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BIOLOGY LIBRAflt






A MANUAL

OF



CLINICAL CHEMISTRY, MICROSCOPY,
AND BACTERIOLOGY



A MANUAL

OF

CLINICAL CHEMISTRY, MICROSCOPY,
AND BACTERIOLOGY

BY
DR. M. KLOPSTOCK AND DR. A. KOWARSKY

OF BERLIN

In their "Institut fur medizinische Diagnostik," in Berlin



ONLY AUTHORIZED TRANSLATION FROM THE LAST

GERMAN EDITION, THOROUGHLY REVISED

AND ENLARGED



ILLUSTRATED WITH FORTY-THREE TEXTUAL FIGURES
AND SIXTEEN COLORED PLATES




NEW YORK

REBMAN COMPANY

1123 BROADWAY






310LOSY
UDRARY



COPYRIGHT, 1912, BY

REBMAN COMPANY

NEW YORK

All Rights Reserved



PRINTED IN AMERICA



PREFACE TO THE GERMAN EDITION

THIS book owes its existence to the desire of the
authors to place a concise manual in the hands of those
taking part in the course in Clinical Chemistry, Micro-
scopy, and Bacteriology held in their ' ' Institut fur medi-
zinische Diagnostic, " in Berlin. It is in nowise intended
to replace larger and more elaborate text-books, but aims
to present in concise form the essential features of the sub-
jects treated. As the book is intended especially for the
practitioner, we have assumed that the reader has an
elementary chemical and bacteriological education. For
the same reason the needs of daily practice have been
especially considered in the choice of the methods of
examination. Wherever it has been possible the simplest
and quickest methods have been chosen.

It would be a source of gratification to us if this book
should find favor in wider medical circles.

THE AUTHORS.



248545



PUBLISHERS' ANNOUNCEMENT

HAVING found this book a valuable laboratory guide,
it is a pleasure to comply with the request of the
Authors to publish an English Translation of this New
Edition. The Authors have retained all those matters
which by experience have proved themselves to be of
genuine, practical value to the student as well as to the
medical man in General Practice. The pages relating to
Typhoid Fever and to the Meningococci have been re-
written with great care; whilst those dealing with the
Spirocheta pallida and with the Wassermann Reaction,
especially, are entirely new.

The book has been brought up to date. The Index
at the end of the volume is a new feature, and the refer-
ences to the pages where they are quoted will prove of
real value when consulting the colored plates.

REBMAN COMPANY.
NEW YORK, 1123 BROADWAY.



CONTENTS



CHAPTER I

BACTERIOLOGICAL EXAMINATION OF THE SECRETIONS
AND DEPOSITS IN THE MOUTH AND PHARYNX

PAGE

Collection of Material to be Examined 1

Morphological and Staining Characteristics of Diphtheria

Bacilli , 2

Cultural Characteristics 4

Animal Inoculation 5

Differential Diagnosis 5

Order of Examination 8

Oidium albicans "Soor fungus" 10

Angina Vincenti (s. Plautii) 11

CHAPTER II

BACTERIOLOGICAL EXAMINATION OF NASAL SECRETIONS

Diphtheria Bacilli 17

Tubercle Bacilli 17

Lepra Bacilli 18

Diplobacillus of Friedlaender 18

CHAPTER III

BACTERIOLOGICAL EXAMINATION OF CONJUNCTIVAL
SECRETION

Diphtheria Bacilli 19

Tubercle Bacilli 19

Gonococci 20

Bacilli of Koch and Weeks 20

Diplobacillus of Morax and Axenfeld 21

Other Exciters of Conjunctivitis 21



CONTENTS
CHAPTER IV

EXAMINATION OF THE SPUTUM

PAGE

Method of Obtaining Material for Examination 22

General Characteristics 23

Especially Prominent Constituents of the Sputum 23

Composition of Sputum 24

Especially Prominent Ingredients of the Sputum 26

Microscopical Examination 28

Curschmanri 's Spirals 29

Fibrin Coagula 30

Dittrich's Plugs 30

Portions of the Echinococcus 31

Actinomyces Granules 32

Cellular Elements of the Sputum 33

Elastic Fibres 35

Crystalline Bodies 36

Bacteriological Examination of the Sputum 37

Examination of Stained Smears 38

Cultural Examination 40

Animal Inoculation 40

Detection of Tubercle Bacilli 40

Pneumococci 48

Streptococci 49

Staphylococci 50

Micrococcus tetragenus 50

Micrococcus catarrhalis 51

Influenza Bacillus 51

Diplobacillus of Friedlaender 52

Bacillus pyocyaneus 53

Bacillus of Bubonic Plague.. 53



CHAPTER V

EXAMINATION OF THE GASTRIC CONTENTS

General Characteristics 56

Qualitative Chemical Examination 58

Reaction.. 58



CONTENTS

PAGE

Qualitative Chemical Examination :

Free Acids 58

Free Hydrochloric Acid 58

Lactic Acid 61

Volatile Fatty Acids 61

Pepsin and Pepsinogen 62

Renin and Reninogen 65

Bile Pigment 66

Blood 66

Hydrogen Sulphide 68

Quantitative Chemical Examination of the Gastric Con-
tents 68

Estimation of Total Acidity 68

of Free Hydrochloric Acid 69

of Total Hydrochloric Acid , . . . 70

of Lactic Acid 71

Microscopical Examination of the Gastric Contents.. 72



CHAPTER VI

EXAMINATION OF THE FAECES

General Characteristics 74

Qualitative Chemical Examination of the Faeces 80

Reaction 80

Mucin 80

Fat 81

Blood . 81

Biliary Constituents 82

Quantitative Chemical Examination of the Faeces 84

Estimation of Dry Matter 84

of Total Nitrogen 85

of Fat 85

of Carbohydrates 86

Direct Estimation of Starch According to Liebermann and

Allihn 87

Fermentation Test According to Schmidt 88

Examination of Gall-Stones and Biliary Concretions 90

Fecal Concretions, Enteroliths, and Pancreatic Stones . . 92



CONTENTS

PAGE

Microscopical Examination of the Faeces 94

Food Particles 94

Pathological Products of the Intestinal Wall 96

Intestinal Parasites and their Eggs 97

Bacteriological Examination of the Faeces 104

Typhoid Bacilli 104

Characteristics of Typhoid Bacilli 105

Biological Characteristics of Typhoid Bacilli 108

Order of Examination of the Faeces for Typhoid Bacilli 113

Planting of Cultures from the Faeces 113

Examination of the Plates 114

Bacilli in Meat-Poisoning 117

Dysentery Bacilli 117

Cholera Vibriones 120

Pfeiffer's Test 122

Detection of Cholera Vibriones in the Faeces 125

Tubercle Bacilli 126

Staphylococci and Streptococci 127

Anthrax Bacilli 128

Plague Bacilli 128



CHAPTER VII

EXAMINATION OF THE URINE

Collection of the Urine 129

The Identification of a Fluid as Urine 130

Chemical and Physical Characteristics of the Urine 132

Color : 132

Transparency 133

Reaction 135

Specific Gravity 136

Freezing-point 137

Quantity 140

The Chemical Examination of the Pathological and Abnor-
mal Constituents of the Urine 141

Albumin 141

Albumoses and Peptones 148

Method of Freeing the Urine from Albumin 149

Albumins Precipitated by Cold Acetic Acid 150



CONTENTS

PAGE

The Chemical Examination of Fibrin 151

Glucose 151

Lactose 160

Levulose 160

Pentose 161

Glycuronic Acid 162

Acetone 163

Diacetic Acid 164

/8-Oxybutyric Acid ' 165

Indican 165

Urobilin 167

Biliary Pigments 168

Blood Pigments 170

HaBmatoporphyrin 173

Melanin 174

Diazo Reaction 174

Adventitious Constituents of the Urine Iodine, Mer-
cury, etc 175

Quantitative Chemical Examination of the Urine 180

Estimation of Albumin 180

of Sugar 182

of Total Nitrogen 187

of Urates 188

of Uric Acid 193

of Chlorides 199

of Phosphates 199

of Sulphates 201

of Oxalic Acid according to Salkowski 202

Examination of Urinary Calculi and Concretions 206

Microscopical Examination of the Urinary Sediment 209

Organized Sediments 224

Bacteriological Examination of the Urine 237

Collection and Preparation of the Urine for Examination 237

Method of Examination 238

Bacterium coli 239

Staphylococci and Streptococci 240

Tubercle Bacilli 240

Typhoid Bacilli 244

Gonococci 244

Proteus vulgaris . 245



CONTENTS
CHAPTER VIII

EXAMINATION OF THE URETHRAL AND PROSTATIC
SECRETIONS

Prostatic Secretion . .



CHAPTER IX

EXAMINATION OF THE BLOOD

Determination of the Specific Gravity 250

of the Freezing-point 251

Estimation of Hemoglobin ] '251

Enumeration of Blood-Corpuscles '253

Histological Examination ' '255

Examination of Fresh Specimens

of Stained Specimens

Sketch of the Mbrphology of the Blood '259

Bacteriological Examination of the Blood 263

Examination of the Blood in Stained Smears. . 263

Malaria 263

Spirilla of Relapsing- Fever

Examination of the Blood by Means of Cultural Pro-
cedures geg

Cultivation of Typhoid Bacilli ............ ' .' .' * ' 2 69

of Staphylococci and Streptococci 271
Examination of the Blood by Means of Animal Inoc-
ulation 271

Serum Diagnosis 272

Macroscopic, Quantitative Agglutination Test!!

Exploratory Agglutination Test 272

Widal's Reaction ... ' 979

Pfeiffer'sTest .'.'.'.'.'.'.'.'.'.'.'.' ' 375

The Bactericidal Test-tube Test ' 276

Serum Diagnosis of Syphilis According to Wasser-

mann 27g

Wassermann's Reaction ] 278



CONTENTS
CHAPTER X

EXAMINATION OF FLUIDS OBTAINED BY PUNCTURE

PAGE

General Characteristics and Chemical Examination 286

Microscopical Examination 290

Bacteriological Examination 291

Collection of Material for Examination 291

Method of Examination 292

The Most Important Bacteriological Findings 295

CHAPTER XI

BACTERIOLOGICAL EXAMINATION OF DISEASES OF THE
SKIN

Purulent Affections of the Skin 299

Glanders 300

Anthrax 301

Tetanus 302

Bacillus of ulcus molle 304

Tuberculosis of the Skin 305

Diseases of the Skin excited by Hyphomycetes (Dermato-

mycosis) 305

Favus 309

Trichophytosis 311

Tinea sycosis 312

' ' circinata 313

Pityriasis versicolor 315

Erythrasma 316

Spirocheta pallida 317

CHAPTER xn

THE USUAL METHODS OF BACTERIOLOGICAL EXAMINATION,
FORMULAE OF STAINS, AND CULTURE MEDIA

Examination in a Hanging-Drop 327

Examination in Stained Smears 328

Preparation of the Specimens 328



CONTENTS

PAGE

Examination in Stained Smears :

Staining Methods and Staining Solutions 329

Stock Solutions 329

of Tubercle Bacilli and other Acid-fast Bacilli 330

of Diphtheria Bacilli 332

" of Gonococci 334

of Spores 334

of the Capsules of Anthrax Bacilli 335

of Flagella 336

of Fungi 337

' ' of Blood Specimens 338

Examination of Cut Sections 339

Universal Staining Methods for Demonstrating Bac-
teria in Sections 341

Special Staining Methods 342

Cultural Methods 344

Preparation of Culture Media 344

The Cultural Methods most frequently Employed 356

Aerobic Cultures 356

Anaerobic Cultures 359

Determination of the Biological Characteristics of Bacteria 361
Methods of Animal Inoculation 363

INDEX.. . 365



LIST OF FIGURES IN THE TEXT

FIG. PAGE

1. Oidium Albicans "Soor Fungus" 10

2. Fibrin Clots from Pneumonic Sputum 27

3. Spirals from the Sputum (natural size) 29

4. Spirals from the Sputum (magnified) 29

5. Hyaline Membrane of an Echinococcus Cyst 31

6. Echinococcus Hooks 31

7. Actinomyces Granules (low power) 32

8. Actinomyces Granules (unstained specimen) 33

9. Elastic Fibres from the Sputum 35

10. Ley den Crystals (magnified 300 times) 36

11. Boas' Fecal Sieve 77

12. . Schmidt's Fermentation Apparatus 89

13. Amoeba Coli 98

14. Balantidium Coli 98

15. Tsenia Solium 99

16. TaBnia Saginata 100

17. Scolex of Bothriocephalus Latus 100

18. Oxyuris Vermicularis 101

19. Ascaris Lumbricoides 102

20. Tricocephalus Dispar 102

21. Anchylostoma Duodenale 103

22. Beckmaryi's Cry oscope 138

23. Einhorn's Saccharometer 157

24. Jolles' Azotometer 191

25. Amorphous Urates 213

26. Calcium Oxalate 215

27. Neutral Calcium Phosphate ... .216



LIST OF FIGURES IN THE TEXT

PIG. PAGE

28. Triple Phosphate 218

29. Tyrosin Cystin Leucin 219

30. Cystin Crystals 220

31. Hippuric Acid 221

32. Fatty Acid Needles 223

33. Epithelial Cells . . . .- 224

34. Squamous Epithelial Cells 226

35. Cylindroids 232

36. Urinary Filaments 233

37. Substances Found in the Sediment of Urine 236

38. Ehrlich's Copper Plate 257

39. Favus Fungi 309

40. Hair in Sycosis 313

41. Epidermal Scales 314

42. Spirochetse 317

43. Migrating Cells and Papillary Bodies . . .325



LIST OF COLORED PLATES

REFERRED TO

PLATE FIG. ON PAGE

I, A. Diphtheria Bacilli 3

I, B. Diphtheria Bacilli 3

II, C. Sputum with Typhoid Bacilli 40

II, D. Pneumonic Sputum 48

III, E. Pneumonic Sputum 48

III, F. Bronchial Sputum 51

IV, G. Influenza Bacilli 51

V, H. Cholera Dejection 120

V, I. Uric Acid 211

VI, J. Ammonium Urate 213

VII, K. Nephritis in Pregnancy Hematoidin Crystals. 222

VIII, L. Casts from Icteric Urine 230

IX, M. Leucocytes 227

IX, N. Red Blood-Corpuscles 229

X, O. Hyaline Casts 230

X, P. Spermatozoa 235

XI, Q. Echinococcus Booklets 235

XI, R. Bacterium Coli 239

XII, S. Tubercle Bacilli 240

XII, T. Gonococci 247

XIII, U. Macrocytes, Microcytes, etc 262

XIII, V. Tertian Parasite 265

XIV, W. Tertian Parasite .... 265

XIV, X. Chronic Tropical Malaria 267

XV, Y. Spirilla of Relapsing Fever 268

XV, Z. Anthrax Bacilli in Rabbit's Blood 301

XVI, a. Tetanus Bacilli.. .302



CHAPTER I

BACTERIOLOGICAL EXAMINATION OF THE

SECRETIONS AND DEPOSITS IN THE

MOUTH AND PHARYNX

In the bacteriological examination of pathological
products found in the mouth and pharynx, the place of
first importance belongs to the diphtheria bacilli. Of
secondary importance only are the staphylo-, strepto-,
pneumo-cocci, influenza bacilli, and the diplobacillus of
Friedlaender j which appear as independent exciters of
inflammation in angina, as well as producers of mixed
infection in diphtheria. Finally, the "soor fungus,"
Of (Hum albicans, should be mentioned. In coatings on
the tonsils, which are excited by the Oidium albicans,
diphtheria bacilli are also not infrequently found.

Collection of Material to be Examined

For the collection of material from the mouth and
pharynx it is best to use the small apparatus which can
be obtained from any supply station. This consists of a
strong test-tube containing a piece of wire, one end of
which is placed in the plug which closes the tube, while
the other carries a swab of common cotton. The test-tube
and contents are sterilized by dry heat for half an hour at
a temperature of 160 C., 1 and then placed in a suitable
box, containing directions for use and a blank to be filled
out by the physician, stating duration of illness, locality
from which the material to be examined is taken, etc.

1 All degrees of temperature quoted in this book are Celsius.

1



S CHEMISTRY, MICROSCOPY, AND BACTERIOLOGY

To obtain the material for examination, the swab is
brushed firmly across the suspected coating and returned
immediately to the test-tube (care being taken that it
touches nothing but the coating in question). No anti-
septic (gargle or application) should be used for some
time before the collection of the material, as the growth
of the diphtheria bacillus is inhibited by even mild anti-
septics.

Morphological and Staining Characteristics of
Diphtheria Bacilli

Diphtheria bacilli are non-motile rods which show
differences in their morphological characteristics depend-
ing upon the nature of the culture media, the age of the
culture, and the temperature at which they are cultivated.
They vary considerably in length, so that short, medium,
and long forms may be differentiated. A six to ten hours'
growth on pure serum, or Loeffler's. blood serum, consists
principally of long, partly straight, partly slightly curved
bacilli, club-shaped or pointed at both ends. In older
cultures the spindle, dumb-bell, and lancet forms are seen.
Small, highly refractive points can be seen in the proto-
plasm of the long forms. The formation in colonies of
the diphtheria bacilli, of loosely arranged groups, in which
the individual bacilli lie crosswise over each other, is
characteristic. Especially in A7/scA-preparata from
young serum cultures and in membranes in which the
diphtheria bacilli are present in great numbers, a picture
is seen, which may be imitated by holding the outspread
fingers of one hand, in various combinations, over or be-
side those of the other (Neisser).

Diphtheria bacilli stain easily with dilute aniline
dyes. Dilute ZieliPs solution (1:9), and Loefflcr's methy-
lenp. blue, are especially adaptable. The former stains in



MOUTH AND PHARYNX 3

one minute, the latter in two minutes, without heating.
Diphtheria bacilli stain according to Gram. Bacilli from
young cultures stain evenly, while in smears from older
cultures they usually show one or more unstained spots.
Bacilli stained with Loeffler's methylene blue, as a rule,
show either at one or both ends, granules more deeply
stained than the rest of the protoplasm. These polar
granules (Babes- Ernsts bodies) are especially distinct in
specimens stained according to the Roux or Neisser
methods (Plate I, Fig. A). Roux' s solution (cf. p. 333)
is made by mixing 1 part dahlia-violet solution with 2
parts methyl-green solution. The mixture keeps well and
produces no precipitate. It stains in two minutes without
heating. Neisser's is a double staining method (formula
cf. p. 333).

1. Old Method. Stain with acetic acid methylene blue
twenty seconds, wash with distilled water, counterstain
with Bismarck brown ten seconds, wash, etc.

2. New Method. Stain with Solution I (cf. formulae
of stains) for about thirty seconds, wash with distilled
water, counterstain with Solution II, also for about thirty
seconds. The bacilli then appear brown, the oval polar
granules dark blue (Plate I, Fig. B). As a rule, each
bacillus shows two granules, one at each end. Some,
however, have but one, at one end, while still others have
a third in the middle. It is common to find two bacilli
at an obtuse angle to each other, having together three or
four granules. This method of staining is only sure of
success when the smears are made from serum cultures,
which are at least nine and not more than twenty to
twenty-four hours old, and have been grown at a tempera-
ture of 34 to 37 C. It is further important that the
smears be very thin. It is necessary to test newly made
solutions for Neisser's method, as the staining power of



4 CHEMISTRY, MICROSCOPY, AND BACTERIOLOGY

different solutions varies. Neisser's method is of value
in differentiating between diphtheria bacilli and other
bacilli resembling them.

Cultural Characteristics

For the cultivation of diphtheria bacilli a temperature
of at least 20 C. is necessary. They grow best between
33 and 37 C. They flourish on all the usual culture
media which have a slight or distinct alkaline reaction.
For diagnostic purposes bouillon, glycerine agar (5 to 7
per cent.), and especially blood serum, are used.

After one to two days' growth, bouillon is either evenly
clouded, with flakes in it, or a fine granular precipitate
has developed, which collects on the sides and bottom of
the glass. Not infrequently a thin, granular, and easily
destroyed film forms on the surface of the bouillon. The
bouillon, originally faintly alkaline, becomes acid after
forty-eight hours' growth of diphtheria bacilli in it. The
growth on agar is scanty, but that on glycerine agar is
richer. The surface colonies appear transparent and gray-
ish-white; examined with low power they show a charac-
teristic granular surface and an irregular delicate border.
The most luxuriant and quickest growth of diphtheria
bacilli is that on blood serum. For their cultivation from
membranes it is especially suitable, as it has a selective
action against the other bacteria of the mouth. On blood
serum the diphtheria bacilli grow first, while other organ-
isms, which may at the same time be present, develop
later.

Either pure blood serum, which may come from cattle,
sheep, or horses is used, or the so-called Loeffle^s serum,
which is a mixture of 3 parts calf or sheep serum with 1
part of 1 per cent, grape-sugar bouillon (cf. p. 354).

On solidified serum the diphtheria bacilli have often,



MOUTH AND PHARYNX 5

after but six hours' growth, developed very small trans-
parent colonies. After twenty- four hours' growth the col-
onies are about the size of a pin's head, round, prominent,
and yellowish-white. If the colonies lying close together
coalesce, a yellowish-white coating is formed, which still
presents a distinctly granular appearance.

Animal Inoculation

Inoculation of guinea-pigs is used for diagnostic pur-
poses as a means of differentiating the diphtheria bacilli
from bacteria resembling them. Guinea-pigs weighing
200 to 800 grammes are inoculated subcutaneously with
2 cc of a forty-eight hours' bouillon culture. The animals
become sick quickly. After twelve to twenty-four hours
a distinct infiltration can be felt at the point of inocula-
tion; after two to three days the animal dies. The post
mortem shows at the site of inoculation a jellylike, cedem-
atous, blood-stained swelling of the subcutaneous tissue.
The peritoneal and pleural cavities contain serous and fre-
quently hemorrhagic 'exudates. The condition of the ad-
renal bodies is characteristic. They are enlarged and
hyperaemic, and their tissue is permeated by small punc-
tiform hemorrhages. In the cedema fluid at the site of
inoculation diphtheria bacilli can usually be detected by
cultural methods.

If the bacilli were less virulent, the animal dies later,
and the post-mortem appearances are not so typical. If
still less virulent, merely a local inflammation at the site
of inoculation is produced, which causes necrosis of the
skin and eventually heals.

Differential Diagnosis

In the differential diagnosis pseudo-diphtheria bacilli
(Hoffmann} and xerosis bacilli come into consideration.



6 CHEMISTRY, MICROSCOPY, AND BACTERIOLOGY

The former are among the normal inhabitants of the
mouth, the latter are found on the conjunctiva and in the
nose. The morphological differences between pseudo- and
true diphtheria bacilli are most evident in Klatsch-prQ-
parata from young (six to ten hours') blood-serum cul-
tures. Here the pseudo-diphtheria bacilli appear usually
as short, plump, often wedge-shaped rods ; the characteris-
tic long forms, which diphtheria cultures of the same age
show, are missing. The typical grouping of the bacilli is
also lacking. Pseudo-diphtheria bacilli lie usually with
the long sides parallel, side by side, arranged like pali-
sades. In smears from older (sixteen to twenty-four
hours') cultures the differentiation may be difficult.

Pseudo-diphtheria bacilli differ from true diphtheria
bacilli, in their staining properties, by their failure to
stain according to Neisser^s method. Although some-
times pseudo-diphtheria bacilli, if stained by this method
show pole kernels, they are always found but scarcely,
never as regularly as in diphtheria bacilli. It is best to
use cultures of from twelve to twenty- four hours for the
differential diagnostical staining, because the absence of
the pole kernels in cultures of from six to twelve hours
means just as little as the presence in older colonies.

In their cultural characteristics they differ by their
luxuriant growth on agar and, at first, slower development
on serum. The colonies are grayish-white, moistly glis-
tening, and somewhat waxy in appearance. It is remark-
able what soft and melting consistency they show, when
touched with the platinum needle in contradistinction to
the more rigid diphtheria bacillus colonies.

The faculty of diphtheria bacilli of forming acid in
culture media containing sugar may also be of differential
diagnostical value.

Diphtheria bacilli decompose dextrose and levulose



MOUTH AND PHARYNX 7

under acid formation, pseudo-diphtheria bacilli, as proven
in numerous types of various origin are almost always in-
active in both kinds of sugar, and, but in rare cases, they
are active in one, never in both.

The following culture media are used for testing acid
formation: Three parts of ox serum, which is rendered
sterile or made germ free by discontinuing sterilization at
55, are added to one part of sterile bouillon, which is
free of sugar. To each 90 parts of this mixture are added
10 parts of a litmus solution, which contains 10 per cent,
dextrose, resp. levulose, in order to obtain 2 media, of
which one contains 1 per cent, dextrose, the other 1 per
cent, levulose. The litmus sugar solution before being
added, has to be sterilized on two consecutive days, for
five minutes each day. The culture media are then filled
in test-tubes and are kept on three to five consecutive days,
for two hours each day, in the incubator at 55.

Procedure. A test-tube of levulose and a test-tube of
dextrose are each inoculated with a loop of the pure cul-
ture to be examined. After twenty-four hours in the in-
cubator at 87 the litmus solution appears red in the test-
tubes, which have been inoculated with diphtheria bacilli
and the serum albumin is precipitated.



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