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OF THE
IRew IPorF? State IDctcrinar^ CoUefie
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CORNELL UNIVERSITY LIBRARY
3 1924 104 224 864
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cooperation witli Cornell University Libraries, 2007.
You may use and print this copy in limited quantity
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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 â– . . . 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 â– 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 ....
â– Apparatus for the determination of ammonia according
to Shaffer
•Digesting rack for the Kjeldahl nitrogen determination
â– 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