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3 1924 104 224 864



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CLINICAL
LABORATORY METHODS



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CLINICAL
LABORATORY METHODS

A MANUAL OF TECHNIQUE AND MORPHOLOGY

DESIGNED FOR THE USE OF STUDENTS

AND PRACTITIONERS OF MEDICINE



BY

ROGER SYLVESTER MORRIS, A.B., M.D.

AB30CIATE PROFEaSOH OF MEDICINE IN WASHINGTON TJNIVEHSITT, ST. LOUIS. FORMERLY ASSOCIATE IN
MEDICINE, THE JOHNS HOPKINS UNIVEHSITY; ASSISTANT RESIDENT PHYSICIAN, THE JOHNS
HOPKINS hospital; INSTRUCTOR IN MEDICINE AND DEMONSTRATOR OF
CLINICAL UEDICINE, THE UNIVERSITY OF MICBIQAN.




D. APPLETON AND COMPANY
NEW YORK AND LONDON
1913



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Copyright, 1913, bt
D. APPLETON AND COMPANY







Printed in the United States of America



' /./



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TO

WILLIAM SYDNEY THAYER

AND

GEORGE DOCK

THIS VOLUME IS DEDICATED

•WITH THE AFFECTION AND GRATITUDE OF

THE AUTHOR



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PEEFACE



Coincident with the improvement in medical education in this
country there has been a widespread increase in the use of labora-
tory methods as aids to diagnosis. Not only is this true in the
case of the more recent graduates in medicine, but, what is more
hopeful, the older practitioners — those whose college days pre-
ceded the introduction of Clinical Pathology into the medical cur-
ricula — are quite generally realizing the necessity of the laboratory
in their daily work. Probably no one thing has done more to
bring about this much desired result than the discovery by Wasser-
mann and his co-workers of the well-l?nown serum reaction for
the diagnosis of syphilis; unconsciously, perhaps, but none the
less effectively, attention has been focused upon laboratory diag-
nostic methods.

The present volume is not a text-book of Clinical Pathology ; it
is a manual of laboratory technique and morphology, dealing merely
with methods and with morphological elements which are of diag-
nostic importance. It attempts to give in detail the means of de-
tecting the abnormal in urine, gastric contents, feces, blood, spu-
tum, and puncture fluids. Unlike the text-books, the significance
of the abnormal is not discussed.

That there is need for such a work the author has long believed.
There is much which it is absolutely essential that the student of
mediciue-^graduate and undergradute — remember. He must
know, for example, under what conditions albuminuria may occur,
whether it be of nephritic, cardiac, toxic, physiologic, or whatever
origin. He must be aware of the possible significance of a second-
ary anemia, of an atypical reduction test in the urine, of Charcot-
Leyden crystals in sputa, of a hydrochloric acid deficit in the
gastric contents. But it is useless to try to burden the memory
with the details of the various laboratory methods, by which such
abnormalities are detected, and with the sources of error in the
methods.



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PREFACE

No attempt lias been made to include within the present volume
a multiplicity of methods; in fact, the aim of author has been to
select one method or more of proved value. Nor have the more
exact, time-consuming methods of physiological chemistry been
drawn upon; in his daily work the average clinician has not the
time, if he has the ability, to employ them.

Free use has been made of the following works: Emerson's
"Clinical Diagnosis," Wood's "Chemical and Microscopical Diag-
nosis," Simon's "Clinical Diagnosis," Sahli's "Klinische Unter-
suchungsmethoden, " Hoppe-Seyler's "Handbuch der chemischen
Analyse, ' ' Hammarsten 's ' ' Lehrbuch der physiologischen Chemie, ' '
Neubauer-Huppert's "Analyse des Hams," Schmidt and Stras-
burger's "Die Paezes des Mensehen," Braun's "Thierische Parasi-
ten des Mensehen," Blanchard's "Traite de Zoologie Medicale,"
Cabot's "Clmical Pathology of the Blood," Naegeli's "Blutkrank-
heiten und Blutdiagnostik, " and Tiirk's "Vorlesungen ueber klin-
ische Haematol ogie." Other authors have been consulted less
freely. To the more recent literature direct reference has been
given in the form of footnotes ; in all instances the writer has en-
deavored to give proper credit to authors.

It is hoped that the volume will prove helpful to medical
students who have completed a course in Clinical Pathology ^and to
practitioners of medicine, or that it may serve as a supplement
to a course of laboratory lectures.

For the original illustrations in black and white the author
is indebted to Dr. James S. Brotherhood. It is a pleasure, also,
to acknowledge his indebtedness to his wife and to his mother for
assistance in the preparation of the index and in other ways.

Roger Sylvester Morris.
St. Louis;



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CONTENTS



CHAPTER I
THE UEINE

PAGE

Collection of the Urine 1

Preservation op the Urine 2

Macroscopic Examination op the Urine .... 3

Color op Urine ... .... 4

Quantity op Urine 4

Reaction op Urine 4

Quantitative determination of urinary acidity: Folin's

method 5

Specipic Gravity 6

Urea 7

Hiifner's hypobromite method 8

Uric Acid 9

Qualitative determination of uric acid .... 9
Quantitative determination of uric acid: Method of

Folin and Shaffer 10

Ammonia 12

Quantitative determination of ammonia: Folin's method
— the vacuum distillation method — the formalin titra-
tion method — Shaifer's modification of Schlosing's

method 13-19

Nitrogen .20

Kjeldahl's method for determination of total nitrogen . 20

Chlorids 23

Qualitative test 23

Quantitative determination of chlorids: Harvey's modi-
fication of Volhard's method 24

ix



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X CONTENTS

PAGE

Sulphates 26

Obermayer's test 27

Jaffe's test 27

Albumin .28

Qualitative tests: Heat and acetic acid test — Heat and
nitric acid test — Heller's test — Potassium ferrocyanid

and acetic acid test 28-34

Quantitative determination of albumin : Tsuchiya 's mod-
ification of the Esbach method — Removal of albumin

from the urine 35-36

Bence- Jones' Body 36

Albumose 37

Glucose 38

Qualitative tests: Trommer's test — Fehling's test — Al-
men-Nylander 's test — The fermentation test — Cippo-
lina 's modification of the phenylhydrazin test . . 38-42
Quantitative estimation of glucose: Benedict's first
method — Benedict's second method — Polariscopic deter-
mination — Robert's specific gravity method — Measure-
ment of carbon dioxid gas formed during fermentation 44-51



Levulosb


. 53


Seliwanoff's test, as modified by Borehardt


. 53


The phenylhydrazin test


. 55


Maltose .........


. 56


Lactose


. 56


Sacchaeose


. 57


Pentose


. 57


The phloroglucin test


. 57


The orcin test


. 58


Bial's modification of the orcin test


. 58


Glycuronic Acid


. 59


B. Tollens' test . ....


. 60


Alkaptonuria


. 61


Acetone . . . . .


. 62


Qualitative tests: Gunning's test — Lieben's test-


-Legal 's


test — Lange's test ......


. 62-64


DiACETic Acid


. 65


Gerhardt's test


. 65



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CONTENTS



XI



DiACETic Acid {Continued)

Arnold's test

^^ OxYBUTYRic Acid

Black's test

Hart's test

Urobilinogen

Ehrlich's aldehyd test

Urobilin

Spectroscopic determination

Schlesinger 's test

Jaffe's test

Bile Pigments

Qualitative tests: Foam test — Gmelin's test — Rosen

bach's modification of Gmelin's test — Huppert's test—

Hammarsten's test
Hematoporphyrin

Garrod's test ....
Hemoglobin . . . .

Spectroscopic determination .

The guiac test .

Heller's test ....

Teichmann's hemin-crystal test
The Diazo Reaction
Chyluria ...

LiPURIA ...

Ferments in the Urine

Wohlgemuth 's method for the determination of diastase
Determination of lipase according to Hewlett

The Urinary Sediments

The unorganized sediments: The quadriurates of sodium
and potassium — Uric acid — Calcium oxalate — Calcium
sulphate — Monocalcium phosphate — Plippuric acid —
Cholesterin — Xanthin — Hematoidin — Tyrosin —
Leucin — Cystin — Tricalcium and trimagnesium phos-
phates — Calcium carbonate — Ammoniomagnesium phos-
phate — Ammonium biurate — Neutral magnesium phos-
phate — Neutral calcium phosphate . . . .



66
67
67
68
70
70
70
71
71
72
73



73-75
76
76
77
78
81
82
82
83
84
86
86
87
88
89



91-99



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xu



CONTENTS



The Ubinaet Sediments (Continued)

The organized sediments : Epithelial cells — Pus — Blood —
Casts — Mucous threads — ' ' Clap threads ' ' — Gonococcus
— Treponema pallidum — Bacillus tuberculosis . 100-115



Animal Parasites in Urinary Passages

Trichomonas vaginalis

Filaria bancrofti

Dioctophyme renale .

Schistosoma hematobium
Prostatic Fluid
Functional Diagnosis op the Kidney

The phthalein test of Rowntree and Geraghty



116
116
117
117
118
119
119
120



CHAPTER II

THE GASTRIC JUICE

Test Breakfasts 125

Examination of the Fasting Stomach .... 127
Macroscopic Examination of the Gastric Contents . . 128

Quantity 128

Odor 128

Mucus 128

Color 129

Food 129

Chemical Examination of the Gastric Contents . . 131

Reaction 131

Hydrochloric acid: Qualitative tests for free hydrochlo-
ric acid (von den Velden's methyl violet test; Giinz-
berg's test; Tropeolin test; Congo-paper test; Topfer's
test; Sahli's desmoid test) — Quantitative determination
of gastric acidity (Topfer's method for free hydrochloric
acid; Other indicators; Titration of total acidity) — The
hydrochloric acid deficit . . 131-138
Lactic acid: Qualitative tests for lactic acid (Ueffel-
mann's test; Strauss' test; Kelling's test) . . 141-142
Butyric acid 142



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CONTENTS



Xlll



Chemical Examination of the Gastric Contents (Continued)

PAGE

Acetic acid 143

Pepsin: Qualitative test for pepsin — Quantitative meth-
ods (Mette's method as modified by Nierenstein and

Schiff) 143-144

145
146
146
148
149



Eennin: Qualitative test for rennin

Eennin zymogen

Pathological enzyme in the gastric contents .

Mucus

MiCEOscopic Examination op the Gastric Contents



CHAPTER III



THE FECES



Macroscopic Examination op the Feces .... 152

Amount 153

Form 153

Color 153

Mucus 154

Gallstones 155

Parasites 155

Intestinal Test Diet ... .... 155

Diet No. I 156

Diet No. II 156

Weight of Dried Feces . 157

Chemical Examination op the Feces 157

Reaction 157

Pigments • . 157

Urobilin (Hydrobilirubin) : Schmidt's test — Schlesin-

ger's test — Spectroscopic determination .... 158
Bilirubin: Schmidt's test — Gmelin's test . . 158-159
Blood: Weber's test — The guiac test — Teichmann's
hemin crystal test . ... . 159-161
Trypsin: Method of Gross for the determination of

trypsin 162



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xiv CONTENTS

PAGE

Chemical Examination op the Feces (Continued)

Amylase: Wohlgemuth 's method for the determination

of amylase, as modified by liawk . . . 163

Microscopic Examination of the Feces . . . 166

Pood remnants . ...... 167

Bacteria ■ . . . 168

Cells. . . 169

Crystals 170

Intestinal parasites: Protozoa, Bhizopoda — Flagellata —
Infusoria — Nematodes — Trematodes — Cestodes . 172-194
Preservation of gross specimens of cestodes and other
parasites : Permanent preparations of flat worms
(Method of Mink and Ebling — Boggs' method^Creosote

method) . . 199-203

Accidental contaminations ...... 203

CHAPTEE IV

THE SPUTUM

Amount 205

Reaction 205

Character 205

Odor 205

Consistence 205

Air Bubbles 205

Dittrich's Plugs 205

Bronchial Casts 206

Curschmann's Spirals 206

Layer Formation 206

Microscopic Examination 206

Examination of fresh sputum 206

Yellow elastic tissue 208

Curschmann's spirals ....... 208

Alveolar epithelial cells 209

Dust cells . 210

" Heart- failure cells" . 210



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CONTENTS XV

PAGE

Microscopic Examination {Continued)

Red blood corpuscles 210

Pus cells 211

Eosinophilic leukocytes 211

Lymphocytes 211

Chareot-Leyden crystals 211

Microorganisms in sputa: Bacillus tuberculosis (Ziehl-
Neelsen method; Antiformin method for the detection of
tubercle baciUi) — Diploeoccus pneumoniae (Gram's
method of staining; "Welch's capsule stain) — Bacillus in-
fluenzae — Bacillus diphtherias (Neisser's staining method;
Beall's method) — Actinomyces bovis — Streptothrix ep-
pengeri — Blastomyeetes . . ... 212-223

Animal parasites in the sputum: Entamoeba histolytica
— Entamoeba tetragena — Trichomonads — Paragonimus
westermanii — Echinococcua cyst .... 224-225



CHAPTER V

THE BLOOD

Obtaining Blood for Examination 226

Blood Stickers 226

Counting the Blood Corpuscles 227

The hemoeytometer 227

Procedure in counting the erythrocytes : Diluting fluids — -
Filling the pipette — Filling the counting chamber — The
enumeration of the cells — Calculation of the result —

Cleaning the apparatus 229-234

Counting the leukocytes : Diluting fluid — Filling the pi-
pette — Filling the counting chamber — The enumeration
of the leukocytes — Calculation of the result — Biirker's
modification of the Thoma counting chamber . 236-239
Counting the eosinophilic leukocytes: Dunger's method 241
Counting the blood platelets: Method of Wright and
Kirmicutt 242



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XVI



CONTENTS



PAGE

Hemoglobin Determinations 244

Tallqvist's method 244

Sahli's hemometer 245

The Fleisehl-Mieseher hemoglobinometer . . . 249

Haldane's hemoglobinometer 252

Dare's hemoglobinometer 252

Sulphhemoglobinemia 252

Methemoglobinemia 252

Color Index 253

Volume Index 253

Measuring the Diameter op Cells 255

Viscosity op the Blood and Other Fluids . . . 256

Method of Hess 256

The Specific Gravity op the Blood 259

The Coagulation Time of the Blood 260

Method of Brodie and Eussell, as modified by Boggs . 260
Milian's method, as modified by Hinman and Sladen . 263
The Resistance op the Red Blood Corpuscles . . . 264
The Examination op Fresh and Stained Preparations of

Blood 265

The cleaning of cover glasses and slides .... 265
Examination of the fresh blood : Sealing the fresh speci-
men — The preparation of dry (permanent) blood smears

— Fixation of blood smears 265-271

Staining the blood: Vital staining of the blood —
Vaughan's method; Method of Widal, Abrami, and
Brule ; The ' ' dry ' ' method of vital staining ; The stain-
ing of dried blood films — Methylene blue ; Eosin ; Hema-
toxylin; Carbol-thionin. Staining mixtures of two or
more stains — Ehrlich's triacid stain; The Romanowsky
stains (Wilson's stain, Leishman's stain, Giemsa's
stain); Jenner's stain; Methyl green-pyronin mixture
of Pappenheim; The iodin reaction of the leukocytes

273-295
Differential counting of the leukocytes: Normal leuko-
cytes — Pathological leukocytes .... 296-299



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CONTENTS xvii

PAGE

The Examination of Fresh and Stained Preparations of
Blood (Continued)
The normal and pathological red blood corpuscles:
Erythrocytes — Erythroblasts — Abnormalities in the
staining of the red corpuscles .... 301-302
Demonstration of protozoa in the blood: Malarial para-
sites 305

Examination of the blood for animal parasites: Filaria
bancrofti — Trichinella spiralis . . . 308-310



CHAPTER VI

PUNCTURE FLUIDS

Specific Gravity 312

Albumin Content 312

Incoagulable Nitrogen . .' 313

A Protein Peecipitable in the Cold by Dilute Acetic

Acid 315

Cytology 315

Cerebrospinal Fluid 318

Cells of the cerebrospinal fluid 318

Method of counting the cells 318

Bacteriology of the cerebrospinal fluid . . . 319
Globulin content: Method of Noguchi — Method of Ross

and Jones ■ 319-321

The Wassermann reaction in cerebrospinal fluid . . 321

Index 323



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LIST OP ILLUSTRATIONS



1. — Apparatus for the quantitative determination of am-
monia according to Folin ....
■Apparatus for the determination of ammonia according

to Shaffer

•Digesting rack for the Kjeldahl nitrogen determination
■Distilling apparatus for the Kjeldahl nitrogen deter-
mination

5. — Lohnstein's areometer . . ....

6. — Lohnstein's fermentation saccharometer for undiluted



2.-

3.-

4.-



FIG. PAGE

14

16
21

22
51

urine 52

7. — Absorption spectra 79

8. — The Sydenham sedimenting glass 89

9. — Treponema pallidum; Spirochseta refringens . . 112

10. — Embryo of Filaria bancrofti 117

11. — Ova of Dioctophyme renale 118

12. — The Autenrieth-Konigsberger colorimeter as modified by
Rowntree and Geraghty for the determination of

phenolsulphonephthalein 123

13. — Parasitic amebae 173

14. — Trichomonas intestinalis 177

15. — Lamblia intestinalis 178

16.— Balantidium coli 179

17. — Ovum of Necator americanus 181

18. — Necator americanus 185

19. — The rhabditiform embryo of Strongyloides stercorals . 186

20. — Ovum of Strongyloides stercoralis 187

21. — The rhabditiform embryo of Strongyloides stercoralis

and the embryo of the hookworm .... 188

22. — Ovum of Oxyuris vermicularis 188

23. — Ovum of Trichuris trichiura 189

xix



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XX LIST OF ILLUSTRATIONS

FIG. PAGE

24. — Ovum of Ascaris lumbricoides ; the same under high

focus, showing the albuminous coating . . . 190

25. — Unfertilized ovum of Ascaris lumbricoides . . 191

26. — Ovum of Schistosoma hajmatobium .... 193

27. — Ovum of Schistosoma japonicum 193

28. — Gravid proglottis of Tenia saginata; ovum of Tenia

saginata; gravid proglottis of Tenia solium . . 195

29. — Ovum of Dibothriocephalus latus ..... 196

30. — Gravid proglottis of Dibothriocephalus latus . . . 197

31. — Ovum of Hymenolepis nana ...... 197

32. — Ovum of Hymenolepis diminuta ..... 198

33. — Dipylidium caninum, showing an egg capsule and a

free ovum 199

34. — Tyroglyphus siro, the cheese-mite and ovum . . . 202
35. — Actinomyces hominis, showing club-shaped extremities

to the rays 222

36. — Blastomycetes in sputum 223

37. — Ovum of Paragonimus westermanii from the sputum . 224

38. — Sediment from echinococcus cyst 225

39. — The Thoma-Zeiss hemocytometer 227

40. — The Neubauer ruling of the hemocjrtometer . . . 228

41. — Burker's hemocytometer 239

42. — The Sahli hemometer 245

43. — The Pleisehl-Miescher hemoglobinometer . . . 250

44. — The viscosimeter of Hess 257

45. — Boggs' modification of the coagulometer of Brodie and

Russell .261

46. — ^Diagram to illustrate the movement of the cells during

coagulation 262



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CLINICAL LABORATORY
METHODS

CHAPTER I

THE URINE

Collection of the Urine.— For chemical examination, as
a general rule, the total amount of urine for the twenty-
four hours should be preserved. The reason for saving all
the urine is that different voidings may vary greatly in
their chemical composition. In the morning, for example,
an albuminuric may excrete urine which is normal chemi-
cally, whereas specimens obtained after the patient has
had more or less exercise may contain albumin. Thus, it
becomes necessary that a mixture of all the urine passed
during the twenty-four hours be. obtained in order to avoid
the possibility of error, for the examination of the early
morning specimen alone, in the case just cited, would be
entirely misleading. Special circumstances arise at times,
nevertheless, which make it desirable to break the rule and
to secure one or more voidings at special hours of the day.
For the purpose of quantitative chemical analysis, it is, of
course, absolutely essential to have the total amount of
urine for twenty-four hours collected.

For microscopic examination a perfectly fresh specimen
should always be insisted upon. The organized elements
of a urinary sediment rapidly deteriorate, especially with
high temperatures, so that within a few hours after the
urine has been passed they may be unrecognizable, or com-
pletely disintegrated. When it is not possible to make an

1



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2 PEESBRVATION OF THE URINE

immediate examination a preservative (thymol, toluol)
should be added to the specimen, which is kept in an ice-
chest as a further safeguard.

Preservation of the Urine.— To all twelve or twenty-
four-hour specimens of urine a preservative should be
added to prevent decomposition through bacterial growth.
The urine should also be kept on ice when possible. The
receptacle for the urine must be perfectly cleaned and
tightly stoppered.

(1) Toluol (toluene) is, on the whole, one of the most
satisfactory preservatives. It apparently interferes with
none of the urinary tests. Bacterial growth is successfully
inhibited by means of it. The one disadvantage in its use
results from the fact that toluol floats on the urine; it is
necessary to pipette or siphon the urine to obtain it with-
out admixture of the preservative. The objection is a
minor one. Diacetic acid may be preserved for weeks,
whereas it disappears in a short time when other preserva-
tives are used. In acidosis, therefore, toluol should be used
as the preservative. Organized sediments are often beau-
tifully preserved, but it must be remembered that casts or
cells, becoming attached to droplets of toluol, rise to the
surface and may be missed, when only a few are present;
spontaneous sedimentation cannot be relied on if toluol
is employed. The pipette used for withdrawing the sedi-
ment must be wiped to remove the toluol before placing the
drop on a slide for examination.

(2) Chloroform is the most generally used preservative
for chemical work. It is a fairly strong reducing agent,
and urine preserved with it must be boiled to drive it off
before any of the reduction tests for sugar are performed.
If the sediment is to be examined, care should be exercised
to avoid drawing up chloroform with it.



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THE UEINB 3

(3) Thymol is very satisfactory, and with it the formed
elements of the urine are often very well preserved. As
Weinberger ^ has shown, many urines to which thymol has
been added give a positive Heller's test, though albumin
be absent, a source of error which must be kept in mind. A
positive test for bile may also be obtained after thymol
preservation (Emerson).

Gum camphor and formaldehyde are used occasionally
as preservatives. Formaldehyde, like chloroform, is a re-
ducing agent. When available, toluol, chloroform, or thy-
mol is to be preferred.

Macroscopic Examination of the Urine.— As a general
rule, normal, freshly voided urine is perfectly clear; the
same is true of the majority of pathological urines. Occa-
sionally, if the reaction of the urine be alkaline when
voided, a turbidity may result from the precipitation of the
phosphates and carbonates in the bladder, in the absence
of a cystitis. Ordinarily, however, fresh urine, when cloudy
or turbid, contains pathological ingredients, such as blood,
pus, bacteria in large number, phosphates, etc. Normal
and pathological urines will become turbid and produce
a macroscopic deposit, more or less abundant, if allowed
to stand for some hours. Concentrated urine often fur-
nishes an abundant precipitate of urates on cooling; the
urates may be redissolved by warming the specimen. More
frequently bacterial decomposition is the cause of the tur-
bidity.

The nubecula is a translucent cloud, composed chiefly
of mucin (mucous threads) enmeshing epithelial or other
cells, which forms in the urine a short time after it is
passed.

'Weinberger, W. "Thymol as a source of error in Heller's test for
urinary protein." Jour. A. M. A., 1909, LII, 1310.



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4 EBACTION OP UEINB

The color of the urine is usually dependent on the quan-
tity of water excreted in the twenty-four hours ; the smaller



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