.11 6.24 6.37 6 6.50 6.33 6.76 6.89 7.02 7.15 7.28 7.41 7..54 7.67 6 7.80 7.93 8.06 8.19 8,32 8.45 8.58 8.71 8.84 8.97 7 9.10 9.23 9.36 9.49 9.62 9.75 9.88 10.01 10.14 10,27 8 10,40 10.53 10.66 10.79 10.92 11,05 11.18 11.31 11.44 11,57 9 11.71 11.84 11.97 12.10 12,23 12.36 12.49 12.62 12.75 12.88 in 13.01 13.14 13.27 13.40 13.53 13,66 13,79 13,92 14,05 14.18 11 14.30 14.44 14.57 14.70 14,83 14.96 15.09 15.22 15.35 15.48 12 15.60 15.74 15.87 16.00 16,13 16.26 16.39 16 62 16.65 16.78 13 16.91 17.04 17.17 17.30 17.43 17..56 17.69 17.82 17.96 18.08 14 18.21 18.34 18.47 18.60 18.73 18.86 18.99 19.12 19,25 19.38 16 19.51 19.64 19.77 19.90 20.03 20.10 20.29 20.42 20.65 20.68 16 20.81 20,94 21.07 21.20 21.33 21.46 21.69 21.72 21.85 21.98 17 22,11 22.24 22.37 22,60 22.63 22,76 22.89 23,02 23.15 23 28 18 23,41 23.54 23.67 23.80 23:93 24.06 24.19 24,82 24.45 24.68 19 24.72 24.85 24 98 2.5.11 26.24 25.37 26.50 25.63 25.76 25,89 20 26.02 26.15 26.28 26.41 26.64 26.67 26.80 26.93 27.06 27.19 21 27.32 27,45 27.58 27.71 27.84 27.97 2810 28.23 28.36 28.49 22 28.62 28.75 28.88 29.01 29.14 29.27 29.40 29.53 29.66 29.79 23 29.92 30,05 30.18 30.81 30.44 80.57 .so,-o 80.83 30.96 31.09 24 31.22 81.35 31.48 31.61 31.74 31.87 32,00 32.1.! 82.26 82,39 25 82,52 32.65 32.78 32.91 83.04 33.17 33,30 3:(,4:! 38.66 33.69 26 33.82 33.96 84.08 84,21 34.34 . 84,47 34.60 84,73 34,86 3499 27 85.12 36.25 86.38 3.5.51 35.64 35.77 35.90 36,03 86.16 36.29 28 36.42 36,6.5 36.68 36.81 36.94 37,07 37.20 37,33 37.46 37.69 29 37.73 37.86 87.99 38.12 38,25 38.88 88.51 38.64 38.77 38.90 30 39.03 39.16 89.29 39.42 89.55 89.68 89.81 39.94 40.07 40,20 CHEMISTRY OF TEE URINE. 359 Ubea. Table for a Temperature of 15° C. 1 iV ^ A A A A A A A 1 1.28 1.41 1.53 1.66 1.79 1.92 2,04 2.17 2.30 2.4S 2 2.56 2.69 2.81 2.94 3.07 3.20 3.33 3.46 ■ 3.58 3.71 3 3.84 3.97 4.10 4.22 4.35 4.48 4.61 4.74 4.87 4.99 4 5.12 6.25 5.38 5.50 6.63 5.76 6.89 6.02 6.14 6.27 6 6.40 6.53 6.60 6.79 6.91 7.04 7.17 7.30 7.43 7.55 6 7.68 7.81 7 94 8.07 8.19 8.32 8.45 8.58 8.71 8.83 7 8.96 9.09 9.22 9.35 9.48 9.60 9.73 9.86 9.99 10.12 8 10.24 10.37 10.60 10.63 10.76 10.88 11.01 11.14 11.27 11.40 9 11.63 11.65 11.78 11.91 12.04 12.17 12.29 12.42 12.65 12.68 10 12.81 12.93 13.06 13.19 13.32 13.45 13.67 13,70 13.83 13.96 11 14.09 U.22 14.34 14.47 14.60 14.73 14.86 14,98 15,11 15.24 12 15.37 15.50 15.62 16.73 1.5.88 16,01 16.14 16.26 16.39 16.52 13 16.66 16.78 16.91 17.03 17.16 17.29 17.42 17.55 17.67 17.80 14 17.93 18.06 18.19 18.31 18.44 18.,57 18.70 18.83 18.95 19.08 15 19.21 19.34 19.47 19.60 19.72 19.85 19.98 20.11 20,24 20,36 16 20.49 20.62 20.75 20.88 21.00 21.13 21,26 21.39 21.52 21.64 17 21.77 21.90 22.03 22.16 22.29 22.41 22.54 22.67 22.80 22.93 18 23.05 23.18 23.31 23.44 23.57 23.69 23.82 23.95 24.08 24.21 19 24.34 24.46 24.69 24.72 24.85 24.98 26.10 25.23 25.36 26,49 20 25.62 26.74 25.87 26.00 26.13 26.26 26.38 26.51 26.64 26.77 21 26.90 27.03 27.15 27.28 27.41 27.64 27.67 27 79 27.92 28.05 23 28.18 28.31 28.43 28.56 28.69 28.82 28.95 29.07 29.20 29.33 23 29.46 29.59 29.72 29.84 29.97 30.10 30.23 30.36 30.48 30.61 24 30.74 30.87 31.00 31.W 31.25 31.38 31.51 31.64 31.76 31.89 25 32.02 32.15 32.28 32.41 32.53 32.66 32.79 32.92 33.05 33.17 26 33.30 33.43 33.56 33.69 33.81 33.94 34.07 34.20 34 33 34.45 27 34.58 34.71 34.84 34.97 35.10 36.42 35.35 35.48 35.61 35.74 28 35,86 35.99 36.12 36.25 36.38 36.50 36.63 36.76 86.89 37.02 29 37.15 37.27 87.40 37.63 37.66 37.79 37.91 38.04 38.17 38.30 30 38.43 38.55 38.68 38.81 38.94 39.07 39.12 39,32 39.45 39.58 Urea. Table for a Temperature op 20° C. 1 A A A A A iV A A A 1 1.26 1.38 1.51 1.63 1.76 1.89 2.01 2.14 2.26 2.39 2 2.52 2.64 2.77 2.90 3.02 3.16 3.27 3.40 3.63 3.65 3 3.78 3.91 4.03 4.16 4.28 4.41 4.54 4.66 4.79 4.91 4 5.04 5.17 5.29 5.42 5.54 5.67 5.80 5.92 6.05 6.17 6 6.30 6.43 6.55 6.68 6.81 6.93 7.06 7.18 7.31 7.44 6 7.66 7.69 7.81 7.94 8.07 8.19 8.32 8.44 8.67 8.70 7 8.82 8.95 9.08 9.20 9.33 9.45 9.58 9.71 9.83 9.96 8 10.08 10.21 10.34 10.46 10.59 10.71 10.84 10.97 11,09 11.22 9 11.35 11.47 11.60 11.72 11.85 11.98 12.10 12.23 12,35 12.48 10 12.61 12.73 12.86 12.98 13.11 13.24 13.36 13.49 13,61 13.74 11 13.87 13.99 14.12 14.25 14.37 14.60 14,62 14.75 14.88 16.00 12 15.13 16.26 15.38 16.61 15.63 15.76 15.88 16.01 16.14 16.26 13 16.39 16.52 16.64 16.77 16,89 17.02 17.15 17,27 17.40 17.52 14 17.65 17.78 17.90 18.03 18.15 18.28 18.41 18.53 18.66 18.78 15 18.91 19.04 19.16 19.29 19.42 19.64 19.67 19.79 19.92 20,05 16 20.17 20.30 20.42 20.55 20.68 20.80 20.93 21.05 21,18 21.31 17 21.43 21.56 21.69 21.81 21.94 22.06 22.19 22.32 22,44 22.57 18 22.69 22.82 22.95 23.07 23.20 23.32 23.45 23.63 23,70 23.83 19 23.96 24.08 24.21 24.33 24.46 24.69 24.71 24.84 24.96 25.09 20 25.22 25.34 25.47 25.59 25.72 25.85 25.97 26,10 26.22 26.35 21 26.48 26.60 26.73 26.86 26.98 27.11 27.23 27.36 27.49 27.61 22 27.74 27.86 27.99 28.12 28.24 28.37 28.49 28.62 28.75 28.87 23 29.00 29.13 29.26 29.38 29.60 29.63 29.76 29.88 30,01 30.13 24 20.26 30.39 30.51 30.64 30.76 30.89 31.02 31.14 31.27 81.39 25 31.52 31.65 31.77 31.90 32.03 32.15 32.28 32.40 32.53 32.66 26 32.78 32.91 33.03 33.16 33.29 33.41 33.64 33.06 33.79 33.92 27 34.04 34.17 34.30 34.42 34.56 34.67 34.80 34.93 35.05 35.18 28 36.30 35.43 35.56 36.68 35.81 35.93 36.06 36.19 36.31 36.44 29 36.57 36.69 36.82 36.94 37.07 37.20 37.32 37.45 37.57 37.70 30 37.83 37.95 38.08 38.20 38.33 38.46 38.58 88.71 38.83 38.96 360 THE URINE. Ueea. Table for a Tempeeatctre or 25° C. 1^ •1^ A A tV io i^ iV ^ 1 1.24 1.36 1.49 1.61 1.73 1.86 1.98 2.11 2.23 2.35 2 2.43 2.60 2.73 2.85 2.97 3.10 3.22 3.3S 3.47 3.59 3 3.72 3.84 3.97 4.09 4.22 4.34 4.46 4.59 4.71 4.84 4 4.96 5.08 5.21 5.33 5.46 5.58 5.70 5.83 6.96 6.08 5 6.20 6.33 6.45 6,67 6.70 6.82 6.95 7.07 7.19 7.32 6 7.44 7.57 7.69 7.81 7.94 8.06 8.19 8.31 8.43 8.50 7 8.68 8.81 8.93 9.06 9.18 9.30 9.43 9.55 9.68 9.80 8 9.92 10.03 10.17 10.30 10.42 10..'i4 10.67 10.79 10.92 10.04 9 11.17 11.29 11.41 11.54 11.66 11.79 11.91 12.03 12.16 12.28 )0 12.41 12.53 12.65 12.78 12.90 13.03 13.15 13.27 13.40 13.62 11 13.65 13.77 13.89 14.02 14.14 14.27 14.39 14.52 14.64 14.76 12 14.89 15.01 16.14 15.26 15.38 15,51 15.63 15.76 15.88 16.00 13 16.13 16.25 16.38 16.50 16.63 16.75 16.87 17.00 17.12 17.26 14 17.37 17.49 17.62 17.74 17.87 17.99 18.11 18.24 18.36 18.49 15 18.61 18.74 18.86 18.98 19.11 19.23 19.36 19.48 19.60 19.73 16 19.85 19.98 20.10 20.22 20.35 20.47 20.60 20.72 20.84 20.97 17 21.09 21.22 21.34 21.47 21.59 21.71 21.84 21.96 22.09 22.21 18 22.33 22.46 22.58 22.71 22.83 22.95 23.08 23.20 23.33 28.45 19 23.S3 23.70 23.82 23.95 24.07 24.20 24.32 24.44 24.57 24.69 20 24.82 24.94 25.06 25.19 25.31 25.44 25.56 26.68 25.81 25.93 21 26.06 26.18 26.30 26.43 26.55 26.68 26.80 26.92 27.05 27.17 22 27.30 27.42 27.55 27.67 27.79 27.92 28.04 28.17 28.29 28.41 23 28.54 28.66 28.79 28.91 29.04 29.16 29.28 29.41 29.53 29.66 24 29.78 29.90 30.03 30.15 30.28 30.40 30.52 30.65 30.77 30.90 25 31.02 31.15 31.27 31.39 31.52 31.64 31.77 31.89 32.01 32.14 26 32.26 32.39 32.61 32.63 32.76 32.88 33.01 33.13 33.25 33.38 27 33.50 33.63 33.75 33.88 34.00 34.12 34.25 34.37 34.50 34.62 28 34.74 34.87 34.99 35.12 35.24 35.36 35.49 35.61 35.74 36.86 29 35.99 36.11 36.23 36.36 36.48 36.61 36.73 36.85 36.98 37.10 30 37.23 37.35 37.47 37.60 37.72 37.85 37.97 38.09 38.22 38.24 Urea. Table fob a Temperature of 30° C. ^ -h 1^ tV ^ A 1^0 A A 1 1.22 1.34 1.46 1..68 1.71 1.83 1.95 2.07 2.19 • 2.32 2 2.44 2.56 2.68 2.80 2.93 3.05 3.17 3.29 3.41 2.64 3 3.66 3.78 3.90 4.03 4.15 4.77 4.39 4.51 4.64 4.76 4 4.88 6.00 5.12 5.25 6.37 6.49 5.61 5.73 6.86 6.98 5 6.10 6.22 6.35 6.47 O..^ 6.71 6.83 6.90 7.08 7.20 6 7.32 7.44 7.57 7.69 7.81 7.93 8.05 8.18 8.30 8.42 7 8.54 8.67 8.79 8.91 9.03 9.15 9.28 9.40 9..32 9.64 8 9.76 9.89 10.01 10.18 10.25 10.37 10.50 10.62 10.74 10.86 9 10.99 11.11 11.23 11.35 11.47 11.60 11.72 11.84 11.96 12.08 10 12.21 12..S3 12.46 12.57 12.69 12.82 12.94 12.06 13.18 13..30 11 13.43 13.56 13.67 13.79 13.92 14.04 14.16 14.28 14.40 14.53 12 14.65 14.77 14.89 16.01 15.14 15.26 16.38 15.50 1.^62 15.75 13 15.87 16.99 16.11 16.24 16.36 16.48 16.60 16.72 16.86 16.97 14 17.09 17.21 17.33 17.46 17.58 17.70 17.82 17.94 18.07 18.19 16 18.31 18.43 18.56 18.68 18.80 18.92 19.04 19.17 19.29 19.41 16 19.53 19.65 19.78 19.90 20.02 20.14 20.26 20.39 20.61 20.63 17 20.75 20.88 21.00 21.12 21.24 21.36 21.49 21.61 21.73 21.85 18 21.97 22.10 22.22 22.34 22.46 22.68 22.71 22.83 22.95 23.07 19 23.19 23..S2 23.44 23.56 23.68 23.81 23.93 24.05 24.17 24.29 20 24.42 24.54 24.66 24.78 24.90 25.03 25.15 26.27 26.39 25.51 21 26.65 26.76 26.88 26.00 26.13 26.26 26.37 26.49 26.61 26.74 22 26.86 26.98 27.10 27.22 27.36 27.47 27.,59 27.71 27.83 27.96 23 28.08 28.20 28.32 28.45 28.57 28.69 28.81 28.93 29.06 29.18 24 29.30 29.42 29.54 29.67 29.79 29.91 30.03 30.15 30.28 80.40 25 30.52 30.64 30.77 30.89 31.01 31.13 31.23 31.38 31.50 31.62 26 31.74 31.86 31.99 32.11 32.23 32.35 32.47 32.60 32.72 82.84 27 32.96 33.09 33.21 33,33 33.45 33.67 33.70 33.82 33.94 34.06 28 34,18 34.31 34.43 34.55 34.67 34.79 34.92 86.04 35.16 35.28 29 36.41 36.53 85.66 35.77 38.89 36.02 36.14 36.26 36.38 36.50 30 86.03 36.76 86.87 86.99 37.11 87.24 37.36 87.48 37.60 , 37.72 CHEMISTRY OF THE URINE. 361 directly the number of grammes or grains of urea contained in the amount of urine employed.^ Green's apparatus (Fig. 87) consists of a tube, graduated in cubic centimeters, which is blown out at the bottom into a wider portion, and holds in all about 50 to 60 c.c. The bulb is provided with a side-tube, into which a bent funnel-tube can be inserted for the purpose of equal- izing the pressure. The side-tube having been detached, the apparatus is filled with sodium hypobromite solution, when 2 c.c. of urine (di- luted if necessary) are introduced by means of a graduated and bent pipette. After all bubbles of Fig. 89. gas have disappeared the funnel-tube is inserted into the side-opening and filled with hypobromite solution Fig. 87 Fig. Green's ureometer. MarshaU's ureometer. Hiifher's ureometer. until the level in both tubes is the same. The volume is then noted, corrected, and the corresponding amount of urea calculated as described. Marshall's apparatus is a conveniently modified form of Green's, and is used in the same manner (Fig. 88). Hwffher's apparatus is excellent (Fig. 89). It consists of a small bulb. A, of 5 c.c. capacity, which is separated from a larger bulb, C, holding about 100 c.c, by a well-oiled gl*ss stopcock. The upper end of C is drawn out to such an extent that the eudiometer D, which is about 30 cm. long, 2 cm. wide, and divided into fifths ' Instead of employing the solution described on page 355, it is sufficient to fill the long arm of the tube with a 40 per cent, solution of caustic soda, and to add 1 c.c. of bromine and a sufficient amount of water to fill the bend of the tube. 362 THE URINE. of a cubic centimeter, can be passed over it for a short distance. The bowl E, fitted over C by means of a cork, serves to hold a portion of the hypobromite solution. The exact capacity of A and of the lumen of the stopcock must be separately determined for each instrument. Method. — The bulb A and the lumen of the stopcock are filled with urine (which has been diluted, if necessary). The stopcock having been closed, C is washed out carefully with distilled water and filled with the hypobromite solution until the liquid in the dish stands several centimeters above the mouth of C. The eudiometer is next filled with the same solution, carefully submerged in the liquid contained in the dish, and adjusted over the mouth of C. The urine in A is then allowed to mix with the hypobromite solution very gradually, by opening the stopcock. After all bubbles of gas have disappeared the eudiometer is transferred to a cylinder filled with water and thoroughly immersed. After twenty to thirty minutes the level of the liquid in the tube and that of the outside water are equalized and the reading taken. The temperature of the water being likewise noted, the volume of the gas is corrected and the corresponding amount of urea calculated. Squibb's Method. — This method, like that of Doremus, may be highly recommended to the practitioner for its simplicity. The apparatus (Fig. 90) consists of two ordinary medicine-bottles, A and Fig. 90. Squibb'a ureometer. B. In A the nitrogen is evolved. B is closed by a doubly perforated rubber stopper, a straight tube passing through the upper aperture and connecting with the bottle A. Another tube, bent downward and carrying a clamp, as seen in the figure, leads to a graduated cylinder, E. B conteiins a sufficient amount of water for the bent tube to dip into ; 25 to 30 c.c. of the hypobromite solution and a CHEMISTRY OF THE VBINE. 363 small tube containing 2 c.c. of urine (diluted if necessary, according to the specific gravity) are placed in A, the clamp at E being closed. The rubber stopper is now firmly inserted and E opened, when a few drops of water, which may be disregarded, will escape. The graduated cylinder is then placed beneath the outflow-tube and the bottle A inclined. The nitrogen collecting in B displaces its own volume of water, which flows out and is collected in E, whence the corresponding amount of urea may be calculated or read ofi" from the accompanying tables (pages 358-360). It should be mentioned that sodium hypobromite liberates nitro- gen not only from urea, but also from the other nitrogenous con- stituents of the urine ; the error thus incurred, however, appears just to counterbalance the deficit in the amount of nitrogen obtained, and corresponds to 1 gramme of urea. If greater accuracy is required, the method recently suggested by Folin may be employed.^ Method of Folin. — This is based upon the following considera- tions : At a temperature of about 160° C. crystallized magnesium chloride, MgCl2.6H20, boils in its water of crystallization. In such a solution urea is quantitatively decomposed into ammonia and carbon dioxide within one-half hour. If the process is carried out in acid solution, the ammonia can subsequently be distilled off" after rendering the mixture alkaline, and is then titrated. The cor- responding amount of urea is ascertained by calculation. At the same time, however, the preformed ammonia is obtained, and it is hence necessary to eliminate this source of error by a separate estimation of this form. This is conveniently done according to the method which has likewise been suggested by Folin (see below). Method. — Three c.c. of urine are placed in an Erlenmeyer flask of 200 c.c. capacity, together with 20 grammes of magnesium chloride and 2 c.c. of concentrated hydrochloric acid. (The magne- sium chloride usually contains a small amount of ammonia, which must be separately determined.) The flask is closed with a per- forated stopper through which a straight glass tube passes, measur- ing 200 mm. in length, with a diameter of 10 mm. The mixture is now boiled until the drops flowing back through the tube produce a hissing sound on coming in contact with the solution. After this point has been reached, the boiling is continued more moderately for twenty-five to thirty minutes. The solution while still hot is care- fully diluted to about 500 c.c. — at first by allowing the water to flow drop by drop through the tube ; it is then transferred to a 1000 c.c. retort, treated with about 7 or 8 c.c. of a 20 per cent, solution of sodium hydrate, and the ammonia distilled off into a measured amount of a decinormal solution of sulphuric acid. The distillation may be interrupted when about 350 c.c. have passed over (viz., after ' O. Folin, Zeit. f. physiol. Chem., vol. xxxii. p. 504. 364 THE UBINE. about sixty minutes). The distillate is boiled for a moment to remove any carbon dioxide which may be present in solution, and on cooling is titrated to determine the excess of acid. Each cubic centimeter of the decinormal ammonia present in the distillate cor- responds to 0.003 gramme, viz., to 0.1 per cent, of urea. From this result the amount of preformed ammonia and that present in the 20 grammes of magnesium chloride must be deducted. . Estimation of Nitrogen. — For the purpose of estimating the total amount of nitrogen in the urine, the method of Kjeldahl or that of Will-Varrentrapp is most conveniently employed. Kjeldahl's Method.' — Principle. — The organic matter of the urine is decomposed by means of sulphuric acid, when all the nitrogen which is not present in combination with oxygen is transformed into ammonia. After adding sodium hydrate in excess the ammonia is then distilled off and received in a known quantity of titrated acid, the excess being retitrated with sodium hydrate. In this manner the amount of ammonia and the corresponding quantity of nitrogen are ascertained, it being remembered that 17 grammes of ammonia correspond to 14 grammes of nitrogen. Reagents required : 1. Gunning's mixture. This consists of 15 c.c. of concentrated sulphuric acid, 10 grammes of potassium sulphate, and 0.5 gramme of cupric sulphate. 2. A solution of sodium hydrate containing 270 grammes in the liter (sp. gr. 1.243). 3. Pulverized talcum or granulated zinc. 4. A one-fourth normal solution of sulphuric acid. 6. A one-fourth normal solution of sodium hydrate. Apparatus required (see Fig. 91) : This consists of a retort of about 750 c.c. capacity (^A), which is connected with a Kjeldahl distilling tube (£), and through this with a Stadeler condenser (C). The ammonia is received in the nitrogen bulb at D. In addition a Kjeldahl digesting flask of 200 to 300 c.c. capacity is required. Method. — Five or 10 c.c. of urine are placed in the digesting flask and treated with Gunning's mixture. To this end, it is best to add the sulphuric acid and cupric sulphate first, to heat until sul- phuric acid vapors are given off in abundance, and then to add the potassium sulphate. The heating is continued until the solution becomes entirely clear and almost colorless, the flask being inclined at an angle of about 45 degrees. Vigorous ebullition should be avoided. Upon cooling, the contents of the flask are transferred to the re- tort with the aid of^a little water, and slowly treated with a moder- ate excess of the sodium hydrate solution. As a general rule, 40 1 J. Kjeldahl, " Neue Methode zur Bestimmung des Stickatoffes in organischen Korpern," Zeit. f. analyt. Chem., 1883, vol. xxii. p. 366. CHEMISTRY OF THE URINE. 365 c.c. for each 5 c.c. of sulphuric acid are suificient. A little pulver- ized talcum or a few pieces of granulated zinc are finally added ; the retort is connected with the condenser, and the distillation begun. This is continued until about two-thirds of the solution have passed over. The distillate is received in the nitrogen bulb, which should contain a carefully measured quantity of the one-fourth normal solution of sulphuric acid. As a general rule, 30 c.c. are sufficient. As soon as the distillation is completed the condenser is discon- nected, washed out with a small amount of distilled water, and the washings added to the distillate. After the addition of a few Fig. 91. Kjeldahl's nitrogen apparatus. drops of tincture of cochineal or dimethyl-amido-azo-benzol the excess of sulphuric acid is retitrated with the one-fourth normal solution of sodium hydrate, and the amount found deducted from the 30 c.c. used. The titration should be continued until every trace of yellow has disappeared and a pure rose color is obtained, or, in the case of the dimethyl-amido-azo-benzol, until the last trace of red has disappeared and the solution has turned yellow. The difference multiplied by 0.0035 will then indicate the amount of nitrogen present in the 5 or 10 c.c of urine. The corresponding amount of urea is found by multiplying this figure by 20. 366 THE URINE. As Kjeldahl's method presupposes a thorough knowledge of chemical technique, it is well to make at least two parallel estima- tions in every case. "Will-Varrentrapp's Method (as modified by Seegen-Schneider).' — Principle. — If nitrogenous organic material is heated in intimate contact with soda-lime, all the nitrogen is given off in the form of ammonia, which is received in a known quantity of acid ; the excess, not used in the neutralization of the ammonia, is then determined by titration with a solution of sodium hydrate of known strength. Fig. 92. Apparatus for the determination of nitrogen. The amount held by the ammonia is thus ascertained, and from it the corresponding amount of nitrogen, it being remembered that 17 grammes of ammonia correspond to 14 grammes of nitrogen. Reagents required : 1. A quantity of thoroughly fused soda-lime, which, while still hot, should be placed in a well-stoppered bottle, where it may be kept ready for use for a long time. 2. A normal solution of sulphuric acid. 3. A normal solution of sodium hydrate. Apparatus required : As is apparent from the accompanying dia- gram (Fig. 92), the apparatus consists of a Kjeldahl digesting flask, A, of about 100 c.c. capacity, and provided with a neck 10 to 12 cm. long ; this is placed in a copper crucet, B, and imbedded in sand. 1 Will-Varrentrapp, see Leube-Salkowski, Die Lehre vom Ham. CHEMISTRY OF THE UMINE. 367 The crucet is placed upon a pipe-stem triangle over the flame. The neck of the flask is surrounded by a hood of copper or tin plate, C, moulded to the flask and reaching not higher than 1.5 cm. below the rubber stopper. The latter is doubly perforated, a tube, e, drawn out to a point and closed at the free end, passing through one aperture and extending about half-way down the flask, while the second passes through the other opening. This second tube, c, is connected by means of a short piece of rubber tubing, upon which a clamp is placed, with a Will-Varrentrapp apparatus. The latter is connected by rubber tubing, upon which a clamp is likewise placed, with an aspirating-bottle filled with water and provided with a siphon tube. Method. — ^Ten c.c. of the normal sulphuric acid solution are placed in the Will-Varrentrapp apparatus, together with a few cubic centimeters of a 1 per cent, alcoholic solution of phenol- phthalein. A layer of sand about 1 cm. in height is placed in the crucet, the clamp a closed, and the flask filled to about one-half its height with the soda-lime, when the hood is adjusted and 6 c.c. of urine are allowed to flow upon the soda. The rubber stopper is quickly adjusted, the rubber tube having been previously connected with the "V^ll-Varrentrapp apparatus. The clamp a is now opened, the crucet filled with sand, and the heating begun. This is at first done carefully with a small flame, but increased gradually until a full heat is applied. This is continued for one-half to three-quarters of an hour. When drops of moisture are no longer visible in the tube e, or when the evolution of gas has entirely ceased, the rubber tube of the aspirating-bottle d is slipped on to the Will-Varrentrapp apparatus, the clamp b slightly opened, the tip of e broken ofi", and air allowed to pass slowly through the entire system for a quarter of an hour, when the flame is extinguished. The Will-Varrentrapp apparatus is then detached and its contents titrated with the normal solution of sodium hydrate. The number of cubic centimeters of the sodium hydrate solution employed is deducted from 10 (the number of cubic centimeters of the normal sulphuric acid solution, 1 c.c. of the latter being equiva- lent to 1 c.c. of the former), the difFerence giving the number of cubic centimeters of the normal sulphuric acid solution neutralized by the ammonia evolved from 5 c.c. of urine. This number multi- plied by 20 will then represent the number of cubic centimeters required to neutralize the ammonia contained in 100 c.c. of urine. As 1000 c.c. of the normal solution of sulphuric acid correspond to 17 grammes of ammonia, or 14 grammes of nitrogen, the number of cubic centimeters of the sulphuric acid solution corresponding to 100 c.c. of urine will be found from the equation : 1000 •.14::x:y; and y ^0.014 X, in which x represents the number of cubic centi- meters required to neutralize the amount of anamonia evolved from 368 THE URINE. 100 C.C. of urine, and y the corresponding amount of nitrogen — i. e., the percentage of nitrogen. If the nitrogen is to be calculated in terms of urea, this is done according to the equation : 1000 : 30 ( = 14N) : :x:y; and y = 0.03 X = percentage of urea, in which x represents, as above, the number of cubic centimeters of sulphuric acid neutralized by the ammonia, viz., nitrogen, contained in 100 c.c. of urine, and y the urea corresponding to this amount. Ammonia. Every urine contains a small amount of ammonia, which normally varies but little, and corresponds to from 4.1 to 4.64 per cent, of the total amount of nitrogen, viz., to about 0.7 gramme in the twenty- four hours. It is present in combination with the various acids of the urine, and in all likelihood represents a small amount of the ammonia which has not been transformed into urea, but has been utilized to saturate the affinities of a slight excess of acid, formed during the nitrogenous metabolism of the body, over the available fixed alkalies. In this manner indeed the body is capable of guard- ing against the appearance of free acid in the blood, and it is for this reason, as I have already pointed out, that free acid cannot occur in the urine. This safeguard, however, does not exist in the herbivorous animals, in which the fixed alkali only is apparently available for the neutralization of acids, and we consequently find that whereas in dogs, for example, an acid intoxication occurs only after the administration of very large quantities of acid, the herbivora rapidly succumb after the ingestion of comparatively small amounts. In man an increased elimination of ammonia is observed when- ever an increased formation of acids occurs, or whenever a sufficient supply of oxygen is not available. In the latter case, no doubt, the increased elimination is owing to the fact that in consequence of the deficient supply of oxygen the synthetic formation of urea from ammonium lactate is impeded in the liver. As this organ, moreover, is the principal seat of the synthesis of urea, we can readily understand that extensive parenchymatous degeneration, as in acute yellow atrophy, in phosphorus poisoning, etc., will lead to an increased elimination of ammonia. In any event, the relative increase of the ammonia is the essential factor, while variations in its absolute quantity are of secondary importance. Some of the results which have been obtained in various diseases are given in the following table : Per cent. Normal values 4.10- 4.64 Febrile diseases 5.72- 6.70 Carcinoma of the liver 6.40-24.50 Liver abscess (actinomycosis) 10.60 Circulatory dyspnoea 13.10-32.20 Respiratory dyspnoea 6.60-14.30 CHEMISTRY OF THE URINE. 369 Abnormally high absolute values are quite constantly observed in diabetes, in which an elimination of from 4 to 5 grammes may be regarded as common. In one instance 5.94 grammes were excreted in twenty-four hours. Very curiously, diminished elimination of ammonia is observed in many cases of nephritis so long as symptoms of venous stasis do not exist. In a case of pernicious ansemia relative amounts, varying between '3.3 and 6.6 per cent., were obtained during the days immediately preceding death. Quantitative Estimation. — Schlosing's Method. — Principle. — ^A carefully measured amount of urine is treated with milk of lime and placed under a bell, together with a vessel containing a known Fig. 93. Desiccator. amount of a normal solution of sulphuric acid. In the course of time the ammonia is liberated and absorbed by the acid. This is then titrated, and the deficit expressed in terms of ammonia. Method. — ^Twenty-five c.c. of perfectly fresh, filtered urine are placed in a flat dish, upon the plate of a desiccator, as shown in Fig. 93. Above this is a smaller dish containing 10 c.c. of a nor- mal solution of sulphuric acid. The urine is treated with 10 c.c. of milk of lime, the bell is carefully adjusted after lubrication with tallow, and the apparatus allowed to stand for at least three or four days. The excess of acid remaining is then titrated with a one-fourth normal solution of sodium hydrate, using as an indicator a few drops of a saturated aqueous solution of methyl-orange until the red color has turned to yellow. To neutralize the 10 c.c. of the acid, 40 c.c. of the one-fourth normal solution are required. The difference is referable to the partial neutralization by the ammonia, 24 370 THE URINE. and is expressed in milligrammes. One c.c. of the one-fourth normal solution corresponds to 4.25 mgrms. of ammonia. Precautions: 1. In every case the urine must be perfectly fresh. Decomposition is best guarded against during its collection by adding about 10 to 20 c.c. of chloroform to the portion first voided. 2. Urines which are undergoing ammoniacal decomposition should not be utilized for examination. 3. Concentrated or albuminous urines must be kept under the bell for from five to eight days, new portions of acid being used when in doubt as to the complete liberation of the ammonia. Owing to a slight deposition of moisture on the inner surface of the bell and a consequent retention of traces of ammonia in this form, the resulting figures are too low. The error thus incurred, however, is insignificant. More satisfactory than this older method is the following, which has recently been suggested by Folin : Folin's Method. — Ten c.c. of urine are diluted to about 450 c.c, treated with a small amount of burnt magnesia (0.5 gramme), and boiled for forty-five minutes, the distillate being received in decinor- mal sulphuric acid. The ammonia is then determined by titration as above. As a small amount of urea, however, is decomposed during the prolonged ebullition, it is necessary to ascertain separately the quantity of ammonia which is referable to this source. To this end, the retort is opened at the expiration of forty-five minutes,, and an amount of water added which is approximately equivalent to that of the distillate. The distillation is then continued for another period of forty-five minutes ; the distillate is received in decinormal sulphuric acid, and the ammonia referable to decomposi- tion of the urea estimated as before. The difference between the two results indicates the amount of preformed ammonia that was origi- nally present. LiTESATUKE. — Hallervorden , Arch. f. exper. Path,, vol. xii. p. 237. Stadelmann, Deutsch. med. Wooh., 1889, p. 942. Miohaelis, Ibid., 1900, p. 276. O. Folin, Zeit. f. physiol. Chem., vol. xxxii. p. 575. Uric Acid. According to our present views, uric acid, in man, is not formed during the decomposition of all albuminous substances, as was for- merly supposed, but constitutes a specific product of decomposition of one class of albumins only, namely, the nucleins.^ It appears, moreover, that the mother-substance of uric acid is confined to the nuclear nucleins, viz., to those containing a nucleinic acid radicle ; while the paranucleins, in which this is lacking, are without effect upon the elimination of uric acid. According to Kossel,^ four differ- 'C. E. Simon, Physiological Chemi.'itry, Lea Bros. & Co., 1901. ' A. Kossel u. A. Neumann, " Ueber Nukleinsaure u. Thyminsaure," Zeit. £ physiol. Chem., vol. xxii. p. 74. CHEMISTRY OF THE VRINE. 371 ent forms of nucleinic acid exist, viz., adenylic acid, guanylic acid, sarcylic acid, and xanthylic acid, and the supposition is that each of these contains one base, viz., adenin, guanin, sarcin or hypoxanthin, and xanthin. These basic substances are collectively spoken of as the xanthin, alloxur, or purin bases. According to Emil Fischer,! they are derived from a hypothetical compound which he terms purin, and which he supposes to be constituted as shown in the formula (6) (1)N=^CH I I (7) (2)HC (5)C NH\ II II ^CH(8). (3)N C N'^^ (4) (9) By substituting the group NHj for the H atom at 6, adenin thus results, and is hence also spoken of as 6-aminopurin : N= HO C NHx II 11 ^^H. N C ^^ Hypoxaqthin, according to this conception, would be 6-oxypurin ; xanthin 2, 6-dioxypurin, and guanin 2-amino-6-oxypurin, as shown by the structural formulae : HN CO HN CO II XI HC C NHv CO C NH- II II ^CH. I II JiCH. K C N*^^ HN C N**^ Hypoxanthin. Xanthin. NH CO I I • HN=C C NH\ >CH. HN- Guanin. From the structural formula of purin it is also apparent that still other derivatives of this substance may exist, and as a matter of fact others are known, viz., mono-methylxanthin or heteroxanthin, di-methylxanthin or paraxanthin, tri-methylxanthin, the isomeric compounds of paraxanthin, viz., theophyllin and theobromin, and others. Their relation to xanthin is shown in the formulae : HN CO HN CO II II CO C NH\ CO C N.CHjx I II JiCK. I II ^^CH. HN C N**^ HN C N==*^ Xanthin. Heteroxanthin. • E. Fischer, Ber. d. Deutsch. chem. Ges., 1897, vol. xxx. p. 549. 372 THE URINE. ,N CO CH3.N CO II II CO C N.CHjx CO C NH HN C N:^=^ CH3.N C N: Paraxauthin. Theophyllin HN CO CHj-N CO CO C N.CHav CO C- I II CHj.N C N* Theobromin. -N.CH3\ Two of these bodies, namely, heteroxanthin and paraxanthin, have also been found in urine. From these basic substances, then, which are found in the nucle- inic acid radicle of the nuclear nucleins, uric acid is supposedly derived, and there are numerous facts which go to show that this supposition is in all likelihood correct. It will thus be observed that structurally uric acid is intimately related to the bodies in ques- tion, and, like these, contains the purin radicle : HN CO I I CO C NH. I il >co. HN C NH/ Uric acid. It may hence be regarded as 2, 6, 8 tri-oxypurin. Uric acid and the xanthin-bases, moreover, qualitatively, all yield the same decom- position-products when treated with fuming hydrochloric acid or hydriotic acid under high pressure ; only the quantitative relations vary, as shown in the equations : C5H5N5 + 8H2O = 4NH5 + CO, + CHj.NH2.COOH + 2H.C00H. Adenin. GlyoocoU. Formic acid. CsH^N.O + 7H.,0 = 3NH3 + COj + CHj.NHj.COOH + 2H.C00H. Hypoxanthin. CsHsNjO + 7HjO = 4NH3 + 2C0j + CHj.NHj.COOH + H.COOH. Guanin. C5H,N40, + 6H2O = 3NH, + 2C0, + CHj.NHj.COOH + H.COOH. Xanthin. CjH.N^O, + 5HjO = 3NH3 + SCO, + CHj.NHj.COOH. Ilrio acid. In accordance with this supposed origin of uric acid we find an increased elimination in the urine following the ingestion of all sub- stances which contain purin bases either as such or in the form of nuclear nucleins. At the same time it must be remembered that uric acid may also result from the nucleins of the body-tissues ; and we find, as a matter of fact, that during starvation uric acid does CHEMISTRY OF THE URINE. 373 not disappear from the urine. The principal source of the uric acid under such conditions are the nucleins of the leucocytes ; and accord- ing to Horbaczewski ^ and others, this source is indeed more impor- tant than the nucleins of the food. According to his idea, the latter call forth an increased elimination of uric acid in only an indirect manner — i. e., by stimulating more strongly than other food-stufFs the cell-formation and cell-destruction of the body. However this may be, there can be no doubt that the amount of uric acid elimi- nated in the urine depends, in the first instance, upon the amount of nucleins or purin bases as such which are ingested, and upon the degree of nuclear destruction which takes place in the body. Other factors, however, also enter into consideration. We thus know that the body is capable of transforming a certain amount of uric acid into urea. This fact was pointed out long ago by Frerichs and Wohler, and has recently again been confirmed. It was found that after the ingestion of large amounts of nucleins only a certain por- tion of the nuclear nitrogen is eliminated as uric acid, and that this amount is extremely variable. Whether individual peculiarities have any part in determining this amount is unknown, but is not improbable. Oxidation on the part of the body-tissues must also be taken into consideration, and it unquestionably varies not only in different people, but also in the same individual at different times. Then again there is evidence to show that under certain conditions uric acid may be formed synthetically in the body. That this is the usual mode of formation in birds and reptiles has been conclusively shown by Minkowski,^ who found that after extirpation of the liver in geese the greater portion of the urinary nitrogen was eliminated in the form of ammonia in combination with lactic acid. In the human being very little uric acid is in all likelihood formed in this manner under normal conditions, but the possibility of its occur- rence, in disease more particularly, should not be overlooked. As uric acid, moreover, may in part at least be eliminated in the feces, it is clear that the amount which appears in the urine cannot be regarded as an accurate index of the degree of nuclear destruc- tion or of the amount which is formed in the body-tissues. That retention of uric acid can further occur in the body, which may or may not be followed by increased elimination, is likewise undoubted. According to our present knowledge, uric acid is formed in all the organs of the body, including the bone-marrow, the muscles, the spleen, the liver, the kidneys, etc. Under pathological conditions it may also originate in the joints and tendons. Under normal conditions the daily elimination of uric acid varies between 0.2 and 1.5 grammes, thus constituting -^ to y^ part of ' J. Horbaczewski, "Beitrage zur Eenntniss . der Bildung von Harnsaure,'' etc., Monatshefte fiir Chem., 1891, vol. xil. p. 221 ; and Wien. Sitzungsber., vol. o. '^ Minkowski, "Ueber den Einfluss d. Leberextirpation auf den Stoffwechsel,"' Arch. f. exper. Path. u. Pharmakol., 1886, vol. xxi. p. 41. 374 THE URINE. the total urinary nitrogen. It is largely influenced by the character of the diet, the amount of exercise taken, the general health of the individual, etc. After the ingestion of large amounts of food rich in nuclear nucleins, such as thymus gland, liver, kidneys, and brain, a corresporiding increase in the amount of uric acid is observed. Generally speaking, animal food causes a greater elimination of uric acid than vegetable food, and it is supposed that this difference is essentially due to the presence of the extractives of the meat.^ Of special interest is the increase in the elimination of uric acid which is observed five hours after the ingestion of a full meal. This in- crease, according to Horbaczewski,^ is associated with the disappear- ance of the digestive leucocytosis and consequent leucolysis. Some observers have attached much importance to the relation existing between the elimination of uric acid and urea, and are in- clined to assume the existence of a special urio add diathesis when this relation continuously exceeds the usual standard of 1 : 50 or 1 : 60. This question is, however, an extremely intricate one, and we are scarcely in a position at the present time to speak definitely of the significance of such variations. On the one hand, there can be no doubt that an unusually high uric acid coefficient may be met with in individuals who are apparently in good health, while in others, in whom larger amounts of uric acid are eliminated than are usual, normal or even subnormal values may be found. The entire ques- tion of the uric acid diathesis is in a chaotic condition, and it would perhaps be well to speak of such a diathesis only when a distinct absolute increase is eontinuously observed. That numerous symptoms of a neurasthenic type are often seen when the uric acid coefficient is increased, is a matter of daily observation, but it would be pre- mature to regard this symptom as a causative factor of the disease in question.^ Even in gout it can scarcely be said that uric acid has been proved the materia peccans, and our knowledge concerning the etiology of the disease is still as obscure as when Garrod ■* showed that an accumulation of uric acid occurred in the blood of such pa- tients. Hitherto it has been supposed that the deposition of urates in the joints and periosteum of gouty patients is referable to a diminished alkalinity of the blood, and that acute paroxysms result whenever an increase in its alkalinity occurrs, leading to a resorp- tion of the urates previously deposited and a consequent flooding of the system with the material in question. As a matter of fact, a ' A. Hermann, " Abhiingigkeit der Harnsaureaussoheidung von Nahrnngs- und Ge- nuaamitteln," Deutsch. Arch. f. klin. Med., 1888, vol. xliii. p. 273. See also W. Camerer, Zeit. f. Biol., N. F., 1896, vol. xv. p. 140. ' Horbaozewski, Harnsaureaussoheidung u. Leucocytose, Sitzungsber. 'd. Wiener Akad. d. Wissensch., 1891, Abth. 3. See also Lowit, Studien z. Physiol, u. Path. d. Blutes, 1892. W. Kiihnau, " Das Verhaltniss d. HarnsaureausBCheidnng zur Leuco- cytose," Zeit. f. klin. Med., vol. xxviii. p. 534. P. F. Eichter, "Ueber Harnsaure- ausscheidung und Leucocytose," Ibid., vol. xxvii. p. 290. s C. E. Simon, Am. Jour. Med. Sci.. 1899, p. 139 , and N. Y. Med. Jour., 1895, p. 330. * A. B. Garrod, On the Nature and Treatment of Gout, 1817. CHEMISTRY OF THE URINE. 375 considerable diminution in its excretion is observed immediately preceding the attack, while during the paroxysm and immediately following it a corresponding increase is noted. Numerous investi- gations, however, have shown that distinct changes in the alkalinity of the blood do not occur in gout, and that an increase in the amount of uric acid in the blood is not only observed in this disease, but in other diseases as well which are not associated with gouty symptoms. The conclusion is hence justifiable that the presence of uric acid in the blood per se cannot be offered as an explanation of the occur- rence of a gouty atta;ck.^ The greatest increase in the elimination of uric acid is observed in .leukaemia, in which amounts of 5 grammes and even more may be observed in the twenty-four hours. That the increased elimina- tion in this disease is referable to the enormous increase in the number of the leucocytes and consequent leucolysis can scarcely be doubted. In other diseases which are associated with a high grade of leucocytosis, and especially those in which the disease terminates by crisis or hastened lysis, such as erysipelas and pneumonia, a con- siderable increase is likewise observed, and is referable to the same origin. This increase is especially marked immediately after crisis has occurred, but it not infrequently precedes this by several hours. In the other febrile diseases an absolute increase is less marked and inconstant. In diabetes a diminished amount of uric acid is usually found. Cases may be seen, however, in which, associated with a diminution or an entire disappearance of the sugar, a most marked increase occurs, amounting in some cases to 3 grammes in the twenty-four hours. To this condition the term diabetes alternans has been applied. In acute articular rheumatism an increased elimination is observed so long as the temperature remains high, while with approaching convalescence the amount returns to normal, and may even fall below normal. In chronic rheumatism, on the other hand, no con- stant relations have been observed. In the ordinary forms of ansemia and chlorosis the amount of uric acid is quite constantly diminished, as also in chronic inter- stitial nephritis, chronic lead poisoning, progressive muscular atro- phy, and pseudohypertrophic paralysis. Properties of Uric Acid. — The close relation existing between uric acid and the xanthin-bases has been already considered. By oxidation uric acid is transformed into urea or into substituted ureas, such as allantoin and alloxan, which latter in turn is closely related to parabanic acid or oxalyl-urea and barbituric acid or malonyl-urea. ' B. Laquer, Ueber die Ausscheidungsverhaltnisse der Alloxurkorper. Bergmann, 1896. (Full literature.) C. von Noorden, Lehrbnch d. Pathologie d. Stoffwechsels, Berlin, 1893. W. Ebstein, " Die Natur u. Behandluug der Giclit," Verhandl. d. VIII. Congr. f. inn. Med., 1889, p. 133. 376 THE URINE. CsH^NA + O Uric acid. + H,0 = C,H,NA + CO<^g' Alloxan. -fj^a- Urea. CsH.NA + H,0 + O = C-HeNA + CO,. Uric acid. Allantoin. Pure uric acid forms a white crystalline powder which is almost insoluble in cold water (1 : 40,000), with difficulty soluble in boiling water (1 : 1800), and insoluble in alcohol and ether. In concentrated sulphuric acid it dissolves with ease, but is precipitated upon dilu- tion with water. In aqueous solutions of the alkaline carbonates and hydrates it dissolves, with the formation of neutral or acid salts, as represented by the equations : CsHjN^Oj + NajCOa = CjHaNaNA + NaHCOj. C5H4NA + 2Na3COj = CsHjNa^NA + 2NaHC03. In freshly voided urine uric acid is said to occur as a quadriurate, viz., as a compound in which one molecule of sodium is in combina- tion with two molecules of uric acid. The quadriurate, however, is readily decomposed with the formation of uric acid and acid urates Fig. 94 £3 Various forms of uric acid crystals. (Finlayson.) (biurates). Its solubility in the urine depends upon the amount of water present, the reaction, and the presence of inorganic salts. When acid sodium phosphate preponderates the biurate is precipi- tated, while free uric acid is thrown down when disodic phosphate only is present, and along with this still other acid compounds which are most likely of organip nature. Neutral urates cannot occur in the urine. The basic substances which may occur in the urine in combination with uric acid are sodium, potassium, ammonium, and possibly also calcium and magnesium. These salts may be deeom- CHEMISTRY OF THE URINE. 377 posed by the addition of a sufficiently large quantity of a stronger acid, such as hydrochloric acid, when uric acid is set free. The acid salts are soluble with great difficulty, and are hence precipitated whenever the urine is markedly acid or concentrated, and also when it is exposed to a low temperature. This holds good especially for the acid ammonium compound, and upon this fact Hopkins' quan- titative estimation of uric acid is based. Pure uric acid crystallizes in transparent, colorless, rhombic plates, while that which usually separates from the urine is of a reddish-brown color and may assume a great variety of forms (Fig. 94). Of these, the so-called whetstone-form is the most character- istic (see Sediments). Colorless rhombic platelets may, however, also be seen. Of the compounds which uric acid forms with the heavy metals, the silver salt is especially important. When a solution of uric acid in ammonia is treated with an ammoniacal solution of silver nitrate (see below) the solution remains clear ; but if calcium chloride, sodium chloride, or magnesia mixture is then added, a precipitate forms, which contains the uric acid in combination with silver. Tests for Uric Acid. — 1. Murexid Test. — A few crystals are dis- solved by means of a few drops of concentrated nitric acid, with the application of heat, upon a porcelain plate, such as the cover of a crucible. The nitric acid is then carefully evaporated, when a yel- lowish-red spot will remain. Upon cooling, a drop of ammonia is placed upon this spot, when in the presence of uric acid a beautiful purplish-red color develops, owing to the formation of ammonium purpurate (murexid). If now a drop of sodium hydrate solution is added, the color changes to a reddish blue, which disappears upon heating ; the reaction thus differs from the somewhat similar xanthin reaction. 2. Copper Test. — A few crystals are dissolved in sodium hydrate solution and treated with a few drops of Fehling's solution. Upon the application of heat white copper urate separates out, while red cuprous oxide appears if a relati\'ely large amount of cupric sulphate is present — a point to be remembered in testing for sugar. The reduction of Fehling's solution is due to the formation of allantoin. 3. When treated with sodium hypobromite solution uric acid gives up about 47 per cent, of its nitrogen. Quantitative Estimation of Uric Acid. — Hopkins' Method. — This method is now commonly used in the clinical laboratory, and is to be preferred to the more complicated pl-ocedures hitherto employed. It is much simpler and equally as accurate as the older methods of Ludwig-Salkowski and of Haycraft. Various modi- fications of the original method have been suggested. Principle. — The method is based upon the complete precipitation of uric acid by ammonium salts, and the possibility of accurately 378 THE URINE. titrating the uric acid with potassium permanganate in the presence of sulphuric acid. Folin's Modification of Hopkins' Method.' — To precipitate the uric acid, and also to remove the small amount of mucoid substance which is found in every urine, the following reagent is employed : 500 grammes of ammonium sulphate and 5 grammes of uranium acetate are dissolved in 650 c.c. of water, to which solution 60 c.c. of a 10 per cent, solution of acetic acid are further added. The resulting solution measures about 1000 c.c. Seventy-five c.c. of the reagent are added to 300 c.c. of urine in a flaskjiolding 500 c.c. After standing for five minutes the mixture is filtered through twp folded filters, and thus freed from the mucoid body, which is carried down with the uranium phosphate in acid solution. The filtrate is divided into two portions of 125 c.c. each, which are placed in beakers and treated with 5 c.c. of concentrated ammonia. After stirring a little the solu- tions are set aside until the next day. The supernatant fluid is then carefully poured off through a filter (Schleicher and Schiill, No. 597) ; the precipitated ammonium urate is collected with the aid of a small amount of a 10 per cent, solution of ammonium sulphate and washed with the same reagent. Traces of chlorides do not interfere with the subsequent titration, and the process of filtration and washing can be completed in from twenty to thirty minutes. The ammonium, urate is washed into a beaker, after opening the filter, using about 100 c.c. of water. Fifteen c.c. of concentrated sulphuric acid are then added, and the solution is titrated at once with a one-twen- tieth normal solution of potassium permanganate. Toward the end of the titration Folin suggests to add the permanganate in portions of two drops at a time, until the first trace of a rose color is apparent throughout the entire fluid. Each cubic centimeter of the reagent corresponds to 0.00375 gramme of uric acid. A final correction of 0.003 gramme for each 100 c.c. of urine employed is necessary, owing to the slight extent to which ammonium urate is soluble. Preparation of the One-twentieth Normal Solution of Potassium Permanganate. — As the molecular weight of potassium perman- ganate is 157.67, one would expect that a normal solution of the salt should contain this amount in grammes dissolved in 1000 c.c. of water. But the substance generally acts in the presence of free acids, upon deoxidizing substances, by losing 5 atoms of oxygen of the 8 atoms contained in 2 molecules, as is seen in the following equation : 2KMiiOi + 5ll,C,0, + SH^SO, = K^SO, + 2MnS04 + lOCOj -|- mf). It follows that two-fifths of the molecular weight, or 63.068 grammes, are the equivalent of 1 oxygen atom. But as oxygen is diatomic and the volumetric normal is calculated for monatomic • 0. Folin a. A. Shaffer, Zeit. f. physiol. Chem., vol. xxxii. p. 552. CHEMISTRY OF THE VBINE. 379 values, this number must be divided by 2, and 31.534 grammes of potassium permanganate should therefore be present in 1 liter of normal solution. A one-tenth normal solution would hence contain 3.1534 grammes, and a one-twentieth normal solution 1.576 grammes pro liter. This amount is weighed off and dis- solved in 950 CO. of water, when the solution is brought to the proper degree of dilution (see page 322) by titration with a one- twentieth normal solution of oxalic acid. A one-twentieth normal solution of oxalic acid contains 3.142 grammes of the acid in 1000 C.C. of water. One c.c. of the one-twentieth normal solution of potassium permanganate should correspond to 1 c.c. of the oxalic acid solution. The titration is best conducted by diluting 10 c.c. of the oxalic acid solution to 100 c.c. with distilled water and add- ing 15 c.c. of concentrated sulphuric acid, so as to bring the tempera- ture of the liquid to from 55° to 65° C. The potassium perman- ganate solution is then added drop by drop until the red color no longer disappears on stirring, but persists for at least thirty seconds. Titration with Sodium Hydrate Solution. — This method is not as accurate as the one just described, but suffices for ordinary purposes. The uric acid is precipitated with an ammonium salt, as above. After standing for two hours the ammonium urate is filtered off, washed with a 10 per cent, solution of ammonium sulphate, and brought into a beaker with the aid of a small amount of hot water. The salt is then decomposed by the addition of from 10 to 15 c.c. of a one-tenth normal solution of hydrochloric acid. The mixture is brought to the boiling-point, and the hydrochloric acid not used in the decomposition of the ammonium urate retitrated with a one- tenth normal solution of sodium hydrate, using dimethyl-amido- azo-benzol as an indicator. The amount of hydrochloric acid found is deducted from the 10 or 15 c.c. added, and the result multiplied by 0.0168. The amount of uric acid contained in the original quantity of urine is thus ascertained, to which 0.003 gramme is added for each 100 c.c. of urine used, as above. Gravimetric Method. — The process is begun as described above. The ammonium urate is decomposed by the addition of from 2 to 3 c.c. of a 25 per cent, solution of hydi-ochloric acid. This solution is evaporated until crystals of uric acid begin to separate out. These are collected on a dried and weighed filter, and washed successively with water, alcohol (90-95 per cent.), and absolute alcohol, aud finally with ether. The mother-liquor and water used in washing are carefully measured, and 0.0004 gramme added to the final result for each 10 c.c Haycraft's Method.^ — This method is based upon the precipitation of uric acid with an ammoniacal silver solution and magnesia mixt- ure, 1 molecule of silver corresponding to 1 molecule of uric acid. ' Haycraft, Zeit. f., analyt. Chem., vol. xxv. 380 THE UBJNE. As the amount of silver thus precipitated can be determined by titra- tion with a solution of potassium sulphocyanide, the corresponding amount of uric acid is readily found. Solutions required: 1. An ammoniacal silver solution. 2. An ammoniacal magnesia mixture. 3. A one-fiftieth normal solution of silver nitrate. 4. A one-fiftieth normal solution of potassium sulphocyanide. Preparation of these solutions : 1. The ammoniacal silver solution is prepared by dissolving 26 grammes of silver nitrate in distilled water, and adding enough ammonia to redissolve the brown precipitate of argentic oxide which is first formed ; distilled water is then added in sufficient amount to make the total quantity 950 c.c. This solution is brought to the proper strength by titrating a known amount of sodium chloride, as described elsewhere. Each cubic centimeter then contains 0.026 gramme of silver nitrate, which is equivalent to 0.0169 gramme of silver. 2. The ammoniacal magnesia mixture is prepared by dissolving 100 grammes of crystallized magnesium chloride in a sufficient amount of water ; to this a cold saturated solution of ammonium chloride is added in excess, and sufficient strong ammonia to impart a decided odor. Should the mixture not be perfectly clear, more ammonium chloride solution is added. The solution is then diluted with water to 1 liter. 3. The one-fiftieth normal solution of silver nitrate is prepared by dissolving 3.4 grammes of silver nitrate in 950 c.c. of distilled water, the degree of further dilution being determined as described elsewhere. 4. To prepare the one-fiftieth normal solution of potassium sul- phocyanide, about 2 grammes of the salt are dissolved in 950 c.c. of water ; the solution is brought to the required strength, so that 1 c.c. shall correspond to 1 c.c of the silver solution. For filtering the uric acid, a perforated platinum cone is placed in a small funnel and packed with a thin layer of glass-wool, upon which in turn a layer of finely scraped asbestos is placed. The asbestos is previously thoroughly washed with dilute hydrochloric acid and subsequently with distilled water until every trace of chlo- rine has disappeared. When properly prepared, the asbestos forms a mould of the cone. Method. — Five c.c. of the ammoniacal silver solution are mixed with 5 c.c. of the ammoniacal magnesia mixture. Ammonia is then added until the solution is clear, when it is poured into 50 c.c. of urine. As soon as the precipitate has settled the supernatant liquid is passed through the prepared filter with the aid of a suction-pump. About 4 grammes of sodium bicarbonate in coarse pieces are now placed upon the filter and the precipitate is added ; the sodium bicar- CHEMISTRY OF THE URINE. 381 bonate serves the purpose of aiding filtration by loosening the pre- cipitate. This is now washed free from chlorine and silver by means of ammoniacal water, using the suction-pump until the precipitate appears broken in places, then without the pump, using this only at last to remove the last drops of liquid. (Test for silver with very dilute hydrochloric acid, and for chlorine with a solution of silver nitrate and nitric acid.) The precipitate is now dissolved on the filter by means of nitric acid of 20 to 30 per cent. The nitric acid must be free from nitrous acid. This is secured by allow- ing it to stand in contact with pure urea until any evolution of gas has ceased. The filter is washed with very dilute nitric acid and then with distilled water until this no longer shows an acid reaction. The solution thus obtained is titrated with the one-fiftieth normal solution of potassium sulphocyanide, using ammonio-ferrio alum as an indicator. As each cubic centimeter of this solution indicates 0.0169 gramme of silver, and as 1 molecule of silver indicates 1 molecule of uric acid — i. e., 108 grammes of silver 168 grammes of uric acid — 0.0169 gramme of silver, corresponding to 1 c.c. of the potassium sulphocyanide solution, represents 0.2629 gramme of uric acid. Ludwig-Salkowski Method. — Princvple. — The method is based upon the formation of insoluble magnesium-silver urate when a solution of uric acid in sodium carbonate is treated with a solution of silver nitrate after the previous addition of an excess of ammonia. This is then decomposed, with the liberation of free uric acid. Method.^ — Two hundred and fifty c.c. of urine are treated with 60 c.c. of ammoniacal magnesia mixture (see above) to remove the phosphates. The magnesia mixture is employed for the reason that the compound of uric acid with magnesium and silver which is formed later on is not decomposed so easily as the sodium or the potassium compound, which would occur if the urine were pre- cipitated only with ammonia. The mixture is then immediately filtered, as otherwise a little magnesium urate would be precipitated. Two hundred and fifty c.c. of the filtrate, corresponding to 200 c.c. of urine, are measured off as soon as possible, and treated with a few cubic centimeters of a 3 per cent, solution of silver nitrate. If the precipitated silver chloride formed in the beginning does not dis- appear on stirring, a little more ammonium hydrate is added. A flaky precipitate next separates out, and is allowed to settle. In order to test whether enough of the silver nitrate solution has been added, a few cubic centimeters of the supernatant fluid are acidified with nitric acid. If a distinct cloudiness, referable to silver chloride, appears, enough has been added. Otherwise the few cubic centimeters that were employed for this test are rendered alkaline 1 E. Salkowski, Salkowski u. Leube, Die Lehre vom Ham. E. Ludwig, Wien. med. Jahrbucher, 1884, p. 597. 382 THE URINE. again with ammonia, poured back, and treated with more silver solution until the required amount has been reached. The liquid is then rapidly filtered through a folded filter of rather loose paper, a feather or rubber-tipped glass rod being used for the purpose of removing all the precipitate from the beaker. The precipitate is washed with ammoniacal water until a specimen of the washings is no longer rendered turbid by nitric acid, and only faintly so by the addition of a drop of silver solution. The filter with the pre- cipitate is next placed in a wide-mouthed flask, containing about 200 c.c. of distilled water, and thoroughly agitated. Hydrogen sulphide is then passed through the mixture. It is now brought to the boiling-point and rendered distinctly acid by means of a few drops of hydrochloric acid, when the argentic sulphide and the paper are rapidly filtered off, as otherwise there will be an admixture of sulphur with the uric acid. The contents of the filter are washed a few times with hot water. Filtrate and washings are quickly evaporated to a few cubic centimeters, treated with a few drops of hydrochloric acid, and set aside in a cool place for twenty-four hours. Occasionally it happens that upon addition of the hydro- chl oric acid a cloudiness appears, which is due to an admixture of sulphur. In such a case the dried uric acid must be washed with carbon disulphide. Otherwise the uric acid that has separated out is directly collected on a dried and weighed filter, and washed suc- . cessively with water, 90 to 94 per cent, alcohol, and finally with absolute alcohol and ether. The water used in washing should be collected separately, and for each 20 c.c. used 0.0048 gramme added to the weight of the uric acid obtained. Precautions : 1 . Rapidity in working is essential. 2. Very concentrated urines must be diluted one-half before com- mencing the examination. 3. If the specific gravity of the urine is low, it should be con- centrated to a specific gravity of about 1.020. 4. If the urine shows a sediment of uric acid, this should be separately collected and weighed, and the weight obtained added to the final result. 5. Any albumin that may be present must be previously removed. 6. If sugar is present in the urine, about 500 to 1000 c.c. are treated with a solution of neutral lead acetate, filtered, and the filtrate precipitated with mercuric acetate. The precipitate thus formed, which consists essentially of mercuric urate, is filtered off after having stood for twelve to twenty-four hours, then washed, and later suspended in water. The mercury is removed by means of hydrogen sulphide, the mercuric sulphide filtered off, and the filtrate collected and set aside. The precipitate itself is thoroughly boiled with water and again filtered, the washings thus obtained being added to the filtrate set aside, as just described. The total amount CHEMISTRY OF TRE URINE. 383 of fluid Is then evaporated to a small volume and acidified with hydrochloric acid, when the uric acid will separate out and may be treated as previously directed. The Xanthin-bases. The xanthin-bases which have been found in the urine are xanthin^ hypoxanthin, heteroxanthin, paraxanthin, guanin, and adenin. Con- jointly- they are also spoken of as the alloxur bases, or purin bases. Together with uric acid they are termed alloxur or purin bodies. Their relation to uric acid and the nucleins has already been con- sidered (see page 371). Unlike uric acid, they also occur as such in animal as well as vegetable tissues. The amount which appears in the urine under normal conditions is very small, constituting about 10 per cent, of the uric acid. Larger quantities may be met with in various diseases, and, generally speaking, an increase in the amount of uric acid is associated with an increase of the xanthin- bases. This is, however, not invariably the case, and at times it may be observed that an increase of the uric acid is accompanied by a diminution of the xanthins, and vice versa. These varying rela- tions can, of course, be readily understood if we remember that uric acid is an oxidation-product of the xanthin-bases, and that their ultimate origin is the same. The literature which deals with the elimination of the xanthin- bases in various diseases has during the past few years assumed enormous proportions. This has largely been owing to the publica- tion by Kriiger and Wulff of a relatively simple method for their quantitative estimation. Unfortunately, however, this method has proved unreliable and the results obtained incorrect. Our knowl- edge of the relation of the xanthins to pathological processes is hence as defective at the present time as it was years ago. Individually the xanthin-bases are of little clinical interest. Xanthin has once been found in a urinary sediment, and has in several instances been encountered as the principal constituent of vesical calculi. Its normal quantity is said to vary between 0.02 and 0.03 gramme. Larger quantities are found after a meal rich in nucleins, in leukaemia, nephritis, pneumonia, etc. Paraxanthin and heteroxanthin are present only in traces, as is apparent from the fact that Kruger and Salomon were able to obtain but 7.5 grammes of heteroxanthin from 10,000 liters of urine. Both apparently are distinctly toxic. Xanthin sediments may be recognized by means of the following test : a small amount of the material is treated with a few drops of concentrated nitric acid on a porcelain plate, and evaporated to dry- ness. In the presence of xanthin a yellow residue is obtained, which turns red upon the addition of a few drops of sodium hydrate solu- 384 THE URINE. tion and the application of heat. The reaction is common to all the xanthine. Quantitative Estimation. — Salkowski's Method.* — Six hundred c.c. of urine are precipitated with 200 c.c. of magnesia mixture (see page 380), when a 3 per cent, ammoniacal solution of silver nitrate is added to from 700 to 750 c.c. of the filtrate. The proportion should be 6 c.c. for each 100 c.c. of urine. The silver nitrate solution should be added as described on page 381. After standing for one hour the mixture is filtered, and the precipitate washed with water until all the free silver has been removed. The filter is then perforated, the precipitate washed into a flask with from 600 to 800 c.c. of water, acidified with hydro- chloric acid, and decomposed with hydrogen sulphide. The excess of hydrogen sulphide is removed by heating on a water-bath, when the silver suphide is filtered off and the filtrate evaporated to dryness. The residue is treated with from 25 to 30 c.c. of dilute sulphuric acid (1 : 100). This solution is brought to the boiling-point and is allowed to stand over night. The uric acid which has then sepa- rated out is filtered off, washed with a small amount of dilute sul- phuric acid (not more than 50 c.c), then with alcohol and ether, and weighed. To the resulting weight 0.0005 gramme is added for each 10 c.c. of the acid filtrate, to allow for the trace of uric acid which is thus lost. After having filtered off the uric acid the filtrate is again treated with ammonia and silver solution, and the xanthin-bases thus pre- cipitated. The precipitate is collected on a small filter, washed with water, dried, and incinerated. The ash is dissolved in nitric acid, and the silver estimated by titration with a solution of potassium sulphocyanide, using ammonio-ferric alum as an indicator (see page 320). The solution of potassium sulphocyanide employed in the estimation of the chlorides may be used, and is of such strength that 1 c.c. corresponds to 0.00734 gramme of silver. As 1 atom of silver in a mixture of the silver compounds of guanin, xanthin, hypoxanthin, etc., represents 0.277 gramme of nitrogen, or 0.7381 gramme of the alloxur bases, it is apparent that 1 c.c. of the potas- sium sulphocyanide solution will represent 0.002 gramme of nitro- gen and 0.00542 gramme of alloxur bases. In every case an accu- rate record must, of course, be kept of the amount of urine and filtrate used. The amount of alloxur bases found by Salkowski in the normal urine of twenty-four hours varied between 0.0286 and 0.0561 gramme. Literature. — M. Kriiger u. G. Salomon, "Die Alloxurbasen d. Hams," Zeit. f. phyaiol. Chem.. vol. xxiv. p. 364, and vol. xxvi. p. 343; Deutsch. med. Woch., 18H9, p. 97. Bondsynski u. Gottlieb, " Ueher Xanthinkorper im Harn des Leukamiker," Arch, f. exper. Path. u. Pharmakol., 1895, vol. xxxvi, p. 132. F. Gumprecht, " Alloxurkorper u. Leukocyten," Centralbl. f. allg. Path. u. path. Anat., 1896, vol. vii. p. 820. ' E. Salkowski, Pfliiger's Archiv, vol. Ixix. p. 268. CHEMISTRY OF THE URINE. 385 Hippuric Acid. Hippuric acid is a constant constituent of normal urine, 0.1 to 1 gramme being excreted in the twenty-four hours. That it is derived to some extent at least, from albuminous material is proved by the fact that its elimination is not suspended during starvation nor during the administration of a purely albuminous diet. The manner in which hippuric acid is formed in the body-economy, however, has not been definitely ascertained. In vitro it may be obtained from glycocoll and benzoic acid, according to the equation CeHj CHjNHj CH^NH — QHsCO I +1 =1 +H,0. COOH COOH COOH Benzoic acid. Glycocoll. Hippuric acid. It has been shown that phenyl-propionic acid, which differs from benzoic acid by the group CjH^, and which latter may be regarded as phenyl-formic acid, is produced during the process of intestinal putrefaction. The relation between the two bodies is seen from the formulse : H CjIIs CHj CHj.CjH, COOH COOH CH, »-^ CH, Formic Phenyl-formic | | acid. acid. COOH COOH Propionic Phenyl-propionic acid. acid. Phenyl-propionic acid is then absorbed into the blood and there, according to our present ideas, transformed into phenyl-formic acid or benzoic acid. When the latter comes in contact with glycocoll, which is probably also produced during the process of intestinal putrefaction, an interaction between the two substances occurs in the body, hippuric acid resulting, as shown in the above equation. This view is supported by the fact that phenyl-propionic acid, just as benzoic acid, when introduced into the circulation of certain ani- mals, reappears in the urine as hippuric acid. The final proof of the possible synthesis of hippuric acid from glycocoll and benzoic acid in the body has been furnished by Bunge and Schmiedeberg,i who obtained this substance, when arterialized blood containing^ glycocoll and sodium benzoate was allowed to pass through isolated kidneys of dogs. Not all the hippuric acid eliminated, however, is referable to albu- minous decomposition, but a considerable portion is derived from benzoic acid or its derivatives, which occur in many fruits, and are transformed into hippuric acid in the body. Among those which are particularly rich in these substances may be mentioned ' Schmiedeberg u. Bunge, Arch. f. exper. Path. u. Pharmakol., vol. vi. 25 386 THE URINE. the red bilberry, prunes, coffee-beans, reinesclaudes, etc., and in all cases in which an increased elimination of hipparic acid is observed the possibility of this source must always be taken into account. As to the seat of this synthesis there appears to be some uncer- tainty, as it is apparently not the same in all animals. In the dog and the frog the kidneys, according to the researches of Bunge and Schmiedeberg, must be regarded as the principal and possibly the only organs in which this process occurs. As Salomon, however, has demonstrated the presence of hippuric acid in the muscles, liver, and blood of nephrectomized rabbits, still other organs must, in the herbivora at least, be concerned in its production. Very little is known of the pathological variations in the excre- tion of hippuric acid ; this is principally owing to the fact that until recently suitable methods for its quantitative estimation were not available. It is an interesting fact that, in accordance with Bunge's experiments in dogs, the formation of hippuric acid appears to be suspended in cases of acute as well as chronic parenchymatous nephritis, for the benzoic acid which is then ingested reappears in the urine unchanged. In amyloid degeneration a marked dimi- nution in its amount has likewise been demonstrated. Large quan- tities of hippuric acid, on the other hand, have been noted in acute febrile diseases, hepatic diseases, diabetes mellitus, chorea, etc. The data, however, are insufficient to warrant any definite conclusions at the present time.' Properties of Hippuric Acid. — Chemically, hippuric acid must be regarded as benzoyl-amido-acetic acid, CgHgNOj — (C5H5.CONH. CH3.COOH). It crystallizes in long rhombic prisms when allowed to separate from its solutions gradually, while it forms long needles if crystallization takes place rapidly and the amount is small (Fig. 95). In cold water and ether it is soluble with difficulty, while it dissolves readily in hot water, in alcohol, and in aqueous solutions of the hydrates and carbonates of the alkalies, with which it forms salts, and from which the acid may again be separated and caused to crystallize out by adding a stronger acid. When hippuric acid or one of its salts is evaporated to dryness with concentrated nitric acid and the residue is heated, the odor of bitter almonds is noticed ; this is due to the formation of nitro- benzol. When boiled with hydrochloric acid or dilute sulphuric acid hippuric acid is decomposed into glycocoll and benzoic acid. A similar decomposition is effected during the process of putrefaction, and hence no hippuric acid is found in decomposing urine, benzoic, add taking its place. The latter is always found in the uriue together with hippuric acid, but has no clinical significance. In ' Th. Weyl u. B. von Anerep, "Ueber die AusscheidunK der Hippursaure und Ben- zoesaure wahrend des Fiebers," Zeit. f. physiol. Chem., 1880, vol. iv. p. 169. CHEMISTRY OF THE URINE. 387 larger amounts it has recently been encountered in a case of diabetes. It crystallizes in needles or lustrous laminae, presenting ragged edges, which resemble plates of cholesterin. It is soluble with ditiiculty in cold water, but easily soluble in ether, alcohol, and solu- tions of the alkaline carbonates and hydrates, forming salts with the latter. Hippuric acid in the urine occurs in combination with sodium, potassium, calcium, and magnesium. Quantitative Estimation of Hippuric Acid. — The following method, which may be employed for the quantitative estimation of hippuric acid, although tedious, must also be employed when it is desired to test for its presence. Principle. — ^Hippuric acid readily dissolves in solutions of the alkaline hydrates and carbonates, forming salts. These are decom- posed by means of a stronger acid, when the hippuric acid which separates out is collected and weighed. Fig. 95. Hippuric acid crystals. Method. — Five hundred to 1000 c.c. of fresh urine are evap- orated to a syrupy consistence on a water-bath, care being taken to keep the urine neutral by the addition of sodium carbonate. The residue is extracted with cold alcohol (90 to 95 per cent.), using about half of the quantity as that of the urine employed. The mixture is then set aside for twenty-four hours. The alcoholic filtrate, which contains the salts of hippuric acid, is freed from alcohol by distillation. The remaining solution is strongly acidified with acetic acid and extracted with at least five times its volume of alcoholic ether (1 part of alcohol to 9 parts of ether). From the combined extracts the ether is distilled off and the remaining solu- tion evaporated on a water-bath. The resinous residue is boiled with water, set aside to cool, and filtered through a well-moistened 388 THE VBINE. filter. The hippuric acid, which is easily soluble in boiling water, is thus separated from other constituents which are soluble in alco- hol and ether. The filtrate is rendered alkaline with a little milk of lime, any excess of calcium being removed by passing carbon dioxide through the mixture. This is then brought to the boiling- point and filtered. Any impurities which may be present are re- moved by shaking with ether. The calcium salts remaining in solu- tion are decomposed by means of an acid, when the solution is again extracted with ether. The remaining solution is evaporated to a few cubic centimeters, when the hippuric acid will separate out ou stand- ing. The crystals are dried on plates of plaster of Paris, shaken with benzol or petroleum-ether to remove any benzoic acid, and finally weighed. These crystals may be shown to be hippuric acid by their microscopical appearance, their solubility in alcohol, and their behavior when evaporated with concentrated nitric acid, as indicated above. Hofmeister's Method. — Two hundred to 300 c.c. of urine are evap- orated in a glass dish to one-third of the original volume, and treated with 4 grammes of disodium phosphate, to transform the acid into its sodium salt. The mixture is evaporated to a syrupy consistence, the residue treated with burnt gypsum, dried thoroughly, and pulverized together with the dish. The powder is extracted in a Soxhlet apparatus with freshly rectified petroleum-ether (boiling- point 60° to 80° C.) for forty-six hours, and then for six to ten hours with pure ether (free from water and alcohol). After dis- tilling ofi" the ether the residue is dissolved in boiling water and decolorized with animal charcoal, the latter being subsequently thoroughly washed with boiling water ; the solution and washings are evaporated to about 1 or 2 c.c. at a temperature of from 50° to 60° C, and set aside to crystallize. The crystals of hippuric acid are finally washed with a few drops of water and ether, and weighed. Kreatin and Kreatinin. Kreatin, which is constantly present in muscle-tissue, is in all probability the immediate and constant antecedent of kreatinin, so that two sources of this body must be recognized, viz., the muscle- tissue of the body and the muscle-tissue ingested as food. Beyond this, however, practically nothing is known, and as the artificial pro- duction of kreatinin from albuminous material has never been accomplished, it is hardly warrantable to venture an hypothesis as to its mode of formation in the body. Kreatinin is a constant constituent of the urine, about 1 gramme being excreted daily by a healthy adult. Pathologically, variations in this amount have been observed, but the data obtained possess little value. Before drawing conclusions from observations made in CHEMISTRY OF THE VBINE. 389 the clinical laboratory it is necessary to take into account the quan- tity of meat ingested, as a meat-diet will greatly increase the amount of kreatinin. If then in patients aifected with acute febrile dis- eases, such as pneumonia, typhoid fever, etc., a large increase is observed, the patient being at the same time upon a milk-diet an increased destruction of muscle-tissue may be inferred, as a milk- diet in itself, oceteris paribus, causes a diminished elimination. A decrease would logically be expected to occur during convalescence from such diseases. In the various forms of ansemia, marasmus, chlorosis, phthisis, etc., a diminished amount is observed.' The transformation of kreatin into kreatinin has been supposed to take place in the kidneys, a view which accords with the greatly diminished excretion of kreatinin in advanced cases of chronic parenchymatous nephritis. In progressive muscular atrophy, in pseudohypertrophic paralysis, and in progressive ossifying myositis a diminution has also been noted. Properties of Kreatin and Kreatinin. — Chemically, kreatin may be regarded as a methyl derivative of glucocyamin, which latter is guanidin in which 1 NHj group has been replaced by glycocoU. Kreatinin, on the other hand, is the methyl derivative of glucocy- araidin, which differs from glucocyamin only in the absence of 1 molecule of water, so that kreatinin is kreatin minus 1 molecule of water, both being derivatives of guanidin. The relation between the various bodies is shown below : C=NH \NH, Guanidin. /NH, /NH, C=NH C=NH \NH.CHj.COOH \]Sr(CH3).CHj.C00H Glucocyamin. Kreatin. C=NH C=N \NH.CH2.C0 \N(CH.,).CH2.C0 Glucocyamidin (glucocyamin minus water). Kreatinin (kreatin minus water). Kreatinin crystallizes without water of crystallization in colorless, glistening prisms. At times, when the crystals are not well devel- oped, it also appears in the form of whetstones. It is readily soluble in hot and also quite soluble in cold water and hot alcohol ; in cold alcohol and ether it dissolves with difficulty. It forms salts with acids, and double salts with some of the salts of the heavy metals. Among these may be mentioned kreatinin hydrochloride, C^HyNjO. HCl, which is easily soluble in water and crystallizes in the form of transparent prisms or rhombic plates. Most important is the com- ^ C. E. Simon, Physiological Chemistry, Lea Bros. & Co., 1901. Senator, Vlrchow's Archiv, 1876, vol. Ixvii. p. 422. Neubauer u. Vogel, Harnanalyse, Pt. ii. 390 THE VBINE. pound of kreatinin with zinc chloride, {C^.j'^fi^.Tio.Cl^ (Fig. 96). This is produced when a watery or alcoholic solution of kreatinin is treated with zinc chloride. The crystalline form of this compound depends greatly upon the purity of the kreatinin solution. When obtained from alcoholic extracts of the urine it occurs in the form of varicose conglomerations which often adhere firmly to the walls of the vessel. If the solution of kreatinin is perfectly pure, how- ever, it is seen in the form of fine needles grouped in rosettes or sheaves. Kreatinin-zinc chloride is soluble with much difficulty in water and insoluble in alcohol. The compound is especially impor- tant, as upon its formation and properties the quantitative estimation of kreatinin in the urine is based. Silver nitrate and mercuric chlo- ride cause a precipitation of kreatinin, and may, therefore, also be employed for the purpose of obtaining the substance from the urine. Fig. 96. Crystals of Icreatinin-zinc chloride. (Salkowski.) Test for Kreatinin in the Urine. — ^A few cubic centimeters of urine are treated with a few drops of a very dilute solution of sodium nitroprusside and then drop by drop with a dilute solution of sodium hydrate. In the presence of kreatinin the urine assumes a ruby-red color, which is particularly well seen in the lower portion of the tube. This color disappears after a few minutes, and is replaced by an intense yellow, which on warming with glacial acetic acid in pure solutions turns to green, then to blue, and on standing a deposit of Prussian blue is ohidiineA. {WeyVs test)? The presence of albumin or sugar does not interfere with the reaction. Quantitative Estimation of Kreatinin in the Urine.^ — Pn»i- ciple. — When an alcoholic extract of urine is treated with an alco- 1 Th. Weyl, Ber. d. deutsoh. chem. Gesellsoh., 1878, vol. xi. p. 217 ; and Jafffi, Zeit. f. physiol. Chem., 1886, vol. x. p. 399. ' Leube u. Salkowski, Die Lehre vom Harn, Hirsohwald, Berlin, 1882, p. 111. CHEMISTRY OF THE URINE. 391 holic solution of zinc chloride kreatinin-zinc chloride separates out. This, as has been mentioned, is almost insoluble in alcohol. Know- ing the molecular weight of kreatinin and kreatinin-zinc chloride the calculation of the amount of kreatinin becomes a simple matter. The molecular weight of kreatinin is 113, that of kreatinin-zinc chloride 362. In 362 parts by weight of the latter there are, hence, 226 parts by weight of kreatinin, so that the amount of the kreatinin may be calculated from the weight of the kreatinin-zinc chloride according to the following equation : 362 : 226 : : y: x; and x =^ 0.6243 y, in which y indicates the weight of the kreatinin-zinc chloride found, and x the corresponding amount of kreatinin. The phosphates must, of course, first be eliminated, as insoluble zinc phosphate would otherwise be precipitated. Method. — In 200 c.c. of urine the phosphates are first removed by alkalinizing with milk of lime, and then adding calcium chloride so long as a precipitate forms. If the volume is now less than 300 c.c, water is added to that amount. The mixture is filtered after having been allowed to stand for from one-quarter to one- half hour, and washed with a little water. Filtrate and washings are slightly acidified with dilute hydrochloric acid, so as to prevent the transformation of kreatinin into kreatin, and evaporated on a water-bath to a syrupy -consistence, and then tiioroughly mixed with 20 to 30 c.c. of absolute alcohol. The mixture is poured into a stoppered flask provided with a 100 c.c. mark, and after thor- oughly rinsing out the evaporating-dish with absolute alcohol the washings are also placed in the bottle, and absolute alcohol is added to the mark. The bottle is thoroughly shaken and set aside in a cool place for twenty-four hours, the mixture being agitated from time to time. It is now filtered and rendered slightly alkaline with a drop or two of a sodium carbonate solution, as kreatinin hydrochloride is not precipitated by zinc chloride. The reaction, however, should be only faintly alkaline, as otherwise zinc oxide will be precipitated. The mixture is now slightly acidified with acetic acid and treated with 0.5 c.c. of an alcoholic solution of zinc chloride, prepared by dissolving the salt in 80 per cent, alcohol and diluting with 95 per cent, alcohol to a specific gravity of 1.2. The mixture is well stirred and set aside in a cool place for two or three days. The crystals, which are usually deposited on the sides of the vessel in the form of wart-like masses, are then collected upon a dried and weighed filter, always using portions of the filtrate to bring the crystals completely upon the filter. These are washed with a small amount of 90 per cent, alcohol, until the washings are without color and give only a slight opalescence when treated with a drop of silver nitrate solution. The crystals are finally dried at a temperature of 100° C, and weighed. By multiplying the weight thus found by 0.6243 the amount of kreatinin is obtained. 392 THE URINE. Precautions : 1. Albumin and sugar, if present, must first be removed. In diabetic urines it is best, after having removed the sugar by fermentation, to take one-fifth of the total quantity elimi- nated in twenty-four hours, and to evaporate this to about 300 c.c. before removing the phosphates. 2. The weighed material should be examined microscopically, to see whether notable quantities of sodium chloride are present. Should this be the case, it is necessary to determine the amount of zinc present, and to estimate the kreatinin from this. To this end, the kreatinin-zinc chloride is evaporated to dryness after the addition of a little nitric acid. The residue is incinerated, extracted with water, washed, dried, fused, and finally weighed. As 100 parts of kreatinin-zinc chloride correspond to 22.4 parts by weight of zinc oxide, the corresponding amount of the compound is found according to the following equation : 22.4 : 100 :: y : x; and X = 4.4642 y, in which y represents the amount of zinc oxide found, and x the corresponding amount of kreatinin-zinc chloride. By multiplying the number thus ascertained by 0.6243 the amount of kreatinin is found. 3. Instead of doing this, the precipitate in the alcoholic solution may be examined microscopically before filtering. If sodium chlo- ride crystals are found, providing that the kreatinin-zinc chloride adheres to the sides of the vessel, the sodium chloride may be dis- solved in a little water and poured offi 4. If the crystals of kreatinin-zinc chloride adhere very firmly to the sides of the vessel, so that their removal would be incomplete, it is perhaps best to dissolve them in a little hot water, to evaporate to dryness, and to weigh the kreatinin compound directly. 5. If the urine shows an alkaline reaction, it is best to acidify with sulphuric acid, and to boil for half an hour before removing the phosphates, so as to transform any kreatin that may be present into kreatinin, when the examination is continued as described. Oxalic Acid. The origin of oxalic acid in normal urine is twofold. The greater portion, no doubt, is derived from the ingested food, but there is evidence to show that a certain amount is also formed during the metabolism of the body-tissues, and in the last instance is derived from the nucleins. Salkowski has shown that the albumins per se do not enter into consideration. Uric acid, however, which, as we have seen, is itself derived from the nucleinic bases, can be readily oxidized to oxalic acid, with the intermediary formation of parabanic acid and oxaluric acid. The latter indeed has been repeatedly dem- onstrated in the urine, and it is conceivable that the same process may occur in the animal body. But even supposing that the oxaluric CHEMTSTMY OF THE URINE. 393 acid which is obtained from the urine is formed artificially during the lengthy process of separation, and that the substance did not exist preformed, there is no reason for the assumption that uric acid may not be the normal antecedent of the oxalic acid after all. For Salkowski has demonstrated conclusively that on oxidation with ferric chloride in aqueous solution uric acid yields oxalic acid and urea directly. These various changes may be expressed by the equations : (1) C^H^NA + H,0 + 20 = CO<^ I + CO/ + CO, \nh.co \nh, Uric acid. Parabanic acid. Urea. /NH.CO CO.NH.CONHj (2) C0< I +H,0 =1 \nh.co COOH Parabanic acid. Oxaluric acid. C0.NH.C0NH, CO.OH NH, (3) I +H,0 =1 +C0<^ COOH do.oH \nh, Oxaluric acid. Oxalic acid. Urea. CO.OH /NHj (4) CsH^NA 4-3H,0+20= | + 2C0/ + CO, CO.OH \nH2 Uric acid. Oxalic acid. Urea. Under pathological conditions, further, oxalic acid may also be formed in the digestive tract from the ingested carbohydrates, as a result of a peculiar fermentative process. This has been- well shown by Helen Baldwin in Herter's laboratory. In some of these cases no free hydrochloric acid could be demonstrated in the gastric contents, and it was observed that inoculation of a digestive mixt- ure, which was originally free from oxalic acid, resulted in its ap- pearance if a few drops of such stomach contents were added. In dogs prolonged feeding with excessive quantities of glucose together with meat was seen to lead eventually to a state of oxaluria, which was associated with a mucous gastritis and the absence of free hydro- chloric acid. Oxalic acid could then also be demonstrated in the stomach contents. Very curiously the ingestion of quite small and non-toxic amounts of oxalic acid is followed by a fairly intense indicanuria. It does not seem likely to me, however, that as Harnack and v. d. Leyden suggest, the indicanuria is here referable to a toxic action upon the tissue-albumins, and I am personally inclined to explain the phe- nomenon upon the basis of increased intestinal putrefaction. The amount of oxalic acid which is normally eliminated in the twenty-four hours fluctuates with the amount ingested, and varies from a few milligrammes to 2 or 3 centigrammes being usually less than 10 milligrammes (Baldwin). 394 THE URINE. Foods rich in oxalic acid are spinach, tomatoes, carrots, celery, string-beans, rhubarb, potato, dried figs, plums, strawberries, cocoa, tea, coffee, and pepper. Foods which contain little or no oxalic acid, on the other hand, are meat, milk, eggs, butter, corn- meal, rice, peas, asparagus, cucumbers, mushrooms, onions, lettuce, cauliflower, pears, peaches, grapes, melons, and wheat, rye, and oat flour. Before drawing conclusions as to the existence of abnormal oxaluria it is hence imperative to eliminate the possibility of an increased ingestion, by placing the patient upon a diet which con- tains little or no oxalic acid. An increased elimination is notably observed in association with various dyspeptic and nervous manifestations, and constitutes the condition commonly spoken of as the oxalic acid diathesis, or as idiopathic oxaluria. In such cases a copious deposit of calcium oxa- late crystals is very frequently observed. From this occurrence, however, it is not permissible to assume that an increased amount is present unless an actual quantitative estimation has been made. At the same time we must remember that a tendency to the de- position of oxalates favors the formation of gravel or calculi, and is hence a symptom which merits due consideration. Not infrequently oxaluria of this type is associated with an increased elimination of uric acid and a mild grade of albuminuria, as has been shown by Senator, v. Noorden, DaCosta, myself, and others. Whether or not the oxaluria in these cases can be explained upon the basis of abnormal fermentations in the gastro-intestinal tract, as is suggested by the observations of Baldwin, remains to be seen. In some this may be the case, but in others I am inclined to asso- ciate the oxaluria with the coexistent lithuria, and rather imagine that both conditions may be referable to impairment' of the normal oxidation-processes in the liver. That this explanation holds good also of the apparent vicarious oxaluria which is at times observed in diabetes, appears quite likely. Properties of Oxalic Acid. — Oxalic acid occurs in the urine as calcium oxalate, CaCjO^, and is held in solution by the diacid sodium phosphate. It can, hence, be precipitated by diminishing the acidity of the urine by the addition of a little ammonia or by allowing it to stand exposed to the air. "When allowed to crystallize out slowly, calcium oxalate occurs in the form of well-defined, strongly refrac- tive octahedra, in which the principal axis of the crystals is placed at right angles to the plane of the microscope slide (Fig. 97). These are very characteristic. Other forms, however, are also quite com- monly observed, such as single and double dumb-bells, spheroids and prisms, etc. (Fig. 105). They are insoluble in ammonia and alcohol, almost insoluble in hot and cold water, and very slightly soluble in acetic acid, but dissolve with ease in the mineral acids. CHEMISTRY OF THE URINE. 395 When strongly heated, the salt is decomposed into calcium oxide, carbon dioxide, and carbon monoxide, according to the equation CaCjO^ = CaO + COj + CO. Tests for Oxalic Acid. — For the detection of calcium oxalate it is frequently only necessary to examine the sediment of the urine after standing for twenty-four to forty-eight hours. No oxalate crystals, however, may be found even when an abnormally large amount can be demonstrated by chemical methods. In such cases it is usually possible to bring about the crystallization of the salt by carefully neutralizing the urine with a little ammonia. Should this procedure not lead to the desired end, it is best to treat the urine with one-third its volume of 95 per cent, alcohol. The mixture is set aside for twenty-four to forty-eight hours, when the sediment is Fig. 97. Calcium oxalate crystals. centrifugalized and examined with the microscope. This method, Baldwin states; represents a more delicate test for oxalic acid than the complicated methods of quantitative analysis which are available. Quantitative Estimation. — Heretofore the old method of ISTeu- bauer has been in general use, but it is at best unsatisfactory. It is still described at this place, as the more recent methods of Dunlop and Salkowski are as yet but little known. At the same time it must be admitted that these, more modern procedures are likewise not free from objections, but they are nevertheless to be preferred to that of Neubauer. Neubauer's Method. — Principle. — The calcium oxalate in the urine is held in solution by the diacid sodium phosphate. If this is re- moved by means of calcium chloride and ammonia, the calcium oxalate is precipitated. By heating this strongly it is transformed into calciiun oxide. As 56 parts by weight of calcium oxide correspond to 128 parts by weight of calcium oxalate, the amount of the latter can be readily calculated according to the equation : 56 : 128 :: y -.x; and 396 THE URINE. X = 2.2857 y, in which y indicates the amount of calcium oxide found in a given amount of urine, and x the corresponding amount of calcium oxalate. As 1 molecule of oxalic acid, moreover, corre- sponds to 1 molecule of calcium oxalate, the amount of the former can be found from that of the latter according to the equation : 128 -.dO-.-.y-.x; and x = 0.703 y, in which y represents the amount of calcium oxalate found, and x the amount of the corresponding acid. Method. — A large amount of urine (600 to 1000 c.c.) is thy- molized, so as to guard against putrefactive processes, and is treated with an excess of calcium chloride solution and rendered strongly alkaline with ammonia. The diacid sodium phosphate which holds the oxalic acid in solution is thus removed. The precipitate of phos- phates is then carefully treated with an amount of acetic acid just sufficient to dissolve it, without filtering. As calcium oxalate is almost insoluble in acetic acid, it gradually separates out. To this end, the mixture is allowed to stand for twenty-four hours, the addition of the thymol preventing the development of bacteria. At the end of this time the calcium oxalate is filtered off through a small filter. It is washed with water and treated with a small amount of warm hydrochloric acid, any uric acid that may have separated out being left behind. The filtrate is further treated with a small amount of very dilute ammonia, so as to render the solution slightly alkaline. After standing for twenty-four hours the calcium oxalate will have separated out, and is collected upon a smaller filter, the weight of the ash in this being known. After washing with water the contents of the filter are dried and incinerated in a cruci- ble, heating strongly for about twenty minutes, whereby the oxalate is transformed into the oxide. From the weight of this the corre- sponding amount of oxalic acid is readily calculated according to directions given above. Dunlop's Method (slightly modified by Baldwin). — In this case the calcium oxalate is precipitated from an acid solution by means of alcohol, instead of from an alkaline solution by calcium chloride. The urine is thymolized, and if alkaline acidified with a trace of acetic acid. Five hundred c.c. of a well-mixed specimen of the collected urine of twenty -four hours are treated with 150 c.c. of over 90 per cent, alcohol, to precipitate the calcium oxalate. The mixture is set aside for forty-eight hours. It is then filtered, care being taken to insure the entire removal of the crystals from the beaker. The sediment is thoroughly washed with hot and cold water, and finally with dilute acetic acid (1 per cent, solution). The filter is placed in a small beaker and soaked in a small amount of dilute hydrochloric acid. It is then washed with hot water until the washings no longer give an acid reaction. The acid solution and washings CHEMISTRY OF THE URINE. 397 are filtered, and the filtrate evaporated to about 20 c.c. This is treated with a very small amount of a solution of calcium chloride, to insure the presence of an excess of calcium. The solution is neutralized with ammonia, slightly acidified with acetic acid, and treated with strong alcohol, so that the mixture contains 50 per cent. After forty-eight hours the sediment is collected on a filter free from mineral ash, and is washed with cold water and dilute acetic acid until free from chlorides. The filter with its contents is then in- cinerated, first over a Bunsen burner, and afterward for five minutes in a blow-pipe flame. On cooling over sulphuric acid the ash is weighed; the result multiplied by 1.6 represents the amount of oxalic acid in the volume of urine examined. Salkowski's Method. — In the case of human urine of moderate concentration 500 c.c. of the non-filtered urine are evaporated to about one-third. On cooling, the liquid is acidified with 20 c.c. of hydrochloric acid (sp. gr. 1.12), and extracted three times with new portions of 200 c.c. each of a mixture of 9 to 10 volumes of ether and 1 volume of alcohol. The ethereal extracts, which contain the liberated oxalic acid, are carefully separated from the urine and filtered through a dry filter. The ether is distilled off; the re- maining alcoholic solution, which still contains a little ether, is placed in a deep evaporating-dish, diluted with 10 to 15 c.c. of water, and evaporated on a water-bath. The resulting milky fluid is concen- trated, more water being added if necessary, until it becomes clear and a gummy material separates out. On cooling, the liquid, which should measure about 20 c.c, is passed through a small filter. This is washed once or twice with a little water, when filtrate and washings are rendered slightly alkaline with ammonia, treated with 1 to 2 c.c. of a 10 per cent, solution of calcium chloride, and acidified with dilute acetic acid. The reaction should be distinctly acid, but an excess should be avoided. An indication that a sufiicient amount has been added is afforded by the dissolution of the precipitate of phosphates, which occurs after the addition of the calcium chloride solution. After standing for twenty-four hours, or still better forty- eight hours, the calcium oxalate that has separated out is collected on a filter free from ash, washed with hot and cold water, dried, and incinerated as usual (see above). The resulting weight, multi- plied by 1.6 indicates the corresponding amount of oxalic acid in grammes. LiTEEATUKB.— p. Furbringer, "Zur Oxalsaureausscheidung durch d. Ham," Deutsch. Arch. f. klin. Med., 187fi, vol. xviii. p. 143. J. C. Dunlop, " The Elimination of Oxalic Acid in the Urine," etc., Jour. Path, and Baet., 1896 (an historical review of the subject of oxaluria is here also given). H. Baldwin, " An Experimental Study of Oxaluria," Jour. Exper. Med., vol. v. p. 27. E. Salkowski, Berlin, klin. Woch., 1900, p. 434 ; and Zeit. f. physiol. Chem., vol. xxix. p. 437. E. Harnack, " Ueber Indican- urie in Folge von Oxalsaurewirkung," Zeit. f. physiol. Chem., 1900, vol. xxix. p. 205. 398 THE URINE. ALBUMINS. The albumins which may be met with in the urine are serum- albumin, serum-globulin, albumoses (peptones), the albumin of Bence Jones, hsemoglobin, nucleo-albumin, fibrin, histon, and nucleo- histon. Of these, serum-alburaia is the most important from a clinical standpoint. Ser'um-albamin. — The question whether or not serum-albumin occurs normally in the urine — i. e., under strictly physiological con- ditions — has been much disputed. It is claimed by some that traces may be temporarily met with in apparently healthy individuals after severe muscular exercise, cold baths, mental labor, severe emotions, during menstruation, digestion, etc. This so-called physiological albu- minuria mostly occurs in young adults, and is usually, if not always, of brief duration. The urine, it is claimed, is otherwise normal — i. e., of normal amount, appearance, specific gravity, and composi- tion, and free from abnormal morphological constituents, such as casts, red corpuscles, leucocytes, and epithelial cells.' The existence of a physiological albuminuria, on the other hand, is denied, and the occurrence of serum-albumin at least regarded as pathological in every case. I have never been able to convince my- self of the occurrence of serum-albumiu in the urine under strictly physiological conditions, and it has been pointed out elsewhere that severe muscular and mental labor, severe mental emotions, cold baths, etc., can hardly be regarded as physiological stimuli for all "persons. The albuminuria, so often observed during the first days of life, at which time sediments of uric acid and urates, mucus, epithelial cells from the different portions of the urinary tract, and even casts may also be seen — i. e., constituents which in adults would rightly be regarded as abnormal — has also been brought for- ward in support of the theory of a physiological albuminuria. There can be no doubt, however, that this form of albuminuria is referable to the profound changes that take place in the circulatory system after birth, and to some extent perhaps also to the well- known uric-acid infarctions so frequently seen in the kidneys of the newly born, so that it would probably be better and more in accord with the teachings of pathology to regard this form of albuminuria also as abnormal.^ The more closely the subject of the so-called physiological albu- minuria is studied the more improbable does its physiological nature appear, and a more detailed study of the metabolic processes, it may be confidently asserted, will ultimately lead to the conclusion that the presence of albumin in every case is a pathological phenomenon. The association of an increased elimination of urea and uric acid ' C. E. Simon, " Functional Albuminuria," N. Y. Med. Jour., 1895, p. 330. ' L. Landi, L'albuminuria nel parto, Morgagni, 1890, vol, xxxii. ALBUMINS. 399 with albuminuria in apparently healthy individuals was noted twenty- five years ago, but received comparatively little attention. More recently, Da Costa ^ has pointed out the existence of albuminuria associated with lithuria and oxaluria. Personal observations have led me to look upon this form of albuminuria as of common occur- rence, and while in almost every case the albumin can be caused to disappear from the urine by proper diet and exercise, there can be no doubt that, if neglected, granular atrophy may ultimately result. An albuminuria may at times be observed in anaemic children and adolescents, and particularly in masturbating boys of the mouth- breathing type, but can hardly be regarded as physiological. The same may be said of the albuminuria of pregnancy and parturition. The course which may be taken by these various forms of what should be termed functional albuminuria, in which the amount of albumin rarely exceeds 0.1 per cent., is very interesting. The elimi- nation of albumin may thus be quite transitory on the one hand, as when following severe muscular exercise, cold baths, and the like. It may, however, also last for several days, or even weeks, and be followed by a disappearance of the albumin for a variable length of time, and again by its reappearance and continuance for days and weeks. The term intermittent albuminuria ^ has been applied to this latter type. At times the albuminuria may follow a definite course, disappearing and reappearing with, such regularity that it has not improperly been styled cyclic albuminuria? In this form the albu- min generally disappears from the urine during the night or during prolonged rest in bed, and reappears during the day, the erect posture apparently favoring its reappearance ; the term postural pr orthostatic albuminuria has hence also been suggested for this form. Oswald, who made a careful study of cyclic albuminuria in Riegel's clinic, regards its occurrence as distinctly pathological, and as indi- cating the existence of nephritis. Remembering the importance of the subject, it may not be out of place to enumerate the reasons which led Oswald * to this conclusion : 1. The patients generally come to the physician complaining of certain definite symptoms which are similar to those noted in cases of true nephritis. At times, however, no complaints are made, be- cause the patients have reasons for concealing them (as in examina- tions for life-insurance), or because they are temporarily absent. 2. The subjective complaints, as well as the anaemia so frequently observed in such cases, generally disappear, together with the albu- ' Da Costa. "The Albuminuria and Bright's Disease of Uric Acid and Oxalic Acid," Am. Jour. Med. Sci., 1895. ' Bull, Berlin, klin. Woch., 1886, vol. xxiii. p. 717. Mareau, Eev. de m^d., 1886, vol. vi. p. 855. Klemperer, Zeit. f. klin. Med., 1887, vol. xii. p. 168. 'A. Keller, Beitrage z. Kenntniss d. cyklischen AlbUminurie, Diss., Breslan. 1896. * K. Oswald, ■' CyMische Albnminurie u. Nephritis," Zeit. f. klin. Med., vol. xxvii. p. 73. 400 THE URINE. min, under suitable treatment, and reappear when the ansemia again becomes marked. 3. In many, a history of an antecedent nephritis the result of scarlatina or diphtheria may be obtained, as in three cases of Heub- ner, in fourteen cases out of twenty described by Johnson, etc. In some also a direct transition from an acute nephritis to the cyclic form of albuminuria has been noted. Where this was not possible the history of an acute infectious disease or an angina that had been overlooked in the clinical history must be regarded as a possible cause. 4. The absence of morphological elements, especially tube-casts, does not exclude a nephritis. A large number of cases, moreover, have recently been observed in which casts were repeatedly found. 5. A cyclic albuminuria may be observed in many cases of chronic nephritis. 6. Marked organic abnormalities (such as heart-lesions) need not be demonstrable, as they may be absent for a long period of time or may be unrecognizable. Senator's ' statement, that the existence of a physiological albu- minuria is proved by the fact that the morphological constituents of the primitive nubecula contain albumin, requires no further criticism, and should be regarded as a misconstruction of the main point at issue — a mere sophism ; and Posner's ^ observations, in view of the researches of Malfatti,^ which tend to show that the body obtained by Posner was not serum-albumin, but a nucleo-albumin, may now be regarded as erroneous. In oonolusion, it may be safdy asserted that a transitory, intermit- tent, and cyclic albuminuria is not infrequently observed in apparently healthy individuals, but that the facts so far brought forward do not warrant the assumption that such forms of albuminuria are physio- logical.^ It would lead too far to enter into a detailed consideration of the various causes that have from time to time been suggested as an ex- planation of the fact that albumin does not occur in the urine under normal conditions. There can be no doubt, however, that the integ- rity of the epithelial lining of the glomeruli and the convoluted tubules must be regarded as the principal factor which prevents the albumin of the blood from passing into the urine. When the readi- ness with which the glandular structures of the kidney respond to any abnormal stimulation is considered, it is easily understood how an albuminuria may be produced in many different ways. Aside ^ Senator, Die Albuminurie, Hirschwald, Berlin, 1882. '' 0. Posner, Berlin, klin. Woch., 1885, vol. xxii. p. 654 ; Virchow's Arcliiv, 1886, vol. civ. p. 497 ; Arch. f. Anat. u. Physiol., 1888. ' Malfatti, Interna*. Centralbl. f. d. Physiol, u. Pathol, d. Ham- u. Sexualorgane, 1839, vol. i. p. 266. * V. Noorden, Deutsoh. Arch. f. klin. Med., vol. xxxviii. pp. 3 and 205. Leube, Zeit. f. klin. Med., 1887, vol. xiii. p. 1. Winternitz, Zeit. f. physiol. Chem., 1891, vol. XV. p. 189. C. E. Simon, loc. cit. ALBUMINS. 401 from acute and chronic inflammatory processes in the widest sense of the word, an albuminuria may be the result of circulatory dis- turbances in the kidneys of whatever kind — i. e., the result of anaemia as well as of hypersemia. In many and perhaps the majority of cases in which what Bamberger ^ terms a hcematogenous albuminuria occurs, we have direct evidence of the existence of cir- culatory disturbances, as in cases of uncompensated valvular lesions, weak heart, emphysema, hepatic cirrhosis, etc. In other cases, how- ever, the existence of such disturbance can only be surmised, and the question, whether or not the albuminuria observed in the various infectious diseases, for example, is referable to circulatory abnormali- ties or to a direct irritative action of microbic poisons upon the renal parenchyma, must still remain open. From personal studies in connection with the functional albu- minuria of Da Costa, it seems not unlikely that in many cases in which obscure circulatory disturbances are supposed to exist and are held responsible for an existing albuminuria, this is referable rather to the strain thrown upon the kidneys by the continued elimination of abnormally large quantities of organic material, the quantity of water being at the same time proportionately small. If it is remembered, furthermore, that injuries affecting certain portions of the brain are followed by albuminuria, and that this may be artificially produced by a piqure, analogous to the glucosuric piqure of C. Bernard, still another factor is given which may pos- sibly enter into the causation of albuminuria. Obstruction to the outflow of urine from the kidneys has also been experimentally shown to lead to albuminuria, an observation with which clinical experience is in perfect accord. Finally, an abnormal composition of the blood may at times cause the albuminuria. In passing on to a more detailed study of the various pathological conditions in which an elimination of albumin may be noted, an attempt will be made to classify the various forms of albuminuria in accordance with the more general considerations set forth above. It should be remembered, however, as already indicated, that it may be very difficult, if not impossible, to assign one single cause to a given clinical case, as several factors may at the same time be operative in the production of the albuminuria. 1. Functional Albijminueia — Under this heading may be. comprised the various forms of "physiological" albuminuria, which, have already been considered. 2. The albuminuria associated with oeganic diseases OF THE KIDNEYS, viz., acute and chronic nephritis, renal arterio- sclerosis, amyloid degeneration of the kidneys.^ In acute nephritis, albuminuria, usually of great intensity, is a, ' V. Bamberger, Wieu. med. Woch., 1881, pp. 145 and 177. ^ Senator, loo. cit. 26 402 THE URINE. constant and most important symptom. The amount eliminated is generally proportionate to the intensity of the disease, but varies within fairly wide limits, generally from 0.3 to 1 per cent., corre- sponding to a daily excretion of from 5 to 8 grammes. Much larger quantities, it is true, are at times excreted, but it may be definitely stated that the daily loss of albumin seldom exceeds 20 grammes. In chronic parenchymatous nephritis the elimination of albumin is likewise constant, and the amount excreted in severe cases may even exceed that observed in the acute form. An elimination of from 15 to 30 grammes, viz., 1.5 to 3 per cent, by weight, is frequently observed. In the ordinary form of chronic interstitial nephritis the elimina- tion of albumin is, as a general rule, slight, and rarely amounts to more than 2 to 5 grammes pro die. At the same time it is not unusual to meet with an apparent absence of albumin if the more common tests (see below) are employed. If it is remembered that very often the diagnosis of the disease is dependent upon the demon- stration of the presence or absence of albumin, the necessity of/re- querd examinations and the employment of more delicate tests, par- ticularly of the trichloracetic acid test, as well as of a microscopical examination, is at once apparent. This is even of greater impor- tance in the renal arteriosclerosis of Senator, in which albumin by the ordinary tests is probably not demonstrable in the majority of cases, and in which even the trichloracetic acid test may not be of service, and casts are absent. Amyloid degeneration of the kidneys, in the absence of inflamma- tory processes, is accompanied by a condition of the urine closely resembling that observed in the ordinairy form of chronic interstitial nephritis. A total absence of albumin, however, is less frequently noted, while an amount varying between 1 and 2 per cent, is not uncommon. It will be shown later on that in this condition con- siderable amounts of serum-globulin are excreted in addition to the serum-albumin ; larger amounts, in fact, than are generally observed in this form of chronic renal disease, so that Senator sug- gests that such a relation, in the absence of an acute nephritis, or an acute exacerbation of a chronic nephritis, may be of a certain diagnostic value. 3. Febrile Albuminuria.' — That albuminuria may occur in almost any one of the various febrile diseases is a well-known fact, but it is important to remember that, while such an albuminuria may at times be referable to a true nephritis developing in the course of or during convalescence from an acute febrile disease, such is the exception, and not the rule. Under this heading, only that form will be considered which is not associated with distinct changes 1 Leyden, Zeit. f. klin. Med., 1881, vol. iii. p. 161. H. Lorenz, Wien. klin. Woch., 1888, vol. i. p. 119. ALBUMINS. 403 affecting the renal parenchyma, and which generally appears during the height of the disease only, and disappears with a return of the temperature to normal. As has been mentioned, it is often difficult, if not impossible, to assign a definite cause for an albu- minuria of this character, and in all probability several factors are in operation at the same time. In the beginning of the disease, when the blood-pressure, as a rule, is increased, the albuminuria may be referable to an ischsemia of the kidneys, as the increased pressure in fever, according to Cohnheim and Mendelson, is largely referable to spasm of the arterioles. Later on, or in the begin- ning of cases in which especially severe intoxication exists, the blood-pressure may be subnormal, and the albuminuria be due to this cause — i. e., a hypersemic condition of the kidneys. As a mat- ter of fact, it has been experimentally demonstrated that both anaemia and hyperaemia of the kidney structure may lead to albuminuria. On the other hand, it is not unlikely that the strain thrown upon the kidneys by an excessive elimination of organic material, in the absence of a correspondingly large quantity of water, may produce albuminuria. I have repeatedly seen the functional albuminuria of the type described by Da Costa disappear during the administration of a diet relatively poor in nitrogen, while an increased diuresis was at the same time effected by the consumption of large amounts of water. In those grave cases of typhoid fever, furthermore, which are characterized by high fever and pronounced nervous symptoms it would appear quite likely that the albuminuria, which in these cases is particularly marked, is referable to a direct influence upon the central nervous system, and in some cases, at least, also dependent upon an irritant action upon the renal epithelium on the part of the microbic poisons circulating in the blood. The character of the albu- minuria will largely depend upon the intensity of the intoxication ; in other words, upon the amount of bacterial poison present at any one time in the blood. Notwithstanding statements to the contrary, albuminuria may be regarded as a constant symptom of typhoid fever, as has been defi- nitely demonstrated by Gubler and Robin. It is difficult to say why other observers have found albumin in only a comparatively small percentage of cases, but it is not unlikely that this is owing to a lack of uniformity in methods, it being presupposed also that questions of this kind can only be decided by daily examinations. According to Robin, the trace of albumin which is at times observed during the first week of the disease is an albumose, while later on serum- albumin is constantly found ; the amount increases with the inten- sity of the morbid process, and the highest figures are reached in fatal cases. The more severe the disease the earlier does albumin appear in the urine, it being remembered, however, that reference is had 404 THE UJtINE. only to those cases in which distinct renal changes are not demon- strable. Toward the termination of the fastigium the amount of albumin generally undergoes a certain diminution, and may even disappear entirely. This diminution, however, is only temporary, and in severe cases the albumin again increases in amount during the period of great variations in the temperature. In light cases an increased elimination also takes place at this stage, but is soon followed by a decrease, after which only traces can be demonstrated. In some cases it disappears entirely, but it is rare, according to Robin, to meet with cases in which at least a trace does not reappear during convalescence. In light cases the albuminuria rarely persists longer than the fifth or eighth day of convalescence, and Robin even goes so far as to say that a relapse may be anticipated if the albuminuria does not disap- pear at that time. A limited number of personal observations have borne out the correctness of this view, and in one case in which a relapse occurred so late as the fifteenth day of convalescence traces of albumin could be demonstrated during the entire period. In severe cases, on the other hand, the albumin persists for a variable length of time, and rarely disappears before the tenth day of con- valescence. At times an increase is seen during convalescence when traces only have previously been observed. It is this form which the French generally speak of as colliquative albuminuria. While this is principally observed in typhoid fever, it is not unusual to meet with it during convalescence from various other acute diseases. Care must be taken not to confound the albuminuria so frequently seen during convalescence from typhoid fever, referable to a pyelitis, with the form just described. From the following summary, constructed from data given in Robin's ' monograph on the urine of typhoid fever and other acute infectious diseases which may be associated with a typhoid condition, an idea may be formed of the occurrence of albuminuria, as well as of its degree of intensity in these diseases : Acute miliary tuberculosis : albumin is much less frequent than in typhoid fever ; when present, it is rarely found in the abundance so characteristic of the fatal cases of the latter disease. Pneumonia : albumin is as uniformly present as in typhoid fever, and at times very abundant. Grippe : albumin is infrequent ; present in about 20 per cent, of the cases, and only in traces. Herpetic fever : albumin never present in large amounts. Embarras gastrique : albumin rarely present. Adynamic enteritis of adults : albumin almost always present, but usually only in traces. Cerebrospinal meningitis : albumin in fairly large amounts. ' A. Eobin, Urologie clinique de la fi^vre typhoide, Paris, 1877. ALBUMINS. 405 Vegetative endocarditis : albumin very abundant in about 14 per cent., evident in 44 per cent., and traces in 42 per cent, of all cases. Acute articular rheunaatism : albumin present in about 40 per cent. Rubeola : albumin usually absent in light cases, but present in the more severe and complicated forms. Intermittent fever : albumin variable. In conclusion, it may be said that practically every acute febrile disease, even simple follicular tonsillitis, may be accompanied by albuminuria in the absence of definite changes affecting the renal parenchyma. Its occurrence in an individual case is probably dependent to a very large degree upon the intensity of the intoxica- tion. While it is generally an easy matter to distinguish between this form of albuminuria and that associated with distinct organic changes in the kidneys, considerable difficulty may at times be experienced ; this question will be dealt with later on. 4. Albtjmintjeia eefbeable to Cieculatory Distuebances.' — To this class belongs the albuminuria so frequently observed in cardiac insufficiency referable to valvular lesions, degeneration of the heart-muscle from whatever cause, disease of the coronary arteries, etc., as well as in cases of impeded pulmonary circulation affecting the general circulation through the right heart, and, finally, in con- ditions associated with local circulatory disturbances, such as com- pression of the renal veins by a pregnant uterus, tumors, etc. It has been pointed out that febrile albuminuria also may, to a certain extent at least, be referable to such causes — i. e., an ischsemia or hyperaemia of the kidneys produced by an increased or diminished blood-pressure. The albuminuria observed in cases of cholera infantum, the simpler forms of intestinal catarrh, and in cholera Asiatica particularly, are undoubtedly dependent upon such causes. The occurrence of albuminuria after cold baths, as stated above, is regarded by many as a " physiological " phenomenon ; but this view should be rejected, as there can be little doubt that this form is also referable to circulatory disturbances. The quantity of albumin found under these circumstances varies considerably, but rarely exceeds 0.1-0.2 per cent, unless the disease has advanced to a stage where distinct changes in the renal parenchyma have resulted. 5. Albuminueia eepeeable to an Impeded Outflow of Urine. — Clinically, albuminuria referable primarily to an impeded outflow of urine from the kidneys is probably of more frequent occurrence than is generally supposed, and especially in women, in whom Kelly and others have demonstrated the frequent existence of ureteral stenoses. A complete blocking of the excretory duct, on the other hand, is rarely seen, but may be caused by the impac- tion of a renal calculus, the pressure of a tumor, or following cer- 1 Senator, loc. cit. 406 THE UBINE. tain gynsecological operations in which the ureter is accidentally caught in a suture, etc. It has also been suggested that the albu- minuria of pregnancy may be due to compression of a ureter, but it is more likely that other factors are here at play, such as com- pression of the renal arteries and veins. 6. Albuminueia of H^mic Okigin.^ — It was formerly sup- posed that Bright's disease was dependent upon certain abnormalities of the blood, and as a matter of fact this view has not only never been disproved, but is actually gaining ground from day to day. According to Semmola, Bright's disease is primarily due to an abnormal power of diffusion on the part of the albumins of the blood, which are eliminated by the kidneys as waste material. As a result of the excessive amount of work thus done definite renal changes are finally produced. According to his theory, then, the albuminuria is the primary factor in the causation of nephritis. Should this hypothesis hold good, Senator is correct in asserting that an albuminuria of functional origin, so to speak, must precede the occurrence of the nephritis propsr. He, however, doubts the occur- rence of a prenephritic albuminuria ; but others have noted the occur- rence of definite renal changes which manifestly followed an appar- ently functional albuminuria (Da Costa). Further researches in this direction are urgently needed, and Semmola's view can at present only be regarded as an hypothesis. But even if such blood-changes as those which Semmola suggests should not exist, there can be little doubt that true nephritis is dependent upon an acute or chronic dyscrasia of the blood, either in the sense of an abnormal mixture of the nor- mal elements or of the presence of abnormal constituents, and not- ably of poisons. The same considerations undoubtedly also apply to various other forms of albuminuria, in so far as these are not the direct result of circulatory disturbances. Clinically, albuminuria of hsemic origin is observed in various diseases of the blood, such as purpura, scurvy, leukaemia, pernicious anaemia, as also in cases of poisoning with lead and mercury, in syphilis, jaundice, diabetes, following the inhalation of ether and chloroform, etc. The albuminuria associated with an excessive elimination of uric acid and oxalic acid, and, according to personal observations, with an excessive elimination of organic material in general, notably of urea, probably also belongs to this class. 7. Toxic Albumhsttjria. — It has already been stated that the albuminuria of acute febrile diseases may, to a certain extent, be referable to a direct irritant action on the part of bacterial poisons upon the renal parenchyma. Poisoning with cantharides, mustard, oil of turpentine, potassium nitrate, carbolic acid, salicylic acid, tar, iodine, petroleum, phosphorus, arsenic, lead, antimony, alcohol, and mineral acids produces albuminuria. In all probability, however, • V. Bamberger, loc. oit. ALBUMINS. 407 the albuminuria here observed is referable not only to a direct irri- tant action upon the glandular epithelium of the kidneys, but also to circulatory disturbances. 8. Neurotic Albuminuria. — It is claimed by some that -albu- min, usually in small amounts, is eliminated in epilepsy after every attack, while others either deny its occurrence under such conditions or regard it as exceptional. In a number of cases in which I had occasion to examine urine voided after an attack albumin was usually absent. It should be stated, however, that the seizures in these cases were comparatively slight, and that unfortunately an exam- ination for semen was not made in those urines in which traces of albumin were demonstrated. An examination of the urine voided by a patient, after having been in the epileptic state for more than forty- eight hours, showed the presence of a small amount of albumin associated with an enormous elimination of uric acid, as well as a large excess of urea. Semen was absent.' A transient albuminuria has also been noted in cases of progressive paralysis, mania, tetanus, delirium tremens, apoplexy, migraine, Basedow's disease, brain tumor, etc. Although albuminuria may apparently be produced artificially by injuries affecting a certain area in the floor of the fourth ventricle, analogous 'to the production of glucosuria (see Glucosuria), it would probably be going too far to assume the existence of a certain spe- cific centre, stimulation of which causes the appearance of albumin in the urine. While the influence of the nervous system in prevent- ing the passage of albumin through the glomeruli under normal conditions is undoubted, it would appear more likely that the albu- minuria following injuries to the central nervous system is referable to circulatory disturbances in the kidneys secondary to lesions of the brain, and especially of the medulla. The albuminuria observed in certain neurotic individuals, on the other hand, is probably more fre- quently associated with metabolic abnormalities, and is of hsemic origin. 9. A DIGESTIVE ALBUMINURIA has also been described, but need not be considiered in detail. Suffice it to say that it may follow the ingestion of excessive amounts of cheese, eggs — particularly when taken raw — ^beef, etc. I have seen albuminuria follow free indul- gence in root beer. It is, of course, difficult to explain suoh oc- currences ; but bearing in mind the fact that albuminuria very often follows the ingestion of such articles almost immediately, and before they have become absorbed, it is hardly justififible to refer this form to the existence of a hyperalbuminosis. It would appear more rational, as Senator has suggested, to think of reflex vasomotor or trophic changes affecting the kidneys ; while in other cases, in which the albuminuria does not follow the ingestion of such articles of food immediately, it is quite probable that this 1 M. Huppert, Vlrchow's Archiv, 1874, vol. lix. p. 305. 408 THE URINE. may be dependent upon certain metabolic abnormalities affecting the normal composition of the blood.' In the account thus given of the occurrence of albuminuria and its possible causes, reference has been had to only a pwrdy renal albu- minuria. It should be remembered, however, that the origin of the albumin may often be extremely difficult to determine, as albuminous material, such as blood and pus, may become mixed beyond the glandular portion of the kidneys with what would otherwise have been a perfectly normal urine, and that such an admixture may take place not only in the ureters, the bladder, and the urethra, but even in the pelvis of the kidney. The term aocidental albuminuria is applied to a condition in which albuminous material becomes mixed with a urine beyond the kidneys, as in cases of cystitis and urethritis, or whenever semen has entered ths urine while the renal urine proper is free from albumin. An ad- mixture of pus, blood, lymph, or chyle may, however, also occur in the kidneys, when the albuminuria is termed aocidental renal albuminuria, an example of which is frequently seen in the slight degree of albu- minuria referable to pyelitis during convalescence from typhoid fever. By a mixed albuminuria and a mixed renal albuminuria, on the other hand, we are to understand conditions in which the source of the albumin is twofold, renal and extrarenal in the first instance, parenchymal and extraparenchymal in the second, examples being the albuminuria of cystitis combined with nephritis and pyelonephritis, respectively. It is manifest, of course, that in every instance in which albumin is found in ths urine its origin should be ascertained. While this question is usually readily decided by a microscopical examination of the urine, considerable difficulty may occasionally be experienced. It is a well-known fact that in the urine of women a trace of albu- min may frequently be detected, which is not due to any lesion of the urinary organs, but to an admixture of vaginal discharge, of ^ blood during the process of menstruation, and, in married women, of semen. Whenever, therefore, doubt is felt as to the origin of the albumin, the specimen for examination should be obtained by the catheter, care being taken previously to cleanse the vulva. In men albumin may be referable to a gonorrhceal urethritis. In such cases it is well to let the patient flush out his urethra first, and to make use for examination of the portion last voided. Very often, how- ever, the conditions are more complex, it being uncertain whether the albumin is referable to the presence of pus only, or whether its origin is in the renal parenchyma. In such cases, as in cystitis, pyelonephritis, etc., a careful microscopical examination and enumer- ation of the pus-corpuscles with the Thoma-Zeiss instrument are • The albumin which is eliminated after the ingestion of much egg-albumin, how- ever, does not belong to this category. ALBUMINS. 409 called for, and will in the majority of instances decide the question. Generally speaking, the amount of albumin found in uncomplicated cases of cystitis does not exceed 0.15 per cent., while in cases of pyelitis of the same intensity the amount of albumin is from two to three times as large. Of late, attention has repeatedly been drawn to the occasional presence in the urine of an albuminous body which is soluble in acetic acid, and which Patein regards as a modification of common serum-albumin. It has thus far been observed in only eight cases, viz., twice in chronic nephritis, three times in eclampsia, once in a cystic kidney, once in tonsillitis following an injection of diphtheria antitoxin, and once in a pregnant woman in whom typhoid fever developed. I should suggest that the substance be spoken of as Patdn's albumin ^ until its chemical identity has been established. The term aceto-soluble albumin is, of course, likewise admissible. So far as the amount of albumin which may be eliminated in the twenty -four hours is concerned, an excretion of less than 2 grammes may be regarded as insignificant, 6 to 8 grammes as a moderate amount, and 10 to 12 grammes or more as excessive. An excretion of 20 to 30 grammes is exceptional. Serum-globulin. — It has been pointed out that in cases of amyloid degeneration of the kidneys serum-globulin is found in the urine together with serum-albumin in large amounts, and, according to Senator, a ratio between the two albumins of 1 : 0.8 : 1.4 may be regarded as a fairly constant symptom of the disease, and is of diag- nostic importance. There seems to be no doubt, however, that serum-globulin occurs in the urine, although in much smaller quan- tities than in the disease mentioned, whenever serum-albumin is eliminated.^ A most remarkable instance of globulinuria has been recorded by Noel Paton,^ in which the globulin separated out in crystalline form and was found in extraordinarily large quantity, amounting on one day to 70 grammes. Albumoses. — Albumoses have frequently been encountered in the urine, but are probably more frequently overlooked, as the bodies in question are not precipitated on boiling. In former years they were commonly regarded as peptones. At present, however, it appears to be a well-established fact that true peptones, in the sense of Kuhne, viz., true albumins which are not precipitated by salting with ammonium sulphate, do not occur in the urine, and the term peptonuria should accordingly be abandoned. 1 Patein, "Aceto-soluble Albumin in the Urine," Compt. rend, de I'Acad. des Sci., 1889. Coplin, Phila. Med. Jour., 1899, p. 957. . '■' Edlefsen, Deutscb. Arch. f. klin. Med., vol. vii. p. 67. Senator, Virchow's Archiv, Tol. Ix. p. 476. Petri. Diss., Berlin, 1876. 3 B. Bramwell and N. Paton, Laboratory Reports of the Royal College of Physicians, Edinburgh, 1892, vol. iv. p. 47. 410 THE UEINE. Albumosuria is observed under a great variety of conditions. It is thus noted in association with large accumulations of pus within the body, and there can be little doubt that the albumo- suria is in such instances referable to a disintegration of the pus- corpuscles and a resorption of the resulting albumoses. This form has hence been termed pyogenic albumosuria. A hepatogenic form is noted in connection with diseases of the liver, notably acute yellow atrophy. Of its origin, however, nothing is known. For- merly, when the condition was looked upon as a peptonuria, and when it was thought that peptones were retransformed into native albumins in the liver, the "peptonuria" was explained upon the assumption that the liver had lost this power, and that the "peptones " accumulated in the blood, and were consequently eliminated in the urine. Later researches showed that the transformation of peptones into albumins takes place in the intestinal mucous membrane, and that the liver probably has no part in the process whatsoever. The explanation given had therefore to be aban- doned, and, as I have just indicated, we know nothing whatever of the origin of this hepatic albumosuria. Possibly it is of an enzymatic nature. An enterogenie form of albumosuria has been noted in various diseases of the intestinal tract, such as typhoid fever, tubercular ulceration, carcinoma, etc.; and it is possible that in these cases the albumoses are either directly absorbed from disintegrating pus, or that the intestine perhaps has in part lost the power of preventing the resorption of albumoses as such into the blood. A histogenie or hwmatogenio origin has been ascribed to the albu- mosuria which is seen in cases of scurvy, in dermatitis, in various forms of poisoning, during the puerperal period and pregnancy, par- ticularly following death of the foetus, in various psychoses, etc. A renal or vesical form of albumosuria is further noted in which the albumoses are derived from contained albumins, owing either to the presence of the common proteolytic ferments of the urine or to bacterial action, as in decomposing albuminous urines. Aside from the conditions already mentioned, albumosuria has been observed in various infectious diseases, such as septicasmia, pysemia, diphtheria, measles, scarlatina, phthisis ; further, in associ- ation with leukaemia, nephritis, puerperal parametritis, endocarditis, caries, pleurisy, heart-disease, apoplexy, myxcedema, carcinomatous peritonitis, pneumonia, liver abscess, etc. In the differential diagnosis of suppurative meningitis a positive peptone-reaction in the older sense of the word, according to Senator, speaks strongly in favor of the existence of this disease. In sup- port of this view he cites the case of a young man, the subject of a median otitis of long standing, in which symptoms pointing to a meningitis — viz., fever, headache, and pains in the neck — were ALBUMINS. 411 present, but in which no " peptonuria " was found to exist, and in which an operation revealed the presence of a cholesteatoma. A digestive form of albumosuria has recently been described, in which albumoses appear in the urine after their ingestion in large quantities, and it is claimed that this is observed only in cases of ulcerative disease of the intestinal tract. Only a positive result, however, is of value. Very frequently albumosuria accompanies albuminuria, a condi- tion which Senator has termed mixed albuminuria, and it is interest- ing to note that the albumosuria may alternate with the albuminuria, and may precede or follow the latter. In any case in which albu- moses can be demonstrated in the urine the appearance of albumin should accordingly be anticipated. LiTEEATUEE.— Hofmeister, Prag. med. Woch., 1889, vol. v. pp. 321 and 325. V. Noorden, Lehrbuch d. Path. d. Stoffwechsels, Hirsehwald, Berlin, 1893, p. 215. Senator, Deutsch. med. Woch., 1895, vol. xxi. p. 217. Stadelmann, Untersuchungen liber Peptonurie, Bergmann, Wiesbaden, 1894. v. Jaksch. Prag. med. Woch., vol. v. pp. 292 and 303, and vol. vi. pp. 61, 74, 86, 133, 143; Zeit. f. klin. Med., 1883, vol. vi. p. 413. Krehl u. Matthes, Arch. f. klin. Med., 1895, vol. xlv. p. 54. Maixner, Zeit. f. klin. Med., 1884, vol. viii. p. 234. Fischel, Arch. f. Gynaek., 1884, vol. xxiv. p. 27. V. Jaksch, Prag. med. Woch., 1895, vol. xx. p. 430. Katz. Wien. med. Blatter, 1890, vol. xiv. L. V. Aldor, Berlin, klin. Woch., 1899, pp. 765 and 785. Bence Jones' Albumin. — In association with the occurrence of multiple myeloma of the bones, notably when affecting the thora- cic skeleton, a peculiar albuminous body is found in the urine, which is apparently pathognomonic of the disease in question. It was first observed by Bence Jones, and has heretofore been regarded as an albumose. From the researches of Magnus Levy and my own investigations, however, it appears that the substance is in reality a true albumin, as it yields a proto-albumose on peptic diges- tion ; but it differs from all known albumins in its relative solu- bility on boiling, and in the readiness with which it dissolves in dilute ammonia after precipitation with alcohol. Like casein, it contains no hetero-group, but is distinguished from it by the pres- ence of a carbohydrate radicle and the probable absence of phos- phorus. It is crystallizable, and may occur in the urinary sediment in the form of typical spheroliths. The amount of the substance which may be found in the urine is variable. Some observers have noted an elimination of from 0.25 to 6.0 pro mUle, while others report much larger quantities. In Bence Jones' case the elimination rose on one occasion to 6.7 per cent., corresponding to a total output of 70 grammes in the twenty- four hours — i. e., to nearly as much as the entire amount of the albumins of the blood-plasma. As regards the origin of the albumin, nothing definite is known, but there is reason to suppose that it is not derived from the myel- omatous tissue as such. "We may imagine, however, that through 412 THE URINE. the agency of the cells of the abnormal tissue, viz., their products of metabolism, the normal transformation of the ingested albumins into tissue-albumins is impeded, resulting in the production of the substance in question, which is then eliminated as foreign matter. The disease seems to be comparatively rare, and thus far only seventeen cases have been reported in jvhich due attention has been paid to the condition of the urine. Besides these there are a few additional cases in which no special note has been made of this point, though Zahn states that in his case " sometimes more and sometimes less albumin" was found. Runeberg also reports that the urine of his patient contained much albumin, while the kidneys were found practically normal at autopsy. As the diagnosis of the disease, in its early stages at least, is altogether dependent upon the demonstration of the albumin in question, a special examination should be made in this direction in all cases of obscure bone-pain, as also in obscure cases of ansemia, since EUinger has shown that at times the disease may take its course without the occurrence of local symptoms, while a marked anaemia may exist. Of special interest in this connection is the fact that Ziilzer claims to have succeeded in bringing about the appearance of Bence Jones' albumin in the urine of animals by feeding with pyrodin, which is known to be a distinct hsemolytic poison. LiTEEATURE. — Bence Jones, Med. and Chir. Trans., 1850, vol. xxxiii. ; and Phil. Trans. Eoyal Soc. of London, 1848. Kiiline, "Ueber Hemialbumose im Harn," Zeit. f. Biol., vol. xxix. p. 209. EUinger, " Ueber d. Vorkommen d. Bence Jones'scben Korper im Harn," Arch. f. klin. Med., 1898, vol. Ixii. p. 255. Magnus Levy, Zeit. f. pbysiol. Chem., 1900, vol. xxx. p. 200. Hamburger, Johns Hopkins Hosp. Bull., Feb., 1901. Ziilzer, Berlin, klin. Wooh., 1900, p. 894. Haemoglobin (Methsemoglobin). — Under normal conditions the disintegration of the red blood-corpuscles which is constantly taking place in the body never results in such a degree of hsemoglobinaemia as to be followed by an elimination of hsemoglobin in the urine. Whenever the destruction of red corpuscles is so extensive, how- ever, that the liver is unable to transform into bilirubin all the blood-coloring matter set free, hcemoghbinuria occurs. While these factors, then — i. e., an excessive destruction of the red blood-cor- 23U3oles and an insufficiency on the part of the liver — must be regarded as explaining every case of hfemoglobinuria, our knowledge of the ultimate causes of such excessive disintegration, as well as the manner in which these operate, is limited. Formerly the term hcematinuria was applied to this condition. It was shown, however, that the pigment eliminated is in reality not hsematin, but usually methsemoglobin, and only at times hsemoglobin, so that the term hsemoglobinuria is also ill chosen. Most common is the hsemoglobinuria produced by certain poisons, such as potassium chlorate, arsenious hydride, hydrogen sulphide, ALBUMINS. 413 pyrogallic acid, naphtol, hydrochloric acid, tincture of iodine, carbolic acid, carbon monoxide, etc., and also by morels (Helvella esculenta). Quite familiar is the hsemoglobinuria observed following trans- fusion of the blood of animals into man, such as that of the calf and lamb ; also the form seen in extensive burns and in insolation. While hsemoglobinuria may occur in the course of any one of the specific infectious diseases, such as scarlatina, icterus gravis, variola hsemorrhagica, typhoid fever, yellow fever, etc., it is said to be espe- cially frequent in cases of malarial intoxication. This view is not accepted by many ; Osier, among others, believes that it has fre- quently been confounded with malarial hsematuria. I have never seen an instance of malarial hsemoglobinuria, and believe that in our more temperate zones it scarcely ever occurs. Bastianello asserts that it is likewise rare in Italy, but more common in Sicily and Greece, and very common in the tropics. According to the same observer, hsemoglobinuria occurs only in infections with the ffistivo-autumnal parasite. A hssmoglobinuria due to quinin is like- wise said to exist, but is certainly rare, excepting in patients who are suffering or have recently suffered from malarial fever. I have seen but one instance of hsemoglobinuria following the ingestion of quinin. To judge from the literature upon the subject, there can be no doubt that syphilis may under certain conditions be a potent fac- tor in the production of hasmoglobinuria. This appears to be par- ticularly true of those cases of so-called paroxysmal hsemoglobinuria in which bloody urine is voided from time to time, the attacks being frequently preceded by chills and fever, so as closely to simulate malarial fever. Other factors, also, notably cold, appear to be con- cerned in the production of this form. The occasional occurrence of hsemoglobinuria in cases of Ray- naud's disease, coincident with attacks of an epileptiform character, has been referred to in the chapter on the Blood (see page 41). Hsemoglobinuria has been observed in a case of leuksemia com- plicated by icterus. Finally, an epidemic hsemoglobinuria has been described as occur- ring in the newborn associated with jaundice, cyanosis, and nervous symptoms ; of its causation we are in ignorance. While hsemoglobinuria is rather uncommon, hoematuria is fre- quently observed, and will be considered later on, as its recognition is not dependent upon the demonstration of the albuminous body, " hsemoglobin," alone in the urine, but upon the presence of red corpuscles, which in hsemoglobinuria are either absent or present in only very small numbers. LiTEKATURE. — Hsemoglobinuria : Eosenbach, Berlin, klin. Woch., 1880, vol. xvii. pp. 132 and 151. Ehrlicb, Zeit. f. klin. Med., 1881, vol. iii. p. 383. Boas, Arch. f. klin. Med., 1885, vol. xxxii. p. 355. Kobler u. Obermayer, Zeit. f. klin. Med., 1888, vol. xiii. p. 163. 414 THE URINE. Fibrin. — The occurrence of fibrin in the urine presupposes the presence of fibrinogen and a fibrinogenic ferment. It is seldom seen. According to Neubauer and Vogel, the fibrin may occur either as coagulated fibrin or in solution. In the former con- dition it is at times observed in the form of blood-coagula, when its significance is essentially the same as that of hsematuria in general, although it must be remembered that the usual form of hematuria is not associated with the presence of coagula. Colorless coagula of fibrin are seen only in cases of chyluria or diphtheritic inflammation of the urinary passages. On the other hand, urines containing fibrinogenic material in solution are likewise seen but rarely, and are characterized by the fact that fibrinous coagula sepa- rate out only on standing, when they usually cover the bottom of the vessel ; but at times they may change the entire bulk of urine into a gelatinous mass. So far this condition has been observed only in cases of chyluria (which see). Nucleo-albumin. — The question whether or not nucleo-albumin is a normal constituent of the urine is still under dispute. Per- sonal investigations have led me to the conclusion that with com- plicated methods and large amounts of urine — from 5 to 25 liters — it is always possible to demonstrate its presence both under physio- logical and pathological conditions. With the usual tests and smaller amounts of urine, however, negative results only are obtained in strictly normal individuals. According to my experience, tri- chloracetic acid, with which Stewart ' claims to have obtained posi- tive results in every one of the one hundred and fifty normal urines which he examined, does not precipitate nucleo-albumin when this is present in normal amounts. A nuoleo-alhuminuria recognizable by the available tests does not exist under normal conditions. Even under pathological conditions nucleo-albumin is by no means always found. Sarzin ^ thus was unable to demonstrate its presence in two hundred cases which he examined in Senator's clinic. Citron' arrived at similar results, and of several thousand urines which I have exam- ined in this direction positive results were obtained in only a small percentage of cases. Its presence always indicates increased epi- thelial desquamation in some portion of the urinary tract. It is essentially met with in diseases which directly or indirectly involve the integrity of the epithelial lining of the uriniferous tubules, or of the bladder. It has thus been frequently found in cases of acute nephritis and associated with febrile albuminuria, although its pres- ence even then is not constant. In chronic nephritis it is more fre- quently absent than present. In cases of renal hypersemia and cystitis the results are variable. In thirty-two icteric urines Obermayer^ ' D. D. Stewart, Med. News, 1894. ' D. Sarzin, Ueber Nucleo-albuminausscheidung, Diss., Berlin, 1894. ' Ueber Mucin im Harn, Diss., Berlin, 1886. * Obermayer, Centralbl. f. klin. Med., 1892, vol. xili. p. 1. ALBUMINS. 415 obtained positive results without exception, and it appears that in leuksemia nucleo-albumin is also ^ quite constantly present. During the administration of pyrogallol, naphtol, corrosive sublimate, tar preparations, arsenic, etc., as well as in cases of poisoning with anilin and illuminating-gas, large amounts of the substance may be found. According to my experience, nucleo-albumin is frequently ob- tained in cases of so-called functional albuminuria, and it is not uncommon to find that this is still present when serum-albumin and serum-globulin can no longer be demonstrated, even with the trichloracetic acid test. Nucleo-albuminuria may thus exist inde- pendently of the presence of the more common forms of albumin. This observation has also been made by Strauss, who found nucleo- albumin only in several cases of cystitis, in one case of chronic in- terstitial nephritis, and in one case of emphysema pulmonum with renal hypersemia. The existence of a hsematogenic form of nucleo-albuminuria has thus far not been satisfactorily demonstrated. Histon and Nucleohiston. — Kolisch and Burian ^ were able to demonstrate the presence of histon in a case of leuksemia in which it was constantly present. More recently Krehl and Matthes ^ claim to have isolated the same substance in various febrile diseases, such as acute peritonitis, following appendicitis, in croupous pneumonia, erysipelas, and scarlatina. It is an albuminous body, and was first discovered by Kossel in the red blood-corpuscles of the goose. It exists in the leucocytes of human blood in combination with the acid leukonuclein, constituting the so-called nucleohiston of Lilienfeld. It is not clear in what manner the histonuria is produced ; so much, however, seems certain, that it is not solely dependent upon increased destruction of leucocytes. Nucleohiston itself has been found in the urine in a case of pseudoleuksemia, by Jolles.^ Tests for Albumin. — The recognition of the various albuminous bodies which may occur in the urine is based partly upon their direct precipitation and partly upon color-reactions when treated with certain reagents. The number of tests which have from time to time been sug- gested is large ; many of them after a brief period of use has^e been discarded as useless or uncertain, while others have been employed only occasionally, and have not received the recognition which they deserve, from the fact that simpler tests exist, that they do not possess sufficient delicacy, or that in some instances it is too great. 1 R. Kolisch u. E. Burian, " Ueber d. Eiweisskorper d. leukamischen Hams," etc., Zeit. f. klin. Med., vol. xxix. p. 374. ' L. Krehl u. M. Matthes, ''Ueber febrile Albumosurie," Deutsch. Arch. f. klin. Med., vol. liv. p. 508. „ , . ,, ^ 2 A. JoUes, Ber. d. deutsch. chem. Gesellsch., vol. xxx. p. 172; Zeit. f. khn. Med., vol. xxxiv. F- 53. 416 THE VRINE. In the following pages no attempt is made to describe all of these tests, and attention will be directed only to those which are generally used, and which clinical experience has proved to be of value, precedence being given to those which have been longest in use. While some of these are applicable for demonstrating the presence of more than one form of albumin, special tests will also be described whereby the various albumins may be individually recognized. In every case the urine should be carefully filtered, so as to free it from any morphological elements, etc., present. To this end, it is generally sufficient to pass the urine through one or two layers of Swedish filter-paper. Frequently, however, a clear specimen cannot be obtained in this manner ; it is then advisable to shake the urine with burnt magnesia or talcum, or to mix it with scraps of filter-paper, when it is filtered as usual. Tests for Serum-albumin. — The Nitric Acid Test ' (Fig. 98). — The value of this test, properly ap- ^lo- 98. plied, cannot be overestimated, as it is not only simple, but yields an amount of information that can otherwise be gained only with dif- ficulty. Usually the student is ad- vised to make use of a test-tube par- tially filled with urine, along the sides of which concentrated, chemi- cally pure nitric acid is allowed to flow, so as to form a layer at the bottom of the tube, when in the presence of serum-albumin a distinct white ring appears at the zone of con- tact between the two liquids (Heller's test). The pictures thus obtained can- not be compared, however, with those seen when the apparently trivial change is made of using a conical glass of about 2 ounces capacity instead of the test-tube. About 20 c.c. of urine are Nitric acid test. placed in the glass, when 6 to 10 c.c. of nitric acid are added by means of a pipette, which is carried to the bottom of the vessel ; the acid is slowly allowed to escape by diminishing the pressure of the finger upon the tube. When this is carefully done the nitric acid forms a distinct zone beneath the urine. In the presence of albumin the white ring then appears, and varies in extent and intensity with the ' J. F. Heller, Arch. f. physiol. u. path. Chetn. u. Micros., 1852, vol. v. p. 169. A. Bobin, Urologie clinique de la fi^vre typhoide, Paris, 1877. ALBUMINS. 417 amount of albumin present (Plate XV., Fig. 1). If now the con- tents of the glass are allowed to stand undisturbed — and if small amounts are present, these appear only on standing for several minutes — it will be observed that the cloudiness gradually extends upward; and if much albumin is present, it may be seen to rise into the supernatant liquid in the form of small, irregular col- umns. This appearance is possibly referable to the partial decom- position of uric acid by means of nitric acid, nitrogen and carbon dioxide being set free, which, rising to the surface in the form of small bubbles, carry the nitric acid upward ; coming into contact with albumin in solution, this is then precipitated. An excess of uric acid is indicated by the appearance, within five to ten minutes after addition of the nitric acid, of a distinct ring in the clear urine, about 1 to 2 cm. above the zone of contact, which is similar in appearance to that due to albumin. If this ring (Plate XV., Figs. 1, 2, and 3), which has been appropriately compared to a communion wafer, does not appear within five to ten minutes, it may be assumed that uric acid is present in diminished amount. The degree of increase, on the other hand, may be determined by the size of the ring, it being presupposed that the same quantities of urine and of the reagent are employed in every case. Should more than 25 grammes of urea be contained in a liter of the urine examined, an appearance like hoarfrost will be noted on the sides of the vessel, which is due to the formation of urea nitrate. Spangles of the same substance appear only in the presence of at least 45 grammes ; and if 50 grammes or more of urea are contained in the liter, a dense mass of urea nitrate may be seen to separate out. Biliary urine, when treated with nitric acid containing a little nitrous acid, shows the color-play referable to the action of nitric acid upon bilirubin (Plate XV., Fig. 4). The production of the colors (red, yellow, green, blue, and violet) takes place from above down- ward, the green color being the most characteristic ; in the absence of the latter the presence of biliary pigment may be positively ex- cluded. The presence of albumin is not objectionable, as the color- play takes place beneath the albuminous disk. In normal urine a transparent ring is also obtained, presenting a peach-blossom red ; the intensity of this may vary, however, from a faint rose to a pronounced brick color, and is referable to normal urinary pigment (Plate XV., Fig. 5). In the presence of urobilin,, on the other hand, this ring presents a distinct 'mahogany color. Indican is indicated by the appearance of a violet ring (Plate XV.,. Fig. 2) situated above that referable to the normal urinary pigment. Its intensity varies with the amount present, from a light blue to a deep indigo. The milky cloud at the zone of contact of the two fluids may be referable not only to the presence of serum-albumin, but also of 27 418 THE URINE. globulin and albumoses (propeptones), while a negative reaction will generally indicate the absence of these bodies. That the uric acid ring will be mistaken for albumin is hardly likely if it is remem- bered that this never iirst appears at the zone of contact of the two fluids, but always in the uppermost portion of the urine. It is true that urines are occasionally observed in which the separation of uric acid takes place so suddenly that within a minute or two the entire urinous portion of the mixture is completely clouded, resembling the appearance presented by a highly albuminous urine. Such an exces- sive elimination of uric acid is uncommon, however, and it is to be remembered that with uric acid the cloudiness extends from above downward, and never from below upward, as is the case with albu- min. Should any doubt be felt, it is only necessary to remove a few cubic centimeters of this cloudy urine by means of a pipette and to heat it gently in a test-tube, when the urine will clear up entirely if the precipitate is due to uric acid, while if caused by albumin it will remain or become more intense. Should the precipitate caused by nitric acid consist of albumoses, it will also clear up entirely, to reappear on cooling, the fluid at the same time assuming a markedly yellow color. The occurrence of a distinctly yellow color in the urine, moreover, which is only partially cleared upon the application of heat (and be it remembered that a much higher tem- perature is necessary for the solution of a precipitate referable to albumoses than of one due to urates), will indicate the existence of a mixed albuminuria — i. e., the presence of coagulable albumin and albumoses. Nitric acid may also cause a precipitation of certain resinous bodies, such as those contained in turpentine, balsam of copaiba and tolu, etc. If any doubt is felt, the mixture should be shaken with alcohol, when the precipitate caused by these substances is at once dissolved. The mucinous body — nucleo-albumin — which is at times found in DESCRIPTION OF PLATE XV. The Nitbio Acid Test as Appued to the Urine. Fig. 1. — The light, colorless ring in the clear urine above shows a slight increase in the amount of uric acid ; the large white band denotes a large amount of albumin, bordering upon a colored ring, referable partly to indican (blue) and partly to uro- rose'in. Fig. 2. — The light ring in the clear urine above denotes a slight increase in the amount of uric acid. Tlie bluish-black band is referable to an enormous increase in the amount of indican. (Ileus.) Fig. 3. — The broad, light band in the clear urine above is referable (o an enor- mous increase in the amount of uric acid. (Laparotomy.) Fig. 4 — The color-play referable to the presence of bilirubin is shown in a dia- grammatic manner. Fig. 5. — The colored ring is referable to the presence of normal urinary coloring- njatter. PLATE XV. FIG. 2. ALBUMINS. 419 the urine is also precipitated by nitric acid, but need not occupy our attention at this place. From what has been said, it is manifest that the employment of the nitric acid test in the manner indicated furnishes much valuable information, and the adoption of the method as described not only by hospital students, but by general practi- tioners as well, cannot be too strongly urged. Boiling Test. — A few cubic centimeters of urine are boiled in a test-tube and then treated with a few drops of concentrated nitric acid, no matter whether a precipitate has occurred upon boiling or not. If albumin is present, this will separate out as' a flaky precipitate, which consists of serum-albumin frequently mixed with serum- globulin. It is true that albuminous urines will generally yield a precipitate on boiling alone ; but it must be remembered that unless the reaction is decidedly acid a precipitation of normal calcium phosphate may occur, owing to the fact that the reaction of the urine upon boiling becomes less acid from escape of the carbonic acid held in solution. In urines presenting an alkaline or amphoteric reac- tion this is very frequently noted, and might give rise to confusion, as the precipitate due to calcium phosphate closely resembles that referable to albumin. Care must hence be taken to insure a dis- tinctly acid reaction, which is best accomplished by the addition of nitric acid, when a precipitate referable to phosphates is at once dis- solved, wliile one due to albumin remains, and may even become more marked. The quantity to be added should usually be equiva- lent to about 0.05 to 0.1 of the volume of urine. Under no con- dition should the acid be added before boiling, nor should the urine be boiled after its addition, as small amounts of albumin will otherwise be overlooked, owing to the fact that hot nitric acid dis- solves the precipitate to a certain degree. If, after addition of the nitric acid the urine turns a distinct yellow, and if then upon cooling a white precipitate appears, the presence of albumoses may be inferred. Uric acid will probably never cause confusion, as this separates out only upon cooling, and then presents a dark-brown color. As in the case of the nitric acid test, so also here, a pre- cipitation of certain resins is noted at times which may be recognized by their solubility in alcohol. Albumoses are also precipitated upon the application of heat, but such precipitates again dissolve when the temperature approaches the boiling-point (see page 425). Should acetic acid be used instead of nitric acid, great care must be taken to avoid an excess, as otherwise the albumin will be dis- solved. As this danger diminishes the greater the quantity of salts contained in the urine, it is advisable to treat the urine first with a few drops of acetic acid until a distinctly acid reaction is obtained, and then to add one-sixth its volume of a saturated solution of sodium chloride, magnesium sulphate, or sodium sulphate, when upon boiling a precipitation of the albumin will occur. Carried 420 THE URINE. out in this manner, the test is absolutely certain and will dem- onstrate even minimal amounts of albumin. If an equal volume of a saturated solution of common salt is added to the acidified urine, albumoses are also precipitated, but the precipitate dissolves on boiling. The Potassium Fbeeocyanide Test. — A few cubic centi- meters of urine are strongly acidified with acetic acid (sp. gr. 1.064) and treated with a few drops of a 10 per cent, solution of potassium ferrocyanide, when, in the presence of but little albumin, a faint turbidity, or, if much albumin is present, a flaky precipitate, is noted, which is best recognized by comparison with a tube contain- ing some of the pure filtered urine, both tubes being held against a black background. Concentrated urines should be previously diluted with water, as albumoses, like serum-albumin and serum- globulin, which may be precipitated in this manner, otherwise re- main in solution. Here, also, as in the tests described, the presence of albumoses may be inferred if the precipitate disappears upon boiling, while a partial clearing up, on the other hand, indicates the presence of albumoses and coagulable albumin. At times the addition of acetic acid by itself is followed by the appearance of a cloud in the urine, which may be due to urates or to urinary mucin (nucleo-albumin), as already mentioned. In such cases the urine should be refiltered, diluted with water, and the test again applied. v. Jaksch advises the careful addition, by means of a pipette, of a few cubic centimeters of fairly concentrated acetic acid, to which a little potassium ferrocyanide has been added, when the albumin, as in Heller's test, is seen to form a ring at the zone of contact between the two fluids. Instead of potassium ferrocyanide, potassium plat- inocyanide may also be employed, and has the advantage that the test-solution is colorless. The Trichloracetic Acid Test.' — This test is undoubtedly the most delicate of those so far described, but not so delicate that a trace of albumin or nucleo-albumin can be demonstrated in every urine. An experience based upon the examination of several thou- sand urines with this reagent warrants my speaking with a certain degree of confidence upon the subject. Very frequently it is pos- sible with this method to demonstrate albumin in urines in which the more common tests yield negative results, but in which tube- casts may nevertheless be found upon microscopical examination. The test is applied as follows : by means of a pipette 1 or 2 c.c. of an aqueous solution of the reagent (sp. gr. 1.147) are carried to the bottom of a test-tube containing the carefully filtered urine, so as to form a layer beneath the urine. In the presence of albumin a white 1 F. Obermayer, Wien. med. Jahrbuch, 1888, p. 375. D. M. Eeese, Johns Hopkins Hosp. Bull., 1890. ALBUMINS. 421 ring will be seen to form at the zone of contact between the two fluids, varying in intensity with the amount of albumin present. So far as the test for albumin is concerned, this reagent possesses an advantage over nitric acid in that the colored rings, which are so confusing to the inexperienced, are commonly not observed. Serum-albumin, serum-globulin, and albumoses are precipitated, the presence of the latter being recognized, as in the previous tests, by the fact that the precipitate disappears upon boiling and reappears on cooling. A cloud, referable to uric acid, also appears if this is present in exces- sive amounts, but disappears upon the applicatioq of gentle heat. A previous dilution of the urine, moreover, guards against its occur- rence. Other tests have also been suggested for the detection of albumin in the urine, such as the metaphosphoric acid test, the phenol, tannic acid, and picric acid tests, that with Tanret's reagent, phospho- tungstic and phosphomolybdic acids, and quite recently Spiegler's reagent. Of these, only the picric acid and Spiegler's test will be con- sidered. Picric Acid Test. — The picric acid test is not applicable as a test for albumin as such, and is mentioned in this connection only because the same reaigent is employed with Esbach's quantitative method. This is composed of 10 grammes of picric acid and 20 grammes of crystallized citric acid, dissolved in & liter of distilled Avater. If to this solution albuminous urine is added, the mixture is rendered turbid, and after some time a sediment which consists not only of albumins, but also of uric acid, kreatinin, and other, extractives, will form at the bottom of the tube (see Quantitative Estimation of Albumin). Spiegler's Test.' — Spiegler's reagent consists of 8 parts by weight of mercuric chloride, 4 parts of tartaric acid, and 200 parts of water, in which 20 parts of cane-sugar are further dissolved, so as to increase the specific gravity of the reagent and permit of its being employed, like Heller's test, even in concentrated urines. One-third of a test-tube is filled with the reagent, and the urine carefully placed above this by allowing it to flow slowly down the side of the tube ; in the presence of albumin a sharply defined white ring will be observed where the two liquids are in contact. Peptone gives no reaction, while albumoses are precipitated and may be recognized as indicated above. Special Test for Serum-albumin. — Should it be desired, for any reason, to demonstrate serum-albumin alone, the urine is ren- dered amphoteric or faintly alkaline with sodium hydrate, and is then saturated with magnesium sulphate in substance, in order to remove any globulin. The filtrate is strongly acidified with acetic acid, iSpiegler, Wien. kllu. Woch., 1892, vol. v. p. 26. 422 THE URINE. when a flaky precipitate, appearing upon boiling, will indicate the presence of serum-albumin. Paiein's albumin differs from the common serum-albumin in being soluble in acetic acid.' Very often, as in the examination for sugar, it is necessary to remove any coagulable albumin that may be present, to which end the urine is rendered distinctly acid with acetic acid and boiled. An examination of the filtrate with potassium ferrocyanide, if the amount of acetic acid added was just sufficient, will then yield a negative result (see page 420). Quantitative Estimation of Albumin. — For the quantitative esti- mation of albumin a large number of methods have been devised, which fact in itself is sufficient to indicate that the majority of them, at least, are unsatisfactory. Old Method by Boiling. — If only comparative results are desired, the old method of boiling a definite amount of urine, after the addition of acetic acid, and allowing the albumin to settle for twenty-four hours, may be employed. For this purpose Neu- bauer suggests the use of glass tubes measuring one-half to three- quarters of an inch in diameter, which are closed at the lower end with a cork. Ordinary test-tubes answer perfectly well, but care should be taken that the same quantity of urine is used in each case. The tubes are corked and kept for several days for com- parison. The results, of course, express only the relative amount of albumin present, and it should be remembered that the error incurred may amount to as much as 30 or even 50 per cent, of the quantity that is found by gravimetric analysis. This is owing to the fact that sometimes the albumin separates out in large flakes, and at other times in small flakes, and that the degree of precipita- tion is also influenced by the specific gravity of the supernatant urine. VoLUMETBic Method of Wassiliew.^ — This method can be recommended for the quantitative estimation of albumin, as it is both simple and accurate. Ten to 20 c.c. of urine, which are best diluted to 50'c.c. with distilled water, are treated with 2 drops of a 1 per cent, aqueous solution of tcuo yelfew , and then titrated with |a 25 per cent, solu- tion of salicyl-sulphonic acid until a distinct brick-red color is obtained. The number of cubic centimeters of the reagent em- ployed, multiplied by 0.0^06, will indicate the amount of albumin in the 10 or 20 c.c. of urine examined. If the urine is alkaline, it should first be slightly acidified with acetic acid. ' Patein, "Aceto-soluble Albumin in the Urine," Compt. rend, de I'Acad. des Sci., 1889. Coplin, Phila. Med. Jour., 1899, p. 957. ' Wassiliew, Eshenedelnik, 1896, No. 26 ; St, Petersburg, med. Wooh., 1897, Beilage, p. 4. ALBUMINS. 423 P Esbach's Method.!— For clinical purposes, Esbach's method is the most convenient. As stated above, his reagent is composed of 10 grammes of picric acid and 20 grammes of citric acid, dissolved in 1000 c.c. of distilled water. Special tubes. Fig- 99. termed albuminimeters (Fig. 99), are employed, which bear two marks, one, U, indicating the point to which urine must be added, and one, R, the point to which the reagent is added. The lower portion of the tube up to U bears a scale reading from 1 to 7. The tube is filled to Z/with the filtered albuminous urine, and the reagent added until the point R is reached. The tube is then closed with a stopper, inverted twelve times, and set aside for twenty- four hours. At the expiration of this time serum-albu- min, serum-globulin, and albumoses, as well as uric acid and kreatinin, will have settled, when the amount pro mille in grammes may be directly read off from the scale. A few precautions must, however, be observed in order to obtain as accurate results as possible. The reaction of the urine should be acid, and if this is not the case acetic acid is added. Its specific gravity should, furthermore, not exceed 1.006 or 1.008, the proper density being ob- tained by diluting with water. The temperature also appears to play an important r6le, the reading generally being higher with a low than with a more elevated tem- ^'^"ter" perature; 15° C is best adapted to the purpose. The Differential Density Method.^ — More accurate results may be obtained with the following method, which is based upon the diminution in the specific gravity of the urine after the removal of all albumin, and its comparison with the specific gravity observed before. To this end, the urine is treated with a sufficient amount of acetic acid to insure complete precipitation of the albumin (see below), when its specific gravity is noted. It is then brought to the boiling-point, care being taken to guard against evaporation by placing the urine in an ordinary medicine-bottle ; this is closed with a rubber stopper that has been previously boiled in a solution of sodium hydrate and washed free from alkali, the stopper being tightly fastened with a cord or wire. Thus prepared, the bottle is kept in boiling water for ten to fifteen minutes. The urine is filtered on cooling, evaporation being again carefully guarded against by filter- ing into a bottle through a funnel which has been passed through a closely fitting stopper ; the funnel is kept covered with a plate of glass. The specific gravity is then again determined, and it is best in both cases to use a pyknometer. (An accurate hydrometer, grad- uated to the fourth decimal, may, however, also be used.) The 1 Guttmann, Berlin, klin. Woch., 1886. vol. xxiii. p. 117. '' Huppert u. ZShor, Zeit. f. physiol. Chem., 1886, vol. xii. pp. 467 and 484. 424 THE URINE. decrease in the specific gravity, multiplied by 400, will indicate the number of grammes of albumin in 100 c.c. of urine. Geavimeteic Method. — If special accuracy is required, the amount of albumin must be determined gravimetrically as follows : a certain amount of urine, after having been acidified with acetic acid, so as to insure complete precipitation of all albumin, is boiled ; the albumin is then filtered off, dried, and weighed. For this pur- pose, 500 to 1000 c.c. of carefully filtered urine should be available. A specimen of this, if already acid, is placed in a test-tube, in boil- ing water, until coagulation takes place, when it is further heated over the free flame and filtered. The filtrate is then tested with acetic acid and potassium ferrocyanide. Should no albumin be thus demonstrable, the entire amount of urine is treated in the same manner, and requires no further addition of acetic acid. If, how- ever, the test yields a positive result, it is apparent that the urine was not sufficiently acid. The entire volume is then treated with a 30 to 50 per cent, solution of acetic acid, drop by drop, the mixture being thoroughly stirred and specimens tested from time to time, as described. When, finally, the urine remains clear or shows only a faint turbidity, 100 c.c. or less, according to the amount of albumin present, are first heated in boiling water until the albumin begins to separate out in flakes, and then carefully brought to the boiling-point over the free flame. The supernatant urine is decanted through a filter, which has been previously dried at 120° to 130° C. and accurately weighed, when the whole amount of the precipitate is brought upon the filter. Any albumin remaining in the beaker is detached from its sides by means of a glass rod tipped with a piece of rubber tubing, and collected by the aid of hot water. The entire precipitate is now thoroughly washed with hot water until the wash- ings no longer become turbid when treated with a drop of nitric acid and silver nitrate; in other words, until the chlorides have been completely removed. The precipitate is further washed with alco- hol and finally with ether to remove any fats that may be present, when it is dried at 120° to 130° C. until a constant weight is reached. If still greater accuracy is required, the dried and weighed precipitate is incinerated to determine the amount of mineral ash in combination with the albumin, which is then deducted from the total weight. The most accurate results are obtained if not more than 0.2 to 0.3 gramme of albumin is contained in the amount of urine employed. A smaller quantity than 100 c.c. should hence be used if a previous test with Esbach's albuminimeter shows a higher percentage. A glass-wool filter insures a more rapid process of drying — twenty- four to thirty hours ; but care must then be had that this is properly prepared, so as to guard against a loss of the wool while washing. Test for Serum-globulin and its Quantitative Estimation. — ^To test for serum-globulin the urine is rendered alkaline by the addition of ALBUMINS. 425 ammonium hydrate, any phosphates that may thus be thrown down being filtered off on standing. The urine is then treated with an equal volume of a saturated solution of ammonium sulphate, when the occurrence of a precipitate wiU indicate the presence of the globulin. Ammonium urate may likewise separate out, but this occurs later. According to Paton, the following test may also be employed : the urine after having been rendered alkaline with sodium hydrate, — any phosphates which may separate out are filtered off, — is carefully poured down the side of a test-tube containing a saturated solu- tion of sodium sulphate, so as to form a layer above this, when in the presence of serum-globulin a white ring will appear at the zone of contact. If a quantitative estimation of the globulin is to be made, the pre- cipitate thus obtained, after about one hour's standing, is collected on a dried and weighed filter, and washed thoroughly with a one- half saturated solution of ammonium sulphate until a specimen of the washings treated with acetic acid and potassium ferrocy- anide no longer gives a precipitate. It is then treated as directed in the method employed for the quantitative estimation of serum- albumin. Tests for Albiunoses. — ^A small amount of urine is strongly acidi- fied with acetic acid and treated with an equal volume of a saturated solution of common salt. In the presence of albumoses a precipitate occurs, which dissolves on boiling and reappears on cooling. If serum-albumin also be present, which is usually the case, the hot liquid must be filtered. The albumoses are found in the filtrate and appear on cooling. If the hot filtrate, moreover, is rendered alkaline with a solution of sodium hydrate, a red color develops upon the addition of a very dilute solution of cupric sulphate, added drop by drop (biuret reaction). On boiling with Millon's reagent a red color is also obtained. This reagent is prepared by dissolving 1 part of mercury in 2 parts of nitric acid of a specific gravity of 1.42, and diluting with 2 volumes of distilled water. Salkowski's Method.' — Fifty c.c. of urine are acidified in a beaker with 5 c.c. of hydrochloric acid, and precipitated with phos- photungstic acid, the mixture being heated over the free flame, when in a few minutes the precipitate will form a resinous mass which closely adheres to the bottom of the vessel. The supernatant fluid is decanted, and the mass at the bottom, which now becomes granular, washed twice with distilled water, which is likewise removed by decantation. The precipitate is then covered with about 8 c.c. of distilled water, and treated with 0.5 c.c. of a sodium hydrate solu- tion (sp. gr. 1.16). Upon shaking the beaker the mass will dissolve, ' E. Salkowski, " Ueber d. Naohweis d. Peptons (Albumosen) im Ham u. d. Darstel- Inng d. Urobilins," Berlin, klin. Wooh., 1887, p. 353. 426 THE URINE. the solution assuming a dark-blue color. This is heated on the free flame until the blue color turns to a dirty, grayish-yellow ; the solu- tion at the same time becomes turbid, but at times may turn yellow and remain clear. This discoloration may be hastened by the further addition of a few drops o/ sodium hydrate solution. As soon as this point has been reached, some of the liquid is placed in a test- tube, allowed to cool, and then treated with a very dilute solution of cupric sulphate (1 to 2 per cent.) drop by drop ; in the presence of peptones the solution assumes a bright-red color, which may be brought out still more strongly if the specimen is now filtered. If albumin or much mucin is present, these bodies must first be re- moved (see pages 422 and 428); but the quantity of urine employed is so small that the mucin can usually be disregarded. With this method, which occupies only about five, minutes, 0.015 gramme of peptones pro 100 c.c. may be demonstrated without dif&culty. Salkowski has recently pointed out that urines which are very rich in urobilin, as in pneumonia, may give rise to the biuret reac- tion even when albumoses are absent. The coloring-matter, it is true, may be removed entirely by precipitation with lead acetate or subacetate, but unfortunately a portion of the albumoses is also carried down, and the substance may thus escape detection when present only in small amounts. He hence suggests that smaller quantities of urine, such as 10 c.c, be employed in the test. The reaction is then not so well marked, but the results are more re- liable. Bang's Method. — This method has recently been introduced, and is said to be free from the objections attaching to the one pro- posed by Salkowski. Ten c.c. of urine are heated in a test-tube with 8 grammes of finely powdered anlmonium sulphate until the salt has been dissolved ; the fluid is then boiled for a moment. The hot fluid is centrifugated for one-half to one minute, the supernatant fluid poured ofi', and the sediment stirred with alcohol in an agate mortar. The alcohol is poured off, and the residue dissolved in a little water ; the solution is boiled and filtered, and the filtrate tested with sodium hydrate solution and cupric sulphate as described. Should the urine be especially rich in urobilin — i. e., manifesting a well-marked fluorescence with zinc chloride and ammonia — it is best to extract the final aqueous solution with chloroform by shak- ing, and to pour off the supernatant" fluid, when this is tested with cupric sulphate. In this manner it is possible to demonstrate the presence of albumoses in a dilution of 1 : 4000-5000. Other con- stituents of the urine, with the exception of hsematoporphyrin, do not interfere with the test. Should hsematoporphyrin be present, however, which may be suspected if a red alcoholic extract is obtained, the urine must first be precipitated with barium chloride. The fil- trate, which contains the albumoses, is then examined as described. ALBUMINS. 427 If a centrifuge is not available, the urine is boiled with the ammo- nium sulphate, when a portion of the album oses will remain on the sides of the tube as a sticky mass. This is washed with alcohol, and if necessary with chloroform, dissolved in water, and tested for biuret. The alcoholic extract may also be used for testing for urobilin. To this end, it is only necessary to add a few drops of a solution of zinc chloride, when in the presence of urobilin a beautiful fluores- cence will be observed. The test is extremely delicate.^ Tests for Bence Jones' Albumin. — The presence of Bence Jones' albumin is usually discovered on slowly heating the urine to the boiling-point. It will then be noted that at a temperature of from 60° to 60° C. a more or less intense, milky turbidity develops, which on subsequent boiling either disappears entirely or partially, and reappears on cooling. The degree to which the urine clears on boiling differs in different cases. As I have just stated, the turbid- ity may disappear entirely ; but, on the other hand, urines are met with in which even a partial clearing can scarcely be made out. This is apparently dependent upon the degree of acidity of the urine, the amount of mineral salts and of urea present, and probably also upon other and stUl unknown factors. Upon the addition of a drop of nitric acid to a few cubic centi- meters of such urine a temporary turbidity develops, which disap- pears on shaking, but persists if a little more of the acid is added. If now the mixture is heated, the albumin first coagulates to a dense mass ; on boiling, this dissolves, and after a while the liquid becomes almost entirely clear, while the turbidity returns, as before, on sub- sequent cooling. Similar reactions are obtained with all the common reagents for albumin. For its complete identification, the albumin should be isolated and further examined as follows : larger amounts of urine are pre- cipitated by the addition of one and one-half to two volumes of 96 per cent, alcohol, or by treating with two volumes of a saturated solution of ammonium sulphate. In either event the total amount of albumin is thrown down. This is then washed with alcohol and ether, and dried over sulphuric acid. To purify the substance, it is dissolved in boiling water, by the aid of a few drops of a dilute solution of sodium carbonate, and dialyzed to running and then to distilled water until free from mineral salts. It is then reprecipi- tated with alcohol (if necessary, after the addition of a drop or two of a dilute solution of hydrochloric acid), washed with absolute alcohol and ether, and dried. Thus purified, the albumin is prac- tically insoluble in distilled water or saline solution at ordinary tem- perature, and only sparingly so at the boiling-point. In boiling 'E. Bang, "Eine iieue Methode zum Nachweia d. Albumosen im Ham," Deutsch. med. Woch., 1898, p. 17. 428 THE URINE. water, however, it dissolves with comparative ease after the addi- tion of a few drops of sodhitn carbonate solution. On neutraliza- tion no precipitate occurs if a sufficient amount of water is present. If such a neutral solution is heated, no change occurs ; but if it is now acidiiied and a certain amount of salt added, the typical reaction appears on heating, viz., precipitation between 50° and 60° C (even between 40° and 50° C. if a sufficient amount of salt is present), clearing on boiling, and reprecipitation on cooling. On digestion with pepsin-hydrochloric acid, as I have said, a proto-albumose is obtained among the early products of digestion, while a hetero-albumose is not formed. Test for (Mucin) Nucleo-albumin. — The carefully filtered urine is treated in a test-tube, drop by drop, with an excess of concentrated acetic acid, when the occurrence of a turbidity will indicate the presence of nucleo-albumin. If the urine contains albumin, this must first be removed by salt- ing with ammonium sulphate in substance. The precipitate is then dissolved and tested in the usual manner, after dialyzing out the salts. Dilution of the urine (1 part to 3 of water) should also be practised when doubt is felt, as urates will then not interfere with the reaction, nor will the urinary salts be so apt to exert a solvent action upon the mucin if they are present in large amounts. Ott's test may also be advantageously employed.' To this end, a few cubic centimeters of urine are treated with an equal volume of a saturated solution of common salt, when Alm6n's solution, which consists of 5 grammes of tannic acid, 10 c.c. of a 25 per cent, solution of acetic acid, and 240 c.c. of 40 to 50 per cent, alcohol, is slowly added. In the presence of nucleo-albumin a precipitate develops at once. Nucleo-albumin is characterized by its insolubility in acetic acid, by the fact that it is precipitated by magnesium sulphate, and that it does not give rise to the formation of a reducing substance when boiled with dilute acids. It is thus readily distinguished from globulin and true mucin, with which it has frequently been con- founded. Globulin precipitates are easily soluble in acetic acid, and mucin when boiled with acid gives rise to the formation of a reduc- ing substance. In order to remove nucleo-albumin from the urine, this is treated with neutral lead acetate, an excess of the reagent being carefully avoided. If it is desired to test for peptones, the filtrate is then treated with hydrochloric acid and the process continued as described above. Test for Haemoglobin. — The diagnosis of hsemoglobinuria is based upon the demonstration of haemoglobin, viz., methsemoglobin, in the urine in solution, in the absence of red corpuscles, or at least in the 1 A. Ott, Oentralbl. f. inn. Med., 1895, vol. xvi. p. 38. ALBUMINS. 429 presence of only a very small number, so that an examination in the latter direction is also an important factor. Bloody urine is generally turbid, and may vary in color from bright red to almost black. Oxyhsemoglobin, as such, can only be recognized by the spectro- scope ; it gives rise to the appearance of two bands of absorption, situated between D and E, as described in the chapter on the Blood. The urine to be examined spectroscopically should be rendered feebly acid by means of acetic acid, and placed before the open slit of the spectroscope in a test-tube, beaker, or similar vessel, when the two bands of oxyhsemoglobin will be seen, either at once or upon carefully diluting with distilled water. If ammonium sulphide is now added, the spectrum of reduced haemoglobin will be obtained. It must be remembered, however, that more commonly the spectrum of methaemoglobin is seen in cases of haemoglobinuria. The following tests, which will also indicate the presence of blood coloring-matter, cannot be employed to decide the nature of the pigment present, as methaemoglobin and oxyhsemoglobin will both react in the same manner. Heller's Test.' — ^A small amount of the urine, or still better a portion of the sediment, is made strongly alkaline with sodium hy- drate and boiled. On standing, a deposit of basic phosphates forms, which in the presence of blood coloring-matter presents a bright-red color. This is referable to the formation of haemochromogen, as may be shown by spectroscopic examination. Thus controlled, the test is extremely sensitive, and still yields a positive result when the chem- ical test alone leaves one in doubt.^ The deciding band is the first between D and E. Care should be had, however, that the solution is cold, as otherwise the haemochromogen is transformed into haematin in alkaline solution. At times, when the urine contains a large amount of coloring-matter (bile-pigment, etc.), it may be. difficult to determine the exact color of the sediment. In such cases the sub- sequent examination with the spectroscope, — the lensless instrument of Hering or that of Browning suffices, — is invaluable. In the absence of such apparatus the procedure of v. Jaksch may be em- ployed. To this end, the phosphatic deposit is filtered off and dis- solved in acetic acid, when if blood-pigment is present the solution becomes red, the color gradually vanishing upon exposure to the air. The delicacy of the test is such that oxy haemoglobin can still be demonstrated in a dilution of 1 : 4000. The Gtjaiacxjm Test.* — A mixture of equal parts of tincture of guaiacum and oil of turpentine (which has been ozonized by expos- ure to the air) is allowed to flow slowly along the side of a test- 1 J. F. Heller, Zeit. d. K. K. Gesellseh. d. Aerzte zu Wien, 1858, No. 48. '' V. Arnold, Berlin, kiln. Wooh., 1898, p. 383. ' Alm^n, see Hammarsten, Lehrbuch der physiol. Chem., 3d ed. p. 488. 430 THE URINE. tube upoD the urine to be examined, in such a manner as to form a distinct layer above the urine. In the presence of blood-pigment a white ring, which gradually turns blue, will be seen to form at the zone of contact. Dojstogany's Test.' — About 10 c.c. of urine are treated with 1 c.c. of a solution of ammonium sulphide and the same amount of pyridin, when in the presence of blood a more or less intense orange color develops, especially if looked at from above, against a white background. In doubtful cases the examination is to be controlled by a spectroscopic examination of the resulting mixture. If blood- pigment is present, the spectrum of hsemochromogen is obtained. Should the ammonium sulphide and pyridin be old, a green or brown color is imparted to the urine, which changes to yellow upon the addition of ammonium hydrate. Test for Fibrin. — Fibrin usually occurs in the urine in the form of distinct clots, the nature of which may be determined by thor- oughly washing with water, when they are dissolved by boiling in a 1 per cent, solution of soda or a 5 per cent, solution of hydrochloric acid. On cooling, this solution is tested as for serum-albumin. Test for Histon. — The urine of twenty-four hours is first examined for albumin, and this removed if present. It is then precipitated with 94 per cent, alcohol, the precipitate washed with hot alcohol and dissolved in boiling water. Upon cooling, the solution thus obtained is acidified with hydrochloric acid and allowed to stand for several hours. During this time a cloudiness, referable to a large extent to uric acid, develops, which is filtered off, and the filtrate is precipitated with ammonia. The precipitate is collected on a small filter and washed with ammoniacal water until the washings no longer give the biuret reaction. It is then dissolved in dilute acetic acid and the solution tested with the biuret test ; if this yields a positive result, and if coagulation occurs upon the application of heat, the coagulum being soluble in mineral acids, the presence of histon may be inferred. i CARBOHYDRATES. The carbohydrates which may occur in the urine are glucose, lac- tose, maltose, dextrin, levulose, certain pentoses, and animal gum. Glucose. — ^Through the researches of Wedenski, v. Udranszky, and others,^ we know that traces of glucose may be encountered in the urine under strictly normal conditions. The amount, however, is extremely small, and special methods are necessary in order to ' Z. Donogany, " Darstellung d. Hsemochromogen als Eeaction auf Blut," etc., Vir- cliow's Archiv, vol. cxlvili. p. 234. ^ A. Baumann, Ber. d. Deutsch, chem. Ges., 1886, vol. xix. p. 3218. N. Wedenski, Zeit. f. physiol. Chem., 1889, vol. xiii. p. 122. K. Baisch, Ibid., 1894, vol. xviii. p. 193, and 1895, vol. xix. p. 348. CARBOHYDRATES. 431 demonstrate its presence. With the usual clinical tests normal urine is apparently free from sugar unless unduly large amounts have recently been ingested. In that event a certain amount of glucose is eliminated in the urine, constituting the so-called digestive gluoo- suria of Claude Bernard.^ The normal limit to the assimilation of glucose on the part of the body economy is subject to considerable variation. Some observers thus report that the iagestion of such large amounts as 200 and 250 grammes does not lead to glucosuria, while others have found sugar in the urine after the administration of 100 grammes. In view of the possible relation existing between diabetes and a lowered limit to the assimilation of glucose in apparently normal individuals, or at least in persons in whose urine glucose cannot be constantly demon- strated, this question has created much interest within the last few years and has called forth a large amount of work. The major- ity of investigators are now in accord in regarding as abnormal a glucosuria that follows the ingestion of 100 grammes of chemically pure glucose. The method usually employed in order to ascertain the power of assimilation for glucose on the part of an individual is the following :. The patient receives 100 grammes of glucose, in substance, dis- solved in SDO c.c. of water, on an empty stomach, and is instructed to pass his water hourly during the following four to five hours. During this time, moreover, no food is to be taken. The individual specimens, as well as the urine which has been passed during the night, are then tested with Trommer's and Nylander's tests, with the fermentation test, and with phenyl-hydrazin. A positive result, how- ever, is recorded only when sugar can be demonstrated with the fer- mentation test. Cane-sugar and larger amounts of glucose have also been used ; but it is better, on the whole, as Strauss has pointed out, to give glucose, and not to exceed the dose of 100 grammes. Especially interesting are the results which have been obtained in various diseases of the liver, to which organ the important function of preventing an undue accumulation of sugar in the blood has been repeatedly ascribed. Bierens de Haen ^ thus reports that of twenty- nine cases of various hepatic diseases he found sugar in eighteen after the administration of 150 grammes of cane-sugar; and v. Jaksch ^ claims to have obtained positive results in fifteen cases of phosphorus poisoning out of forty-three. Strauss,* on the other hand, states that he found sugar in only two of his thirty-eight cases, and has collected one hundred and seven additional cases from 1 Claude Bernard, Compt. rend, de I'Acad. des Sci., 1859, vol. xlviii. p. 673. * J. C. Bierens de Haen, " XTeber alimeutare Glycosurie bei Leberkranken," Arch, f. Verdauungskrank., vol. iv. p. 4. ' V. Jaksch, " Alimentare Glycosurie," Prag. med. Woch., 1895, Nos. 27, 31, and 32. * H. Strauss, " Leber und Glycosurie," Berlin, kiln. Woch., 1898, p. 1122. 432 THE URINE. the literature, in only fourteen of which could sugar be demon- strated. If we add these together, we have one hundred and forty- five cases of various hepatic diseases, with negative results in 88.9 per cent. Referring to the contradictory results obtained, Strauss points out that these may have been accidental in part, but that the interpretation which has been offered by v. Jaksch and de Haen may not have been correct. It is thus possible that in his cases of phosphorus poisoning other factors besides the changes in the liver, such as the action of the poison upon the nervous system, etc., played a rdle, as a digestive glucosuria may also occur in con- nection with other forms of intoxication, as in fevers, following the administration of large doses of diuretin, in acute alcoholism, etc., in which the liver is not the only organ that is involved. Strauss further shows that great care must be exercised in the selection of the material for such investigations, and believes that errors referable to this source may have been incurred by Bierens de Haen. He thus cites two cases of hypertrophic cirrhosis, associated with delirium tremens, in which small amounts of sugar could be demonstrated in the urine a few days after recovery from the delirium, while shortly after negative results only could be obtained. The lowering effect of alcoholism upon the limit to the assimilation of glucose is a well- known phenomenon, and it would be erroneous to conclude that because alcoholism may call forth organic changes in the liver the digestive glucosuria in such cases is referable to such alterations. Without entering further into the question at this place, it appears that diseases of the liver per se do not materially lessen the as- similation of glucose, and that other forces are at the disposal of the body to supply the glycogen-forming or retaining power of the liver when this becomes insufficient, and that these also must be at fault when a digestive glucosuria is observed in association with hepatic disorders. The association of digestive glucosuria with various diseases of the nervous system has been carefully studied by v. Jaksch,' Striimpell, Strauss,^ von Oordt, Geelvink, and Arndt.^ From the work of these investigators, it appears that digestive glucosuria is rarely seen in spinal diseases, and is decidedly more common in functional diseases of the central nervous system than in organic affections. Of thirty cases of tabes examined by Strauss, digestive glucosuria resulted in only one after the administration of 100 grammes of glucose, and in that one case a family history of diabetes existed. In the neuroses a positive result was noted in forty-two out of two hundred and ten cases which I have been able to collect ' V. Jaksch, loc. cit. ' H. Strauss, " Zur Lehre v. d. neurogencn n. d. thyreogenen Qlycosurie," Deutsch. med. Wooh., 1897, pp. 275 and 309. ' M. Arndt, " Ueber alimentare Glycosurie bei Neuropsychosen," Berlin, klin. Woch., 1898, p. 1085. CARBOHYDRATES. 433 from the literature. Most frequently it is met with in the traumatic neuroses, in which Strauss observed the phenomenon in 37.5 per cent, of his forty cases ; while in the non-traumatic forms only 14.4 per cent, were insufficient in this respect. Of the organic diseases of the central nervous system, it appears that diffuse cerebral lesions referable to alcohol and syphilis are more likely to give rise to this form of glucosuria than the more localized lesions. A digestive glucosuria is further observed in numerous febrile dis- eases, such as pneumonia, typhoid fever, acute articular rheumatism, scarlatina, tonsillitis, etc. The amount of sugar usually found, varies from 0.5 to 3 per cent. ; larger amounts may, however, also be encountered, and one case is on record in which 8 per cent, was present.' Very common also, as I have indicated, is the digestive glucosuria of drinkers, and there can be little doubt that the habitual ingestion of large quantities of beer and spirits will in the course of time lead to a more than temporary enfeeblement of the carbohydrate metabolism. In the course of his investigations in this direction, Krehl ^ found that among the Jena students the proportion of those in whose urine sugar appeared apparently varied with different kinds of beer, but was much greater after morning drinking. Of fourteen who drank' bock or export beer in the morning, five had glucosuria. After the evening drinking, amounting in one case to 7 liters, of nineteen only one had sugar in the urine, and with Bavarian beer one of eleven. Of diseases of the skin, digestive glucosuria is notably associated with psoriasis ; and it is interesting to note that the same disease is not infrequently seen in diabetic patients. Gross thus records five cases, in four of which the psoriasis had existed for many years before the appearance of diabetic symptoms. Similar instances are recorded by Strauss, Grube, Polotebuoff, Nielssen, Schiitz, and others. Nagelschmidt * was able to produce glucosuria by the ingestion of 100 grammes of glucose in eight cases out of twenty -five. During pregnancy digestive glucosuria is also frequently observed,, and is by some regarded as a fairly constant symptom and of diag- nostic importance. The amount is variable, and while Lanz * records one case in which 29.6 grammes of glucose were found after the ingestion of 100 grammes, such figures are certainly uncommon, and as a general rule less than 3 grammes are recovered from the urine. After delivery the power of assimilation for glucose no longer appears to be subnormal. ' E. V. Bleiweis, " Ueber alimentare Glycosurie e saceliaro bei aouten, fieberhaftea Infektionskrankheiten," Centralbl. f. inn. Med., 1900, No. 2. ^ Krehl, "Alimentare Glycosurie nach Biergenuss," Centralbl. f. inn. Med., 1897, No. 40. ' Nagelschmidt, "Psoriasis und Glycoisnrie," Berlin, klin. Wuch., 1900, No. 2. * Lanz, Wien. med. Presse, 1895, vol. xxxvi. 28 434 TSE URINE. Of other pathological conditions in which a digestive glucosuria has been observed, may be mentioned acute and chronic lead poison- ing, poisoning with nitrobenzol, anilin dyes, opium, atropin, and carbon monoxide ; further, the febrile form of embarras gastrique, etc. In these conditions, however, the phenomenon has received little attention. Very important is the fact that in diabetes mellitus the sugar may at times disappear from the urine, while its elimination is replaced by an excessive excretion of uric acid or phosphates. In such cases glucosuria may be produced with ease by the ingestion of 100 grammes of glucose, a point which may be of value in diagnosis. The exhibition of such amounts of sugar in true diabetes while glucosuria already exists ^vill cause an increased elimination, while this apparently does not occur in other forms of glucosuria. Inter- esting further is the fact that in diabetic patients an increased elim- ination of sugar can be produced by the administration of full doses of copaiba. That this drug is in itself capable of lowering the limit to the assimilation of glucose has recently been shown by Bettmann. A digestive glucosuria was thus produced in four patients out of twelve to whom copaiba had been given for one week in amounts varying from 1 to 2 grammes. The digestive glucosuria to which reference has been made in the preceding pages is generally spoken of as the digestive glucosuria e saceharo. Similar results have been obtained after the administra- tion of starches in excess, viz., 160—200 grammes. But while a digestive glucosuria e saceharo is regarded only as a possible indica- tion of a pathological alteration of the carbohydrate metabolism, it is generally thought that every glucosuria ex amylo ^ is indicative of a definite disturbance in the sense of diabetes, unless special factors, such as an increase of the surrounding temperature, dimin- ished radiation of heat, or complete lack of muscular activity, are active. Strauss, however, has shown that in cases in which a some- what more than temporary predisposition toward glucosuria e sac- eharo exists, as in alcoholics, for example, a coincident tendency toward glucosuria ex amylo may likewise be demonstrated. As a result of his experiments he concludes that the difference between the digestive glucosuria e saceharo and glucosuria ex amylo is essen- tially a question of degree. Goeteris paribus, it appears that harm- ful influences of a slight character lead to glucosuria e saceharo, while grave insults call forth glucosuria ex amylo. It results prac- tically that the prognosis in those cases in which digestive glucosuria follows a temporary insult is far better than when the carbohydrate metabolism is permanently damaged, and especially when a gluco- suria ex amylo accompanies a glucosuria e saceharo. In the first > E, Kiilz, Beitrage zur Pathol, u. Therap. d. Diabetes, Marburg, 1874, vol. i. p. 110. CARBOHYDRATES. 435 instance it is scarcely likely that true diabetes will develop in the course of time, while in the latter this is at least possible. Aside from the digestive form of glucosuria which has just been considered, and which is produced artificially, an idiopathic transi- tory form is also known to occur. A transitory glucosuria, appar- ently of central origin, is thus noted in connection with lesions affecting the central as well as the peripheral nervous system, such as tumors and hemorrhages at the base of the brain, lesions of the floor of the fourth ventricle, cerebral and spinal meningitis, concus- sion of the brain, fracture of the cervical vertebrse, tetanus, sciatica ; following epileptic, hystero-epileptic, and apoplectic seizures, mental shock produced by railroad accidents (traumatic neuroses), etc. ; mental strain and worry, fatigue, and anxiety. Glucosuria follow- ing epileptic and apoplectic attacks, however, does not appear to be so common as is generally believed, v. Jaksch was unable to de- monstrate the presence of sugar in fifty recent cases of hemiplegia, and in a large number of cases of epilepsy, with urines voided within the first few hours following the seizure I have reached only negative results. Siegmund noted a transitory glucosuria in 52.38 per cent, of general paretics, in 7.4 per cent, of epileptics, and in 3.77 per cent, of dementia cases, while it was not observed in other mental diseases. It is a well-known fact that Claude Bernard experimentally pro- duced a transitory glucosuria by puncturing a certain spot in the floor of the fourth ventricle, the supposed origin of the hepatic vasomotor nerves, and it is not improbable that this neurotic form of glucosuria is due to some direct or reflex influence affecting that portion of the medulla. The transitory glucosuria occasionally observed in acute febrile diseases, such as typhoid fever, scarlatina, measles, cholera, diph- theria, influenza, and especially malaria, particularly during conva- lescence, may possibly be referable to the action of ptomaiins or leukomains upon this centre. Seegen reports five cases of malaria with " diabetes " in which both conditions disappeared under the administration of quinin. In diphtheria glucosuria appears to be of common occurrence. Binet thus obtained a positive result in twenty-nine cases out of seventy ; twenty-seven times in severe in- fections out of thirty-eight, and twice in mild cases out of thirty- two. I have personally found a transitory glucosuria in four cases out of thirty-two ; the infection in these was of moderate severity. Hibbard and Morrissey arrived at similar results.' A glucosuria of toxic origin has been noted in cases of poisoning with curare, chloral hydrate, sulphuric acid, arsenic, alcohol, carbon monoxide, morphin, etc., and even after simple transfusion of nor- ' C. M. Hibbard and M. J. Morrissey, " Glycosuria in Diphtheria," Jour. Exper. Med., vol.iv. p. 137. 436 THE URINE. mal salt solution into the blood. Phloridzin, a glucoside obtained from the bark of the root of the apple tree, will likewise cause sugar to appear in the urine. The glucosuria thus produced is, however, only temporary, and ceases upon withdrawal of the drug.' In patients afflicted with disease of the heart, liver, and kidneys Gobbi ^ observed a digestive glucosuria, after the ingestion of from 100 to 200 grammes of glucose, if diuretin was at the same time administered. The occurrence of a transitory glucosuria under the conditions above mentioned, and which may be met with in almost any disease, moreover, while interesting from a theoretical standpoint, must in the majority of instances be regarded as a medical curiosity only, and it is but rarely possible to draw either diagnostic, prognostic, or therapeutic conclusions from its existence. A persistent form of glucosuria is noted in connection with certain lesions of the brain, especially those affecting the floor of the fourth ventricle, and is at times of considerable value in diagnosis. This is also observed after removal of the thyroid gland, and in cases in which thyroid extract has been administered in unduly large amount. A continuous elimination of sugar, however, is noted principally in the complex of symptoms to which the term diabetes mdlitus has been applied, and it is this condition to which the greatest practical and theoretical interest attaches. Diabetes mellitus is essentially a persistent form of glucosuria associated with the occurrence of a more or less intense polyuria and a greatly increased elimination of all the metabolic products normally found in the urine, with the exception of uric acid, which is usually present in diminished amount. In the more advanced cases aceto- nuria, lipuria, and lipaciduria may also exist. Diabetes, however, is not a persistent form of glucosuria in an absolute sense of the word, as periods may occur in the course of the disease when glucose is temporarily absent. The quantity of sugar excreted may be very large, and 180 to 360 grammes pro die are amounts which may be frequently observed. This quantity may diminish to zero under various conditions, such as the occurrence of intercurrent diseases, but often also without any apparent cause, and not infrequently in the condition which has been termed diabetic coma. Cases are also observed in which from begin- ning to end mere traces are eliminated, the total amount of sugar not exceeding a few grammes, while the course of the disease rapidly tends toward a fatal termination, so that the severity of the pathological process cannot be measured by the amount of sugar eliminated. A few years ago I had occasion to observe a diabetic patient in whom for months a daily examination of the urine never revealed the ^ Zuntz, " Zur Kenntniss d. Phloridzindiabetes," Du Bois' Archiv, 1895, p. 570. ' Q. Gobbi, "La glicosuria da dluretiua," II Policlinioo, 1900, No. 5. CARBOHYDRATES. 437 presence of more than 5 to 10 grammes of sugar, and in whom death occurred after eighteen months. At the same time it should be remembered that diabetes cannot be excluded by one or even more negative urinary examinaitions, and the value of repeating such examinations three or four hours after the exhibition of 100 grammes of glucose, as indicated, cannot be too strongly urged. Clinicians are in the habit of determining the severity of a case, to a certain extent at least, from the condition of the urine under a diet free from starches and sugars, and generally regard those cases as the more serious in which the glucosuria does not disappear under a diet of this character, while a more favorable prognosis is given if the sugar disappears. It should be remembered, however, that there are numerous exceptions to this rule, and that a light case, — i. e., one in which the sugar disappears under appropriate dietetic treat- ment, — may suddenly exhibit symptoms seen only in the most severe forms, or succumb to one of the numerous intercurrent maladies, while apparently severe cases may assume the more benign type. It may not be out of place in this connection to say a few words regarding the specific gravity of the urine. While usually very high, varying bfetween 1.030 and 1.060, as pointed out in the chapter on Specific Gravity, comparatively low figures are noted at times, such as 1.012, corresponding to a quantity of urine not exceed- ing 1000 c.c, and implying, of course, a diminished elimination of solids. This is especially marked in those cases described by Hirschfeld,"^ in which, as pointed out in the chapter on Urea, the resorption of nitrogenous material from the digestive tract is below the normal. Polyuria, a fairly constant symptom of the more com- mon types of diabetes mellitus, is much less pronounced in Hirsch- feld's form, and may be altogether absent, although it is true that this may occur in ordinary diabetes also. The simultaneous occurrence of glucosuria, acetonuria, lipuria, and lipaciduria (which see) is probably always indicative of true diabetes. It is, of course, impossible to enter here into a detailed considera- tion of the origin of diabetes. Suffice it to say that a persistent glu- cosuria, aside from nervous influences, may be referable, on the one hand, to an inability on the part of the liver to transform into gly- cogen all of the sugar which is carried to this organ ; or, on the other hand, to an inability on the part of the muscular system of the body to utilize all the sugar sent to it. Accordingly, we may distinguish between a hepatogenic and a myogenio diabetes. As a matter of fact, cases are seen, usually belonging to the milder form of the disease, ' F. Hirschfeld, "Uebereine neue klinische Form d. Diabetes," Zeit. f. klin. Med., Vol. xix. pp. 294 and 325. 438 THE VBINE. in which the sugar may be temporarily caused to disappear from the urine by muscular exercise. On the other hand, again, cases are seen, and unfortunately only too frequently, in which, notwithstand- ing a total abstinence from carbohydrates and a free indulgence in muscular exercise, the sugar does not disappear from the urine. In such cases it is permissible to. speak of a hepatogenic combined with a myogenic diabetes. Within recent years it has been shown that pancreatic disease is frequently associated with diabetes, and while the number of cases in which no pancreatic lesions are discovered is still too large to war- rant the conclusion that disease of this organ is invariably associated with glucosuria, it still must be admitted that lesions of the pancreas are the more frequently met with in diabetes the more carefully the organ is examined. So much appears to be certain, that diabetes may be produced by pancreatic disease. As to the manner, how- ever, in which such a result can occur we are in ignorance. In this connection it is interesting to note that, according to Opie, dis- ease of the areas of Langerhans more especially is associated with the clinical picture of diabetes, while lesions affecting the secreting portion of the gland only do not influence the carbohydrate metab- olism.' Hirschfeld pointed out the fact that while in the majority of diabetic patients the proteid food ingested is quite satisfactorily utilized, the assimilation of fats and albumins is much below nor- mal in others, and particularly so in cases of diabetes associated with pancreatic disease (see also Urea). Observations in this direction are as yet very scanty, so that a definite opinion cannot be expressed regarding the utility in diagnosis of investigations similar to those of Hirschfeld. I have had occasion to observe a diabetic patient for some time in whom, notwithstanding that conclusions were reached similar to those of Hirschfeld, the existence of pancreatic disease could not be determined post mortem. Whether or not a renal arid a thyroigenic diabetes also exists, as has recently been suggested, remains an open question.^ Tests for Sugar. — The tests for sugar usually employed in the clinical laboratory depend upon the following properties of sugar : 1. In the presence of alkalies it acts as a reducing agent upon certain metallic oxides, such as those of copper and bismuth (Feh- ling's, Trommer's, Bottger's, and Nylander's tests). 2. In the presence of yeast (Saocharomyces cerevisise) it under- 1 Opie, Jour. Exper. Med., 1901, vol. v. p. 527. 'Diabetes; J. Seegen, Die Zuckerbildung im Thierkorper, Berlin, 1890, p. 260. T. Noorden, Pathol, d. Stoffweohsels, Berlin, 1893. Seegen, " Ueber d. Zuckergehalt d. Blutes von Diabetikern," Wien. med. Wooh., 1886, Nos. 47 and 48. P. W. Pav.v, " Ueber die Behandlung von Diabetes mellitus," Verhandl. d. X. interna*, med. Congr. 1891, II., Abt. 5, p. 80. P. F. Eichter, " Nierendlabetes," Deutsob. med. Wooh., 1899, p. 840. CARBOHYDRATES. 439 goes fermentation, with the formation of alcohol, carbonic acid, succinic acid, glycerin, and a number of other bodies, such as amyl alcohol, etc. (fermentation test). 3. With phenylhydrazin sugar forms an insoluble crystalline compound — phenylglucosazon. _ 4. Solutions of glucose turn the plane of polarized light to the right, from which property glucose has also received the name dextrose. In every case the urine should first be tested for the presence of albumin, which should be removed by boiling. Teommee's Test.i — ^A few cubic centimeters of urine are strongly alkalinized with sodium hydrate solution, and treated with a 5 per cent, solution of cupric sulphate, added drop by drop, until the cupric oxide formed is no longer dissolved. The mixture is care- fully heated, when in the presence of sugar a yellow precipitate of cuprous hydroxide is formed, which gradually settles to the bottom as a sediment of red cuprous oxide. It is important to note that while sugar, unless present in mere traces, can readily be detected in this manner, other substances are or may be present in the urine, such as uric acid, kreatin and krea- tinin, allantoin, nucleo-albumin, milk-sugar, pyrocatechin, hydro- chinon, *and bile-pigment, which likewise reduce cupric oxide. Following the ingestion of benzoic acid, salicylic acid, glycerin, chloral, sulphonal, etc., reducing substances also appear. These may generally be disregarded, it is true, if care is taken not to boil the urine after the addition of the cupric sulphate, as the precipitation of cuprous oxide in the presence of sugar takes place before this point is reached. Unfortunately, however, , the test when thus applied yields negative results, or results which are doubtful, if traces only are present, so that it cannot be utilized, as a rule, in the study of transitory or digestive glucosuria. Fehling's Test.^ — ^This is a modification of the test just described, and can be recommended only with the same restrictions. Two solutions are employed, which must be kept in separate bottles, the one containing 34.64 grammes of crystallized cupric sul- phate, dissolved in 500 c.c. of distilled water, and the other 173 grammes of potassium and sodium tartrate and 125 grammes of potassium hydrate, dissolved in an equal volume of water. Equal parts of the two solutions, mixed in a test-tube and diluted with four times as much water, are boiled, when a small amount of urine is added. In the presence of sugar a precipitate of the yellow hydroxide of copper or of red cuprous oxide will be produced ; but care should be taken only to warm, and not to boil the solution after addition of the urine. ' C. Trommer, Annal. d. Chem. u. Pharm., 1841, vol. xxxix. p. 361. 2 H. Fehling, Ibid., 1849, vol. Ixxii. p. 106. 440 THE URINE. Not infrequently it will be observed that upon standing, when no precipitation has occurred previously, the blue color of the mixture changes to an emerald green, while the solution at the same time becomes turbid. Such a phenomenon should not be referred to the presence of sugar, as it is in all probability due to the action of other reducing substances, such as those mentioned above. Bottger's Test with Nylandee's MoDincATiON.' — A few cubic centimeters of urine are treated with Almin's solution in the proportion of 11:1. This is prepared by dissolving 4 grammes of potassium and sodium tartrate, 2 grammes of bismuth subnitrate, and 10 grammes of sodium hydrate in 90 c.c. of water, heating the solution to the boiling-point and filtering upon cooling, when it should be kept in a colored glass bottle. The mixture of urine and Alm6n's fluid is thoroughly boiled, when in the presence of sugar a grayish, dark-brown, and finally a black precipitate, con- sisting of bismuthous oxide or of metallic bismuth, is obtained. Albumin, if pressnt, must first be removed, as, owing to the sulphur contained in the albuminous molecule, alkaline sulphides would be formed upon boiling, and, acting upon the bismuth, give rise to the formation of black bismuth sulphide, which might be mistaken for metallic bismuth. Rhubarb-pigment, as well as melanin and melan- ogen (which see), and free hydrogen sulphide must also be absent, as misleading; results will otherwise be obtained. Nylander's test, as well as those of Trommer and Fehling, is, however, not without objections, as a partial reduction of the bis- muth subnitrate may be produced by other substances, such as kairin, tincture of eucalyptus, turpentine, and large doses of quinin. Fermentation Test.^ — A small piece of ordinary compressed .yeast is shaken with some of the suspected urine and a test-tube filled with the mixture, to which some mercury is added. The tube is then inverted into a vessel containing mercury, and allowed to stand in a warm place (22°-28° C). If sugar is present, fermentation will occur in the course of twelve hours, and the carbon dioxide formed rise to the top of the tube, gradually displacing more and more of the urine or mercury as the amount of the gas increases. It is easy to demonstrate that the gas thus formed is carbon dioxide by introducing a small piece of caustic soda into the urine, when, owing to absorption of the carbon dioxide, the liquid will again rise in the tube. Very convenient for this purpose also are the saccha- rimetric tubes of Einhorn (Fig. 100) or Lohnstein' (Fig. 102), which are employed as just described, a little mercury being poured into the bent limb to guard against escape of gas. As the yeast itself, however, may give rise to the formation of a little gas in the 1 E. Nylander, Zeit. f. physiol. Chem., 1883, vol. viii. p. 175. 2 M. Einhorn, Virchow's Arohiv, 1885, vol. cii. p. 263. ' Lohnsteiu, Berlin, klin. Woch., 1898, p. 866. I ^ '' .•.\^ PLATE XVI. Phenyl-Glucosazon Crystals obtained froni a Diabetic Urine. CARBOHYDBA TES. 441 absence of sugar, it will always be well to make a control-test with normal urine — i. e., to prepare a similar tube with normal urine mixed with yeast, and to allow this to stand at the same temperature. If a positive result is thus obtained, there can be no doubt as to the pres- ence of a fermentable substance in the urine. This, however, is not necessarily glucose, as other carbohydrates, such as lactose, maltose, and levulose, may likewise undergo fermentation. Still, if large amounts of gas are obtained, and if Trommer's test also yields a positive result, it will be fairly safe to regard the substance present as glucose. Fig. 100. m EinhoTn's saooharimeter. PHENTfLHYDEAZiN Test.' — As Originally proposed by v. Jaksch, the test is conducted as follows : 6 to 8 c.c. of urine are treated with 0.4 to 0.5 gramme of phenylhydrazin hydrochlorate and 1 gramme of sodium acetate, and warmed until the salts have been dissolved, a little water being added if necessary. The tube is placed in boiling water for twenty to thirty minutes, and then transferred to a beaker filled with cold water. If sugar is pres- ent in moderate amounts, a bright-yellow crystalline deposit will at once be thrown down and partly adhere to the sides of the tube. But even in the presence of mere traces a careful microscopical ex- amination will reveal the presence of crystals of phenylglucosazon (Plate XVI.). These are seen singly or arranged in bundles and sheaves composed of very delicate bright-yellow needles which are insoluble in water. ' V. Jaksch, Zeit. f. klin. Med., 1886, vol. xi. p. 20. 442 THE VBINE. Still more convenient is the following modification of the test, as suggested by Kowarsky : ^ 5 drops of pure phenylhydrazin are mixed in a test-tube with 10 drops of glacial acetic acid and 1 c.c. of a saturated solution of common salt. A white caseous mass results, which consists of phenylhydrazin hydrochlorate and sodium acetate. To this, 3 c.c. of urine are added, when the mixture is boiled for two minutes and then set aside to cool. Should more than 0.5 per cent, of sugar be present, the typical crystals begin to sepa- rate out after two minutes, and may be recognized with the naked, eye. In the presence of smaller amounts the mixture should be allowed to stand for from fifteen to twenty minutes, or, if traces only are present, for one hour. This test, properly applied, is undoubtedly not only the most deli- cate, but at the same time the most reliable, as no other substances which may be present in the urine, excepting maltose and certain pentoses, will give rise to the formation of an osazon. Hence, when- ever doubt is felt as to the nature of a substance reacting in a posi- tive manner with the reagents described above, recourse should be had to this test. It has been stated that maltose forms an exception ; this, however, will never become embarrassing, as the microscopical appearance of the maltosazon crystals differs from that of the phenyl- glucosazon. The melting-point of phenylglucosazon (206° C.), moreover, is about 15 degrees higher than that of the maltosazon — 190°-191° C To determine this point, it is necessary to filter off the osazon, and, after washing with water, to dissolve it upon a filter by means of a little hot alcohol. From this alcoholic solution it is reprecipitated by water, when it may be collected and dried over sulphuric acid. The melting-point is then determined according to the usual methods. The pentosazons also can be readily distinguished from glucosazon by their melting-points (which see). • The amount of lactose which may be found in the urine is far too small to give rise to the formation of an osazon when the test is directly applied to the urine. With the conjugate glucuronates phenylhydrazin also combines to form crystalline compounds, but these may likewise be distinguished by their melting-points and the form of the crystals. Such com- pounds, moreover, are usually not present in amounts sufficient to give rise to confusion (see Glucuronic Acid). PoLAEiMBTRic Test. — Glucose tums the plane of polarized light to the right, but the same may be said of maltose, the degree of polarization of which is even more marked, so that it may be impos- sible to state in a given case whether such rotation is referable to a large quantity of glucose or to a smaller quantity of maltose. The 'A. Kowarsky, "Zur Vereinfaohung d. Phenylhydraziiiprobe," Berlin, klin. Wooh., 1899, p. 412. CARBOHYDRATES. 443 latter substance, however, occurs in the urine but rarely, and may be recognized not only by the microscopical appearance of its osazon, but also by the fact that its power of reduction is increased in the presence of sulphuric acid and by the application of heat. An error which may further arise with the employment of the polarimetric method is referable to the fact that if glucose is pres- ent in only small amounts, while the urine contains large quantities of j9-oxybutyric acid, the latter turning the plane of polarized light to the left, it may happen that the rotation in this direction will neu- tralize or even counterbalance any rotation to the right which may be due to glucose. In such cases, however, the urine will react in a positive manner with the other reagents described, and the fermented urine wUl, moreover, turn the plane of polarization still more strongly to the left, indicating the presence of a dextrorotatory substance, and in all probability of glucose. The delicacy of this method varies with the instrument employed ; the figures given below were obtained with the apparatus of Lippich, which yields the best results. (For a description of this method see the Quantitative Estimation of Sugar by Means of the Polarimeter.) Table showing the DEucAcy or the Tests described. Troramer's test . . . Fehling's test . Nylander's test . . . Fermentation test . Phenylhydrazin test Polarimetric test 0.0025 percent, 0.0008 0.025 0.1-0.05 •• 0.025-0.05 " 0.025-0.05 " Table showing the Behavior of the Various Forms of Sugar which MAY OCCUR IN THE UrIHE TOWARD THE TESTS DESCRIBED. Trommer's, viz., Fehling's test. Nylander'i test. Fermenta- tion test. Phenylhydrazin test. Polarimetric test. Glucose. Levulose. Maltose. Lactose. Laiose. Positive reaction. Positive reaction. Positive reaction. Positive reaction. Positive reaction on boiling only ; 1.2-1.8 per cent, more is obtain- ed than by the polarimeter. Positive reaction. Positive reaction. Positive reaction. Positive reaction. Positive reaction. Positive reaction. Positive reaction. Positive reaction. No re- action or only a very faint one. No reac- tion. Positive reaction; melting-point 205° C. Same osazon ob- tained as with glucose, only more rapidly. A maltosazon is formed; melting- point 190°-191° C. No reaction in the concentration in which It maj; oc- cur in the urine ; melting-point 200° 0. With phenylhy- drazin a yellow- ish brown, non- crystallizable oil is obtained. Rotation toward the right. Eolation toward the left. Eotation toward the right. Rotation toward the right; in- creased by boil- ing with a 2.6 per cent, solution of sulpliurlc acid. No reaction, or ro- t a 1 1 o n toward the left. 444 THE URINE. Clinically, it is unimportant to search for minute traces of sugar, such as may be found in every normal urine, and the reader is referred ft) special works on physiological chemistry for a considera- tion of the methods generally employed (see method of Baumana and V. Udranszky. Quantitative Estimation of Sugar. — The methods used in the quantitative estimation of sugar are essentially based upon the quali- tative tests described. Fehling's Method.' — Fehling's solution prepared as described above is of such strength that the copper contained in 10 c.c. is completely reduced by 0.05 gramme of glucose. If then urine is carefully added to this quantity until complete reduction takes place, the amount of sugar contained in a given specimen of urine can be readily calculated according to the following equation : y : 0.05 : : 100 : a; ; and a; = — , y in which y indicates the number of cubic centimeters of urine required to reduce the 10 c.c. of Fehling's solution, and x the amount of sugar contained in 100 c.c. of urine. As the best results are obtained only if from 5 to 10 c.c. of urine are used in one titration, it is usually necessary to dilute the urine to the required degree ; in the determination of this point the specific gravity may serve as a guide. As a general rule, urines of a speciiio gravity of 1.030 should be diluted five times, and if the density is still higher ten times. To be certain that the proper degree of dilution has been reached, 5 c.c. of Fehling's solution are treated with 1 c.c. of the diluted urine, a little caustic soda solution and distilled water being added to make in all about 25 c.c. This mixt- ure is thoroughly boiled ; if the fluid still remains blue, another 1 c.c. of diluted urine is added, and so on, until the last two tests differ by 1 c.c. of urine, the last cubic centimeter added causing a separation of cuprous oxide. In this manner the percentage of sugar may be approximately determined. Albumin, if present, must first be removed by boiling. Ten c.c. of Fehling's solution diluted with 40 c.c. .of water are placed in a porcelain dish and boiled. While boiling, the diluted urine is added from a burette, 0.5 c.c. at a time, when, as a rule, the precipitated cuprous oxide will rapidly settle, so that gradually a white bottom may be seen through the blue field, the color of which becomes less and less intense upon the further addition of urine until finally the solution is almost colorless. When this point is reached the urine is added drop by drop until the decolorization is complete. The degree of dilution multiplied by 5 and the result divided by the number of cubic centimeters of diluted urine em- ' Loc. cit. OABBOBYDRA TES. 445 ployed will then indicate the percentage-amount of sugar. In the following table the percentage results corresponding to the number of cubic centimeters of undiluted urine employed will be found. Sugar. — Qwmtity of Glucose pro liter, corresponding to the number of cubic centimeters v^ed for the complete reduction of 10 cubic centimeters of FeUing's solution. 1 ^ 41.68 A ^ ^0 ^ iV io 1^ 1 50.00 45.44 38.46 35.70 33.32 31.24 29.40 27.76 26.30 2 25.00 23.80 22.72 21.72 20.84 20.00 19.22 18.50 17.84 17.24 3 16.66 16.00 16.62 15.14 14.15 14.28 13.88 13.50 13.14 12.82 4 12.50 12.18 11.90 11.62 11.36 11.10 10.86 10.62 10.40 10.20 5 10.00 9.80 9.60 9.42 9.24 9.08 8.92 8.76 8.62 8.50 B 8.32 8.18 8.06 7.92 7.80 7.68 7.66 7.44 7.34 7.24 7 7.14 7.04 6.94 6.86 6.78 6.66 6.66 6.48 6.40 6.32 8 6.24 6.16 6.08 6.02 . 5.94 5.88 5.80 5.74 5.68 6.60 9 ,5.54 6.48 6.42 5.36 5.30 5.24 5.20 5.16 5.12 6.06 10 6.00 4.94 4.90 4.82 4.78 4.76 4.70 4.66 4.62 4.58 11 4.54 4.50 4.46 4.42 4.38 4.34 4.30 4.26 4.22 4.20 12 4.16 4.14 4.12 4.08 4.04 4.00 3.98 3.96 3.92 3.86 13 3.84 3.80 3.78 3.76 3.74 3.70 8.68 3.66 3.62 3.58 14 3.66 3.54 3.52 3.48 3.46 3.44 3.42 3.40 3..36 3.34 15 3.32 3.32 3.28 3.26 3.24 3.22 3.20 3.18 3.16 3.14 16 3.12 3.10 3.08 3.04 3.04 3.02 3.00 2.98 2.96 2.94 17 2.94 2.92 2.90 2.88 2.86 2.84 2.82 2.82 2.80 2.78 18 2.76 2.76 2.74 2.72 2.70 2.70 2.68 2.64 2.64 2.64 19 2.62 2.62 2.60 2.60 2.58 2.66 2.56 2.64 2.52 2.62 20 2.60 2.50 2.48 2.48 2.44 2.42 2.42 2.40 2.40 2.38 21 2.38 2,36 2.34 2.34 2.32 2.32 2.30 2.30 2.28 2.28 22 2.26 2.26 2.24 2.24 2.22 2.22 2.20 2.20 2.18 2.18 23 2.16 2.16 2.14 2.14 2.12 2.12 2.12 2.10 2.10 2.10 24 2.08 .2.08 2.06 2.06 2.06 2.04 2.04 2.02 2.02 2.02 25 .2.00 •l.98 1.98 1.96 1.96 1.96 1.94 1.94 1.92 1.92 26 1.92 1.92 1.90 1.90 1.88 1.88 1.88 1.86 1.86 1.86 27 1.84 1.82 1.82 1.82 1.82 1.80 1.80 1.80 1.80 1.80 28 1.78 1.76 1.74 1.74 1.74 1.74 1.74 1.74 1.74 1.72 29 1.72 1.70 1.70 1.70 1.70 1.68 1.68 1.68 1.68 1.66 30 1.66 1.66 1.65 1.63 1.63 1.62 1.62 1.62 1.62 1.62 Unfortunately, it is difficult, as a general rule, to determine ex- actly the point when all the copper has been reduced — i. e., the point at which the blue color has entirely disappeared. When it is thought that this has been reached, about 1 c.c. should be filtered through thick Swedish filter-paper, and the filtrate (which must be absolutely clear) acidified with acetic acid and treated with a drop or two of a solution of potassium ferrocyanide. If unreduced copper is still present in the solution, a brown color will result, indicating that sufficient urine has not been added. But if, on the other hand, no brown discoloration is noted, it is possible that the desired point has been passed, when the titration should be repeated. At times the precipitate will not settle at all, and even pass through the filter, so that it is practically impossible to determine the end of the reaction. In such cases the following procedure, suggested by Cause, will be found of value : Ten c.c. of Fehling's solution are diluted with 20 c.c. of distilled water and treated with 4 c.c. of a 0.05 per cent, solution of potas- sium ferrocyanide. While boiling, the diluted urine is added drop by drop until the blue color entirely disappears. A precipitate does not form with this method. 446 THJE VBINE. In order to obtain reliable results, however, the Fehling solution must be prepared with great care and its strength determined. This may be done in the following manner : 0.2375 gramme of pure crystallized cane-sugar, dried at 100° C, is dissolved in 40 c.c. of distilled water, to which 22 drops of a 0.1 per cent, solution of sul- phuric acid have been added. This solution is kept on the boiling water-bath for an hour, when it is allowed to cool and diluted to 100 c.c. with distilled water. Twenty c.c. of this solution will then contain exactly 0.05 gramme of glucose, corresponding to 10 c.c. of Fehling's solution, if this is of the required strength. If too strong, so that 21 c.c. of the sugar solution, for example, are required to obtain a complete reduction of the copper, the strength of Fehling's solution may be determined according to the equation : 20 : 0.05 : : 21 : a;; and x = 0.0525. If too weak, on the other hand, so that 19 c.c, for example, are required, its strength is similarly deter- mined : 20 : 0.05 : : 19 : a; ; and a; = 0.0475. Knapp's Method.^ — This method is said to be more satisfactory than that of Fehling. Daylight is not necessary ; the method is simpler, and it is applicable even in cases in which the amount of sugar is small ; and the solution keeps for a long while. The principle of the method depends upon the observation that mercuric cyanide in alkaline solution is reduced to metallic mercury in the presence of sugar. The solution required should contain 10 grammes of chemically pure, dry mercuric cyanide and 100 c.c. of a solution of sodium hydrate (sp. gr. 1.145) to the liter. Twenty c.c. of this solution correspond to 0.05 gramme of glucose. Method. — Twenty c.c. of the solution are placed in a small retort and diluted with 80 c.c. of water. If we have reason to suppose that the urine contains less than 0.5 per cent, of sugar, 40 to 60 c.c. are sufficient. The solution is then heated to the boiling- point, when the diluted urine (see below) is added, at first 2 c.c. at a time, then 1 c.c, 0.5 c.c, 0.2 c.c, and 0.1 c.c, as the final point is approached. After each addition the solution is boiled for One-half minute. As the end-reaction is approached the solution clears, and the mercury, together with the phosphates, settles to the bottom. The final point is determined by placing a drop of the supernatant fluid upon a piece of clean, white Swedish filter-paper, and holding this first over a bottle containing concentrated hydro- chloric acid and then over one containing a saturated solution of hydrogen sulphide. If all the mercuric cyanide has not been reduced, a yellow spot will result, the color of which becomes the more manifest if it is compared with one which has not been ex- posed to the action of hydrogen sulphide. As soon as the mercury is entirely reduced the reading is taken. Example. — Supposing that 1 5 c.c. of urine have been required, the 1 K. Knapp, Annal. d. Chem. u. Pharm., 1870, vol. cliv. p. 252. CARBOHYDRATES. 447 corresponding amount of sugar is then found according to the fol- lowing equation, 20 c.c. of Knapp's solution requiring 0.05 gramme of sugar for its reduction : 15: 0.05 :: 100: x; 15a; = 5; and i = 0.333 per cent. Precautions : 1. Albumin must first be removed. 2. The urine should not contain more than 0.5 to 1 per cent, of sugar. The urine is hence diluted, if necessary, as with Fehling's method. Differential Density Method.^ — This method is very useful in clinical work, and should be preferred to the more uncertain titra- tion with Fehling's solution, unless considerable experience has been acquired with the method. The specific gravity of the urine is accurately ascertained by means of a pyknometer, or a hydrometer graduated to the fourth decimal and provided with a thermometer indicating tenths of a degree. The temperature at which the specific gravity is taken should be that for which the hydrometer has been constructed, the urine being heated or cooled to the desired degree. One hundred to 200 c.c. are then set aside in a flask, after the addition of some yeast which has been washed free from mineral material, loosely stoppered or provided with an arrangement like the one shown in the accompanying figure (Fig. 101). After twenty-four hours if but little sugar is present, or forty-eight hours if there is much, the specific gravity is again determined under the precautions given, after having filtered the urine. The dificrence in the specific gravity is multiplied by 230, an empirical factor which has been found by dividing the amount of sugar ascertained by titration or polarization with the difference in the density of the urine after fermentation. The result indicates the percentage of sugar. The process may be hastened if to each 100 c.c. of urine 2 grammes of potassium and sodium tartrate and 2 grammes of diacid-sodium phosphate are added, with 10 grammes of compressed yeast, and the mixture is allowed to stand at a temperature of from 30° to 34° C. If but little sugar is present, two to three hours will be sufficient. That portion of the urine of which the specific gravity is deter- mined before fermentation should really be treated in the same man- ner. It will suffice, however, to add 0.022 to the specific gravity found, to make up for the increase that would otherwise be observed in the second specimen owing to addition of the salts. In every case the urine must be perfectly fresh, as fermenta- tion generally begins spontaneously, even after standing a short time. Einhoen's Method. — This will answer very well for ordinary 1 Roberts, Lancet, 1862, i. p. 21. Worm-MuUer, Pfluger's Archiv, 1884, vol. xxxiii. p. 211, and 1885, vol. xxxvii. p. 479. 448 THE URINE. Fio. 101. purposes. Two especially constructed and graduated saccharimetric tubes (Fig. 100) are used, one of which is filled with a mixture of the suspected urine and yeast, and the other with normal urine and yeast, as a control. The tubes are set aside at a temperature of from 30° to 34° C, when the percentage-amount of sugar in the urine is read off from the column of carbon dioxide formed. Should the second tube also show a small amount of gas, the figure corresponding to this amount is deducted from the first. Lohnstein's Method. — A very conve- nient modification of Einhorn's instrument, and one furnishing more accurate results, has been introduced by Lohnstein.^ As will be seen from the accompanying figure (Fig. 102), this is essentially a U-tube open at both ends. The longer limb is closed during the process of fermentation by a ground-glass stopper. This stopper is pro- vided with an air-hole, to which a similar hole corresponds in the drawn-out portion of the tube. The apparatus is filled with the urine to be examined, through the bulb A, while the two air-holes at B are in commu- Care should be had that the liquid stands exactly at the The stopper is then turned so that all communication A little mercury is finally Flask for the approximate estimation of sugar by fer- mentation. (V, Jaksch.) nication mark 0, between the air and the urine is cut off. poured into the saccharimeter, when the instrument is placed in a vessel containing water at 35°-40° C, and maintained at a temper- ature of about 30° C. After twelve hours the percentage of sugar is read off directly. Precautions : 1. As every urine contains traces of free carbon dioxide, it is well to remove this by boiling if we have reason to suppose that only a small amount of sugar is present. Before adding the yeast the urine is, of course, cooled to the surrounding temperature. 2. As the instrument yields satisfactory results only if the urine contains less than 1 per cent, of sugar, it is necessary to dilute it with water when more is present. The specific gravity may here serve as an index; urines of a specific gravity up to 1.018 are examined directly; from 1.018 to 1.022 they are diluted twice, from 1.022 to 1.028 five times, and those above 1.028 ten times. 3. A test-tube, provided with the necessary marks for diluting the urine, accompanies the instrument. In every case a globule of yeast, ' T. Lohnstein, " Kin neues Gahrungsflacoharometer, " Berlin, kiln. Woch., 1898, p. 866. CARBOHYDRATES. 449 Fig. 102. approximately 6-8 mm. in diameter, is added to the urine and shaken in the tube until an even suspension has been reached.' PoLAEiMETKic METHOD. — For this purpose the saccharimeter of Soleil-Ventzke is very convenient (Fig. 103). This consists essen- tially of a Nicol prism, A, which may be rotated about the axis of the apparatus ; a second Nicol prism, at D ; vertically placed com- pensating prisms, consisting of dextrorotatory quartz, at E, which may be moved horizontally by means of a rack-and-pinion adjustment, turned by a milled head at K, so that light can pass through a thicker or thinner layer of the dextrorotatory quartz. At F is a plate of Isevorotatory quartz cut perpendicularly to the optical axis, and covering the en- tire field of vision ; at H biquartz plates of Soleil, and at I an Iceland-spar crystal ; BC represents a small telescope, by means of which the biquartz plates can be accu- rately focussed. When the compensation- prisms of this apparatus are in a certain position the Isevorotation of the plate F will be exatetly compensated, and the two halves of the field of vision present the same color, while the zero of the scale X will coincide with the zero of the vernier Y, arranged on the upper surface of the compensators. Any change in this posi- tion produced by turning the screw K will cause the appearance of a different color in each half of the field of vision. If now, with a zero-position, an optically active dextrorotatory or laevorotatory sub- stance is interposed, the color of each half of the field of vision will become altered, but may be equalized again by changing the position, of the compensators, the degree of change necessary to produce this result constituting an index of the power of rotation of the solution interposed in the tube M. Soleil- Ventzke's apparatus is constructed in such a manner that if a solution of glucose is employed, the length of the tube M being 10 cm., every entire line of division on the scale will indicate 1 per cent, of sugar. The tube of the saccharimeter should be carefully washed out with distilled water, and at least once or twice with the filtered urine,, when it is placed on end upon a flat surface and filled with the ' Lohnstein's sacchaiimeter may be procured from B. Kallmeyer & Co., Oanrein- burger Str. 45, Berlin. 29 Lohnstein's saccharimeter. 450 THE URINE. urine, so that -this forms a convex cup at the end. The glass plate is now carefully adjusted, so as to guard against the admission of bubbles of air. The metallic cap is placed in position, care being taken to avoid undue pressure. The examinations are made in a dark room ; an ordinary lamp is used, and several readings are taken, until the differences do not amount to more than 0.1 or 0.2 per cent. The tubes should be thoroughly cleansed immediatdy after the experiment. In every case the filtered urine should be free from albumin, and, if markedly colored, should be previously treated with neutral lead acetate in substance and filtered. If it is desired to demonstrate only the presence of sugar, the compensators are first brought to the zero-position. If now, upon Fig. 103. Soleil-Ventzke'a eaccharimeter. interposition of the tube filled with urine, a difference in the color of the two halves of the field of vision is noted, the presence of an optically active substance in the urine may be assumed ; and if the deviation is at the same time to the right, the presence of glucose is rendered highly probable, while a deviation to the left will generally be referable to levulose or j8-oxybutyric acid. Indican, peptones (albumoses), cholesterin, and certain alkaloids, it is true, also turn the plane of polarization to the left ; but as a rule these substances need not be considered, as cholesterin occurs but rarely, and indican is usually present in only small amounts in diabetic urines. Albu- moses, if present, must first be removed. Lactose and maltose, which also turn the plane of polarization to the right, may be dis- CARBOHYDRATES. 451 tinguished from each other and from glucose by the phenylhydrazin test. Levulose turns the plane of polarization to the left. Oxy- butyric acid is practically always associated with the presence of glucose, and may be recognized by allowing the urine to undergo fermentation, when the filtered urine will become distinctly Isevo- rotatory. Bkemee's Diabetic Urine Test.^ — The test is based upon the different behavior toward certain anilin dyes of diabetic, as compared with non-diabetic, urine. If a trace of a mixture of 2 parts of eosin and 3 parts of gentian-violet, for example, is added to non-diabetic urine, it will be observed that the urine gradually dissolves the eosin and assumes a yellowish or bright-red color, while the gentian-violet fails to dissolve. If diabetic urine, on the other hand, is treated in the same manner, the eosin will likewise dissolve, but a solution of the gentian-violet also occurs, and the entire specimen eventually assumes a violet color. Of late, Bremer has advised the use of Merck's gentian-violet B, or of methyl-violet 5B. The test is extremely simple : two well-dried test-tubes are filled to about one-half, the one with normal urine and the other with the urine to be examined. About 0.5 mgrm. of either of the above reagents is then placed upon the surface of the urine ; the tubes ' are kept in a warm place or immersed in warm water. On standing, streaks of blue gradually appear in both specimens, but on shaking the color disappears in the normal specimen, while the entire bulk of the diabetic urine assumes a blue or violet color. A reddish-purplish color is often observed in non-diabetic specimens, but is of no significance. Bremer admits that doubtful results may be obtained with urines presenting a specific gravity below 1.014 or 1.015, and that in such cases it may be impossible to distinguish non-diabetic from diabetic urine. He claims, on the other hand, that a positive result with a urine of high specific gravity is pathog- nomonic of diabetes, and that this may be obtained even at a time when the sugar has temporarily disappeared from the urine. The substance which gives rise to this peculiar reaction is un- known. Sugar in itself, as also acetone and diacetic acid, are not concerned in its production. The reaction of the urine also is unim- portant. Bremer is inclined to believe that in non-diabetic urines one of the coloring principles helps to render the urine refractory. As he says, colorless diabetic urines yield the most striking color- reactions, and especially those in which a greenish shimmer is apparent. On the whole, Bremer's observations have been confirmed so far ' L. Bremer, " Anilinfarbenproben d. flarns bei Diabetes," Centralbl. f. inn. Med., vol. xix. p. 307. T. B. Futcher, Phila. Med. Jour., 1898. L. Bremer, " On tbe Chemi- cal Behavior of Eosin and Gentian-violet toward Normal and Diabetic Urines," N. Y. Med. Jour., 1897. 452 THE VBINE. as diabetic urine is concerned. Exceptions, however, occasionally occur even in cases of true diabetes, and, as Bremer admits, positive results are frequently observed in urines of a low specific gravity. The test is of interest, and may possibly be further modified so as to be of value in diagnosis, but as yet it would scarcely be warrant- able to draw definite conclusions from its occurrence, even when the specific gravity is high. Lactose. — Lactose may be found in the urine toward the end of gestation, but it occurs more especially in nursing-women in whom the flow of milk is impeded. It is generally stated, however, that lactosuria also occurs in nursing-women who have well-developed breasts, in the absence of any obstruction, and that the good qual- ities of a wet-nurse are indicated by a copious and persistent elim- ination of milk-sugar. Its presence may be inferred if a positive result is obtained with Trommer's and Nylander's tests, while the phenylhydrazin and fermentation tests give negative results, although an osazon can be obtained from the isolated substance, and although lactose undergoes a certain form of alcoholic fermentation. Lemaire, who has recently investigated this subject, found that the urine of nineteen women examined in this direction apparently contained no sugar during the last twelve days preceding confine- ment (Trommer's and Nylander's tests), while a positive reaction was obtained with Trommer's reagent in two cases and with Nylander's reagent in thirteen cases after confinement. The phenylhydrazin test was negative in all nineteen before and positive after confine- ment, when this was directly a/pplied to the substance isolated according to Baumann's method. The percentage varied between 0.013 and 0.438, and appeared to be uninfluenced by the act of nursing.^ Levulose.^ — It is claimed that levulose is occasionally found in diabetic urines together with glucose. Such urines show a deviation to the left or none at all, while the other tests for sugar indicate the presence of a reducing substance. Maltose. — Maltose, together with glucose, was found in the urine of a patient supposedly the subject of pancreatic disease, associated with an acholic condition of the stools. Its recognition is practi- cally dependent upon the formation of its osazon and a determina- tion of the melting-point of the latter. Dextrin.' — In one case of diabetes dextrin appeared to take the place of glucose. It may be recognized by the fact that upon the application of Fehling's test the blue liquid first becomes green, then yellow, and sometimes dark brown. Traces of dextrin are 1 De Sinety, Maly's Jahresber., 1874, vol. iii. p. 134. Hempel, Arch. f. Qynaelc, 1875, vol. viii. p. 312. Ney, Ibid., 1889, vol. xxxv. p. 239. F. Hofmeister, " Ueber Laktosurie," Zeit. f. physiol. Chem., 1877, vol. i. p. 101 (lit.). F. A. Lemaire, Ibid., 1896, vol. xxi. p. 442. '' Seegen, Centralbl. f. d. med. Wiss., 1884, vol, xxii, p. 753. ' Eeiohard, Maly's .Tahresber., 1876, vol. v. p. 60. CABBOBYDRATES. 453 probably present in every urine, but cannot be demonstrated with the common tests. Laiose.' — Laiose has been found in the urine of a diabetic patient. It is essentially characterized by the fact that on titration with Fehling's solution from 1.2 to 1.8 per cent, more sugar is indicated than by the polarimetric method. Pentoses. — To judge from recent observations, traces of pentoses, viz., xylose, arabinose, and rhamnose, may be found in every urine. Larger quantities were first observed by Salkowski and Jastrowitz, in the urine of a morphin habitui, in which the pentosuria alter- nated with glucosuria. A similar case was reported by Real. Kiilz and Vogel found larger quantities in a case of diabetes ; and still more recently Bial has reported two instances which occurred in apparently healthy individuals. A digestive pentosuria has also been described. Such urines reduce Fehling's solution and Nylan- der's solution, and give rise to the formation of an osazon when treated with phenylhydrazin. The osazon, however, can be readily distinguished from that obtained from glucose, maltose, or lactose, etc., by the melting-point (159°— 160° C). The fermentation test is negative. Xylose and rhamnose turn the plane of polarization to the right, while arabinose is optically inactive. The presence of pentoses ca;n be readily detected with Tollens' orcin test. ToUens' Orcin Test. — A few granules of orcin are dissolved in 4 to 5 c.c. of concentrated hydrochloric acid by the aid of heat, so that a slight excess is present. This solution is divided into two equal parts and allowed to cool. To one portion 0.5 c.c. of the urine to be examined is added, and to the other an equal amount of normal urine of the same speciiic gravity. Both speci- mens are placed in a beaker containing boiling water, when in the presence of pentoses a green color will first be observed at the top, which gradually extends throughout the mixUire, while the normal specimen scarcely changes in color. In the presence of 0.1 per cent, a positive reaction is still obtained, which is especially marked if the urine has been previously decolorized with animal charcoal. The green pigment which results can be extracted by shaking with amyl alcohol, and on spectroscopic examination it gives rise to a well-deiined band of absorption in the red portion of the spectrum near the yellow border. ToUens' phloroglucin test, in which phloroglucin is substituted for the orcin, and in which a deep-red color is obtained in the presence of a pentose, may also be used, but the reagent indicates the presence of glucuronates as well. Very curiously, the pentosuria persists even though no carbo- hydrates are ingested ; and there is evidence to show that pentoses are formed within the body. As a matter of fact, Hammarsten has ' Leo, Virchow's Arohiv, vol. cvii. 454 THE URINE. succeeded in demonstrating the presence of a pentose among the decomposition-products of a nucleo-glucoproteid which is found in the pancreas ; and Blumenthal arrived at similar results in the case of various nucleinic acids which occur in the animal body. It is possible, on the other hand, that the pentoses may result from the metabolic products of glucose which are formed under normal con- ditions by a process of oxidation, and are then eliminated as such under still unknown influences. Aside from the traces normally present in the urine, pentosuria must be regarded as a metabolic anomaly, analogous to glucosuria, cystinuria, alkaptonuria, etc. LiTERATUKE. — E. Salkowski u. M. Jastrowitz, " Ueber eine bisher nicht beobachtete Zuckerart iin Ham," Centralbl. f. d. med. Wiss., 1892, No. 19. E. Salkowski, " Ueber d. Pentosurie," Berlin, klin. Woch., 1895, No. 17. F. Blumenthal, Ibid., No. 26 ; and Zeit. f. klin. Med., vol. xxxvii. p. 415. E. Kulz u. J. Vogel, Zeit. f. Biol., N. F., 1896, vol. xiv. p. 189. E. Salkowski, " Ueber d. Vorkommeu von Pentosen im Ham," Zeit. f. physiol. Chem., 1899, vol. xxvii. p. 507. Animal Gum. — Landwehr's animal gum, according to modern researches, is a constant constituent of normal urine, but is of no clinical interest. Of the chemical nature of the substance not much is known, but there is evidence to show that in all probability the body is a derivative of chondroitin-sulphuric acid. GLUCURONIC ACID. Glucuronic acid is derived from glucose, and constitutes an inter- mediary product of the normal metabolism of the body. In the urine it is found only in combination with certain fatty and aromatic alcohols, forming compounds . which are related to the glucosides and are generally spoken of as the conjugate glucuronates. Such bodies have been observed in the urine following the ingestion of chloral, camphor, naphtol, oil of turpentine, menthol, phenol, mor- phin, antipyrin, etc., and traces may also be obtained from nor- mal urines. The normal glucuronates are undoubtedly compounds of glucuronic acid with phenol, paracresol, indoxyl, and skatoxyl. Their amount is exceedingly small, as the greater portion of these bodies is normally eliminated in combination with sulphuric acid. Of the quantitative variations of the normal glucuronates and their relation to disease, next to nothing is known. Their clinical interest centres in the fact that certain glucuronates are capable of reducing copper and bismuth in alkaline solution, and may thus be confounded with glucose. Such urines, however, do not undergo fermentation. The glucuronates turn the plane of polarization to the left, while glucuronic acid itself is dextrorotatory. Like the pen- toses, the glucuronates give a positive reaction with phlorogluoin, URINARY PIGMENTS AND CHBOMOGENS. 455 while they do not react with orcin (see page 453). With the free acid phenylhydrazin forms crystalline compounds (see page 442). Literature.— H. Thierfelder, "TJeberd. Bildung v. Glykuronaaure," etc., Zeit. f. physiol. Chem., 1886, vol. x. p. 163 ; "Untersuchungen fiber d. Glykuronsiiure," Ibid., 1887, vol. xi. p. 388. P. Mayer, "Ucber d. Ausscheidung u. d. Nachweis d. Glyku- ronsaure," Berlin, klin. Woch., 1899, pp. 591 and 617. P. Mayer u. C. Nenberg, Zeit. f. physiol. Chem., 1900, vol. xxix. p. 256. INOSIT. According to Hoppe-Seyler, traces of inosit may be found in the urine under normal conditions. Somewhat larger quantities are eliminated following the ingestion of large amounts of water, and for this reason possibly inosituria is notably observed in cases of diabetes insipidus, in diabetes mellitus, and in chronic intersti- tial nephritis. The substance is devoid of clinical interest. It is not a carbohydrate, but belongs to the aromatic series, and is commonly regarded as hexa-hydroxybenzol. Its formula is CgHijOj + HjO. For methods of isolating the substance from the urine, the reader is referred to special works.' URINARY PIGMENTS AND CHROMOGENS. Under normal conditions urochrome and uroerythrin, to which latter the red color of urate sediments is due, are the only known pigments which occur preformed in the urine, while indigo-red and indigo-blue, derived from indoxyl sulphate and indoxyl glucuronate, may be artificially produced. In disease, on the other hand, various other pigments may be found, which occur in the urine either free or in the form of chromogens. Among the former may be mentioned haemoglobin, methsemoglobin, hsematin, hsematoporphyrin, urorubro- hsematin, urofuscohsematin, urobilin,the biliary pigments, and melanin; while abnormal chromogens are met with following the ingestion of certain drugs, such as santonin, senna, rheum, iodine, etc., as also in cases of poisoning with carbolic acid, creosote, etc. The occurrence of some of these substances, such as the various forms of blood-pig- ment, the biliary pigments, and indigo, viz., indican, is of considerable clinical interest, while others again are of only minor importance. Normal Pigments. — Urochrome. — To the presence of this pig- ment, which appears to be identical with the normal urobilin of MaoMunn, but which should not be confounded with ihe pathological urobilin of Jaff&, the normal yellow color of the urine is probably largely due. It is supposedly derived from bilirubin, which in turn is referable to hsematin, and thus to the haemoglobin of the blood. From the bilirubin secreted into the intestinal tract it is derived by a process of oxidation, and not of reduction, as is generally stated (Gautier). Such a transformatiou, according to our present knowledge, may, 1 C. E. Simon, Physiological Chemistry, Lea Bros. & Co., 1901. 456 THE UBINE. however, also occur directly, without the intervention of bilirubin, as urochrome is found in the urine of dogs in which the bile is prevented from entering the intestinal tract by the establishment of a biliary fistula. An increased amount is similarly found in cases in which resorption of large extravasations of blood is taking place — in short, whenever an increased destruction of red corpuscles occurs. Under the opposite circumstances — i. e., in conditions associated with a new formation of red corpuscles, as in certain forms of anaemia, chronic parenchymatous nephritis, diabetes, dis- eases of the bone-marrow, etc. — it occurs in diminished amount. Urochrome, moreover, is present in urobilin-free feces, and even in those of infants with congenital atresia of the biliary ducts. In order to obtain urochrome from normal urine, this is acidulated with 1-2 grammes of dilute sulphuric acid pro liter, filtered, and saturated with ammonium sulphate in substance, when the flakes which are formed, if an excess of the salt has been added, are dried and treated with warm, slightly ammoniacal absolute alcohol ; the pigment is then obtained upon evaporation of the alcohol. An alcoholic solution of urochrome, like the urobilin of JafPg, is said to exhibit a beautiful greenish fluorescence when treated with ammonia and a few drops of a solution of zinc chloride ; but, unlike the latter substance, its acidulated alcoholic solutions present a broad band of absorption at F, which extends more to the left than to the right of this line, while the remainder of the spectrum is at the same time absorbed to the right end, from a point some- what to the left of G. Garrod, on the other hand, states that by acting upon urochrome with acids he did not succeed in obtaining any product showing the urobilin band or yielding the well-known fluorescence with zinc chloride and ammonia. But a substance having both these properties was readily obtained by the action of aldehyde upon an alcoholic solution of the pigment. In a short time — shorter still when the liquid is warmed — an absorption-band appears like that of urobilin, and the tint of the solution deepens to a rich orange-yellow. With zinc chloride and ammonia a bril- liant green fluorescence appears, and the band is shifted toward the red, as that of urobilin is under like circumstances. The process can be stopped at this point by the simple addition of water, for aldehyde has no such action upon aqueous solutions of urochrome. If, however, the action be allowed to continue, a further change ensues ; the liquid reddens, and a second band appears in the violet. The fluorescence can still be obtained with zinc chloride and am- monia, and both bands are shifted toward the red and are closer together than before. The reaction with aldehyde, according to Garrod, aflbrds a very delicate test for the presence of urochrome in alcoholic solutions. The product of the earlier stage, although it is not identical with urobilin, resembles that pigment quite as URINARY PIGMENTS AND CHROMOQENS. 457 closely as the products obtained from bilirubin and hsematin by the action of reducing agents ; but no second band is developed when aldehyde is added to an alcoholic solution of urobilin.' By the action of potassium permanganate upon urobilin Riva and Chiodera ^ obtained a substance closely resembling urochrome, and a similar product is formed when an aqueous solution of uro- bilin containing ether is evaporated upon a water-bath. Neither product shows any absorption-band, and both behave as urochrome does when it is acted upon by aldehyde. Uroerjrthrin. — Uroerythrin is the pigment which imparts the red color to crystals of uric acid and the pink tint to urate sediments. Under strictly normal conditions it probably does not occur in the urine, but it readily appears with the slightest deviation from health, and when present in larger amounts imparts a deep-orange color to the urine. Under pathological conditions it is seen espe- cially in cases of hepatic insufficiency, in which the liver, owing to a greatly increased destruction of red corpuscles, is unable to transform into bile-pigment all the blood-pigment which is carried to it. It also occurs when an absolute insufficiency on the part of the hepatic cells exists, so that the organ is not even capable of causing the transformation of a normal amount of haemoglobin. Uroerythrin is thus seen in notable quantities in cases of cirrhosis and carcinoma of the liver, in passive congestion resulting from hearl^disease, in acute articular rheumatism, gout, pneumonia, malarial fever, erysipelas, spinal curvature, etc. In typhoid fever a marked excretion of uroerythrin is exceptional, and its occurrence has been associated with pulmonary complications. In nephritis it is seldom found in the urine, but Garrod cites an instance of pneumonia in which an abundant excretion of the substance accom- panied conspicuous albuminuria. In certain diseases, such as hepatic cirrhosis, the excretion of uroerythrin, as also of urobilin, is said to be much diminished when the patient is placed upon a milk-diet (Riva). Chemically, its relation to haemoglobin, hsematoidin, and bilirubin is seen from the following analyses of the various pigments : C H N O S Fe Bfcmoglobin, 53.85 7.32 16.17 . . . 0.39 0.43 Hsematoidin, 65.05 6.37 9.51 Bilirubin, 67.83 6.29 9.79 16.79 . . . ' Uroerythrin, 62.51 5.79 31.70 When present in large amounts uroerythrin is readily recognized by the salmon-red color which it imparts to urinary sediments. Otherwise it is best to precipitate the urine with neutral lead acetate, ' A. E. Garrod, " The Bradshaw Lecture on the Urinary Pigments in Their Patho- logical Aspects," Lancet, Nov. 10, 1900. 'Eiva and Chiodera, Arch. ital. diClin. Med., 1896, toI. xxxv. p. 505. 458 THE URINE. barium chloride, or a similar reagent, when in the absence of uro- erythrin a milky-white precipitate is obtained, while a pale rose- colored sediment indicates the presence of the piginent in appreciable amounts ; a more pronounced rose color is produced if large quan- tities are present. In every case at least ten to fifteen minutes should be allowed to elapse before forming a definite conclusion, so that the sediment may have abundant time to settle. The pigment itself .is unstable. Its solutions in alcohol or chloroform are rapidly decolorized by light, and even when kept in the dark quickly undergo change. Alkalies destroy the pigment readily, with the production of a green tint. Neutralization of the alkali does not restore the original color or bring back the absorption spectrum, which is characteristic, though ill-defined, consisting of two faint bands in green and blue, united by a fainter shading. One of these bands has the position of the urobilin band, but both alike disappear when the solutions are decolorized by light. The pigment is readily soluble in amyl alcohol and acetic ether (Grarrod).' Normal Chromogens. — The chromogens occurring in normal urine are indican, urohsematin, and an unknown chromogen which yields urorosein when treated with mineral acids. Indican. — It has been pointed out (see Sulphates) that the indol formed during intestinal putrefaction is oxidized to indoxyl in the blood ; this, entering into combination with sulphuric acid, is elimi- nated in the urine as sodium or potassium indoxyl sulphate, or indican, as represented by the equations : V (1) C8H,N + O =C8H,N0 Indol. Indoxyl. /OH /CsHeNO (2) CjHjNO +80/ =S0»< +H.0 \0H \0H Indoxyl. Indoxyl sulphate. /CsH^NO /CsHeNO (3) S02< + NajHPOi = SO/ + NaH^PO^ ^OH \ONa Indoxyl sulphate. Indoxyl-sodlum sulphate. Formerly it was thought that indican was also formed within the tissues of the body in the absence of putrefactive organisms.^ Further researches, however, have demonstrated that micro-organ- isms are always concerned in the production of indican, and that in health the large intestine is its sole source. Baumann, who succeeded in absolutely disinfecting the intestinal tract of a dog by means of large doses of calomel, thus observed that all traces of 1 A.E. Garrod.loe. cit. A. Eobin, Urologie cliniqnede la FiSvretyphoide, Paris, 1877. 2 E. Salkowski, Ber. d. deutsch. chem. Gea., 1876, vol. ix. pp. 138 and 408. Bau- mann, Zeit. f. physiol. Chem., 1886, vol. x. p. 123. Senator, Centralbl. f. d.med. Wiss., 1877, vol. XV. pp. 357, 370, and 388. URINARY PIGMENTS AND CHROMOQENS. 459 indican, as also of phenol and paracresol, disappeared from the urine. According to Senator, moreover, indican does not occur in the urine of newly born infants which have not as yet received nourishment.- This observation is a strong point in favor of Nencki's teachings that indol is a specific product of albuminous putrefaction in the presence of organized ferments, as putrefiable substances are here present, but no putrefactive organisms. Tuczek's observations on abstinence from food in cases of insanity, in which indican was observed in the urine only when albumins, though in minimal amounts, were ingested, also speak very strongly against Salkowski's theory. Finally, it has been demonstrated that in cases in which an artificial anus is established near the distal end of the ileum the conjugate sulphates disappear almost entirely from the urine, while they reappear in normal amount as soon as the connection between the small and large intestines has been re-established.' The amount of indican which is normally eliminated in the urine varies somewhat with the character of the diet. Jaff6 ^ obtained 6.6 mgrms. from 1000 c.c. of urine, as an average of eight obser- vations. The largest quantities excreted in health are found after a liberal indulgence in animal food, particularly the so-called red meats, wBile the smallest amounts are observed during a milk- or kefir-diet. By means of the latter article, indeed, the greatest dimi- nution in the degree of intestinal putrefaction may be eifected in man. In pathological conditions an increased elimination of indican is observed : 1. In aU diseases which are associated with an increased degree of intestinal putrefaction. As there appears to be little doubt that this is largely regulated by the acidity of the gastric juice, an in- creased indicanuria, according to personal observations, is encountered when anachlorhydria or hypochlorhydria exists. It has been pointed out elsewhere that it is possible to form a fairly accurate idea of the amount of free hydrochloric acid in the gastric juice by an examina- tion of the urine in this direction. Large quantities of indican are thus eliminated in cases of carcinoma of the stomach, and exceeded only by those observed in cases of ileus, so that this symptom, in my estimation, is of considerable value in difFerential diagnosis, and is one, moreover, which has not received the attention it deserves. Exceptions to this rule are at times, though rarely, met with, for which it is, however, impossible to account at present. Large quantities of indican are also observed in cases of acute, subacute, and chronic gastritis. In the course of personal ' Nencki, Macfadyen u. Sieber, Arch. f. exper. Path. u. Pharmakol., 1891, vol. xxix. ^ Jaff6, Centralbl. f. d. med. Wiss., 1872, vol. x. pp. 2, 481, and 497 ; and Virchow'a Archiv, 1877, vol. Ixx. p. 72. 460 THE URINE. observations in this direction I was impressed with the curious phenomenon that in cases of ulcer of the stomach, notwith- standing the simultaneous occurrence of hyperchlorhydria, an increased elimination of indican, contrary to what is usually seen in hyperchlorhydria referable to other causes, is quite constantly found. Possibly the existence of muscular atony which was noted in those cases may serve to explain this apparent incongruity, but it is as yet impossible to offer a satisfactory explanation of the phenomenon. Remembering the origin of indican, and the relation which the amount eliminated bears to the degree of intestinal putrefaction, it will be unnecessary to enumerate the long list of diseases in which an increased indicanuria has been observed, as it wUl be found that in the majority of these cases the indicanuria is merely an index of the condition of the gastric juice and the motor power of the stomach.' 2. It should be noted that in cases in which the peristaltic move- ments of the small intestine have become impeded, as in ileus, acute and chronic peritonitis, an increased elimination of indican will inva- riably take place, no matter what the state of the gastric juice may be. In such conditions, and especially in ileus, the largest quanti- ties are observed, a point which may be of decided value in differ- ential diagnosis, as diseases of the large intestine alone are Tieoer associated with an increase in the amount of indican. In simple, unoomplioated constipation inoreased indicanuria is not seen; and should an examination in such cases reveal the presence of more indican than normal, it wUl be safe to assume the existence of disease elsewhere, and especially of the stomach. 3. As albuminous putrefaction may also take place within the body, an increased indicanuria is observed in cases of empyema, putrid bronchitis, gangrene of the lung, etc. ; but whUe in the con- ditions mentioned above the indol-producing organisms appear to be especially active, the elimination of phenol in the latter condition may be more pronounced at times than that of indican. Bearing in mind the points here set forth, I cannot agree with others in saying that the study of indicanuria possesses no importance from a clini- cal standpoint. I maintain, on the other hand, that an examina- tion of the urine in this direction is at least as important as the testing for albumin and sugar, and that points of decided importance, not only in diagnosis, hut also in prognosis and treatment, may thus be gained. When indican is treated with hydrochloric acid it is decomposed into sulphuric acid and indoxyl ; should an oxidizing substance be present at the same time, indigo-blue, the blue coloring^matter of the urine, results : ' C. E. Simon, " Indicanuria," Am. Jour. Med. Sci. (full literature), 1895, vol. ex. p. 48. URINARY PIGMENTS AND CHROMOQENS. 461 2CeH6NKS04 + 20 = C,eHioNA + 2HKSO4. Potassium indoxyl Indigo-blue, sulpliate. Indigo-blue in small amounts may be found free in the sediment of almost every decomposing urine, usually occurring in the form of small, amorphous granules, and more rarely in crystalline form. Urines have, however, also been observed which were blue when passed, or which turned blue as a whole upon standing. Such a phenomenon must be regarded as a medical curiosity. The blue pigment which may be obtained from urines has been variously described as Prussian-blue, urocyanin, cyanurin, Harn- blau, uroglaucin, choleraic urocyanin, but it has been shown to be indigo-blue, and derived from a colorless mother-substance which is present in every urine to a greater or less extent, and which has been named indican. This has been shown to be identi- cal with the uroxanthin of HeUer and Thudichum's choleraic uro- cyaninogen. Tests foe Indican. — The urine of twenty-four hours is care- fully collected and a specimen taken for examination. A few cubic centimeters are then mixed with an equal volume of Obermayer's reagent, and shaken with a small amount of chloroform. Ober- mayer's r&igent is a 2 pro mille solution of ferric chloride in concen- trated hydrochloric acid.^ Stokvis' modification of Jaffa's test may also be employed.^ To this end, a few cubic centimeters of urine are treated with an equal volume of concentrated hydrochloric acid, and two or three drops of a strong solution of sodium or calcium hypochlorite. The mixt- ure is shaken with 1 or 2 c.c. of chloroform as above. The indigo which is set free in this manner is taken up by the chloroform, and colors this blue to a greater or less extent, the degree of increase, as compared with the normal, being determined by the intensity of the color. Albumin need not be removed. Bile-pigment, which inter- feres with the reaction, is removed by means of a solution of lead subacetate, which is carefully added in order to avoid an excess. Urines presenting a very dark color may be cleared in the same manner. Potassium iodide, owing to the liberation of free iodine, will color the chloroform more or less of a carmine. For the sake of comparison, it is well to employ the same quantities of urine and reagente in every case, marked tubes being very convenient for this purpose. The method last described I have also found to be a fairly sensi- tive test for albumin, in the presence of which a well-marked cloud appears near the surface of the mixture and gradually extends downward. 1 Obermayer, Wien. klin. Woch., 1890, vol. iii. p. 176. 2 See Senator, Centralbl. f. d. med. Wiss., 1877, vol. xv. p. 257. 462 THE URINE. Quantitative Estimation. — Womg's Method} — The method is based upon the decomposition of potassium indoxyl sulphate by means of concentrated hydrochloric acid and the oxidation to indigo- blue of the indoxyl which is thus formed. The indigo-blue is fur- ther transformed into indigo-sulphuric acid, and this titrated with a solution of potassium permanganate of known strength. The various changes which take place are represented by the following equations : (1) CgHeNSO^K + HjO = CbHbN.OH + HKSOj. Indican. Indoxyl. (2) 2C8H6lir.OH + 20 = C,6H,„NA + SHjO. Indoxyl. Indlgo-blue. (3) CieH,„NA + 2H,S04 = C,,H8( HSO, ),N,Oj + 2H,0. Indigo-blue. Indigo-sulpliuric acid. (4) 5C,,H,„N,A + 4KMnO, + 6H,SO,= Indigo-blue. SCsHioNA + 2K2SO4 -|- ^MnSO^+eHA Eeagents required : 1. A 20 per cent, solution of lead acetate. 2. Obermayer's reagent. This is a 2 pro mille solution of ferric chloride in concentrated hydrochloric acid (sp. gr. 1.19). 3. Chloroform. 4. Concentrated sulphuric acid. 5. A mixture of equal parts of alcohol (96 per cent.), ether, and water. 6. A concentrated solution of potassium permanganate — i. e., a solution containing about 3 grammes pro liter. The titration is conducted with this solution diluted in the proportion of 5 c.c. to 195 c.c. of water. Its titre is ascertained before each titration by comparing it with a dilute solution of oxalic acid of known strength ; for example, one containing 0.1 gramme of the acid dissolved in 100 c.c. of water, as described on page 379. The amount of indigo-blue which each cubic centimeter will represent is ascertained by multi- plying the corresponding amount of oxalic acid by 1.04. Example. — Supposing that the permanganate solution is found of such strength that 1 c.c. represents 0.00014 gramme of oxalic acid ; the corresponding amount of indigo would be 0.00014 X 1.04 = 0.00015 gramme. Method. — The urine is first examined for indican, as described above. Should a very intense reaction be thus obtained, only 25 or 50 c.c. are used for the quantitative estimation, while larger amounts are taken (200-500 c.c.) if the reaction is of only moderate intensity or negative altogether. The urine is precipitated with lead acetate solution, care being taken to avoid an excess. A large and accurately measured ' E. Wang, " Ueber d. quantitative Bestimmung d, Harnindikans," Zeit. f. physlol. Chem., vol. xxv. p. 406. URINARY PIGMENTS AND CHROMOQENS. 463 portion of the clear filtrate is treated in a separating funnel with an equal volume of Obermayer's reagent and extracted with chloroform. To this end, 30 c.c. are added at a time and shaken for one minute. Two or three extractions are usually sufficient to remove the entire amount of indigo. The extract is placed in a small flask, and the chloroform distilled off. The residue is dried for a few minutes on a water-bath until traces of remaining chloroform have been re- moved. It is then washed with the alcohol-ether-water mixture to remove the reddish-brown pigment which is present together with the iudigo-blue. The latter remains undissolved. After filtering off any particles of indigo that may be in suspension, through a small filter, this is dried and repeatedly extracted with boiling chloroform. The chloroform extract is filtered into the original indigo flask, the chloroform distilled off, the residue dried as before, and while still warm treated with 3 or 4 c.c. of concentrated sulphuric acid. The entire residue should be brought into solution by careful agitation. After standing for twenty-four hours the contents of the flask are poured into 100 c.c. of cold water ; the flask' is rinsed and the washings added to the solution. This is filtered once more and titrated with the permanganate solution. At first the blue color of the solution changes but little ; later it turns greenish, and finally becomes yellowish or entirely colorless — not red. As a rule, the end-reaction is quite distinct, but the titration requires experience. The best results are obtained when from 10 to 15 c.c. of the dilute permanganate solution are used. The resulting amount of indigo contained in the measured-off quantity of the first filtrate is then ascertained as described above. Example. — Amount of urine : 1780 c.c. The stock solution of potassium permanganate contains 3 grammes to the liter; 1 c.c. = 0.00596 gramme of oxalic acid = 0.0062 gramme of indigo. Diluted solution (5 : 200) ; 1 c.c. = 0.00015 gramme of indigo. 300 c.c. of urine were precipitated with 25 c.c. of the lead solution; 250 c.c. of the filtrate, corresponding to 230.7 c.c. of urine, treated with 250 c.c. of Obermayer's reagent. Extracted twice with chloroform. 4.3 c.c. of the permanganate solution were used in the titration = 0.00065 gramme of indigo, corresponding to 0.005 gramme in the 1780 c.c, according to the equation 230.7 : 0.00065 : : 1780 :%; x = 1:1^ = 005. 230.7 Other methods for the quantitative estimation of indican which have heretofore been used, with the exception of the spectroscopic method of Miiller, are not only inaccurate, but, like this, too time- consuming and complicated to be of value to the practising physician. As a consequence almost all observers have based their conclusions upon an approximative estimation only. For practical purposes this 464 THE UJRINE. is sufficient, and even Wang's method, though accurate and simple, will hardly find a ready entrance into the clinical laboratory, as it is still too time-consuming and too expensive for daily use. For scientific purposes, however, it may be recommended. Urohaematin.' — Urohsematin appears to be the chromogen of the red pigment of the urine, and is very likely closely related to in- doxyl. Little is known of its chemical composition or of its mode of formation. In all probability the red pigment which may be obtained from this substance is identical with other red pigments which have been described from time to time as occurring in the urine, such as that of Scherer, the urrhodin of Heller, the urorubin of Plosz, Schunk's indirubin, Bayer's indigo-purpurin, Giacosa's pigment, and also the indigo-red obtained by Rosenbach and Rosin by careful oxidation of the urine with nitric acid. Further investigations are necessary before this subject can be fully understood ; but bearing in mind the probable origin of urohsematin from indoxyl, it would possibly be best to speak of the red pigment as indigo-red. In accordance with the view that urohsematin is an indoxyl derivative, its clinical significance is similar to that of indican (which see). The presence in normal urine of urohsematin — i. e., a chromogen yielding a red pigment when treated with certain reagents — may be demonstrated by shaking urine with chloroform and decanting after several days, when the addition of a drop of hydrochloric acid to the chloroform extract will cause the appearance of a beautiful rose color ; this varies in intensity according to the amount of the chromogen present. The purplish color so often obtained in the chloroform extract when Stokvis' modification of Jaff6's indican test is employed is due to a mixture of indigo-blue and indigo-red. Indican, however, is generally present in larger amounts than urohsematin. In normal and, usually also, in pathological urines a red color is not obtained with the test mentioned. In a few isolated cases of ileus, peritonitis, and carcinoma of the stomach I have found more indigo-red than indigo-blue. The so-called " Reaction of Rosenbach " is a convenient test for indigo-red when this is present in increased amounts : the boiling urine is treated drop by drop with concentrated nitric acid, when in the presence of large amounts of indigo-red it assumes a dark Bur- gundy color, which sometimes takes on a bluish tinge when held to the light. Owing to a precipitation of the pigment the mixture at the same time becomes cloudy and the foam assumes a blue color. In well-marked cases the Burgundy color does not appear to be changed by the further addition of nitric acid, but will sometimes suddenly change from red to yellow when 10—20 drops of the acid ' 6. Harley, Verhandl. d. physik. med. Ges. z. Wiirzburg, 1855, vol. v. p. 1. UBINABY PIGMENTS AND CHBOMOGENS. 465 have been added. This reaction Rosenbach ' regarded as symptomatic of various forms of severe intestinal disease associated with an impeded resorption throughout the entire intestinal tract. Ewald ^ likewise noted this reaction in cases of extensive disease of the small intestine, in carcinoma of the stomach, and in acute and chronic peritonitis ; but he obtained negative results in carcinoma of the colon, stricture of the oesophagus, chronic diarrhoea, etc. Eosenbach's reaeUon should be viewed in the same light as a highly increased elimi- nation of indioan. I have met with the reaction in all conditions associated with greatly increased intestinal putrefaction, and, like Ewald, failed to note the reaction in a few cases of occlusion of the large intestine, in which an increased elimination of indican is like- wise never observed. Uroroseinogen.^ — In addition to indican and urohsematin, still another chromogen, which yields a rose-red pigment when treated with mineral acids, appears to occur in normal urine, although in small amounts. Beyond the fact that the chromogen is not a conjugate sulphate, practically nothing is known of its chemical nature. The pigment, which has received the name urorosein, or Harnrosa, appears to be identical with Heller's urophain. Urorosein is best demonstrated by treating 5—10 c.c. of urine with an equal amount of concentrated hydrochloric acid, and 1 or 2 drops of a concentrated solution of sodium hypochlorite, when in the presence of much indican the mixture assumes a dark-greenish, blackish, or dark- blue color, owing to the formation of indigo. When the mixture is shaken with chloroform the supernatant fluid exhibits a beau- tiful rose color, which is due to the urorosein. This may now be extracted with amyl alcohol and separated from other pigments which are present at the same time, by shaking with sodium hydrate, whereby the solution is decolorized. Upon the addition of a drop or two of hydrochloric acid to the alcoholic extract the rose color reappears. Such solutions, however, soon become decol- orized upon sfeinding. A rose-red ring, referable to this pigment,, is also frequently obtained in pathological urines when the ordi- nary nitric acid test is employed. While normally urorosein is obtained only in traces, appreciable amounts are often met with in pathological conditions associated with grave disturbances of nutrition, as in nephritis, diabetes, carcinoma, dilatation of the stomach, pernicious anaemia, typhoid fever, phthisis, and at times in profound chlorosis, etc. A vege- table diet also appears to cause an increase in the amount of the; chromogen. • Eosenbach, Berlin, klin. Woch., 1889, vol. xxvl. pp. 5, 490, and 520, and 1890; vol. xxvii. p. 585. 2 Ewald, Ibid., 1889, vol. xxvi. p. 953. ' H. Eosin, Deutsch. med. Woch., 1893, p. 51. 30 466 THE URINE. Pathological Pigments and Chromogens. — The Blood-pigments. — The blood-pigments proper which may occur in the urine have already been considered (see page 412), and in this connection it will only be necessary to refer briefly to the occasional presence of hsematin, urorubrohsematin, urofuscohsematin, and haematopor- phyrin. HiEMATiN is only rarely found. In order to demonstrate its pres- ence, the urine is rendered strongly alkaline with ammonia, filtered, and the filtrate examined spectroscopically, when the spectrum shown in Fig. 6 will be noted ; this may be changed into the spectrum represented in Fig. 7 by the addition of ammonium sulphide. IjBOKTJBEOHiEMATiN and UHOFUSCOii^MATiN Were observed by Baumstark ^ in the urine of a case of pemphigus leprosus compli- cated with visceral lepra; they appear to be closely related to hsematin. The color of the urine in this case varied between dark red and brownish red, strongly suggesting the presence of blood. In order to separate the pigments, the urine was dialyzed and the contents of the dialyzer dissolved in sodium hydrate solu- tion. Upon the addition of hydrochloric acid to this solution a brown pigment separated out in flakes, while a second pigment remained in solution, imparting to it a beautiful red color. Upon filtration the acid filtrate was again subjected to dialysis, when the red pigment likewise separated out. The former substance Baum- stark termed urorubrohsematin, and the latter urofuscohsematin. UeohjEMATOPORPHyrin has the formula CjjHjgJSTjOj, and is probably identical with the hsematoporphyrin resulting from the action of sulphuric acid upon hsematin. McMunn found a pigment answering the description of this substance in the urine in cases of rheumatism, Addison's disease, pericarditis, and paroxysmal hsemoglobinuria, which he termed urohsematin, but which in all probability was hsematoporphyrin. Le Nobel found the same pigment in two cases of hepatic cirrhosis and in one case of crou- pous pneumonia. Others have likewise met with hsematoporphy- rinuria in various forms of hepatic disease, as also in phthisis, exophthalmic goitre, typhoid fever, and hydroa sestivalis ; further, in association with intestinal hemorrhages, in cases of lead poisoning, and especially during long-continued use of sulphonal, trional, and tetronal. Nebelthau records the history of a female patient, the subject of congenital syphilis, who had passed dark-red urine as long as she could remember, and continued to do so while under observa- tion. Recent researches, moreover, have shown that in traces at least the substance is present in everj' urine. As regards the origin of these normal traces, the evidence is in favor of the view that they are formed within the body during its normal metabolism, and 1 F. Baumstark, Pfliiger's Archiv, 1874, vol. ix. p. 568. See, also, J. W. Schultz, Diss., Greifswald, 1874. URINARY PIGMENTS AND CHROMOGENS. 467 most likely in the liver, whence the substance is eliminated in the bile. A portion then escapes with the feces, while a similarly small amount is resorbed and eliminated in the urine. Increased amounts would accordingly suggest the existence of a hepatic insufficiency ; and, as a matter of fact, we find that actual anatom- ical lesions then not infrequently occur. Taylor and Sailer thus report that in their case of sulphonal poisoning widespread degener- ation of the hepatic cells existed ; and Neubauer was able to isolate the pigment from the liver of rabbits to which sulphonal had been administered, while it was absent in all other organs. On the other hand, it is difficult to ascribe all the phenomena of such hsemato- porphyrinuria to hepatic changes, seeing that changes of like degree may occur without conspicuous urinary abnormality, and there is still much that is obscure in this condition. Stokvis attributed the increased elimination of haematoporphyrin in cases of lead poisoning and following the continued use of sulphonal to the occurrence of hemorrhages into the intestinal mucosa, and suggested that the transformation of the haemoglobin into hsematoporphyrin was favored by the sulphonal. But while intestinal hemorrhages may occur in the sulphonal cases, they are not always observed, and, as Garrod points out, Kast and Weiss, as also Neubauer, were unable to verify the recorded experiments of Stokvis, in which he claims to have obtained a small amount of hsematoporphyrin when fresh blood was digested with pepsin-hydro- chloric acid and sulphonal at from 38° to 40° C. Urines which contain much hsematoporphyrin are usually dark red in color, but the shade may vary from a sherry or port^wine tint to a dark Bordeaux. It is noteworthy, however, that this color is not primarily due to the exaggerated degree of hsematoporphy- rinuria, but, as Hammarsten first pointed out, to other abnormal pigments which are but little known, but which are probably closely related to haematoporphyrin. As Garrod says, the removal of the hsematoporphyrin from such urines causes little or no change of color, and when this pigment is added to normal urine until on spectroscopic examination bands of similar intensity are seen the change of tint produced is comparatively slight. In one such case, not due to sulphonal, he was able to isolate a purple pigment which differed in its properties from any known urinary coloring-matter, and to which the color of the urine in question was obviously in the main due. Neumeister also states that in sulphonal intoxication an iron-containing derivative of hsemoglobin occurs in the urine, which presents a reddish- violet color and shows a single band of absorption in the blue portion of the spectrum immediately bordering on the green. Albumin is not present in uncomplicated cases of haematopor- phyrinuria, and the pigment itself does not give the albumin reactions. 468 THE URINE. To test for hsematoporphyrin, the following procedure may be employed : Thirty c.c. of urine are treated with an alkaline solution of barium chloride. The precipitate, after having been washed with water and then with absolute alcohol, is extracted with ordinary alcohol acidu- lated with hydrochloric acid, by rubbing in a mortar. The solution thus obtained will present a reddish color in the presence of hsema- toporphyrin, and its filtrate yields the characteristic spectrum of the latter substance — i. e., four bands of absorption, of which two are broad and dark and two light and narrow. The former alone are characteristic, and frequently the only ones visible. One of these extends beyond D into the red portion of the spectrum, while the other is situated between 6 and F. Of the other two bands, one may be seen between C and D and the other between D and E, nearer E (Fig. 10). Oarrod's Method. — To demonstrate the presence of hsematopor- phyrin under normal conditions, or when small amounts only are present in the urine, Garrod's method should be employed. To this end, several hundred c.c. of urine (500-1500) are treated with a 10 per cent, solution of sodiuni hydrate in the proportion of 20 c.c. of the alkali solution for 100 c.c. of urine. The precipitated phosphates are filtered off and thoroughly washed by repeatedly suspending them in water. Should the precipitate be of a reddish color, or if it shows the spectrum of hsematoporphyrin in alkaline solution, when examined on the filter in the moist state, we may con- clude that much hsematoporphyrin is present. In this case it is washed until the filtrate is colorless. If traces only are present, however, one washing must suffice. The precipitate is then treated with alcohol, which is acidified with hydrochloric acid to such an extent that the phosphates are entirely dis- solved. The resulting solution should not exceed 15 to 20 c.c. in volume. This is then examined in a layer, of not less than 3 to 4 cm. in thickness, for the spectrum of acid hsematoporphyrin, using a spectroscope with slight dispersion. The solution is now rendered alkaline with ammonia and treated with an amount of acetic acid which just suffices to redissolve the precipitated phosphates. On shaking with chloroform this extracts the pigment, and the chloro- form solution then gives the spectrum of the alkaline hsematopor- phyrin, since organic acids do not change the pigment to the form which yields the acid spectrum. The residue which remains after evaporating the chloroform can finally be washed with water and dissolved in alcohol, when a nearly pure solution is obtained, which is comparable with a solution of hsematoporphyrin obtained from hiematin. Precautions : If a preliminary test shows that the urine con- tains but little phosphates, a small quantity of calcium phosphate URINARY PIGMENTS AND CHROMOGENS. 469 in acetic acid is added before the urine is rendered alkaline with the sodium hydrate solution. As haematin and chrysophanic acid are also precipitated with the phosphates, their absence must be insured. For this reason the urine should contain no rhubarb or senna. In conclusion, it may be said that a chromogen of hsematopor- phyrin is also usually present in urines containing the free pigments, which probably explains why such urines gradually become darker on standing. LiTERATUEE.^A Complete account of the literature on liEematoporphyrinuria up to 1893 is given by E. Zoja, "Su gualche pigmento di alcune urine," etc, Arch ital. di Clin, med., 1893, vol. xxxii. p. 63. A. E. Garrod, loc. cit. ; and Cen- tralbl. f. inn. Med., 1897, No. 21. Taylor and Sailer, Contributions from the William Pepper Laboratory, Philadelphia, 1900, p. 120. 0. Neubauer, Arch. f. exper. Path, u. Pharmakol., 1900, vol. xliii. p. 455. B. J. Stokvis, " Zur Pathogenese d. Htemato- porphyrinurie," Zeit. f. klin. Med., vol. xxviii. p. 1. Kast u. Weiss, Berlin, klin. Woch., 1896, vol. xxxiii. p. 621. Hammarsten, "Skandin. Arch. f. Physiol.," 1891, vol. iii. p. 31. Neumeister, Physiol. Chem., Jena, 1897. Nebelthau, Zeit. f. physiol. Chem., 1899, vol. xxvii. p. 324. B. Ogden, Boston Med. and Surg. Jour., 1898. Biliary Pigments. — Of the four biliary pigments, viz., bilirubin, biliverdin, bUiprasiu, and bilifuscin, the former alone is met with in freshly voided urines, while the others may form upon standing, being oxidation-products of bilirubin. The pigment is never found in normal urine, and its occurrence may be regarded as a positive symptom, of disease. In health it will be remembered that bilirubin, CigHigNjOg, formed in the liver from blood-pigment, is eliminated into the small intestine, in which it is transformed into hydrobilirubin and largely excreted as such in the feces, while a small portion is reabsorbed into the blood and eliminated in the urine as urochrome or normal urobilin. Whenever, then, the outflow of bile into the intestines becomes impeded bilirubin is absorbed by the lymphatics and elimi- nated in the urine. Among the numerous causes which give rise to eholuria under such conditions may be mentioned obstruction of the biliary ducts, and especially of the common duct, referable to simple swelling of its mucous membrane, as in the ordinary forms of catarrhal jaun- dice. It may also be due to the presence of a biliary calculus, to parasites, compression of the duct by tumors of the liver, the gall- bladder, the duct itself, and of neighboring structures, and particu- larly of the pancreas, stomach, and omentum. Whenever the blood-pressure in the liver is lowered, so that the tension in the smaller biliary ducts becomes greater than that in the veins, ehol- uria likewise results. , The icterus occurring under these conditions has been termed hepatogenic icterus, in contradistinction to the form observed in cases in which the liver has either totally or partially lost the power of forming bile, be this owing to the existence of degenerative processes affecting its glandular epithelium, as in cases of acute yellow atrophy, or to destruction of red corpuscles 470 THE URINE. going on so rapidly and so extensively that the organ is incapable of transforming into bilirubin all the blood-pigment which is carried to it. This occurs in pernicious ansemia, malarial intoxication, typhoid fever, poisoning with arsenious hydride, etc. Icterus neonatorum is probably to a certain extent also dependent upon the latter cause. To this form the term hoeinatogenio icterus has been applied. In such cases the occurrence of bilirubin in the urine can only be explained by assuming that a transformation of blood coloring-matter into bilirubin has taken place in the blood itself or in other tissues of the body. As a matter of fact, it appears to be generally accepted that such a transformation can actually occur outside of the liver, as the hsematoidin which may be found in old extravasations of blood seems to be identical with bilirubin. On the other hand, however, the existence of a hsematogenic icterus is positively denied, especially by Stadelmann. In accordance with his view, it may be demon- strated that in cases of pernicious ansemia, malaria, etc., the urine does not contain bilirubin, but usually urobilin. In cases of this kind which I had occasion to examine, bilirubin was never found. Further investigations are necessary to settle this question definitely. Usually the presence of biliary pigment may be recognized by direct inspection, as urines which contain this in notable amounts present a color varying from a bright yellow to a greenish brown. Any morphological elements which may occur in the sediment are stained a golden yellow, and the same color is imparted to the foam of the urine as well as to the filter-paper used in the filtration. At times, however, and particularly in cases in which the icterus is only beginning to appear, the presence of bilirubin is not infrequently overlooked, and urines containing urobilin in large amounts may be similarly mistaken for icteric urines. In doubtful cases, therefore, whether icterus exists or not, but in which the urine presents an intense yellow color, it is necessary to have recourse to chemical tests. A large number of these have been devised for the purpose of demonstrating the presence of bilirubin, all of which are fairly reliable. Only those will be described which I have examined myself and which are especially delicate. Smith's Test} — Five to 10 c.c. of urine are placed in a test-tube and treated with 2 or 3 c.c. of tincture of iodine (which has been diluted with alcohol in the proportion of 1 : 10) in such a manner that the iodine solution forms a layer above the urine. In the pres- ence of bilirubin a distinct emerald-green ring is seen at the zone of contact. This test can be highly recommended, as it is exceedingly simple and not surpassed in delicacy by any other. Huppert's Test.^ — Ten to 20 c.c. of urine are precipitated with milk of lime (a solution of barium chloride is, perhaps, still more > W. G. Smith, Dublin Med. .Tour., 1876, p. 449. ' Huppert, Arch. d. Heillt., 1867, vol. viii. pp. 351 and 476. URINARY PIGMENTS AND CHROMOGENS. 471 convenient), and the precipitate after filtering brought into a beaker by perforating the filter and washing its contents into the latter with a small amount of alcohol acidulated with sulphuric acid. The mixture is boiled, when in the presence of bilirubin the solution assumes a bright emerald-green color. Huppert's test is as delicate as is that of Smith, but is not so convenient for the needs of the practising physician. Ghnelin's Test (as modified by Rosenbaoh).^ — The urine is filtered through thick Swedish filter-paper, when the latter is removed and a drop of concentrated nitric acid, which has been allowed to stand exposed to the air for a short time, is placed upon its inner surface. In the presence of bilirubin a prismatic play of colors will be seen to occur around the nitric acid spot. Gmelin's Test.^ — The urine is treated with nitric acid, which is carried to the bottom of the test-tube by means of a pipette, so as to form a layer beneath the urine, when a color-play, as already described (page 417), will take place at the line of contact between the two fluids ; the green color is the most characteristic. In this connection a few words may also be said of the occurrence in the urine of biliary acids and cholesterin. Biliary Acids. — These may usually be found in the urine whenever bile-pigment is present, so that their clinical significance is essenti- ally the same as that attaching to bilirubin. Their demonstration is, however, attended with such difficulties that the methods devised for this purpose may well be omitted at this place (see also page 228). Cholesterin. — Cholesterin has never been found in icteric urines, and is only rarely seen in other pathological conditions. It has been observed in cases of chyluria, fatty degeneration of the kidneys, diabetes, in one case of epilepsy, and in two cases of pregnancy. V. Jaksch has noted the presence of cholesterin crystals in a urinary sediment in a case of tabes and cystitis. I have found cholesterin crystals in the sediment in a case of acute nephritis. The urine was of a dark amber-color, cloudy, of an acid reaction, and a specific gravity of 1.028. In the sediment numerous hyaline and epithelial casts and some red blood-corpuscles were found. Giiterbock described a urinary calculus obtained from the bladder of a woman which con- sisted almost entirely of cholesterin (see also Feces). Langgaard noted the presence of the substance in a case of chyluria.^ Pathological Urobilin. — This pigment should not be confounded with the urochrome or normal urobilin described above, to which it is closely related, but from which it may be distinguished by means of the spectroscope. Gautier states that pathological urobilin ^ Eosenbach, Centralbl. f. d. med. Wiss., 1876, vol. xiv. p. 5, ^ Tiedemann u. Gmelin, Die Verdanung nach Versucheii, Heidelberg, 1831, I. p. 79. ' T. Jaksch, Klinische Diagnostik, p. 339. Glinski, Maly's Jabresber., 1894, vol. xxiii. p. 484. Langgaavd, Virchow's Arcbiv, vol. Ixxvi. 472 THE URINE. may be obtained from urochrome by submitting the latter to the action of reducing agents ; and, as I have already pointed out, Riva and Chiodera obtained a substance from urobilin by the action of potassium permanganate, which closely resembles urochrome. It is said to be identical with the stereobilin found in the feces, but differs from Maly's hydrobilirubin in containing a much smaller percentage of nitrogen, viz., 4.11, as compared with 9.22 (Garrod and Hop- kins). While its occurrence in the urine is essentially a pathological phenomenon, it is at times also met with in normal urine, and appears to be derived from a special chromogen, urobilinogen, from which it may be set free by the addition of an acid. Both urobilin and its chromogen are precipitated by saturating the urine with ammonium sulphate, and both are soluble in chloroform. Accord- ing to Maly, urobilin is formed by the reduction of bilirubin in the intestine, and is then in part resorbed and eliminated in the urine. Hayem, on the other hand, proposed the hypothesis that the sub- stance originates in a diseased or disordered liver, as bilirubin does in the same organ in health, and accordingly he regards the appear- ance of much urobilin in the urine as evidence of hepatic insuf- ficiency. Others, again, maintain that urobilin is formed in the tissues at large either by the reduction of bilirubin or directly from the blood-pigment. The first view is notably held by Kunkel, Mya, Giarr6, and others, while the hsematogenous theory is notably rep- resented by Gerhardt. Garrod discusses these various hypotheses at some length in his most interesting lecture on the urinary pig- ments in their pathological aspects, in which he personally inclines to the intestinal theory, as now held by Miiller, Schmidt, Esser, and others. In a work of this scope it would lead too far to discuss the various investigations which lend themselves in support of this view, and I can here quote only the following from Garrod's paper : the chief seat of the formation of urobilin (for it is conve- nient to employ this term as including both pigment and chromogen) is undoubtedly the intestinal canal. This can only be gainsaid by denying the identity of the urinary and fecal pigments. The quantity normally present in the feces is far larger than that which enters the intestine with the bile (when a small amount is found), and there is strong evidence that the urobilin in bile is itself of intestinal origin. This being so, it is clear that theories other than the intestinal and its modifications merely attempt to trace a second source for the urobilin of the urine. It is equally clear that the substance from which the intestinal urobilin is formed is the bile- pigment. Under ordinary conditions the bile-pigment is destroyed in its passage along the intestine, and does not appear as such in the feces. In its place we find large quantities of urobilin, which in its turn disappears when occlusion of the common duct prevents the entrance of bile into the intestine. Again, when under certain URINARY PIGMENTS AND CHROMOGENS. 473 morbid conditions the bile-pigment passes along the intestine unal- tered, urobilin is absent from the feces. However, the conversion of bilirubin into urobilin is no mere process of reduction, but in- volves a much more radical change, with elimination of. nitrogen. That the change is brought about by bacterial action there is much evidence to show. When bile is inoculated with fecal material and kept in an incubator a formation of urobilin rapidly takes place, and at the same time the bile-pigment diminishes, and ultimately dis- appears. From its frequent occurrence in febrile urines pathological urobilin has also received the name febrile urobilin. It is, however, also observed in many other conditions, and especially in cases present- ing the so-called hsematogenic form of icterus, from which fact, indeed, and the usual absence of bilirubin at the same time, this form has been termed urobilin icterus. Urobilinuria has further been observed in certain hepatic diseases. In twelve cases of atrophic aud hypertrophic cirrhosis v. Jaksch was able to demonstrate the presence of urobilin in every instance, a point which may at times be of considerable diagnostic importance, providing that other causes which are known to lead to urobilinuria can be eliminated. I have observed urobilin in a few cases of he- patic cirrhosis, chronic malaria, and pernicious anaemia, in all of which the skin presented a light icteric hue, and in which bile-pigment was absent from the urine. Unfortunately, an examination of the blood was not made, and I have hence not been able to con- firm the statement of v. Jaksch that bilirubin occurs in the blood in almost every case in which urobilin is present in the urine. Urobilin has also been noted in cases of carcinoma, scurvy, Addi- son's disease, haemophilia, in cases of retro-uterine hsematocele, in extra-uterine pregnancy, following intracranial hemorrhages, etc. According to Bargellini, the degree of constipation in simple atony of the bowel is without influence upon the amount of urinary urobilin, but he states that in typhoid fever it causes an obvious increase ; whereas disinfection or emptying of the large bowel pro- duces a notable diminution in the amount. Urines rich in urobilin usually present a dark-yellow color which is strongly suggestive of the presence of bilirubin ; even the foam in such cases may be colored, making the resemblance between the two pigments still more complete, v. Jaksch points out, however, that urines containing indican in large amounts often likewise present a very dark-yellow color, a statement with which my own observations are in perfect accord. In every case a more detailed chemical examination should hence be made. Geehaedt's TEST.^If the urine contains much urobilin, which the color will indicate, 10—20 c.c. are extracted with chloroform by shaking, and the extract treated with a few drops of a dilute solu- 474 THE URINE. tion of iodo-potassic iodide. Upon the further addition of a dilute solution of sodium hydrate the chloroform extract is colored a yellow or yellowish-brown, and exhibits a beautiful green fluorescence ; this is even more intense than that noted in the case of normal urobilin. LlTERATUEB. — A. E. Garrod, loo. cit. A. E. Garrod and F. G. Hopkins, "On Urobilin," Jour, of Ptiysiol., 1898, vol. xxii. p. 451. Maly, Centralbl. f. d. med. Wiss., 1871, vol. ix. p. 849. Hayem, Gaz. hebdom., 1887, vol. xxiv. pp. 520 and 534 ; and Gaz. des Hop., 1889, vol. Ixii. p. 1314. Kunkel, Virchow's Arohiv, 1880, vol. Ixxir. p. 655. Mya, Arch. ital. di olin. med., 1891, vol. xxx. p. 101 ; and Lo Sperimentale, 1896, vol. 1. p. 71. GiarriS, Ibid., 1895, vol. xlix. p. 89, and 1896, vol. 1. p. 81. F. Miiller, Schlesische Gesellsch. f. vaterland. Kultur, January, 1892. A. Schmidt, Verhandl. d. XIII. Con- gress, f. inn. Med., 1895, p. 320. Esser, Untersuchungen fiber d. Entstehungsweise d. Hydrobilirubius, etc., Diss., Bonn., 1896. Bargellini, Lo Sperimentale, 1892, vol. xlvi. p. 119. V. Jaksch, Zeit. f. Heilk., 1895, vol. xvi. p. 48. D. Gerhardt, Zeit. f. klin. Med., 1897, vol. xxxii. p. 313. Specteoscopic Examination. — This is necessary when Ger- hardt's test yields a doubtful result. The urine is then best examined as follows : 50 c.c. of urine are extracted in a separation funnel with amyl alcohol, which takes up both the pigment and its ehromogen. After standing for several hours the urine is allowed to flow away, by opening the stopcock, when the alcoholic extract is decanted from above, and is treated with a concentrated alcoholic and ammoniacal solution of zinc chloride. In the presence of urobilin the liquid shows a beautiful fluorescence, and on spectroscopic examination a single band of absorption is seen between b and F. In acid solu- tions, on the other hand, a single band is likewise obtained between b and F, but this extends to the right beyond F, and is much darker. Should the urine contain much urobilin, its special extraction is not necessary. In such an event the acid urine shows the acid spectrum, while the alkaline band is obtained after the addition of ammonia (see also Bang's Test). Melanin and Melanogen. — In cases of melanotic disease it has been repeatedly observed that the urine, which usually and probably always presents a normal yellow color when voided, gradually becomes darker upon exposure to the air, and finally turns black. This phenomenon indicates without a doubt that such urines contain a ehromogen, melanogen, which, upon oxidation, yields the black pigment noted in these cases, viz., melanin. As yet, it has not been possible to isolate this substance in pure form, and it is, indeed, not definitely determined that the black color in such urines is refera- ble to a single pigment. Such urines generally contain melanin and its ehromogen in solution ; deposits of melanin granules by them- selves are only occasionally seen, and are not characteristic, as they may also be found iu cases of chronic malarial intoxication, etc., when they may, indeed, be met with in the blood, constituting the condition spoken of as melancemia. While the occurrence of melanin in the urine is probably indica- tive in most cases of the existence of melanotic tumors, it should URINARY PIGMENTS AND CHROMOOENS. 475 be stated that this symptom cannot be regarded as pathognomonic, as it may be absent in the case of melanotic tumors, and present in wasting diseases and inflammatory affections, and may at times, though very rarely, even be associated with the presence of non-pig- mented growths. Nevertheless, its occurrence should always be regarded with suspicion, and, taken in conjunction with other symp- toms, will often lead to a correct diagnosis. Urines which darken upon standing should be subjected to the following tests : 1 . A few cubic centimeters of urine are treated with bromine- water, when in the presence of melanin or melanogen a precipitate is obtained, which is yellow at first, but gradually turns black. 2. The addition to melanotic urine of a few drops of a strong solution of ferric chloride will cause the appearance of a gray color, which is imparted to the precipitate of phosphates occurring at the time. LiTEKATUEE.— T. H. Eisclt, "Die Diagnose d. Pigmentkrebses durch d. Harn," Prag. Vierteljahrschr. f. praktische Heilk., 1858, iii. p. 190, and 1862, vol. iv. p. 26. Senator, "Ueber schwarzen Urin," Charite Anna)., 1891. Hoppe-Seyler, Zeit. f. physiol. Chem., 1891, vol. xv. p. 179. F. Grohe, " Zur Gesch. d. Melauaemie," Vir- chow's Archiv, 1861, vol. xx. p. 306. Phenol. Urines. — The development of a dark-brown or black color upon standing is not always due to the presence of melanin, as a similar appearance may be noted in cases of poisoning with carbolic acid, following the ingestion of salol, hydrochinon, pyrocatechin, salicylic acid, etc., in large amounts. The color in such cases is due in all probability to the presence of various oxidation-products of hydrochinon, and in the last instance possibly to the so-called humin-substances. The test referred to above will prevent confusion as to the origin of the color as far as melanin is concerned, and with the his- tory of the case given, moreover, further chemical examination is generally unnecessary. In suspected cases of carbolic acid poison- ing, however, the mineral as well as the conjugate sulphates should be quantitatively determined, when the factor — (see Sulphates) will be found greatly diminished. If at the same time other fac- tors, which might cause a greatly increased elimination of conjugate sulphates, can be excluded, the diagnosis of poisoning with carbolic acid or one of its derivatives may be inferred. Salol and salicylic acid may be recognized from the fact that such urines when treated with a solution of ferric chloride develop a marked violet color which does not disappear on standing. The reaction thus differs from that obtained with diacetic acid (see also page 489). Alkapton. — Urines are at times, though very rarely, seen which, like the phenol urines, turn dark on standing, but in which the 476 THE URINE. change in color is neither referable to the presence of phenol or its derivatives, nor to melanin. Such urines are of a normal color when passed, but gradually turn reddish brown upon exposure to the air. Treated with a small amount of alkali, this change occurs almost immediately. Fehling's solution is reduced on the applica- tion of heat, while bismuth is not affected. Ammoniacal silver solution is reduced in the cold, and a temporary bluish-green color develops when the urine is treated with a feri-ic salt. The fermenta- tion test is negative, and examination with the polarimeter shows that the substance in question is not glucose. With phenylhydrazin no osazon is formed. Bodeker, who first observed a urine of this kind, termed the sub- stance giving rise to the reactions just described alkapton, and sub- sequently expressed the belief that his alkapton might possibly have been pyrocateohin. Subsequent investigators succeeded in isolating substances from such urines which have been variously termed pyro- catechuic acid, urrhodinic acid, glucosuric acid, uroleucinic acid, and uroxanthinic acid. Baumann and Wolkow later were able to iso- late homoffentisinio add in pure form from the urine of such a case, and expressed the belief that some of the substances obtained by previous observers were in reality the same. Since that time this acid has also been found by Ogden, Stange, Stier, and others. There is reason to believe, however, that the reaction is not always due to one and the same substance. Of the origin of alkapton very little is known. Baumann ex- pressed the opinion that homogentisinic acid might be derived from tyrosin, and that the condition is referable to the activity of special micro-organisms in the upper portion of the intestines. Others oppose this view and regard alkaptonuria as evidence of a definite metabolic anomaly taking place in the tissues of the body. Tyrosin, moreover, belongs to the para-series, while homogentisinic acid is an ortho-compound, and has never been encountered in the feces. Culture-experiments from ordinary stools and from those passed after the administration of castor oil have yielded negative results, no alkapton acid being formed by culture in either broth, meat-juice, or tyrosin-broth (Garrod). Embden also observed that when an alkaptonuric individual took homogentisinic acid by the mouth a far larger proportion appeared in the urine than when the same substance was administered to a healthy individual. However this may be, alkaptonuria can scarcely be regarded as a pathological phe- nomenon, although it may occur in disease. It has thus been ob- served in connection with glucosuria, acute gastro-intestinal catarrh, phthisis, acute miliary tuberculosis, in one case of brain tumor, carcinoma of the prostate, etc. More frequently the condition is accidentally discovered by life insurance physicians in apparently healthy individuals, and has repeatedly been confounded with glu- VBINARY PIGMENTS AND CHROMOGENS. 477 cosuria. Like cystinuria and diaminuria, it may occur in families ; it may appear in childhood, and persist through years, and perhaps a lifetime. The amount of homogentisinic acid eliminated in the twenty-four hours is variable, hut is usually large. Baumann thus found an average elimination of 4.6 grammes, which in one case could be increased to 14 grammes by the administration of tyrosin. Larger quantities are also obtained after a liberal diet of meats. Phenyl- acetic acid, phenyl-amido-acetic acid, and benzoic acid, on the other hand, do not cause an increased excretion of homogentisinic acid. To isolate homogentisinic acid from alkapton urines, and to deter- mine its amount, Baumann's method may be employed. The col- lected urine of twenty-four hours is acidified with 250 c.c. of a 12 per cent, solution of sulphuric acid and extracted three times with an equal volume of ether. The ethereal extract is evaporated to a syrup. The crystals which separate out on standing are dis- solved in 250 C.C. of water. This solution is brought near the boil- ing-point, and is then treated with 30 c.c. of a neutral lead acetate solution (1:5) and rapidly filtered. In the filtrate the lead salt crystallizes out in transparent needles and prisms. This is then decomposed with hydrogen sulphide and the filtrate carefully evap- orated on a water-bath until the fluid begins to darken, when it is further concentrated in the vacuum to the point of crystallization. The resulting prismatic crystals are almost colorless and transparent. They melt at a temperature of 146.5°-147° C, and are readily soluble in water, alcohol, and ether, and are almost insoluble in chloroform, benzol, and toluol. A solution of the acid, which may thus be isolated in pure form, presents the same characteristics as the urine from which it was obtained. The following method, suggested by Garrod, may also be employed, and has the advantage of greater simplicity. Gareod's Method. — The urine itself is heated nearly to boiling without any preliminary treatment, and for each 100 c.c. of urine at least 5 or 6 grammes of solid neutral lead acetate are added. As soon as the acetate is dissolved, the bulky gray precipitate which forms is removed by filtration, and the filtrate, which has a pale-yellow color, is allowed to stand for twenty-four hours in a cool place. If the urine be very rich in homogentisinic acid, or if the flask containing it be placed upon ice, minute acicular crystals, which are almost colorless, quickly form ; but as a rule crystallization does not commence until several hours have elapsed. The crystals are then much larger, are grouped in stars or rosettes, and are more deeply colored. In summer weather it would probably be desirable to start the crystallization by artificial cooling ; but although the process is greatly 478 THE URINE. accelerated at a low temperature, the total yield is not materially increased. If formation of the crystals be long delayed, the liquid may be warmed again and more lead acetate added. After the lapse of twenty-four hours crystals cease to form, even when the liquid is placed upon ice. The crystalline product so obtained is lead homogentisinate. When the crystals are dissolved in hot water the solution assumes a deep- brown color with alkalies; it reduces Fehling's solution readily with the aid of heat, and yields a transitory deep-blue color with a dilute solution of ferric chloride. From the lead salt free homo- gentisinic acid may be obtained by decomposing it with hydrogen sulphide. LiTEKATUEE. — Bodeker, Annal. d. Chemie ii. Pharmakol., 1861, vol. oxvii. p. 98. Baumann u. Wolkow, Zeit. f. physiol. Chem., 1891, vol. xv. p. 228. Stier, Berlin, klin. Woch., 1898, vol. XXXV. p. 185. Embden, Zeit. f. physiol. Chem., 1893, vol. xvii. p. 182, and vol. xviii. p. 304. Ogden, Zeit. f. physiol. Chem., 1895, vol. xx. p. 280. Futcher, N. Y. Med. Jour., 1898, vol. Ixvii. p. 69. Garrod, Jour. Physiol., 1899, vol. xxiii. p. 512; and Med.-Chir, Trans. Eoyal Med. and Chir. Soc, vol. Ixxxil. p. 367. Blue Urines. — Blue urines are sometimes seen, the color of which is due to indigo formed from urinary iudican, in all probability within the urinary passages. Their occurrence can only be regarded as a medical curiosity. Formerly, when indigo was employed in the treatment of epilepsy, blue urines were frequently seen. At the present time, when methylene-blue is occasionally used in the treat- ment 'of malaria and chyluria, this pigment is found in the urine. Green Urines. — ^Green urines have also been described ; the cause of the color, however, has not been definitely ascertained. Pigments referable to Drugs. — Certain drugs may also cause changes in the normal color of urine, and in doubtful cases inquiry in this direction should be made. It has been pointed out that car- bolic acid, hydrochinon, pyrocatechin, and salol cause the appearance of a dark-brown color, and that after the administration of indigo and methylene-blue blue urines are voided. Santonin, rheum, and senna color urines a bright yellow, so that they may resemble icteric urines in appearance. The yellow color in such cases is changed to an intense red by the addition of an alkali, and, if ammoniacal fer- mentation is going on at the same time in the bladder, the patient may believe himself to be suffering from hsematuria. The red color thus produced is due to the action of the alkali upon chryso- phanic acid. When urines containing copaiba are treated with hydrochloric acid a red color results, which changes to violet upon the application of heat. During the administration of potassium iodide, or the use of iodine in any form, a dark mahogany color is obtained when the urine is treated with nitric acid. In doubtful cases Stokvis' modification of Jaffa's test for indican should be em- PLATE XVII. I H Tf- y^J Ehrlich's Diazo-Reaetion, as niodified by the aLithor. The orange eolor in the lower portion of the test tube may be obtained in any urine; the dark earnnine ring indicates the presence of the reaction in a well-pronouneed degree; the colorless zone above is intended to indicate the am- monia that has l^een added. j^ URINARY PIGMENTS AND CHROMOOENS. 479 ployed, when in the presence of an iodide the chloroform assumes a beautiful rose-red color. For the detection of other drugs and poisons in the urine the reader is referred to special works. Ehrlich's Reaction. — Under certain pathological conditions, and especially in typhoid fever, a chromogen may be present in the urine, which, when treated with diazo-benzene-sulphonic acid and ammonia imparts a distinct red color to the urine, varying from eosin to a deep garnet-red (Plate XVII.). This reaction, which is generally spoken of as Erhlich's reaction, or the diazo-reaction, was at one time regarded as pathognomonic of typhoid fever. Subsequent examinations, however, have shown that it may also be present in other diseases. Michaelis, who has made an exhaustive study of this question, divides into four groups the diseases in which the reac- tion has been observed. In the first group, comprising diseases of the nervous system, chronic diseases of the heart and kidneys, malignant tumors, etc., the reaction is rarely seen. When present, it usually indicates a secondary infection. The second group in- cludes those diseases in which the reaction is almost always present, namely, typhoid fever and measles. In the diseases of the third group it is often, though not invariably, observed. Under this heading are classed scarlet fever, erysipelas, pneumonia, diphtheria, pysemia, acute miliary tuberculosis, etc. The fourth group comprises pulmonary tuberculosis, and includes acute caseous pneumonia. The value of Ehrlich's reaction in typhoid fever was at first overesti- mated, but is at present certainly underestimated. I have personally studied this problem with great care, and after many years' experience maintain, as I did years ago, that the test is a most important diagnostic aid in the disease in question. As a general rule the reaction is present as early as the fifth or sixth day, and may persist into the third week ; it then disappears, but may reappear when a relapse occurs. Excepting in children, its absence from the fifth to the ninth day usually indicates a mild case. This rule, however, is not without exception, and I have seen a case of typhoid fever in which notwithstanding exceedingly high temperatures (106.5° at 6 A. M.) the reaction was not obtained until the beginning of the third week, and then persisted for only a few days. When the reaction is continuously present after the third week I am inclined to suspect acute tuberculosis. Of late much attention has been paid to the occurrence of Ehrlich's reaction in pulmonary phthisis. As a result of his investigations Michaelis concludes that its presence in such cases indicates either that the process is very extensive or that it will progress very rap- idly, and that the prognosis is grave. A cure, he thinks, is impos- sible, and improvement, if any, only temporary. His conclusions in the main coincide with the results obtained by others, but it must 480 THE URINE. be admitted that exceptions occur. Personally I regard the outlook as very bad in those cases in which the reaction is almost constantly present, even if the physical signs are but little pronounced. Of the nature of the body which gives rise to Ehrlich's reaction nothing is known, v. Jaksch regards the test as an uncertain indi- cation of the presence of acetone, but that this is not the case can be easily shown. As the preparation of chemically pure, crystalline diazo-com- pounds is a difficult process, Ehrlich uses sulphanilic acid, which, when treated with nitrous acid in a nascent state, gives rise to the formation of diazo-benzene-sulphonic acid, as is shown by the equations : (1) NaNOj + HCl = NaCl +HNOj. (2) CeH/ +HN02 = C5H/ ^N + 2H A \sO3H ^SOj/ Para-amido- Diazo-benzene- benzene-sulphonic acid. Bulpbonic acid. This is the active principle in the mixture employed. Other compounds may, of course, also be used, such as meta-amido- benzene-sulphonic acid, ortho- and para-toluidin-sulphonic acid, etc. ; but of all these, Ehrlich found the common sulphanilic acid the most convenient. Two solutions, which must be kept in separate bottles, are employed. The one is a 5 per cent, solution of hydrochloric acid, to which sulphanilic acid is added in the proportion of 1 gramme for each 100 c.c. The other is a 0.5 per cent, solution of sodium nitrite. The two solutions are mixed immediately before using in the pro- portion of 40 to 1. A few cubic centimeters of urine are then treated with an equal volume of the reagent, the mixture is shaken and rendered alkaline with ammonium hydrate. This is best allowed to flow down the sides of the tube, so as to form a layer above the mixture. At the junction of the two fluids a colored ring will now be observed. With urines which do not contain the chromogen this will be a more or less distinct orange, while in its presence a red color is obtained. The intensity of this color may vary from eosin to a deep garnet-red. If the mixture is now agi- tated and the reaction is positive, the foam will likewise be colored red, and upon pouring the solution into a porcelain basin containing much water a beautiful salmon color is obtained, even if only traces of the chromogen. are present. Carried out in this manner no question will arise as to the presence or absence of the reaction. Ehrlich states that on standing a green sediment forms in the alkalinized mixture, and he regards this sediment as especially char- acteristic. My experience has been that this becomes manifest only when the color-reaction is well pronounced, and I am inclined to attach URINARY FIGMENTS AND OHROMOOENS. 481 Cases Tested. Typhoid fever . . Malarial fever Tetanus .... Acute miliary tuberculosis . Joint tuberculosis Pulmonary tuberculosis . . . Septicsemia Ulcerative endocarditis . . . Secondary syphilis Erysipelas Scarlatina Measles Carcinoma Pneumonia . Rheumatism, chronic . . . . Eheumatism, acute Diphtheria . . . . . Diarrhoea . . . . . . Appendicitis Albuminuria of pregnancy . . Chronic nephritis Cystitis Urethritis, specific . . . . Oxaluria and lithsemia . . . Pleurisy « Pysemic abscess of lung .... Tuberculosis of prostate . . . . Necrosis of long bones . . . . Rotheln Syphilis (third stage) Alcoholic neuritis Hysteria Epilepsy Leg ulcer, varicose Fractures, long bones Fracture, skull Burns, severe Gunshot wounds, aseptic . . . Morphin poisoning . . . . Sciatica .... . . . . Cirrhosis, hepatic Simple enteritis . . . . . Angioneurotic oedema ... Endometritis Pericarditis Meningitis ... Vulvitis and vaginitis, specific . Orchitis, gonorrhoeal . . . . Valvular heart-disease . . . Quinsy and tonsillitis . . . . Normal urines Varicella Typhoid relapse .... Gastric \ilcer . Acute bronchitis Chronic constipation Total Number. 64 4 2 3 4 16 4 1 4 2 3 2 4 11 10 5 3 4 3 6 19 2 7 11 5 1 3 2 1 5 3 6 2 7 5 2 2 2 1 3 2 3 2 3 ■ 1 1 2 1 7 3 30 1 3 2 3 7 Reaction. Present. 315 611 2 3 2 1 3 1 95 per cent. 31 482 THE URINE. more importance to the salmon color obtained upon copious dilution. With normal urines this is never obtained, and it can still be seen when inspection of the fluid in the test-tube would leave in doubt. The older method of Ehrlich I have abandoned, as the test just described is simpler, and, in my experience, just as reliable. He advised the addition of about 50 c.c. of absolute alcohol to 10 c.c. of urine, subsequent filtration, and examination of the filtrate, as just described. Greene states that if 1 part of the sodium nitrite solution is added to 100 instead of 40 parts of the sulphanilic acid solution, a positive reaction is no longer obtained in cases of croupous pneu- monia and of pulmonary tuberculosis, while in typhoid fever the reaction occurs with the same intensity. It is thus possible that the test may be still further modified, and become even more valuable. On page 481 are given some of the results which Greene obtained with this method. While in the absence of the chromogen, as I have already stated, a more or less pronounced orange color is usually obtained, excep- tions have been noted. Ehrlich thus records that in urines contain- ing biliary coloring-matter an intensely dark, cloudy discoloration occurs at times, which upon boiling is changed to a well-marked reddish violet. In rare instances of ulcerative endocarditis, hepatic abscess, and intermittent fever, Ehrlich further observed an intense yolk-yellow color, which was even imparted to the foam. Of interest is the observation of Burghart, that after the adminis- tration of tannic acid, gallic acid, and certain iodine preparations, Ehrlich's reaction disappears from the urine. But, as Burghart himself suggests, it is likely that this inhibitory eifect is not exerted upon the diazo-forming substance, but upon the reagent employed. LiTBKATUEE. — Ehrlich, Zeit. f. klin. Med., 1882, vol. v. p. 285; Charit6 Annal., 1883, vol. viii. p. 28, and 1886, vol. xi. p. 139. Goldsclimidt, Miinch. med. Woch., 1886, vol. xxxiii. p. 35. Eutimeyer, Corresp. Blatt. f. Schweizer Aerzte, 1890, vol. xxvi. Greene, Med. Eecord, Nov. 14, 1896. C. E. Simon, Jolins Hopkins' Hosp. Bull., 1890. J. Friedenwaia, N. Y. Med. Jour., 1893. M. Michaelis, Berlin, klin. Woch., 1900, p. 274; and Deutsoh. med. Wooh., 1899, p. 156. J. E. Arneill, Am. Jour. Med. Sci., 1900, p. 296. CONJUGATE SULPHATES. In addition to indoxyl (see Indican), skatoxyl, phenol, paracresol, and pyrocatechin occur in the urine in combination with sulphuric acid. Skatoxyl. — Skatoxyl results from the skatol formed during the process of intestinal putrefaction, as indoxyl is derived from indol, and is partly eliminated in the urine as skatoxyl sulphate. Clini- cally it is of little interest, as the amount excreted is very small, and it is not necessary to enter into a further consideration of its chemi- cal properties or mode of detection at this place (see Feces). CONJUGATE SULPHATES. 483 Phenol. — Phenol, according to Brieger, occurs only in very small amounts in human urine, the usual phenol reactions being largely referable to paracresol. Normally, about 0.03 gramme is eliminated in the twenty-four hours, but in pathological conditions much larger quantities may be found. Remembering the origin of phenol, it is clear that an increased elimination may be observed whenever putre- factive processes are going on in the tissues and cavities of the body, or whenever there is an increase in the degree of intestinal putre- faction, though in the latter condition the indican is usually the only conjugate sulphate that is found increased. In peritonitis, diph- theria, erysipelas, scarlatina, empyema, pulmonary gangrene, putrid bronchitis, etc., an increased elimination of phenol is commonly seen. Important from a diagnostic standpoint, further, is the fact that in uncomplicated cases of typhoid fever no increase is observed, while this is common in tubercular meningitis.' The largest amounts, of course, are seen in cases of poisoning with carbolic acid or one of its derivatives. As the quantitative estimation of phenol is too complicated for the purposes of the general practitioner, Salkowski's qualitative test is here also described. From the intensity of the reaction certain con- clusions may be drawn as to' the amount present. It is especially serviceable in cases of suspected poisoning with carbolic acid. Salkowski's Test. — About 10 c.c. of urine are boiled in a test- tube with a few cubic centimeters of nitric acid, and, on cooling, treated with bromine-water. The development of a pronounced turbidity or the occurrence of a precipitate indicates the presence of an increased amount of phenol. Quantitative Estimation. — Principle. — When potassium-phenyl sulphate is treated with hydrochloric acid, phenyl sulphate results, which further takes up one molecule of water, giving rise to the formation of sulphuric acid and phenol, according to the following equations : (1) so/ +HC1= KCl +S0/ /O.CeH, /OH (2) S0,< -KH,0 = SO/ -FCsHj.OH. ^OH ^OH From the action of bromine-water upon phenol a yellowish-white crystalline precipitate of tribromophenol results : C6H5.OH + 6Br = 3HBr + CeH^Brj.OH. ' A. Strasser, " Ueber d. Phenolausscheidung bei Krankheiten," Zeit. f, klin. Med., vol. xxiv. p. 543. Brieger, Zeit. f. klin. Med., 1881, vol. iii. p. 468. Kast u. Boas, Miinch. med. Wooh., 1888, vol. xxxv. p. 55. 484 THE URINE. As 331 (molecular weight) parts by weight of tribromophenol correspond to 94 (molecular weight) parts by weight of phenol, the amount of the latter contained in a certain volume of urine is readily determined according to the equation 331 : 94 : : a: « ; and y =?^ = 0.28398 j, "' 331 » in which x indicates the weight of the tribromophenol found in the amount of urine employed, and y the corresponding quantity of phenol. Method. — From 500 to 1000 c.c. of urine are treated with one-fifth of an equivalent amount of dilute hydrochloric acid (1 : 4), and dis- tilled so long as a specimen of the distillate is rendered cloudy upon the addition of bromine-water (1 : 30), the specimens used fo this purpose being carefully preserved. The total quantity of the filtered dis- tillate, together with the specimens which have been set aside, is now treated with bromine-water, shaking the mixture after each addition of the reagent until a permanent yellow color results. Beyond this point further addition is beset with danger, as compounds will be formed which contain more bromine, the presence of which would indicate a smaller amount of phenol than that actually contained in the urine. After two or three days the precipitate is collected on a filter which has been dried over sulphuric acid, washed with water containing a trace of bromine, and then dried over sulphuric acid and weighed. Pyrocatechin. — Urines containing pyrocatechin, like those con- taining hydrochinon (see above), darken upon standing, though presenting a normal color when voided. ACETONE. The amount of acetone which may be found in the urine under normal conditions varies between 0.008 and 0.027 gramme, and is greatly influenced by the character of the diet. Whenever the car- bohydrates are withdrawn the quantity rapidly increases, and reaches its maximum about the seventh or eighth day. At this time from 200 to 700 mgrms. may be eliminated in the twenty-four hours. If, then, carbohydrates are again added to the diet, the acetonuria soon disappears. This result is not reached, however, if fats are sub- stituted for the carbohydrates. The acetonuria is greatest when but little albuminous food and no carbohydrates at all are ingested, and during starvation the same amounts are essentially found. There can hence be no doubt that acetone is derived from proteid material. Increased amounts are accordingly found whenever, as in fevers, the various cachexias, in conditions associated with inanition, etc., large ACETONE. 485 quantities of circulating albumin are broken down, or whenever car- bohydrates are not furnished in sufficient amount.' Most important is the diabetic form of acetonuria, and it may be stated, as a general rule, that the diagnosis of diabetes mellitus is justifiable whenever sugar and notable quantities of acetone are found in the urine. The amount of acetone, moreover, stands in a direct relation to the intensity of the disease, the maximum excretion being usually observed toward the fatal end.^ Whether or not this form of acetonuria can always be explained upon the basis given above remains an open question. There can be no doubt, however, that the threatening symptoms which are so commonly associated with a greatly increased elimination of acetone will often disappear when carbohydrates are administered in large amounts. It is certain, moreover, that diabetic coma is more apt to occur when the old- fashioned plan of excluding carbohydrates entirely from the dietary of diabetic patients is adopted. Hirschfeld ^ suggests that in every case of diabetes the excretion of acetone be carefully followed, and that large amounts of carbohydrates be administered whenever the acetonuria approaches a dangerous extent. This agrees with my experience. Of the febrile diseases in which acetonuria has been observed may be mentioned typhoid fever, pneumonia, scarlatina, measles, acute miliary tuberculosis, acute articular rheumatism, and septi- caemia. In those of short duration, on the other hand, even if the fever is high, as in acute tonsillitis, intermittent fever, the hectic fever of phthisis, etc., an increased elimination of acetone is rarely observed. In the continued fevers the acetonuria is largely referable to the character of the diet, as carbohydrates are usually excluded entirely, and I have repeatedly observed that a return to the normal occurred as soon as sugar was administered in amounts varying from 50 to 100 grammes. In certain nervous and mental diseases, as in general paresis, mel- ancholia, following epileptic seizures, and in tabes, acetonuria is fre- quently observed. During the second stage of general paresis in- creased amounts are indeed quite constantly found, but Hirschfeld is probably correct in stating that the psychotic form of acetonuria is largely referable to improper feeding. In the primary diseases of the stomach, and notably in carcinoma, acetonuria is frequently observed, and it is possible that the acetone in these cases is to some extent at least formed in that organ directly from the proteids ingested. The fact that in carcinoma acetone may ' V. Jaksch, Ueber AcetoDurie u. Diaceturie, Hirschwald, Berlin, 1885. Eosenfeld, Centralbl. f. inn. Med., 1895, vol. xv. Waldvogel, " Zur Lehre von der Acetonuria," Zeit, f. klin. Med., vol. xxxviii. p. 506. '^ V. Jaksch, Zeit. f. klin. Med., 1885, vol. x. p. 362. Lorenz, Ibid., 1891, vol. xix. p. 19. ' F. Hirschfeld, " Beobachtungen liber d. Acetonurie u. das Coma diabeticum," Zeit. f. kiln. Med., vol. xxviii. p. 176, and vol. xxxi. p. 212. 486 THE URINE. be observed at a time when marked loss of flesh has not as yet occurred, and tluit larger amounts of acetone may be found in the stomach than in the urine, is certainly in favor of this view/ The acetonuria following chloroform narcosis is probably refer- able to an increased destruction of organized albumin. Finally, the possibility of the occurrence of an enterogenic form of acetonuria must be borne in mind. The cases of so-called asthma acetonicum probably belong to this class. Tests for Acetone. — Legal's Test.^ — This test may be applied to the freshly voided urine, but is not conclusive. Several cubic centimeters of urine are treated with a few drojis of a strong solution of sodium nitroprusside and sodium hydrate ; the mixture assumes a red color, which rapidly disappears, and in the presence of acetone is replaced by a purple or violet i*ed when acetic acid is added. As a rule, it is safer to distil the urine (500—1000 c.c.) after the addi- tion of a little phosphoric acid (1 gramme pro liter), and to employ the first 10-30 c.c. of the distillate for the Ibllowing two tests. Lieben's Test.'^ — A few cubic centimeters of the distillate are treated with several drops of a dilute solution of iodo-potassic iodide and sodium hydrate, when in the presence even of traces of acetone a precipitation of iodoform in crystalline form occurs, A^aich may be readily recognized by its odor when the solution is heated. Reynolds' Test.* — A few cubic centimeters of the distillate are treated with a small amount of freshly precipitated yellow mercuric oxide. This is ])repared by precipitating a solution of mercuric chloride with an alcoholic solution of sodium hydrate. If acetone is present, a black color, due to the formation of mercuric sulphide, will result in the clear filtrate upon the addition of a few drops of ammonium sulphide. Dennigfes' Test (as modified by Oppenheimer).* — The reagent is prepared as follows : 20 grammes of concentrated sulphuric acid are poured into 100 c.c. of distilled water, when 5 grammes of freshly prepared yellow mercuric oxide (see Reynolds' test) are added. The mixture is allowed to stand for twenty-four hours and is then ready for use. This reagent is added to about .3 c.c. of urine, drop by drop, until the precipitate which is thus formed no longer disappears on stirring. When this ]ioint is reached a few more drops arc added. After two to three minutes the precipitate is filtered off. The clear filtrate is further treated with about 2 c.c. of tlie reagent, and 3-4 c.c. of a 30 per cent, solution of sulphuric acid, and boiled for a minute or two, or, still better, placed in a vessel with boiling water. ' H. Lorenz, Inc. cit. '' Le Nobel, Arch. f. exper. Path. u. Pharmakol., 1884, vol. xviii, p. H. ■' Taniguti u. Salkowski, Zeit. f. physiol. Clieni., 1890, vol. xlv. p. 476. * Gunning, .Jour, de Pharmacol, et fleChim., 1881, vol. iv. p. 30. s Oppenheimcr, Berlin, klin. Woch., 1899, p. 828. ACETONE. 487 In the presence of an abundant amount of acetone a copious white precipitate forms immediately ; while in the presence of traces only (less than 1 : 50000), a slight cloud develops on standing for several minutes. The precipitate is almost entirely soluble in an excess of hydrochloric acid. If albumin is present, the urine becomes turbid at once when the reagent is added. In that case the test is continued as described, attention being directed to the coarser precipitate which occurs later. To such urines large amounts of the reagent must be added, the idea being to precipitate everything that can be precipitated with the reagent, before heating. It will be observed that Dennigfes' test is much simpler than the tests already described, and Oppenheimer claims that it is as delicate as that of Lieben, viz., giving a well-pronounced reaction with a dilution of 1 : 20000, and being still discernible with a dilution of 1 : 60000. As diacetic acid yields acetone when treated with mineral acids, a positive result is always obtained when this is pres- ent. But as diacetic acid is usually found only in association with acetone, this fact does not lessen the value of the test, and is an error, moreover, which is common to the other tests as well. Quantitative Estimation of Acetone. — For the purpose of estimating the amount of acetone the method of Messinger, as modified by Huppert, is now employed, and is greatly to be preferred to the older procedure of v. Jaksch.' Prindple. — The method is based upon the observation of Lieben that acetone gives rise to the formation of iodoform when treated with iodine in an alltaline solution. If, then, a solution of acetone is treated with a known amount of iodine, it is a simple matter to determine the quantity present by retitrating the iodine which was not used in the formation of iodoform. Solutions required : 1. Acetic acid (50 per cent, solution). 2. Sulphuric acid (12 per cent, solution). 3. Sodium hydrate solution (50 per cent.). 4. A decinormal solution of iodine. 5. A decinormal solution of sodium thiosulphate. 6. Starch solution (see page 189). Preparation of the solutions : 1. The decinormal solution of iodine is prepared as described elsewhere (see page 188). 2. As the molecular weight of sodium thiosulphate — Na^Sp, + 5Hp— is 248, a decinormal solution of the salt would contain 24.8 grammes to the liter. This quantity is dissolved in about 950 c.c. of distilled water, and brought to the proper strength by titration 1 See Nenbaueru. Vogel, Analyse des Hanis, 9th ed., p. 470. 488 THE URINE. with a decinormal solution of iodine. As 1 c.c. of the thiosulphate solution should correspond to 1 c.c. of the iodine solution, the neces- sary amount of water which must be added to the former is then determined. Method. — One hundred c.c. of urine, or less if much acetone is present, as determined by Legal's test, are treated with 2 c.c. of the acetic acid solution and distilled until seven-eighths of the total amount have passed over. The distillate is received in a retort which is connected with a bulb-tube containing water. As soon as seven-eighths of the urine have distilled over, a small amount of the distillate of the remainder is tested for acetone according to Lieben's method. Should a positive reaction be obtained, it will be necessary either to repeat the entire process with less urine, diluted to about 200 c.c, or to add about 100 c.c. of water to the residue and to distil until all the acetone has passed over. The distillate is then treated with 1 c.c. of the sulphuric acid and redis- tilled. The addition of the acetic acid and of the sulphuric acid, respectively, serves the purpose of holding back phenol and am- monia. Should the first distillate contain nitrous acid, moreover, which is recognized on the addition of a little starch paste contain- ing a trace of potassium iodide, when the solution turns blue, the acid is removed by adding a little urea. The second distillate is re- ceived in a bottle provided with a well-ground glass stopper, and holding about 1 liter. The distillate is then treated with a carefully measured quantity of the one-tenth normal solution of iodine, — about 10 c.c. for 100 c.c. of urine, — and sodium hydrate solution until the iodoform separates out. To this end, a slight excess of the solution must be added. Should ammonia be present, a blackish cloud will be observed at the zone of contact of the sodium hydrate and the iodine solution, and it will be necessary to repeat the entire process. The bottle is closed and shaken for about one minute. The solution is then acidified with concentrated hydrochloric acid, when the mixture assumes a brown color if iodine is present in excess. If this does not occur, more of the iodine solution must be added, and the process repeated until an excess is present. The excess is then retitrated with the thiosulphate solution until the fluid presents a faint-yellow color. A few cubic centimeters of starch solution are now added, and the titration continued until the last trace of blue has disappeared. The number of cubic centimeters employed in thfe titration is finally deducted from the total amount of the iodine solution added, and the result multiplied by 0.976. The figure thus obtained indicates the amount of acetone contained in the 100 c.c. of urine, in mgrms., as 1 c.c. of the thiosulphate solution is equivalent to 1 c.c. of the iodine solution, or to 0.967 mgrm. of acetone. DIACETIC ACID. 489 DIACETIO ACID. The occurrence of diacetic acid in urine must always be regarded as abnormal. Its pathological significance is identical with that of acetonuria. It is met with especially in diabetes, in various digestive diseases, and in febrile diseases. In the continued fevers of childhood it is almost constantly present. In order to demonstrate the presence of diacetic acid a few cubic centimeters of urine are treated with a strong solution of ferric chloride added drop by drop. Should a precipitation of phosphates occur, these are filtered oif, when more of the iron solution is added to the filtrate. If now a Bordeaux-red color appears, another por- tion of the" urine is boiled and similarly treated. If in the second test no reaction is obtained, a third portion of the urine is treated with sulphuric acid and extracted with ether. A positive reaction, when the ethereal extract is tested with ferric chloride, the color dis- appearing upon standing for twenty-four to forty-eight hours, will indicate the presence of diacetic acid, particularly if the urine is rich in acetone. Arnold's Test. — Two solutions are employed, viz., a solution of para-amido-aceto-phenon and a 1 per cent, solution of sodium nitrite. The first is prepared by dissolving 1 gramme of para- amido-aceto-phenon in from 80 to 100 c.c. of distilled water, and adding hydrochloric acid drop by drop until the solution, which at first is yellow, becomes colorless ; an excess, however, should be avoided. Immediately before using, the two solutions are mixed in the proportion of two to one. A few cubic centimeters of the reagent are then treated with an equal volume of urine, and a few drops of ammonia added. Thus treated, all urines give a more or less marked brownish-red color on shaking ; and if much diacetic acid is present, an amorphous reddish-brown sediment is thrown down. A small amount of the colored solution is then placed in a conical glass and treated with an excess of concentrated hydrochloric acid (10—12 c.c. for each 1 c.c). In the presence of diacetic acid the mixture assumes a beautiful purplish-violet color. According to Arnold, the test is more delicate than that of Ger- hardt, and does not respond with acetone or oxybutyric acid. With bilirubin and the common antipyretics, as well as salicylic acid, no reaction is obtained. Highly colored urines should first be filtered through animal charcoal. According to Lipliawski, the following modification of Arnold's test is even more sensitive : two solutions are employed, viz., a 1 per cent, solution of para-amido-aceto-phenon and a 1 per cent, solution of potassium nitrate. Six c.c. of the first solution and 3 c.c. of the second are added to an equal volume of urine, to which a drop of ammonia has been added. The mixture is shaken until it assumes 490 . THE URINE. a brick-red color, when a small amount (10 drops to 2 c.c.) is added to a solution of 15—20 c.c. of concentrated hydrochloric acid, 3 c.c. of chloroform, and 2-4 drops of an aqueous solution of ferric chloride. After gently shaking this mixture, care being taken not to emulsify the chloroform, a beautiful and very characteristic violet tinge results if diacetic acid is present. LiTERATUEE. — V. Jaksoh, Ueber Acetonurie u. Diaceturie, loc. cit. Idem., Zeit. f. Heilk., 1882, vol. iii. p. 34. Schraek, Jahrbuch f. Kinderheilk., 1889, vol. xxix. p. 411. V. Arnold, Wien. klin. Woch., 1899, p. 541. OXYBUTYRIC ACID. The fact that in some cases of diabetes an excessive elimination of ammonia was observed led to the belief that there must be present an unknown acid ; this was shown to be /9-oxybutyric acid. The occur- rence of this acid in the urine of diabetic patients is of great clini- cal interest, as a possible connection has been established between its presence in the blood and diabetic coma. The latter condition is explained by assuming that the diabetic patient is unable to furnish sufficient ammonia to neutralize the acids formed in the tissues of the body, the alkalies of the blood being consequently attacked. A prophylactic treatment with alkalies, such as intravenous injections, has hence been suggested in severe cases. This, however, is a mere theory, and the fact that a case of diabetic coma has been reported in which the alkalinity of the bipod was not diminished, and in which recovery took place without the use of alkalies, renders the correctness of the hypothesis doubtful. Possibly the cause of the coma is due to the presence of toxins circulating in the blood, which produce an increased tissue-destruction, with a simultaneous forma- tion of oxybutyric acid, from which diacetic acid and acetone may further result. However this may be, the presence of oxybutyric acid may always be regarded as indicating a severe type of the dis- ease, and, when associated with marked acetonuria and diaceturia, as indicating danger of coma. The presence of oxybutyric acid may be inferred in diabetic urines if after fermentation a rotation of the plane of polarized light to the left is observed. LiTEKATURE. — V. Jaksch, Ueber Acetonurie u. Diaceturie, loc. cit. H. Wolpe, Arch. f. exper. Path. u. Pharmakol., 1886, vol. xxi. p. 131. LACTIC ACID. Sarcolactic acid is normally absent from the urine, but is met with in pathological conditions, and particularly in hepatic diseases, as the liver is normally concerned in the decomposition of lactic acid and of the lactates that have been ingested with the food. As has been pointed out, moreover, there is evidence to show that by far VOLATILE FATTY ACIDS. 491 the greatest portion of the nitrogen eliminated from the body reaches the liver as ammonium lactate, and is here synthetically transformed into urea. As a consequence, lactic acid appears in the urine when- ever, as in phosphorus poisoning, acute yellow atrophy, etc., an extensive destruction of the hepatic parenchyma occurs, and the formation of urea is consequently impaired. In such cases the elimination of lactic acid is associated with an increased excretion of ammonia. The same will occur when, owing to insufficient oxy- genation of the blood, the power of oxidation on the part of the liver is interfered with. We accordingly find lactic acid in the urine in the chronic anaemias, in cases of poisoning with carbon monoxide, in association with the various forms of circulatory and respiratory dyspnoea, in cases of epilepsy immediately after the attack, following excessive muscular exercise, as in soldiers after forced marches, etc. In order to test for lactic acid, the urine is evaporated on a water- bath to a thick syrup and extracted with 95 per cent, alcohol. This is decanted off after twenty-four hours, evaporated to a syrup, acidi- fied with dilute sulphuric acid, and extracted with ether so long as this presents an acid reaction. The ether is then distilled off and the residue dissolved in water. This solution is treated with a few drops of a solution of basic lead acetate, filtered, the excess of lead removed by means of hydrogen sulphide, and the filtrate evaporated to dryness on a water-bath, when the lactic acid will remain behind as a slightly yellowish syrup. This is then dissolved in a little water, the solution is saturated with zinc carbonate, and boiled. Zinc lactate will Separate out upon evaporation, especially if a little alcohol is added, and may be recognized by the form of its crystals, viz., small prisms. These crystals are Isevorotatory, soluble in alco- hol (1 : 1100), and contain two molecules of water of crystallization, which is lost at 105° C, so that the loss of weight after heating to this temperature must correspond to 12.9 per cent. LiTEEATUEE. — O. Minkowski, "Ueber den Einfluss d. Leberextirpation auf d. Stoffwechsel," Arch. f. exper. Path. u. Pharmakol., vol. xxi. p. 41 ; and " Ueber d. Ur- sache d. Milcbsaureaussoheidung naoh Leberextirpation," Ibid., vol. xxxi. p. 214. G. Colosanti u. E. Moscatelli, " Ueber d. Milchsauregehalt d. menscMichen Hams, Ibid., vol. xxvii. p. 158. VOLATILE FATTY ACIDS. The term Upaeiduria has been applied to an increased elimina- tion of volatile fatty acids in the urine, and may be observed in various hepatic diseases affecting the glandular structure of the liver, in leukaemia, in diabetes, in purulent peritonitis, phlegmonous ton- sillitis, erysipelas, etc. Traces of fatty acids are also found under normal conditions, and are probably formed in the lower segment of the small intestine. The fatty acids which have thus far been isolated from the urine are formic, acetic, butyric, and propionic acid. They may be demonstrated in the same manner as described 492 THE URINE. in the chapter on Feces. According to some observers, the amount of fatty acids in the urine may be regarded as an index of the degree of carbohydrate fermentation in the intestinal tract. Under normal conditions this may be the case, but in disease the ques- tion is probably more complicated. LiTEEATUEE. — V. Jaksch, Zeit. f. klin. Med., 1886, vol. xi. p. 307 ; and Zeit. f. physiol. Chem., 1886, vol. x. p. 5.S6. FAT. Under strictly normal conditions the urine contains no fat, while variable amounts may be found in disease. When present in large quantities, so that it is possible to recognize it with the naked eye, the condition is termed lipuria. Such cases, however, are rare, and the diagnosis should only be made when it is possible to exclude an accidental contamination of the urine. Smaller quantities of fat, recognizable only with the microscope, are much more common, and are indeed quite constantly observed whenever fatty degeneration of the renal epithelial cells, of pus-corpuscles, or of tumor-particles is taking place in the urinary tract. The fat-droplets may then be found floating in the urine or attached to or imbedded in any morphological elements that may be present. Lipuria may also occur when ab- normally large quantities of fat are circulating in the blood. It is thus observed after the administration of cod-liver oil in large quan- tities, following oil inunctions, in cases of fracture of the long bones with extensive destruction of the bone-marrow, in cases of eclampsia, as also in such diseases as diabetes mellitus, chronic alcoholism, phthisis, obesity, leukaemia, in certain mental diseases, in aifections of the pancreas and heart, etc. The term chyluria or galaoturia has been applied to a condition in which a turbid urine presenting the macroscopical appearance of milk is excreted. Upon microscopical examination it may be de- monstrated that the turbidity in such cases is owing to the presence of innumerable highly refractive globules of fat, which may be removed by shaking with ether. Of morphological constituents, leucocytes are occasionally encountered in large numbers. Red blood-corpuscles are also seen at times, and when present in large numbers impart a rose color to the urine. Fibrinous coagula are often observed when the urine has stood for some time, and the entire bulk of urine may even become transformed into a gelatinous mass. Albumin is present in most cases in the absence of other constituents pointing to renal disease, such as tube-casts and renal epithelial cells. Leucin, tyrosin, and cholesterin may also at times be found, particularly the latter. Formerly it was quite gen- erally accepted that this condition was due to the presence of the Filaria sanguinis hominis ; but while filarise are undoubtedly pres- FERMENTS— QASES. 493 ent in the blood in the majority of instances, and may also be pres- ent in the urine, it has been demonstrated that cases occur in which filariasis does not exist, and Gotze expressed the opinion that chy- luria may be owing to a distinct anatomical lesion affecting the renal parenchyma. Further observations, however, are necessary in order to clear up not only the etiology of the disease, but also the manner in which the fat and albumin enter the urine. Literature.— Lipuria : Schiitz, Prag. med. Woch., 1882, vol. vii. p. 322. Ebstein Arch. f. klin. Med., 1879, p. 115. Chyluria: Huber, Virchow's Archiv, 1886 vol' cvi. p. 126. Eossbaoh-Gotze, Verhandl. d. Congr. f. inn. Med., 1887, vol. vi. p 212 Brieger, Zeit. f. physiol. Chem., 1880, vol. iv. p. 407. Grim, Langeubeck's Archiv 1885, vol. xxxii. p. 511. ' FERMENTS. Ferments may be demonstrated in every urine, both under physio- logical and pathological conditions, but are of little clinical impor- tance, excepting, perhaps, pepsin, which is said to be absent in cases of typhoid fever, carcinoma of the stomach, and possibly also in nephritis. In order to demonstrate its presence, a small flake of fibrin is placed in the urine, and after several hours removed to a 2 to 3 pro mille solution of hydrochloric acid. The pepsin, if present, will be deposited upon the fibrin, and effect the digestion of the latter in the hydrochloric acid solution. Diastase, a milk-curdling ferment, and a ferment causing decomposition of urea into carbon dioxide and ammonia, have also been observed. GASES. Every urine contains a small amount of gases, notably carbon dioxide, oxygen, and nitrogen, which may be withdrawn by means of an air-pump. Under pathological conditions hydrogen sulphide is at times also found, constituting the condition known as hydrothionwria. In some instances this is referable to a diffusion of the gas into the bladder from neighboring organs or accumulations of pus ; but this is rare. In others an abscess has ruptured into the bladder, or a direct communication exists between it and the bowel. Under such con- ditions it can, of course, not be surprising that hydrogen sulphide together with other products of albuminous putrefaction are elimi- nated in the urine. More commonly, however, the hydrothionuria occurs idiopathically, and is then referable to the action of certain micro-organisms. This can be readily demonstrated by adding a few cubic centimeters of such urine to normal urine, when upon standing the formation of hydrogen sulphide may be demonstrated in the normal specimen. The common organisms, however, which cause ammoniacal decomposition apparently have no part in this process, and the formation of the hydrogen sulphide may be ob- 494 THE URINE. served before ammoniacal decomposition has set in and while the reaction is yet acid. If a small amount of ordinary decomposing urine, moreover, is added to fresh normal urine, no hydrogen sul- phide is as a rule produced. The character of the organisms in question is variable ; sometimes micrococci are found, at other times bacilli, and in still other instances both. Besides being capable of producing hydrogen sulphide from the sulphur bodies of the urine, some of them also cause the formation of ammonium carbonate in dilute solutions of urea. The source of the hydrogen sulphide in cases of hydrothionuria is in most cases probably the so-called neutral sulphur, but it is pos- sible that the oxidized sulphur is at times also attacked. Very in- teresting is the fact that in cystinuria, in which the neutral sulphur is more or less increased, hydrothionuria is commonly observed. Its occurrence in such cases is indeed so frequent that I am in- clined to suspect cystinuria, although crystals of cystin are not found in the sediment. Further work in this direction, however, is needed, and especially to determine the relative frequency with which the two conditions are associated. In a few recorded instances the hydrothionuria accompanied indigosuria, viz., the presence of free indigo-blue in the urine ; and this Miiller has likewise shown to be referable to the action of cer- tain micro-organisms. One case of this kind I saw several years ago, but made no examination for the presence of cystin. Owing to the well-known poisonous effect of hydrogen sulphide upon the blood, it is well in every case to ascertain whether its formation occurs in the bladder, or whether it takes place only on standing. The formation of hydrogen sulphide in decomposing urines containing albumin is, of course, common, and should not be con- fused with the idiopathic hydrothionuria here described. The chemical test for hydrogen sulphide is very simple : a strip of filter-paper is moistened with a few drops of sodium hydrate and lead acetate solution and clamped into the neck of the bottle containing the urine. After a variable length of time, in some instances immediately, in others only after twelve to twenty-four hours, a discoloration of the paper will be observed, varying from a grayish brown to black according to the amount present. When this is large it is, of course, also recognized by its characteristic odor. LlTEKATURE.—F. Miiller, " Sohwefelwasserstoff im Ham," Berlin, klin. Woch., 1887, Nos. 23 and 24. Rosenheim u. Gutzmann, Deutsch. med. Woch., 1888, No. 10. Kahler, Prag. med. Woch., 1888, No. 50. PTOMAINS. Numerous researches have shown that traces of toxic alkaloidal substances may be encountered in the urine under the most diverse pathological conditions, and may be present evea in health. Of PTOMAlNS. 495 the nature of these bodies, however, little is known. Thudichum claims to have isolated three distinct basic substances from normal urine, which he has termed reducin, pararedudn, and arorriin. Pouchet and Mme. Eliacbeff, working in Gautier's laboratory, have likewise extracted toxic bodies from normal urines ; and Adduco states that after fatiguing exercise, especially, he could demonstrate in the urine a substance which was extremely toxic, and was not iden- tical with cholin, as was first supposed. All this work, however, must be repeated with great care before the results obtained can be regarded as conclusive. This is also true of the work which has been done in various diseases. Some observers have here described bodies which they regard as specific toxins. Griffith thus reports the presence of a specific poison of scarlatina, of measles, mumps, etc. Others again have obtained only negative results. The only substances belonging to the class of ptomains which have thus far been obtained from the urine in amounts sufficient to estab- lish their identity are cadaverin and putrescm. They were originally discovered by Brieger in putrefying cadavers, and subsequently also found in cultures of the bacillus of Asiatic cholera, the Finkler- Prior bacillus of cholerina, the bacillus of tetanus, and in the rice- water stools of cholera patients. From the urine cadaverin, putrescin, and a third diamin isomeric with cadaverin, and which has been regarded as saprin or neuridin, were first obtained by Baumann and V. Udranszky in a case of cystinuria, and it appears that dia- minuria occurs only in association with this disease. All attempts to isolate diamins from the urine under other pathological conditions at least have given rise to negative results. Whether or not diamin- uria is invariably associated with cystinuria is, however, an open question. Putrescin has thus far been found in only three cases, viz., in the first case of Baumann and v. Udranszky, in Bodtker's case, and in a recent, as yet unpublished, case by Garrod. Brieger, Stadthagen, Leo, Garrod, Lewis, and I have succeeded in isolating cadaverin from such urines. Others have been less successful, and the theory which was announced shortly after Baumann's discovery, and quite generally accepted, namely, that the formation of the diamins in question is in some manner responsible for the appear- ance of cystin in the urine, was certainly premature. This is even more true of the inference drawn from this supposed association, viz., that cystinuria is a specific infectious disease of the intestinal canal. This conclusion was based upon the belief that diamins are formed from albuminous material only in the presence of certain bacteria. I have shown, however, that this is not necessarily the case, and that putrescin at least may be formed in the absence of micro-organisms. Further investigation will show whether or not cystinuria is invariably accompanied by diaminuria. Per- sonally I incline to the belief that this is the case; but I have 496 THE URINE. also shown that while cystinuria and diaminuria may coexist, this is not always so, and that the two conditions may alternate, and that the one may temporarily disappear while the other continues. Like Moreigne, I have been led to the conclusion that diaminuria is a metabolic anomaly analogous to diabetes and gout, and that both diaminuria and cystinuria are the expression of a marked impairment of the normal oxidation-processes of the body. The amount of diamins which may be met with in the urine of cystinuric patients is extremely variable. In one case I was able to isolate as much as 1.6 grammes of the benzoylated cadaverin from the collected urine of twenty-four hours.' On other days traces only were present, and at times, as I have already stated, no diamins at all could be found. A few observers who have investigated this question, state that they were unable to find even traces of diamins in their cases ; but as single examinations only were made, their conclusion that diaminuria does not always accompany cystinuria is scarcely justifiable. When single negative results are obtained, the examination should be repeated at frequent intervals or larger quan- tities of urine employed. In general, I should advise those who wish to investigate the question of ptomainuria to experiment with large quantities of urine only, as some of the bodies belonging to this order exhibit a degree of toxicity which is out of all proportion to the amount present. Where specific alkaloids are to be sought for, it is scarcely worth while to use less than 100 or 200 liters of urine, and even with such amounts the results are frequently disap- pointing. In cases of cystinuria much smaller quantities will usually suffice, and an initial experiment may be made with the collected urine of twenty-four hours. Isolation of Diamins. — ^Method of Baumann and v. Udranszky. — The collected urine of at least twenty-four hours is shaken with a 10 per cent, solution of sodium hydrate and benzoyl chloride in the proportion of 1500 : 200 : 25 until the odor of the benzoyl chloride has entirely disappeared. The resulting precipitate contains phos- phates, the benzoyl compounds of the normal carbohydrates of the urine, and a portion of the benzoylated diamins. These are filtered oflF with the aid of a suction-pump and digested with alcohol. The filtered alcoholic extract is concentrated to a small volume and poured into about 30 times its amount of water. Upon standing for from twelve to forty-eight hours the benzoylated diamins separate out in the milky fluid in the form of a more or less voluminous sediment composed of fine, intensely white crystals. In order to remove the benzoylated carbohydrates likewise present, the precipitate is redissolved in alcohol, the solution concentrated to a small volume, and diluted with water as described. This process is repeated several • In the case of Dr. Lewis, which was examined in my laboratory, 0.3 gramme only could be obtained from 12,000 c.c. PTOMAINS. 497 times. The resulting crystals, if both diamins are present, will lose their water of crystallization at 120° C. and melt at 140° C. A smaller portion of the benzoyl diamins remains in the first fil- trate. In order to recover this, the filtrate is acidified with sulphuric acid and extracted with ether. The ethereal residue, before congeal- ing, is placed in as much of a 12 per cent, solution of sodium hydrate as is required for its neutralization, when from 3 to 4 times the volume of the same solution is added. This mixture is placed in the cold, when long needles and platelets separate out, which consist of the- sodium compound of benzoyl cystin and the benzoy* lated diamins. The sediment is filtered off and placed in cold water, in which the sodium-benzoyl cystin dissolves, while the benzoylated diamins remain undissolved. In order to separate the putrescin from the cadaverin, the crystals are dissolved in a little warm alcohol and treated with 20 times the volume of ether. Benzoyl-putrescin is thus thrown down, and may be recognized by its melting-point, viz., 175°-176° C, while the ethereal residue contains the benzoyl-cadaverin, which melts at from 129° to 130° C. The diamins may then be separated from the benzoyl radicle by heating the crystals on a water-bath with a mixture of equal parts of alcohol^ and concentrated hydrochloric acid until a specimen is entirely dissolved by sodium hydrate. The separation is complete after from twenty-four to forty-eight hours, according to the amount present. The solution is then diluted with water, when the benzoic acid, which has been formed, separates out and is filtered off. After extracting with ether, in order to remove any benzoic acid still remaining, the filtrate is evaporated to dryness. A crystalline mass remains, which is easily soluble in water but with difficulty in alcohol. This consists of putrescin and cadaverin hydrochlorates, from which the various double salts with platinum, silver, mercury, etc., can be readily obtained. The platinum salt of cadaverin is formed by adding an alcoholic solution of platinum chloride to a solution of the hydrochlorate in alcohol ; it occurs as a voluminous yellow crystalline mass, which can be purified by recrystallization from hot water. When this salt is decomposed by hydrogen sulphide the hydrochlorate again results, from which the free base is obtained by distillation with caustic potash. During this distillation water passes over at first; and above 160° C. a colorless oil appears, the boiling-point of which is about 173° C. This constitutes the free base, which may be identified by its sperm-like odor and the avidity with which it attracts carbon dioxide from the air to form a carbo- nate. LiTERATUEE.— Stadthagen, "Ueber d. Harngift," Zeit. f. klin. Med., 1889, vol.xv. p. 383. Bouchard, Compt. rend. Soc. de Biol., 1884 ; and Compt. rend, de 1 Acad, des Sci., vol. cii. p. 1127. Lupine et Aubert, Ibid., vol. oi. p. 90. Adduce, Arch. ital. d. Biol., vol. ix. p. 203, and x. p. 1. .^2 498 THE URINE. Diaminuria ; v. Udranszky u. Baumann, Zeit. f. physiol. Chem., 1889, vol. xill. p. 562. Stadthagen u. Brieger, Berlin, klin. Wooh., 1889, vol. xxvi. p. 344. Bodtker, Norsk. Mag. f. Laegevidensk., 1892, vol. vii. p. 1220. Moreigne, Arch.de M^d. exp^r. et d'Anat. path., 1899, p. 254. Simon, Am. Jour. Med. Sci., 1900, vol. cxix. p. 39. Garrod and Cammidge, Jour. Path, and Bact., Feb., 1900. MICROSCOPICAL EXAMINATION OF THE URINE. Sediments. In the chapter treating of the general physical characteristics of the urine it was stated that, on standing, every urine gradually be- comes cloudy owing to development of the so-called nubecula. This was shown to consist of a few mucous corpuscles, a small number of pavement epithelial cells derived from the urinary and genital passages, and under certain conditions of a few crystals of uric acid, of calcium oxalate, or of both. It was further pointed out that owing to a diminution in the acidity of the urine on standing, in consequence of an interaction between the neutral sodium urate and the acid sodium phosphate, a sediment is thrown down which con- sists of acid sodium urate, and at times of free uric acid (see Reac- tion). Should the reaction of the urine be alkaline, however, when freshly voided, a condition which may occur physiologically, when it is dependent upon the ingestion of large quantities of vegetables rich in organic salts of the alkalies, but which may also be due to ammoniacal decomposition, those constituents of the urine which are held in solution merely in consequence of the presence of acid sodium phosphate are also thrown down. In that case the sediment consists essentially of calcium, magnesium, and ammonium salts. Crystals of ammonio-magnesium phosphate, it is true, may also be observed in alkaline urines of the first variety, but they are then almost always due to an increased elimination of ammonia, and hence are rarely observed under physiological conditions. Normally calcium is found only in combination with phosphoric acid and carbonic acid. Of the three possible calcium salts of phos- phoric acid — i. e., C&J^O^.^, CaHP04, and Ca(H2P04)2 — only the first two are found in an alkaline urine, but they may also be observed in specimens which are either neutral or but faintly acid. The acid calcium phosphate, Ca(H2P04)2, is seen but rarely in sediments, and its occurrence always presupposes the existence of a high degree of acidity ; it is precipitated together with uric acid and under similar conditions. Calcium carbonate, CaCO,, is seen only in neutral or alkaline urines. As soon as ammoniacal fermentation has begun, ammonium salts are, of course, formed, viz., ammonium urate and ammonio-magnesium phosphate. The following table shows the various mineral constituents usually observed in sediments, the reaction of the urine being in every case the all-important factor : MICROSCOPICAL EXAMINATION OF THE URINE. 499 Reaction acid : Uric acid. Sodium urate. Calcium oxalate. Primary calcium phosphate. Ammonio-magnesium phosphate. Reaction alkaline (referable to fixed alkalies) : Secondary calcium phosphate. Tricalcium phosphate. Calcium carbouate. Ammonio-magnesium phosphate. Reaction alkaline (referable to ammonia) : Ammonium urate. Ammonio-magnesium phosphate. Tricalcium phosphate. Calcium carbonate. In pathological conditions still other unorganized substances may be observed, viz., cystin, xanthin, hippuric acid, indigo, urorubin, bilirubin, hsematoidin, magnesium phosphate, calcium sulphate, cholesterin, leucin, tyrosin, fats, soaps of magnesium and calcium, etc. Of these, cystin, xanthin, hippuric acid, tyrosin, calcium sul- phate, bilirubin, hsematoidin, magnesium phosphate, leucin, and the soaps of magnesium and calcium occur principally in acid urines, while indigo, urorubin, and cholesterin are usually only found in alkaline specimens. Before considering these various constituents in detail, a few words regarding sediments in general and the method to be followed in their microscopical examination may not be out of place. An idea of the nature of a deposit may often be formed by simple inspection, especially if the reaction of the urine is known. A crystalline sediment, presenting a brick-red color and appear- ing to the naked eye like cayenne pepper, is usually referable to uric acid. On the other hand, a deep-red amorphous deposit occurring in an acid urine consists essentially of urates, the color in this case, as in the former, being due to uroerythrin. Further proof is hardly required. Should doubt be felt, however, it will only be necessary to heat the urine, when the deposit will dissolve. A white floccu- lent sediment in an alkaline urine is usually referable to a mixture of phosphates and carbonates, and will dissolve without difficulty upon the addition of acetic acid, but remains unaffected by heat. A sediment consisting of pus, and occurring in alkaline urines, is frequently mistaken for a phosphatic deposit by the beginner. Aside from a microscopical examination, this question may be settled by the addition of a small piece of caustic soda and stirring, when in the presence of pus the liquid becomes mucilaginous and ropy. If much pus is present, a tough, jelly-like mass will be formed, which 500 THE URINE. escapes from the vessel en masse when the urine is poured out. Such a sediment, moreover, does not disappear upon the addition of an acid, and is rendered still more dense upon the application of heat. Blood when present beyond traces may also be recognized. As a general rule, the non-organized elements of a sediment are of little clinical interest. Students are frequently in the habit of diagnosing an excessive, normal, or subnormal elimination of one or another urinary con- stituent from the result of a microscopical examination. This is unwarrantable, and it should always be remembered that no con- clusions whatsoever can be drawn in this manner as to the amount actually eliminated. Nothing would be more erroneous than to infer an excessive excretion, not to speak of an exces- sive production, of uric acid or of oxalic acid from the fact that crystals of these substances are seen in large numbers under the microscope. Again and again cases are observed in which an ex- cessive elimination of uric acid, oxalic acid, or phosphates is diag- nosed by mere inspection, and in which a careful chemical analysis shows not only no increase, but even a diminution of the normal quantity, A urine which is turbid when passed may be examined micro- scopically at once. As a rule, however, it is necessary to wait until a sediment has formed. To this end, the urine should be kept in a clean and well-stoppered bottle. A small amount of chloroform is added if necessary, and will preserve the specimen almost in- definitely. A few drops of the sediment are then removed by means of a dean pipette, carried down to the sediment, with the distal end tightly closed by the finger, care being taken not to allow the urine to rush into the tube by suddenly releasing the pressure, but withdrawing an amount just sufficient for an examination. This is then spread over a clean slide that has been moistened by the breath, when the specimen may be exam- ined at once. Covering the specimen with a slip is not only vmnec- essary, but even vmdesirable. A low power of the microscope should always be employed, and the high power only used to study details of structure. If a centrifugal machine is available, it is, of course, not necessary to let the urine stand until a sediment has formed. An amount sufficient for a microscopical examination can then be obtained in a few minutes. Non-organized Sediments. Sediments occurring in Acid Urines. — Uric Acid. — The form which uric acid crystals may present in a deposit varies greatly, the most common being the so-called whetsto.ne-form shown in Fig. 94. MICROSCOPICAL EXAMINATION OF THE VBINE. 501 The crystals may occur singly or arranged in groups. Accidental impurities, such as threads or hairs, are at times covered with such crystals, forming long cylinders. Very frequently uric acid crystal- lizes in the form of large rosettes composed of drawn-out whetstone- crystals, presenting a deep-red color, referable to uroerythrin, when they are often visible to the naked eye, and form the" well-known brioh-dust sediment. While it is generally stated that uric acid crystals can always be recognized by their color, which may vary from a light yellow to a dark brown, this is, in my experience, not the case. I have often seen uric acid sediments in which the crystals formed small rhombic plates with rounded edges, and were absolutely devoid of coloring-matter, so far as a microscopical examination could show (Fig. 104). Uric acid " dumb-bells " are also at times observed, and may be mistaken for calcium oxalate. Fig. 104. Colorless crystals of uric acid. Hexagonal plates of uric acid have been similarly confounded with oystin. A uric acid sediment may be observed in cases in which an in- creased excretion of uric acid occurs ; but it should be remembered that, as a rule, it is not permissible to infer an increased production or elimination from the presence of an abundant deposit of this sub- stance alone. Brick-dust sediments are frequently observed during cold weather ; but it would be erroneous to infer an increased elimi- nation from such an occurrence, as the phenomenon is owing to the fact that uric acid is less soluble in cold than in warm water. During the summer months, for the same reason, a deposit of uric acid is less frequently observed, although an increased amount may nevertheless be present, being held in solution owing to the higher temperature. The more concentrated the urine and the more uric acid it contains, the more readily will such a deposit form. It is hence noted after profuse perspiration, following severe muscular exercise, in acute rheumatism with copious diaphoresis, in acute 502 THE URINE. gastritis and enteritis associated with copious vomiting or diarrhoea, during the crisis of pneumonia (particularly if accompanied by much sweating), etc. In all these conditions, however, an increased elimination of uric acid does not necessarily take place, the all- important factors being the reaction of the urine, its degree of con- centration, and the surrounding temperature. Should formed concretions of uric acid — i. e., uric acid gravel — be found in the urine, a direct indication is afforded to diminish the acidity of the urine and to increase the amount of water, so as to guard against the formation of renal or vesical calculus. Chemically, the nature of a uric acid sediment may be recognized by the fact that the crystals dissolve upon the addition of sodium hydrate, and reappear in the rhombic form upon acidifying with hydrochloric acid. When heated with dilute nitric acid the beauti- ful red color of ammonium purpurate is obtained upon the subsequent addition of ammonia (murexid test), as described elsewhere (see page 377). Amorphous Urates. — Sodium and potassium urate frequently, and especially in fevers, form sediments of such density that upon microscopical examination it is almost impossible to discern anything but innumerable amorphous granules scattered over the entire field and obscuring all other elements that may be present. Cells or casts will frequently be seen studded with these granules. In such cases it is best to heat the urine to a temperature of 60° C, and to filter it as rapidly as possible while hot, the contents of the filter being subsequently used for a microscopical examination. Urate sediments are always colored, the tint varying from a dirty brown to a bright salmon-red, owing to the presence of uroerythrin. Difficulties can hence never arise in determining the nature of the sediment, as a colored deposit appearing in an acid urine which dis- solves upon the application of heat cannot be due to anything but urates. If a drop of the sediment, moreover, is treated upon a slide with a drop of hydrochloric acid, characteristic whetstone- crystals of uric acid separate out, but the greater portion appears in the form of rhombic platelets. Calcium Oxalate. — This substance generally appears in urinary sediments in the form of colorless, highly refractive octahedra (Fig. 105), which vary greatly in size; some appear as mere specks under even a comparatively high power, while others may attain the dimensions of a large leucocyte. Frequently one axis is longer than the other. From the fact that their diagonal planes are highly refractive, apparently dividing the superficial plane into four triangles, they have been compared to envelopes, and it is this envelope-form of the crystals which is especially character- istic. In the same specimen of urine so-called dumb-bell forms may be seen, which appear to be made up of two bundles of needle-hke MICROSCOPICAL EXAMINATION OF THE URINE. 503 crystals united in the form of the figure 8. These, according to Beale, originate in the uriniferous tubules, and are frequently found adherent to or imbedded in tube-casts. Other forms may also be seen, and are shown in the accompanying figure. While the envelope crystals are highly characteristic and can hardly be mistaken, for any other substance, the student may at times confound them with crystals of ammonio-magnesium phosphate. This error may be avoided if it is remembered that the calcium oxa- late crystals are usually not so large as those of the magnesium salt, and that the latter dissolve upon the addition of acetic acid, in which calcium oxalate is insoluble. The distinction from uric acid, if we are dealing with the dumb-bell form, cannot always be made by Fig. 105 a a Of □ a # o a # ^ i ^^^ #^ m IS3 ^ ^ / Less common forms of calcium oxalate crystals. (Finlayson.) mere inspection. A drop of caustic soda should be added, which will dissolve the crystals if these are uric acid, while calcium oxalate remains unchanged. It has been pointed out that under strictly normal conditions a few isolated crystals of calcium oxalate may be found in the primi- tive nubecula, so that their presence in urinary sediments cannot be regarded as pathological. After the ingestion of certain vegetables and fruits, notably rhubarb, garlic, asparagus, and oranges, or follow- ing the continued administration of sodium bicarbonate or the salts of vegetable acids, calcium oxalate crystals may be observed in large numbers ; so also in certain diseases, such as diabetes mellitus, catar- rhal jaundice, phthisis, emphysema, etc. As in the case of uric acid, no inference as to the quantity eliminated can be drawn from a microscopical examination of the sediment. The frequent occurrence of abundant sediments of this substance may, however, generally be regarded as abnormal, pro- viding that such an occurrence cannot be explained by the nature of the diet. It is very suggestive to note the frequency with which such sediments are observed in cases of neurasthenia, asso- ciated with a mild degree of albuminuria, as also in various di- 504 THE UBINE. gestive neuroses. Finally, as with uric acid, the possibility of the formation of renal calculi should be borne in mind whenever abun- dant sediments of calcium oxalate are encountered upon frequent examination. Ammonio-magnesium phosphate, usually spoken of as triple phos- phate, crystallizes in large prismatic crystals of the rhombic system ; Fig. 106. Various forms of triple phosphates. (Finlayson.) it is most abundantly observed in alkaline urines, but may also occur in feebly acid specimens. Of the various forms which may occur, that resembling the lid of a German coffin is the most characteristic (Fig. 106). At times these crystals attain considerable size ; very small specimens, however, also occur which may be mistaken for Fig. 107. Orystalllne phosphates. (Finlayson.) oxalate of calcium, but from these they are readily distinguished by_ the ease with which they dissolve in acetic acid, as has been pointed out. Here, as elsewhere, it should be remembered that no conclusions MICROSCOPICAL EXAMINATION OF THE URINE. 505 as to the amount actually eliminated can be drawn from a micro- scopical examination, and the diagnosis " phosphaturia " should be based only upon the results of a quantitative analysis. The continued elimination of a turbid urine, the turbidity of which is referable to phosphates, is notably observed in neurasthenic indi- viduals with a predominance of cerebral symptoms. Very curiously, the phosphaturia is not influenced by diet. Monocalcium phosphate crystals are rarely seen, and only in speci- mens presenting a highly acid reaction, when uric acid crystals are also frequently observed in large numbers. I have seen only a few cases of this kind, occurring in patients the subjects of functional albuminuria. The urine was highly acid, in one case of a specific gravity of 1.036, and on standing deposited a sediment which con- sisted largely of monocalcium phosphate crystals (Fig. 108), with a considerable number of uric acid crystals, from which they are Fig. 108. Monocalcium phosphate crystals. readily distinguished by the absence of pigment and their solubility in acetic acid. Neutral Calcium Phosphate. — These crystals may be found in alka- line, neutral, and feebly acid urines. They are at times of large size, but more commonly acicular, occurring either singly or united in a star-like manner (Fig. 107). They are colorless, readily solu- ble in acetic acid, and insoluble in warm water, so that they can be easily distinguished from uric acid. Basic magnesium phosphate crystals occurring in the form of large, highly refractive plates (Fig. 109), are at times seen in alkaline, neutral, or faintly acid and highly concentrated urines. They are readily recognized by treating a drop of the sediment upon a slide with a drop of ammonium carbonate solution (1 : 4), when the crys- tals become opaque and their edges assume an eroded aspect. In acetic acid they dissolve with ease and may then be reprecij^itated by means of sodium carbonate.* 1 stein, Arch. f. Mln. Med., 1876, vol. xviii. p. 207. 506 THE VBINE. Hippuric acid crystals have been observed, although rarely, in uri- nary sediments, in acute febrile diseases, diabetes, and chorea ; while their occurrence following the ingestion of large amounts of prunes, mulberries, blueberries, or the administration of benzoic acid and salicylic acid, is more common. Fig. 109. Basic magnesium phosphate crystals, (v. Jaksch.) Hippuric acid occurs in the form of fine needles or rhombic prisms and columns, the ends of which terminate in two or four planes, at times resembling the crystals of ammonio-magnesium phosphate and of uric acid. From the former they may be readily distinguished by their insolubility in hydrochloric acid, and from the latter by the fact that they do not give the murexid reaction when treated with nitric acid and ammonia (see page 377). In the case of urines rich in hippuric acid in which the substance does not appear in the sedi- ment, it is well to add a small amount of hydrochloric acid, when the crystals will gradually separate out. Their presence does not appear to possess any clinical significance. Calcium sulphate, in the form of long colorless needles or elon- gated prismatic tablets (Fig. 110), has been observed in urinary Fig. 110. Calcium sulphate crystals, (v. Jaksch.) sediments in only two cases. In both the urine, especially on standing, deposited a milky-looking sediment, the reaction being MICROSCOPICAL EXAMINATION OF THE URINE. 507 strongly acid. It may be recognized by its insolubility in acids and ammonia.^ Cystin (CjHjP^Sj) is rarely seen in urinary sediments. It occurs in the form of colorless hexagonal platelets, which are very charac- teristic (Fig. 111). The crystals are soluble in ammonia and hydro- chloric acid, and insoluble in acetic acid, water, alcohol, and ether. Fig. 111. Crystals of eystin spontaneously voided with urine. (Roberts.) They can thus be readily distinguished from certain forms of uric acid, with which they might possibly be confounded at first sight. When heated upon platinum foil they burn with a bluish-green flame without melting. Cystin-containing urines may be of normal appearance, but they often present a peculiar greenish-yellow color. Their reaction is mostly neutral or alkaline. Upon exposure to the air a marked odor of hydrogen sulphide develops, owing to decomposition of the cystin ; but at times urines are met with in which a distinct odor of hydrogen sulphide is noticeable, although crystals of cystin are not seen in the sediment. It may then be demonstrated by strongly acidifying the urine with acetic acid or by allowing it to undergo ammoniacal decom- position. In either case cystin crystals will separate out on standing. It should be remembered, however, that not all urines in which hydrogen sulphide is formed contain cystin (see Hydrothionuria). The amount of cystin which may be found in urinary sediments is variable. Sometimes a few centigrammes only are obtained, while at others from 0.5 to 1 gramme may be recovered. As is the case with the other non-organized constituents of sediments, however, the amount deposited does not necessarily indicate the total amount 1 V. Jaksch, Zeit. f. klin. Med., 1892, vol. xxii. p. 554. 508 THE URINE. present. Where a quantitative estimation of cystin is to be made, it is best to filter oif that which is deposited and to estimate the amount of neutral sulphur in the filtered urine. An increase beyond the normal may be referred to the cystin remaining in solution (see Neutral Sulphur). Clinical interest in connection with cystinuria centres in the fre- quent association of cystin sediments with cystin gravel or calculi ; but it is curious to note that the cystinuria, notwithstanding the removal of the calculus, may persist for years without giving rise to symptoms denoting the existence of a pathological process. Very remarkable is the not uncommon occurrence of cystinuria in families. Cases of transient cystinuria likewise occur, and it is hence scarcely admissible to speak of a " cured " cystinuria when the condition disappears under treatment. Of the origin of the condition little is known. It has been sup- posed that the appearance of cystin in the urine is in some manner connected with the formation of certain diamins in the intestinal canal. I have pointed out, however, that in all probability the for- mation of cystin and diamins takes place in the tissues of the body, and that the appearance of both is the expression of a definite meta- bolic anomaly rather than of a specific infection (see page 496). LiTKKATURE. — C. E. Simon, "Cystinuria and its Eelation to Diaminuria," Am. Jour. Med. Scl., 1900, vol. oxix. p. 39. See also the literature on page 498. Leucin and tyrosin belong to the group of amido-acids, and are represented by the formulae C5H13NO2 and CgHjiOj. They are never found in urinary sediments under normal conditions, while traces of both substances may be present in solution. Larger amounts are notably found in acute yellow atrophy, of which disease their presence in sediments is almost pathognomonic. In acute phosphorus poison- ing leucin and tyrosin are usually not found. The fact that urea may be altogether absent from the urine in acute yellow atrophy or present in greatly diminished amount has been previously referred to (see Urea, page 345), and the elimination of leucin and tyrosin in its stead, as it were, has been regarded not only as indicating the probable origin of urea from amido-acids, but also the formation of urea, to a large extent at least, in the liver. The albuminous origin of these substances has also been noted (see Urea). Traces of leucin and tyrosin are said to be constantly present in cases of cirrhosis and carcinoma of the liver, in cholelithiasis, catar- rhal jaundice, Weil's disease, nephritis, cystitis, gout, bronchitis, tuberculosis, typhoid fever, hysteria, erysipelas, glucosuria, etc. In connection with cystinuria, the elimination of tyrosin has also been observed, but in two cases which! examined in this direction I obtained negative results. In diabetic urines both are supposedly absent. MICROSCOPICAL EXAMINATION OF THE URINE. 509 As leucin is hardly ever found in the sediment, and tyrosin only when present in large quantities, the urine in every case should first be concentrated upon a water-bath and examined on cooling. At times, however, when these substances are present in only very small quantities, this procedure may not lead to the desired end, and in doubtful cases the following method should be employed : The total amount of urine voided in twenty-four hours is pre- cipitated with basic lead acetate and filtered, when the filtrate, from which the excess of lead has been removed by means of hydrogen sulphide, is evaporated to as small a volume as possible, and is set aside for crystallization. The residue thus obtained is then examined with the microscope; if crystals are detected which answer the description of tyrosin and leucin, they should be subjected to further chemical tests. Fig. 112. Tyrosin crystals. (Chaeles.) Ulrich advises to evaporate the urine to dryness and to heat the residue gently while the vessel is covered with a plate of glass or a funnel. The tyrosin is then said to sublime, and is deposited on the cool glass in crystalline form, the crystals giving the characteristic reactions. Tyrosin crystallizes in the form of very fine needles (Fig. 112), which are usually grouped in sheaves or bundles crossing each other at various angles. They are insoluble in acetic acid, but soluble in ammonia and hydrochloric acid. . Leucin (Fig. 113) occurs in the form of spherules of variable size, which closely resemble globules of fat, but may be distinguished from these by their insolubility in ether. In the urine they present a more or less pronounced brownish color, and upon close examination concentric striations as well as very fine radiating lines can at times be made out, which are especially characteristic. If crystals resembling tyrosin and leucin are found, the following tests should be made : Tests for Tyrosin. — The sediment is filtered off, washed with water and dissolved in ammonia to which a little ammonium car- 510 THE URINE. bonate has been added. The solution is allowed to evaporate, when the tyrosin remains behind. Firia's Test} — A bit of the tyrosin is moistened on a watch-crys- tal with a few drops of concentrated sulphuric acid, covered, and set aside for half an hour. It is then diluted with water, heated, and while hot saturated with calcium carbonate and the solution filtered. The filtrate is colorless, but when heated with a few drops of a very dilute solution of ferric chloride, which must be free from hydrochloric acid, it assumes a violet tint (v. Jaksch). Fig. 113. Crystals of leucin (different forms). (Crystals of kreatinin-zino chloride resemble the leucin crystals depicted at a.) The crystals figured to the right consist of comparatively impure leucin. (Chaeles.) Hoffmann's Test? — A small amount of tyrosin is dissolved in hot water and treated, while hot, with mercuric nitrate and potassium nitrite. The solution assumes a beautiful dark-red color and yields a voluminous red precipitate. Tests fok Leucin. — Seherer's Test.^ — To test for leucin, this is separated from tyrosin by the addition of a little alcohol (see below). The alcohol is allowed to evaporate, and a portion of the residue treated upon platinum foil with nitric acid, when a colorless residue is obtained which, upon the application of heat and the addition of a few drops of a solution of sodium hydrate, forms a droplet of an oily fluid which does not adhere to the platinum. Hofmeister's Test* — A small amount of leucin dissolved in water causes a deposit of metallic mercury when heated with mercurous nitrate. In order to separate the leucin from the tyrosin, the sediment is treated with a small amount of alcohol, in which leucin is more readily soluble than tyrosin. LlTEKATHRE.— Frerichs, Wlen. med. Wooh., 1854, vol. iv. p. 465. Schultzen u. Eiess, Charity Annal., vol. xv. Pouchet, Maly's Jahresber., 1880, vol. x. p. 248. Irsai, Ibid., 1885, vol. xiv. p. 451. Prus, Ibid., 1888] vol. xvii. p. 345. Frankel, Berlin, klin, Woch., 1878, vol. XV. p. 265. ' Piria, Liebig's Annal., 1852, vol, Ixxxii, p. 251. 2 Hoffmann, Ibid., 1857, vol. Ixxxvii. p. 124. ' Scherer, Jour. f. prak. Chem., 1887, vol. Ixxix. p. 410. * Hofmeister, Liebig's Annal., 1877, vol. cxxxix. p. 6. MICROSCOPICAL EXAMINATION OF THE URINE. 511 Xanthin crystals (Fig. 114) are very rarely observed in urinary sediments, and, so far as I have been able to ascertain, the case observed by Bence Jones ^ is the only one on record. Care should be had not to confound certain forms of uric acid with xanthin, and I well remember an instance in which crystals were observed Fig. 114. •ftM 1^ '^W^W^^ ;-o^ ^>^o a, Crystals of xanthin (Salkowski) ; 6, Crystals of cystin (Robin). identical in appearance with those here pictured, but which upon chemical examination proved to be uric acid. The necessity of disre- garding the statement generally made that uric acid crystals found in urinary sediments are invariably colored cannot be insisted upon too strongly. I| has been stated elsewhere that colorless uric acid Fig. 115. Lime and magnesium soaps, (v. Jaksch.) -crystals may be encountered, and in the case just cited such were observed. Clinically, xanthin sediments are of interest only in so far as this substance may give rise to the formation of calculi ; in the case observed by Bence Jones attacks of renal colic had occurred several years previously. 1 Bence Jones, Chem. Centralbl., 1868, vol. xiii. 512 THE URINE. Soaps of Lime and Magnesia. — v. Jaksch has pointed out that in various diseases crystals may be found which " closely " resemble tyrosin in appearance, and pictures such crystals (Fig. 115), which from their behavior toward reagents he is inclined to regard as cal- cium and magnesium salts of certain higher fatty acids. Should doubt arise, the question may be readily decided by a chemical examination (see tests for tyrosin and fatty acids). Bilirubin crystals in the form of yellow or ruby-red rhombic plates or needles, as well as amorphous granules, have been seen in the urine in rare cases, but are of no special interest. They are easily soluble in alkalies and chloroform, but not in ether. When treated upon a slide with a drop of nitric acid a green ring will be seen to form around them (Gmelin's reaction).^ Such crystals have been found in icteric urine and in a case of pyelonephritis. Haematoidin crystals are likewise only rarely seen. They cannot be distinguished from bilirubin, with which, indeed, they are sup- posedly identical.^ They may be found either free or imbedded within cells or tube-casts, in cases of scarlatinal nephritis, the nephritis of pregnancy, in granular atrophy, amyloid degeneration of the kidneys, and in carcinoma of the bladder, of which latter condition they have been regarded by some as pathognomonic. Fat. — When small, strongly refractive globules of fat, which may be readily recognized by their solubility in ether, are observed either floating on the urine or held in suspension, it is necessary to ascer- tain first of all whether such fat may not be present accidentally, owing to the use of a bottle or vessel not absolutely clean, or previous catheterization, etc. The diagnosis lipuria should only be made when all possible precautions have been taken to insure against the aaaidental presence of this substance. Every phy- sician who has frequent occasion to examine urines has undoubtedly met with instances in which fat-globules were found, and in which careful inquiry showed that these were accidentally present. True lipuria — i. e., an elimination of fat usually in the form of droplets floating on the urine — has been noted in various cachectic conditions, in cases of heart-disease, affections of the pancreas and liver, in gangrene and pysemia, in diseases of the bones, especially following fractures, in diseases of the joints, etc. Fat has also been observed in the urine following the ingestion of large amounts of cod-liver oil and inunctions with fats and oils. In fatty degeneration of the kidneys, in Brighf s disease, phos- phorus poisoning, etc., droplets of fat may be seen in the epithelial cells and tube-casts. This, however, does not constitute lipuria. The nature of the droplets may be recognized by their solubility in 1 Kussmaul, Wiirzburger med. Zeit., 1863, vol. iv. p, 64. Ebstein Arch. f. klin. Med., 1879, vol. xiil. p. 115. '' Hoppe-Seyler u. Thierfelder, Handb. d. physiol. u. path., chem. Analyse. MICROSCOPICAL EXAMINATION OF THE URINE. 513 ether, benzol, chloroform, carbon disulphide, xylol, etc., and by the fact that they are colored black when treated with a 0.5 to 1 per cent, solution of osmic acid, and red when a drop of tincture of alcanna is added to the specimen. A very convenient method of demonstrating the presence of fat is also the following : a few cubic centimeters of the urine are mixed with an equal volume of 96 per cent, alcohol and a concentrated solution of Sudan III. in 96 per cent, alcohol. The sediment which collects is then ex- amined under the microscope ; the excess of stain is removed by allowing a few drops of 60 or 70 per cent, alcohol to run under the cover-slip and removing it with filter-paper placed at the edge of the preparation. The fat-droplets are thus colored an intense scarlet red, while granules of albuminous origin are unstained. Free fat can, of course, be demonstrated in the same manner. The largest amounts of fat are observed in chyluria, a condition which is usually due to the presence of a specific paijasite in the blood, viz., the Filaria sanguinis hominis, or more rarely the Distoma haematobium, which have been described in the chapter on the Blood (see also Chyluria). Sediments occurring in Alkaline Urines.— Basic Phosphate of Calcium and -Magnesium. — The most common sediments observed in alkaline urines consist of amorphous phosphates of calcium and magnesium. They are usually as abundant as the urate sediments which have been described, but may be readily distinguished from these by the fact that they do not dissolve upon the application of heat, but readily disappear upon the addition of acetic acid, and are never colored. In this manner it is also easy to distinguish such a sediment from one due to pus, with which it might possibly be con- founded at first sight. Upon microscopical examination a drop of the sediment will be seen to contain innumerable transparent granules scattered over the entire field, and closely resembling those of urate of sodium and potassium. Phosphatic sediments are observed, as mentioned elsewhere, when- ever the reaction of the urine is alkaline, whether this be owing to the presence of fixed alkali or to ammoniacal fermentation. Ammonium urate is observed only in urines which are undergoing ammoniacal decomposition. Its presence should always call for a careful investigation in order to ascertain whether this has taken place after the urine has been voided or before (see Reaction). The salt occurs in the form of colored spherical bodies of variable size, which are sometimes composed of delicate needles, while at others they are amorphous, but may be beset Avith prismatic spicules. They are not easily mistaken for any other substance which may be present in urinary sediments (Fig. 116). Ammonium urate is characterized, moreover, by its solubility in acetic and hydrochloric as 514 TEE URINE. acids, and by the subsequent separation of rhombic crystals of uric acid. Magnesium phosphate has been described above (see page 505). Ammonio-magnesium Phosphate. — ^While the well-known coffin-lid crystals are commonly seen in feebly acid urines, as pointed out, ammonio-magnesium phosphate presents a great variety of forms in alkaline urines, and especially in specimens undergoing ammoniacal decomposition (see Fig. 107). Ammonium urate crystals. Calcium carbonate frequently occurs in alkaline urines, and appears under the microscope in the form of minute granules, occurring singly or arranged in masses ; dumb-bell forms are also seen (Fig. 117). They may be recognized by the fact that they readily dis- solve in acetic acid with the evolution of gas. Fig. 117. Calcium caibonate crystals. Indigo in the form of delicate blue needles (Plate XVIII.), ar- ranged in a stellate manner or in plates, visible only with the micro- scope, is rarely seen, and a specimen such as the one which v. Jaksch pictures can be regarded only as a medical curiosity. In an amorphous condition, however, indigo may be met with in almost PLATE XVII Indigo Crystals from a Urine Rich in Indiean, after standiny for Eight Days at Ordinary Tennperature. (V. Jakwch.) MICROSCOPICAL EXAMINATION OF THE URINE. 515 every decomposed urine, occurring in the form of small granules, and frequently staining the morphological elements that may be present a distinct blue. Sediments presenting a bluish-black color were noted in the time of Hippocrates already, and have been described since by numerous observers, but the nature of the color- ing-matter has only been determined within the last fifty years. Clinically, the occurrence of indigo in the urine is of interest, as renal calculi have been observed which consisted almost entirely of this substance. But little is known of the causes which give rise to its appearance in the urine, but there can be no doubt that its occurrence is referable to the action of certain micro-organisms upon urinary indican (see page 494).' Organized Constituents of Urinary Sediments. Epithelial Cells (Fig. 118). — Bearing in mind the fact that desquamative processes are constantly going on in the epithelial Fig. 118. Epithelium from the urinary passages, o, Round cells ; b, conical and caudate cells ; e, flat cells. lining of the various cavities and channels of the body, one should expect to find in every urine representatives of the different forms 1 V. Jaksch, Prag. med. Woch., 1892, vol. xvii. p. 602. 516 THE URINE. of epithelium occurring in the urinary organs, from the Malpighian tufts down to the meatus urinarius. To a certain extent this actu- ally happens, and cells apparently derived from the meatus, the urethra, bladder, ureters, and pelvis of the kidneys may be met with in almost every specimen, although it may at times be difficult to refer to their origin the individual cells observed. Bizzozero even claims that it is impossible to distinguish between the cells of the bladder and those of the meatus and renal pelvis, while as a class they may readily be differentiated in most cases from the cells of the urethra, the ureters, the prepuce of the male, and the vulva and vagina of the female. Cells from the uriniferous tubules of the kidneys are seldom seen in normal urines, and when they do occur it is impossible to determine their exact origin — i. e., the particular portion of the tubule from which they have been detached. Cells presenting the characteristic striated appearance seen in the irregu- lar, and to a less evident degree in the convoluted, portions of the uriniferous tubules, are never observed in the urine. This fact, as well as the usual absence of true glandular cells, remains to be explained. It is not improbable that the absence of these cells may be referable to a less marked desquamation going on in those parts in which the mechanical injury to which the epithelium is subject must of necessity be far less severe than in the remaining portions of the urinary tract, and particularly in the bladder and urethra. As stated elsewhere, the number of epithelial cells occurring in urinary sediments under physiological conditions is small, and the presence of large numbers may hence always be regarded as abnormal, and indicating the existence of a circulatory or inflammatory dis- turbance affecting some portion of the urinary tract. Were it possible in every case to determine the exact origin of the cells, it is evident that information of great value would thus be obtained. Unfortunately, this is not always possible, as the form of the cells is dependent to a certain extent upon the reac- tion of the uriue, an alkaline or neutral reaction causing the cells to swell and to appear larger and rounder than is the case in acid urines. As has been mentioned, the cellular type is practically the same, moreover, in the bladder, ureters, and pelvis of the kidneys. Definite conclusions should hence be drawn only exceptionally from a microscopical examination alone, but there can be no doubt that in conjunction with other factors and the clinical history the demonstration of a normal or increased number of epithelial cells may frequently be of decided value in a differential diagnosis, and taking these factors into consideration it may even be possible to localize the seat of the lesion. If attention is directed to the struct- ure of the individual cell — and this holds good more especially for the cells derived from the uriniferous tubules — an idea may at times even be formed of the character of the lesion (see below). MICROSCOPICAL EXAMINATION OF THE UEINE. 517 Ultzmann recognizes three forms of epithelial cells which may be found in urinary sediments, viz.: 1. Round cells. 2. Conical and caudate cells. 3. Flat cells. Round cells are usually derived from the uriniferous tubules and the deeper layers of the mucous membrane of the pelvis of the kid- neys. In the urine they present a more or less rounded form and are provided with a distinct nucleus ; they are not much larger than pus-corpuscles. From the latter they are distinguished by the pres- ence of a well-defined nucleus, which in pus-cells becomes distinct only upon the addition of acetic acid, and is, moreover, polymor- phous. Whenever such cells are found adhering to urinary casts, which may at times consist entirely of these structures, it is clear that they represent the glandular elements proper of the kidneys. As similar cells are found in the male urethra, confusion may possibly arise. Should albumin, however, be present, the cells are probably of renal origin. The presence of such cells in large numbers together with pus, in the absence of tube-casts and albumin beyond traces, will usually indicate the existence of a simple pyelitis, particularly if round cells are found joined in a shingle-like manner. Should the pyelitis be associated with a ne- phritis, tube-casts and albumin in larger amounts will at the same time be present. In such cases it may be impossible to determine the origin of the cells, excepting of such that may adhere to casts. In simple circulatory disturbances affecting the renal parenchyma no special abnormalities can be discovered in the structure of the cells, while in cases of fatty degeneration of the kidneys they will be seen to contain fatty particles in greater or less abundance, so that it may be possible to determine the existence of degenerative processes which may be of inflammatory or non-inflammatory origin. The same may be said to hold good if the epithelial elements are markedly granular and occur in fragments. Conical and caudate cells are mostly derived from the superficial layers of the pelvis of the kidneys, and are hence especially seen in cases of pyelitis. Similar cells are also found in the neck of the bladder, and may usually be distinguished from those of the pelvis by the greater length of their processes. Flat cells may come from the ureters, the bladder, the prepuce of the male, and the vulva and vagina of the female. These cells pre- sent the usual characteristics of squamous epithelium, being large, polygonal in form, and provided with a well-defined nucleus ; the extra-nuclear protoplasm is only slightly granular. Other more or less rounded forms are also seen, which are derived from the deeper layers of the mucosa, but may be distinguished from the small round cells of the kidneys proper. Irregular or conical cells, often 518 THE URINE. provided with one or more protoplasmic processes, likewise come from the lower layer of the mucosa of the bladder and ureters. While the cells of the bladder may thus be confounded with those of the ureters and vagina under the microscope, it is not likely that a vaginitis or vulvitis will be mistaken for a cystitis or a ureteritis. In doubtful cases specimens of urine should be procured by means of the catheter, care being taken to first thoroughly cleanse the vulva. The warped appearance so frequently seen in vaginal epithelial cells, and the fact that they often and indeed usually appear in masses, may further aid in the differential diagnosis. It has been pointed out by Peyer that the presence of pavement- epithelial cells, together with mucus and leucocytes, in the urine of hysterical and anaemic girls may be regarded as indicating an irrita- ble condition of the genitals, possibly in consequence of masturba- tion. Bearing in mind the moist and sensitive condition of the vulva of female masturbators, such a view is plausible. A ureteritis, notwithstanding the fact that the ureteral cells closely resemble those of the bladder, may be inferred indirectly, the presence of squamous cells in abundance pointing to a cystitis, a small increase in their number to ureteritis. In conclusion, it should be stated that the so-called mucous corpuscles present in every urine are young vesical cells. From what has been said, it is clear that, with due precautions and taking other factors into consideration, the discovery of epi- thelial cells in large numbers in urinary sediments may be of decided value in diagnosis. LiTEEATUBE. — Bizzozero, loc. cit. Eichhorst, Lehrbuch d. physikal. Untersuch. inn. Krankheit., 2d ed., p. 336, Braunschweig. Leucocytes. — Leucocytes are encountered in only very small numbers in normal urines. A marked increase should, hence, always be regarded as indicating the existence of disease somewhere in the course of the urinary tract, excepting in females, where their presence may be owing to an admixture of leucorrhoeal discharge. In that case the source of the pus will generally be recognized by the simul- taneous occurrence of pavement epithelial cells of the vaginal type in correspondingly large numbers. In doubtful cases the urine should always be obtained with the catheter, care being taken to thoroughly cleanse the vulva before the introduction of the instru- ment. Occasionally the pus is derived from a neighboring abscess that has opened into the urinary passages. The amount of pus which may be found in urines is most varia- ble. On the one hand, deposits several centimeters in height are not uncommon, and closely resemble deposits of phosphates in appear- ance, for which they are indeed frequently mistaken ; on the other MICROSCOPICAL EXAMINATION OF THE URINE. 519 hand, it may only be possible to discover the presence of pus by means of the microscope, which should be employed in every case. The appearance of the pus-corpuscles likewise varies in different cases. In acid urines their form is usually well preserved, and in feebly alkaline and neutral specimens it may even be possible to observe amoeboid movements when the slide is carefully warmed. In alkaline urines, however, they usually swell up and become opaque, so that it is impossible to discern a nucleus unless they are treated with acetic acid. At other times, and particularly when pus has remained long in the body, as where an abscess has burst into the urinary passages, it may be almost impossible to make out a nucleus, and in extreme instances nothing but a mass of granular and fatty detritus is left. While with a certain amount of experience it is hardly likely that a sediment of pus will be mistaken for anything else, such as a deposit of phosphates, it should be remembered that if pus is exposed to the action of ammonia or an ammonium salt the pus- corpuscles become disintegrated. In such cases, as in cystitis, in which ammoniacal decomposition of the urine has taken place in the bladder, a deposit may be obtained which macroscopically resembles mucus, and in which pus-corpuscles may not even be demonstrable with the microscope. The sediment then escapes as a gelatinous, slippery mass when the urine is poured from one vessel into an- other. Recourse must then be had to certain chemical tests, as a pyuria might otherwise be overlooked. To this end, the following procedure, suggested by Vitali,^ may be employed : The urine, after having been acidified with acetic acid, is filtered, and the contents of the filter treated with a few drops of tincture of guaiacum which has been kept in the dark, when in the pres- ence of pus the filter-paper is colored a deep blue. The reaction is supposedly due to the presence in the leucocytes of specific nucleo- proteids. A solution of iodo- potassic iodide may be employed in less extreme instances. A drop of this solution is added to a drop of the sediment upon a slide, when the pus-corpuscles, owing to the presence of glycogen, are colored a dark mahogany-brown, while epithelial cells, with certain forms of which they might possibly be mistaken, assume a light color. Donne's pus-test is based upon the fact that the transformation of pus into a gelatinous, mucus-like mass, observed in cases of cystitis, owing to the action of ammonium carbonate, may also be artificially produced by the addition of a small piece of caustic soda and stir- ring, when in the presence of pus in small amounts the liquid becomes mucilaginous and ropy, while a gelatinous mass is obtained if it is abundant. » Vitali, Maly's Jahresber., 1890, vol. xviii. p. 326. 520 THE UBINE. From a clinical point of view it is most important to establish the source of the pus in every case of pyuria. This may at times be difficult, but the following data will be found of value in a diiferen- tial diagnosis : 1. In diseases affecting the renal parenchyma the amount of pus, as a rule, is small, except where a large abscess located in the kidney structure proper has suddenly burst into the pelvis of the kidney. In uncomplicated cases it is a comparatively easy matter to recog- nize the renal origin of the pus, as other constituents, such as renal epithelial cells, and especially tube-casts, are usually present at the same time, and, as was noted in the case of renal epithelial cells, leucocytes are frequently found adhering to the tube-casts, and at times apparently compose these entirely, when they are spoken of as pus-casts (see Casts). In nephritis, according to Bizzozero, the number of pus-corpuscles stands in a direct relation to the intensity and acute character of the morbid process, the greatest number being found in cases of acute nephritis, while in the chronic forms their number is usually insignificant. Whenever in the course of a chronic nephritis large numbers of pus-corpuscles appear, they may be regarded as indicating either an acute exacerbation of the disease or a complicating inflammation of some portion of the urinary tract. In such cases errors may be guarded against by carefully observing the number and character of the epithelial cells present at the same time, when it will often be found that what at first sight appears as an acute exacerbation of a chronic process, judging from the number of pus-corpuscles, is in reality a secondary pyelitis, ureteritis, or cystitis. In cases of simple renal hypersemia pus-corpuscles never occur in notable numbers. 2. In pyelitis the amount of pus eliminated may vary consider- ably, and at times even perfectly normal urine may be voided. This is pro.bably owing to the fact that the ureter of the aifected side, if the disease is unilateral, becomes obstructed temporarily, when sud- denly large quantities may again appear. The diagnosis of pyelitis is often difficult, and should be based not only upon the condition of the urine, but upon the clinical symptoms as well. Very significant is the fact that the urine in pyelitis is usually acid, a point to be remembered in the differential diagnosis between this condition and cystitis, with which pyelitis is frequently confounded. A careful examination of the epithelial elements may also be of value, and should never be neglected. Bacteria in large numbers are generally present. When pyelitis is associated with nephritis it may at times be almost impossible to determine the origin of the pus ; but if the rule set forth above is remembered, that in chronic nephritis the number of leucocytes is always small, it is not likely that a pyelitis will be MICROSCOPICAL EXAMINATION OF THE URINE. 521 overlooked, particularly if the clinical symptoms are taken into consideration. Matters may become still more complicated when a cystitis is accompanied by a pyelitis or a pyelonephritis. Catheterization of the ureters, which was first practised in the United States by the late Dr. James Brown, should then be resorted to, and it is highly desirable that this most valuable method of diagnosis should become common property as soon as possible. Fischl regards the presence of cylindrical masses composed of pus-corpuscles, formed in all probability in the papillary ducts, as highly characteristic of pyelitis. 3. A pyuria referable to ureteritis can hardly be diagnosed from the appearance of the urine, and in suspected cases catheterization of the ureters should be resorted to, which may possibly elicit informa- tion of value. 4. In mild cases of cystitis pus may be altogether absent, while in the more severe forms its presence is constant. In cystitis the largest amounts, referable to disease of the urinary organs, are observed, and are exceeded only in those rare conditions in which a neighboring abscess has suddenly opened into the urinary pas- As the urine in cystitis is usually alkaline, and always so in the more severe>forms, the alkalinity being due to ammoniacal fermen- tation, it may happen, owing to the disintegrating action of the ammonium carbonate upon the pus-corpuscles, that these may not even be demonstrable with the microscope, and that a gelatinous, mucoid sediment appears instead, which escapes from the vessel en masse when the urine is poured out. Vitali's test for pus (referred to on page 579) should be employed in such cases. 5. In urethritis pus may be present in the urine in considerable amounts. The source of the pus is recognized by the fact that a drop may be manually expressed from the urethra, particularly in the morning upon awaking. Mucoid gonorrhoeal threads, — the " Tripperfaden " of the Germans, — which are largely composed of pus-corpuscles, will almost always be detected in the urine in such cases (Fig. 129). In order to distinguish between a simple urethritis and a urethritis complicated with cystitis, the urine should be obtained in two portions and allowed to settle. In simple urethritis affecting the anterior portion of the urethra the first specimen is cloudy, while the second one is clear. If the urethritis, however, has extended to the neck of the bladder, in the absence of cystitis, the first portion will, of course, be cloudy, while the second may present a variable appearance, being clear at times and cloudy at others. This phenomenon is explained by the fact that a portion of the pus contained in the posterior portion of the urethra has found its way into the bladder. A cystitis may, however, be excluded by the acid reaction of the second specimen, and the fact that the latter 522 THE URINE. is never so cloudy as the first. In cases of urethritis complicated with a purulent cystitis the second portion of the urine contains at least as much pus as the first, and usually more, owing to the fact that the pus (which is heavier than the urine) falls to the floor of the bladder, in which case also the last drops passed "will often be found to be pure pus. The reaction of the urine, moreover, will then be generally alkaline. 6. A sudden elimination of large quantities of pus with a urine which up to that time has presented a normal or nearly normal ap- pearance may almost always be referred to rupture of an abscess into the urinary passages. Exceptions to this rule have been noted in rare instances in which large amounts of pus suddenly appeared, the origin of which could not be demonstrated upon post-mortem inves- tigation. Whether such a phenomenon, as v. Jaksch suggests, is dependent upon " unusual conditions favoring diapedesis " remains an open question. Enumeration of the Pus-corpuseles in the Urine. — In order tp determine the relation existing between the degree of pyuria and albuminuria, as well as to watch the progress of an individual case, an enumeration of the number of pus-corpuscles is at times neces- sary. To this end, a specimen of the urine is thoroughly shaken and the number of corpuscles contained in one cubic millimeter ascertained with the aid of the Thoma-Zeiss blood-counter. Dilu- tion with a 3 per cent, solution of common salt is necessary when a preliminary examination has shown the presence of more than 40,000 corpuscles per cbmm. A dilution of five times is usually sufficient. In every case one hundred squares at least should be counted. Some of the results which have thus been obtained are extremely interesting. In cases of mild cystitis 5000 pus-corpuscles are found on an average in the cubic millimeter ; in cases of moderate severity from 10,000 to 20,000 ; while in severe cases 50,000 and even more may be seen. In one case of cystitis complicating carcinoma of the bladder Hottinger obtained 152,000 per cbmm. In the presence of less than 50,000 a mere trace of albumin is found, and with 80,000- 100,000 only 1 pro mille is referable to this source.^ Red Blood-corpuscles. — The presence of red blood-corpuscles in the urine, constituting the condition usually spoken of as hcematuria, is observed only in pathological conditions, and is, in contradistinc- tion to hsemoglobinuria (which see), a very common occurrence. Urine containing blood-corpuscles in notable numbers presents a color which may vary from a bright red to a dark brown verging upon black. Upon standing, a sediment of a corresponding color is obtained in which distinct coagula of variable size are at times seen. ' E. Wnnderlich, Ueber d. Werth d. ZaUung d. weisseu Blutkorperchen im Ham, etc., Diss., Wurzburg, 1885. MIOBOSGOPICAL EXAMINATION OF THE URINE. 523 If the urine should contain only a small number of red corpuscles, however, no deviation from its normal appearance will be noted, and the diagnosis of hsematuria can then only be made with the micro- scope, which should be employed in every case. The appearance of the red corpuscles varies greatly, being influenced especially by the length of time during which they have remained in the urine. In cases of hsematuria of urethral or vesical origin it will be found that they have mostly retained their normal appearance fairly well, or have become crenated, when they may be recognized without diffi- culty. Other corpuscles, however, will probably also be seen which are no longer biconcave, but which have become spherical or shrunken and present an irregular outline. In cases, on the other hand, in which the corpuscles have remained in the urine for a longer time, as in hsematuria of renal origin, the inexperienced will frequently be puzzled by the presence of bodies of the size of red corpuscles, or somewhat smaller, which are entirely devoid of coloring-matter, and appear as faint, transparent rings, often presenting a double contour, and in which no nucleus can be discovered. These formations are red blood-corpuscles from which the hsemoglobin has been dissolved. They are usually spoken of as blood-shadows. Chemical tests are rarely necessary, but may be employed if doubt should arise (see page 429). • Clinically it is, of course, all-important to determine the source of the blood. This may at times be accomplished without much diffi- culty by a urinary examination, but at other times it may almost be impossible, when the clinical symptoms and physical signs must be taken into consideration. 1. Hematuria of urethral origin, due to urethritis or traumatism incident to catheterization, for example, is a common event, and readily diagnosed, as in such cases blood either escapes of itself from the urethra or it may be squeezed out manually. The last portion of the urine voided, moreover, will always be found free from blood, unless it is referable to disease of the neck of the bladder, when the blood appears only toward the end of micturition, or at least more markedly then than in the beginning. 2. The diagnosis of vesical hsematuria is not always easily made. It should be remembered, however, that the blood-corpuscles here present a normal appearance, as has been mentioned, unless ammo- niacal decomposition is occurring in the bladder, in which case blood- shadows are seen in large numbers. The blood, moreover, is less intimately mixed with the urine than in cases of renal hsematuria, so that the corpuscles rapidly settle after the urine has been passed. Blood-clots of an irregular form and considerable dimensions can only be of vesical origin. A careful examination for the presence of any other morphological constituents which may be observed in urinary sediments, when considered in conjunction with the clinical 524 THE URINE. symptoms, will usually lead to a correct diagnosis so far as the seat of the hemorrhage is concerned. Hsematuria of vesical origin may be due to numerous causes, among which may be mentioned diph- theritic cystitis, ulcers of the bladder caused by calculi and carci- noma, traumatism, the presence of parasites, and, more rarely, rupture of varicose veins in the bladder. In determining the cause of the hemorrhage in a given case more reliance should be placed upon the clinical history than upon the urinary examination. 3. In hsematuria of ureteral origin characteristic blood-coagula, corresponding in diameter and form to the ureters, are occasionally seen. Their presence, however, does not necessarily indicajte that the blood has come from the ureters ; more frequently the hemor- rhage will be found to be due to disease of the pelvis of the kidney. 4. The diagnosis of hemorrhage into the pelvis of the kidney must" be based upon the clinical symptoms taken in conjunction with the results of a urinary examination. In nephrolithiasis only a small number of red corpuscles is usually found, which is important from the standpoint of differential diagnosis. 5. Hsematuria of purely renal origin is of common occurrence, and may be due to numerous causes. In simple hypersemic conditions of the organs and in acute nephritis the passage of smoky-looking urine containing blood-corpuscles, usually in large numbers, is thus a fairly constant symptom. In chronic nephritis the number of the red cor- puscles may be taken to indicate the intensity of the morbid process. Hsematuria may also be due to renal abscess, nephrophthisis, renal carcinoma, and, in rare instances, to aneurism and embolism of the renal artery, thrombosis of the renal vein, etc. In the malignant forms of the acute infectious diseases, such as small-pox, yellow fever, malaria, etc., in scurvy, hsemophilia, and purpura, in leukaemia, filariasis, and distomiasis, renal hsematuria is common. It is also observed in cases of poisoning with turpentine, carbolic acid, can- tharides, etc. 6. An idiopathic form of hsematuria has also been described, in which hemorrhage from the kidneys occurs without apparent cause. To this form Senator applied the term " renal hsemophilia." I have seen three cases of this kind in which no lesion existed which could be made responsible for the hemorrhage. In all three the attacks of hsematuria were invariably associated with anachlorhydria, while normal values were found between the attacks. Two of the patients were males, and undoubtedly neurasthenics. The third was a hys- terical chlorotic female, in whom hsematemesis, pulmonary hemor- rhages, and melaena were also at times observed. Hsematuria of renal origin is usually recognized without much difficulty, as in such cases tube-casts bearing red blood-corpuscles, and at times apparently consisting of these altogether, as well as numbers of renal epithelial cells, will usually be found upon careful MICROSCOPIGAL EXAMINATION OF THE URINE. 525 examination. The blood, moreover, is intimately mixed with the urine, and the individual corpuscles have mostly lost their haemoglo- bin and appear as mere shadows. The clinical history should, of course, always be talien into consideration, especially in determining the primary cause of the hemorrhage. Urine containing red blood-corpuscles is always albuminous, so that it may sometimes be difficult to decide in a given case whether the albumin found is due solely to the presence of blood or whether the hsematuria is complicated with an albuminuria per se. Fre- quently it is possible to arrive at some conclusion by comparing tlie amount of albumin with the number of the red corpuscles, the presence of a large amount of the former in the presence of only a small number of the latter indicating that the albumin is not alto- gether due to the blood. At other times it is impossible to gain information in this manner, when the only expedient left is to deter- mine the quantity of albumin and of iron separately, and to ascertain whether the amount of iron found is sufficient to combine with that of the albumin. As a rule, however, the presence of serum-albumin, aside from that contained in the blood of the urine, may be inferi-ed whenever tube-casts are present, although the amount can only be estimated approximately in this manner. Tube-casts. — In various pathological conditions, and it is claimed even in health, curious formations are seen in the urine, which repre- sent moulds of diiferent portions of the uriniferous tubules. To these the term tube-casts or urinary cylinders has been applied, and it may be said that there is hardly a subject of greater importance in urinary analysis, from a clinical point of view, than that of oylindruria ; but it must also be admitted that notwithstanding numerous investiga- tions our knowledge of their nature and mode of formation is still defective, and the same may be said of their clinical significance. The term " tube-casts," however, is not altogether appropriate, as it is applicable to only one great division of such formations — i. e., to those consisting of a uniform, transparent, gelatinous matrix, to which other elements, such as epithelial cells, red blood-corpuscles, leucocytes, and salts in a crystalline or amorphous form, may acci- dentally have become attached — the tube-casts proper. From these the so-called " pseudocasts " must be sharply differ- entiated, a pseudocast being characterized essentially by the absence of a uniform matrix. Closely related apparently to the true casts are the so-called cylindroids — i. e., band-like formations which resemble the former in appearance, and like these may carry various morpho- logical elements as well as salts. It is thus necessary to distin- guish between true casts, pseudocasts, and cylindroids. Of these, the true casts are by far the most important. They may be divided into hyaline and waxy casts, the two forms being readily differentiated by the fact that the former readily dissolve in 526 THE VRINE. acetic acid, while the waxy casts are either not affected at all by this reagent, or, if so, at least not so rapidly. The latter, moreover, are more strongly refractive, to which property their waxy appearance is due ; their color is slightly yellow or yellowish gray, while the hyaline casts are colorless and usually very pale and transparent.' Mode of Examination. — Unless a urine can be examined within a few hours after being voided, it is well to add a small amount of chloroform, so as to guard against bacterial decomposition. The use of conical glasses is unsatisfactory, and I find it more convenient to keep the urine in well-stoppered bottles. Preserved with chloro- form it will keep almost indefinitely. Where a centrifugal machine is available the specimen can, of course, be examined at once. As soon as a sufficient amount of sediment has been obtained,, a few drops are spread on a slide and examined, uncovered, with a low power. It is essential, however, to make use of the flat mirrm' and to avoid a bright light. If this is borne in mind, no difficulty what- ever mil be found in demonstrating even the most hyaline specimens, though they may be present in very small numbers. In many text- books on urinary analysis the writers speak of the difficulty at- tending the search for hyaline casts, and the advice is frequently given to color the preparations with a drop of a dilute aqueous solu- tion of iodo-potassic iodide, or of some other staining reagent, such as gentian-violet, picrocarmin, methylene-blue, or osmic acid. This is unnecessary if the directions just given are strictly followed. If a bright light is used, however, I am willing to admit that even the most experienced examiner may be unsuccessful in his search. For the preservation of mounted specimens the following method, devised by Kronig, may be employed, though I personally prefer to keep the urine itself and to mount a fresh specimen when necessary. A drop of the sediment, best obtained by centrifugation, is spread on a cover-glass and allowed to dry in the air. It is then placed in a 10 per cent, solution of formalin, for ten minutes, rinsed in water, and stained for about ten minutes in a concentrated solution of Sudan III. in 70 per cent, alcohol. The excess of stain is removed by immersion for one-half to one minute in 70 per cent, alcohol, when the specimen is counterstained with Ehrlich's hsema- toxylin, rinsed in water, and mounted in glycerin. Evaporation is guarded against by ringing the specimen with asphaltum. The tube-casts are thus stained a more or less pronounced blue, the nuclei of the leucocytes dark blue, and any fatty granules or needles of fatty acids that may be present a bright red. True Casts. — 1. Hyaline Casts (Fig. 119). — Upon careful ex- amination it will be seen that with rare exceptions the matrix of hyaline casts is not altogether homogeneous, as small granules may 1 Eovida, see J. Moleschott, Unterauchung. i.. Naturlehre d. Menschen u. d. Thiere, 1867, vol. xl., I. p. 182. MICROSCOPICAL EXAMINATION OF THE URINE. 527 almost always be detected imbedded in or adhering to the matrix. As these granules occur in greater or less numbers, hyaline casts are spoken of as being finely granular (Fig. 120), coarsely granular. Fig. 119. Hyaline tube-oasts. finely dotted, etc. Should true morphological elements be detected, the casts are termed blood-casts, epithelial casts (Fig. 121), or pus- casts (Fig. 122). It would be better, however, to add the term hyaline in every instance, so as to distinguish them from pseudo- casts, which consist of these elements entirely, and lack a uniform Fig. 120. Granular tube-casts. matrix. It would thus be proper to speak of hyaline epithelial casts, hyaline blood-casts, etc., and to apply the collective term — compound hyaline casts — ^to these various subvarieties. 528 THE URINE. The nature of these various forms can probably always be made out without much difficulty, and even in those cases in which the hyaline matrix is apparently concealed beneath cellular elements it will usually be possible, upon closer observation, to detect a fine boundary-line at some portion of the structure. Not infrequently Fig. 121. Epithelial casts. also the end of the cast will be seen to be more or less distinctly hyaline. In others, again, a hyaline zone may be observed along the sides of a central organized thread, so to speak, this being fre- quently seen in specimens which are very broad and long. Should doubt arise, however, a drop of acetic acid is added to a drop of the sediment on the slide ; the acid dissolves the hyaline matrix, the organized constituents are set free, and the differential diagnosis between a pseudocast and a compound hyaline cast is thus readily established. Fig. 122. Pus-casts. The length of hyaline casts varies greatly. It may scarcely exceed the breadth, on the one hand, whUe on the other, although rarely, the cast may traverse the entire microscopical field. In breadth they vary between 0.01 and 0.05 mm. As a rule, the breadth of a cast is uniform throughout jts entire length, but speci- MICBOSOOPWAL EXAMINATION OF THE URINE. 529 mens are not infrequently observed in which one. end tapers con- siderably and presents a spirally twisted appearance. This may be so marked that the entire cast appears transversely striated. It is generally supposed that this results from the adhesion of one end of the cast to the walls of a tubule, the lumen of which it does not fill, so that the free end becomes twisted in the downward course. A dichotomous branching of one end is also at times seen in very broad hyaline specimens. "Fatty globules are found upon the surface of granular casts (Fig. 123), but they also form by themselves short, strongly refrac- tive casts, which are often beset all over with needles of fatty crys- tals. These, however, are not composed exclusively of fat, but probably to some extent of lime and magnesium salts of the higher Fig. 123. a. Fatty casts. 6 and c, Blood-oasts, d, Free fatty molecules. (Robekts.) fatty acids and allied compounds, for they are not all soluble in ether. They have their origin doubtless in fatty degeneration of the renal epithelium " (v. Jaksch). Granules of melanin, indigo, and altered blood-pigment may at times be observed in casts. Eiedel regards the occurrence of dark- brown casts as pathognomonic of fractures. 2. The waxy casts (Fig. 124) may be divided into two groups — true waxy casts and amyloid, casts ; but as the latter are not neces- sarily indicative of the existence of amyloid degeneration of the kid- neys, such a classification is at the present time at least of only theoretical interest. They are readily distinguished from the hyaline casts by the characteristics mentioned above — i. e., their higher degree of refraction, their yellow or yellowish-gray color, and the fact u 530 THE URINE. Fig. 124. I that they are either not attacked at all by acetic acid or only very gradually. As a rule, only small fragments are found, but these are broader and more compact than the largest hyaline casts. Waxy casts may also contain celliflar elements, crystals, and amorphous mineral matter ; but, as a rule, such compound casts are not so commonly observed as are those of the hyaline variety. From the latter they differ furthermore in frequently presenting a clpudy appearance, which in some cases is undoubtedly due to the presence of innumer- able bacteria, and it has been suggested that these may be directly concerned in their production. As has just been stated, some waxy casts give the amyloid reaction — i. e., they assume a mahogany color when treated with a dilute solution of iodo-potassic iodide, which changes to a dirty violet upon the ad- dition of dilute sulphuric acid. It should be remembered, however, that this reaction in casts does not necessarily indicate the exist- ence of amyloid disease of the kidneys, as the reaction may be absent on the one hand in this condition, and present on the other where amyloid degeneration does not ex- ist. This curious phenomenon is usually explained by assuming that such easts have remained in the uriniferous tubules for a long time, and have there undergone certain chemical changes analogous to the so-called " amyloid metamorphosis " of old precipitates of fibrin, and it is indeed possible that waxy casts are originally hyaline. Frerichs has pointed out that fibrin which has remained in the uriniferous tubules for a long time becomes denser and yellowish in appearance, which would explain the fact that these casts are only with difficulty attacked by acetic acid.^ Before leaving this subject it should be stated that " cast-like " formations consisting entirely of amorphous urates are not infre- quently encountered in urines, and according to Leube they may be obtained from any urine if it is concentrated in a vacuum at a tem- perature of 37° to 39° C.2 Students frequently regard such forma- tions as coarsely granular casts, an error which may be guarded against if the characteristics of hyaline casts set forth above are borne in mind. Bacteria (in cases of infectious pyelonephritis), hsematoidin, and 1 Bovida, loc. cit. ICobler, Wien. klin. Wocli., 1890, vol. iil. pp. 531, 557, 574, 576. 2 Leube, Zeit. f. kliu. Med., 1887, vol. xiii. u Different forms of waxy casts: a, With a coating of urates, b, Waxy cast covered with crystals of calcium oxal- ate, c, Fragments of waxy casts, (v. Jaksch.) MICROSCOPICAL EXAMINATION OF THE URINE. 631 granular detritus frequently occur grouped in a cast-like manner; their nature is readily ascertained, as in the case of the so-called urate casts just described.^ PsEUDOCASTS, Consisting of epithelial cells or blood-corpuscles and fibrin, are not often found in urinary sediments. The epithelial pseudocasts are probably seen only in cases of desquamative nephritis, and, unlike true casts, are hollow, the epithelium of the uriniferous tubules being thrown off en masse. Blood-casts (Fig. 123) consist of fibrin, within the meshes of which red corpuscles are generally found ; these either present a normal appearance or occur as mere shadows, owing to the fact that their haemoglobin has been dissolved. They are seen whenever extensive hemorrhage has taken place in the renal parenchyma, and are far more frequently observed than the epithelial pseudocasts. Hyaline casts are probably always met with in urinary sediments in which pseudocasts are found, and may be readily distinguished from the latter even when beset with numerous epithelial cells or red corpuscles (see above). Cylindeoids (Fig. 125) resemble hyaline tube-casts somewhat in general appearance, but differ from them in being much larger and band-like. Like true casts, they have a uniform breadth, and are often beset with crystals and cellular elements, such as leucocytes, red corpuscles, and epithelial cells. They are readily dissolved by acetic acid, thus differing from the mucous cylinders or pseudo- cylinders (Fig. 126) which may be observed in any urine containing mucus ; the latter probably never contain morphological or mineral constituents, and are never of uniform breadth throughout their length. The cylindroids proper are undoubtedly of renal origin and closely related to true casts ; formations are indeed not infre- quently seen in which a tube-cast terminates in a cylindroid at one or both ends (see Fig. 1 1 9).^ Formation of Tube-casts. — Several hypotheses have been advanced to explain the formation of tube-casts — reference is here had only to true casts, and not to pseudocasts, the origin of which is sufficiently obvious — and until recently it was quite generally accepted that they consist of coagulated albumin which has transuded into the tubules. According to this view, a cylindruria would always be indicative of the existence of albuminuria. In Neubauer and Vogel's Urinary Analysis (ninth edition) it is stated that "as to the significance of tube-casts, it must be remembered that these, according to our present knowledge, consist of albumin, which coagulates under the influence of the acid reaction of the urine, in the renal paren- chyma, in a peculiar hyaline manner. They represent merely a 1 Martini, Arch. f. klin. Chir., 1884, vol. xvi. p. 157. v. Jaksch, Deutsoh. med. Woch., 1888, vol. xiii. Nos. 40 and 41. 2 Bizzozero, loc. cit. Thomas, Arch. f. Heilk., 1870, vol. xi. p. 130. PoUak u. Torok, Arch. f. exper. Path. u. Pharmakol., 1888, vol. xxv. p. 87. 532 THE URINE. solidified portion of the albumin held in solution by the urine ; their elimination essentially indicates the existence of an albuminui-ia." More recently, however, it has been suggested that tube-casts are the product of a faulty metamorphosis, or of inflammatory irrita- tion of the renal epithelium, and that a secretion from these cells or Fig. 125. o and b, Cylindrolds from the urine In congested kidney, (v. Jaksoh.) Fig. 126. Mucous cylinders. a disintegration of their protoplasm occurs, which results in the formation of cylindroids or true casts.^ Clinical Significance of Tube-casts. — Formerly the occurrence of tube-casts in urine was held to indicate the existence of nephritis. 'See also Elndfleisoh, Lehrbuch d. path. Gewebelehre, Leipzig, ]875, p. 438. Langhans, Virohow's Arohiv, 1879, vol. Ixxvi. p. 85. Bovida, loc. cit. Kobler, loc. cit. Eibbert, Centralbl. f, d. med. Wiss., 1880, vol. xix. p. 30.5. MICBOSOOPIOAL EXAMINATION OF THE UBINE. 533 This view has been abandoned, however, for the same reasons which led to the rejection of the theory that albuminuria invariably indi- cates Bright' s disease (see above). The statement is frequently made in text-books that tube-casts may occur in the urine of perfectly healthy individuals, following severe muscular exercise, cold baths, etc. — in short, all stimuli which may cause the appearance of albumin in apparently normal individ- uals. It has been indicated elsewhere (see Functional Albuminuria), however, that such stimuli cannot be regarded as " physiological " in every instance, and the presence of tube-casts in the urine similarly should he regarded as a, pathological enent} It is not necessary in this connection to enumerate the various diseases in which cylindruria is observed, as they are the same as those which give rise to albuminuria ; and just as a nephrangiogenie albuminuria is more frequently observed than a nephritidogeriic albu- minuria, so also is the presence of tube-casts in the urine more fre- quently due to circulatory disturbances in the kidneys than to true nephritis. In every case in which tube-casts occur in the urine it may be assumed that the accompanying albuminuria is, to a certain extent at least, of renal origin. While the existence of cylindruria is not necessarily associated with definite pathological alterations of the renal parenchyma, this statement should be restricted to the occurrence of purely hyaline casts, and their presence in only small numbers. A few renal epi- thelial cells may be found at the same time, occurring either free in the urine or adhering to the casts, but never presenting an atrophic or otherwise altered appearance in the absence of definite renal lesions. The presence of compound hyaline and coarsely granular casts, as well as of waxy and amyloid casts, on the other hand, may probably always be regarded as indicating definite changes in structure, so that, so far as the diagnosis of nephritis is concerned, a microscopical examination of the urine will furnish information of more value than the simple demonstration of albumin. Hyaline casts are those most frequently seen, — reference is here had only to the purely hyaline or, at least, but faintly granular form, — ^and are found in all conditions in which albuminuria occurs. When present in only small numbers, and particularly when occur- ring but temporarily in the urine, it may be assumed, in the absence of other symptoms pointing to renal disease, that we are dealing with a mild circulatory disturbance of the kidneys. Kenal epithelial cells are absent, or present, in only small numbers. The albumin- uria at the same time is trifling. If, however, hyaline casts are continuously present in large numbers, and if the amount of albumin exceeds a trace, the existence of a nephritis may usually be inferred. ' Nothnagel, Deutsoh. Arch. f. Win. Med., 1874, vol. xii. p. 326. Burkhart, Die Hamcylinder, 1884. Fischel, Prag. Vlerteljahrschr., 1878, vol. exxxix. p. 27. 634 THE URINE. In such cases granular casts and compound hyaline casts, particu- larly the former, will be found if the nephritis is chronic, while in the acute form the hyaline type prevails. Should blood-casts be present at the same time, the probabilities are that we are dealing with an acute nephritis or an acute exacerbation of a chronic process ; in the latter case coarsely granular casts will also be present in large numbers. Waxy casts always indicate a chronic or, at least, a subacute process. The fatty casts described by Knoll and v. Jaksch " are most commonly associated with subacute or chronic inflammations of the kidney of protracted course, with a tendency to fatty degen- eration of the renal tissue. Post-mortem examination has shown that they form most frequently in cases of large white kidney. In some cases in which they were present, however, the organ was found to be more or less contracted ; but when this was so, it was invariably far advanced in fatty degeneration." It has been stated that from a careful examination of the renal epithelial cells it is often possible to determine whether an inflamma- tory process affecting the kidneys is at the same time complicated with degenerative changes. As a matter of fact, the cells found on the tube-casts under such conditions no longer present a normal appearance, but are shrunken and atrophied, and in cases of fatty degeneration studded with fatty granules. Epithelial casts, in the absence of distinct changes affecting the renal parenchyma, are prob- ably never seen. The occurrence of pus-oasts presupposes the existence of suppura- tive inflammation in the kidneys, while the presence of only a small number of leucocytes on hyaline casts may be observed in the ordi- nary forms of nephritis, and particularly in the acute form. The pathological significance of the so-called amyloid casts and pseudocasts has' already been considered. Cylindroids are present whenever hyaline casts are seen, and have es.sentially the same import. They are said to occur most frequently in the urine of children. So far as the constancy with which tube-casts occur in the urine in nephritis is concerned, it is well known that in the chronic inter- stitial form of the disease they, as well as the albumin, are frequently absent for a long time, so that it may only be possible to make the diagnosis from the clinical history and the physical signs. It is a well-known fact, moreover, that pathological alterations of the kid- neys, particularly in men past middle age, are observed again and again in the post-mortem room, where a previous examination of the urine showed no evidence of the existence of renal disease. In the acute and subacute forms of nephritis, as well as in the ordinary parenchymatous form, tube-casts are probably always found, and it would further appear that acute circulatory disturbances affecting MICBOSCOPIOAL EXAMINATION OF THE URINE. 535 the renal parenchyma quite constantly lead, not only to albuminuria, but also to cylindruria. Spermatozoa. — Spermatozoa, for a description of which the reader is referred to the chapter on the Semen, are frequently observed in the urine of healthy adults, and are quite constantly met with in the first urine passed after coitus or nocturnal emissions, when their presence is, of course, of no significance (Fig. 127). Such urines are always cloudy, but it is impossible to recognize the source of the turbidity by simple inspection. A sediment composed of phosphates is popularly often regarded as due to semen, and no doubt every physician has seen patients, — usually sexual neurasthenics, — who were greatly alarmed at find- ing a white deposit in the chamber, and who imagined themselves Fig. 127, Human spermatozoa. " sufferers from loss of manhood." The microscope is necessary in every case to determine the presence of spermatozoa. In females semen may be found in the urine whenever the external genitals have been polluted during or after coitus, as well as in the exceptional cases in which connection has been effected by the urethra. From a medico-legal standpoint the discovery of spermatozoa in the urine of women may be of the greatest importance, but otherwise it is without significance. In a few instances it is stated that trichomonads have been mis- taken for spermatozoa. I am convinced, however, that such an error can only occur if the observer is totally unacquainted with the subject under consideration. In pathological conditions spermatozoa are not infrequently tound in the urine. In cases of obstinate constipation, owing to pressure 536 THE URINE. of hard scybalous masses upon the seminal vesicles, a partial evacu- ation of semen may occur, which may or may not be accompanied by sexual excitement. Horowitz has pointed out that a discharge of semen may be noted in cases of peri-urethral abscess with per- foration into the ejaculatory ducts, giving rise to spermato-oystitis, the condition being due to a tight stricture of the urethra with dilatation beyond the constricted portion. I have observed a case of cystitis in which spermatozoa could almost always be detected in the urine. An operation revealed a tight stricture of the urethra and a sacculated bladder ; the constant passage of semen was apparently owing to the irritating action of the ammoniacal urine. It should be noted that in this case, as well as in those in which semen is frequently passed during the act of defecation in the absence of sexual excitement, no deleterious effects referable to such loss were noted. In the urine voided during and after epileptic and, more rarely, hystero-epileptic seizures spermatozoa may be found. Such an event is undoubtedly due to muscular spasm, and is identical in origin with the emission of semen observed so frequently in the death agony, and especially during strangulation. In certain spinal diseases semen may be found in the urine, and Fiirbringer relates a case in which, following fracture and dislocation of the vertebral column, with partial destruction of the middle dorsal cord, spermatorrhoea associated with partial erection occurred thirty hours later, and continued until death, which took place after three days. More important is the loss of semen noted in cases of true sperma- torrhoea due to venereal excesses or masturbation, when spermatozoa may be found almost constantly, and the diagnosis indeed will often be dependent upon such an observation. So far as the question of sterility in the male is concerned, reliance should not be placed upon an examination of the urine, but the semen should be obtained as soon as possible after ejaculation, and exam- ined as indicated elsewhere (see page 566). Parasites. — Vegetable Parasites. — It has been shown by numerous investigations that bacteria are always jiresent both in the male and female urethra, and that they may at times gain entrance to the bladder. The weight of evidence, however, is in favor of the view that the urine iintra vesioam is under normal conditions free from micro-organisms, and that any bacteria which may have found their way into the bladder are rapidly killed in healthy individuals. In every urine, on the other hand, that has been exposed to the air, bacteria are always present. Whenever, then, it is desired to deter- mine whether or not the urine of the bladder contains micro-organ- isms, every precaution should be taken to guard against accidental contamination. To this end, the following method should be em- ployed : if the patient is a male, he is instructed to hold his urine MICROSCOPICAL EXAMINATION OF THE URINE. 537 until a fairly large amount has accumulated. The glans is then thoroughly washed with soap and water, and further cleansed with cotton soaked in mercuric chloride solution (1 : 1000). The fossa navicularis is also thoroughly cleansed with the same solution. The urine is then voided under as great pressure as possible. The first portion (about 100 c.o.) is thrown away, and the second received in a sterilized vessel, when cultures should be made at once, agar or gelatin plates being inoculated with 1 or 2 c.c. of the urine. In the female the vulva is cleansed with soap and water, and the urethral aperture disinfected with bichloride ' solution. After then washing with sterilized water and drying with sterilized cotton the urine is evacuated through a sterilized metallic or glass catheter, and received in a sterilized vessel. Brown describes the method which is in use in Dr. Kelly's department at the Johns Hopkins Hospital as follows : the external urethral orifice being carefully cleansed with mercuric chloride solution, followed by sterile water, a sterilized glass catheter, whose external end is covered by a sterile rubber cufi", extending several centimeters beyond the end of the catheter, is introduced, the fingers of the operator being allowed to touch only the distal end of the rubber cuff. The urine is allowed to flow for a short time, when the rubber cuff is pulled off by traction on its distal end. A small amount of urine is then collected in a sterile test-tube, and the cotton plug immediately inserted. Brown states that an extended series of experiments with normal urines has shown that this method is absolutely reliable.^ Fig- 128. Of the bacteria which may be found in every ^^ \ ^ urine that has been exposed to the air, the Micro- ---•v",-Cf"'^> coccus ureoe is of special interest, as ammo- }/^ o'V^"^^ niacal fermentation is largely due to its presence. 'Ov^'* When fermentation has commenced, it is readily ■^—^ recognized, occurring in almost pure culture upon Micrococcus urea;, the surface of the urine, mostly in the form of characteristic chains (Fig. 128). The individual coccus is colorless and quite large, so that it may be mistaken by beginners for a blood- shadow. It is a common error to infer from the occurrence of ammoniacal decomposition very soon after micturition that this process has already begun in the bladder. It should be remembered that urine may un- dergo fermentation, particularly in warm weather, shortly after having been voided, and especially if the vessel employed is not perfectly clean and the urine has been exposed to the air. The diagnosis of ammoniacal fermentation in the bladder should hence only be made when the presence of ammonia can be demonstrated in the urine immediately upon being voided. Under pathological conditions various pathogenic bacteria may be ' T. E. Brown, loc. cit. 538 THE URINE. found in the urine. Their presence usually indicates the existence of definite changes in the renal parenchyma, although these changes are not necessarily of an inflammatory character. Pyogenic cocci are especially prone to settle in the kidneys, and there give rise to focal inflammations ; but even in the absence of such lesions they are frequently found in the urine. In all forms of infectious nephritis an abundant elimination of bacteria may generally be observed, v. Jaksch states that in erysipelas the bacteriuria and nephritis dis- appear, together with the cessation of the disease, and in various suppurative processes taking place in the body the specific bacteria disappear from the urine within twenty-four to forty-eight hours after evacuation of the pus. Most interesting observations on the occurrence of bacteria in the urine of nephritic patients have been reported by Engel. Thirty- one cases were examined. In sixteen the Staphylococcus albus and aureus were found, in eight pyogenic streptococci, in four the tubercle bacillus, in five the Bacillus coli communis, and in one the typhoid bacillus, while negative results were obtained in only two instances. In the same series Engel also found a pyogenic coccus in seventeen cases. This coccus was larger than the known forms ; it could be stained according to Gram's method, and did not liquefy gelatin. Intravenous injections of large numbers of the organism caused nephritis in rabbits. In pneumonia and pneumococcus infections in general the corre- sponding diplococcus may be found, and in erysipelas and strepto- coccus infections streptococci. Very common is the presence of the Bacillus coli communis in cases of pyelonephritis ; it is usually found in pure culture, but is at times associated with the Staphy- lococcus aureus and the Proteus vulgaris. In some instances the latter organism has also been met with in pure culture. Of great interest is the frequent occurrence of the typhoid bacillus in the urine of typhoid fever patients. Bouchard^ in 1881 drew attention to the elimination of the bacillus through this channel, and stated that he was able to demonstrate its presence in 50 per cent, of his typhoid fever cases. Other observers were less successful, but with improving technique and more general investigation a larger number of positive results is being obtained every year.^ At the present time it may be said that the typhoid bacillus can be found in the urine of from 20 to 30 per cent, of all typhoid fever patients. The organism usually appears in the second or third week of the disease, and may persist for months and even years. "When present it usually occurs in pure culture, and often the bacilli are so numerous as to render cloudy a freshly voided specimen of urine. Symptoms 1 Bouchard, Eev. de M«d., 1881, p. 671. Tor an account of the literature, see T. E. Brown, "Cystitis due to the Typhoid Bacillus," etc., Med. Eecord, March 10, 1900. MICROSCOPICAL EXAMINATION OF THE URINE. 539 of cystitis and marked renal involvement often occur, but in a con- siderable number of cases there are no indications of local disease. The elimination of the organism in the urine is of no prognostic significance, but is important from the standpoint of prophylaxis. Of special interest is the fact that the organism may be found in the urine although the patient is not the subject of typhoid fever at the time. Brown ^ thus reports the case of a woman in whom a cystitis developed on the ninth day following an abdominal operation, and in whom it was thought that the typhoid bacillus was accidentally introduced by the catheter. The patient had had typhoid fever thirty -five years previously. Young ^ gives the history of a patient in whom cystitis developed during an attack of typhoid fever, owing to infection with the ' typhoid bacillus. The organism could still be demonstrated in the urine after seven years. A double infection with the gonococcus subsequently occurred, and four months later typhoid bacilli and gonococci were both present in considerable numbers. Cystoscopic examination showed a chronic ulcerative cystitis. Two additional cases of chronic cystitis due to the typhoid bacillus are reported. The bacillus may be isolated and identified according to the usual method (see page 254). Very important further is the fact that in tubercular disease of the urinary organs tubercle bacilli may be found in the urine. The search for them, however, is always tedious and frequently fruitless. In suspected cases it is best to centrifugate the urine, and to spread the sediment upon slides or cover-glasses. The preparations are then fixed by heat, and are best stained with Pappenheim's reagent (see page 285). GreiMs method, which was formerly used to differentiate the two, is less reliable. With this method the specimens are stained with a concentrated alcoholic solution of fiachsin, the staining fluid being brought to the boiling-point on the slide. They are then washed in water and counter stained with a concentrated alcoholic solution of methylene-blue without the application of heat. The excess of stain is washed off, when the preparations are dried with filter-paper and examined as usual. As with Pappenheim's method, the tubercle bacilli are colored red, while the other morphological elements which may be present, including the smegma bacillus, are stained blue. The usual methods of staining are not admissible, as the smegma bacillus, which may also be present in the urine, is likewise stained, and may readily be mistaken for the tubercle bacillus. If, in suspected cases, notwithstanding repeated examination and the preparation of numerous specimens, tubercle bacilli are not found, it is best to inject a few drops of the sediment into the anterior ' H. H. Young, " Chronic Cystitis due to the Bacillus Typhosus," Maryland Med. Jonr., Nov., 1901, p. 456. 540 THE URINE. chamber of the eye of a rabbit, and to watch for the development of miliary tubercles in the iris. The number of bacilli which may be found in the urine in tuber- cular disease of the urinary organs is extremely variable. Fre- quently none at all are found, notwithstanding careful search ; in other cases they are present in small numbers ; while in still others they are extremely numerous, and are often bunched to form par- ticles visible to the naked eye. Isolated tubercle bacilli have also been found in the urine in cases of acute miliary tuberculosis, in the absence of renal changes ; such observations, however, are rare. The gonoooccus of Neisser ^ is rarely found free in the urine, but for sake of convenience is described at this place. The organism (Plate XIX.) occurs in the form of small oval or round granules, usually grouped in twos and fours, resembling a German biscuit or the figure 8. As a rule, it is found enclosed within pus-corpuscles and epithelial cells ; but it may also occur free in the pus obtained from the urethra, in the vaginal discharge, and more rarely in uri- nary sediments, as in cases of complicating prostatitis, peri-urethritis, etc. In cover-glass preparatioris account should be taken only of those organisms which are enclosed within cellular elements, as these alone may be regarded as characteristic. To this end, a drop of the discharge is spread in a thin layer upon a slide or cover-glass, dried in the air, and fixed by passing three or four times through the flame of a Bunsen burner. The specimens may then be stained with any one of the basic anilin dyes. In my laboratory the eosinate of methyliene-blue is almost exclusively used for this purpose (see page 99). The organisms are thus colored blue, while the granules of the eosinophilic leucocytes, which are commonly present at the same time, appear a bright red or a brownish red. After five minutes the excess of stain is washed off, the preparations are rinsed in water, dried with filter-paper, and examined with a high power. Of special interest is the observation of Unna and Plato,^ that the gonococcus can be stained in the living leucocyte with Ehrlich's neutral red. The method employed is simple. A small drop of the fresh pus is mixed with an ose of a dilute solution of neutral red in normal salt solution (1 c.c. of a saturated aqueous solution to 100 c.c), and examined either as hanging drop or mounted on a slide as usual. Thus prepared, a certain number of the intracel- lular gonococci are stained a deep red, while others are not stained ; and it may be observed on warming the slide, so as to elicit amoeboid movements, that some of the gonococci which are stained so long as they remain within the granular portion of the leucocytes, are gradually decolorized when they come to lie in the homogeneous ' Neisser, Centralbl. f. d. med. Wiss., 1879, vol. xvii. p/497. ' J. Plato, " Ueber Gonokokkenfarbung mit Neutralroth," etc., Berlin, klin. Woch., 1899, p. 1085. PLATE XIX. L. SCHMIDT, FEC. Urethral Discharge fron-i a Case of Gonorrhcea, showiiig Gonoeoeei Enclosed in Pus Corpuscles, and Lyiiig Free in the Discharge. Stained with Methylene Blu.e. (Personal Observation.) MICROSCOPICAL EXAMINATION OF THE URINE. 541 ectosarc, and are colored again on returning to the granular pro- toplasm. Plato states that he has examined numerous other intra- cellular organisms, including pseudogonococci, but that he has never observed as rapid and intense staining as with the true gonococci. He therefore suggests that with neutral red it may be possible to differentiate the gonococcus from similar organisms. Extra-cellular gonococci, as well as numerous other bacteria, are not stained, even after an exposure of several days. When no discharge can be obtained from the urethra, or an ex- amination of such discharge is negative, positive results may at times still be obtained if some of the gonorrhoeal threads are examined which may be found floating in the urine. In these the organisms can occasionally be demonstrated after months and even years have . elapsed since the primary infection.' In doubtful cases, and especially in women and children, cultures should be made, as the organisms may be confounded with pseudo- gonococci, which are frequently present both in the diseased and normal urethra of males and females. The organism grows best on a mixture of human blood-serum and nutrient agar (1 : 2 or 3 parts). The surface colonies are pale, grayish, translucent, and iinely granu- lar, with finely notched borders. In bouillon and blood-serum mixed it forms a membrane, while the fluid remains clear. On agar the organism does not grow. Like the pseudogonococci, the gono- cocci are decolorized by Gram's method. In cases of cystitis a great variety of micro-organisms has been met with in the urine. Among the more important may be men- tioned the Staphylococcus aureus, albus, and citreus, streptococci, the Bacillus coli communis, the Bacillus pyocyaneus, the Bacillus typhosus, the Proteus vulgaris, etc. In many cases of cystitis organisms are found, moreover, which are apparently non-patho- genic, and are capable of causing the formation of hydrogen sul- phide from certain sulphur bodies of the urine (see Hydrothionuria). Actinomyces kernels may be observed in the urine when the disease in question has attacked the genito-urinary tract or when the organism has found its way into the urine from other organs. In conclusion, reference should be made to the occasional occur- rence of a form of bacteriuria which is not associated with any pathological process, and has hence been termed idiopathic hacteriwria. Of its causation and significance nothing is known, but it is pos- sible that in these cases a few bacteria enter the bladder either through the anterior rectal wall or are eliminated through the kid- neys from the blood-current. Finding a suitable medium for their growth in the urine, they here multiply and may thus be constantly present. Of late, the Bacillus lactis aerogenes has been found in 1 E. E, Owings, " The Infectiousness of Chronic Urethritis," Bull. Johns Hopkins Hosp., 1897, p. 210. 542 THE URINE. such a case. The diagnosis "idiopathic bacteriuria" should, of course, only be made if every possible source of contamination of the urine can be definitely excluded.' Urines containing bacteria in large numbers are always cloudy, and usually present an acid reaction when voided unless cystitis exists at the same time. Attention is directed to their presence by the fact that such specimens cannot be cleared by simple filtration. Yeast-cells in large numbers are usually only seen in urines con- taining sugar. Whenever a chemical examination has not been made their demonstration will be of importance, as suggesting the pos- sible existence of glucosuria. Moulds are usually seen in old diabetic urines after alcoholic fer- mentation has taken place, but they may also occur, though far less frequently, upon the surface of putrid urines that have contained no sugar. The urinary saroina which is at times met with is smaller than the sarcina of the gastric contents, but closely resembles it in appear- ance. It is of no clinical significance. Whenever a urine is to be examined bacteriologically, special pre- caution should be taken to guard against its accidental contamination. The safest procedure, of course, is to obtain the urine by suprapubic puncture. This is, however, only exceptionally necessary, and as a general rule the method of disinfection which I have described above (see page 537) will suffice. Animal Parasites. — The organism which Hassal saw in a urine that had been " freely exposed to the air " and was alkaline, and which he termed Bodo urinarius, was in all probability an infusorial monad and of no pathological significance. Salisbury was the first to point out that the Triohomonas vaginalis of Donn6 may at times occur in the bladder, but he gave no detailed account of his cases: Kiinstler, Marchand, Miura, and Dock subsequently reported cases in which flagellate protozoa were found, and modern research leaves no doubt that the organisms described by these observers are identical with the trichomonas of Donn6. In Miura's case the habitat of the parasite was the urethra, and an examination of the patient's wife revealed the presence of similar organisms in the vagina. Kiinstler's case was one of pyelitis following cystotomy. Marchand's patient had a fistula in the perineum following suppuration in the pelvis, of unknown origin ; cystitis did not exist. Dock's case was associated with hsematuria. During the past few years I have seen the same organism in six eases, two of which occurred in the practice of Dr. W. M. Lewis, of Baltimore. Five were women, and I have no doubt that the parasite found its way into the bladder from the vagina, 1 Roberts, "On Bacilluria," Trans. Internat. Med. Cong., London, 1881, vol. il. p. 157. Schottelius u. Eeinhold, Centralbl. f. klin. Med., 1886, vol. viii. p. 635. Boss, Baumgarten's Jahresber., 1891, vol. vi. p. 360. MICROSCOPICAL EXAMINATION OF THE URINE. 543 where it could be demonstrated in two instances. Curiously enough, a history of hsematuria was obtained from three of the six patients. In one case the urine contained blood at the time of the examina- tion. Evidence of nephritis or well-marked cystitis did not exist. The number of the parasites was variable, and in four cases large.' Balz observed numerous amcebse in the turbid urine of a girl the subject of phthisis, which he described as being of larger size than the Amoeba coli. Ciliated infusoria have also been found in the urine in isolated cases. The ova of Distoma hsematobium and the Filaria sanguinis hominis are at times found in the urine, their elimination being usually accompanied by hsematuria and chyluria. Echinococcus booklets and fragments of cysts may also be found, and in rare in- stances ascarides find their way into the urinary passages when a fistulous opening exists between the rectum and the bladder. Both- riocephalus linguloides (Leuckart) was found in the urine in a case Fig. 129. ®-^^-~ A gouorrhoeal thread. occurring in Eastern Asia. Eustrongylus gigas is likewise found very rarely. Moscato records one case in which chyluria existed at the same time. In Dr. Clark's case, which was recently reported in this country, the passage of the worm was accompanied by hsematuria. Tumor-particles.— Tumor-particles are so rarely seen in the urine that a detailed account of their occurrence may be omitted, particularly as it is seldom possible to base the diagnosis of tumor upon the presence of fragments in the urine, the chnical history and the physical signs being usually sufficient to reach a satisfactory diagnosis. i • ii. Foreign Bodies. — Of foreign bodies which may be tound in the urine may be mentioned particles of fat, fibres of silk, linen, and wool, etc. ; in short, material the presence of which is owing to the use of unclean vessels for reception of the urine. Fecal matter may ' Dock, Am. Jour. Med. Soi., Jan., 1896. 544 THE URINE. be passed by the urethra ; such an occurrence, of course, always in- dicates the existence of an abnormal communication between the bowel and the urinary passages. Hair derived from a dermoid cyst may similarly be found. In hysteria foreign bodies of almost any kind, such as hair, teeth, fish-bones, wood, etc., and even snakes and frogs, may be shown the physician as having been passed in the urine. I had occasion to examine " gravel " " passed " from time to time by a hysterical patient in large amounts, " every attack being accom- panied by agonizing pains shooting down into the lower abdomen" ; the gravel upon examination proved to be mortar, obtained from the cellar of the patient's house. CHAPTEK VIII. TRANSUDATES AND EXUDATES. In health the so-called serous cavities of the body contain very httle fluid, and quantities sufficient for analytical purposes can nor- mally only be obtained from the pericardial sac. In pathological conditions, on the other hand, large accumulations of fluid may be observed, not only in the serous cavities, but also in the areolar con- nective tissue, beneath the skin, and beneath the muscles. "When due to circulatory disturbances, a hydraemic condition of the blood, or an insufficient elimination of water through the kidneys, such accumulations of fluid are spoken of as transudates, while the term exudates is applied to similar accumulations of inflammatory origin. Clinically, it is frequently difficult to distinguish between trans- udates and exudates, and large ovarian, pancreatic, and hydatid cysts, as well as cystic kidneys, may at times be mistaken for ascites. In such cases a careful chemical and microscopical examination of the fluid in question may be of decided value. Very frequently, moreover, it is possible only in this manner to determine the nature of the disease, and the free use of the trocar and the aspirating- needle in diagnosis cannot be too strongly advocated. TRANSUDATES. General Characteristics. Transudates are usually serous in character, when they present a Ught-straw color ; at times, however, owing to admixture of blood, they have a reddish tinge, and are then said to be sanguineous ; in rare instances they are chylous. Specific Gravity. The specific gravity varies somewhat according to the origin of the fluid, but is usually lower than that of serous exudates occurring in the same cavities — one of the most important points of difference between the two kinds of fluid. Thus, in acute pleurisy the specific gravity of the exudate is usually higher than 1.020 ; and in chronic pleurisy, if an accumulation of pus exists at the same time, higher than 1.018, reaching even 1.030. In transudates into the pleural cavity, on the other hand, referable to circulatory disturbances, for 35 545 546 TMANSVDATES AND EXUDATES. example, as in cases of hepatic cirrhosis or cardiac insufficiency, the figures obtained are usually lower than 1.015. Transudates of peri- toneal origin similarly present a specific gravity varying between 1.005 and 1.015, while that of exudates frequently reaches 1.030. As the chemical composition, in so far as the mineral constituents and extractives are concerned, is practically the same in both classes of fluid, the difference in the specific gravity appears to be essen- tially due to the amount of albumin present, viz., serum-albumin and serum-globulin. It may be demonstrated, as a matter of fact, that exudates contain far more albumin than transudates, the amount varying between 4 and 6 per cent, in the former, as compared with 1 and 2.5 per cent, in the latter. The largest amounts of albumin in transudates are found in those of pleural origin, while in cedema not more than 1 per cent, is usually present. In the table below, taken from Reuss, the relation between the percentage-amount of albumin and the corresponding specific gravity is shown. Reuss suggests the following formula for the purpose of determining from the specific gravity the amount of albumin in transudates and exudates : m E = i (-S— 1000) —2.8, in which E indicates the percentage-amount of albumin and 8 the specific gravity taken by means of an accurate urinometer. Specific gravity. Albumin. Specific gravity. Albumin 1.008 0.2 1.019 ... 4.3 1.009 .... 0.6 1.020 .... . , 4.7 1.010 ... . ... 1.0 1.021 .... ... 5.1 1.011 . . . . ... 1.3 1.022 . ... 5.5 1.012 ... 1.7 1.023 ... 5.8 1.013 ... 2.1 1.024 . . . ... 6.2 1.014 . . . . . . 2.-5 1.025 .... . . 6.6 1.015 .... 2.8 1.026 . . . ... 7.0 1.016 ... 3.2 1.027 .... ... 7.3 1.017 . . . . ... 3.6 1.028 .... ... 7.7 1.018 .... . . 4.0 The following table shows the percentage-amount of albumin obtained by Runeberg in ascitic fluid under various pathological conditions : Average Maximum. Minimum. Hydrsemia (Bright' s disease, tuberqulosis, etc., with amyloid degeneration) . . 0.21 Portal stasis (referable to liepatic cirrhosis or stenosis) 0.97 General venous stasis (referable to or- ganic heart-disease) 1.67 Carcinoma of the peritoneum (compli- cated with carcinoma of the stomach). 3.51 Chronic peritonitis (one case complicated with heart-disease) 3.71 The fact that transudates do not coagulate spontaneously in the absence of blood may further serve to distinguish them from exu- 0.41 0.03 2.68 0.37 2.30 0.84 5.42 2.70 4.25 3.36 TRANSUDATES. 547 dates, in which a coagulum is frequently observed after standing for twenty-four hours. Not much reliance should be placed upon this point of difference, however, as exudates likewise do not always coagulate, and clotting of transudates in the presence of blood may take place within the body. LiTERATDBB. — Ecuss, Deutscli. Arch, f. klin. Med., vol. xxviii. p. 317. Eune- berg, Ibid., 1884, vol. xxxiv. pp. 1 and 266; and Berlin, klin. Wocb., 1897, No. 33. Citron, Ibid., 1897, p. 854 ; and Deutsch. Arch. f. klin. Med., vol. xlvi. Eanke, Mit- theil. a. d. med. Klin. z. Wiirzburg, 1886, vol. ii. p. 189. Chemistry of Transudates. An idea of the chemical composition of the various forms of transudates may be formed from the following tables, taken from Hoppe-Seyler and Hammarsten, the figures corresponding to 1000 parts by weight of fluid ; the specimens were taken from one individual r Water . . . Solids . . . . . Albumin . . Ethereal extract Alcoholic extract Aqueous extract Inorganic salts Errors of analysis Pleura. . 957.59 . 42.41 . 27.82 14.59 Peritoneum. 967.68 32.32 16.11 5.27 10.94 Analysis op Hydkocele Flttid. Water . . . Solids Fibrin (formed) . Globulins .... Serum-albumin . Ethereal extract . Soluble salts . . . Insoluble salts . Sodium chloride . Sodium oxide . ■ 938.85 61.15 0.59 13.52 35.94 4.02 8.60 0.66 6.19 1.09 Sugar and uric acid in small amounts are also, as a rule, found in transudates, and in one case of hepatic cirrhosis Moscatelli succeeded in demonstrating the presence of allantoin. v. Jaksch states that he has frequently been able to demonstrate the presence of urobilin in both transudates and serous exudates, even though red blood-cor- puscles and blood-coloring matter in solution were absent. ^ Peptone is never found; and Paiykull states that nucleo-albumin is not present in transudates of non-inflammatory origin. LiTEKATUBE.— Moscatelli, Zeit. f. physiol. Chem., 1889, vol. xiii. p. 202. v. Jaksch, Zeit. f. Heilk., 1891, vol. xi. p. 440. Eichhorst, Zeit. f. klin. Med., 1881, vol. ni. p. 537. 548 TRANSUDATES AND EXUDATES. Microscopical Examination of Transudates. Upon microscopical examination only a few isolated leucocytes and endothelial cells derived from the serous surfaces and under- going fatty degeneration are usually seen. Mast-cells and eosinophilic leucocytes have been observed in the ascitic iluid in cases of myeloge- nous leuksemia. Charcot-Leyden crystals were present at the same time. In cases in which the transudates have been confined for a long time plates of cholesterin are frequently found. They are especially abundant in hydrocele fluid. EXUDATES. Exudates may be serous, serofibrinous, seropurulent, purulent, putrid, hemorrhagic, chylous or chyloid, terms which do not require further definition. The purulent, seropurulent, and putrid forms are manifestly of inflammatory origin ; while it may at times be diifiault to decide the nature of serous, serofibrinous, and serosanguineous fluids. In such cases the points of difference already described between transudates and exudates should be borne in mind, and will, when taken in con- junction with the physical signs and the clinical history, generally lead to a correct diagnosis of the origin of the fluid. Serous Exudates. Serous exudates are clear, of a light-straw color, and present a specific gravity usually exceeding 1.008. On standing, a white, fibrinous coagulum is generally formed. Such exudates, as indi- cated, differ from the corresponding transudates in presenting a higher specific gravity, and in the fact that clotting in transudates is observed only in the presence of blood. Exudates, however, do not invariably coagulate, and hence too much importance should not be attached to this point (see also page 547). Upon microscopical examination some red corpuscles, which are probably referable to the puncture, polynuclear leucocytes, and endo- thelial cells undergoing fatty degeneration are found.-^ Widal reports that in three cases of acute rheumatism he found polynuclear leucocytes in the serous exudate, while these were absent in traumatic cases of arthritis. He maintains that an examination of the cellular elements ■ysrhich may be found in pleural effusions may furnish valuable information from the standpoint of diagnosis, pathogenesis, and etiology. To this end, a few cubic centimeters of the fluid are withdrawn with a hypodermic syringe, defibrinated, and centrifugated. The residual material is then spread upon cover- glasses, fixed and stained with thionin, haematoxylin-eosin, Ehrlich's ' Bizzozero, loc. cit. EXUDATES. 549 triacid stain, or with eosinate of methylene-blue, which I personally prefer. In idiopathic pleurisy small lymphocytes, together with a few isolated red corpuscles, are exclusively found. In the various forms of tubercular pleurisy morphological elements are essentially absent ; only a few partially broken-down polynuclear leucocytes are seen. In a case of serofibrinous pleurisy refera- ble to streptococcus infection neutrophilic polynuclear leucocytes were found. Especially noteworthy are the findings in pneumo- coccus cases : besides red corpuscles and a few leucocytes, numer- ous polynuclear cells are seen, as also large numbers of mono- nuclear cells of endothelial origin, some of which may be very large and enclose polynuclear leucocytes in their interior. In cases of traumatic and aseptic pleurisy, in association with diseases of the heart and the kidneys, on the other hand, large endothelial cells from the surface of the serous coat, occurring either singly or in groups of two, or three, or four, are especially characteristic' Hemorrhagic Exudates. Hemorrhagic exudates are essentially serofibrinous in character, the color depending upon the amount of blood-pigment present. Microscopical* examination reveals the presence of a large number of red corpuscles, polynuclear leucocytes, and endothelial cells. Cholesterin-crystals may also at times be seen, though rarely in large numbers. When numerous, attention is readily drawn to them, dur- ing the macroscopical examination of the fluid, by the peculiar glis- tening appearance of its surface. Tuberculosis. — As hemorrhagic exudates are most commonly observed in cases of tuberculosis and of carcinoma of the lungs and pleura, the specimen should be carefully examined for tubercle bacilli and cancer-cells. In every case it will be best to subject portions of the fluid to centrifugation and to examine the sediment thus obtained. Usually tubercle bacilli are not found, even when tuberculosis of the pleura exists. If in such cases culture-experiments likewise prove negative and cancer-cells are not found, the diagnosis of probable tuberculosis will nevertheless be warrantable. Cancer. — The diagnosis of cancer should be based upon the demonstration of cancer-cells in the fluid. The physician, however, is warned not to mistake endothelial cells for cancer-cells. The diagnosis should hence only be made when epithelial cells of vari- able form, measuring at times 120 /u in diameter, are found in large numbers, especially when arranged in groups, unless, indeed, can- cerous nodules presenting the characteristic alveolar structure are at once found. ^Widal and Eavaut, " Cystodiagnostic des epanchements s^ro-fibrineux de la plevre," Trans. XIII. Internat. Med. Congress, Paris, 1900. 550 TRANSUDATES AND EXUDATES. Rieder has lately called attention to the occurrence of cells under- going division, their nuclei presenting atypical karyokinetic figures, which he regards as pathognomonic of carcinoma. Cover-slip prep- arations are made from the sediment, dried in the air, fixed by immersion for an hour in a mixture of equal parts of absolute alcohol and ether, and stained with a dilute solution of hsematoxylin. In cases of neoplasm Quincke ^ has drawn attention to the occur- rence in the fluid of large numbers of fat-droplets, which may attain a diameter of from 40 /i to 50 ji. At times, however, the fat- droplets are so small and numerous as to give a chylous appearance to the exudate. At other times a similar appearance is due to the presence of minute albuminous granules, which may be readily dis- tinguished from the former by their insolubility in ether. The occurrence of numerous fatty acid crystals arranged in groups should likewise be regarded as favoring the diagnosis of carcinoma. It is also claimed by Quincke that carcinoma probably exists if a marked glycogen reaction can be obtained in the endothelial cells. This test has been described in the chapter on the Blood (see page 52). Putrid Exudates. Putrid exudates are observed following perforation of a gangren- ous focus or of a gastric or intestinal ulcer into one of the body- cavities. At other times they are encountered in cases of neoplasm, and at times even without apparent cause. The material obtained in such cases has a brown or brownish-green color, and emits an odor which in itself indicates the character of the exudate. Micro- scopically, cholesterin, hsematoidin, and fatty acid crystals, as well as degenerating leucocytes, are found. In cases in which aspiration of. a higher intercostal space reveals the presence of serous fluid, while putrid material is obtained at a lower point, the existence of a subphrenic abscess should be suspected. In such cases a pure cult- ure of the Bacillus coli communis has been obtained. The reaction of putrid exudates is usually alkaline, but an acid reaction may be obtained in cases of perforation of a gastric ulcer ; the Sarcina ven- triculi and saccharomyces may then also be found. Pus. General Characteristics of Pus. — If pus, which usually pre- sents a color varying from yellowish gray to greenish yellow, is allowed to stand for some time, a liquid gradually appears at the top, and increases in amount until it is finally possible to distinguish two distinct layers, the one above — the pus-serum, the other at the bottom — the pus-corpuscles. Upon the number of the latter the ' Quincke, Deutsch. Arch. f. klin. Med., 1882, vol. xxx. pp. 369 and 580. Rieder, Ibid., 1895, vol. liv. p. 544. EXUDATES. 551 consistence as well as the specific gravity of the pus is dependent. This may vary between 1.020 and 1.040, with an average of 1.031 to 1.033. Fresh pus has always an alkaline reaction, which may become neutral or slightly acid upon standing, owing to the develop- ment of free fatty acids, glycerin-phosphoric acid, and lactic acid. The color of pus-serum may be a light straw, a greenish or a brownish yellow. Chemistry of Pus.— The chemical composition of pus-serum and pus-corpuscles may be seen from the following tables : Analysis of Pus-serum. Water 9]3>0 905.(55 . 86.30 94.35 63.23 77.21 1.50 0.56 0.26 0.29 • • • 0-53 0.87 Alcoholic extract . ... . 1.52 0.73 Aqueous extract ... . 11.53 6!92 Inorganic salts .... 7.73 7.77 Solids Albumins Lecithin Fat . . Cholesterin Analysis of Pus-cobpuscles. I. XL 2Sfucleip . . . . 342.37' Insoluble matter . . ... 205.66 \ 673.69 Albumins Lecithin \ Fat ( Cholesterin Cerebri n . Extractives , 1.37) 1.66 \ .62j 137 143.83 ( 75.64 1 75.00 74.00 72.83 4li} 102.84 Albumoses are usually present, and are derived from the pus-cor- puscles. Leucin and tyrosin are likewise frequently met with in the pus of old abscesses ; and fatty acids, urea, sugar, glycogen, biliary pigments and acids (in catarrhal jaundice), acetone, uric acid, xanthin- bases, cholesterin, etc., have occasionally been observed.^ Microscopical Examination of Pus. — Leucocjrtes. — If a drop of pus is examined with the microscope, it will be seen to contain innumerable leucocytes, the diameter of which varies from 8 /z to 10 IX, and which in fresh pus exhibit amoeboid movements. It is curious to note that the so-called lymphocytes do not occur in pus, and even in the rare cases in which a predominance of this variety is met with in the blood, as in cases of lymphatic leukaemia, only the larger forms occur in the pus of abscesses which may have formed. While the leucocytes of fresh pus usually present a nor- mal appearance, specimens may be observed in which amoeboid move- ments can no longer be observed, even upon the application of heat, ' M. Pickardt, " Z. Kenntniss d. Chemie path. Ergiisse," Berlin, klin. Woch., 1897, p. 844. 552 TRANSUDATES AND EXUDATES. and in which rounded vacuoles, filled with a clear liquid, aud fatty granulations in moderate numbers, may be seen. A predominance of such dead leucocytes usually indicates that the pus is old or has formed in greatly debilitated subjects. Owing to resorption of water from accumulations of pus of long standing, such material finally assumes a caseous aspect, and the leucocytes will be seen to have greatly diminished in size, and to have assumed an angular, shrunken appearance ; it is then hardly possible to demonstrate the presence of a nucleus, even after the addition of acetic acid. It is noteworthy that in cases of hepatic abscess referable to the Amoeba coli it is seldom possible to demonstrate any normal leuco- cytes, and it will be seen that under such conditions the pus consists essentially of granular and fatty detritus, while iu liver-abscesses due to other causes the leucocytes usually present a fairly normal appearance. In gonorrhoeal pus eosinophilic leucocytes are frequently found. Dr. E. Owings, who studied this question in my laboratory, was led to the following conclusions : 1. Eosinophilic leucocytes are present in gonorrhoeal pus in a large percentage of cases. They may be absent, however, even when a marked hyperleucocytosis and eosinophilia exist in the blood. 2. Their number varies pan passu with the number present in the blood, and the percentage in the pus is never in excess of the percentage in the blood. 3. Gonococci are rarely found in eosinophilic leucocytes. As has been pointed out, eosinophilic leucocytes are also found in the sputum, and are especially abundant in cases of bronchial asthma and emphysema. Mast-cells are only exceptionally seen in pus. Giant Corpuscles. — So-called giant pus-corpuscles, measuring at times from 30 ^ to 40 /^ in diameter, have been observed in ab- scesses pf the gum, hypopyon, and in the contents of suppurating ovarian cysts, but they do not appear to have any special significance. Upon careful examination these bodies will be seen to contain one oval nucleus, usually located eccentrically within the cell, and from one to thirty or even forty pus-corpuscles.* Detritus. — Fatty and albuminous detritus in variable amount may be observed in every specimen of pus, and increases with the length of time it has been confined within the body. The same holds good for the presence of free nuclei, which were formerly re- garded as young pus-corpuscles, but which have now been definitely recognized as originating during disintegration of the corpuscles. Red Corpuscles. — Red blood-corpuscles in variable numbers are usually seen in every specimen, their appearance depending upon the ' Bottcher, Virchow's Archiv, 1867, vol. xxxiz. p. 512. Bizzozero, loc. cit. EXUDATES. 553 length of time they have been confined. Pus-corpuscles may at times contain a red corpuscle. In doubtful cases it is always well to search carefully for the presence of tissue-elements, as only in this manner is it possible at times to recognize the character of the morbid process. As the data of importance have been detailed in other sections of this book (viz., Sputum and Urine), it is unnecessary to recapitulate at this place. Pathogenic Vegetable Parasites. — Of the pathogenic organisms which are of especial interest from a clinical standpoint may be mentioned the true pus-organisms, notably the Staphylococcus pyogenes aureus and the Streptococcus pyogenes ; furthermore, the tubercle bacillus, the Actinomyces hominis, the bacillus of glanders, the bacillus of anthrax, leprosy, tetanus, influenza, and Frankel's pneumococcus, etc. The majority of these have already been described, and the reader is referred for more detailed informa- tion to special works on bacteriology. In this connection it will suffice to state that, so far as pleural exudates are concerned, an absence of micro-organisms is usually indicative of tuberculosis, while the presence of Frankel's pneumococcus in exudates forming in the course of a pneumonia appears to be a favorable omen as regards the origin of the pleuritic eifusion.' Protozoa, with the exception of the Amceba coli, have only rarely been found. Kunstler and Pitres ^ observed numerous large spores with from ten to twenty crescentic corpuscles in pus taken from the pleural cavity of a man, which closely resembled the coccidia of mice. Litten^ observed cercomonads in fluid withdrawn from a pleural cavity. Trichomonads have been found in a case of em- pyema. Most important in this connection is the demonstration of the Amoeba coli in the pus, and in cases of liver-abscess an examination with this view should never be neglected, as the prognosis will to a large extent depend upon the results obtained. So far as the occur- rence of amoebae in pus is concerned, the observation of Flexner, who demonstrated their presence in an abscess of the lower jaw, shows that they should not be looked for in the pus of abscesses of the liver or lung only. Vermes.— Of these, the filaria and hydatids are rarely observed in this country. Bothriocephalus leguloides has been found in the pleural cavity of a Chinese patient. Crystals. — As has been stated, crystals of cholesterin are fre- quently found in old pus and in exudates of long standing, but are 1 Ludwig Ferdinand v. Bayern, Arch. f. klin. Med., 1892, vol. 1. p. 1. Frankel, Charity Annal., 1888, vol. xiii. p. 147. 2 Kunstler u. Pitres, Compt. rend, de la Soc. de Biol., 1884, p. 5-«. ' Litten, Verhandl. d. Cong. f. inn. Med., 1886, vol. v. p. 417. 554 TRANSUDATES AND EXUDATES. rarely seen in recent exudates. They may be recognized by their characteristic form and their chemical reactions, as described in the chapter on the Feces (page 218). Triple phosphates, fatty acid crystals, and hsematoidin are likewise frequently seen, the presence of the latter, of course, indicating a previous admixture of blood. Chylous and Ghyloid Exudates. Chylous and chyloid exudates have been repeatedly observed. They are most frequently met with in the abdominal cavity (one hundred and four times out of the total number of one hundred and fifty-five, which have thus far been reported), less commonly in the pleural cavity (forty-nine times), and only rarely in the pericardial sac (twice only). Quincke believes that the two forms can be etiologically distinguished from one another by means of a micro- scopical examination, as the cloudy appearance in the chyloid form is usually referable to the presence of endothelial or epithelioid cells undergoing fatty degeneration. Later observations, however, have shown that the differentiation of the two forms cannot be made upon this basis, as the same anatomical lesion, such as carcinoma, may at times give rise to the formation of a chylous exudate, at others to that of the chyloid form, and both, moreover, may coexist. Senator claimed that the presence of more than mere traces of sugar is strongly suggestive of the chylous nature of the exudate. Possibly this observation may be of some value, but it must not be forgotten that sugar is commonly met with in all forms of trans- udates and exudates. Only the presence of more than 0.2 per cent, is of value. Chylous exudates in their general appearance resemble milk, while chyloid fluid is more suggestive of pus. The turbidity in both cases is usually referable to the presence of innumerable fat-globules, which are especially abundant in the chylous form. In chyloid exudates the origin of the fat from cellular elements is often appar- • ent at once ; but, as has been said, it is impossible to draw definite etiological conclusions from that difference. Some chyloid exudates contain no fat at all, and Lion has shown that the milky appearance in such cases is owing to the presence of a curious albuminous substance, belonging to the class of nucleo-albumins. LiTEEATURE.— Quincke, loc. oit. Boulengier, Schmidt's Jahrb., 1890, vol. coxxvi. p. 28. CHAPTEE IX. THE EXAMINATION OF CYSTIC CONTENTS. CYSTS OF THE OVARIES AND THEIR APPENDAGES. The material obtained from cysts of the ovaries or their appen- dages varies greatly in character. On the one hand, it may be fluid, clear, of low specific gravity, and contain little albumin; while, on the other, it may be dense, viscous, of colloid appearance, and have a specific gravity varying between 1.018 and 1.024, owing to the presence of a large amount of albumin, viz., serum-albumin, serum-globulin, and, most important of all, metalbumin or paralbu- min. The latter is almost constantly met with in ovarian cysts, and its presence is characteristic of fluids derived from this source.' Test for Metalbumin. — The fluid is mixed with three times its volume of .alcohol and set aside for twenty-four hours, when it is filtered and the precipitate suspended in water. This is again filtered and the filtrate tested in the following manner : 1. A few cubic centimeters are boiled, when in the presence of metalbumin the liquid will become cloudy, without the formation of a precipitate. 2. With acetic acid no precipitate is obtained. 3. Upon the appli- cation of the acetic acid and potassium ferrocyanide test the liquid becomes thick and assumes a yellowish color. 4. When boiled with Millon's reagent a few cubic centimeters of the filtrate will yield a bluish-red color, while the addition of concentrated sulphuric acid, without boiling, gives rise to a violet color. The color of cystic fluids may vary from a light straw to a reddish brown, or even a chocolate ; the latter color may be observed when hemorrhage has taken place into the cyst. Of morphological elements, ovarian cysts contain red blood-cor- puscles, leucocytes, and at times fatty granules in large numbers, crystals of cholesterin, hsematoidin, and fatty acids. Most im- portant, however, from a diagnostic standpoint is the presence of cylindrical or prismatic ciliated epithelial cells, derived from the internal lining of the cyst, in the presence of which the diagnosis may be definitely made (Fig. 130). At times such cells cannot be demonstrated, as they may have undergone fatty degeneration ; moreover, if the epithelium lining the cyst is squamous in character, it may be difficult, if not impossible, to arrive at a satisfactory con- 1 Hammersten, Zeit. f. physiol. Chem., 1882, vol. vi. p. 194. 555 556 THE EXAMINATION OF CYSTIC CONTENTS. elusion from an examination of the morphological elements alone. Colloid conoretions, which may vary in size from several micromil- limeters to 0.1 mm., are occasionally observed, and more particu- larly in colloid cysts. They may be recognized by their irregular form, homogeneous appearance, slightly yellow color, and delicate outlines. In dermoid cysts, epidermal cells and occasionally hairs are observed. The differential diagnosis of ovarian, parovarian, and fibrocystic (uterine) cysts cannot always be made from the character of the fluid withdrawn by puncture, but at times it is possible. The most im- portant pointe of difference are here given : 1. The fluid in ovarian Contents of an ovarian cyst. (Eye-piece III., obj. 8 A, Eeichert.) (v. Jaksch.) a, Squamous epithelial cells; b, Ciliated epithelial cells; c, Columnar epithelial cells r d, Various forms of epithelial cells ; e, Fatty squamous epithelial cells; /, Colloid bodies; g, Cholesterin-crystals. cystomata is usually more or less viscid, and often contains non- nucleated granular corpuscles of about the size of leucocytes, the granules of which do not dissolve in acetic acid nor disappear when treated with ether. In all probability they are free nuclei ; in the United States they are often called Drysdale's corpuscles. 2. In parovarian cysts the fluid is thin, watery, of low specific gravity (under 1.010), and contains very few morphological elements. Cylindrical epithelium is very rarely found during life in the fluid withdrawn by aspiration from either ovarian or parovarian cysts. ■ 3. The fluid from fibrocystic tumors of the uterus is thin, watery, and coagulates spontaneously, while that from ovarian and paro- varian cysts never coagulates spontaneously unless blood is present. Fibrocystic tumors of the uterus have no epithelial lining. HYDATID AND PANCREATIC CYSTS. 557 HYDATID CYSTS. Hydatid cysts are scarcely ever seen in the United States. The fluid in question is clear, alkaline, of a specific gravity varying between 1.006 and 1.010, and contains no albumin. Succinic acid is usually present, and may be demonstrated by acidifying a small amount of the fluid with hydrochloric acid and evaporating to dryness. The residue is extracted with ether and the ether evaporated ; the aqueous solution of the second residue, in the presence of succinic acid, will yield a rust-colored gelatinous precipitate when treated with a few drops of a solution of ferric chloride. Sodium chloride is always present in notable amounts, and may be recognized by evaporating a drop of the liquid upon a slide, when the characteristic crystals of salt will be found.^ Most important, of course, is the microscopical examination, which may reveal the presence of booklets and shreds of membrane, and at times of scolices (see Sputum). HYDRONEPHROSIS. The diagnosis of hydronephrosis can usually be made without diffi- culty if a sufficient amount of fluid can be obtained ; the presence of urea and uric acid in notable quantities, as well as of renal epithelial cells, which latter especially should be sought for, is quite character- istic. Small amounts of uric acid, however, may also be present in ovarian cysts. PANCREATIC CYSTS. These cysts may be recognized by the fact that the fluid possesses the power of digesting albumin in alkaline solution. A small amount of the liquid is added to milk, when after precipitation of the casein the biuret test is applied ; a positive reaction indicates the presence of trypsin. Unfortunately, however, the test does not always yield positive results, even if the fluid in question is derived from a pancreatic cyst, as the trypsin is apparently destroyed in the course of time. The larger the cyst, the less likely will it be pos- sible to obtain the reaction. A positive result is hence only of value, while a~ negative result does not exclude the existence of the disease.^ 1 J. Munk, Virohow's Arohiv, 1875, vol. Ixiii. p. 255. „ ^ . , ^Karewski, Deutsch. med. Woch., 1890, vol. xvi. pp. 1035 and 1069. HofineisteT Prag. med. Woch., 1891, vol. xvi. pp. 365 and 377 (see Gussenbauer). v. Jaksch, Zeit. f. Heilk., 1888, vol. ix. p. 126 (see Wolfler). CHAPTEE X. THE CEREBROSPINAL FLUID. AccOEDiNG to our present knowledge, the cerebrospinal fluid is secreted by the choroid plexuses into the lateral ventricles. Passing through the foramina of Monro, the third ventricle, and the aque- duct of Sylvius, on the one hand, it reaches the fourth ventricle and enters the cistern-like subarachnoid spaces at the base of the brain, through the foramen of Magendie and the lateral clefts of the fourth ventricle. On the other hand, a certain portion of the fluid reaches the same destination directly through the cleft in the descending horn of each lateral ventricle. The larger portion of the fluid then passes upward through the subarachnoid spaces along the convexity of the brain to the Pacchionian granulations, while the smaller portion enters the vertebral canal through the subarachnoid spaces of the spinal arachnoid membrane. Within recent years puncture of the vertebral canal has been frequently resorted to, both for therapeutic and diagnostic purposes. The practical value of this method of diagnosis is now beyond ques- tion, and it is to be hoped that ere long physicians will resort to spinal puncture in obscure cases of cerebrospinal disease with as little hesitancy as puncture of the thoracic and abdominal cavities is now practised.^ The operative method to be employed is the following : with the patient placed upon his left side, — some observers prefer the sitting posture, — and the body bent well forward, a long aspirating-needle is introduced upon a level with the lower third of the third or fourth lumbar spinous process, and about 1 cm. to the side of the median line, the needle being directed slightly upward and inward. The depth to which it is necessary to puncture will, of course, vary with the age of the patient. In a child two years of age the vertebral canal may be reached at a depth of 2 cm., while in the adult it is necessary to insert the needle for a distance of from 4 to 8 cm. As soon as the subarachnoid space is reached cerebrospinal fluid will flow from the needle. Aspiration should always be avoided. Some writers have advised that the operation be performed under ' H. Quincke, Verhandl. d. X. Cong. f. inn. Med., 1891. A. Hand, " A Critical Summary of the Literature on the Diagnostic and Therapeutic Value of Lumbar Puncture," Am. Jour. Med. Soi., 1900, vol. cxx. p. 463. A. Stadelmann, "Klinische Erfahrungen rait d. Lumbalpunction," Deutsch. iiied. Woch., 1897, p. 745. 558 THE CEREBROSPINAL FLUID. 559 narcosis ; and without doubt this may be necessary at times, particu- larly when contracture of the dorsal muscles exists. In the majority of cases, however, it is not necessary. Amount. — So far as I have been able to ascertain, no observations have been made regarding the amount of fluid which may be obtained by puncture in normal individuals. In all probability, however, this is small. Under pathological conditions the amount may vary from a few drops to 100 c.c, and even more. In general terms it may be stated that the amount is directly proportionate to the degree of intracranial pressure. Exceptions, however, are frequent. Small amounts of cerebrospinal fluid or none at all may thus be obtained when owing to the formation of a thick exudate or the existence of a cerebral tumor communication between the basilar subarachnoid spaces of the brain and those of the spinal cord has been interrupted. Whenever, then, symptoms of intracranial pressure exist, while no fluid or minimal amounts only can be obtained by puncture, the conclusion will usually be justifiable that we are dealing with a purulent meningitis or with a tumor of the brain, and more especially of the cerebellum. It should be remembered, however, that the same result may be obtained in cases of obliteration of the aqueduct of Sylvius, or when sclerotic processes in\'olve the foramen of Magendie, which is occasionally observed in certain forms of hydro- cephalus. Adhesions of the pia mater to the arachnoid and the dura mater may, by interfering with the flow of cerebrospinal fluid, also lead to the formation of hydrocephalus, but in these cases a tumor can usually be excluded, as the changes in question always develop as sequelse to a meningitis. A serous or tubercular menin- gitis, as well as acute hydrocephalus and tetanus, can, however, always be excluded when only minimal amounts of fluid are obtained by puncture. The largest amounts, on the other hand, are seen in cases of serous meningitis, tubercular meningitis, and cerebral tumors, which do not interfere with the circulation of the cerebrospinal fluid. Appearance. — Normal cerebrospinal fluid, as well as that obtained in cases of serous meningitis, tubercular meningitis, hydrocephalus, and tumors of the brain, is perfectly clear, and as a rule colorless unless a small blood-vessel has been punctured, when the fluid may present a slightly reddish tinge. More or less pronounced yellow shades are, however, at times observed. Important from the stand- point of diagnosis is the fact that in cases of hemorrhage into the ventricles pure blood is obtained, while such a result is, of course, a mechanical impossibility in cases of epidural hsematoma. In subdural hffimatoraa, on the other hand, blood may also find its way into the subarachnoid space, but the amount is always small, and cannot be compared with that seen in cases of ventricular hemorrhage. When- ever, then, as in traumatic cases with severe cerebral symptoms, the 560 THE CEREBROSPINAL FLUID. surgeon is confronted with the question whether or not to trephine, puncture of the subarachnoid space may furnish much valuable information. If in such cases no blood at all is found, it may be inferred that an epidural hsematoma or a subdural haematoma of slight extent only exists ; an operation may then be performed. If, however, pure blood is encountered, it would be justifiable to assume the existence of extensive injury to the brain-substance proper, or, in cases in which the history is obscure, an intracerebral hem- orrhage with rupture into the ventricles. In such cases the idea of an operation would, of course, be entertained only under excep- tional conditions. If, further, the fluid is only tinged with blood, a subdural haematoma probably exists, and an operation should be advised. Accidental hemorrhage, viz., hemorrhage referable to the puncture itself, can be readily recognized, as the first few drops only are then tinged with blood, or the blood appears only after the flow has been definitely established ; the amount, moreover, is insig- nificant. Cloudy fluid is obtained in all cases of purulent meningitis unless the disease is limited to a very small area. This is, of course, most imj^ortant from a diagnostic standpoint. Cases of abscess of the brain or sinus thrombosis occur again and again in which the question as to the advisability of operative interference is largely dependent upon the presence or absence of a complicating purulent meningitis. In certain instances a satisfactory conclusion may, of course, be reached without puncture ; but in many others this is impossible, and Lichtheim's dictum, that an operation should never be under- taken in such cases unless the integrity of the meninges has been established by spinal puncture, should be borne in mind. The degree of cloudiness naturally varies in different cases, and while in some instances the character of the fluid is seropurulent, pure, creamy pus may be found in others. Generally speaking, a cloudy fluid indicates the existence of an acute inflammatory process or an exacerbation of a chronic process. Important, furthermore, is the fact that the fluid in non-inflam- matory diseases of the brain, such as tumor or abscess, rarely undergoes coagulation, while this is the rule in all inflammatory diseases. In tubercular meningitis the coagula are very delicate, and may be well compared to spider-webs extending throughout the fluid, while in purulent meningitis the coagula are much firmer. Specific Gravity. — ^The specific gravity of cerebrospinal fluid normally varies between 1.005 and 1.007, corresponding to the presence of from 10 to 15 pro mille of solids. Under pathological conditions variations from 1.003 to 1.012 may be observed, the specific gravity, generally speaking, being higher in the inflamma- tory than in the non-inflammatory diseases of the brain. From a diagnostic standpoint, however, the determination of the specific CHEMICAL COMPOSITION. 561 gravity is of little value, as numerous exceptions to the above rule occur. The reaction is always alkaline. Chemical Composition. — An idea of the chemical composition of the cerebrospinal fluid may be formed from the following analysis, taken from Gautier : Water . . ... 987.00 Albumin . 1 jO Fat . o!o9 Cholesterin . . .... ... . 0.21 Alcoholic and aqueous extract, minus salts 1 Sodium lactate f ' • ^- '^ Chlorides . . . . 6.14 Earthy phosphates ... .0.10 Sulphates . . . 0.20 In addition, urea is at times found, as also a substance which reduces Fehling's solution and gives rise to a brown color when boiled with caustic potash, but which neither undergoes fermentation nor forms an osazon when treated with phenylhydrazin. The sub- stance in question is generally regarded as pyrocatechin. Its amount varies between 0.002 and 0.116 per cent. According to C. Ber- nard, glucose may also be present, but it is questionable whether this is the case under normal conditions (see below). Nawratzki discovered a reducing substance in his cases, which was demon- strated to be glucose ; his subjects, however, were unfortunately not normal, but general paretics with fever. Pyrocatechin was absent. So far as the albuminous bodies are concerned which may be found in the cerebrospinal fluid, serum-albumin is said to be present only under exceptional conditions, while normally a mixture of globulin and albumoses is found. The question whether or not mucin may also be present is still undecided.* Under pathological conditions the amount of albumin may vary considerably, and is of diagnostic importance. According to the majority of observers, the figure given in the above analysis is too high, and it is doubtful whether 1 pro mille may be regarded as normal. The lowest values have been obtained in cases of chronic hydrocephalus (traces only), meningitis serosa (0.5 to 0.75 pro mille), and tumors of the brain (traces to 0.8 pro mille) ; while the largest amounts have been found in chronic hydrocephalus the result of hypersemia (1 to 7 pro mille), and in tubercular meningitis (1 to 3 pro mille). Nawratzki in recent examinations found amounts varying between 0.047 and 0.170 per cent., but the subjects of his investi- gation had fever at the time. Lichtheim claims to have found glucose — ^by means of the phenyl- hydrazin test — in all cases of tumor which he examined. In cases ' Stadelmann, Mitth. a. d. Grenzgeblet. d. Med. u. Chir., vol. ii. Comba, Clin, med., 1899 (cited in Arch. d. M^d. d. Enfants, 1900). Lenhartz, Verhandl. d. XIV. Cong, f. inn. Med., 1900. 36 562 THE CEREBROSPINAL FLUID. of tubercular meningitis, on the other hand, a positive result was only exceptionally obtained. Quincke also reports that he was able to demonstrate the presence of sugar whenever the liquid obtained was sufficient in amount for the necessary tests. Unfortunately, however, he does not detail his cases. Concetti found no sugar in hydrocephalic fluid. The experience of other observers does not agree with that of Lichtheim and Quincke ; and Fiirbringer,^ who has thus far reported the largest number of spinal punctures, found sugar in only two cases of diabetes associated with tuberculosis. According to Gumprecht, the normal cerebrospinal fluid also con- tains traces of cholin. Microscopical Examination. — The microscopical examination of the fluid withdrawn by spinal puncture is most important. Under normal conditions, as well as in cases of tubercular men- ingitis, tumor, abscess, acute and chronic hydrocephalus, only a few leucocytes and endothelial cells from the subarachnoid spaces are usually found, enclosed in extremely delicate meshes of fibrin. In purulent meningitis, on the other hand, leucocytes are present in large numbers, and in some instances even pure pus may be obtained. Most important from a diagnostic standpoint is the fact that patho- genic micro-organisms may be found. Lichtheim, Fiirbringer, Frey- han, Dennig, and Frankel were thus able to demonstrate the presence of tubercle bacilli in a fairly large number of cases of tubercular meningitis. Other observers, it is true, have been less fortunate, but the fact that Fiirbringer found tubercle bacilli in thirty cases out of thirty-seven is certainly significant. Schwarz states that he ob- tained positive results in sixteen out of twenty-two cases, and Slawyk and Manicatide found bacilli in all of nineteen cases (six- teen times by direct microscopical examination, and three times by the animal experiment). In order to examine for tubercle bacilli, the fluid should be placed on ice for from six to twenty-four hours, until a slight coagulum has formed, when the fine, spider-web-like threads of fibrin are transferred to a cover-slip, spread in as thin a layer as possible, and stained as described in the chapter on the Sputum. If a centrifugal machine is available, the examination may, of course, be made at once ; the chances of finding the bacilli are then also much greater. In every case a large number of speci- mens should be prepared before the search is abandoned. Only a positive result, however, is of value, and in doubtful cases recourse should be had to the animal experiment.^ In the diagnosis of epidemic cerebrospinal meningitis lumbar puncture is of signal value, as the Diphcocous meningitidis intracellu- laris of Weichselbaum-Jager can be demonstrated in a large per- > Furbringer, Verhandl. d. XV. Cong. f. inn. Med., 1901. 2 Fiirbringer, loe. cit. Wentworth, Arch, of Pediat., Nov., 1899. MICROSCOPICAL EXAMINATION. 563 centage of cases. Councilman^ thus states that during a recent epidemic of the disease in Boston lumbar puncture was performed in fifty-five cases, and that in the fluid obtained the diplococci were found on microscopical examination or in culture in thirty-eight cases. The average time from the onset of the disease before spinal puncture was made was seven days in the positive cases, and seven- teen days in the negative cases. The longest time after the onset in which a positive result was obtained was twenty-nine days. Similar results have also been reached by other observers.^ The organism in question is a diplococcus, each hemisphere being of about the same size as the ordinary pathogenic micrococci. It is readily stained with the usual dyes, and decolorized by Gram's method. Short chains of from four to six and tetrads may at times be seen. It grows best upon Loffler's blood-serum mixture, form- ing round, whitish, shining, viscid-looking colonies, with smooth, sharply defined outlines, which may attain a diameter of from 1 to IJ mm. in twenty-four hours. Their cultivation upon plain agar, glycerin-agar, and in bouillon is less reliable. In order to obtain the best results, it is necessary to use large amounts of the exudate, and to make a number of cultures, as many of the organisms are usually dead, or at least will not grow. In ordinary cover-slip preparations they are often numerous, and are found enclosed in the polynuclear leucocytes. Their number then varies considerably. On the one hand, only one or two may be present in a cell, while in others they may be so closely packed as to obscure the nucleus. Mixed infections are not uncommon in epidemic cerebrospinal meningitis. Councilman thus found the pneumococcus in seven cases, and Friedlander's bacillus in one. Terminal infections with staphylococci and streptococci also occur. In other forms of purulent meningitis a large variety of organisms has been found. Wolf gives the following figures, resulting from an analysis of 174 cases, in which epidemic cerebrospinal meningitis is, however, included : in 44.23 per cent, the pneumococcus was found ; in 34.48 per cent, the Diplococcus meningitidis intracellularis ; in 3.45 per cent, staphylococci; in 8.03 per cent, streptococci, in 1.13 per cent, the bacillus of Friedlander ; in 2.87 per cent, the Bacillus typhosus; in 1.72 per cent, the bacillus of Neumann-Schaifer, and in 2.87 per cent, the Bacillus coli communis, the Bacillus pyogenes foetidus, the Bacillus aerogenes meningitidis, and the Bacillus mallei, while no bacteria were found in 1.15 per cent, of the cases. 1 W. T. Councilman, " Cerebrospinal Meningitis," Johns Hopkins Hosp. Bnll., 1898, p. 27 ; and Phila. Med. Jour., 1898, p. 937. W. T. Councilman, F. B. Mallory, and J. H. Wright, " Epidemic Cerebrospinal Meningitis," Am. Jour. Med. Sci., 1898, p. 252. 2 W. Osier, "The Cavendish Lecture on the etiology and Diagnosis of Cerebro- spinal Fever," Phila. Med. Jour., 1899, p. 26. E. Stadelmann, " Meningitis Cerebro- spinalis," Zeit. f. klin. Med., vol. xxxviii. p. 46. E. Neurath, Centralbl. f. d. Grenz- gebiete d. Med. u. Chir., 1897, vol. i. CHAPTEK XI. THE SEMEN. The ejaculated semen is a mixture of the secretions furnished by the testicles, the prostate gland, the seminal vesicles, and the glands of Cowper. GENERAL CHARACTERISTICS. Semen is white or slightly yellowish in color, semifluid, sticky, and of an opaque, non-homogeneous, milky appearance, which is due to the presence of white, opaque islets floating in the otherwise clear fluid ; these consist almost entirely of the specific morphological elements of the semen, the spermatozoa. Its odor, which strongly resembles that of fresh glue, is characteristic, and is owing to the presence of spermin. It is generally attributed to an admixture of prostatic fluid, as the semen obtained from the vasa deferentia is odorless. According to Robin, however, this odor is produced only at the moment of ejaculation, and cannot be ascribed to any single one of the secretions present. The reaction of human semen is slightly alkaline, and its specific gravity greater than that of water, in which it sinks to the bottom. CHEMISTRY OF THE SEMEN Accurate analyses of human semen or of mammalian semen do not exist, and only the old analyses of Vauquelin and KoUiker can be given : Man. Water 90 Albuminous material 1 Extractives .... [•.,.. g Ethereal extract . . J Mineral material 4 The mineral matter consists largely of calcium phosphate. If semen is kept, or if it is slowly evaporated, crystals of phos- phate of spermin separate out, which are commonly known as Bottcher's crystals, and which were long regarded as identical with the so-called Charcot-Leyden crystals that are found in the sputum of bronchial asthma, in the blood of leukaemia, in the stools in cases of helminthiasis, etc. 564 Horse. Ox. 81,90 82.10 " 15.30 16.45 2.20 iiei 2.60 MICROSCOPICAL EXAMINATION OF THE SEMEN. 566 Spermin is a basic substance, and, according to Ladenburg and Abel, is closely related to, if not identical with, diethylene diamin (piperazin) : The phosphate crystallizes in the form of monoclinic four-sided spindles or prisms, which appear as flattened needles of variable size. Some are scarcely visible even with a fairly high power of the microscope, while others attain the length of 40 /z to 60 fi. The substance is soluble in formol, thus differing from Charcot- Leyden crystals. In water it dissolves with difficulty ; it is slowly soluble in acids and alkalies, even in ammonia, while it is insoluble in alcohol, ether, chloroform, and dilute saline solution. Florence's reagent (see below) colors the crystals a bluish black. According to Cohn, the Bottcher crystals are formed exclusively in the prostate gland, the gland itself furnishing the basic component, while the necessary phosphoric acid is derived from other portions of the reproductive apparatus.^ MICROSCOPICAL EXAMINATION OF THE SEMEN. Upon microscopical examination normal semen is seen to contain mnumerable actively moving thread-like bodies, measuring from 50 /I to 60 // in length — the spermatozoa. These consist, of an egg- FiG. 131. Human semen. a, Spermatozoa; 6, Cylindrical eplthelinm; «■ B°^iff, .«°«'°4'°f„ii^"' ^Ssllt?"^^^^^ Squamous epithelium from the uretlira; d', Testicle-cells ; e. Amyloid corpuscles , j, oper matic crystals ; g, Hyaline globules, (v. Jaksch.) shaped head, when seen from above, which is from 3 // to 5 ^ in length, the broader end being directed anteriorly ; a middle portion, 4 /i to 6 /^ in length, with which the head is united by its smaller 1 Th. Cohn, "Zur Kenntniss d. Spermas," Centralbl. f. allg. Path. u. path. Anat., vol. X. pp. 940-949. 566 THE SEMEN. end ; and a posterior piece or tail, into which the middle piece grad- ually fades (Fig. 131). In addition to the spermatozoa a few hyaline bodies are seen which are derived from the seminal vesicles ; further, numerous small pale granules of an albuminous nature, some testicular and urethral epi- thelial cells, lecithin-corpuscles, and so-called prostatic or amyloid corpuscles, which at first sight resemble starch-granules in appearance, owing to their concentric striations. A few leucocytes and occasion- ally a few red corpuscles may also be found. PATHOLOGY OF THE SEMEN. The study of the semen has received little attention from clini- cians, and gynecologists frequently hold the wife responsible for sterility when an examination of the husband's semen would — according to Kehrer,^ in 40 per cent. — reveal an absence of sperma- tozoa, constituting the condition usually spoken of as azoospermaMsm. This may be temporarily observed following veliereal excesses, when the fluid finally ejaculated is almost entirely of prostatic origin ; their absence then possesses no significance, but persistent azoosper- matism must of necessity be associated with sterility.^ Cases have been recorded in which, notwithstanding the presence of spermatozoa and apparently normal sexual conditions in both husband and wife, sterility existed nevertheless, but in which it was observed that the spermatozoa lost their motile power almost imme- diately after ejaculation. Under normal conditions, following inter- course actively moving spermatozoa may be found in the vagina after hours, days, and even weeks. Whenever it is deemed advisable to make an examination of the semen, this should be done immediately following ejaculation, or as soon as possible thereafter. Note should then be taken, not only of the presence, but also of the degree of motility of the spermatozoa ; a drop of the semen is mixed with a drop of normal (0.6 per cent.) saline solution, and examined at once with the microscope. Bloody semen, constituting the condition spoken of as hcenw- spermia, has been observed on several occasions. It may follow excessive sexual indulgence, but may also occur in connection with gonorrhoeal epididymitis. The blood is readily recognized upon microscopical examination. THE RECOGNITION OF SEMEN IN STAINS. In medico-legal cases the physician may be called upon to decide whether or not certain stains on body-linen are caused by spermatic ' Kehrer, Beitrage z. klin. u. exper. Gynaek., 1879, vol. ii., Giessen. » Furbringer, Zeit. f. klin. Med., 1881, vol. iii. p. 310. THE RECOGNITION OP SEMEN IN STAINS. 667 fluid, whether or not a rape has been committed, etc. In such cases it is frequently only necessary to examine a drop of the vaginal fluid in order to arrive at a positive result at once. At other times, however, recourse must be had to the following method : a fragment of the linen or scrapings from the vulva or vagina are placed in a watch-crystal and allowed to soak for at least one hour in from 27 to 30 per cent, alcohol, when a bit of the material is teased in a solution of eosin in glycerin (1 : 200), and examined. The heads of the spermatozoa are thus stained a deep red, while the tails, which are often broken, exhibit a pale-rose tint, and can readily be distinguished from vegetable fibres, which do not take the stain at all. A positive statement can thus be made in every case, even after months and years, as spermatozoa not only resist the action of reagents, but also the process of putrefaction ; this is prob- ably owing to the large proportion of mineral matter which enters into their composition, and which insures preservation of their form. Instances have been recorded in which it was possible to demon- strate spermatozoa in stains after eighteen years. The semen test of Florence^ has attracted much attention, and may be recommended in doubtful cases ; only a negative result, however, is of value (see below). It is based upon the observation that very characteristic crystals of iodospermin are formed when spermatic fluid is treated with a solution of iodo-potassic iodide containing 1.65 grammes of pure iodine and 2.54 grammes of potassium iodide, dissolved in 26 c.c. of water. When a drop of this solution is added to a drop of spermatic fluid or an aqueous extract of a seminal stain, dark-brown crystals of iodospermin sepa- rate out at once, and may be readily recognized under the microscope. They occur in the form of long rhombic platelets or fine needles, often grouped in rosettes, but also occurring singly or as twin crystals. The examination with the microscope should be made at once after addition of the reagent, as the crystals disappear on standing. As l£e reaction may also be obtained in cases of azoosperma- tism, and with pure prostatic secretion, while a negative result is obtained with the fluid from spermatoceles, it is manifest that the test is not applicable for the determination of the presence or ab- sence of spermatozoa per se. Posner^ states that he obtained similar crystals when the test was applied to a glycerin extract of ovaries. More recently Richter ^ has shown that Florence's reaction is also obtained with a decomposition-product of lecithin, viz., cholin, ' Florence, " Du sperme et des taches de sperme en m^decine legale," Arch. d'Anthrop. crimin., vols. X. and xi. 2 C. Posner, " Die Florence'sche Eeaktion," Berlin, klm. Wocb., 1897, P- 60^- ' M. Eichter, "D. mlkrochemische Nachweis v. Sperma," Wien. klin. Woch., 1897, p. 569. 568 THE SEMEN. which would explain the observation that better results are com- monly obtained with dried semen than with fresh material. But it follows also that the reaction cannot be a specific semen reaction, and Richter accordingly concludiss that a negative result only is of value, and indicates that the material under examination is not semen. He states that he obtained positive results with vaginal and uterine mucus, with decomposing brain-substance, and other organs as well. CHAPTER XII. VAGINAL DISCHARGES. GENERAL CHARACTERISTICS. The secretion which is normally furnished by the vaginal glands is small in amount, and just sufficient to keep the mucous mem- brane moist. It is a clear or somewhat milky-looking, semiliquid material, in which numerous epithelial laminae, which have been thrown off during the normal process of desquamation, may be found. It has been stated that the reaction of the vaginal secretion in virgins is invariably acid, while an alkaline reaction is the rule in the dtfxyr&es. During pregnancy, however, the secretion is probably always acid. In iive hundred cases which Kronig examined in this direction an alkaline reaction was never observed. According to Zweifel, the vaginal secretion contains traces of trimethylamin.' Microscopically, numerous epithelial cells, mucous corpuscles, a few large mononuclear leucocytes cellular detritus, and bacteria are found (Fig. 132). Doderlein^ has described a non-pathogenic Fi8. 132. Vaginal Becretion. u, Mucous corpuscles ; 6, Vaginal epithelium ; c, Epithelium from vulva. bacillus or a group of bacilli which are characterized by the fact that they give rise to marked acid fermentation of sugar, and he regards these organisms as the only ones which are constantly present in the normal vagina. Kronig and Menge, however, state 1 Zweifel, Arch. f. Gynaek., 1881, vol. xviii. p. 359. 2 DodeTlein, Ibid., 1887, vol. xxxi. p. 412. 569 570 VAGINAL DISCHARGES. that they are often absent. These observers have found, on the other hand, that under normal conditions there are various bacilli and cocci present which belong to the class of obligatory anaerobes, and are likewise non-pathogenic. Unfortunately they have not described these organisms in detail. Near the outlet they found bacteria which may be cultivated upon alkaline aerobic culture- media, but which are usually absent in the upper portion of the vagina. It is important to note that various diplococci may also be found under normal conditions, and care should be taken not to confound these with gonococci. Like the gonococci, they are decolorized by Gram's method. If the various characteristics of the former be borne in mind, however, mistakes may probably always be avoided ; but in married women and in children it would be best to make the diagnosis of gonorrhoea only when the gonococcus has been iso- lated by cultivation. The question whether or not pathogenic bacteria niay occur in the normal vagina of pregnant or non-pregnant women, may be an- swered in the affirmative ; although it must be admitted that with the exception of the gonococcus they are only exceptionally found.^ The vaginal secretion has been shown to possess powerful bac- tericidal properties, so that pathogenic organisms, even when artificially introduced into the vagina, are rapidly killed. Kronig thus found that the Bacillus pyocyaneus disappears from the vagina of pregnant women in from ten to thirty hours, the staphylococci in from six to thirty-six hours, and the Strepto- coccus pyogenes within six hours. Important from a practical standpoint is the fact that the bacteria disappeared less rapidly when irrigation of the vagina with water or even antiseptics was employed. Of animal parasites, the Trichomonas vagiyialis is apparently the only one which may be encountered in the vaginal discharge. The organism is identical with the trichomonas found in the feces and the urine. In this country it is not often observed, while it is common among the peasant population of Central Europe. As far as is known, the organism is of no pathological significance, and may occur both under normal and pathological conditions. From a medico- legal standpoint, however, its presence may not be unimportant, as cases are on record in which trichomonades have been confounded with spermatozoa. In my judgment, however, such a mistake can only occur if the observer is without microscopical training. 1 Doderlein, Das Scheidenseoret, Leipzig, 1892. J. W. Williams, " Bacteria of the Vaginal Secretion of the Pregnant Woman," Am. Jour. Obstet., 1898, vol. xxxviii. "The Bacteria of the Vagina and Their Practical Significance," Trans. A.m. Gyn. Soc, 1898. " The Cause of the Conflicting Statements concerning the Bacterial Con- tents of the Vaginal Secretion of the Pregnant Woman," Am. Jour. Obstet., 1898. VAGINAL BLENNORRH(EA—THE LOCHIA. 571 VAGINAL BLENNORRHCEA. In physiological conditions an increased vaginal secretion is ob- served during sexual excitement, especially during coitus, just pre- ceding and at the beginning of menstruation, and during preg- nancy, when a profuse blennorrhoja is frequently seen, which often assumes a virulent character. The secretion under' such conditions readily becomes purulent. When not dependent upon a gonorrhceal infection the secretion is thicker than normal, white, and creamy. At times also the vaginal catarrh observed in pregnancy is com- plicated with mycosis, when white or yellowish-gray patches may be seen at the orifice of the vagina ; the latter may, indeed, even be filled with particles which consist entirely of fungi. MENSTRUATION. At the beginning of menstruation, as has been pointed out above, an increase in the amount of vaginal secretion is observed, in which leucocytes, prismatic epithelial cells coming from the uterus, as well as the usual vaginal cells, may be seen upon microscopical exami- nation. Later the secretion becomes sanguineous in character, and finally only epithelial cells, leucocytes, and gi-anular detritus are encountered, the cells usually showing evidence of fatty degenera- tion. The amount of blood lost at each menstrual period amounts to about 200 grammes in perfectly healthy females. THE LOCHIA. The lochia during the first day following parturition are red in color — the lochia rubra — and emit the characteristic sanguineous odor. At this time a microscopical examination will reveal an abundance of red corpuscles, some leucocytes, and a variable number of epithelial cells, which are almost exclusively of vaginal origin. On the second and third days the number of red corpuscles dimin- ishes, while the leucocytes increase in number. Still later the dimi- nution in the red and the increase in the white corpuscles become more marked, and the discharge at the same time assumes a grayish or white color, until about the tenth day the red . corpuscles have almost entirely disappeared, while the leucocytes and epithelial cells are abundant. Finally, the secretion becomes thicker, mucoid, and milky white in color — the lochia alba^ which condition may persist for from three to four weeks in nursing-women, and still longer in those who do not nurse, until finally the normal secretion is again established. Numerous bacteria are encountered in the lochia, and it is curious to note that among these pus-organisms are quite con- stantly present without giving rise to symptoms. When a portion 572 VAGrNAL DISCHARGES. of the placenta or membranes have been retained the lochia soon give off a fetid odor, and assume a dirty brownish color ; the reten- tion of blood-clots alone may also produce this result. In such cases the lochia swarm with bacteria of all kinds.^ VULVITIS AND VAGINITIS. In cases of vulvitis and vaginitis a marked increase is observed in the number of the leucocytes and epithelial cells, the character of the latter depending, essentially of course, upon the portion of the genital tract affected. Red corpuscles are also met with at times ; their number generally stands in a direct relation to the intensity of the inflammatory process. In some instances epithelial casts of the entire vagina have been observed, constituting the condition termed vaginitis exfoliativa. The condition, however, is rare. The discharge of large amounts of pure pus through the vagina points to perforation of an abscess of the genital organs or of the neighboring structures into the uterus or the vagina ; it is of rare occurrence. Much more common is the discharge of fecall matter or of urine through this channel, indicating the existence of a vagino-reetal or vagino-vesical fistula. MEMBRANOUS DYSMENORRHCEA. While ordinarily, during menstruation, shreds of desquamated uterine lining are frequently encountered, it is rare to meet with large pieces or complete casts of the uterus, the elimination of which is usually associated with the symptoms of a severe dysmenorrhcea, constituting the condition spoken of as membranous dysmenorrhcea. CANCER. While the diagnosis of malignant growth of the uterus is probably never based upon a microscopical examination of the vaginal dis- charge alone, it may be mentioned that in advanced cases this is pos- sible, as fragments of an epithelioma of the cervix, for example, may frequently be detected upon microscopical examination (Fig. 133). In suspected cases small pieces of tissue should be removed and examined according to usual histological methods.^ GONORRHCEA. In suspected cases of gonorrhoea an examination of the vaginal and urethral discharge for the presence of gonococci is important, as it is practically impossible to diagnose this condition posi- tively in any other manner. Care should be taken, however, not to ' Doderlein, loc. eit. Thomen, Centralbl. f. d. med. Wlss., 1890, vol. xxviil. p. 537; and Arch. f. Gyn., 1889, vol. xxxvl. p. 231. 2 T. S. CuUen, Cancer of the Uterus, Appleton & Co., 1900. ABORTION. 573 Fig. 333. Vaginal eecretion from a case of epithelioma of the cervix uteri. confound the diplococci which may be normally present in the urethra and vagina with gonococci (see chapter on the Urine). ABORTION. In cases of abortion it is often possible to discover chorion villi in the expelled blood-clots which present the characteristic capillary Fig. 134. Chorion villi. 674 VAGINAL DISCHARGES. network (Fig. 134), and often manifest signs of advanced fatty degeneration. Important also from a diagnostic point of view is the Fig. 135. Decidual cells. presence of decidual cells (Fig. 135), which are characterized by their large size, their round, polygonal, or spindle-like form, and their characteristic nuclei and nucleoli. CHAPTEE XIIL THE SECRETION OP THE MAMMARY GLANDS. THE SECRETION OF MILK IN THE NEWLY BORN. A SECRETION from the mammary glands of the male is observed only in the newly born, if we except those rare cases in which adult males were known to suckle infants. The fluid in question, which may also be obtained from the female infant, is termed " Hexenmilch " (witches' milk) by the Germans. Qualitatively it has the same composition as milk, but may manifest considerable quantitative variations. COLOSTRUM. Aside frcpi those curious instances in which a secretion of milk has been observed in non-pregnant women, mammary activity is essentially connected with the physiological phenomena of pregnancy and parturition. Often as early as the third month a small drop of a serous-looking fluid can be obtained from the nipple by pressure upon the breasts. Immediately after delivery a variable amount of fluid is secreted, which is watery, semi-opaque, mucilaginous, and of a yellowish color. To this secretion, as well as to that observed during pregnancy, the term colostrum has been applied. It is dis- tinguished from true milk by its physical characteristics and by the presence of a greater proportion of sugar and salts. The fluid, moreover, coagulates upon boiling. An idea may be formed of its composition from the appended table : 4 weeks before birth. 17 days be- 9 days be- fore birth. 24 hours after birth. 2 days I. II. Water . . . 945.2 852.0 851.7 858.8 843.0 867.9 Solids . . 54.8 148.0 148.3 141.2 157.0 132.1 Casein . 21.8 Albumin . . 28.8 69.0 74.8 80.7 . . . Fat . 7.3 41.3 30.2 23.5 '48.6 Lactose 17.3 39.5 43.7 36.4 61.0 Salts .... 4.4 4.4 4.5 5.4 5.i Upon microscopical examination fat^droplets, a few leucocytes, some epithelial cells, and so-called oolostrum-oorpuscles are found. 575 576 THE SECRETION OF THE MAMMARY QLANDS. Fig. 136. Colostrum of a woman in sixth month of pregnancy. (Eye-piece III., ohj. 8 A., Reiohert.) {v. Jaksch.) The latter are highly refractive bodies, of irregular size, whose inte- rior is filled with fatty granules (Fig. 136). LiTEEATUEE.— G. Woodward, "Chemistry of Colostrum Milk," Jour. Erper. Med., vol. ii. p. 217. THE SECRETION OF MILK PROPER, IN THE ADULT FEMALE. The secretion of milk proper usually begins about the third day following parturition, and may continue for a variable length of time. On the one hand, the amount of milk secreted may be so small as to be insufficient for the needs of the child, so that lacta- tion may have to cease after several days; on the other hand, women are not infrequently seen who nurse their children for two years and even longer. Usually infants are nursed until six or seven teeth have appeared, which period varies with the individual child^ averaging about the eleventh month. HUMAN MILK. Human milk is of a bluish color, and differs in this respect from the milk of cows. Its reaction is alkaline. The specific gravity may vary between 1.026 and 1.035, one between 1.028 and 1.034 being the most common. The amount of milk secreted in twenty- four hours varies from 500 to 1500 c.c. Microscopically, it is a fairly homogeneous emulsion of fat, and is practically destitute of cellular elements. From the following table an idea may be formed of its chemical composition : Biehl. Gerber. Christenm. Pfeiffer. Pfeiffer. Mendes de Leon Water Solids Albumin .... Fat Lactose Salts 876.00 124.00 22.10 88.10 60.90 2.90 891.00 109.00 17.90 33.00 53.90 4.20 872.40 127.60 19.00 43.20 59.80 2.60 892.00 108.00 16.13 32.28 57.94 1.65 890.60 109.40 17.24 29.15 59.92 2.09 877.90 '25.30 38.90 55.40 2.50 THE MILK IN DISEASE. 577 Upon comparing this table with the following analysis of cows' milk it will be seen that the latter contains more albumin and less sugar than human milk. Human milk, moreover, is relatively deficient in mineral matter, and especially in calcium salts and phosphoric acid : Water > . .... 874.2 Solids . . . . . 125.8 Casein . . 28.8 \„,^ Albumin .... . 5.3 j Fat 36.'6 Lactose. . . . . 48.1 Salts . 7.1 The albumins found in milt-plasma are casein, lactoglobulin, and lactalbumin. It is claimed by some observers that the casein of human milk differs from that obtained from cows' milk. The casein-coagula in human milk are not so large and dense as those observed in cows' milk. Human casein, moreover, is not so readily precipitated by acids and salts ; it does not always coagulate upon the addition of rennet ferment, and while it may be precipitated by the gastric juice, it is readily dissolved by an excess. Although accurate analyses of human casein are not available, it is probable that the two forms are not identical (Hammarsten). The question whether or not normal human milk contains micro- organisms may now be answered in the affirmative. There can be no doubt, however,- that the milk as it is secreted by the healthy gland is sterile, but upon passing along the lacteal ducts in the nipple it is always contaminated by the Staphylococcus epidermidis albus (Welch). This micro-organism must be regarded as a constant inhabitant of the skin, and is the only one of the cutaneous bacteria which regularly penetrates into the deeper layers of the epidermis and into the glandular appendages of the skin. It is thus at once apparent why this organism is so constantly met with, and is prac- tically the only one found in normal human milk. Exceptionally the Staphylococcus pyogenes aureus is found. THE MILK IN DISEASE. The chemistry of the milk in pathological conditions has received little attention. It appears, however, that the milk of women when ill usually contains less fat, and that the proportion of lactose is diminished. In cases of jaundice the presence of bile-pigment and of biliary acids has not been satisfactorily demonstrated. In cases of mammary tumors bloody secretion has been observed in rare cases, the nipple itself being intact. Microscopically, an admixture of leucocytes is observed in various diseases of the breast, and especially in cases of abscess. Of patho- genic micro-organisms, streptococci may be found in cases of puer- 37 678 THE SECRETION OF THE MAMMARY GLANDS. Fig. 137. peral fever ; more commonly, however, they are absent. The typhoid bacillus has occasionally been seen in cases of typhoid fever, and it is interesting to note that the specific agglutinins of typhoid fever have been noted in the milk. Pneumococci have been obtained from the milk of pregnant women affected with lobar pneumonia. The important question whether or not tubercle bacilli are elimi- nated in the milk in cases of phthisis cannot be definitely answered. In cows such an occurrence is certainly common, even when there is no demon- strable tubercular lesion of the udder. So far as I have been able to ascertain, however, tubercle bacilli have never been found in human milk.^ A blue and a red color have been observed in the milk of cows, owing to the presence of the Bacillus pyocyaneus and the Micrococcus prodig- iosus, respectively. A chemical examination of human milk should always be made whenever it is apparent that the nutrition of the baby is below normal. Valuable dietetic suggestions may thus be ob- tained. In other cases, as when the mother is unwilling or unable to nurse her child beyond a certain period, a knowledge of the composition of her milk will enable the physician to give specific instructions regarding the proper modification of cows' milk. If a wet-nurse is to be employed, her milk should likewise be examined. Most important is the determination of the specific gravity and of the amount of fat. The former may vary between 1.029 and 1.033. The amount of fat should not be less than 3 per cent. Determination of the Specific Gravity. The specific gravity is best determined with the lactodensimeter of Quevenne (Fig. 137). As the instrument is graduated for a temperature of 60° F., it is necessary to correct the specific grav- ity whenever the temperature is above or is below this point. In the following tables the corrected specific gravity may be found corresponding to temperatures ranging from 46° to 75° F. : ' Escherich, Fortschr. d. Med., 1885, vol. iii. p. 321. KarUnski, Wien. med. Wooh., 1888, vol. xxxvlii. No. 28. Ott, Prag. med. Woch., 1892, vol. xvii. p. 145. Cohn u. Neumann, Virchow's Archlv, 1880, vol. oxxvl. p. 187. Quevenne's lactoden- bimeter. THE MILK IN DISEASE. CORKECTIONS FOR TeMPERATDRB. 579 Specific grsTity. Degrees of thermometer (Fahrenheit). 46 47 48 49 50 51 52 53 54 65 1020 19.0 19.1 19.1 19.2 19.2 19.3 19.4 19.4 19.5 19.6 20.0 20.0 20.1 20.2 20.2 20.3 20.3 20.4 20.5 20.6 1022 21.0 21.0 21.1 21.2 21.2 21.3 21.3 21.4 21.5 21.0 22.0 22.0 22.1 22.2 22.2 22.3 22.3 22.4 22.5 22.6 1024 22.9 23.0 23.1 23.2 23.2 23.3 23.3 23.4 23.6 23 6 23.9 24.0 24.0 24.1 24.1 24.2 24.3 24.4 24.5 24.6 24.9 24.9 25.0 25.1 25.1 25.2 25.2 25.3 25.4 25.5 25.9 25.9 26.0 26.1 26.1 26.2 26.2 26.3 26.4 26.5 1028 26.8 26.8 26.9 27.0 27.0 27.1 27.2 27.3 27.4 27.5 1029 27.8 27.8 27.9 28.0 28.0 28.1 28.2 28.3 28.4 28.5 1030 28.7 28.7 28.8 28.9 29.0 29.1 29.1 29.2 29.4 29.4 29.6 29.6 29.7 29.8 29.9 .W.O 30.1 30.2 30.3 30.4 1032 30.5 30.5 30.6 30.7 30.9 31.0 31.1 31.2 31.3 31.4 31.4 31.4 31.5 31.6 31.8 31.9 32.0 • 32.1 32.3 32.4 1034 32.3 32,3 32.4 32.5 32.7 32 9 33.0 33.1 33.2 33.3 33.1 33.2 33.4 33.5 33.6 33.8 33.9 34.0 34.2 34.3 Specific Degrees of thermometer (Fahrenheit). gravity. 56 57 68 59 60 61 62 63 64 65 1020 19.7 19.8 19.9 19.9 20.0 20.1 20.2 20.2 20.3 20.4 1021 20.7 20.8 20.9 20.9 21.0 21.1 21.2 21.3 21.4 21.6 1022 2*.7 21.8 21.9 21.9 22.0 22.1 22.2 22.3 22.4 22.5 1023 22.7 22.8 22.8 22.9 23.0 23.1 23.2 23.3 23.4 23.5 1024 23.6 23.7 23.8 23.9 24.0 24.1 24.2 24.3 24.4 24.5 1025 24.6 24.7 24.8 24.9 25.0 25.1 25.2 25.3 25.4 25.5 1026 25.6 25.7 25.8 25.9 26.0 26.1 26.2 26.3 26.5 26.6 1027 26.6 26.7 26.8 26.9 27.0 27.1 27.3 27.4 27.5 27.6 1028 27.6 27.7 27.8 27.9 28.0 28.1 28.3 28.4 28.5 28.6 1029 28.6 28.7 28.8 28.9 29.0 29.1 29.3 29.4 29.5 29.6 1030 29.6 29.7 29.8 29.9 30.0 30.1 30.3 30.4 30.5 30.7 1031 30.5 30.6 30.8 30.9 31.0 31.2 31.3 31.4 31.5 31.7 1032 31.5 31.6 31.7 31.9 32.0 32.2 32.3 32.5 32.6 32.7 1033 32.5 32.6 32.7 32.9 33.0 33.2 33.3 33.5 33.6 33.8 1034 33.5 33.6 33.7 33.9 34.0 34.2 34.3 34.5 34.6 34.8 1035 34.5 34.6 34.7 34.9 35.0 35.2 35.3 35.5 35.6 35.8 Specific Degrees of thermometer (Falirenheit). gravity. 66 67 68 69 70 71 72 73 74 75 1020 20.5 20.6 20.7 20.0 21.0 21.1 21.2 21.3 21.5 21.6 1021 21.6 21.7 21.8 22.0 22.1 22.2 22.3 22.4 22.5 22.6 1022 22.6 22.7 22.8 23.0 23.1 23.2 23.3 23.4 23.5 23.7 1023 23.6 23.7 23.8 24.0 24.1 24.2 24.3 24.4 24.6 24.7 1024 24.6 24.7 24.9 25.0 25.1 25.2 25.3 25.5 25.6 25.7 1025 25.6 25.7 25.9 26.0 26.1 26.2 26.4 26.5 26.6 26.8 1026 26.7 26.8 27.0 27.1 27.2 27.3 27.4 27.6 27.7 27.8 1027 27.7 27.8 28.0 28.1 28.2 28.3 28.4 28.6 28.7 28.9 1028 28.7 28.8 29.0 29.1 29.2 29.4 29.5 29.7 29.8 29.9 1029 29.8 29.9 30.1 30.2 30.3 30.4 30.5 30.7 30.9 31.0 1030 30.8 30.9 m.\ 31.2 31.3 31.5 31.6 31.8 31.9 32.1 1031 31.8 32.0 32.2 32.2 32.4 32.5 32.6 32.8 33.0 33.1 1032 32.9 33.0 33.2 33.3 .33.4 33.6 33.7 33.9 34.0 34.2 1033 33.9 34.0 34.2 34.3 34.5 34.6 34.7 34.9 35.1 35.3 1034 34.9 35.0 3.1.2 3.5.3 35.5 35.6 35.8 36.0 36.1 36.3 1035 36.9 36.1 36.2 36.4 36.5 36.7 36.8 37.0 37.2 37.3 580 THE SECRETION OF THE MAMMARY GLANDS. Estimation of the Fat. The estimation of the fat is most conveniently made by means of the lactoscope of Feser, shown in Fig. 138. Milk is drawn into Fig. 138. Feser's lactoscope. the pipette up to the mark M, when it is emptied into the cylinder C. The pipette is then rinsed with water and the washings added to the milk. "While shaking, water is added mitil the black lines upon the milk-colored glass plug A can just be discerned. The fig- ure upon the right of the scale at the level reached by the mixture indicates the percentage-amount of fat, while the number upon the left indicates in cubic centimeters the amount of water that has been added. Estimation of the Proteids. Woodward's Method.— Two "milk-burettes" (see Fig. 139), each containing 5 c.c. of milk, are kept at a temperature of from 37° to 40° C. for from eighteen to twenty-four hours. At the end of THE MILK IN DISEASE. 581 this time the milk has separated into two layers, viz., an upper layer of viscid yellow fat, and a lower layer of fluid milk, which is quite opaque above and almost translucent below. Clinging to the sides of the tube, and especially at the bottom, a granular precipitate will be seen. The burettes are then cooled, when the milk-serum IS Fig. 139. Woodward's milk-burette. •withdrawn into two tubes graduated to 15 c.c, and treated with Esbach's reagent to the 15 c.c. mark. The mixture in each tube is thoroughly stirred with a glass rod and then centrifugated to a con- stant reading. Woodward ' has checked his analyses by Kjeldahl's method, and has obtained satisfactory results. 1 G. Woodward, "A Clinical Method for the Estimation of Breast-milk Proteids," Phila. Med. Jour.,'l898, p- 956. INDEX. ABORTION, vaginal discharge in, 572 Abscess of the liver with perfora- tion into the lung, 296 pulmonary, 294 Absorption, rate of, in the stomach, 204 Acetic acid, 191, 218 fermentation, 191 tests for, 191, 218 Acetonsemia, 58 Acetone in the blood, 58 in tlie gastric contents, 195 in the urine, 484 quantitative estimation of, 487 tests for, 486 Aoetonuria, 58, 484 Acholic stools, 223 Acliroodextrin, 139, 180 Acid, acetic, 191, 218 benzoic, 386 butyric,'191, 218 carbolic, 216, 483 diacetic, 489 diazo-benzene-sulphonic, 480 glucuronic, 454 hippuric, 385 homogentisinic, 476 hydrochloric, 155 lactic, 183, 490 oxalic, 392 oxaluric, 392 oxybutyrio, 490 phosphoric, 325 propionic, 218 succinic, 557 sulphuric, 334 tauro-carbarainic, 341 uric, 370 uroleuoinio, 476 valerianic, 218 Acids, organic, in the gastric contents, 183 Actinomyces hominis, 289, 541 Actinomycosis, 289, 541 Adenin in the urine, 371, 383 Agglutinins, 117 a-granulation of Ehrlich, 75 Albumin, aceto-soluble, 409, 422 in the feces, 261 in the gastric contents, 181 in the urine, 398 quantitative estimation of, 422 special test for serum-albumin, 421 for serum-globulin, 424 Albumin, tests for, 415 boiling, 419 nitric acid, 416 picric acid, 421 potassium ferrocyanide, 420 Spiegler's, 421 trichloracetic acid, 420 Albuminimeter, 423 Albuniinuriii, 398 accidental, 408 colliquative, 404 cyclic, 399 Da Costa's, 399 digestive, 407 febrile, 402 functional, 399, 401 hematogenous, 401, 406 in organic diseases of the kidneys, 401, 411 intermittent, 399 mixed, 408 neurotic, 407 orthostatic, 399 physiological, 398 postural, 399 referable to circulatory disturbances, 405 to impeded outflow of urine, 405 renal, 401, 408 toxic, 406 transitory, 399 Albumoses in the blood, 48 in the feces, 261 in the gastric contents, 179 in the urine, 409 tests for, 182, 425 Albumosuria, 409 digestive, 410 enterogenic, 409 hsematogenic, 409 hepatogenic, 409 liistogenic, 409 pyogenic. 409 renal, 409 vesica], 409 Alkaliraeter, Engel's, 24 Alkaline stools, 210, 222 urine, 311 Alkalinity of the blood, 20 distribution of, 104 estimation of, 21 Alkapton in the urine, 475 583 584 INDEX. Alkaptonuria, 475 AUoxur bases in tlie urine, 371, 383 estimation of, 384 Ahu^n's solution, 440 Alveolar epithelium, 279 Ammonia in the blood, 53 in the gastric contents, 194 in the urine, 368 estimation of, 369 Ammoniacal fermentation, 312 Ammonisemia, 53 Ammonio-magnesium phosphate, 504 Ammonium urate, 513 Amoeba coll, 233 in the feces, 233 in the sputum, 283 Amoebae in the urine, 543 Amoebic colitis, 233 Amoebinse in feces, 233 Amphistoraum hominis, 247 Amphoteric urine, 311 Amyloid corpuscles in the semen, 566 Anachlorhydria, 162 Anacidity, hysterical, 163 Anadeny of the stomach, 196 Anaemic degeneration of the red corpus- cles, 63 Anchylostomiasis, 249 Anchylostomum duodenale, 249 Anguillula intestinalis, 251 stercoralis, 251 Angiiilluliasis, 137, 251 Anilin dyes, classification of, 70 water, gentian-violet, 146 Animal gum in the urine, 454 Animal parasites in the blood, 125 in the feces, 231 in the sputum, ^81 in the urine, 542 Annelides, 247 Anthomyia, 232 Anthracosis of the lungs, 297 Anthrax, bacillus of, 121 Arnold's test for acetone, 489 Aronsolin-Philips' stain, 100 Ascarides in the feces, 247 in the urine, 543 Ascaris lumbricoides, 247 maritima, 248 mystax, 248 Asiatic cholera, bacillus of, 252 feces in, 265 Asthma, bronchial, Charcot-Ijeyden crys- tals in, 294 Azoospermatism, 566 BACILLI of Booker, 253 Bacillus acidophilus, 257 butyricus, 201 coli communis, 256 dyaenteriffi, 259 lactis aerogenes, 257 melitensis, 124 Bacillus of anthrax, 121 of cholera Asiatica, 252 of diphtheria, 146 of dysentery, 259 of Finkler and Prior, 253 of glanders, 122 of influenza, 122, 288 of leprosy, 285 of Le Sage, 253 of Malta fever, 124 of Oppler and Boas, 201 of tuberculosis, in the blood, 121 in the feces, 256 in the meningealfluid, 562 in the milk, 578 in the mouth, 143 in the nasal discharge, 267 in the sputum, 283 in the urine, 539 methods of staining, 285 of typhoid fever, in the blood, 114 in the feces, 254 in the urine, 538 of whooping-cough, 288 of yellow fever, 124 ' pyocyaneus, 257 smegma, 285, 288 Bacteria in blood, 113 in exudates, 553 in feces, 213, 251 in gastric contents, 201 in milk, 577, 578 in mouth, 140 in nasal secretion, 267 in pus, 553 in sputum, 283 in urine, 536 in vagina, 570 Bacterial decomposition of the urine, 537 Bacteriuria, 536 idiopathic, 541 Bacterium lactis aerogenes, 257 Balantidium coli, 239 Bang's test for albumoses, 426 for urobilin, 426 Barfoed's reagent, 182 Basic anilin dyes, 71 (foot-note) double stain, 102 phosphate of magnesium, 505, 513 Basophilic leucocytes in the blood, 76 in the sputum, 278 perinuclear granules, 77 Baumann and v. Udranszky's method of isolating diamins, 496 Bence Jones' albumin, 411 tests for, 427 Benzoic acid in the urine, 386 Benzopurpurin lest for hydrochloric acid, 166 _ Bile-pigment in the blood, 57 in the feces, 261 in the gastric contents, 198 INDEX. 585 Bile-pigment in the urine, 469 tests for, 470 Gmeliu's, 471 Huppert's, 470 Eosenbach's, 471 Smith's, 470 Bilharzia hsematobia, 136 Bilharziasis, 136 Biliary acids in the blood, 57 in the feces, 219 in the urine, 471 tests for, 57, 220 concretions, 227 analysis of, 228 Bilirubin, 57, 469, 512 Biuret test, 352 Blood, 17 acetone in, 68 albumins in, 26, 48 albumoses in, 48 alkalinity of, 20 ammonia in, 53 ■ bacteriology of, 113 biliary constituents in, 57 carbohydrates in, 49 cellulose in, 52 chemical examination of, 24 coagulation of, 26 color of, 17 color-ipdex, 34 drying and staining of, 96 fat in, 28, 55 fatty acids in, 55 fibrin in, 26, 48 gases in, 28 general characteristics of, 17 chemistry of, 25 glycogen in, 51 hsemokonia of, 105 in the feces, 223, 230 in the gastric contents, 198 in the sputum, 271, 278 in the urine, 413, 522 .lactic acid in, 55 leucocytes of, 17, 69 medico-legal test for, 44 microscopical examination of, 58 nucleated corpuscles in, 67 odor of, 18 parasites in, 113 parasitology of, 113 peptone in, 48 pigments of, 29 proteids in, 47 protozoa in, 125 reaction of, 20 solids of, 20 specific gravity of, 18 staining of, 96 sugar in, 49 tests for, 44, 198 Donogany's, 199 guaiacum, 429 Blood, tests for, Heller's, 429 Korczynski and Jaworski's, 224 Miiller and Weber's, 198 urea in, 52, uric acid in, 53 xanthin-bases in, 53, 55 Blood-corpuscles, red, 58 ansemic degeneration, of, 63 behavior toward anilin dyes, 63 granular degeneration of, 65 enumeration of, 106 nucleated, 67 variations in color, 62 in form, 59 in number, 60 in size, 58 white (see Leucocytes). Blood-crisis, 67 Blood-iron, 36 Blood-plasma, 17, 24, 27 Blood-plates, 104 Blood-serum, 24, 26, 27 Blood-shadows, 523 Boas' bulbed stomach-tube, 151 method for estimating lactic acid, 188 test for hydrochloric acid, 165 for lactic acid, 187 Boas-Oppler bacillus, 201 Bodo urinarius, 542 Bothriocephalus latus, 243 Bottcher's crystals, 291, 565 Bottger's test for sugar, 440 Bremer's diabetic blood test, 64 urine test, 451 Brodie and Bussell' s method of enumerat- ing the plaques, 110 Bronchial asthma, 275, 293 Bronchitis, acute, 293 chronic, 293 fibrinous, 293 putrid, 293 Browning's spectroscope, 47 Buccal secretion (see Saliva), 138 Butyric acid fermentation, 191 in the feces, 218 in the gastric contents, 191 test for, 191 CADAVERIN, 262,341,495 Cahn-Mehring's method of estimat- ing fatty acids, 192 Calcium carbonate, crystals of, 514 oxalate, crystals of, 292, 503 phosphate, crystals of, 505 sulphate, crystals of, 506 Calliphora erythrocephala, 232 Calomel stools, 208 Carbohydrates, digestion of, 179 in the blood, 49 in the feces, 261 in the urine, 430 tests for, 438 Carbol-fuchsin, 286 586 INDEX. Carbolic acid, estimation of, 483 test for, 216, 483 Carbolo-chloride of iron test for lactic acid, 185 Carbon dioxide haemoglobin, 42 monoxide haemoglobin, 41 Caries of the teeth, 138 Casein, digestion of, 179 in the milk, 577 test for, Leiner's, 229 Casts, classification of, 525 examination of, 526 fatty, 529 fibrinous, 273 formation of, 531 granular, 527 hyaline, 526 pus, 627 significance of, 532 staining of, 526 urinary, 525 waxy, 529 Catarrh, acute intestinal, 262 bronchial, 293 chronic intestinal, 263 duodenal, 262 intestinal, of infants, 263 of ileum, 262 of jejunum, 262 of large intestine, 262' Cause's method of estimating sugar, 445 Cellulose in the blood, 52 Cenomonadina, 235 Cercomonas intestinalis, 236 Cerebrospinal fluid, 558 amount of, 559 appearance of, 559 chemical composition of, 561 microscopical examination of, 562 reaction of, 561 specific gravity of, 560 Cestodes, 232 Chalicosis, 297 Charcot-Leyden crystals, in the feces, 231 in the nasal discharge, 268 in the sputum, 275, 276, 290 Chemical examination of blood, 24 of cystic fluids, 555 of feces, 214, 260 of gastric juice, 154 of milk, 576 of pus, 551 of saliva, 138 of semen, 564 of sputum, 292 of transudates, 547 of urine, 315 Chenzinsky-Plehn's stain, 101 Chlorides in the urine, 317 estimation of, 320 according to Neubauer and Salkowski, 325 Chlorides in the urine, estimation of, ac- cording to Salkowski and Volhard, 320 direct method, 324 test for, 320 Chloroform-benzol mixture, 19 Cholsemia, 57 Cholera Asiatica, 265 bacillus of, 252 infantum, 263 nostras, 263 bacillus of, 253 Cholesterin in the blood, 28 in the feces, 218 in the sputum, 292 in the urine, 471 isolation of, from the feces, 218 test for, 219 Choluria, 469 Chorion villi, 573 Chromogens in the urine, 455 Chyluria, 136, 414, 492, 513 Chymosin, 176 estimation of, 178 test for, 178 Chymosinogen, 176 estimation of, 178 test for, 178 Ciliated epithelium in cysts, 555 in the sputum, 279 Cladothrix, 289 Coagulation of the blood, 26 Coating of the tongue, 144 Coccidia in the feces, 239 Coflin-lid crystals, 504, 514 Colica mucosa, 226 Colloid concretions in ovarian cysts, 556 Color index of the blood, 34 Colostrum, 575 Comma bacillus, 252 Concretions, biliary, 227 fecal, 228 intestinal, 228 pulmonary, 277 Congo-red test for free acids, 163 Conjugate glucuronates, 454 sulphates, 336, 483 Copper test for uric acid, 377 Coproliths, 228 Corpora amylacea, 566 Cresol in the feces, 217 in the urine, 483 Crystals, ammonio-magnesium phos- phate, 504, 514 bilirubin, 512 calcium carbonate, 514 oxalate, 292,' 502 phosphate, 504, 505, 513 sulphate, 506 Charcot-Leyden, 212, 231,275, 276, 290 cholesterin, 212, 292, 471 cystin, 507 INDEX. 587 Crystals, fatty acids, 210, 292 hsematoidin, 45, 291, 512 hsemin, 43 hippuric acid, 506 indigo, 210, 231, 290, 514 leucin, 508 leucocytic, 291 magnesium phosphate, 505, 513 monocalcium phosphate, 505 neutral calcium phosphate, 505 phenyl-gUicosazon, 441 phosphate of spermin, 564 Teichmann, 44 triple phosphate, 292, 504, 514 tyrosin, 292, 508 urate of ammonium, 513 uric acid, 500 xanthin, 511 Curschmann's spirals, 275 Cylinders, mucous, in the feces, 226 in the urine, 531 urinary, 525 Cylindroids, 525, 531 Cylindruria, 525 Cystein, 340, 341 Cysticercus cellusosse, 242 Cvstin, 341, 507 Cystinuria, 341, 507 Cysts, colloid, 556 contents of, 555 dermoid, 556 fibro-cystic, 556 hydatid, 557 ovarian, 557 pancreatic, 557 parovarian, 556 D ALAND'S hsematokrit, 111 Decidual cells, 574 (J-granulation of Ehrlich, 76 DennigS's test for acetone, 58, 486 Dermoid cysts, 556 Dextrin in the urine, 452 Dextrose in the urine (see Glucose). Diabetes, 436 alternans, 375 Bremer's blood test in, 63 urine test in, 451 elimination of sugar in, 436 of urea in, 349 hepatogenic, 437 Hirschfeld's form of, 349, 438 insipidus, 304, 320 myogenic, 437 phosphatic, 328 Williamson's blood test in, 50 Diacetic acid in the urine, 489 tests for, 489 Diacetnria, 489 Diamins in the feces, 262 in the urine, 341, 495 isolation of, 496 Diarrhoea of infants, 263 Diathesis, oxalic acid, 394 uric acid, 374 Diazo-reaction (seeEhrlich' s reaction), 479 Digestion, gastric, 178 of albumins, 178 of albuminoids, 179 of carbohydrates, 179 of milk, 179 of proteids, 179 products of, 181 Dimethyl-araido-azo-benzol test, 164 Diphtheria, 145 ■Diplococcus meningitidis intracellularis, 562 pneumoniae, 119, 287 in the blood, 118 Distoma Buskii, 246 capense, 136 conjunctum, 247 hffimatobium, 136, 283, 543 hepaticum, 245 heterophyes, 247 lanceolatum, 246 pulmonale, 283 rhatonisi, 246 sibiricum, 246 spatulatum, 247 Distomiasis, 136 Donne's pus test, 519 Donogany's blood test, 199, 430 Doremus' ureometer, 357 Drosophila melanogastra, 232 Drugs, effect of, on the color of the stools, 208 Drysdale's corpuscles, 556 Dunlop's method of estimating oxalic acid, 396 Diist-particles of Miiller, 105 Dysentery, 264 amoebic, 264 Shiga's bacillus of, 259 EARTHY fihosphates, 328 Eberth's bacillus, 254 Echinococcns, 276, 281 membranes in the sputum, 276 polymorphus, 282 e-granulation of Ehrlich, 73 Ehrlich's granulations, 71 haematoxylin-eosin, 101 neutral red, 540 neutral stiiin, 102 reaction, 479 tri-acid stain, 100 tri-glycerin mixture, 102 Einhorn's bucket, 153 saccharimeter, 447 Elastic tissue in the sputum, 273, 280 stain for, 281 Eisner's method, 255 Engel's alkalimeter, 24 method of estimating the alkalinity of the blood, 24 688 INDEX. Enteritis, acute, 262 chronic, 263 membranous, 226, 263 mucous (see Membranous], 226, 263 Enterogenic albumosuria, 409 Enteroliths, 228 Eosin, staining with, 102 Eosin-raethylal and methylene-blue, 103 Eosinate of methylene-blue, staining with, 99 Eosinophilia, 89 Eosinophilic leucocytes in the blood, 75, 79 in the sputum, 275, 277 Epithelial cells, alveolar, 279 ciliated, 279 in the buccal secretion, 140 in the feces, 280 in the gastric contents, 201 in the sputum, 278 in the urine, 515 in the vaginal secretions, 569 Eructatio nervosa, 195 Erythrodextrin, 139, 182 test for, 139, 182 Erythrosin, acid, staining with, 104 Esbach's albnminimeter, 423 method of estimating albumin, 423 reagent, 423 Escherich's stain, 258 Ethyl sulphide, 341 Euchlorhydria, 162 Eustrongylus gigas, 543 Ewald's modification of Mohr's test for hydrochloric acid, 166 Extractives in the blood, 28 Exudates, 545, 548 chyloid, 554 chylous, 554 hemorrhagic, 549 in cancer, 549 in tuberculosis, 549 purulent (see Pus), 550 putrid, 550 serous, 548 FAT in the blood, 28, 55 in the milk, estimation of, 580 in the urine, 492, 512 Fatty acids, clinical significance of, 190 estimation of, 192 formation of, 190 in pus, 554 in the blood, 55 in the feces, 217 in the gastric contents, 190 in the sputum, 292 in the urine, 491 tests for, 191, 218 Fatt^ casts, 529 Febrile acetonuria, 484 albuminuria, 402 urobilin, 220, 455, 473 Fecal matter in the urine, 543 vomiting, 199 Feces, 207 alimentary detritus in, 208, 225 amount of, 207, 222 annelides in, 247 biliary acids in, 219 concretions in, 227 blood in, 230 chemistry of, 214, 260 cholesterin in, 218 color of, 208, 222 composition of, 210 concretions in, 227 consistence of, 207, 222 crystals in, 210, 231 examination of normal, 207 fatty acids in, 217 flagellata in, 235 foreign bodies in, 209 form of, 207, 222 gases in, 215 general characteristics of, 207, 221 indol in, 216 insects in, 251 macroscopiCal constituents of, 208 microscopical constituents of, 209, 228 mucus in, 226, 230 number of stools, 207, 221 odor of, 208, 222 parasites in, 212 animal, 231 vegetable, 212,251 pathology of, 221 phenol in, 216 pigments in, 220 protozoa in, 232 ptomains in, 262 reaction of, 210, 222 skatol in, 216 technique in examination of, 228 trematodes in, 245 vermes in, 239 Fehling's method of estimating sugar, 444 solution, 439 . test for sugar, 439 Ferment, milk-curdling, 176 of saliva, 139 Fermentation test for sugar, 440 Ferments in the gastric juice, 173 Ferrometer, JoUes', 36 Feser's lactoscope, 580 Fibrin, 26, 48 estimation of, 48 ferment, 26 in the blood, 26 in the urine, 414 test for, 430 Fibrinogen, 26 Fibrinoglobulln, 26 Fibrinous casts, 273 coagula in the sputum, 273 INDEX. 589 Fibrinous coagula in the urine (see Chy- luria). Filaria Bancrofti, 135 diurna, 135 Mansoni, 135 noctiirna, 135 perstans, 135 sanguinis liominis, 135, 543 Wucliereri, 135 Filariasis, 135 Finkler-Prior bacillus, 253 Flagellata, 235 Fleisohl's hsemometer, 32 ^ Florence's test for semen, 567 Folin's method of estimating ammonia, 370 urea, 363 uric acid, 378 Foreign bodies in the feces, 209 in the sputum, 277 in the urine, 543 Formic acid, detection of, 218 Freund's method of determining acidity of urine, 314 Furfurol test for bile acids, 220 Futcher's stain, 126 GABETT'S staining method, 286 Galacturia, 492 Gall-stones in the feces, 227 analysis of, 227 Gangrene of the lung, 294 Garrod's test for hsematoporphyrin in the urine, 468 for homogentisinie acid, 477 for uric acid in the blood, 55 Gases in the blood, 28 in the feces, 215 in the gastric contents, 193 in the urine, 493 Gastric contents, examination of (see also Gastric juice), 148 Gastric digestion of albuminoids, 179 of carbohydrates, 179 of native albumins, 178 of protelds, 179 products of, 178 analysis of, 181 Gastric juice, 148 acetic acid in, 192 acetone in, 195 acidity of, 155 amount of, 153 antiseptic properties of, 161 aspiration of, 152 blood in, 198 butyric acid in, 191 cause of acidity of, 155 chemical composition of, 154 examination of, 154 chymosin in, 176 chymosinogen in, 176 expression of, 152 Gastric juice, fatty acids in, 190 ferments in, 173 free acid in, 155, 163 gases in, 193 general characteristics of, 153 hydrochloric acid in, 155, 160 hyperacidity of, 159 hypersecretion of, 154, 159 indirect examination of, 205 lactic acid in, 183 methods of obtaining, 151 microscopical examination of, 200 . milk-curdling ferment of, 176 organic acids in, 190 pepsin in, 173 pepsinogen in, 173' ptomains and toxalbumins in,195 secretion of, 148 zymogens in, 173 Gastrosucorrhoea mucosa, 197 Gigantoblasts (see Megaloblasts), 68 Glanders, bacillus of, 122 Glandular fever, 145 Glucose, 430 in the blood, 49 estimation of, 50 in the urine, 430 Nylander's test for, 440 quantitative estimation of, 444 tests for, 438 Glucosuria, 430 digestive, 431 e saccharo, 434 ex amylo, 434 persistent, 436 transitory, 434 Glucosuric acid, 476 Glucuronic acid in the blood, 49 Glycogen in the blood, 51 test for, 52 in the sputum, 293 in the urine, 454 Gmelin's reaction, 471 Gonococcus in the blood, 120 in the mouth, 143 in urethral diseliarge, 540 of Neisser, 540 staining of, 540 Gonorrhceal stomatitis, 143 threads in the urine, 541, 543 Gowers' hsemoglobinometer, 35 Gram's method of staining, 146 Granular degeneration, 65 y-granulation of Ehrlich, 76 Grape-sugar (see Glucose). Green's ureometer, 361 Gregarina, 239 Grethe's method of staining tubercle ba- cilli in the urine, 539 Guaiacum test for blood, 429 Guanin in the urine, 371, 383 Gum, animal, 454 590 INDEX. Gunning's mixture, 364 Gunzburg's packages, 205 reagent, 164 Gynsecophorus, 136 H^MATEMESIS, 198 Hsematin, 43 Hsematinuria, 466 Hffimatoblasts, 104 Hsematoidin in the blood, 45 in the sputum, 291 in the urine, 152 Hsematokrit, 110 Hsematoporphyrin in the blood, 45 in the feces, 221 in tlie urine, 466 Hsematoporphyrinuria, 46, 466 Hsematuria, 413, 522 Hsemin (see Teichmann's crystals), 43 Hsemocytometer of Thoma-Zeiss, 105 Haemoglobin, 17, 29 carbon dioxide, 42 monoxide, 41 estimation of, with Fleischl's haemom- eter, 32 with Gowers' hsemoglobinome- ter, 35 hydrogen sulphide, 42 nitric oxide, 42 tests for, 44. 198 Hsemoglobjnsemia, 40 Hsemoglobinometer of Gowers, 35 Hsemoglobinuria, 41, 412 Hsemokonia, 105 Hsemometer of Fleischl, 32 Hemospermia, 566 Halitus sanguinis, 18 Hammerschlag's method, 19 Haycraft' s method of estimating uric acid, 379 Hayem's fluid, 106 Pleart-disease cells, 296 Hehner-Seemann's method of estimating organic acids, 192 Heller's test for albumin, 416 for blood, 429 Hepatogenic icterus, 469 Heteroxanthin in the urine, 371, 383 Hippuric acid in the urine, 385, 506 estimation of, 387 properties of, 386 test for, 387 Histon in the urine, 415 test for, 430 Hoffmann's test for tyrosin, 510 Hofmeister's method of estimating hip- puric acid, 388 • test for leucin, 510 Homialomyia, 232 Homogentisinic acid in the urine, 476 isolation of, 477 Hopkins' method of estimating uric acid, 377 Hiifner's ureometer, 361 Huppert's test for bile-pigment, 470 Hydatid cysts, 557 echinococcus membranes and booklets in, 557 sodium chloride in, 557 succinic acid in, 557 Hydrobilirubin, 220 Hydrocele fluid, 547 cholesterin in, 547 Hydrochinon in the urine, 475 Hydrochloric acid in the gastric juice, 156 amount of, 162 combined, 167 estimation of, according to I^eo, 172 according to Mar- tins and Liittke, 170 according to Tiip- fer, 168 free, 155 quantitative estimation _ of, 168 significance of, 160 source of, 159 tests for, 164 Hydrogen sulphide, in the gastric con- tents, 194 tests for, 194 in the urine, 494 Hydronephrosis, 557 Hydrothionuria, 342, 493, 507 Hypalbuminosis, 48 Hyperalbuminosis, 48 Hyperchlorhydria, 163 Hyperinosis, 48 Hyperisotonia, 28 Hyperleucocytosis, 80 active, 80 mixed, 91 passive, 80, 93 pathological, 84 physiological, 81 polynuclear eosinophilic, 89 neulropliilic, 81 Hypersecretio acida et continua, 154, 163 Hypersecretion, 154 Hypinosis, 48 Hypobromite method of estimating urea, 355 solution, 355 Hypochlorhydria, 162 Hypoleucocytosis, 80, 94 Hypoxanthin in the urine, 371, 383 TCTEBUS, 469 JL hematogenic, 470 hepatogenic, 469 neonatorum. 470 nrobilin, 473 Idiopathic bacteriuria, 541 oxaluria, 394 INDEX. 591 Ilasvay's reagent, 140 Indican in the urine, 458 estimation of, 462 tests for, 461 Indicanuria, 458 Indigo-blne in tlie urine, 461, 480, 514 Indig6-red in the urine, 464 Indigosuria, 461, 480 Indol in the feces, 216 tests for, 216 Indoxyl, 458 sulphate (see Indican), 458 Influenza, bacillus of, 122, 288 Infusoria in pus, 553 in the feces, 231 in the urine, 543 in vaginal discharges, 570 Inosit in the urine, 455 Insects in the feces, 251 Intermittent albuminuria, 399 Intestinal catarrh, 262 concretions, 227 putrefaction, 261, 458 Intestines, diseases of, 262 Iodine stain for the Plasmodium malariae, 127 lodoform-test for lactic acid, 186 lodospermin, 567 Iron test, 224 in bloo4, 36 Isotonia, 27 JAFFE'S test for indican, 461 Jaundice (see Icterus), 469 Jenner's stain, 99 JoUes' ferrometer, 36 KELLING'S test for lactic acid, 186 Kjeldahl's method, 364 Knapp's method of estimating sugar, 446 Koplik's bacillus, 288 Korczynski and jaworski's test, 224 Krabbea grandis, 245 Kreatin, 388 properties of, 389 Kreatinin, 388 estimation of, 390 properties of, 389 test for, 390 Kreatinin-zinc chloride, 390 LACMOID paper, preparation of, 25 Lactic acid, 183 bacillus of, 183 clinical significance of, 183 estimation of, 188 fermentation, 185 in the blood, 56 in the gastric contents, 183 in the urine, 490 mode of formation, 1 83, 185 tests for, 185 Boas', 186 Lactic acid, tests for, Kelling's, 185 Strauss', 185 Uffelmann's, 185 Lactodensimeter of Quevenne, 578 Lactoscope of Feser, 580 Lactose in the urine, 452 Laiose in the urine, 453 Landois' estimation of the alkalinity of the blood, 21 Laveran's organism, 125 Laverania malariae, 131 Lecithin in the blood, 28 Legal's test for acetone, 486 Leiner's test for casein, 229 Leo's method of estimating hydrochloric acid, 172 Leprosy, bacillus of, 285 Leptothrix buccalis, 145 pulmonalis, 294 Leube's test of motor power of stomach, 204 Leucin, 508 Leucocytes, 17, 69 basophilic, 76 degenerative changes, 74 differential enumeration, 110 differentiation according to their be- havior toward anilin dyes, 71 Ehrlich's granulations in, 71 enumeration of, 108 eosinophilic, 75, 79 estimation of the number of, 108 general differentiation of the various forms, 69 indirect enumeration of, 109 in the blood, 17, 69 in the exudates, 548, 551 in the feces, 231 in the sputum, 277 in the urine, 518 irritation forms, 79 large mononuclear, 73 lymphogenic, 71 myelogenic, 78 neutrophilic, 73, 78 oxyphilic, 75 polymorphonuclear, 73 polynuclear, 73 pseudolymphocytes, 79 small mononuclear, 71 transition forms, 73 variations in number of, 80 Leucocytic crystals, 291 Leucocytosis (see Hyperleucocytosis), 80 active, 80 digestive form of, 81 passive, 80 Leucopenia, 94 Leuksemia, lymphatic, 94 myelogenous, 91 Leviilose in the urine, 452 Lichen's test for acetone, 486 Lientery, 225 592 INDEX. Lipacidsemia, 56 Lipacidiiria, 491 Lipsemia, 56 Lipuria, 491,512 Lilhsemia, 58 Liver, abscess, 295 aciite yellow atrophy of, 508 diseases of, feces in (see Acholic stools), urine in (see Bile-pigments). Lochia, 571 alba, 571 rubra, 571 Loffler's bacillus, 145 raethylene-blue solution, 146 Lohnstein's saccharimeter, 448 Lowy's method of estimating the alka- linity of the blood, 23 Ludwig-Salkowski's method of estimating uric acid, 381 Lymphocytes, 72 Lymphocytosis, 93 MACROCYTES, 59 Macrocythfemia, 59 Magnesia mixture, 331 soaps of, in the urine, 512 Magnesium phosphate, 505, 513 Malaria, Plasmodium of, 125 Malta fever, bacillus of, 124 Maltose in the urine, 452 JIammary secretion, 575 Marrow cells, 78 Marshall's ureometer, 361 Marsh gas in the gastric contents, 193 Martins and Liittke's method of esti- mating hydrochloric acid, 170 Masons' lung (see Siderosis), 297 Mastzellen, 76 Meconium, 265 Medico-legal test for blood, 43 Megaloblasts, 68 Megalocytes, 59 Megastoma entericnm, 237 Melansemia, 134, 474 Melanin in the urine, 474 tests for, 475 Melanogen, 474 Membranous. dysmenorrhoea, vaginal dis- charge in, 572 Meningeal fluid, examination of, 558 Menstruation, vaginal discharge in, 571 Metalbnmin in ovarian cysts, 555 Methsemoglobin, 45 sulpliide, 42 Methaemoglobiniemia, 41, 45 MethEemoglobinuria, 412 Methane (see Marsh gas), 193 Michaelis' stain, 103 Microblasts, 69 Micrococci in pus, 558 Micrococcus ureaj, 537 Microoytes, 59 MicrocythEemia, 59 Micro-organisms in pus, 553 in the feces, 213, 251 in the milk, 577, 578 in the mouth, 140 in the nasal secretion, 267 in the urine, 536 in vaginal discharges, 570 Microscopical examination of cystic fluids, 555 of exudates, 548, 551 of the blood, 58 of the buccal secretion, 140 of the feces, 209 of the gastric contents, 200 of the nasal secretion, 267 of the sputum, 277 of the urine, 498 of the vaginal secretion, 569 of the vomit, 169, 200 of transudates, 548 Jliescher's hsemometer, 34 Milk, 575 chemical composition of, 576 cows', 577 examination of, 578 fat in, estimation of, 580 human, 576 in disease, 577' proteids of, 580 secretion of, in the adult female, 576 in the newly born, 575 specific gravity of, 578 witches', 575 Milk-curdling ferment in the gastric juice, 170 Millon's reagent, 425 Mohi-'s test for hydrochloric acid, 166 Monadina in feces, 235 Monera in the feces, 232 Monooalcium phosphate, 505 Moro's bacillus, 2-57 Motor power of stomach, examination of, 203 Leube's method, 203 salol test of Ewald and Sievers, 204 Mouth, actinomycosis of, 143 secretions of, 138 tuberculosis of, 143 Mucin in the feces, 260 in the urine, 414 test for, 428 Mucous corpuscles in the urine, 299 cylinders in the feces, 226' in the urine, 531 Mucus in the feces, 226, 230 in the gastric contents, 197 Miiller- Weber test for blood, 198 Murexid test, 377 Myelcemia, 92 Myelin granules in the sputum, 279 INDEX. 593 Myelocytes, eosinophilic, 79 neutrophilic, 78 of Cornil, 78 of Ehrlich, 78 NASAL catarrh, 267 secretion, 267 cerebrospinal fluid in, 267 characteristics of, 267 Charcot - Leyden crystals in, 286 in disease, 267 Neisser, gonococcus of, 540 Nematodes, 232 Nessler's reagent, 187 Neubauer's method of estimating oxalic acid, 395 Neusser's grannies, 77 stain, 101 Neutral phosphate of calcium in the urine, 505 red stain for gonococci, 540 sulphur in urine, 340 Neutrophilic granules in the blood, 73, 78 Nitric acid test for albumin, 416 Nitric oxide hsemoglobin, 42 Nitrites in the saliva, 139 Nitrogen in the urine, 347 estimation of, 364 according to Kjeldahl, 364 according to Will-Varren- trapp, 366 Nitrogenous equilibrium, 347 Nitro-prusside of sodium as a test for acetone (see Legal's test), 486 Nocht-Romanowsky stain, 126 Normal urobilin, 455, 471 Normoblasts, 67 Nose, secretion from, 267 Nubecula in the urine, 299 Nucleated red corpuscles, 67 Nucleo-albumin in the blood, 48 in the urine, 414 test for, 428 Nucleohiston in the urine, 415 Nummular sputum, 272 Ny lander" s test for sugar, 440 OBEBMAYER'S reagent, 461 Obermeier, spirochseta of, 123 (Edema of the lungs, sputum in, 296 Oiilium albicans, 144, 290 defiant gas, 194 Oligochromsemia, 32 Oligocythsemia, 32, 61 Oliguria, 305 Orcin test for pentoses, 453 ii^Organic acids In the blood, 55 in the gastric juice, 183 quantitative estimation of, 192 in the sputum, 290 38 Organized sediments of the urine, 515 Ott's test, 428 Ovarian cysts, 555 Oxalate of calcium crystals in the sputum, 292 in the urine, 502 Oxalic acid, diathesis, 394 in the urine, 392 properties of, 394 quantitative estimation of, 395 tests for, 395 Oxaluria idiopathica, 394 Oxaluric acid, 392 Oxybutyric acid, |8, in the urine, 490 Oxyhseraoglobin, 18, 29 Oxyuris vermicularis, 248 Ozsena, 267 PACINI'S fluid, 106 Pancreatic cysts, 557 trypsin in, 557 juice in the gastric contents, 198 Pappenheim's stain, 285 Paraciesol in the urine, 483 Paramoecium coli, 239 Parasites in the blood, 113 in the feces, 212, 231 in the gastric contents, 200 in the sputum, 281 in the urine, 536 malarial, 113 Paraxanthin in the urine, 371, 383 Patein's albumin, 409, 422 test for, 422 Pathological acetonuria, 484 albuminuria, 398 glucosuria, 436 urobilin, 471 Pentoses in the urine, 453 tests for, 453 Pepsin in the gastric juice, 173 estimation of, 176 tests for, 175 Pepsinogen in the gastric juice, 173 estimation of, 176 tests for, 175 Peptonuria (see Albumosuria). Persistent glucosuria, 436 Pettenkofer's test, 220 Phagocytes, 69 Phagocytosis, 69, 134 Pharyngomycosis leptothrica, 145 Phenol, 216, 475, 483 estimation of, 483 in the feces, 216 in the urine, 475, 483 tests for, 216, 483 Phenylglucosazon, 441 Phenylhydrazin test for sugar, 441 Phlorogluoin test for pentoses, 453 vanillin test for hydrochloric acid, 164 Phosphates in the urine, 325, 504 594 INDEX. Phosphates in the urine, estimation of, 331 removal of, from urine, 334 separate estimation of alkaline and earthy, 334 tests for, 330 Phosphatio diabetes, 328 sediments in the urine, 504, 505, 513 Phthisis pulmonalis, sputum in, 295 Picrio-acid test for albumin, 421 Pigments in the feces, 220 in the urine, 455 Piorkowski's method, 254 Piria's test for tyrosin, 510 Placenta sanguinis, 25 Plaques, 104 enumeration of, 110 Plasma of the blood, 17, 24 Plasmodium malarise, 125 crescentio bodies, 131 flagellate bodies, 132 hyaline bodies, 127 ovoid bodies, 131 pigmented extracellular bodies, 132 intracellular bodies, 128 segmenting bodies, 1 30 spherical bodies, 131 staining of. 126 Plastic bronchitis, 293 Plastodes, 232 Plehn's stain, 126 Pneumoconioses, 296 Pneumonia, diplococcus of, 287 in the blood, 118 sputum in, 295 Poikilocytes, 60 Poikilocytosis, 60 Polarimeter, 449 Pole bacillus, 288 Polychromatophilic degeneration, 63 Polycythaemia, 60 Polymastigina, 235 Polyuria, 302 Propepsin, 174 Prostatic fluid, 564 Protagon, 280 Proteids, formed in the stomach, 178 of the blood, 47 reactions of, 181 Proteus vulgaris, 259 Protozoa, 232 in pus, 553 in the blood, 113 in the feces, 232 in the sputum, 282 in the urine, 542 Pseudocasts, 525, 531 Pseudogonococci, 541 Pseudolymphocytes, 79 Psorospermiasia, 239 Ptomains in the feces, 262 in the gastric contents, 195 in the urine, 494 Ptomains in the urine, isolation of, 496 Ptyalin, 139 test for, 139 Pulmonary abscess, 294 diseases, sputum in, 293 gangrene, 294 oedema, 296 phthisis, 295 Purin, 371 bases, 371, 383 Purulent exudates, 550 Pus, 550 chemistry of, 551 crystals in, 553 detritus in, 552 general characteristics of, 550 giant-corpuscles in, 552 in the feces, 224 in the gastric contents, 199 in the urine, 519 leucocytes in, 551 microscopical examination of, 551 parasites in, 553 red corpuscles in, 552 tests for, 519 Putrescin. 262, 341, 495 Putrid bronchitis, 293 exudates, 550 Pyogenic albumosuria, 409 Pyrocatechin in the urine, 484 Pyrocatechuic acid, 476 Pyuria, 519 AUEVENNE'S lactodensimeter, 578 REACH'S test, 206 Bed blood-corpuscles, 17, 58 anopraic degeneration of, 63 behavior toward anilin dyes, 63 degeneration of, 65 enumeration of, 106 granular, 65 nucleated forms, 67 variations in color, C2 in form, 59 in number, 60 in size, 58 Eelapsing fever, spirillum of, 123 Eenal albuminuria, 401, 408 Eesorcin test, 165 Resorptive power of the stomach, ex- amination of, 204 Reynolds' test for acetone, 486 Rhizopoda, 232 Rice-water stools, 265 Rosenbach' s reaction, 464 test for bile-pigments, 471 Round worms, 239 Roy's method of determining the specific gravity of the blood, 18 Eust-colored expectoration, 271 INDEX. 595 SACCHAKIMETER of Einhorn, 447 of Lohnstein, 448 of Soleil-Ventzke, 450 Saccharomyces cerevisise (see Yeast). Saliva, 138 chemistry of, 138 general characteristics of, 138 in special diseases of the mouth, 143 in the gastric contents, 198 microscopical examination of, 140 nitrites in, 1 39 pathological alterations of, 142 ptyalin in, 139 test for nitrites, 139 for ptyalin, 139 for sulphocyanides, 138 Salivary corpuscles in, 140 Salivation, 142 Salkowski's method of estimating oxalic acid in urine, 397 xanthin-bases in urine, 384 test for albumoses, 425 for phenol, 483 Salkowski-Neubauer method of estimat- ing the chlorides in urine, 325 Salkowski-Volhard method of estimating the chlorides in urine, 320 Salol test of Ewald and Sievers, 204 Sanarelli's Bacillus icteroides, 124 Sarcina pulmonalis, 290 nrinae, 542 ventriculi, 201 Scherer's test for leucin, 510 Sehislosoma, 136 Schi?omycetes in the feces, 212 Schlosing's method of estimating ammo- nia, 369 Schmalz and Peiper's method of deter- mining the specific gravity of the blood, 19 Scybala, 222 Secretions of the mouth, 138 Sediments in acid urines, 500 in alkaline urines, 513 urinary, 299, 498 ammonio-magnesium phosphate in, 504, 514 ammonium urate in, 513 amorphous urates in, 502 basic magnesium phosphate in, 505, 513 biliruljin in, 512 biick-dust, 501 calcium carbonate in, 514 oxalate in, 502 sulphate in, 506 cystin in, 507 epithelial cells in, 515 fat in, 512 foreign bodies in, 543 I hsematoidin in, 512 hippuric acid in, 506 indigo in, 514 Sediments, urinary, leucin in, 508 leucocytes in, 518 mode of examination of, 500 monocalcium phosphate in, 505 neutral calcium phosphate in, 505 non-organized, 500 organized, 515 red corpuscles in, 522 soaps of lime and magnesium in, 512 spermatozoa in, 535 tube-casts in, 525 tumor-particles in, 543 tyrosin in, 508 urates in, 502, 513 uric acid in, 500 xanthin in, 511 Seegen-Schneider method of estimating nitrogen, 366 Semen, 564 chemistry of, 564 general characteristics of, 564 microscopical examination of, 565 pathology of, 566 recognition of, in stains, 566 spermatic crystals in, 564 spermatozoa in, 565 Sepsis, organisms in the blood in, 120 Seropurulent exudates, 548 Serous exudates, 548 Serum albumin in the blood, 26 in the urine, 398 estimation of, 422 tests for, 415, 421 Serum-globulin in the blood, 26 in the urine, 409 estimation of, 424 test for, 424 Shiga's bacillus, 259 Siderosis, 297 Skatol in the feces, 216 tests for, 216 SUatoxyl, 481 sulphate. 481 Smegma bacillus, 288, 539 Soaps of lime and magnesium in the urine, 512 Sodium chloride in hydatid fluid, 557 Spectroscope, 46 Spermatic crystals, 291 Spermatocystitis, 536 Spermatorrhoea, 536 Spermatozoa in the semen, 565 in the urine, 535 Spermin, 565 Spiegler's reagent, 421 Spirals of Curschniann, 275 Spirillum of relapsing fever, 123 Spirochieta Obermeieri, 123 Sporozoa, 239 Sputum, 269 Amoeba coli in, 283 596 INDEX. Sputum, amount of, 270 bacteria in, 283 blood in, 271, 278 cheesy particles in, 273 chemistry of, 292 color of, 271 concretions in, 277 configuration of, 272 consistence of, 270 cruduni, 272 crystals in, 276, 290 Curschmanu's spirals in, 275 Distoma pulmonale in, 283 echinococcus in, 276, 281 elastic tissue in, 273, 280 epithelial cells in, 278 fibrinous casts in, 273 foreign bodies in, 277 general characteristics of, 270 globosum, ;i72 heterogeneous, 272 homogeneous, 272 in various diseases, 293 leucocytes in, 277 macroscopical constituents, 273 microscopical examination of, 277 nummular, 272 odor of, 271 parasites, animal, in, 281 vegetable, in, 283 specific gravity of, 272 technique in the examination of, 269 Squibb's ureometer, 362 Staphylococcus pyogenes albus, 120 aureus, 120 eitreus, 120 Starch, digestion of, 139 solution, 189 Steatorrhcea, 225 Stercobilin, 220, 472 Stercoraceous material in the vomit, 199 Stokes' fluid, 30 Stomach, motor power of, 203 rate of absorption in, 204 washing, 153 Stomach-tube, 150 contraindications to its use, 151 its introduction, 151 Stomatitis, catarrhal, 143 gonorrhoeal, 143 ulcerative, 143 Stools (see Feces). Strauss' test for lactic acid, 186 Streptococcus pyogenes, 120 brevis, 120 conglomeratus, 121 longus, 120 Strongyloides, 232 Strongylus duodenalis, 249 Stycosis, 297 Succinic acid in hydatid fluid, 557 Sudan stain for fat, 513 Sugar in tlie blood, 28, 49 Sugar in the urine, 430 tests for, 438 Sulphanilic acid test (see Ehrlich's reac- tion). Sulphates in the urine, 334 conjugate, 336, 481 estimation of, 338 mineral, 335 tests for, 337 Sulphocyanides in the saliva, 138 in the urine, 340 Sulphur, neutral, in urine, 340 estimation of, 342 TjENIA cucumerina, 243 diminuta, 243 echinococcus, 281 flavapunctata, 243 mediocanellata, 240 nana, 242 saginata, 240 solium, 241 Tartar, 144 Tanro-carbarainio acid in urine, 341 Teichmann's crystals, 43 Test-breakfast of Boas, 150 of Ewald and Boas, 149 Test-dinner of Riegel, 149 Test-meal of Salzer, 150 Test-meals, 149 Thecosoma, 136 Thiosulphates in urine, 342 Thoma-Zeiss' lisemocytometer, 105 Thrush, 144 Toison's fluid, 106 ToUens' orcin test, 453 phloroglucin test, 453 Tongue, coating of, 144 Tonsillitis, 145 ' Tonsils, coating of, 145 Topfer's method of estimating hydro- chloric acid, 168 test for hydrochloric acid, 164 Toxalbumins in the gastric contents, 195 Transitory glucosuria, 434 Transudates, 545 albumin in, 546 chemistry of, 547 coagulation of, 546 general characteristics of 545 microscopical examination of, 548 specific gravity of, 545 Trematodes, 245 Trichina cystica, 135 sanguinis liominis nocturna, 135 spiralis, 251 Tricliocephalus dispar, 250 Trichomonads in the feces, 236 in the sputum, 282 in the stomach contents, 200 in the urine, 542 in vaginal discharges, 570 Trichomonas vaginalis, 236, 282, 542 INDEX. 597 Trichotrachelides, 250 Triple phosphate crystals in the sputum, 292 in the urine, 504, 514 Tripperfaden, 521 Trommer's test, 439 Tropseolin test for hydrochloric acid, ] 66 Trypsin, 587 in pancreatic cysts, 557 test for, 557 Tube-casts in the urine, 525 amyloid, 530 blood, 527 clinical significance of, 532 compound hyaline, 527 epithelial, 527 fatty, 529 formation of, 531 granular, 527 hyaline, 526 mode of examination of, 526 pseudo-, 525 pus, 527 staining of, 526 true, 525, 526 waxy, 529 Tubercle bacillus, 283 detection of, 283 in pus, 553 in the blood, 121 in the cerebrospinal fluid, 562 in the feces, 256 in the milk, 578 in the mouth, 143 in the sputum, 283 in the urine, 539 Tuberculosis, bacillus of, 283 Tumor-particles in the gastric contents, 203 in the urine, 543 Typhoid fever, bacillus of, 114, 254 in the blood, 114 in the feces, 254 stools in, 265 Tyrosin, 508 in the sputum, 292 in the urine, 50S test for, 509 UPFELM ANN' S t?st for lactic acid, 185 Unorganized sediments in urines, 500 Uraemia, 53 Urates in urinary sediments, 502, 513 Urea in the blood, 52 in the urine, 343 estimation of, 355 isolation of, 354 origin of, 343 properties of, 351 tests for, 351 nitrate, 352 oxalate, 353 Ureometers, 355 Ureometers, Doremus', 357 Green's, 361 Hiifner's, 361 Marshall's, 361 Simon's, 356 Squibb's, 362 Urethritis, gonorrhoeal, 540 Uric acid, 370 crystals of, 500 diathesis, 374 estimation of, 54, 377 Folin's method, 378 gravimetric method, 379 Haycraft's method, 379 Hopkins' method, 377 Ludwig - Salkowski's me- thod, 381 in sediments, 500 in the blood, 53 in the urine, 370 properties of, 375 tests for, 377 Urinary cylinders, 525 sediments, 500 Urine, 298 acetone in, 484 acidity of, 314 albumins in, 398 albumoses in, 409 "' alkapton in, 475 alloxur bases in, 383, ammonia in, 368p^^ animal gum in, 454 parasites in, 542 Bence Jones' albumin in, 411 benzoic acid in, 386 bile acids in, 471 pigments in, 468 blood in, 412, 466, 522 carbohydrates in, 430 casts in, 525 chemistry of, 315 chlorides in, 317 cholesterin in, 471 chroraogens in, 455 chyle in, 136, 414, 492, 513 color of, 299 • consistence of, 301 cystin in, 507 dextrin in, 452 diacetic acid in, 489 Ehrlioli's reaction in, 479 epithelium in, 515 fat in, 492, 512 fatty acids in, 491 fecal matter in, 543 ferments in, 493 fibrin in, 414 foreign bodies in, 543 gases in, 493 general appearance of, 299 chemical composition of, 315 glucose in, 430 598 INDEX. Urine, glucuronic acid in, 454 haemoglobin in, 412 hippuric acid in, 385 histon in, 415 indiean in, 458 inosit in, 455 kreatin in, 388 kreatinin in, 388 lactic acid in, 490 lactose in, 452 laiose in, 453 leucocytes in, 518 levulose in, 452 maltose in, 452 melanin in, 474 microscopical examination of, 498 mineral ash, estimation of, 316 neutral sulphur in, 340 nitrogen in, 343, 364 nubecula in, 299 nucleo albumin in, 414 nucleohiston in, 415 odor of, 301 organized sediments in, 515 oxalic acid in, 392, 502 oxaluric acid in, 392 oxybutyric acid in, parasites in, 536 pentoses in, 453 phenol in, 475, 483 phosphates in, 325, 505 pigments in, 455 ptomains in, 494 pus in, 618 pyrocatechin in, 475, 484 quantity of, 301 reaction of, 310 sediments in, 498 serum-albumin in, 398 serum-globulin in, 409 skatoxyl sulphate in, 481 solids in, 310 specific gravity of, 305 spermatozoa in, 535 sugar in, 430 sulphates in, 334 sulphur, neutral, in; 340 tumor-particles in, 542 urea in, 343 uric acid in, 370, 500 urobilin in, 455, 471 urochrome in, 455 uroerythrin in, 457 urohismatin in, 464 urohaematoporphyrin in, 466 urorosein in, 465 vegetable parasites in, 536 xanthin-bases in, 3B3 Urines, blue, 478 green, 478 Urinometer, 308 Urobilin, febrile, 455, 473 identity with stercobilin, 220 Urobilin, normal, 455, 471 pathological, 471 tests for, 473 Bang's, 426 Gerhardt's, 473 Urobilinogen, 471 Urobilinuria, 471 Urochrome, 455 Uroerythrin, 457 Urofuscohsematin, 466 Urohaematin, 464 Urohsematoporphyrin, 466 Uroleucinic acid, 476 Urophain, Heller's, 465 Urorosein, 465 Uroroseinogen, 465 Urorubrohsematin, 466 Uroxanthinic acid, 476 Urrhodinic acid, 476 VAGINAL blennorrhoea, 571 discharges, 569 bacteria in, 569 during menstruation, 571 following parturition, 571 general description of, 569 in abortion, 572 in gonorrhoea, 572 in membranous dysmenorrhcea, 572 in uterine cancel-, 572 in vaginitis, 572 in vulvitis, 572 parasites in, 569, 570 reaction of, 569 Vaginitis exfoliativa, 572 Valeur globulaire, 34 Vermes in the blood, 135 in the feces, 239 in the sputum, 281 in the urine, 543 Vitalli's test for pus, 519 Vomited material, 196 bile in, 198 blood in, 198 food material in, 196 mucus in, 197 odor of, 200 pancreatic juice in, 198 parasites in, 200 pus in, 199 saliva in, 198 stercoraceous material in, 199 Vomitus matutinus, 159, 198 V. Fleischl's haemometer, 32 WANG'S estimation of indiean, 462 Wassiliew's estimation of albumin, 422 Waxy casts, 529 Weigert-Ehrlich stain, 286 Weigert's elastic tissue stain, 281 Weyl's test for kreatinin, 390 INDEX. 599 Whetstone crystals (see Uric acid), 500 White blood-corpuscles (see Leucocytes), 69 Whooping-cough, bacillus of, 288 sputum in, 288 Widal's serum-test, 114 Williamson's blood-test in diabetes, 50 Will- Varrentrapp' s method of estimating nitrogen, 366 Woodward's method of estimating milk- proteids, 580 Worms (see Vermes), 239 XANTHIN-BASES in the blood, 55 in the urine, 371, 383, 511 estimation of, 384 Xanthoproteic reaction, 217 YEAST-CELLS in the gastric con- tents, 201 in the urine, 542 Yellow fever, bacillus of, 124 ZIEHL-NEELSEN stain, 287 Zymogens in the gastric juice, 173