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A MANUAL OF BACTERIOLOGY

WILLIAMS

A MANUAL

BACTERIOLOGY

HERBERT U. WILLIAMS, M.D.

PROFESSOR OF. PATHOLOGY AND BACTERIOLOGY, MEDICAL DEPARTMENT,
UNIVERSITY OF BUFFALO

WITH NINETY-NINE ILLUSTRATIONS

THIRD EDITION, REVISED AND ENLARGED

PHILADELPHIA :
P. BLAKISTON'S SON & CO.

1012 WALNUT STREET
1903

Main Lib.
Depi.

fee LlMAftV Of
COM6NESS,

T*» C*pfcM Itoctiwtf

•CT 24 1903

entry

BUM

BIOLOGY

LIBRARY

PRtSS OF

ERA PRINTING COMPANY,
LANCASTER PA,

PREFACE TO THE THIRD EDITION.

THE plan used in the preceding editions of this manual
has been followed in the preparation of the present one.
The only departures have been in the insertion of a short
historical sketch and the freer use of references to original
articles and reviews. It is hoped that these features will
assist in arousing the interest of students. As far as pos-
sible, reference has been made to articles in American and
English journals likely to be easy of access. Besides the
ones just named, numerous additions have been made which
the recent advances in our knowledge have rendered neces-
sary. Most of the illustrations of apparatus are new. The
photomicrographs also are new and original with a few
exceptions noted in the text. It is probably needless to say
that none of them were retouched. The writer is indebted
to the Gratwick Laboratory of Buffalo for the use of its
facilities in making these photographs.

BUFFALO, NEW YORK, August, 1903.

268453

PREFACE TO THE SECOND EDITION.

ALTHOUGH there has been no lack of works on bacteri-
ology, it seemed to the writer that there was still a field
open for one which sought to give the portions essential to
medical science in a concise manner. It is gratifying, there-
fore, that the first edition of this little book should have been
exhausted so soon.

Whether wisely or not, it is a fact that many medical
schools require their students to absorb an amount of
knowledge that taxes the brain to the utmost. While such
conditions remain, the need is urgent for presenting what
is taught in the accessory branches in as condensed a form
as is consistent with a clear understanding of their great
fundamental principles. It is mastery of such principles,
after all, which is the object of a course in bacteriology,
for they are essential to a correct understanding of most of
the other branches. After that has been accomplished,
(including the applications of bacteriology to diagnosis),
it must be admitted that other branches deserve a larger
amount of the student's time. This may be said without
meaning to minimize the importance of bacteriology in the
training of a physician. In the opinion of the writer it is
neither possible nor desirable that every graduate should
be a trained bacteriologist. However, no instructor can
hope to bring the principles above mentioned home to his
classes except by laboratory work. Very little attempt
has been made to outline the program of a laboratory
course, as that will always need to be planned according
to the circumstances under which it is given.

PREFACE. Vll

The purpose of this book is to give in the smallest pos-
sible space the facts which a physician must know, with
some of those which it is desirable that he should know,
and a little of that which he may learn if his needs or in-
clinations lead him to go further. It is acknowledged,
however, that, in deference to precedent, this purpose has
not been carried to its fullest extent. Much time has been
spent on the index, in order to make the contents quickly
accessible. It is a source of regret to the writer that the
additions which the revision seemed to demand have made
the present book a little larger than the first edition.

BUFFALO, NEW YORK, June, 1901.

PREFACE TO THE FIRST EDITION.

IN this manual the writer has endeavored to describe
the laboratory technique which the beginner must follow,
and at the same time to give a concise summary of the
facts in bacteriology most important to the physician. In
preparing a work of this character, which claims to be
nothing more than a compilation, the standard text-books
were necessarily consulted freely. On account of the need
for brevity it has, in most cases, been impossible to men-
tion authorities.

The writer is glad to have this opportunity to acknowl-
edge his obligation to the works of Sternberg, Fliigge,
Gunther, Eisenberg, Abbott, W. H. Park, Muir and Ritchie,
Vaughan and Novy, and McFarland; and to numerous
papers by Professor Welch and others. It is thought that
the chapters on Germicides, and Surgical Disinfection, by
Drs. Thos. B. Carpenter and Marshall Clinton, will be use-
ful not only for the information presented in them, but
especially in correlating that information with the facts of
bacteriology.

BUFFALO, NEW YORK, October, 1898.

Vlll

CONTENTS.

INTRODUCTION 1 1

Historical Sketch 18

PART I.

BACTERIOLOGICAL TECHNIQUE.
CHAPTER I.

PAGE.

Examination of Bacteria with the Microscope, including Methods of
Staining 29

CHAPTER II.
Sterilization ur

CHAPTER III.
Culture-media 71

CHAPTER IV.

The Cultivation of Bacteria. Tube-cultures ; the Incubator ; Anaer-
obic Methods 84

CHAPTER V.

The Cultivation of Bacteria (Continued). Isolation of Bacteria;

Plate-cultures 0.6

CHAPTER VI.
Inoculation of Animals. Autopsies ; Collodion Sacs 103

CHAPTER VII.
Collection of Material 108

CHAPTER VIII.

Systematic Study of Species of Bacteria. Suggestions for Class-
work; Rules 112

X CONTENTS.

PART II.

CHAPTER I.

Classification; General Morphology and Physiology of Bacteria. ... 117

CHAPTER II.
Products of the Growth of Bacteria 128

CHAPTER III.
Distribution of Bacteria — Soil, Air, Water, Foods 133

CHAPTER IV.
Bacteria of the Normal Human Body 151

CHAPTER V.
Bacteria in Disease 157

CHAPTER VI.
Toxins 17.2

CHAPTER VII.
Immunity 176

CHAPTER VIII.
Disinfectants and Antiseptics. By Thomas B. Carpenter, M.D 194

CHAPTER IX.
Preparation of Instruments, Ligatures, Dressings, etc., for Surgical

Purposes. By Marshall Clinton, M.D 211

PART III.

NON-PATHOGENIC BACTERIA 221

Yeasts and Moulds 231

PART IV.
PATHOGENIC BACTERIA.

Suppuration and Allied Conditions 235

Staphylococcus pyogenes aureus 243

albus 245

epidermidis albus 246

Streptococcus pyogenes 246

of Erysipelas 250

Micrococcus tetragenus 250

lanceolatus (of Pneumonia) 251

melitensis 255

CONTENTS. xi

Diplococcus intracellularis meningitidis 256

Micrococcus of Gonorrhea 258

Bacillus of Soft Chancre 260

pnetimoniae (of Friedlander) 260

of Rhinoscleroma 261

pyocyaneus 262

" proteus 264

of Bubonic Plague 265

aerogenes capsulatus 268

of Malignant Edema 270

of Tetanus 270

of Anthrax 273

" of Influenza 277

of Diphtheria 278

" tuberculosis 287

of Leprosy 296

" mallei (of Glanders) 297

Actinomyces bovis 299

Bacillus of Typhoid Fever 301

coli communis 309

lactis aerogenes 313

of Dysentery 313

Spirillum of Asiatic Cholera 315

Spirilla Allied to the Spirillum of Asiatic Cholera 323

Spirillum of Relapsing Fever 326

APPENDIX.

PATHOGENIC PROTOZOA.

Amoeba of dysentery 328

Malarial parasite 330

Small-pox and Vaccinia 334

Yellow fever 335

Trypanosomes 335

LIST OF ILLUSTRATIONS.

FIG. PAGE.

1. Micrococci, Bacilli, Spirilla 14

2. Test-tube with Culture-medium 17

3. Microscope 30

4. Abbe Condenser 31

5. Platinum Wires 33

6. Hanging-drop 34

7. Cornet Forceps for Cover-glasses 37

8. Stewart Forceps for Cover-glasses 38

9. Kirkbride Forceps for Slides 38

10. Schanze Microtome <o

11. Hot-air Sterilizer 62

12. Arnold Steam Sterilizer 64

13. Massachusetts Board of Health Sterilizer 65

14. Koch Steam Sterilizer 66

15. Autoclave 68

1 6. Kitasato Filter 69

17. Test-tube with Potato 78

1 8. Wire Basket for Test-tubes 82

19. Manner of Holding Test-tubes 85

20. Stab-culture 86

21. Smear-culture 86

22. Incubator 88

23. Reichert Gas-regulator 89

24. Gas-regulator 89

25. Koch Automatic Gas-burner 90

26. Buchner's Method for Cultivating Anaerobes 92

27. Frankel's 93

28. Novy's 94

29. Cover-glass as used to Distinguish Anaerobes from Aerobes 95

30. Petri Dish 98

31. Dilution-cultures in Esmarch Roll-tubes facing 99

32. Appearance of Colonies on Gelatin in a Petri Dish facing 99

33. Esmarch's Roll-tube 100

34. Mouse-holder 103

35. Apparatus for the Subcutaneous Insertion of Solid Substances.. .. 104

xiii

XIV ILLUSTRATIONS.

36. McCrae's Method for making Collodion Capsules 106

37. Cover-glass Preparation of Blood 100

38. Sternberg Bulb no

39. Micrococci of Various Forms 118

40. Bacilli of Various Forms 119

41. Spirilla of Various Forms 119

42. Involution Forms 120

43. Bacteria with Capsules .* 122

44. Bacteria with Spores 122

45. Bacteria Showing Flagella 124

46. Fermentation-tube 131

47. Sedgwick-Tucker Aerobioscope 136

48. Diagram to Illustrate Side-chain Theory of Immunity 185

49. Bacillus subtilis 226

50. Spirilla from Swamp Water 228

51. Spirilla from Swamp Water with Flagella 229

52. Yeast Cells 23 1

53. Penicillium glaucum, Oidium lactis, Aspergillus glaucus, Mucor

mucedo 232

54. Staphylococcus pyogenes aureus in Pus 242

55. Pure Culture 243

56. Culture in Gelatin 245

57. Streptococcus pyogenes, Pure Culture 247

58. in Pus 248

59. " Culture on Agar 249

60. Micrococcus tetragenus in Pus 250

61. lanceolatus (of Pneumonia) in Sputum 252

62. lanceolatus (of Pneumonia) showing Capsules 253

63. Diplococcus intracellularis meningitidis 257

64. Gonococcus in Pus 259

65. Bacillus pyocyaneus 263

66. Bacillus of Bubonic Plague 265

67. Bacillus aerogenes capsulatus 268

68. Culture 269

69. " of Tetanus 271

70. " of Anthrax 273

71. with Spores 274

72. " Colony 275

73. " Culture 275

74. " showing Concave Ends 276

75. " in the Liver 277

76. " of Diphtheria 279

77. Neisser's Stain 280

ILLUSTRATIONS. XV

78. Tubes for Cultivation of Diphtheria Bacillus 281

79. Bacillus of Diphtheria, Culture 282

80. " tuberculosis 287

81. Branching Form of Tubercle Bacillus 288

82. Bacillus tuberculosis, stained, in Sputum 289

83. Ray- fungus of Actinomycosis, Fresh Preparation 299

84. Actinomyces bovis from a Pure Culture 300

85. Bacillus of Typhoid Fever 302

86. " " " with Flagella 303

87. Widal Serum-reaction with Typhoid Bacilli 305

88. Bacillus coli communis 310

89. with Flagella 311

90. Spirillum of Cholera 316

91. Involution Forms 317

92. " Colonies on Gelatin plates 318

93. " Culture in Gelatin 319

94. Vibrio proteus 324

95. Spirillum of Relapsing Fever 327

96. Malarial Parasite 331

97- " " 33i

98. " 33i

99- " 331

INTRODUCTION.

ANYONE who has not himself worked in a bacteriological
laboratory finds it difficult to form a vivid conception of
what bacteria are like, because among the familiar animals
and plants there are none with which a close comparison
can be made. Of the common organisms, perhaps ordi-
nary yeasts and moulds are most like the bacteria. Yeasts
and moulds, as everyone knows, grow on bread, cheese,
meat, syrups and the like. They flourish in moist and
dark places, as do mushrooms, puffballs and the other
fungi. All these fungi, appearing so different in some re-
spects, are alike in one particular, which is the absence of
the green color that we are apt to think of as being the
essential feature of vegetation. Plants that are green owe
their color to a substance called chlorophyll. Upon the
properties of this substance one of the most fundamental
facts in biology depends. Under the influence of sunlight,
by means of chlorophyll, plants are able to use as food the
carbon dioxide which is always present in the atmosphere
in small amounts. Although carbon dioxide is one of the
most simple and stable of compounds, the union of its com-
ponent elements is broken by the plant, and they are em-
ployed in the formation of other much more complex and
unstable compounds, such as starch and cellulose, which
enter into the plant's structure. The work of plants, it will
be noticed, is, in the main, precisely the reverse of that per-
formed by animals. Animals take the unstable carbohy-
drates wTith high potential energy, such as starches and

12 MANUAL OF BACTERIOLOGY.

sugars, as food, and exhale the stable carbon dioxide from
the lungs. At the same time the animal receives the bene-
fit of the energy resulting from the oxidation of the carbo-
hydrates, which may appear indirectly in the form of nerv-
ous or muscular activity or warmth.

Those plants that are devoid of chlorophyll are compelled
to some extent to use the same kinds of food as animals.
They are unable to decompose carbon dioxide (in most
cases), and procure their nourishment from the dead or
living bodies of other plants or animals. Since they have
no chlorophyll, light is of no advantage to them, and is
often a positive detriment. Bacteria contain no chlorophyll,
and are usually classed with the fungi, which they resem-
ble in their inability to decompose carbon dioxide and to
use it as food.1

There is another well-known property, possessed by
yeasts especially, which may be useful in explaining the
work done by bacteria. It is a fact of every-day observa-
tion that, when yeasts grow in dilute solutions of sugar,
alcohol and gas are formed. It not only appears that
bacteria sometimes form alcohol and gas from sugar, but
that with different kinds of bacteria and different kinds of
food material a great number of substances are made, some
of which are powerful poisons. In most of the diseases
caused by bacteria such poisons are produced within the
living body of the patient. The symptoms of the disease
and the changes in the patient's body are due to these
poisons, rather than to the direct action of bacteria.

The extreme smallness of the bacteria prevents us from
seeing them as individuals without the aid of the micro-
scope, although great numbers of them taken together may
form a plainly visible mass or growth. When they are
examined with the microscope they appear as little round,

'See Chapter I, Part II.

INTRODUCTION. 13

rod-shaped or curved bodies, which may be likened to so
many periods, dashes and commas. It is at once perceived
that each bacterium is an individual by itself, and that it
consists of a single cell, not of an aggregation of cells, as
do most of the common plants and animals.

Under favorable conditions bacteria may be seen to mul-
tiply, one organism being divided by a partition into two
parts, which separate and become two new organisms.
The process is called fission.

At times certain bacteria present little bright spots which
enlarge, and from which the rest of the cell breaks away
in fragments. The bright body that remains is called a
spore, and has greater resisting power against injurious
influences than has the fully developed organism. To this
extent these spores are something like the seeds of higher
plants. There are spores that can withstand boiling for
hours, but fortunately that is not true, as far as we know,
of the spores of any of the bacteria that produce disease.
The earlier investigators observed the appearance of bac-
teria in nutrient infusions which they had endeavored to
sterilize by heat. They looked upon this fact as indicating
the possibility of spontaneous generation, and it furnished
the chief support of that theory. Probably their fluids
contained very resistant spores, and were in reality not
sterile.

From these facts, a definition for bacteria may be formu-
lated.

Bacteria (Greek paxTijpeov, meaning a little stick) are ex-
tremely minute, unicellular plants, which have no chloro-
phyll, and zvhich divide by fission. They are sometimes
called schizomycetes. In every-day language they are
known as microbes, and also as germs. They are gener-
ally classed with the fungi. In some respects they seem
quite closely related to the algae or simplest green plants,

14 MANUAL OF BACTERIOLOGY.

and, on the other hand, they have strong points of likeness
with some of the unicellular animals belonging to the in-
fusoria.

Bacteria are divided into three great groups :

Micrococci, or cocci1 (singular, coccus) — spherical forms.

Bacilli (sing., bacillus) — long and straight, or rod-shaped
bacteria.

Spirilla (sing., spirillum) — consisting of spiral filaments
like the turns of a corkscrew, or parts of spirals shaped
like commas.

The extreme smallness of the bacteria is hard of com-
prehension. We may say, of most of them, that from
5,000 to 25,000 placed end to end would make a line about

an inch in length. When one
touches a growth of bacteria

K? ^£^\l "/NT/" with the sterilized platinum wire
• • •• v \ .j*

' ^ % ^ (j r j anc{ spreads the tiny portion

Micrococci. Bacilli. Spirilla. that adheres to the wire upon a

slip of glass, it is found upon ex-
amination with the microscope that the bacteria left on
the glass may be compared to the stars in the sky, the
grains of sand on the shore, or any of the other standards
for numbers that are nearly beyond computation.

It is well known that bacteria are present on most of the
objects about us. They occur on the skins of men and
other animals, as well as in the mouth, stomach and intes-
tines, and on most of the surfaces of the body that open
to the external w^orld. They are found in the water of
rivers and lakes, and in the ocean. They appear in the
soil down to a depth of several feet. They float in the
air, except at high altitudes and over the ocean. Nansen

1 Pronounced kok-si or kok-ke : see Webster's International, Century,
and Standard Dictionaries, and Foster's and Keating's Medical Diction-
aries. The writer knows of no authority for the prevailing pronuncia-
tion kok-kl.

INTRODUCTION. 1 5

found bacteria on the ice of the Polar sea. Investigators
have even reported finding them fossilized, indicating, as
we might expect, that they existed at remote periods in the
earth's history. But the vast majority of them are entirely
harmless as far as we are concerned, and many of them
are indispensable in maintaining the balance existing be-
tween the different kinds of living things.

Were it not for the putrefactive and nitrifying bacteria
the dead bodies of plants and animals would lie practically
unchanged where they fell, and the fertilization of the soil
necessary for the life of most plants, by means of substances
derived from such dead material, would cease.

In northern Siberia the bodies of the extinct species of
elephant called mammoths have been found imbedded in
frozen soil where they appear to have lain for thousands of
years. In this case the growth of putrefactive bacteria has
been prevented by cold, as in the modern refrigerator or
cold-storage plant.

Some bacteria have been made to do work in industries,
like the bacilli whose growth in cream imparts an agree-
able flavor to the butter and cheese.

Bacteria are also made use of in the manufacture of
vinegar.

The study of bacteria has led to the understanding of
many hitherto unexplained facts. The unaccountable de-
velopment of a moist, brilliant red deposit on bread and
other articles of food, which was formerly believed by the
superstitious to be blood, deposited by some miraculous
agency, we know to be due to the growth of a common or-
ganism (bacillus prodigiosus). The emission of light by
decaying substances when seen in the dark may be caused
by bacteria as well as other organisms.

It seems that in some cases in which death was attributed
to the suction of air into the veins, because air appeared to

1 6 MANUAL OF BACTERIOLOGY.

be present inside the heart, the air was in reality a gas,
formed by certain bacilli that invaded the body just before
or just after death (bacillus aerogenes capsulatus).

Woodhead tells us that some savages are in the habit of
smearing the soil of certain localities upon their arrows for
an arrow-poison, which is intelligible in the light of the fact
that soil often contains the bacilli of tetanus (lockjaw).

The comparatively small number of species of bacteria
that cause disease are the ones that interest us most, and
are those which have been most carefully studied. The
necessity that falls upon bacteria, in common with other
fungi, to derive their food from organic matter makes it
easy to understand that they should frequently exist as para-
sites upon living animals and plants. Pear-blight and some
other diseases of plants are caused by bacteria. We find
that frogs, birds, cattle and a great number of animals
besides men suffer from diseases produced by bacteria.

When bacteria are placed upon slips of glass they may be
studied with the microscope while alive. Some of them
when living are motionless ; others wriggle vigorously.
Some dart about like minnows in a stream, or they make
their way slowly across the field of the microscope like a
boat that is being sculled from the stern. By proper methods
it can be shown that the movements are effected through
one or more fine, hair-processes, called flagella.

Often it is expedient to study bacteria after drying them
on slips of glass, when they may be made more conspicuous
by giving them an artificial color (staining). Some of the
substances of which they are composed readily absorb cer-
tain dyes. For this purpose the aniline dyes are used, and
their employment has been one of the important factors in
making progress in bacteriology possible.

With the microscope alone it is not usually practicable to
distinguish accurately between various kinds of bacteria.

INTRODUCTION.

Micrococci, for instance, which are, in reality, extremely
different, may look very much alike. The differences are
usually apparent when the bacteria are grown artificially.
The cultivation is done for the most part in FIG. 2.
test-tubes containing some material which fur- s^"y
nishes suitable food. The nutrient materials c£

£'•':•••' '-•;,. .,. rf£*.

most used are meat-extract and peptone, which,
dissolved with salt in water, constitute nutrient
bouillon. Ordinary gelatin, or a vegetable gel-
atin called agar-agar , may be added to the
bouillon when a solid culture-medium is de-
sired. Before these substances can be used for
the cultivation of bacteria all other bacteria
which they might contain must be destroyed by
heat.

When bacteria are to be conveyed from one
tube to another, or from a tube to a glass slide,
in order to examine them with the microscope,
the manipulation is performed on a platinum
wire fastened into a glass rod. The rules laid
down for the management of the tubes and the
platinum wire (Part I., Chapter VIII.) must ~ .

Test-tube con-
be carefully followed. There is little or no dan- taining cui-

ger in bacteriological work if the proper pre-
cautions are conscientiously observed; but carelessness may
lead to disastrous and even fatal results, as has happened
more than once.

Finally, the effects of bacteria in bringing about disease
may be tested on the lower animals. The proof that a par-
ticular species of bacteria causes a particular disease cannot
be considered complete unless the disease can be repro-
duced by introducing these bacteria into some animal.

The student who wishes to pursue bacteriological study
in any direction farther than it is possible for the limits of

1 8 MANUAL OF BACTERIOLOGY.

a short manual to go, may, besides consulting the large
text-books, and weekly medical journals, obtain much
assistance from technical periodicals. The Journal of Ex-
perimental Medicine, Journal of Medical Research, and the
Journal of Applied Microscopy, published in this country,
and the English Journal of Pathology and Bacteriology and
Journal of Hygiene will give a great deal that is valuable.

A reading knowledge of German and French is very
desirable. The Centralblatt fiir Bakteriologie, etc., a
German weekly, and the Bulletin de I'Institut Pasteur,
published bimonthly in Paris, contain abstracts of most of
the important researches made in all parts of the world.
The Annalcs de I'Institut Pasteur, the Zeitschrift fiir Hy-
giene, and the Archiv fiir Hygiene contain many original
articles on bacteriological subjects.

The whole literature of any specified subject in bacteri-
ology can be most conveniently found in Baiungarten's
Jahresbericht dcr Mikrodrganisinenlclire.

Historical Sketch. — The remarkable growth of mechan-
ical and industrial enterprises which the last half century
has witnessed is held to be characteristic of it. The world
justly takes pride in its achievements along these lines.
Nearly all that we know of bacteria and the part they play in
producing disease has been learned during the same period.
It is but fair to say that the rapid growth of this knowledge
has been equally characteristic of the age.

Nevertheless many facts were known long ago, and even
by the ancients, which were effective in directing the thought
of later years. The epidemic nature of certain maladies
was naturally among the earliest of these to be noticed, and
was, even until recently, attributed to the influence of gods,
demons, or other supernatural agencies. The superstitions
and crude beliefs of the past gave rise to a mass of grotesque

INTRODUCTION. 1 9

theories and fanciful speculations. But with all this we
hear of certain beliefs and practices which plainly fore-
shadowed those of the present day. Latin writers nearly
two thousand years ago recorded a relation between insects
and malaria, which has but lately been proved and ex-
plained. The treatment of lepers by the Hebrews resembles
that now in vogue : " He is unclean : he shall dwell alone ;
without the camp shall his habitation be " (Lev. XIII. 46).
There is, in fact, much in the laws of Moses that points to
some knowledge of the nature of infections. ' This is the
law, when a man dieth in a tent : all that come into the tent
and all that is in the tent shall be unclean for seven days.
And every open vessel which has no covering upon it shall
be unclean" (Numb. XIX. 14, 15).

" Everything that may abide the fire, ye shall make it go
through the fire, and it shall be clean " (Numb. XXXI. 23).

In Homer we read of Ulysses, that, having slain his
wife's troublesome suitors :

" With fire and sulphur, cure of noxious fumes,

He purged the walls and blood-polluted rooms" (Pope's Odyssey).

The massive aqueducts of the Romans still remain to
testify that they understood the importance of a pure water-
supply.

In Rome there were also sewers for the disposal of drain-
age; while the Cretans and Assyrians used sewerage sys-
tems hundreds and even thousands of years before.

About the fourteenth century we find quarantine against
infectious diseases, plague in particular, practiced by certain
Italian cities ; and the word " quarantine " came into use
from the fact that the period of detention was about forty
days (Ital. quarantine*) *

1 The Early History of Quarantine, J. M. Eager, Yellow Fever hist.
Bui., No. 12, U. S. Marine Hosp. Service.

20 MANUAL OF BACTERIOLOGY.

Leeuwenhoek, a citizen of Delft, in Holland (1632-
1723), appears to have been the first who actually saw bac-
teria. Yeast-cells he certainly observed, besides making
many other contributions of great value to biology. Leeu-
wenhoek produced admirable lenses of high magnifying
power, and described what he witnessed with singular accu-
racy and enthusiasm.

Even before this time men had sought to explain the
phenomena of infectious diseases by supposing the body
to have been penetrated by minute parasites, for example
worms. The spread of such diseases through a community
from a single center could readily be accounted for by the
multiplication of a contagious element, itself alive (con-
taghnn vivum). With increasing knowledge of the abund-
ance of microscopic life these speculations took firmer hold.
But long before their truth was finally demonstrated great
advances \vere made in the prevention of infectious diseases.
Much honor is due the clinicians whose accurate observa-
tions and foresight accomplished important results at an
early day, working with what now seems a very meagre
knowledge of the facts.

The production of immunity against small-pox by inocu-
lation was first practiced in oriental countries. The method
had long been in use in the East, when in 1718 it was
brought to the notice of Europeans by Lady Montagu, wife
of the English ambassador at Constantinople. The proced-
ure consisted simply of the introduction of the virus of
small-pox by puncture of the skin. An attack of small-pox
resulted, which was much milder and far less dangerous
than the natural disease.

Lady Montagu stated in a letter : " Every year thousands
undergo the operation; and the French ambassador says
pleasantly that they take the small-pox here by way of
diversion, as they take the waters in other countries." The

INTRODUCTION. 21

mild attacks that followed inoculation were, however, just
as contagious to other persons as the natural disease, so that
the dangers of this practice to the community were very
great.

A much better method was found in vaccination. At this
time a belief was current among farmers that a mild form
of disease, called cow-pox, acquired by milkers, furnished
protection against small-pox. This belief was investigated
and introduced to the world by Edward Jenner. In 1796
he inoculated his first patient with cow-pox. In a few years
the practice of vaccination spread to all parts of the world.1

It was introduced into the United States by Dr. Benjamin
Waterhouse of Harvard. President Thomas Jefferson was
active in bringing it into general use especially in the south.

As early as 1847 Semmelweis of Vienna attributed the
origin of puerperal fever to poisons carried by the fingers of
physicians and students, whose hands had been soiled in
the dissecting room. To this he was led by the death of a
friend from pyemia following a dissection-wound. He
noted the similarity of the course of his friend's case with
cases of puerperal fever. He advocated washing the hands
of the attendant in solutions of chlorin or chlorid of lime,
in addition to cleansing them with soap and water.

The cause of puerperal fever was still unknown. En-
deavors to connect it with atmospheric influences and the
like had been unsuccessful. During the seventeenth and
eighteenth centuries it had been attributed to the absorption
of milk into the blood from the breasts. Semmelweis stood
his ground in spite of opposition and ridicule, though he
somewhat modified his doctrine. His views agree substan-
tially with the practice of the present day which they have
greatly influenced.

1 See the works of Edward Jenner by Dock, N. Y. Med. Jour., Nov.
29 and Dec. 6, 1902 ; also The History of Vaccination, by Dulles, Phila-
delphia Medical Journal, May 30, 1903.

22 MANUAL OF BACTERIOLOGY.

During the same period similar ideas were ad-
vanced by Dr. Oliver Wendell Holmes in the United States.
His paper on " The Contagiousness of Puerperal Fever "
appeared in 1843. A lively controversy lasting several
years was provoked, in which Holmes defended his position
with great vigor. His admirable literary style served him
effectively.1

In the first half of the nineteenth century, with improved
microscopes, knowledge of minute living things grew
rapidly, chiefly with respect to infusoria and other relatively
large forms. In 1840 Henle described the part played by
microorganisms in producing disease in terms surprisingly
in accord with views held at the present time. His deduc-
tions were based almost entirely on knowledge of the gen-
eral nature, spread and course of infections. So too,
Villemin anticipated the discovery of the bacillus of tuber-
culosis, for he transmitted the disease to animals, by inocu-
lating them with material from cases of tuberculosis in
man.

The key to exact knowledge of the microorganisms of
disease was finally discovered in the study of fermentation.
No better illustration could be found of the possible value to
mankind which may lie in any addition whatever to the
common stock of facts. The study of bottles of bad-smell-
ing broth would have seemed, fifty years ago, a most un-
promising beginning for the discovery of the causes of
cholera, plague, and the like, or for an antitoxin for diph-
theria.

Studies on Fermentation and Spontaneous Generation. —
Two observers (Schwann, Cagniard-Latour, 1837) almost
simultaneously stated the proposition that yeast cells were
living organisms, and that the fermentation of solutions of
sugar was clue to their growth. From this time ensued

1 See Medical Essays, O. W. Holmes, Houghton, Mifflin & Co., 1889.

INTRODUCTION. 23

a controversy which lasted more than thirty years. The
agency ascribed to yeasts was energetically denied by many,
prominent among them Liebig; while it was sustained with
vigor by others. The latter extended the original conception
to include other sorts of fermentation and the putrefaction
of albuminous material. Different kinds of fermentation,
with different products, such as acetic acid and butyric acid,
were ascribed to the growth of different kinds of microbes.

These microbes were found to be fungi of various sorts,
and chiefly one or another variety of bacteria. The most
celebrated among the students of fermentation was Pasteur,
the simplicity and kindliness of whose character excite our
admiration equally with his devotion to his work.1

Before the nature of fermentation was understood the
possibility of spontaneous generation had been universally
admitted. When vermin of various sorts appeared in putre-
fying material the conclusion was drawn that they had their
origin directly from it. Although that had long since been
disproved in the case of large organisms like worms and
frogs, still, as late as the middle of the last century, it was
held by many to account for the swarming microscopic life
found in fermenting fluids. A flask of meat broth left ex-
posed to the air will after a few days contain countless tiny
living things, chiefly bacteria. Pasteur and his supporters
showed that these bacteria were the progeny of others
already in the flask or which had fallen in from the air.

When the flask of broth was boiled, no development of
organisms took place, if the entrance of germs from the
atmosphere was prevented. The latter was accomplished
by such devices as heating the air, passing it through sul-
phuric acid, using a flask with a long twisted neck or by
plugging the flask with cotton (Schroder and Von Dusch).

1 See Louis Pasteur, His Life and Labors by His Son-in-Law, trans-
lated by Lady Claude Hamilton.

24 MANUAL OF BACTERIOLOGY.

To prove that boiling had not made the fluid unfit for the
growth of organisms, air was subsequently allowed to have
access to it without such precautions, when putrefaction
took place in the usual manner.

At the same time it was demonstrated not only that bac-
teria are present in all fermenting and putrefying sub-
stances, but that they exist wherever there is animal life or
vegetation.

These principles underlie the methods used daily for the
preservation of meat, fruit and vegetables, in the household
and in factories.

Although even boiling occasionally failed to prevent fer-
mentation, investigators came with practice to have a
smaller number of failures. Such failures it was shown
were due to the presence of the resistant state called spores,
which some bacteria assume. The true nature of spores
was recognized later by Colin. Pasteur found that exposure
to temperatures above the boiling point (no°C.) would
destroy the most resistant microbes and their spores.

The controversies over fermentation and putrefaction
lasted almost until the present day. They were productive
of numerous benefits to the arts and manufactures. But
what is of more importance to our subject, they led to a
vastly better understanding of all kinds of microorganisms.
The study of bacteria was now pursued with great vigor.
In the space of about twenty-five years, most of what we
know concerning the bacteria of disease has been learned.
The period of rapid progress is not yet completed. Nearly
every year yields some advance of great importance.

The discussions concerning fermentation and putrefac-
tion, were still going on when Lister made his brilliant
deduction that suppuration and septic processes in wounds
were a species of fermentation (1867). From this came the
antiseptic and aseptic methods of operating and of dressing

INTRODUCTION. 25

wounds, which have made possible the wonderful results of
modern operative surgery.1

In 1834 the parasite of itch (an insect, Acarus scabei) was
discovered, and the cause of one contagious malady deter-
mined.

Quite early in the nineteenth century also the relatively
large fungi of thrush and some of the parasitic skin diseases
were discovered. The bacilli of anthrax, which are also
large, were seen in the blood of animals by Pollender in 1855
and Davaine in 1863.

Davaine produced anthrax in animals by injecting into
them blood containing anthrax bacilli. But complete proof
that these bacilli were the cause of the disease, required that
they should produce it when injected alone and when freed
from the smallest trace of material derived from the first
diseased animal. Unless these conditions were complied
with, some other material, for example an enzyme or fer-
ment, might be supposed to be carried from the first to the
second animal and to be the real cause of the disease. For
this purpose it was necessary to cultivate the bacilli in
nutrient fluids, such as meat broth, as was done by Pasteur.
It then became possible to demonstrate that their properties
could remain unaltered after being grown in successive
generations on different lots of broth. As bacteria of two
or three species were often encountered in mixtures, it be-
came most important to secure a method by which the differ-
ent species could be separated from one another and be
propagated as separate " pure cultures." This was done
successfully by diluting such mixtures greatly, so that a
drop planted in a new tube of broth should contain only a
single organism. The growth ensuing would of course con-
sist of the same kind of organism exclusively. Such pro-
cedures were uncertain and very laborious.

1 See History of Medicine, Dr. Roswell Park.

2O MANUAL OF BACTERIOLOGY.

Koch introduced in 1881 his method of separating bacteria
by "plating" (described in Part I.), probably the most
important single contribution to bacteriological technique.
He also brought solid culture-media into general use by em-
ploying gelatin. Other important technical improvements
of the same period were the adoption of the illuminating
apparatus of Abbe and immersion objectives, and of aniline
dyes for staining bacteria and making them visible ( Weigert
and Ehrlich). Beginning with the bacillus tuberculosis de-
scribed by Koch in 1882, a large number of pathogenic bac-
teria were discovered during the ensuing years in rapid suc-
cession.

The application of the newly-gained knowledge concern-
ing the bacteria causing infectious diseases to the preven-
tion and cure of these diseases was begun almost imme-
diately by Pasteur. A few facts existed to guide the direc-
tion of the research. It had been known even in ancient
times that one attack of an infectious disease, such as scarlet
fever, may confer immunity from subsequent attacks.

The protection against small-pox which was furnished by
vaccination also was suggestive, although the mechanism by
which this protection came about was not understood.

Pasteur worked on the theory that immunity to a disease
might be secured by producing a mild attack of the disease.
Such a mild attack might be expected to follow if a sus-
ceptible individual were inoculated with microbes of lowered
virulence. Various methods were employed to reduce the
virulence of bacteria, notably cultivation at high tempera-
tures (43°C.). In this manner Pasteur was able to produce
immunity against a number of the diseases of the lower
animals. His method of inoculating sheep and cattle against
anthrax is widely and successfully used. A similar prin-
ciple has led to the preparation of a vaccine for the disease
of cattle called " black leg," and such vaccine is now dis-

INTRODUCTION. 27

tributed gratuitously to farmers by the United States gov-
ernment. Inoculation of human subjects with the atten-
uated living virus of a disease is used only for hydrophobia.
This method also was invented by Pasteur.

The preparations of antitoxins for infectious diseases
(see the chapter on Immunity) we owe to Behring. This
portion of our subject belongs entirely to the present day,
and is now being studied with great energy.

Allusion has already been made to moulds and other
microscopic parasites whose nature makes their study almost
inseparable from that of the bacteria. In this class also
belong the primitive forms of animal life (Protozoa) which
are the causes of amebic dysentery (Losch, 1875) and
malaria (Laveran, 1880). The disease of cattle called
" Texas fever " is also caused by a protozoon. Theobald
Smith in the United States discovered that the parasite of
Texas fever is conveyed from one animal to another by an
insect, the cattle-tick. Since then it has been shown (by
Manson, Ross and others) that malaria is conveyed from a
person having the disease to one not affected by means of
mosquitoes. It now appears probable that a similar rela-
tion exists between mosquitoes and yellow fever. The part
played by flies and other insects in carrying disease germs is
still receiving active attention and the future may have larger
possibilities in store.

It is encouraging to reflect that the progress of bacte-
riology has been made by gradual and logical steps. The
great discoveries have not been lucky accidents, but have
been worked out patiently and with deliberation.

PART I.

CHAPTER I.

EXAMINATION OF BACTERIA WITH THE MICROSCOPE, IN-
CLUDING METHODS OF STAINING.

The Microscope. — The microscope consists of a tubular
body which carries the optical parts, and which can be
raised or lowered for focusing. The objectives should be
three in number, and should be attached to the body by
means of a triple nose-piece, which permits any objective
to be turned into the optical axis at will. The eye-piece
slips into the upper and opposite end of the body or tube.
The arrangements for focusing consist of a rack and pinion
which accomplish the coarse adjustment, and a more deli-
cate fine adjustment. The stage, upon which the objects
to be examined are placed, has an opening in the middle.
In this opening an iris diaphragm and Abbe condenser are
inserted. The iris diaphragm enables one to alter the size
of the opening as desired. Beneath the stage is a mov-
able mirror, of which one side is plane and the other con-
cave. All of these parts are supported on a short, heavy
pillar which is fixed in the horseshoe-shaped base.

The essential parts of the microscope are, of course, the
eye-piece (German, Ocular), and the objective. Objectives
are given various names by different makers, for instance,
A, B, C, etc., or i, 2, 3, etc.; or they are named according
to their focal distances, as f inch, J inch, -J inch, etc. In
bacteriological work a rather " low power " § or J inch

3O MANUAL OF BACTERIOLOGY.

objective, an ordinary " high power " | to ^ inch dry objec-
tive, and a high power ^ inch oil-immersion objective are
needed. The magnification with the f or J inch objective

FIG.

Microscope.

is about 75 to 100 diameters; with the ] to ,1 inch 300 to
500 diameters; with the ^ immersion 750 to 1,000 diame-
ters. The magnification varies according to the eye-piece
used, as well as with the objective. A i inch and 1.1 inch

EXAMINATION OF BACTERIA WITH THE MICROSCOPE. 3!

eye-piece (Zeiss No. 2 and No. 4) serve well for most pur-
poses. The eye-pieces are usually named arbitrarily, like
the objectives. The oil-immersion objective is used in the
examination of bacteria where a very high power is desired.
A layer of thickened oil of cedar-wood is placed between the
lower surface of the objective and the upper surface of the
glass covering the object under examination. The oil must
be wiped away from the surface of the objective when the
examination is finished. For this purpose the soft paper
sold by dealers in microscopical apparatus serves admi-
rably. Care must be taken not to scratch the lower surface
of this objective. Oil of cedar-wood furnishes a medium

FIG. 4.

Abbe Condenser. On the right side the figure gives a sectional view.

having nearly the same refractive index as the glass of the
lens and the glass on which the object is mounted, and it
obviates the dispersion of light which takes place when a
layer of air is interposed between the objective and the
object, as happens with the ordinary dry lens. This ob-
jective is used in connection with the Abbe condenser,
which consists of two or three lenses combined so as to
focus the rays coming from the plane mirror upon the
object. The condenser gives a very intense illumination
over a very small field. The condenser is not necessary
excepting with the oil-immersion objective. If it is used
with the other objectives the illumination must be regulated
by lowering the condenser, closing the diaphragm more or

32 MANUAL OF BACTERIOLOGY.

less, and substituting the concave for the plane mirror.
It is to be remembered that more depends upon securing a
distinct picture than upon a very high magnification of the
object.

The microscope should be placed in front of the observer
on a firm table. The observer should be able to bring the
eye easily over the eye-piece when the tube of the micro-
scope is in vertical position. Daylight should be em-
ployed if possible. When artificial illumination is neces-
sary, an ordinary lamp, a Welsbach burner or an incan-
descent electric light may be used. It is best to modify the
artificial light by inserting a sheet of blue glass between
the light and the mirror.

In order to focus upon any object, having first secured a
satisfactory illumination with the mirror, it is best, begin-
ning with the low power and using the coarse adjustment
for focusing, to bring the objective quite close to the object,
and then, with the eye in position, to raise the tube until
the object comes into focus. The exact focusing is done
with the fine adjustment. The observer should keep both
eyes open when using the microscope, and should be able
to use either eye at will.

All measurements of microscopic objects are expressed
in terms of a micromillimeter. This is one-thousandth of
a millimeter (.001 mm.), which is about .2-t (1MM) of an inch.
It is generally called a micron for short, and is denoted by
the Greek letter ft. For example, 5 IJL == .005 mm. == - (]lQ-$
inch.

The Preparation of Specimens of Bacteria for Exami-
nation with the Microscope. — The substance under ex-
amination is usually placed upon thin slips of glass called
cover-glasses. The material is spread over the cover-glass
by means of a platinum wire which has been fixed in a
glass rod about six inches long. Such a platinum wire is

EXAMINATION OF BACTERIA WITH THE MICROSCOPE. 33

used constantly in doing bacteriological work. It is the
tool by means of which one is able to handle bacteria with
impunity. It serves in fact as a kind of additional finger.
The platinum wire must be stiff enough not to bend too
easily, and yet it should not be so large that it will not cool
rapidly after heating. A good size for most purposes is
number 23, American wire gauge (Brown and Sharp).
The wire may be straight throughout its length, or the tip
may be bent to form a loop (German, Oese). It is well
to follow, from the beginning, certain rules which make
the use of the platinum wire safe and accurate. Every
time it is taken into the hand and before using it for any
manipulation heat it in the flame of a Bunsen burner or

Straight platinum wire and platinum wire loop.

an alcohol lamp to a red heat ; and always, after using and
before putting it down, heat it again to a red heat. After
the needle has become wet by clipping it in a fluid and is to
be sterilized in the flame, it is necessary to avoid " sputter-
ing " of the fluid by bringing the wet needle gradually to
the flame, so as to dry the material adhering to it before
burning it. This procedure must be done with great care
when the wire has been dipped in milk or other substances
containing oil. When the needle " sputters," as it is called,
from too rapid heating, particles that have not yet been
sterilized may be thrown some distance. On no account
should the needle touch any object other than that which

34 MANUAL OF BACTERIOLOGY.

it is intended it should touch. With such a platinum wire,
which has been properly sterilized, one can easily remove
portions from a culture of bacteria, or from a fluid in
which bacteria are supposed to be present. The glass rod
in which the platinum wire is fixed should be held between
the thumb and forefinger of the right hand like a pen. (For
the manner of holding test-tubes, see page 84.)

The Hanging-drop. — Living bacteria may be studied
with the microscope while suspended in some fluid sub-
stance. The needle having been heated to a red heat in
the flame and having been allowed to cool, a small portion
of the culture or other material may be removed with it
and deposited in the center of an ordinary cover-glass.
The needle should again be sterilized in the flame. When

FIG. 6.

Diagram of the hanging-drop.

cultures on solid media arc to be examined, a small particle
may be mixed with a drop of sterilized water or bouillon.
The cover-glass should have been carefully cleaned and
sterilized over the flame. The cover-glass with the small
drop of fluid material held in sterilized forceps is now to be
inverted over a sterilized glass slide, which has a concavity
ground in the middle of it. Around the concavity, the slide
should be smeared with vaseline. In this manner a small
air-tight chamber is made. This slide and cover-glass may
be put upon the stage of the microscope. A good dry lens,
if of sufficiently high power, is more convenient for ex-
amining the hanging-drop than an oil-immersion. If the
latter be used, having placed a drop of cedar-oil on the
center of the cover-glass, and a good light having been
secured, the oil-immersion objective should be brought

EXAMINATION OF BACTERIA WITH THE MICROSCOPE. 35

down upon this drop of oil. The beginner often experi-
ences difficulty in focusing upon a hanging-drop. It is
well to shut off most of the light by means of the iris dia-
phragm. Often it is well to secure the focus roughly upon
the extreme outer edge of the chamber, or to find the edge
of the drop of fluid with the low power and then to focus
upon this edge with the oil-immersion objective. Above all
things guard against breaking the cover-glass by forcing
the objective down upon it. The motility of certain bacteria
is one of the most striking phenomena to be observed in
the hanging-drop. It is not to be confused with the so-
called " Brownian movement " which is exhibited by fine
particles suspended in a watery fluid. It is well for the
beginner to observe the character of the Brownian move-
ment by rubbing up some carmine in a little water, and with
the microscope to study the trembling motion exhibited by
these particles of carmine. It will be noticed that, although
the particles oscillate, no progress in any direction is ac-
complished unless there are currents in the fluid. Such
currents might give rise to the impression that certain
bacteria possessed motility when they were, in fact, power-
less to move of themselves. In the hanging-drop the
multiplication of bacteria can be studied, the formation of
spores and the development of spores into fully formed
bacteria. The hanging-drop has recently been put into
service for the demonstration of the so-called serum-
reaction with the bacillus of typhoid fever. Sometimes
bacteria must be watched in the hanging-drop for hours,
or even days, and it may be necessary to keep it at the
temperature of the human body for this length of time.
Various complicated kinds of apparatus have been devised
for this purpose, but they are needful only with special
kinds of work. When the hanging-drop preparation is no
longer required, the slide and cover-glass should be
4

36 MANUAL OF BACTERIOLOGY.

dropped into a 5 per cent, carbolic acid solution and after-
ward sterilized by steam.

Hanging-block preparations, which \vere introduced by
Hill,1 make use of a cube of nutrient agar instead of a drop
of fluid. Bacteria are distributed on the surface of the agar,
which is then applied to a cover-glass, and mounted like a
hanging-drop. The bacteria are kept in a layer close to the
glass, where growth may be studied.

Cover-glass Preparations. — The study of bacteria with
the microscope is for the most part done by means of smears
made upon thin slips of glass. Such slips of glass are
generally called cover-glasses. It is best to obtain the
kind sold by dealers as No. i, f inch squares.

The cover-glass may be cleaned best by immersion in a
mixture of sulphuric acid and bichromate of potassium so-
lution, and afterward washed thoroughly in distilled water,
and finally in alcohol. A stock of clean cover-glasses may
be kept in a bottle of alcohol.

CLEANING FLUID.

Potassium bichromate 40 grams.

Water 150 c.c.

Dissolve the bichromate of potassium in the
water, with heat ; allow it to cool ; then add
slowly and with care sulphuric acid, com-
mercial 230 c.c.

For most purposes it is sufficient to wash the cover-glass
in alcohol containing 3 per cent, of hydrochloric acid. It
should then be wiped clean with a piece of linen cloth.
"Whenever it is taken into the ringers it should be held by the
edges, never by the flat surfaces. As far as possible it
should be handled with the forceps. It can be used very
conveniently in the form of forceps known as the Cornet
forceps, or in the modification devised by Stewart. Bac-

1 Journal of Medical Research, Vol. VII., March, 1902.

EXAMINATION OF BACTERIA WITH THE MICROSCOPE. 37

teria may be placed upon the cover-glass by allowing the
glass to fall upon one of the colonies of bacteria, on a gelatin
or agar plate (see page 99), which will adhere to it in part,
producing an " impression preparation " (German, Klatsch-
preparat}. Such a preparation, after drying in the air, is
to be fixed by passing it through the flame three times. (See
below.) The forceps with which it is handled should be
sterilized in the flame.

Generally bacteria contained in fluids, like sputum, or
taken from the surface of a culture, are smeared over the
cover-glass by means of the platinum wire or loop, which
must be heated to a red heat before and after the opera-

FIG. 7.

Cornet forceps for cover-glasses.

tion. Such preparations are called smear, cover-glass,
cover-slip, or film preparations. When the material to be
spread is thick or very viscid, a small drop of distilled
water must first be placed in the center of the cover-glass
so as to dilute it. Beginners generally take too much ma-
terial on the wire. As thin a smear as possible is made.
It is allowed to dry in the air; this should occupy a few
seconds. The drying may be hastened by holding the
forceps with the cover-glass a long distance above the
flame, at a point where the heat would cause no discomfort
to the hand. Having dried the preparation, it is to be
passed through the flame of a Bunsen burner or alcohol
lamp three times, taking about one second for each transit.
The heat of the flame serves to dry the bacteria upon the
cover-glass and fix them permanently in position; it is not

3« MANUAL OF BACTERIOLOGY.

sufficient, however, when applied in this manner, to kill all
kinds of bacteria, especially those containing spores. After
it has been passed through the flame three times the
preparation may be stained with one of the aniline dyes,
and after washing in water and drying may be mounted,

FIG. 8.

Stewart forceps for cover-glasses.

face down, in Canada balsam upon a glass slide. It makes
a suitable object to be examined with the oil-immersion
objective. The slide is a thin slip of glass, 3 inches by i
inch, with ground edges.

The smear preparation may equally well be made directly
upon the glass slide. The fixation in the flame must then
occupy a longer time than with the small and thin cover-

FIG. 9.

Kirkbride forceps for holding slides.

glass. Such preparations have the advantage that several
may be made upon one slide, and that after staining them
they" may be examined in cedar-oil, with the oil-immersion
lens, without the use of the cover-glass and Canada balsam.
The forceps of Kirkbride will be found convenient when
staining on the slide. Experiments performed in the

EXAMINATION OF BACTERIA WITH THE MICROSCOPE. 39

writer's laboratory have shown that the ordinary method
of fixation in the flame, when applied to bacteria spread
upon slides, has little effect on the vitality of many species.
The beginner is, therefore, advised to make his preparations
on cover-glasses.

When very resistant or dangerous pathogenic bacteria
are being handled, after fixation by heat upon the slide or
cover-glass, the preparation may, if desired, be immersed in
i-iooo solution of bichloride of mercury long enough to
kill the bacteria, without injuring the preparation or its
staining properties.

Staining. — The staining of bacteria is done for the most
part with the aniline dyes. The object of staining bacteria
is to give them artificially some color which makes them
distinct and easily visible without imparting this color to
the substance or medium in which they are imbedded. The
substances known as aniline dyes are derivatives of coal-tar,
but not always of aniline. These dyes are of great import-
ance in bacteriological work. Their number is very large,
but only a few are in common use. It is important to have
the purest, and those manufactured by Griibler are reliable.

It is simplest to classify the aniline dyes as acid or basic.
Eosin, picric acid and acid fuchsin are acid dyes; they tend
to stain tissues diffusely. Fuchsin, gentian-violet and
methylene-blue are basic dyes ; they have an affinity for the
nuclei of tissues and for bacteria; they therefore are the
dyes used chiefly in bacteriological work. The other va-
rieties may be employed as contrast-stains; another con-
trast-stain frequently used is Bismarck brown. It is best
to keep on hand saturated solutions of the aniline dyes in
alcohol, from which watery solutions may be made when
needed by adding a few drops of the alcoholic solution to
a small dish filled with water. The alcoholic solution is
diluted about ten times, or so as to make a liquid which is

40 MANUAL OF BACTERIOLOGY.

just transparent in a layer about 12 mm. in thickness, after
filtering.

Fuchsin and gentian-violet operate rapidly and intensely.
Methylene-blue works more slowly and feebly; it is to be
preferred where the bacteria occur in thick or viscid sub-
stances, like pus, mucus, and milk.

Method of Staining Cover-glass Preparations. — (a) A
smear preparation of bacteria having been made in the
manner above described, and a watery solution of either
fuchsin, gentian-violet or methylene-blue having been pre-
pared, the cover-glass is to be dropped into a dish contain-
ing the dye, or the dye may be dropped upon the cover-
glass held in the forceps.

(fr) Allow the stain to act for about thirty seconds.

(d) Examine with the microscope in water directly or
after drying and mounting in Canada balsam.

The rapidity and intensity of staining may be increased
by warming the solution slightly. The bacteria will usually
appear more distinct if, directly after pouring off the stain,
the preparation is rinsed for a few seconds in i per cent,
solution of acetic acid, and then thoroughly washed in water.
The acetic acid solution serves to remove in a measure any
color which has been imparted to the background, and which
is undesirable.

Preparations that are mounted at first in water may be
made permanent by moistening the edge of the cover-glass
so that it may easily be removed from the slide, then dry-
ing and mounting in Canada balsam. Cover-glass prepara-
tions which have been stained are examined with the oil-
immersion objective, employing the plane mirror, having
the iris diaphragm open and the condenser close to the
lower surface of the glass slide. The purpose is to obtain
the most intense illumination possible over a small field.

EXAMINATION OF BACTERIA WITH THE MICROSCOPE. 4!

The watery solutions of aniline dyes prepared as above de-
scribed deteriorate in a short time, and it is best to prepare
them freshly each time they are required. A very useful
solution, which is permanent, is Loffler's alkaline methylene-
blue:

Concentrated alcoholic solution of methylene-blue. . 3Oc.c.
Potassium hydrate (caustic potash) 1-10,000
watery solution 100 c.c.

Loffler's methylene-blue is a good stain for general pur-
poses. It is perhaps more in use than any other formula
for coloring the diphtheria bacillus.

Aniline-water Staining Solutions. — The intensity with
which aniline dyes operate may be increased by adding
aniline oil to the solution :

Aniline oil 5 c.c.

Water 100 c.c.

Mix, shake vigorously, filter; the fluid after filtration
should be perfectly clear; add—

Alcohol 10 c.c.

Alcoholic solution of fuchsin (or gentian-violet, or
methylene-blue) 1 1 c.c.

Aniline-water staining solutions do not keep well, and
need to be freshly prepared about every two weeks. The
applications of the aniline-water stains will be given under
separate headings. In general, however, they are em-
ployed where a stain of unusual power is required.

Gram's Method. — Cover-glass preparations, having been
prepared and fixed in the usual manner (see page 37), are
stained as follows :

(a) Stain in aniline-water gentian-violet solution, from
two to five minutes. The intensity of the stain may be in-
creased by warming slightly.

(b) Iodine solution, one and one-half minutes :

42 MANUAL OF BACTERIOLOGY.

Iodine I gram.

Potassium iodide 2 grams.

Water 300 c.c.

In this solution the preparation becomes nearly black.

(c) Wash in alcohol repeatedly; the alcohol becomes
stained with clouds of violet coloring matter; the alcohol
is used as long as the violet color continues to come away,
and until the preparation is decolorized or has only a faint
steel-blue color.

(d) When desired, the specimens may be stained, by way
of contrast, with a watery solution of Bismarck brown or
eosin.

(e) Wash in water, and examine either in water directly
or after drying and mounting in Canada balsam. [A modi-
fication of this method, sometimes called the Gram-Gunther
method, differs from the preceding by using a 3 per cent,
solution of hydrochloric acid in alcohol for ten seconds to
hasten decolorization, washing in pure alcohol before and
after the acid alcohol. Decolorization is more intense than
by the Gram method; the diphtheria bacillus, which is
stained by Gram's method, is decolorized by the Gram-
Gunther (Kruse).] The advantages of Gram's method are
that with it certain bacteria are stained a violet color witli
more or less intensity and other bacteria are not stained at
all. To some extent, then, it furnishes a means of diagnosis.

List of some of the important bacteria that are stained
by Gram's method :

Staphylococcus pyogenes aureus.

Streptococcus pyogenes,

Micrococcus lanceolatus (of pneumonia),

Micrococcus tetragenus,

Bacillus of diphtheria.

Bacillus of tuberculosis,

Bacillus of leprosy,

EXAMINATION OF BACTERIA WITH THE MICROSCOPE. 43

Bacillus of anthrax,

Bacillus of tetanus,

Bacillus aerogenes capsulatus,

Ray fungus of actinomycosis.

The following bacteria are not stained by Gram's method :

Gonococcus,

Diplococcus intracellularis meningitidis,

Micrococcus melitensis,

Bacillus of chancroid (Ducrey),

Bacillus of dysentery (Shiga),

Bacillus of typhoid fever,

Bacillus coli communis,

Bacillus pyocyaneus,

Bacillus of influenza,

Bacillus of bubonic plague,

Bacillus of glanders (bacillus mallei),

Bacillus of malignant edema,

Bacillus of Friedlander,

Bacillus proteus,

Spirillum of Asiatic cholera,

Spirillum of relapsing fever.

Staining the Bacillus of Tuberculosis. — A very large
number of methods have been proposed for staining the
bacillus tuberculosis, all of which depend upon the princi-
ple that, after adding to solutions of aniline dyes certain
substances, like aniline-water, carbolic acid, or solutions
of ammonia or soda, the bacillus tuberculosis is stained
with great intensity, and gives up its stain with difficulty.
Solutions of acids will remove the stain from all parts of
the preparation excepting from the tubercle bacilli, which
retain the dye having once acquired it. The rest of the
preparation may now be given a different color — contrast-
stain.

Bacilli that resist decolorization by acids are called acid-

44 MANUAL OF BACTERIOLOGY.

proof or acid-fast. The most important are tubercle and
leprosy bacilli. There are various other species however
most of which are less resistant to acids and alcohol than
tubercle bacilli. They are discussed in the article on the
bacillus tuberculosis in Part IV.

Occasionally spores of other bacteria, micrococci and
horny epithelial cells are imperfectly decolorized, but their
forms distinguish them from tubercle bacilli. Minute
crystalline needles which have a shape like that of bacilli,
are often encountered in sputum, but their nature will be
recognized after a little practice.

The stain for tubercle bacilli is most frequently used for
specimens of sputum from cases of suspected pulmonary
tuberculosis ; it may be applied to other fluids and secretions
equally well. It is not reliable, however, when applied to
milk, as the oil present in milk interferes with its operation,
and milk and its products quite often contain other acid-
proof bacilli. The smegma of the external genitals also fre-
quently contains acid-proof bacilli that are not tubercle
bacilli. On this account all fluids and discharges from the
genito-urinary tract need to be examined with particular
care not to confuse tubercle bacilli with smegma bacilli.
(See smegma bacilli in Chapter IV., Part II.)

Patients should be given minute instructions concerning
the collection of sputum. The bottle used should be new,
wide-mouthed, clean, and kept tightly stoppered with a
clean cork. The patient should be cautioned against allow-
ing the expectoration to get on the outside of the bottle.
Probably whatever risk is incurred by those who examine
sputum comes chiefly from the outside of the bottle having
been soiled with sputum containing tubercle bacilli. Often
little white particles may be seen floating in the mucous por-
tions of the sputum. These particles should be selected for
the investigation, and may be spread in a thin film on the

EXAMINATION OF BACTERIA WITH THE MICROSCOPE. 45

cover-glass with the platinum wire, which is sterilized in the
flame before and after using. The selection of the little
white particles will be facilitated if the sputum be poured
into a clean glass dish, which may be placed on a black sur-
face. A form of porcelain dish is furnished by dealers, the
bottom of which is black, and which is convenient, for these
manipulations. The smears must be made thin, or the sub-
sequent decolorization, after staining, will not be uniform.
It is hardly necessary to observe that the operator must be
scrupulously careful not to contaminate the material under
examination with any kind of extraneous matter. The
cover-glasses and slides which are used should be new, and
should have been cleaned with bichromate of potassium and
sulphuric acid (see page 36).

When the work is completed, the bottle containing the
sputum should be sterilized by steam or boiling.

Many different methods for staining the tubercle bacillus
have been proposed. In most of those now in use the fol-
lowing solution is employed—

Fuchsin i gram.

Carbolic acid, pure 5 c.c.

Alcohol 10 c.c.

Distilled water 100 c.c.

The method given below is the one recommended.
Method for staining the tubercle bacillus:

(a) The cover-glass preparation is made, dried, and fixed
by passing through the flame three times.

(b) The cover-glass, held in forceps or in a watch-
crystal is covered with steaming carbol-fuchsin for five
minutes.

(d) Wash in alcohol containing 3 per cent, of hydro-
chloric acid one minute, or longer if necessary to remove
the red color.

46 MANUAL OF BACTERIOLOGY.

(e) Wash in water.

(f) Stain with methylene-blue solution (see page 41)
thirty seconds.

(g) Wash in water.

(h) Examine in water directly, or after drying and
mounting in Canada balsam. Tubercle bacilli take a brilliant
red color; other bacteria and the nuclei of cells are stained
blue.

Gabbett's Method. — This method is very popular and
widely used on account of its convenience. It is not as
reliable as the one just given.

Gabbett's solution :

Methylene-blue I to 2 grams.

25 per cent, watery solution of sulphuric acid. looc.c.

(a) The cover-glass preparation is to be made, dried, and
fixed by passing through the flame three times.

(b) The carbol-fuchsin stain is applied from two to five
minutes to the cover-glass, held in forceps or in a watch-
crystal ; it need not be warmed.

(d) Gabbett's solution is applied for one minute.

(e) Wash in water. The preparation should have a blue
color. It may be examined in water directly or after dry-
ing and mounting in Canada balsam.

Gabbett's method has the advantage of decolorizing the
preparation and staining the background with methylene-
blue at the same time. Tubercle bacilli are colored a
brilliant red; most other bacteria and the nuclei of cells are
colored blue. The acid-proof bacilli mentioned on page 44
would keep the red stain also, in most cases, and would prob-
ably be confused with tubercle bacilli.

Of the numerous methods of staining tubercle bacilli only
a few others can be mentioned. Aniline-water fuchsin,
aniline-water gentian-violet, or carbol-fuchsin may be used.

EXAMINATION OF BACTERIA WITH THE MICROSCOPE. 47

The intensity of the stain must then be increased by warm-
ing the preparation till it steams or boils, then allowing the
warm stain to act on the specimens for from three to five
minutes; the preparation may also be left in the cold stain
over night. Decolorization may be effected with a 25 per
cent, solution of sulphuric acid used till the red color disap-
pears, or a 30 per cent, solution of nitric acid, which operates
very rapidly. If the red color persists after washing in
water, dip in the acid again. After either acid the prepara-
tion is to be washed in alcohol until the' last trace of the
stain has been removed. An excellent decolorizing agent
is a 3 per cent, solution of hydrochloric acid in alcohol,
used for about a minute. With any of these acid solutions
the decolorization can be accomplished more perfectly than
with Gabbett's solution, where the operation of the de-
colorizing agent is masked. The contrast-stain may be
omitted entirely if it is desired. A suitable contrast-stain
after fuchsin staining is a solution of methylene-blue ; after
gentian-violet staining, Bismarck brown.

Those who have had experience in staining tubercle ba-
cilli soon discover that the bacilli exhibit some differences
in their resisting power to strong acids. One encounters
occasionally bacilli that are perfectly stained side by side
with others that are more or less completely decolorized.
These facts show the necessity of practice with any method,
and of exercising caution and judgment in making a' diag-
nosis where the number of bacilli happens to be scanty.
If tubercle bacilli are not found in the first preparation,
other preparations should be made. Sometimes a large
number of cover-glasses must be examined.

Various expedients have been devised to concentrate
tubercle bacilli when only a small number may be present
in a sample of sputum. In Biedert's method about 15 c.c.
of sputum are mixed with 5 c.c. of distilled water, 4 to 8

48 MANUAL OF BACTERIOLOGY.

drops of sodium hydrate solution are added, and the mix-
ture is boiled. After boiling, add about 15 c.c. of distilled
water. The mixture may be set aside in a conical glass
for from twenty-four to forty-eight hours when the sedi-
ment may be collected, smeared on a cover-glass and
stained for tubercle bacilli ; or the sediment may be precip-
itated rapidly by the use of the centrifuge. The sediment
will be found to have little adhesive power, and will not
stick well to the cover-glass. It is convenient to save some
of the original sputum and mix it with the sediment for
this purpose.

Staining Bacteria in Tissues. — Pieces of organs about
i cm. in thickness may be taken. Alcohol is the best
agent for preserving them. The hardening will be com-
pleted in a few days. It is best to change the alcohol.
The amount of the alcohol must be twenty times the bulk
of the tissue to be preserved.

Ten parts of the standard 40 per cent, solution of form-
aldehyde, with 90 parts water make a good mixture for
fixation ; after twenty-four hours change to alcohol.

Imbedding in Collodion or Celloidin. — From alcohol the
pieces of tissue are placed in equal parts of alcohol and
ether twenty-four hours; thin collodion (i^ per cent.),
twenty-four hours ; thick collodion of a syrupy consistency
(6 per cent.) twenty- four hours. The specimen is laid upon
a block of wood and surrounded by thick collodion, and then
inverted in 70 per cent, alcohol. The collodion makes
a firm mass, surrounding and permeating the tissue, and
permits very thin sections to be cut. The soluble cotton
sold by dealers in photographer's supplies serves as well as
the expensive preparation known as celloidin. To make
collodion, dissolve it in equal parts of alcohol and ether.
Soluble cotton is also called pyroxylin, and is a kind of
gun-cotton.

EXAMINATION OF BACTERIA WITH THE MICROSCOPE. 49

Imbedding in Paraffin. — (a) Pieces of tissue 2 to 3 mm.
thick which have already been fixed in alcohol or formal-
dehyde are to be placed in absolute alcohol for twenty-four
hours.

(b) In pure xylol one to three hours.

(d) In melted paraffin having a melting-point of 50° C,
which requires the use of a water-bath or oven, one to three
hours. The xylol must be entirely driven off, and the tis-
sue thoroughly infiltrated.

(e) Change to fresh paraffin for one hour.

(/) Finally, place the tissue in a small dish or paper
box and pour the melted paraffin about it. Harden as
quickly as possible with running water. It is important to
fix the piece of tissue in a suitable position, if the position
is of importance, before pouring in the melted paraffin.

Sections of exquisite thinness may now be cut. The
knife need not be wet. Paraffin imbedding is especially
desirable when serial sections are to be made.

In order to mount the sections, proceed as follows :

(a) Place the sections on water in a porcelain capsule.
Warm slightly, when the sections will flatten nicely. Smear
the surface of a slide with a very thin layer of Mayer's
glycerin-albumen mixture. Dip the slide under the sec-
tions; lift them; and then drain off the water, leaving the
sections in their proper positions. Let them dry for some
hours in the incubator, and they will be firmly fastened to
the slide.

(b) Dissolve out the paraffin in one of the numerous sol-
vents (xylol, a few minutes).

(d) The section is stained.

50 MANUAL OF BACTERIOLOGY.

(e) Dehydrate in absolute alcohol.
(/) Clear in xylol.
(g) Mount in balsam.

GLYCERIN-ALBUMEN MIXTURE (MAYER).

Equal parts of white of egg and glycerin are thoroughly mixed, and
then filtered. Add a little gum-camphor to preserve.

Section Cutting. — Cutting is best done with an instru-
ment called a microtome. The tissues may be imbedded in
collodion or paraffin ; or when they have been hardened with
formaldehyde they may be cut after freezing. Bacteria

Fie. 10.

Schanze microtome.

stain admirably in fro/en sections. Kor routine work col-
lodion imbedding will be found as convenient a process as
any. Paraffin imbedding gives the thinnest sections.

A microtome consists of a heavy, sliding knife-carrier,
which moves with great precision on a level, and of a de-

EXAMINATION OF BACTERIA WITH THE MICROSCOPE. 5!

vice for elevating the object which is to be cut any desired
distance after each excursion of the knife. The thickness
of the section will be the distance which the object is ele-
vated. The knife is kept wet with alcohol during the cut-
ting of collodion sections, otherwise it is left dry. The
microtome is usually provided with a special form of knife.
A razor will serve nearly as well, after having had the
lower side ground flat. If a razor is used, a special form
of razor-holder must be attached to the microtome to re-
ceive the razor. Above all, it is necessary that the knives
should be kept in good condition. Only occasionally will
they need honing, using a fine water-stone or Belgian
hone. The movement in honing should be from heel to
toe, always placing the back of the knife next the hone
when turning. The knife should be stropped frequently.
The leather of the strop should be glued to a strip of wood
to make a flat surface. The movement in stropping should
be from toe to heel. Sections should be cut to a thickness
of not more than 25^. Thinner sections (5 to io/*) are to
be desired.

Staining of Sections. — A watery solution of one of the
aniline dyes is used — fuchsin, gentian-violet or methylene-
blue — made by adding a few drops of the alcoholic solution
to a dish filled with water. Loffler's solution of methylene-
blue serves very well.

By this process most bacteria are stained ; also the nuclei
of cells; frequently, also, certain granules contained within
some cells (German, Mastzcllcn}, which may easily be
mistaken for bacteria by the inexperienced (basophilic
granules).

(a) Place the section in the staining solution from two to
five minutes.

(b) Wash in water.
5

52 MANUAL OF BACTERIOLOGY.

(d) Alcohol, one to two minutes; change to absolute al-
cohol. Touch the sections to blotting-paper to remove the
superfluous alcohol.

(e) Xylol until clear; xylol is to be preferred to other
clearing agents, like oil of cloves, most of which slowly re-
move aniline colors. It has the disadvantage of not clearing
when the slightest trace of water is present ; dehydration in
alcohol must, therefore, be complete. The section should
be removed from the xylol as soon as it is cleared ; otherwise
wrinkling occurs.

(f) The section is placed upon a glass slide; a drop of
Canada balsam is placed upon it and then a cover-glass.
The Canada balsam should be dissolved in xylol.

The section is to be manipulated with straight or bent
needles. The removal from xylol to the glass slide is man-
aged best with a spatula or section-lifter.

The above statements apply to frozen sections or to sec-
tions imbedded in celloidin. Paraffin sections are preferably
attached to the slide with glycerin-albumen. The different
steps in the process follow in the same order. The stain
may be poured on the slide, or the slide may be placed in
a large dish full of staining fluid. (See page 49.) Celloidin
sections may also be stained on the slide. If the section be
well spread and flattened thoroughly with blotting-paper,
it will usually adhere to the slide, and is less likely to
wrinkle. It must not be allowed to dry.

Gram's Method may be applied to the staining of sec-
tions of tissues as well as to smears upon cover-glasses.

(a) Place the section in aniline-water gentian-violet,
one to five minutes.

(&) Rinse briefly in water.

EXAMINATION OF BACTERIA WITH THE MICROSCOPE. 53

(d) Alcohol, until decolorized to a faint blue-gray.

(e) Xylol.

(f) Mount on a slide in balsam.

Weigert's Modification of Gram's Method, or Weigert's
Stain for Fibrin. — (a) Place the section in aniline-water
gentian-violet solution, five minutes or more.

(&) Wash briefly in water.

(c) Place the section upon a slide by means of a section-
lifter; having straightened it carefully, absorb the water
with blotting-paper.

(d) Iodine solution (see page 42) one to two minutes.

(e) Absorb the iodine solution with blotting-paper.

(f) Add aniline oil, removing it from time to time with
blotting-paper, and adding fresh aniline oil until the color
ceases to come away. (Aniline oil serves in this connection
both to decolorize and to dehydrate. It absorbs the water
rapidly and efficiently. However, on account of its decol-
orizing tendency, it must be removed before the specimens
can be mounted permanently.)

(g) Add xylol; remove it with blotting-paper; and add
fresh xylol several times, in order to extract the last trace
of aniline oil.

(h) Mount in Canada balsam.

This method is more convenient for the staining of sec-
tions than the Gram method. The results, however, are
essentially the same as far as the bacteria are concerned ;
fibrin and hyaline material are stained blue, bacteria violet.
It is often impossible to decolorize the nuclei completely
without decolorizing the bacteria also. The parts of the
nuclei which remain stained often present pictures that re-
semble bacteria, and which may lead to error if not recog-
nized. Basophilic granules also retain the stain, as do the
horny cells of the epidermis. These remarks apply also
to Gram's method, except as regards fibrin. Very beauti-

54 MANUAL OF BACTERIOLOGY.

ful preparations can be obtained according to this or the
Gram method when the sections have previously been
stained in carmine; the nuclei will then be colored red,
bacteria violet.

Tubercle bacilli may be stained in sections as follows :

(a) Use carbol-fuchsin, or aniline-water gentian-violet
for one-half to two hours with very gentle warming, or
over night without warming.

(b) Wash in water.

(c) Decolorize with some one of the decolorizing agents
mentioned in connection with the staining of tubercle ba-
cilli in cover-glass preparations, preferably 3 per cent, hy-
drochloric acid alcohol. Decolorization must be continued
until the red color has disappeared, which requires one-half
to several minutes.

(d) Wash in alcohol.

(e) Wash in water.

(/) Use hematoxylin as a contrast-stain for fuchsin
preparations, and carmine for gentian-violet preparations.
(It is better to stain with carmine first of all and before
staining the bacilli. The carmine is not affected by the
subsequent treatment. )
(g) Wash in water.
O) Alcohol,
(i) Xylol.
(/) Balsam.

Nuclear stains, which may be used as contrast-stains for
sections :

DELAFIELD'S HEMATOXYLIX.

Hematoxylin crystals 4 grams.

Alcohol 25 c.c.

Ammonia alum 50 grams.

Water 400 c.c.

Glycerin TOO c.c.

Methyl alcohol 100 c.c.

EXAMINATION OF BACTERIA WITH THE MICROSCOPE. 55

Dissolve the hematoxylin in the alcohol, and the am-
monia alum in the water. Mix the two solutions. Let
the mixture stand four or five days uncovered; it should
have become a deep purple. Filter and add the glycerin
and the methyl alcohol. After it has become dark enough,
filter again. Keep it a month or longer before using; the
solution improves with age. At the time of using, filter
and dilute with water as desired.

LITHIUM-CARMINE (ORTH).

Carmine 2.5 grams.

Saturated watery solution of lithium carbonate. loo.oc.c.

Add a few crystals of thymol. The carmine dissolves
readily in the lithium carbonate solution. Filter the stain
at the time of using. Sections are to be left in the stain
five to twenty minutes.

Sections stained in carmine are placed directly in acid
alcohol (i part hydrochloric acid, 100 parts 70 per cent.
alcohol) for five to ten minutes. They acquire a brilliant
scarlet color. When used as a contrast-stain for tissues
containing bacteria, it is best to use it before staining the
bacteria, which might be decolorized by the acid alcohol.

Staining of Blood-Films. — The method of Wright is
the one recommended. It is applicable to bacteria and to the
parasite of malaria, and is useful as a general stain for
blood. Films of blood are prepared as directed in chapter
VII., Part I., and are allowed to dry.

(a) The stain is poured over the surface of the prepara-
tion till it covers it. This serves to fix the film of blood. It
is allowed to remain for one minute.

(6) Add distilled water, drop by drop, till a reddish tint
appears at the edges and a metallic scum forms on the sur-
face. About six drops are needed for a three-fourths inch
cover-glass. The real staining of the preparation now takes
place, and requires two or three minutes.

56 MANUAL OF BACTERIOLOGY.

(c) Wash in distilled water till the thin parts of the
preparation have a yellowish or pinkish tint, which requires
one to three minutes.

(d) Dry with blotting-paper and mount in Canada bal-
sam.

Bacteria, malarial parasites, and cell-nuclei are stained
blue, red blood-corpuscles are orange-pink, while the specific
granules of the leucocytes (neutrophilic, etc.) appear in
various tints from red to dark blue. The chromatin of the
malarial parasite takes a lilac to red color. The blood-plates
have a bluish or purplish color and must not be confused
with malarial parasites.

The staining fluid is prepared as follows : To 100 c.c. of a one per
cent, solution of sodium bicarbonate in water add i gram of methyl ene-
blue. Place in the steam sterilizer at ioo°C. for one hour. When cool
add one-tenth per cent, watery solution of eosin (Griibler, yellowish,
soluble in water) until the mixture loses its blue color, becomes pur-
ple, and a metallic scum forms on the surface. About 500 c.c. of the
eosin solution are needed. Collect the precipitate on a filter ; let it dry ;
make a saturated solution of the precipitate in methyl alcohol; filter.
To the quantity obtained add one-fourth as much methyl alcohol, so
that the solution may not be completely saturated. The purpose of the
above procedures is to modify the methylene-bluc so that other stain-
ing elements are developed in it (polychromism). The modified
methylene-blue solution is then combined with eosin. For full details
see Wright, Journal of Medical Research, Vol. VII. 1902.

Staining of Spores. — The method is applicable to cover-
glass preparations which may be prepared in the usual
way from material supposed to contain spores.

(a) After drying the smear on the cover-glass, and fixa-
tion with heat by passing through the flame three times,
use as a stain aniline-water fuchsin.

(b) Heat until the preparation begins to boil; remove
for a minute; heat again, and again remove; repeat this
process six times.

f '

EXAMINATION OF BACTERIA WITH THE MICROSCOPE. 57

(d) Wash in water.

(e) Stain with watery solution of methylene-blue half a
minute.

(/) Wash.

(£) Dry.

(h) Balsam.

The spores are intensely stained by the fuchsin. The
stain is removed from everything except the spores by the
acid alcohol. The methylene-blue solution stains the bodies
of the bacteria, the spores remaining brilliant red. There
are various other methods for staining spores, but this pro-
cedure gives good results. The principle is the same as in
staining the tubercle bacillus, except that more pains are
needed to impregnate spores with the dye.

Staining of Capsules. — The capsules which many bac-
teria possess, appear to be made of some gelatinous sub-
stance, which is difficult to stain.

Method of Welch. — (a) Cover-glass preparations are
made in the usual manner. Pour glacial acetic acid over
the film.

(&) After a few seconds, replace with anilin- water gen-
tian-violet, without washing in water. Change the stain
several times to remove all the acetic acid. Allow it to act
three or four minutes.

Bacteria are deeply stained, while their capsules are pale
violet. This method has been recommended for staining
the capsule of the pneumococcus.

Methods of Hiss. — i. (a) Cover-glass preparations are
made in the usual manner, and fixed in the flame.

(&) Stain for a few seconds in a half-saturated watery
solution of gentian-violet.

58 MANUAL OF BACTERIOLOGY.

(d) Mount and study in the same.

2. (a) Cover-glass preparations are made and fixed in
the ordinary way.

(b) Use the following stain, heated till it steams:

Saturated alcoholic solution of gentian-violet or fuchsin. 5 c.c.
Distilled water 95 c.c.

(d) Dry and mount in Canada balsam.

The methods of Hiss are recommended to be used for
bacteria that have been cultivated on serum-agar with i per
cent. of dextrose. They have shown that many streptococci
have capsules. The writer has had good success from the
latter method, with preparations of the pneumococcus from
animal tissues.

Staining of Flagella. — Flagella are among the most
difficult of all objects to stain. The best-known method is
that of Lofllcr. It is important to use young cultures, pref-
erably on agar.

(a) A small portion of the culture is mixed on a cover-
glass with a drop of water. The preparations must be ex-
ceedingly thin. The mixing must be done with care in
order not to break off the delicate flagella. The cover-
glass must be perfectly clean, see page 36.

(b) After drying, fixation is effected by passing through
the flame three times.

Tannic acid, 20 per cent, solution 10 c.c.

Saturated solution of ferrous sulphate 5 c-c-

Saturated alcoholic solution of fuchsin i c.c.

This solution is filtered and a few drops are placed on
the cover-glass, or the cover-glass is placed, face down, in

EXAMINATION OF BACTERIA WITH THE MICROSCOPE. 59

a dish containing the stain ; it is then left tor one to five min-
utes, warming slightly.

(d) Wash in water.

(e) Stain with aniline-water fuchsin, or carbol-fuchsin.
(/) Wash in water.

(£) Dry.

(h) Mount in Canada balsam.

(According to Loffler, certain bacteria require the addi-
tion of an acid solution, and certain others an alkaline
solution, but many observers consider this unnecessary.)

Another and very valuable method is that of Van Er-
mengem.

(a) Make and fix cover-glass preparations as in the pre-
ceding method.

(b) Use the following mordant for one-half hour at
room-temperature or for five minutes at 50° to 60° C.

Osmic acid 2 per cent, solution I

Tannic acid 10 to 25 per cent, solution 2

(d) Place for a few seconds in a 0.25 to 0.50 per cent,
solution of nitrate of silver — " the sensitizing bath."

(e) Without washing transfer to the " reducing and re-
inforcing bath " :

Gallic acid 5 grams.

Tannic acid 3 grams.

Fused potassium acetate 10 grams.

Distilled water 350 c.c.

(/) After a few seconds, replace the preparation in the
nitrate of silver solution, in which it is kept constantly
moving, till the solution begins to acquire a brown or black
color.

Some recommend leaving the preparation in the nitrate
of silver solution for two minutes in the first place, and in
6

60 MANUAL OF BACTERIOLOGY.

the reducing bath for two minutes, without using the nitrate
of silver solution a second time.

(g) Finally wash in distilled water, dry, mount in
Canada balsam. It is difficult to avoid the formation of
precipitates; otherwise the results of this method are usu-
ally good.

(" *

STERILIZATION. 6 1

CHAPTER II.

STERILIZATION.

BY sterilization is meant the killing of all microorgan-
isms found on or in any body or substance. It is possible
to sterilize objects by the use of bichloride of mercury (cor-
rosive sublimate), carbolic acid and other chemical agents.
Sterilization is usually accomplished by heat. The most
effective sterilization is that done by steam and by borling;
they are not, however, suitable for all kinds of material.

The naked flame of the Bunsen burner or the alcohol
lamp is used largely for the sterilization of small articles.
It is evident that no more efficient way of sterilization could
be devised than by burning objects, or subjecting them to
a red heat. The uses of this method will at once suggest
themselves; for instance, surgical dressings that have be-
come soiled with discharges and similar materials can be
most easily disposed of by simply burning them up. In
laboratory work the flame is constantly employed for the
sterilization of the platinum wire, forceps, pipettes and
cover-glasses; occasionally test-tubes are sterilized in this
manner.

Hot-Air Sterilization. — Hot air, at a temperature of 150°
C, or higher, maintained for an hour, is very valuable for
some materials although less effective than steam. It has
been found that the spores of certain bacteria are not killed
even by exposure to this temperature, but it is sufficient for
ordinary conditions. Hot-air sterilization is employed for
glassware such as Petri dishes, flasks and test-tubes.
Flasks and test-tubes are generally plugged with raw cot-

MANUAL OF BACTERIOLOGY.

ton. The sterilization should change the cotton to a light
brown color, but it should not be scorched to a dark brown.
Glassware should be placed within the sterilizer when it is
cold, and after heating should be allowed to cool gradually
in order to avoid breaking. Hot-air sterilization is never
used for culture-media.

The hot-air sterilizer is a box made of sheet-iron, the
walls being double, with an air-space between them. On

FIG. ii.

Hot-air sterilizer.

one side is a door. There are openings at the top to secure
the circulation of air in the air-chamber. A thermometer
passes from the top into the interior of the sterilizer so
that one may read off the temperature that is being attained.
The sterilizer should be placed so that there will be no dan-
ger of its setting fire to inflammable articles, as the heat

STERILIZATION. 63

may occasionally become very intense. It is well, if pos-
sible, to have it fastened to a brick wall.

Boiling. — Boiling is an efficient method of sterilization.
It is often used for instruments. In laboratory work steam
is generally substituted for it.

Steam Sterilization. — Steam sterilization is the most
generally used of all forms of sterilization and is the most
effective. It is employed for perishable bodies which would
be injured by dry air sterilization or by chemical ger-
micides ; for example, it is used for surgical instruments and
for culture-media; in laboratory work, especially for cul-
ture-media. It has been found that there are some forms
of bacteria which, in the resting or spore stage, can resist
even the action of steam for several hours. Such pro-
longed exposure to steam would be very injurious to cul-
ture-media, which are more or less unstable organic sub-
stances. What is called fractional, intermittent or dis-
continuous sterilization is used for such materials. By that
plan the medium is sterilized with steam for fifteen minutes
on each of three consecutive days. The object of inter-
mittent sterilization as explained by Tyndall, who proposed
it, is this : The culture-medium may be supposed to contain
fully developed bacteria, and also bacteria in the spore or
resting stage. The first sterilization of fifteen minutes will
probably be sufficient to destroy all the fully developed bac-
teria; during the twenty-four hours between the first and
second sterilization all of the spores which have survived
the first sterilization may be expected to have become fully
developed into bacteria which can be destroyed by the
second sterilization ; the third sterilization is directed against
any spore forms which may possibly have survived the sec-
ond sterilization.

Although the spore forms which are so extremely resist-
ant are mostly non-pathogenic, as for example the bacilli of

MANUAL OF BACTERIOLOGY.

hay and potato, they nevertheless are capable of ruining
the culture-media with which one works.

It has been shown by T. Smith that the discontinuous
method cannot be relied upon to sterilize fluids in shallow
layers that are freely exposed to the air. For if the spores
of anaerobic bacteria happen to be present in such fluids,
they will not develop into the adult form between the appli-
cations of heat, under aerobic conditions.

The form of sterilizer most widely used in the United
States is that which is known as the Arnold Steam Sterilizer.

FlC. 12.

Diagram of the Arnold steam sterilizer.

The Arnold sterilizer consists of a cylinder of tin or
copper with a cover, which is enclosed in a movable, cylin-
drical outer cover or hood. The inner cylinder has an
opening in the bottom through which steam may enter, the

STERILIZATION. 65

steam coming from a small chamber underneath with a
copper bottom to which the flame is applied. The peculi-
arity of this form of sterilizer consists in the fact that the
steam which escapes from the sterilizing chamber. will be
condensed beneath the outer cover or hood and will fall
back upon the pan over the chamber in which the steam is
generated. The bottom of this pan is perforated with three

FIG. 13.

Steam sterilizer, Massachusetts Board of Health.

small holes which allow the water of condensation to return
into the chamber where the steam is generated. The ster-
ilizer will, therefore, to a certain extent, supply itself with
water, although not by any means perfectly. It is, how-
ever, less likely to boil dry than other forms of sterilizers,
and it has the advantage of being reasonably cheap and

MANUAL OF BACTERIOLOGY.

quite effective. The space enclosed by the hood also serves
as a steam-jacket and helps to overcome fluctuations in tem-
perature. A great improvement upon the ordinary Arnold
sterilizer is the modification of it devised by the Massachu-
setts Board of Health.

In the use of this, or any form of steam sterilizer, the
time when sterilization is supposed to begin must be counted

FIG. 14.

Koch's Steam Sterilizer.

as that when boiling is brisk and it is evident that the ster-
ilizing chamber is filled with hot steam; or, what is better,
when the thermometer registers 100° C, if the sterilizer be
provided with a thermometer. With a large Arnold ster-
ilizer a temperature of 100° C. may not be reached until it

STERILIZATION. 67

has been heated with a rose-burner for twenty to thirty-five
minutes.

The sterilizer invented by Koch is still largely in use.
It is a tall cylindrical tin vessel covered with asbestos or
felt. The lower portion is filled with water; on the side
is a water-gauge indicating the height of the water, in order
that one may observe when there is danger of the sterilizer
boiling dry. Over the top there is a tight-fitting cover.
The steam is generated by a Bunsen burner standing under-
neath. A perforated shelf placed some distance above the
surface of the water is for the reception of the tubes and
flasks that are to be sterilized.

The sterilization of blood-serum sometimes has to be per-
formed in a specially devised sterilizer, when a clear, fluid
medium is desired. In this case the serum is heated for an
hour on each of six consecutive days to a temperature of
only 58° C. To obtain a transparent but solid medium the
serum is kept at a temperature of 75° C. for an hour on
each of four consecutive days. The process must be con-
ducted carefully to avoid clouding of the serum.

Pasteurization. — The name pasteurization has been ap-
plied to the partial sterilization of substances at a compara-
tively low temperature. It is employed particularly for
milk. The temperature used (70° to 75° C. for 20 to 30
minutes) is sufficient to destroy all ordinary pathogenic
bacteria ; for example, the bacilli of tuberculosis and typhoid
fever. Furthermore, the great majority of the saprophytic
bacteria are destroyed, and milk which has been pasteurized
will remain unchanged for several days, if kept cool. Its
application is principally in the feeding of infants when
ordinary milk has been found to produce undesirable results.
Freeman1 has invented a pail of special form for the pas-
teurization of milk in bottles. This pail is filled with hot

1 Medical Record, July 2, 1892, and August 4, 1894.

MANUAL OF BACTERIOLOGY.

water and the bottles are placed in it; it has been found to
keep up a temperature of about 75° C.

The Autoclave. — The autoclave is an instrument de-
signed for sterilization by steam under pressure. It was
invented in France but is now used extensively in all parts
of the world. Steam generated at the ordinary atmospheric
pressure is much less destructive to bacteria, and especially

Vic.. 15.

Autoclave.

to their spores, than steam in the autoclave at a pressure of
an additional one-half to one atmosphere; the steam then
reaches a temperature of about 110° to I2O°C. Under
these conditions culture-media may be sufficiently sterilized
in the autoclave in fifteen minutes, and at a single steriliza-
tion. The autoclave consists of a metal cylinder with a

STERILIZATION.

FIG. i 6.

movable top, which is fastened down tightly during sterili-
zation. It is furnished with a thermometer, a pressure-
gauge, and a safety-valve which allows the steam to escape
if too high a pressure is attained. Heat is furnished by a
gas-burner underneath. The lower part of the cylinder
contains water. The objects to be sterilized are supported
above this water on a perforated bottom or shelf.

It is necessary to follow certain precautions in the use of
the autoclave, especially during cooling. The apparatus
must not be opened while the steam contained within it is
still under pressure, as there may be a
sudden evolution of steam upon the re-
moval of the pressure which may blow
the media out of their tubes and flasks.
The apparatus must, therefore, be kept
closed until the gauge shows that the
atmospheric pressure is as great as the
pressure within, or, what is equivalent,
until the temperature has fallen to 100°
C. Gelatin, especially, may be dam-
aged by sterilization with the autoclave,
if it be heated too long or to too high
a temperature.

Sterilization by Filtration. — Ordi-
nary filters are useless for this purpose,
but the tubes or bougies of unglazed
porcelain devised by Pasteur and Cham-
berland are effective when properly em-
ployed. The Berkenfeld filter employs
bougies made of infusorial earth, and its pores are larger than
those of the Pasteur filter. Both of these are made in several
grades according to the coarseness or fineness of the pores.
The coarser of these filters permit the passage of very small
bacteria. Bacteria of average size, like bacillus coli com-

Kitasato Filter.

JO MANUAL OF BACTERIOLOGY.

munis, may grow through the pores in the walls of both the
Berkenfeld and Pasteur filters if sufficient nutrient material
is present to permit of their multiplication.1

Filters of these kinds are widely used for water, and will
be spoken of in connection with the chapter on water. Simi-
lar tubes are employed for the filtration of certain organic
nutrient media whose ingredients would be damaged by
sterilization with heat, chiefly extracts of organs, such as
the thymus gland. The soluble " toxins " of bacteria may
be obtained by filtration of fluid-cultures through such tubes,
which remove the bacteria (Fig. 16). These fluids usually
filter very slowly, and filtration will have to be assisted by
some form of vacuum-pump ; usually the filter-pump, which
is used in connection with a stream of running water, is
employed. Compressed air or carbonic acid may be' used to
assist in forcing fluids through the filter. The filter
bougies, the flasks and all parts of the apparatus must, of
course, be sterilized by heat before and after using.

1 Wherry, Journal of Medical Research. Vol. VIII., 1902.

CULTURE-MEDIA. 7 1

CHAPTER III.

CULTURE-MEDIA.

CULTURE-MEDIA are substances in which bacteria are
artificially cultivated. The number of such substances is
very large, different materials being suited to different
purposes and to different kinds of bacteria. The most
important ones are nutrient bouillon or beef-tea, nutrient
gelatin, and nutrient agar-agar. The two last have a jelly-
like consistency, owing to the addition of a gelatinizing
substance, but otherwise are of the same composition as
bouillon.

Nutrient Bouillon.

Beef-extract (such as Liebig's) 3 grams.

Peptone, pure (Witte's)1 10 grams.

Sodium chloride (common salt) 5 grams.

Water i liter.

The solid ingredients are dissolved in water, and the
mixture is boiled for a few minutes. It is made neutral or
very faintly alkaline by the addition of a solution of sodium
hydroxide, drop by drop, the reaction being tested at inter-
vals with litmus-paper. The bouillon may now be filtered
through filter-paper. The filter-paper should be folded and
creased as is done by pharmacists; it is usually placed in a
glass funnel, and should be moistened with water before
using. After filtration the medium is to be placed in prop-
erly plugged tubes or flasks, and is to be sterilized once in
the autoclave, or in the steam sterilizer for fifteen minutes

1 Commercial "peptones" are mixtures of albumose and a small
amount of peptone.

72 MANUAL OF BACTERIOLOGY.

or longer on each of three consecutive days. When pre-
cipitates form, they are usually caused by a too alkaline
reaction. That may be corrected by the addition of a little
weak hydrochloric acid, drop by drop, testing frequently
with litmus-paper.

A more accurate way of obtaining the proper reaction is Schultz's
method. Take of the bouillon 10 c.c. ; add a few drops of phenol-
phthalein1 (alcoholic solution i per cent.) ; with a burette add, drop by
drop, a solution of caustic soda 0.4 per cent, until a faint red color ap-
pears, which indicates the beginning of the alkaline reaction. This
procedure is followed with three samples. The amount of soda solu-
tion required in each case is noted and the average taken. If now, on
the average, for each 10 c.c. of bouillon i c.c. of soda solution needs
to be added, for 1,000 c.c. of bouillon 100 c.c. of the soda solution must
be added; only, instead of adding a weak soda solution, one-tenth as
much is taken of a solution ten times as strong.

Another method of making bouillon is to use, instead
of beef-extract, 500 grams (one pound) of finely chopped,
lean beef, which is placed in one liter of water and kept
on ice for twenty-four hours. It is strained, thoroughly
cooked to coagulate the albumen in it, filtered, and a liter
of fluid obtained, adding water if necessary. The peptone
and salt are then added and the medium heated to dissolve
them. It is then neutralized, filtered, and sterilized. Al-
though bouillon made with solid beef-extract is convenient
and serviceable for most purposes, it is advisable to use
fresh meat when the bouillon is to be employed for the
development of bacterial toxins. Fresh meat should also be
used in the preparation of either bouillon, gelatin or agar-
agar when new species of bacteria are being studied for pub-
lication.

1 In neutralizing an acid culture-medium it has been found that when
the medium appears to be neutral or slightly alkaline to litmus, it may
still be acid if phenolphthalein be employed as an indicator. Fuller,
Journal .-lincrican Public llcaltli Association. 1895.

CULTURE-MEDIA. 73

In both of these cases the recommendations of the American Public
Health Association should be followed.1

These also advise that media be neutralized by titration. —

The following solutions are required : $ per cent, phenolphthalein in 50
per cent alcohol, normal2 (£') and twentieth normal (|^) solutions
of sodium hydroxide and of hydrochloric acid.

To 5 c.c. of bouillon in a porcelain evaporating dish add 45 c.c. of dis-
tilled water ; boil three minutes ; add I c.c. of phenolphthalein solution,
and proceed with the titration wrwle still hot. As the reaction will
usually be found acid, add from a burette /^ sodium hydroxide solu-
tion, stirring constantly, until a decided pink color develops in the
entire solution. The color reaction indicates the more or less arbi-
trarily adopted neutral point. Repeat this procedure with three differ-
ent portions of bouillon, and determine the average amount of ^
sodium hydroxide required. It is now possible to calculate the amount

1 See the Report of the Committee of the American Public Health
Association entitled Procedures Recommended for the Study of Bac-
teria. 1898. Rumford Press, Concord, N. H.

2A normal solution of any substance contains, fn a liter, as many
grams of the substance as there are units in its molecular weight, in
case it contains a single atom of replaceable hydrogen. If it has two
atoms of replaceable hydrogen the number of grams used equals the
molecular weight divided by two ; and so on. Thus the molecular
weight of sodium hydroxide is 40, and its normal solution contains 40
grams of sodium hydroxide in a liter. It is not expedient to prepare
normal solutions of sodium hydroxide by weight. For convenience,
crystallized oxalic acid is used as a starting point in making normal
solutions. Its molecular weight, including a molecule of water of
crystallization, is 123. As it is a dibasic acid (having two atoms of
replaceable hydrogen), half of this weight, or 62.5 grams, per liter, is
taken. Any y" acid solution will exactly neutralize an equal volume of
any ~ alkaline solution. To make j" sodium hydroxide solution, add
about 41 grams of pure caustic soda to a liter of distilled water. Find
the amount of this solution needed to exactly neutralize I c.c. of y
solution of oxalic acid; this amount contains the quantity of sodium
hydroxide which should be present in I c.c. of a normal solution. It
is now possible to calculate the amount of distilled water to be added
in order that I c.c. of the sodium hydroxide solution may neutralize
i c.c. of the £ solution of oxalic acid. With an f solution of sodium
hydroxide as a standard, an ^ solution of hydrochloric acid may be
prepared. Twentieth normal solutions have one-twentieth the strength
of normal solutions.

74 MANUAL OF BACTERIOLOGY.

of y sodium hydroxide needed to neutralize the whole quantity of
bouillon. This should be added. The bouillon should then be boiled
for ten minutes, and again titrated. It will usually be found acid.
The deficiency should be corrected by adding the necessary amount of
y sodium hydroxide. It should be boiled again, and again titrated,
and any deficiency made good. It is rarely necessary to repeat the
process, except to determine that the neutral point has been reached.
After neutralizing it is boiled thirty minutes and filtered. Enough y
hydrochloric acid or sodium hydroxide is added to give the degree of
acidity or alkalinity desired. It is then sterilized.

An acid reaction may be denoted by +, an alkaline by — . The
degree of acidity or alkalinity may be indicated by the amount of £
solution required to render the medium neutral to phenolphthalein, thus
+ 1.5 signifies that a medium is acid, and requires 1.5 per cent, of y
sodium hydroxide to neutralize it.

A reaction of +1.5 is recommended as the optimum. There is much
disagreement as to what reaction is most favorable for the growth of
the majority of species of bacteria. In any case the degree of reaction
should be noted in descriptions.

Bouillon may be modified by the addition to it of other
substances, the most important of which are glycerine (6 per
cent.) and sugars, — as dextrose,1 saccharose or lactose (i
per cent.). It is better to sterilize media containing sugars
in the steam sterilizer by the fractional method than in the
autoclave, where decomposition of the sugars may occur.

Dextrose-free Bouillon. — Ordinary bouillon often contains some
muscle-sugar, which is objectionable if fermentation tests with lactose
or saccharose are to be made. To secure bouillon free of sugar, beef-
infusion is prepared from fresh meat, and is inoculated in the evening
with a quantity of bacillus coli commnnis, and kept in the incubator.
Early next morning it is boiled, filtered, peptone and salt added, and
the bouillon is prepared as usual.2

Nutrient Gelatin.

Beef-extract 3 grams.

Peptone 10 grams.

Sodium chloride 5 grams.

Gelatin (best gold label) 100 grams.

\Yuter i liter.

1 Dextrose is the principal ingredient of commercial grape-sugar and
should be obtained in a pure condition.

2 See T. Smith, Journal of Experimental Medicine, Vol. II., p. 546.

CULTURE-MEDIA. 75

Dissolve the ingredients in the water, stirring actively
to prevent burning at the bottom. It is best to conduct the
operations in granite- or enamel-ware vessels over a large
Bunsen or rose-burner. Neutralize with sodium hydroxide
solution (see page 71). The reaction at the beginning will
usually be found to be quite acid. Allow the mixture to cool
until below 60° C, and add the whites of one or two eggs
which have been beaten up with a little water; stir in thor-
oughly. Heat the mixture to the boiling-point; stir at the
bottom to prevent burning and at the same time avoid as far
as possible breaking the coagulum of egg-albumen which
forms at the surface. Boil for ten minutes. Filter while
hot. The nitration may be done through folded filter-paper
which has been moistened. It is well to fasten a piece of
coarse cheese-cloth over the top of the funnel to catch the
large particles of coagulated albumen. Place in suitable
tubes or flasks plugged with cotton, and sterilize once in the
autoclave, or, preferably, in the steam sterilizer for fifteen
minutes on each of three consecutive days. Gelatin is in-
jured by too prolonged boiling and loses its solidifying
qualities. Neutralization may be with litmus paper or by
titration. The remarks on pages 72 to 74 with regard to
the use of fresh beef and the titration method for the prepa-
ration of bouillon apply equally to gelatin.

Instead of filter-paper, some prefer to filter through sev-
eral layers of absorbent cotton placed inside of the moist-
ened glass funnel, the top of which is covered with coarse
cheese-cloth. This expedient answers very well.

If the product appears cloudy after it has been sterilized,
it may be that the egg-albumen was incompletely coagu-
lated in the first place or that the reaction has been made
too alkaline. In any case it will be desirable to melt it
and filter a second time, correcting the reaction with hydro-
chloric acid if necessary. It may be well to stir in another
7

76 MANUAL OF BACTERIOLOGY.

egg to entangle the opaque particles; then to boil a second
time and filter.

The medium is sometimes modified by adding to it other
substances, as sugar, glycerin, etc. The solidifying prop-
erty of the gelatin must be carefully guarded, and too much
boiling is to be avoided. Certain bacteria, it will be found,
have the property of causing gelatin to become fluid.
Gelatin melts at about 25° C. and solidifies at about 10° C.
It cannot be used in the incubator, where it would liquefy
at the temperature of 38° C. In hot weather it may be
necessary to use 150 grams of dry gelatin to the liter.
Nutrient gelatin is usually spoken of simply as " gelatin."

Nutrient Agar-agar. — Agar-agar (French, gelose) is a
kind of vegetable gelatin which comes from the southern
and eastern coast of Asia. It melts with much greater diffi-
culty than gelatin.

The medium is not quite transparent. The finished me-
dium is commonly called " agar.'1

Beef-extract 3 grams.

Peptone 10 grams.

Sodium chloride 5 grams.

Agar 10 grams.

Water I liter.

The dry agar, cut tine, is to be dissolved in water over a
flame. It should be boiled for from one-half hour to two
hours, skimming off the scum which forms on the surface
from time to time. The beef-extract, peptone and sodium
chloride are dissolved in a liter of water, boiled and neu-
tralized. Add the agar now in solution in a small quantity
of water. The reaction of the agar alone is faintly alka-
line. Mix thoroughly; the bulk of the mixture is a little
more than a liter, and should be reduced to a liter after the
subsequent boiling. Cool to about 60° C. ; stir in the whites

CULTURE-MEDIA. 77

of one or two eggs and boil thoroughly. Avoid breaking
the eoagulum of egg which is designed to entangle the solid
particles that make the medium cloudy; stir at the bottom,
however, to prevent burning. Filter while hot, using filter-
paper or absorbent cotton covered with cheese-cloth. The
hot water funnel originally devised for the filtration of agar
is not necessary. If filtration is slow, the funnel and flask
may be placed inside of the steam sterilizer and kept heated
during filtration. The medium is collected in suitable flasks
or tubes plugged with cotton, and sterilized once in the auto-
clave or in the ordinary steam sterilizer for fifteen minutes
on each of three consecutive days. As agar is frequently
used for smear-cultures where a slanted medium is desired,
some of the tubes may be allowed to cool in a slanting posi-
tion. It is not well to keep on hand many tubes which
have been slanted, as the medium dries more rapidly. Agar
is not liquefied by bacteria as is gelatin. Its solidifying
qualities are impaired somewhat if the reaction be acid.

The remarks on pages 72 to 74 with regard to the use of
fresh beef and the titration method for the preparation of
bouillon apply equally to agar-agar.

Glycerin-agar is used extensively. It is agar, made as
above directed, to which 6 per cent, of glycerin is added
before sterilization. It is very useful in cultivating the
bacilli of tuberculosis and diphtheria.

Sugar-cigar. — Before sterilizing, i per cent, of either dex-
trose, lactose, saccharose, or other sugars may be added to
agar. With media containing sugar, litmus forms a useful
indicator of the production of acid. Enough tincture of
litmus is used to give the medium a blue color before ster-
ilization; the litmus is somewhat unstable and prone to
change its color during sterilization. Neutral red may also
be added in the same manner ; its color is said to be changed
by certain bacteria and not by others (see bacillus of typhoid
fever, and bacillus coli communis, Part IV.).

MANUAL OF BACTERIOLOGY.

FIG. 17.

Potato. — The potatoes are washed, a slice is removed
from each end, and with an apple-corer or cork-borer a
cylinder is cut out. This cylinder is divided diagonally into
two pieces. The pieces are washed in running water for
twelve to eighteen hours. They are placed in
test-tubes containing a little water to keep the
potato moist, and are supported from the bot-
tom on a piece of glass tubing about i to 2
cm. in length (or on cotton, or in a specially
devised form of tube with a constriction at the
bottom) . The tubes are plugged, and sterilized
as with other media. Sterilization, however,
must be thorough on account of the danger
of contamination with the extremely resistant
spores of the potato bacillus. Potato is best
when freshly prepared; it is likely to become
dry and discolored with keeping. It is a
very useful medium ; certain growths on it, like
those of the bacillus of typhoid fever or of
glanders, and those of chromogenic bacteria,
are very characteristic.

Milk. — Milk fresh as possible is placed in
a covered jar, sterilized for fifteen minutes,

Tube contain- an(j t]ien k t Qn jce £Qr twenty-four hours,
ing Jrotato.

At the end of that time the middle por-
tion is removed by means of a siphon. The upper and
lower layers must not be taken; the upper part contains
cream, and the lower part particles of dirt, both of which are
to be avoided. About 7 to 10 c.c. are to be run into each
test-tube. The tube is plugged with cotton, and sterilized
as usual. When milk is contaminated with spores of the
hay or potato bacillus it is sometimes very difficult to steril-
ize, a fact of much importance in connection with the feed-
ing of children, where the fractional method of steriliza-
tion and the use of the autoclave are impracticable.

CULTURE-MEDIA. 79

The coagulation of milk, which is accomplished by cer-
tain bacteria, is a very valuable differential point. A little
litmus tincture may be added to the tubes of milk before
sterilizing, until they acquire a blue color, to indicate
whether or not acids are formed by the bacteria which are
afterwards cultivated in the milk.

Dunham's Peptone Solution.

Peptone 10 grams.

Sodium chloride 5 grams.

Water i liter.

Boil, filter, sterilize in the usual manner.

Dunham's solution is valuable to test the development of
indol by bacteria (see Part II., Chapter II.). The develop-
ment of acids may be detected after the addition of 2 per
cent, of rosolic acid solution (.5 per cent, solution in
alcohol) ; alkaline solutions give a clear rose-color which
disappears in the presence of acids.

Blood-serum. — The blood of the ox or cow may be ob-
tained easily at the abattoir. It should be collected in a
clean jar. When it has coagulated, the clot should be
separated from the sides of the jar with a glass rod. It
may be left on the ice for from twenty-four to forty-eight
hours. At the end of that time the serum will have sepa-
rated from the clot and may be drawn off with a siphon
into tubes. These tubes are sterilized for the first time in
a slanting position as the first sterilization coagulates the
serum. The coagulation may be done advantageously, as
advised by Councilman and Mallory, in the hot-air sterilizer
at a temperature below the boiling-point. After coagulation,
sterilize as usual. This serum makes an opaque medium of
a cream color. Blood-serum may be sterilized in the special
form of sterilizer devised for it. A clear blood-serum is to
be obtained by sterilization at a temperature of 58° C. for
one hour, on each of six days, if a fluid medium is desired,

8O MANUAL OF BACTERIOLOGY.

or of 75° G. on each of four days if the serum is to be solidi-
fied. In the latter case the tubes are to be placed in an in-
clined position. ( See page 67. ) Opaque, coagulated blood-
serum has most of the advantages of the clear medium.
Blood-serum may be secured from small animals by collect-
ing blood directly from the vessels, using very great care to
obtain the blood in a sterile condition ; and the serum may
be separated and stored in a fluid state. Human blood-
serum is sometimes obtained from the placental blood, some-
times from serous pleural transudates or from hydrocele
fluid. The preservation of blood-serum is sometimes accom-
plished with chloroform, of which i per cent, is to be added
to the medium ; in this manner the serum may be preserved
for a long time. It may be divided into tubes, solidified and
sterilized as required ; the chloroform will be driven off
by the heat, owing to its volatility. Blood-serum media
which are sterilized at low temperatures should be tested for
twenty-four hours in the incubator to prove that steriliza-
tion has been effective; if it has not, development of the
contaminating bacteria will take place and be visible to the
eye.

It will be impossible to do more than merely mention
some of the most important of the other culture-media.

Lbffler's blood-serum consists of one part of bouillon
containing i per cent, of glucose, and three parts of blood-
serum. It is sterilized like ordinary blood-serum. It is
used largely for the cultivation of the bacillus of diph-
theria.

Blood-scntm-agar is a medium made with considerable
difficulty, but very valuable for the cultivation of the gon-
ococcus. One part of placental blood-serum, or pleuritic
serum, or hydrocele fluid, is mixed with one to two parts
of nutrient agar in the fluid condition. It must be divided
into tubes before solidification. Solidify in a slanting posi-

CULTURE-MEDIA. 8l

tion; subsequently sterilize at 75° C. so as not to coagulate
the albumen of the blood-serum. The nutrient agar in this
case should contain 2 per cent, of dry agar. Another ex-
pedient has also been to smear a little blood over the sur-
face of a tube of nutrient agar — blood-agar — used for culti-
vating the bacillus of influenza. Marmorek's blood-serum
is supposed to assist in maintaining the very evanescent
virulence of the streptococci ; it consists of bouillon mixed
with human blood-serum, ass's serum or horse's serum.

Guarnieris medium consists of a mixture of gelatin and
agar.

Media containing fat were employed by Sommaruga to
test the ability of bacteria to decompose fats. Clarified
beef-suet or olive-oil in the proportion of I or 2 per cent.
is added to gelatin or agar. The fat must be mixed with
the melted medium; it is to be shaken and then rapidly
cooled in a freezing-mixture after the last sterilization.

Fresh eggs in their shells may be used without other
preparation than washing the surface thoroughly with bi-
chloride of mercury solution; or after sterilization by steam,
which of course coagulates the albumen. The egg is easily
inoculated through a small opening made with a heated
needle, which may be closed afterward with collodion.
Hueppe recommended eggs closed in this manner for the
cultivation of anaerobic bacteria. Egg-albumen has been
used as a constituent of various media. Dorset1 states that
good results may be secured when eggs are used as a cul-
ture-medium for tubercle bacilli. The yolk and the white
are mixed, poured into tubes, slanted, coagulated, and ster-
ilized. Just before using pour into the tube a few drops of
sterile distilled water to moisten the medium.

Bread-paste (finely-divided dry bread, mixed with water
and sterilized) is used for the cultivation of moulds. Sa-
1 American Medicine, April 5, 1902.

MANUAL OF BACTERIOLOGY.

FIG. 18.

bouraud recommends the following for the cultivation of
the trichophyton fungus :

Peptone 5 grams.

Maltose 3.8 grams.

Agar 1.3 grams.

Water 100 c.c.

Test-tubes. — Bacteria are generally cultivated in test-
tubes. A convenient size is one f of an inch in diameter
and 5 inches in length. The tubes
should be of a heavier glass than in
those used for ordinary chemical work.
The New York Board of Health, and
some others, use a tube three inches
in length without a flange for the cul-
tivation of the diphtheria bacillus on
Loffler's blood-serum mixture. Test-
tubes should be thoroughly cleaned with
a swab before using; they should be
boiled with washing-soda, rinsed,
filled with hydrochloric acid solution,
rinsed, and inverted to drain away the fluid.

Plugs of raw cotton or cotton batting are employed as
stoppers. Some prefer absorbent cotton, but it is likely to
become soggy after exposure to steam. The plug should fit
smoothly; creases and cracks around the edges are to be
avoided. The plug should be tight enough to sustain the
weight of the tube when held by the plug. These plugs pre-
vent bacteria from entering or leaving the tubes.

Sterilization of Test-tubes. — The tubes are to be steril-
ized in a hot-air sterilizer for one hour, at a temperature
of 150° C. The cotton should acquire a light brown color
but should not be burned. If the plugs touch the sides of
the sterilizer or lie against the bottom they may be scorched.
The necessity for sterilization of the tubes before filling

Wire Basket for Test-
tubes.

CULTURE-MEDIA. 83

them with the medium has been questioned, and it is prob-
ably unnecessary as far as the preservation of the culture-
medium is concerned, but it will be found that the cotton
plugs fit much better after sterilization with dry heat.
During this and subsequent sterilizations the tubes are held
in a wire basket.

Filling of the Tubes. — A special funnel closed with a
stop-cock for filling tubes with liquefied media is often
recommended. They may readily be filled with an ordi-
nary funnel of small size. During the filling, the neck of
the test-tube where it comes in contact with the cotton must
not be wet with the medium. Ordinarily about 7 to 10 c.c.
are placed in a test-tube. For Esmarch's roll-tubes a some-
what smaller quantity is desirable.

The sterilization of tubes containing culture-media is
always done by steam, and has been sufficiently described.
It is to be remembered that the solidifying power of gelatin
is impaired by too prolonged heating, while heating is less
likely to damage other culture-media. The media which
are sterilized at a low temperature (70° C.) should be tested
for two days in the incubator to determine whether steriliza-
tion has been effective. It is the universal experience in
bacteriological laboratories that occasionally culture-media
will become contaminated with extremely resistant spores
which fail to be sterilized by the ordinary processes, an
occurrence which causes great annoyance and calls for the
exercise of much patience. Sometimes, also, moulds attach
themselves to the plugs, especially if they are moist, and send
their filaments down through the cotton ; finally, having
reached the lower edge of the cotton, their spores may fall
upon the medium, grow there and ruin it.

84 MANUAL OF BACTERIOLOGY.

CHAPTER IV.

THE CULTIVATION OF BACTERIA.

Inoculation of the Tubes. — The air of the laboratory
should he as quiet as possible, to lessen the chances of con-
tamination by bacteria clinging to particles of dust. Avoid
working where there may be draughts or gusts of air or
near an open window. Spores are blown from the surfaces
of moulds, like thistle-down, and are constantly being wafted
about in the air. Given any material containing bacteria,
for example a pure culture of some well-known species, a
very minute portion is to be introduced into a tube contain-
ing the sterile culture-medium. The introduction is effected
with a straight platinum wire, or with a platinum wire loop.
The platinum is to be heated red-hot before using, and then
allowed to cool. It is also to be heated red-hot after using.
The plug of the test-tube is to be withdrawn, twisting it
slightly, taking it between the third and fourth fingers of
the left hand, with the part that projects into the tube point-
ing- toward the back of the hand. It must not be allowed to
touch any object while the inoculation is going on. Pass
the neck of the tube through the flame. If any of the cotton
adheres to the neck of the tube, pull the cotton away with
sterilized forceps, while the neck of the tube touches the
flame, so that the threads of cotton may be burned and not
fly into the air of the room. The tube is held as nearly
horizontal as possible. The tube is to be held in the left
hand between the thumb and forefinger, the tube resting
upon the palm, and the neck of the tube pointing upward

THE CULTIVATION OF BACTERIA. 85

and to the right. When two tubes are being used at the
same time, as is often necessary, they are placed side by side
between the thumb and forefinger of the left hand. The
two plugs are held between the second and third and the
third and fourth fingers of the left hand, respectively. The
wire may now, be passed into the first tube, which we will
suppose to hold some material containing bacteria, and a
little of this material may be removed on the tip of the wire
from the first tube to the second. When the needle is intro-
duced into or removed from either tube it should not touch
the side of the tube at any point, and should only come in
contact with the region desired. After inoculation of the
second tube has been effected the wire is to be heated to a
red heat in the flame, the necks of the tubes are to be passed
through the flame, and the plugs are to be returned to their

FIG. 19.

Manner of Holding Tubes.

respective tubes. When the wet wire is to be sterilized in the
flame it should be approached to the flame gradually, so as
to dry the material on it before burning it, in order to avoid
"sputtering" (see page 33). It is well from the start to
train one's self to sterilize the platinum wire every time it is
taken from the table and before it is laid down again. The
platinum wire loop may be used in the same manner as the
straight wire, especially when a substance containing a small
number of bacteria is being handled.

MANUAL OF BACTERIOLOGY.

When a tube of gelatin is to be inoculated the wire is
usually introduced into the medium vertically, " stab-cul-
ture " ; when a medium with a slanted surface is employed,
as agar, potato or blood-serum, the needle should lightly
streak the surface, "smear-culture" (Figs. 20 and 21).

The safety and success of this method of inoculation de-
pend upon a principle which has been established by long
and repeated observation, namely, that bacteria do not of
themselves leave a moist surface. They should not, there-

FIG. 20.

FIG. 21.

Stab-Culture.

A rubber stopper may be

used to prevent drying,

see page 91.

Smear-Culture.

This tube shows the rubber

cap used to prevent

drying.

fore, rise from the surface of the moist culture-medium, nor
drop from the needle during its transit, if proper care be
exercised. They may be thrown into the air if the needle
be allowed to sputter in the flame.

THE CULTIVATION OF BACTERIA. 87

If, by any accident, drops of infectious material should
fall upon a surface like the table, they should be covered
at once with bichloride of mercury solution i-iooo. A
good way is to cover the spot with a piece of blotting-paper
wet with the solution ; place a bell- jar over it and leave for
several hours. If infectious material should reach the hands
or clothing, they should be thoroughly soaked in the bichlo-
ride solution. When working with pathogenic bacteria it
is well to wash the hands in this solution and with soap
and water, as a routine procedure, before leaving the labor-
atory.

To maintain their vitality bacteria need to be transplanted
from one tube to another occasionally; the time varies
greatly with different species. Many bacteria grow on cul-
ture-media with difficulty at the first inoculation, but hav-
ing become accustomed to their artificial surroundings, as
it were, they may be propagated easily afterward; this is
especially true of the bacillus tuberculosis.

Some bacteria flourish better on one culture-medium than
another. The bacillus tuberculosis grows best on blood-
serum and glycerin-agar ; the bacillus of diphtheria grows
best on LofBer's blood-serum; the gonococcus on human
serum-agar.

The virulence of most pathogenic bacteria becomes
diminished after prolonged cultivation upon media. Some-
times the virulence is lost very quickly, for example, the
streptococcus pyogenes and micrococcus lanceolatus of pneu-
monia.

Incubators. — Many bacteria flourish best at a tempera-
ture about that of the human body, 38° C. Some species
will grow only at this temperature. The pathogenic bac-
teria in particular, for the most part, thrive best at a point
near the body temperature.

The incubator is a box made of copper, having double

88 MANUAL OF BACTERIOLOGY.

walls, the space between the two being filled with water.
The outer surface is covered with some non-conductor of
heat, such as felt or asbestos. At one side is a door, which

Tic. 22.

Incubator.

is also double. The inner door is of K"^lss< the outer door
is of copper covered with asbestos. At one side is a gauge
which indicates the level at which the water stands in the

THE CULTIVATION OF BACTERIA.

water-jacket. The roof is perforated with several holes,
some of which permit the circulation of the air in the air-
chamber inside the box; some of them enter the water-
jacket. A thermometer passes through one of these holes
into the interior of the air-chamber, and often another into
the water standing in the water-jacket. A gas-regulator
passes through another hole, and is immersed in the water

FIG. 23. FIG. 24.

Reichert's Gas-regulator. Mercurial Gas-regulator, a. Cham-

ber containing volatile hydrocarbon.
b. Capillary opening.

standing in the water-jacket. There are various forms of gas-
regulators more or less complicated. In general they consist
usually of a tube containing mercury ; into this tube are two
openings, one for the entrance and the other for the exit of
gas. The gas enters through a small tube, which is cut off
diagonally at the bottom, and which projects into the sur-
face of the mercury. Heating the water in the water-jacket

MANUAL OF BACTERIOLOGY.

Fir,. 25.

causes expansion of the mercury, which rises, and, little by
little, cuts off the inflow of gas through this tube. The
flow is never completely cut off, as there is a capillary open-
ing in the tube considerably above any point to which the
mercury could possibly rise, which will always allow the
flow of a small quantity of gas (Fig. 24, b). This diagram
also shows a modification of the simple form of regulator,
in the shape of a partition which divides off a lower cham-
ber, which contains mercury and is connected with the upper

part by a glass tube. The pur-
pose is to make use of the elastic
properties of some volatile fluid,
like ether, which floats on the sur-
face of the mercury at a. The
gas coming from the gas-regulator
passes to a Bunsen burner, which
stands underneath the incubator.
This burner should have some
kind of automatic device for cut-
ting off the flow of gas in case it
becomes accidentally extinguished
by a sudden draught of air or
from any other cause. The auto-
matic burner invented by Koch
is an ingenious, simple and effec-
tive device. A bar of metal
stands above the flame; by its

expansion, through a system of levers, it supports a weight ;
the weight controls a gas-cock. While the flame is burning
the expansion of the metal holds the weight horizontally; if
the flame becomes extinguished, the metal contracts, the
weight falls, and cuts off the flow of gas. Some incon-
venience will arise from irregularities in the flow of gas

Koch Automatic Gas-burner.

THE CULTIVATION OF BACTERIA. 91

from the main supply-pipe. Any incubator will vary a little
from such causes. In the experience of the writer, natural
gas is of such variable pressure as to be entirely useless.
Fluctuations of the temperature within the incubator depend
very largely upon the external temperature. Therefore the
incubator should, as far as is practicable, be protected from
sudden draughts of cold air and should be kept in a room
having as equable a temperature as possible.

Culture-tubes which are being kept in the incubator are
likely to become dry if their stay is prolonged. In such
cases they should be covered with rubber caps, tin-foil,
sealing-wax, paraffin, or some other device to prevent
evaporation. If rubber caps are used, they should be left
in i-iooo bichloride of mercury solution for an hour, and
the cotton plugs should be singed in the flame, before put-
ting them on. (Fig. 21.) The writer prefers rubber
stoppers, which may be boiled and stored in bichloride of
mercury solution. Cut the cotton plug even with the edge
of the tube ; singe it in the flame ; push it into the tube about
i cm. ; and insert the rubber .stopper. (Fig. 20.)

CULTIVATION OF ANAEROBIC BACTERIA.

The cultivation of anaerobic bacteria is done best in a
medium containing i to 2 per cent, of dextrose. The tube
should contain a large quantity of the culture-medium.
Just before using, the medium should be boiled for a few
minutes. Inoculate the tube after cooling, but while the
medium is fluid. Anaerobes may be cultivated in the
closed arm of the fermentation-tube (see Fig. 46), but the
opening between the two arms of the tube must be small.

Buchner's method for the cultivation of anaerobes: Into
a bottle or tube which can be tightly stoppered, pour 10 c.c.

MANUAL OF BACTERIOLOGY.

FIG. 26.

of a 6 per cent, solution of sodium or potassium hydroxide,
for each 100 c.c. of air contained in the jar. Add one gram
of pyrogallic acid for each 10 c.c. of solution. The culture-
tube is placed inside of the
larger bottle or tube, supported
above the bottom, and the stop-
per, smeared with paraffin, is
inserted. The mixture of pyro-
gallic acid and potassium hy-
droxide possesses the property
of absorbing oxygen.

ITright's Modification of
BiicJiucr's method: The tube of
culture-medium is to be plug-
ged with absorbent cotton, us-
ing a plug of large size. The
culture-medium is inoculated
in the usual way. The plug
is cut off close to the neck of
the tube, and is then pushed
into the tube about I centi-
meter. Now allow a watery
solution of pyrogallic acid to
run into the plug, and then a
watery solution of sodium or
potassium hydroxide. Close
quickly and tightly with a rub-
ber stopper. Wright recommends that the first solution
be freshly made and consist of about equal volumes of
pyrogallic acid and water, and that the second solution con-
tain I part of sodium hydroxide and j parts of water. With
6 inch test-tubes, :| inch diameter, the amounts advised are
- .1 c.C. solution of pyrogallic acid, I c.c. solution of sodium
hydroxide.

Arrangement of Tubes for Cul-
tivation of Anaerobes by
Buchner's Method.

THE CULTIVATION OF BACTERIA.

FIG. 27.

Cultivation of Anaerobic Bacieria under Hydrogen:
Method of Frank el: A test-tube containing a large amount
of the liquefied culture-medium is closed with a sterilized
rubber stopper, through which pass two sterilized glass
tubes, bent above the stopper at a right angle. One of
these tubes is cut off just underneath
the stopper, and the other is long
enough to project nearly to the bottom
of the culture-tube. The horizontal
projecting parts are drawn to a small
caliber at some point, although not
quite closed, to facilitate sealing later
on. Through the longer of these
tubes hydrogen gas is passed until the
atmosphere inside of the culture-tube
is pure hydrogen, entirely free from
mixture with air. The horizontal
parts of the small glass tubes project-
ing from the stopper are then sealed
in the flame at the places where they
were previously drawn out to a small
caliber, and the tubes are thus closed.
(Fig. 27.)

The stopper should be surrounded
with melted paraffin,
according to this plan may, if desired, be
converted into an Esmarch roll-tube. The hydrogen is gen-
erated according to the common method with pure zinc and
pure sulphuric acid, 25 to 30 per cent. The precautions
advised by chemists for the generation of hydrogen must
be carefully followed, because when hydrogen mixed with
oxygen or air is ignited a violent and disastrous explosion
may occur.

The well-known Kipp's generator may be used. First

A tube prepared Cultivation of Anaerobes

by Frankel's Method.

MANUAL OF BACTERIOLOGY.

FIG. 28.

let the reservoir fill with hydrogen; then allow its contents
to escape. This should be repeated, after which some of
the hydrogen may be collected in an inverted test-tube under
water. When this sample is ignited, it should burn without
any explosion ; otherwise the hydrogen is not yet ready to
use. The hydrogen should bubble through the medium five

minutes or more.

The inconvenience of
sealing the tubes in the
flame, as has to be done in
Franker s and other meth-
ods for cultivation under
hydrogen, is obviated in
Novy's apparatus. The
tubes or plates are placed
in jars through which
hydrogen may " be con-
ducted. The stopper, hav-
ing been smeared pre-
viously with a soft wax,
is sealed by giving it one-
fourth of a turn.
Novy's Jar for the Cultivation There have been vari-

of Anaerobes. oug Qther kinds Q£ appa_

ratus, usually complicated and expensive, devised for the
growth of plate-cultures under hydrogen.

Other expedients for the cultivation of anaerobic bacteria are less
effective. In cases where a very deep stab-culture is made in gelatin
or agar, where the growth appears in the lower part of the tube by
preference, it is supposed to be anaerobic. Koch covered part of the
surface of a gelatin plate with a bit of sterilized mica or a cover-
glass; bacteria which grew beneath this plate were considered to be
anaerobic. Another method was to cover the surface of the gelatin in
the cultu/e-tube with sterilized oil. W. H. Park has recommended a mix-
ture of solid paraffin with 25 to 50 per cent, of fluid paraffin or albolene
as a covering for the surface of anaerobic cultures. This mixture has a

THE CULTIVATION OF BACTERIA.

semi-solid consistency, and does not retract at the edges on cooling.
The paraffin prevents the absorption of oxygen, except to a small
extent at the edges. The method is useful for large quantities of cul-
ture material, as in flasks. Esmarch advised making roll-tubes, and
after cooling them to fill them with a liquefied gelatin cooled down

FIG. 29.

An Aerobic Organism (Potato Bacillus) which will not grow under a

cover-glass.

to near the point of solidification. Hueppe made use of eggs in their
shells. The eggshell was carefully cleaned, sterilized with a solution
of bichloride of mercury, washed with sterilized water and wiped dry
with sterilized cotton. The end of the eggshell was punctured with a
hot needle. Through the opening thus made the inoculation was accom-
plished. The opening was closed with collodion.

96 MANUAL OF BACTERIOLOGY.

CHAPTER V.

CULTIVATION OF BACTERIA, CONTINUED.

Isolation of Bacteria. — In order to study any kind of
bacteria it is necessary to have the particular species sepa-
rated from other sorts with which it may be mixed. The
earlier bacteriologists endeavored to separate bacteria of
different sorts by successive transplantations through a series
of tubes. The procedure now generally used for this pur-
pose is the so-called plate-method of Koch. The great
progress which bacteriology has made during the last twenty
years is largely owing to this invention.

Pathogenic bacteria may sometimes be isolated through
inoculations into animals. Thus an animal may be inocu-
lated with sputum containing tubercle bacilli mixed with
other bacteria. The animal may die of tuberculosis, and
its tissues may contain tubercle bacilli in pure culture, the
other bacteria having produced no important effect.

Still another method which is occasionally useful is to
subject the mixture of bacteria to steam for a few minutes.
If it contains very resistant spores, like those of the teta-
nus bacillus or hay bacillus, they may be expected to sur-
vive, and may perhaps be propagated in pure culture,
everything else having been killed by the steam.

Plate-Cultures. — It is impossible in most cases to dis-
tinguish between bacteria of different varieties by micro-
scopical examination alone. Bacteria of widely different
species and quite unlike one another in their properties may
present similar appearances under the microscope. The
differences which they exhibit are usually apparent when

THE CULTIVATION OF BACTERIA. 97

they are grown in culture-media. The growth, called a
colony, which results from the multiplication of a single
bacterium, is in many cases quite characteristic for the
species. By the plate-method, the individual bacteria in a
mixture are separated from one another by dilution. They
are fixed in place by the use of a solid medium. They
are allowed to grow, and from each individual there forms
a colony. It is usually possible to distinguish between col-
onies arising from different species when it was not possible
to distinguish between the individual bacteria of these
species. A convenient illustration has been suggested by
Abbott. A number of seeds of different sorts may appear
very much alike, and considerable difficulty may be found
in distinguishing one from another with the eye. Let
them be sown, however, and let plants develop from them,
and these plants will easily be distinguished from one
another.1

Method of Making Plate-cultures. — Melt three tubes of
gelatin or agar. (There is some difficulty in keeping agar
in a fluid state while dilutions are being made. It is best to
have some form of water-bath with a thermometer for the
purpose.) Let the liquefied tubes cool to 40° C. Take a
small portion of the material to be examined — pus, for ex-
ample— and introduce it with a sterilized platinum wire or
loop into one of the tubes. Stir it in carefully. Remove
the needle, sterilize it, and replace the plug. Mix the ma-
terial introduced thoroughly with the liquefied culture-
medium, taking care not to wet the plug. Now remove
the plug again, and, having sterilized the platinum wire,
insert it into the liquefied medium. Carry three loopfuls in

1 It must be understood that no close comparison can be drawn be-
tween higher plants, which simply complete the development of parts
potentially present in the seed, and colonies of bacteria, which are aggre-
gates of individuals, the progeny of one individual of the same kind.

98 MANUAL OF BACTERIOLOGY.

succession from this tube, which is No. i , into tube No. 2 ;
sterilize the needle; replace the plugs; mix thoroughly,
without wetting the plug. Carry three loopfuls from tube
No. 2 into tube No. 3 in the same manner. The original
material will obviously be diluted in tube No. I, more in
tube No. 2, and still more in tube No. 3. The most con-
venient form of plate is that known as a Petri dish, a small
glass dish about 8 cm. in diameter and 1.5 cm. in height,
provided with a cover which is a little larger but of the same

FIG. 30.

Petri Dish.

form. This dish should be cleaned and sterilized for an hour
in a hot-air sterilizer at 150° C. or higher. When it is cool
it may be used.

Such dishes having previously been prepared, the con-
tents of tube No. i are poured into one dish, and those of
tube No. 2 into another, and those of tube No. 3 into a
third. They are to be labeled Nos. i, 2, and 3.1 In pour-
ing proceed as follows : remove the plug of tube No. i ; heat
the neck of the tube in the flame ; allow it to cool, holding it
in a nearly horizontal position. When the tube has cooled,
lift the cover of the Petri dish a little, holding it over the
dish; pour the contents of tube No. I into the dish, and
replace the cover of the dish. The interior of the dish
should be exposed as little and as short a time as possible.

1 The labels should be moistened with the finger, which has been dipped
in water. They should not be licked with the tongue. While working
in the bacteriological laboratory it is best to make it a rule that no ob-
ject is to be put in the mouth.

FIG. 31. Dilution-cultures in Esmarch Roll-tubes.

FIG. 32. Appearance of Colonies on Gelatin in Petri Dish.

THE CULTIVATION OF BACTERIA. 99

Tubes Nos. 2 and 3 are to be treated in the same manner.
Burn the plugs, and fill the empty tubes with 5 per cent.
solution of carbolic acid. They should be sterilized for an
hour in the steam sterilizer on each of three days.

The culture-medium in the Petri dish will soon solidify.
Colonies develop usually in from one to two days. In
plate No. i they will be very numerous, in plate No. 2 less
numerous, and in plate No. 3 still less numerous. Where
the "number is small the colonies will be widely separated
and can readily be studied. They may be examined with
a hand-lens, or the entire dish may be placed on the stage
of the microscope and the colonies be inspected with the
low power. The iris diaphragm should be partly closed
and the concave mirror should be used. Dilution-cultures
prepared as described in the next paragraph, where the
principle is the same, are shown in Fig. 31. In tube No.
i the colonies are so numerous as to look like fine white
dust. In tubes 2 and 3 they become less numerous and
larger.

Esmarch's Roll-tubes. — Use liquefied gelatin or agar.
The dilutions in tubes i, 2 and 3 are made as above.
Tubes containing a rather small amount of the culture-
medium are more convenient. A block of ice should be at
hand, and, with a tube filled with hot water and lying
horizontally, a hollow of the size of the test-tube should be
melted on the upper surface of the ice. In this hollow place
the tube of liquefied gelatin or agar; roll it rapidly with
the hand, taking care that the culture-medium does not
run toward the neck as far as the cotton plug. The medium
is spread in a uniform manner around the inside of the tube,
where it becomes solidified. Gelatin roll-tubes must be
kept in a place so cool that there is no danger of their
melting; in handling them they are to be held near the
neck, so that the warmth of the hand may not melt the

100

MANUAL OF BACTERIOLOGY.

gelatin. Agar roll-tubes should be kept in a position a
little inclined from the horizontal, with the neck up, for
twenty-four hours, so that the agar may stick to the wall
of the tube.

By the plate-method as originally devised by Koch, instead of using
Petri dishes, the gelatin was poured upon a sterile plate of glass. This
plate of glass was laid on another larger plate of glass, which formed
a cover for a dish of ice-water, the whole being provided with a leveling

FIG. 33.

Manner of Making Esmarch Roll-tube.

apparatus. The plate was kept perfectly level until it had solidified,
which took place rapidly on the cold surface. The glass plates were
placed on little benches enclosed within a sterile chamber. The more
convenient Petri dish has displaced the original glass plate to a large
extent.

The isolation of bacteria may sometimes be effected by
drawing a platinum wire containing material to be exam-
ined rapidly over the surface of a Petri dish containing
solid gelatin or agar; or over the surface of the slanted
culture-medium in a test-tube; or by drawing it over the
surface of the medium in one test-tube, then, without steril-

THE CULTIVATION OF BACTERIA. IOI

izing, over the surface of another, perhaps over several in
succession.

Appearance of the Colonies. — The colonies obtained in
the Petri dishes or roll-tubes (Fig. 32) may be studied
with a hand-lens or with a low power microscope. In the
latter case, use the concave mirror with the iris diaphragm
partly closed. The colonies present various appearances.
Some of them are white, some colored; some are quite
transparent and others are opaque; some are round, some
are irregular in outline; some have a smooth surface, others
appear granular, and others present a radial striation.
Surface colonies often present different appearances from
those occurring more deeply. Surface colonies are likely
to be broad, flat and spreading. If the colony consists of
bacteria which have the property of liquefying gelatin, a
little funnel-shaped pit or depression forms at the site of
the colony. The appearance of colonies may be of great
assistance in determining the character of doubtful species.
The appearance in gelatin plates of the colonies of the
spirillum of Asiatic cholera, for instance, is one of the most
characteristic manifestations of this organism.

Pure Cultures. — From these colonies pure cultures may
be obtained by what is called " fishing." Select a colony
from which cultures are to be made; touch it lightly with
the tip of a sterilized platinum wire, taking great care not
to touch the medium at any other point. Introduce the
wire into a tube of gelatin. Sterilize the wire and plug the
tube. In a similar manner, and from the same colony, in-
oculate tubes of agar, bouillon, milk, potato and blood-
serum. At the same time it is well to make a smear prepa-
ration from the colony and to stain with one of the aniline
dyes so as to determine the morphology of the bacteria.
The growths which take place in the tubes should contain
one and the same kind of bacteria. As seen under the mi-

T02 MANUAL OF BACTERIOLOGY.

croscope their bacteria should have the same general form
and appearance as those seen in the colony from which
they were derived. This will be the case, provided the
colony has resulted from the development of a single bac-
terium or from several bacteria of the same kind. Occa-
sionally, however, a colony will develop from several bac-
teria which may not all be alike. In that case a pure
culture will not be obtained, and the process of plating
may have to be repeated.

I
INOCULATION OF ANIMALS. ICK

CHAPTER VI.

INOCULATION OF ANIMALS.

IN the study of pathogenic bacteria, the inoculation of
animals is frequently indispensable. The animals most
often used are white mice, guinea-pigs, rabbits and pigeons.
Larger animals are occasionally employed for special pur-
poses. White mice may be kept in a glass jar covered
with wire netting. They may be fed with moistened bread
or oats. It is important to see that they receive drinking-
water. During inoculation the mouse must be kept in

FIG. 34.

Mouse-holder.

position by some sort of mouse-holder, or may be held by
an assistant, who takes the skin at the back of the neck
between his fingers and at the same time holds the tail.
The hair is cut off from the skin at the root of the tail.
A small V-shaped opening in the skin is made with scis-
sors, and a stiff sterilized platinum wire is passed into this
opening, separating the skin from the muscles for some
distance so as to make a pocket. Into this pocket the ma-
terial is introduced by means of the platinum wire. The

IO4 MANUAL OF BACTERIOLOGY.

wound may be covered with collodion. The peritoneal
p>1G - cavity of the mouse may be inoculated with a fluid
culture introduced with a sterile hypodermic
syringe.

Guinea-pigs and rabbits, after inoculation, are to
be kept in cages of galvanized iron and wire-
netting. The bottom may conveniently be made in
the form of a movable pan which permits of the
disinfection of the excreta. Rabbits and guinea-
pigs may be fed with oats, carrots, cabbage, grass
and the like. Guinea-pigs and rabbits may be held
by an assistant or tied by the legs upon a board.
The hair over a small portion of the abdomen is cut
away and a short incision is made through the skin :
a pocket is produced \vith a stiff wire, and the ma-
terial inserted with a sterile platinum wire. The
wound may be covered with collodion. Sutures
may be used if the wound is large. Solid sub-
stances may conveniently be introduced by placing
them in a sterile glass cannula, which is pushed to
the proper situation through a small incision. The
substance in the cannula is forced out of it with a
stiff sterile platinum wire. (Fig. 35.) The peri-
toneal cavity may be inoculated with a previously
sterilized hypodermic syringe, or an incision may
be made which reaches to the peritoneal cavity,
into which the desired substance may be introduced
with a sterile platinum wire, the incision being
closed with sutures.

Intravenous inoculation is most commonly prac-
ticed upon rabbits. A small vein which is near the
posterior margin of the ear of the rabbit is easily
reached from the dorsal surface; the hypodermic
needle is introduced directly into this vein. In making a

INOCULATION OF ANIMALS. IO5

hypodermic injection, the needle and syringe should of
course be sterilized before and after each operation.

Autopsies upon animals should be held as soon as pos-
sible after death. During the interval the body should be
kept in the ice-box. The autopsy room should be furnished
with screens to keep out flies, so that they may not light on
the infected animal. The animal should be extended on
its back upon a board. The legs may be fastened with
pins or tacks. The animal should be handled with forceps
as far as possible, and after beginning the autopsy the
fingers should not touch it. If the fingers come in contact
with infectious matter, disinfect them at once. Have a
basin of bichloride of mercury solution i-iooo ready for
this purpose. Knives, scissors, platinum wires and forceps
should be sterilized in the flame before and after each ma-
nipulation. Be prepared to make smear preparations on
cover-glasses, and to inoculate tubes of gelatin, agar and
other media as desired. Moisten the hairs over the thorax
and abdomen with bichloride of mercury solution i-iooo,
to prevent them from being carried into the air. Make an
incision, passing through the skin from the sternum to the
pubis along the thorax and abdomen, and diagonal incisions
extending down the fore and hind legs. Dissect away the
skin from the thorax, abdomen, and upper parts of the legs.
With a knife heated in the flame, sear a broad line extend-
ing down the middle of the abdomen. Through this burned
surface make an incision through the muscles of the ab-
domen. In a similar manner make a transverse incision
across the middle of the abdomen through a burned sur-
face. Cultures should be made from the peritoneal cavity,
and smears upon cover-glasses prepared, which are after-
wards to be stained. With a hot knife, scorch a small area
on the surface of the liver; through this surface enter the
liver with a sterilized platinum wire, and with the material

IO6 MANUAL OF BACTERIOLOGY.

withdrawn inoculate the tubes; also make cover-glass prep-
arations. In the same manner inoculate tubes and make
cover-glass preparations from the spleen, the kidneys, the
pleural cavity, the pericardial cavity, the lungs, and the
blood inside the heart. All incisions are to be made through
the burned surfaces, and all material collected for inocula-
tion is to be obtained through burned surfaces. In steriliz-
ing the instruments in the flame avoid sputtering, especially
when they become covered with oil from adipose tissue.
Pieces of lung, liver, spleen, kidney and other organs, as
may be indicated, should be placed in 95 per cent, alcohol
for fixation and hardening. The animal and the board on
which it was extended should be covered with bichloride
of mercury solution i-iooo, and afterwards burned. The
cage or jar and the instruments, dishes and towels used
should be sterilized by steam. The hands of the operator
should be washed thoroughly with soap and water and with
a i-iooo solution of bichloride of mercury.

FIG. 36.

Method of Making Collodion Capsules. (After McCrae.)

Collodion Capsules. — Bacteria may be cultivated in the
living body of an animal, without infecting the animal,
when they are enclosed in collodion capsules. Their soluble
products are able to diffuse through the collodion, while the
animal's fluid may pass into the sac to nourish them. These
capsules were originally made by dipping the round end of
a glass rod into collodion repeatedly. McCrae's method1
is easier and more satisfactory. (Fig. 36.)

A piece of glass tubing is taken, and a narrow neck drawn on it near
one end. This end of the tube is rounded in the flame, and the body

'Journal of Experimental Medicine, Vol. V., p. 635.

INOCULATION OF ANIMALS. IO/

of a gelatin capsule is fitted over it, while still warm, so that the
gelatin may adhere to the glass. The capsule is now dipped into 3 per
cent collodion, covering the gelatin and part of the glass. It is allowed
to dry a few minutes, and is dipped again. In all two or three coatings
may be given. The capsule is filled with water and boiled in a test-
tube with water. The melted gelatin is removed with a fine pipette.
The capsule is partly filled with water or broth and sterilized. The
capsule may now be inoculated. The narrow part of the neck must
then be sealed in the flame, taking care that the neck be dry. The
sealed capsule should be placed in bouillon for twenty-four hours. No
growth should occur outside the capsule if it is tight. It may now be
placed in the peritoneum of an animal.

IO8 MANUAL OF BACTERIOLOGY.

CHAPTER VII.

COLLECTION OF MATERIAL.

SAMPLES of water or milk collected in sterilized tubes or
bottles, when they are not examined immediately, or when
they are to be transmitted any distance, should be kept on
ice. Specimens of sputum may be collected in clean bot-
tles tightly corked. They should be examined as soon as
possible. Although decomposition appears not to interfere
with the staining properties of the tubercle bacilli, the spu-
tum should be fresh in order that the other bacteria con-
tained in it may be studied. Therefore it should be free
from contamination with putrefactive germs. Valuable
information can also be obtained by examination of spu-
tum in a fresh condition before staining (see also page 44).

Samples of urine keep better after the addition of a few
crystals of thymol, which retards the fermentative process,
so that the sedimentation of the bacteria and of other solid
matter in conical vessels is facilitated, although that pur-
pose can be accomplished at once by the centrifuge.
Thymol will also be a useful addition, as far as a bacterio-
logical examination is concerned, in case samples of urine
are to be sent by mail; thymol should not be added if cul-
tures are to be made.

Specimens of sputum, pus or blood may be collected con-
veniently in the form of thin smears upon cover-glasses.
The smears are fixed by passing through the flame three
times. Smears of blood are prepared as follows : Have
two perfectly clean, square cover-glasses. The finger, or
the lobe of the ear, having been carefully washed with

COLLECTION OF MATERIAL.

water, alcohol, and ether, is punctured with a sterilized
needle, and a small drop of blood issues which is wiped
away. The second drop of blood should be taken ; it should
be about the size of a pin's head. No pressure should be
exerted upon the skin. This drop of blood is placed on one
of the cover-glasses. The other cover-glass is laid upon the
first, both being handled with forceps. The drop of blood
becomes flattened out into a thin film. Immediately and
before the blood has had time to coagulate the two are
slipped or slid away from each other in a horizontal plane,
not forcibly pulled apart. The blood, therefore, will be
spread in thin films on the cover-glasses. It is best to place
the cover-glasses so that one does not cover the other ex-
actly, but so that the sides of the one
lie diagonally to the sides of the other,
although their centers coincide (Fig.
37). Films of blood which are to be
examined for the parasite of malaria may
be prepared in this manner. Samples of
blood to be used for the serum reaction ^/

for typhoid fever need to be pretty good-

• ij r ut j u- u u Manner of Placing

sized drops of blood, which may be col- cover-glasses in Mak-
lected on cover-glasses or pieces of un- ing Films of Blood,
sized paper and allowed to dry. To (After Cabot.)
test blood by culture methods, i to 5 c.c. may be drawn
from a vein during life, using a sterilized hypodermic syringe
and all antiseptic precautions. The blood thus taken may
then be used for cultures in various ways. A good method
for general purposes is to empty the syringe quickly into a
flask holding 100 c.c. or more of bouillon or dextrose-
bouillon. The mixture of blood and bouillon should be
placed in the incubator for one to two days. If the bacteria
develop, they may be secured in pure cultures by plating,
and may be studied further, as the occasion requires.

IIO MANUAL OF BACTERIOLOGY.

At autopsies on human subjects plate-cultures should be
made, if possible, directly from the organs. In all cases
organs should be entered by the platinum wire through
burned surfaces. The method of isolation by streaking the
platinum wire containing the material under examination
lightly, several times, over the surface of an agar plate, will
be found convenient. At the same time smears should be
made from the organs upon cover-glasses for microscopical
study, and portions of the organs should be saved and hard-
ened in alcohol.

A convenient device for the collection of infected mate-
rial is a stiff wire wound with a pledget of absorbent cotton
at one end, the whole sterilized in a tube, as recommended
by Warren for collecting pus and other fluids for examina-
tion, and as introduced by W. H. Park for the collection
of material from the throat in cases of suspected diphtheria
(Fig. 78).

The so-called Sternberg bulb is valuable for the collec-
tion of fluid materials for examination. A short piece of

glass tubing is taken; at one end
FIG. 38. is blown a bulb ; the other end is

drawn out to a long, fine point.
To introduce the substance into
the bulb, the expanded end is
Sternberg Bulb. heated in the flame ; the point is

broken and introduced below the

surface of the fluid which is to be collected; as the bulb
cools, the air in it contracts and draws the fluid into it.
When it has taken up as much as it will, the point may
again be closed in the flame.

When infectious material is to be transported, it should
be so packed that breakage or leakage is impossible.

Concerning the transmission of materials containing bac-
teria in the mails, the ruling of the post-office department
of the United States, March 2, 1900, is as follows:

COLLECTION OF MATERIAL. Ill

"That the order of the Postmaster General of December 27, 1897
(Order No. 677), amending Order No. 88 of February 5, 1896, prescrib-
ing the conditions under which specimens of diseased tissues may be
admitted to the mails is hereby further modified in the following
manner :

" Specimens of diseased tissues may be admitted to the mail for trans-
mission to United States, State, or municipal laboratories, only when
enclosed in mailing packages constructed in accordance with the specifi-
cations hereinafter enumerated : Liquid cultures, or cultures of micro-
organisms in media that are fluid at the ordinary temperature (below
45° C. or 113° F.) are unmailable. Such specimens may be sent in
media that remain solid at ordinary temperatures.

" Upon the outside of every package shall be written or printed the
words ' Specimen for Bacteriological Examination. This package to be
treated as letter mail.' No package containing diseased tissue shall be
delivered to any representative of any of said laboratories until a permit
shall have first been issued by the Postmaster General certifying that
said institution has been found to be entitled, in accordance with the
requirements of this regulation, to receive such specimens."

The regulation includes not only cultures but " moist
specimens of diseased tissues." The specifications prescrib-
ing the manner of packing, which are minute and compli-
cated, may be obtained from local postmasters.

112 MANUAL OF BACTERIOLOGY.

CHAPTER VIII.

SYSTEMATIC STUDY OF SPECIES OF BACTERIA.1

IN order to conduct the study of any species of bacteria
it is necessary to have the organism isolated in a pure cul-
ture by the plate-method, or by some other method already
described. Having thus obtained the organism in pure
culture, it is to be examined with reference to its behavior
in certain particulars. It is well for the beginner to study
a few known species of saprophytes obtained from some
reliable laboratory in pure culture. The points which are
to be considered can be illustrated best by presenting them
in tabular form, filling out the items of the table for a
given species of bacteria.

1. Name.

2. Habitat or source.

3. Morphology; grouping, as in chains or in zoogloe^e.

4. Size.

5. Staining proporties. Behavior by Gram's Method.

6. Capsule, present or otherwise.

7. Spore formation.

8. Motility, flagella.

Growth on culture-media.

9. Relation of growth to temperature.

10. Gelatin; observe whether the gelatin is liquefied or
not. Colonies in gelatin plates, study under low
power of microscope.

1 For the identification of unknown species consult "A Manual of
Determinative Bacteriology," by Frederick D. Chester.

SYSTEMATIC STUDY OF SPECIES OF BACTERIA. 113

11. Agar. Colonies in agar plates, study under low

power of microscope.

12. Bouillon, note cloudiness, pellicle, or precipitate.

13. Milk; observe whether or not the milk is coagu-

lated and subsequently peptonized.

14. Production of gas in fermentation-tube with bouillon

containing sugar, as dextrose, or in agar with
sugars.

15. Potato.

1 6. Blood-serum; observe whether or not peptonization

occurs.

17. Production of indol.

1 8. Pigment formation.

19. Production of acid or alkali.

20. Relation to oxygen; observe whether the superficial

or the deep part of the growth is the more luxu-
riant in stab-cultures; use anaerobic methods if
necessary.

21. Pathogenesis.

In commencing the study of bacteriology the pupil should
try the common staining methods and make the most im-
portant culture-media. Having culture-media prepared, it
is customary to study a number of species of non-pathogenic
bacteria. Notes of the work and sketches showing the mor-
phology of the organisms should be made. It is well to
choose species which have properties decidedly different
from one another. The micrococci, bacilli and spirilla
should be represented; forms that are motile and that are
not; species that form spores and others that do not form
spores; some that liquefy gelatin and some that do not.
There should be chromogenic forms, and species that fer-
ment dextrose, and that produce indol, — such species as
some of the sarcinse, the bacillus coli communis, the hay
bacillus, the potato bacillus, bacillus prodigiosus, a bacillus

114 MANUAL OF BACTERIOLOGY.

fluorescens and spirillum rubrum. It is well, when possible,
to obtain material directly from nature rather than from
laboratory cultures. This may readily be done in the case
of the hay bacillus and the potato bacillus. Fecal matter
may be spread on gelatin plates and the bacillus coli com-
munis obtained in pure culture. Fluorescing bacilli are very
common in water. Large spirilla are often found in swamp
water. Some organisms like spirillum rubrum can only be
had from laboratory cultures. The growth of some aerobic
organism, like the potato bacillus, may be tested under a
cover-glass (see Fig. 29). The pyogenic bacteria, which
can easily be isolated from pus, may be studied in this con-
nection with great advantage. The staphylococcus pyogenes
aureus and the streptococcus pyogenes should on no account
be omitted. The diplococcus of pneumonia can most readily
be obtained from a mouse or a rabbit which has died with
pneumococcus infection. Such an animal can best be in-
fected by subcutaneous inoculation, using some of the rusty
sputum of a case of lobar pneumonia. The cultivation of
the pneumococcus will be found to present difficulties in
classes containing large numbers of students.

Representative forms of moulds and yeasts should be
studied at the same time. Moulds are easily obtained by
exposing some nutrient substance to the air, covering it,
and allowing cultures to develop ; yeasts will probably grow
also. Ordinary brewer's yeast may be isolated in pure cul-
ture from gelatin plates. Bacteriological examinations also
should be made of air, soil, water and milk. With such
simple means, all the important properties of bacteria may
be demonstrated.

Experiments in sterilization and disinfection as described
in Chapter VIII. , Part II. , may be performed with the bac-
teria mentioned, which present every variety of resisting
power up to the almost incredible toughness of the spores of
the hay and potato bacilli. The efficiency of the methods

SYSTEMATIC STUDY OF SPECIES OF BACTERIA. 115

used for sterilizing surgical materials, as silk and catgut
(Chapter IX., Part II.), should be tested; also, of the
methods for disinfecting the hands; if possible, of the
methods for disinfecting rooms, as well.

After some proficiency has been acquired, various patho-
genic bacteria may be studied as the circumstances of the
case require. Much judgment has to be used in allowing stu-
dents to work with pathogenic bacteria. Anthrax, glanders,
tetanus, cholera, bubonic plague, Malta fever, and diphtheria
all have occurred in laboratory workers through accidental
infection, sometimes with fatal results. The various rules
for the management of the platinum-wire, hanging-drop
slides and sputum bottles, and for the handling of cultures
and other infectious materials have already been given
(pages 33, 36, 44 and 84 to 88). The most important pre-
caution, perhaps, is observance of the rule that while work-
ing in the laboratory, nothing should be put in the mouth.
Cultures should never be carelessly left in improper places.
Cultures of bacteria should be thoroughly sterilized before
the tubes are cleaned. The writer is in the habit of having
tubes and dishes containing pathogenic bacteria placed in the
steam sterilizer for an hour on each of three days, and of
having the plugs removed and burned and the tubes filled
with 5 per cent, carbolic acid between the second and third
sterilizations. In taking these measures, the same kind of
reasoning applies as that which induces engineers to give
bridges from four to six times the strength they need to
bear the greatest strain likely to be put upon them, or to
make the boiler of a steam engine strong enough to bear
six times the greatest pressure which it is expected that
the steam contained in it will exert.

PART II.

CHAPTER I.

CLASSIFICATION ; GENERAL MORPHOLOGY AND PHYSIOLOGY
OF BACTERIA.

THE relationships existing between bacteria and other
kinds of organisms are not perfectly clear. It is quite
generally conceded, however, that bacteria are plants. They
show affinities with both the lower algse and the lower fungi,
and they have even some points of resemblance with certain
of the protozoa. On account of their extreme smallness it
is impossible to analyze the structure of the individual bac-
teria and to contrast the structure of one with that of
another. The classification cannot therefore be established
on morphological grounds chiefly, as is done with large
animals or plants. We are obliged to rely also upon their
growth with relation to the presence or absence of oxygen
and to temperature, their behavior on culture-media, the
appearances of the growths, and the production of certain
substances with peculiar chemical reactions, when we wish
to establish the points of difference between one species and
another — all of which is extremely unsatisfactory and prob-
ably not perfectly reliable. It is likely that forms which are
now considered as different species are not really such in all
cases, and also that different species may now be included
under one heading as a single species. Notwithstanding the
unsatisfactory condition of the classification of bacteria, it
must not be supposed that the species of bacteria are not

117

Il8 MANUAL OF BACTERIOLOGY.

permanent. For instance, it would be incorrect to imagine
that it is possible for the micrococci and spirilla to become
converted into species of bacilli, or for the bacilli of one
species to be transmuted into those of another. This does
not contradict the statement that we may frequently, through
erroneous and imperfect information, be in the habit of
including unlike species under one name, or of classifying
mere varieties of one species as entirely different species. At
present the simple division of bacteria into three great
generic groups is probably as good as any : micro cocci,
spherical forms; bacilli, rod-shaped forms, one diameter
being in excess of the others; spirilla, twisted like a cork-
screw, making long spirals or simply parts of spirals
( comma-shaped forms ) .

Recent investigations indicate that several species of bac-
teria often are closely related to one another, so as to form

FIG. 39.

Staphylococci. Streptococci. Diplococci. Tetrads. Sarcinae.

a well-marked group. Such a group is constituted by the
bacillus of typhoid fever, bacillus coli communis and similar
forms. The spirillum of cholera and other comma-shaped
spirilla resembling it may be held to constitute another
group. Still another is that containing the tubercle
bacillus and other acid-proof bacilli.

The micrococci are subdivided into Staphylococci, where
the spheres grow in clusters like a bunch of grapes; strep-
tococci, where they are arranged in long rows or chains,
like a string of beads ; diplococci, or pairs of micrococci ;
tetrads, where the individual spheres are grouped in fours;
sarcincr, where they are grouped in eights, making the out-
line of a cube, resembling a bale or package tied with rope.

MORPHOLOGY AND PHYSIOLOGY OF BACTERIA. 1 19

The bacilli are not usually subdivided in this manner,
although their forms vary considerably. The ends are
sometimes square, sometimes round. Sometimes they are
very short. Sometimes they grow in longer, thread-like
forms, in which, however, the transverse markings which

FIG. 40.

»« -&

Bacilli of Various Forms.

indicate the outlines of the individual bacilli can generally
be seen, and which resemble a bamboo rod. Short oval
bacilli may look exceedingly like micrococci. Bacilli with
rounded extremities, placed end to end, look like strings of
sausages. Under exceptional circumstances, branching
forms of the bacilli of diphtheria, tuberculosis, glanders and
bubonic plague and various other species have been en-
countered.1

The word ce bacterium " was formerly used to designate
short bacilli which generally formed no spores, while the

FIG. 41.

Spirilla of Various Forms.

word bacilli was restricted to the longer forms in which
spore formation occurred. This use is no longer common,
although not rarely the name bacterium is still given to a
species — for instance, bacterium coli commune.

1 See Hill, Journal of Medical Research, Vol. VII., January, 1902;
Loeb, ibid., Vol. VIII., 1902.

I2O MANUAL OF BACTERIOLOGY.

Spirilla present a very great variety of form. The short
{f comma-shaped bacilli " are only parts, at most, of spirals,
although the microbes of cholera do sometimes form long
spirals. On the other hand, there are among spirilla large
and long sinuous figures which present most remarkable
pictures under the microscope; for example, the spirillum
of relapsing fever. Formerly spirilla without very marked
windings were called " vibrios " ; and long, wavy forms
with corkscrew-like windings " spirochcetcu " ; and the
rigidly spiral forms were denominated " spirilla." These
definitions have for the most part lost their significance,
although the names still linger in nomenclature.

FIG. 42.

Involution Forms of the Spirillum of Cholera. (Van Ermengem.)

Besides the classification already mentioned, bacteria are
sometimes grouped according to certain other qualities. In
general botany, saprophytes are plants that grow on decay-
ing vegetable matter. In a bacteriological sense, sapro-
phytes are bacteria which grow in external nature on dead
organic matter, and parasites are bacteria which exist upon
the living tissues or fluids of any organism. Nearly synon-
ymous with the above words are those which do not and
those which do produce disease, or non-pathogenic and

MORPHOLOGY AND PHYSIOLOGY OF BACTERIA, 121

pathogenic. The adjectives facultative, or optional, and
obligate, or strict, are used to qualify the above terms and
many others.

Size. — Bacteria vary greatly in size. The micrococci are
usually i [J. or less in diameter. The short diameters of
bacilli and spirilla also are less than i fJ. as a rule, while
the length may be several p. . The anthrax bacillus (1.5 A*
X 3 to 10 p) and the spirillum of relapsing fever are the
largest bacteria known to be pathogenic to man. To say
that a micrococcus is i p. in diameter means that 25,000
end to end would make a line I inch long. It has been
estimated that I milligram of a pure culture of the staphy-
lococcus pyogenes aureus contains 8,000,000,000 micro-
cocci.

There is good reason for believing that organisms exist,
which are too small to be visible with the most powerful
microscopes. The nature of these organisms is not known,
but it is not improbable that some of them are bacteria.
(See pleuro-pneumonia of cattle, etc., Part II., Chapter V.)

In stained preparations the bodies of bacteria frequently
seem to be homogeneous. On the other hand, they may
exhibit certain spots which stain more intensely than others,
the stained spots alternating with clear areas. The dark-
staining granules may take a slightly different shade of color
from the rest (metachromatic granules, Babes-Ernst
bodies). Somewhat similar appearances may result from
changes in the density of the protoplasm of bacteria, leaving
vacuoles that do not stain (plasmolysis).

In old cultures bacteria are likely to show deformed and
twisted outlines called involution forms. It is not uncom-
mon for bacteria to be enclosed in a kind of envelope of
some clear substance, which stains with difficulty or not at
all, called a capsule. The paired micrococci of pneumonia
are inclosed in such capsules. The capsule is more likely to

122 MANUAL OF BACTERIOLOGY.

be demonstrated when the bacteria are obtained from the
fluids derived from an animal's body than when they have
been grown artificially in culture-media. A zoogloea is a
large mass of bacteria in a resting condition held together

by a mucilaginous substance. The

F IG. 43.

composition of bacteria varies con-

<*?> I) siderably with different species.

The basis aPPears to be Proteid
substance.

T*16 multiplication of bacteria

Bacteria with Capsules. takes Place in almost all CaSCS by

transverse fission. The formation

of tetrads or sarcinae from micrococci depends upon fission in
two or three planes. Repeated fissions of micrococci in one
plane result in the formation of streptococci. Micrococci
that have recently divided are likely to be somewhat flat-
tened. Multiplication under favorable circumstances may
take place at a phenomenally rapid rate. Bacilli have been
observed to divide in twenty minutes. If division takes
place once in an hour, the progeny of one organism at the
end of twenty- four hours will be 16,777,216, i. e., (2 X i)24-
The ordinary form of reproduction by fission is called
vegetative, and bacteria that are multiplying in this man-
ner are often spoken of as being in the vegetative condi-
tion.

FIG. 44.

);

Bacteria with Spores.

Spores. — Under certain circumstances the reproduction
of bacteria takes place by means of bodies called spores.

MORPHOLOGY AND PHYSIOLOGY OF BACTERIA. 123

They appear in a typical form in the large bacilli, where,
near the centers of the bacilli, highly refracting, shining
spots may be seen which are found to stain less rapidly
with the "aniline dyes than the rest of the bacilli. They
are not to be confused with the unstained spots described
as vacuoles. On account of their being formed from a
part of the interior of the bacterium, such spores are called
endogenous. These spores are found mostly in the bacilli,
rarely in spirilla and micrococci. They are what is meant
when the word spore is used alone without qualification.
The existence of another kind of spore, described as form-
ing from the whole of the bacterium (called arthrospore) ,
is doubtful. At all events, its significance is not at present
understood. Spores develop generally, though not always,
under adverse conditions of various kinds, as of tempera-
ture and of nutrition. They are more resistant to unfavor-
able influences of all sorts than are the fully developed
bacteria. Spores resist drying, light, heat and chemical
agents to a remarkable degree, at times.

Anthrax spores are said to have been found which could
withstand steam for twelve minutes, i-iooo mercuric chlo-
ride for nearly three days, or 5 per cent, carbolic acid for
more than forty days. The greatest resistance is displayed
by the spores of some of the saprophytic bacteria, particu-
larly those of hay and potato, which are sometimes not
destroyed by several hours of steaming; and bacteria are
said to have been obtained from the soil which resisted
100° C. for sixteen hours. When cultivated at a tempera-
ture as high as 42° C. the anthrax bacillus becomes inca-
pable of forming spores. Spores themselves do not mul-
tiply, nor do they manifest any activity. Spores may be
located at the center of the bacillus, or nearly at one end,
when the end of the bacillus is likely to enlarge, making
a form having the shape of a drumstick, as takes place
ii

124 MANUAL OF BACTERIOLOGY.

with tetanus bacilli (Fig. 44). When a bacillus assumes a
spindle shape on account of having the middle part bulged
through the formation of a spore it is called a clostridium.
With rare exceptions, a single bacillus contains but one
spore. Under favorable conditions the spores germinate, as
it is called, and develop to the adult form of the organism.
This may be witnessed in hanging-drop preparations.

Motility. — Motility is rarely exhibited by micrococci ;
some bacilli possess it and some do not; while nearly all
of the spirilla are motile. The phenomenon is observed in
the hanging-drop. The degree of motility is variable, being

FIG. 45.

Bacteria showing Flagella.

sometimes slight and sometimes very active. When seen
under a high power the little particles taken from a cul-
ture of a motile organism may look like a writhing mass
of maggots or like tadpoles in a pool. The motility is most
active in young cultures. The movement results from the
vibration of little processes, or flagella (Fig. 45). Of these
there may be one or several. They may be placed singly
or in groups, at the end, or scattered around the sides. They
are extremely difficult to demonstrate except by special stain-
ing methods, which, furthermore, are decidedly capricious.
After the flagella have been stained, the bacteria appear
somewhat larger than when stained by the ordinary
methods. The flagella upon the bacilli of typhoid fever are
numerous and form a very striking picture.

Chemotaxis. — Motile bacteria possess the property of
being attracted by certain substances (positive chemotaxis)

MORPHOLOGY AND PHYSIOLOGY OF BACTERIA, 125

and of being repelled by others (negative chemotaxis).
Similar properties are widely distributed among living
cells, both animal and vegetable.

CONDITIONS FAVORABLE FOR THE GROWTH OF
BACTERIA.

Warmth. — Among the different kinds of bacteria forms
are said to exist which multiply at temperatures as low as
o° C, while there are species that multiply at 70° C. Bac-
teria which flourish at a very high temperature (maximum
about 70° C.) are called thermophilic. The pathogenic
bacteria usually flourish better at a point somewhere near
the temperature of the human body. This is not necessa-
rily the case with the non-pathogenic species. Ordinary
water bacteria thrive better at ordinary temperatures.

Sternberg's method for determining the thermal death-
point of a species of bacteria is to draw portions of a pure
culture of the organism into capillary tubes with expanded
ends, when the tubes are sealed in the flame. The tubes
are supported upon a glass plate placed in a water-bath,
whose temperature is indicated by a thermometer, while a
uniform temperature is secured by stirring. The time of
exposure is, as a rule, ten minutes. The tubes should be
removed quickly to cold water. Their contents should
afterwards be inoculated into bouillon to determine whether
or not the organisms have been killed.

Moisture is indispensable to the growth of bacteria, and
drying causes the death of certain kinds, as, for instance,
the spirillum of cholera.

Food. — There are a few species of bacteria that contain
chlorophyll, but it is wanting in most forms. On account
of the absence of chlorophyll, bacteria require, as part of
their food, organic compounds containing carbon, such as
sugar. They are unable, with possibly a very few excep-

126 MANUAL OF BACTERIOLOGY.

tions, like the nitrifying bacteria, to derive their carbon
from the carbon dioxide of the atmosphere, or from inor-
ganic carbon compounds. Although some species are
able to obtain nitrogen from inorganic salts, most bacteria
flourish best if organic substances containing nitrogen, like
peptone and albumen, are furnished them as part of their
food. The complicated, unstable, organic molecules with
high potential energy are converted by them into simple
and more stable compounds like carbon dioxide, ammonia
and water, with the liberation of energy. These facts be-
come manifest in connection with their important work in
decomposition, putrefaction and fermentation. A culture-
medium having a slightly alkaline or neutral reaction is
favorable to most bacteria.

The prolonged artificial cultivation of bacteria may or
may not modify their properties. The pathogenic bacteria
are likely to undergo considerable modification both in the
quality and luxuriance of their growth and the intensity of
their pathogenic characters.

The growth of bacteria may eventually be hindered by
the accumulation of the products of their own metabolism.
Many bacteria refuse to grow on culture-media at all. Some
species are extremely fastidious, and can only be propagated
on particular sorts of nutrient substances.

Relation to Oxygen. — Oxygen is indispensable to the
growth of some bacteria, aerobes. Its absence is equally
indispensable to certain others, anaerobes. Others still are
able to flourish either in the presence or absence of oxygen,
facultative aerobes or anaerobes. The first-named varieties
are sometimes called strict, or obligate aerobes or anaerobes.

Effects of Sunlight. — Direct sunlight kills the- vegeta-
tive forms of bacteria more or less rapidly, and constitutes
one of the most efficient among the natural methods of dis-
infection. Diffuse daylight acts much more slowly. Elec-

MORPHOLOGY AND PHYSIOLOGY OF BACTERIA. 127

trie light acts like sunlight or daylight, the results being de-
pendent on the intensity of the light. The violet part of the
spectrum is most active.

The influence of electricity upon bacteria has not yet been
fully studied. Apparently the destruction of bacteria re-
ported as having been effected by electricity was the result
of electrolysis of the medium.

It appears probable that X-rays do not produce impor-
tant effects on bacteria, although further investigation of
this subject is needed. The success which has attended the
use of light rays and X-rays in the treatment of lupus and
other diseases is not necessarily to be explained as the result
of bactericidal action of the rays.

128 MANUAL OF BACTERIOLOGY.

CHAPTER II.

PRODUCTS OF THE GROWTH OF BACTERIA.

Phosphorescence. — Bacteria whose cultures exhibit phos-
phorescence have been found in the ocean and in fish.

Chromogenic Bacteria. — Many bacterial growths display
brilliant coloring. The different species of sarcinse are
remarkable for forming highly-colored growths; some of
them are rose-red, some orange-yellow, some lemon-yellow,
and so on. The bacillus prodigiosus presents a brilliant red
growth whose rapid development is said to have formed the
basis for the so-called " Miracle of the Bleeding Host " (see
page 15). The bacillus pyocyaneus in culture gives a bril-
liant green fluorescence and is responsible for the color of
blue or green pus.

Bacilli which exhibit a green fluorescence in cultures are
common in water. In cultures on potato or agar the colors
of the chromogenic forms are usually well shown.

Ferments or Enzymes.1 — Many bacteria form ferments
which have the power of dissolving proteid substances in
a manner similar to trypsin. The liquefaction of gelatin
is a familiar example of this process. The property of
liquefying gelatin, or otherwise, is used in classifying bac-
teria and in determining the nature of unknown species.

Some bacteria, as the bacillus coli communis, form fer-
ments which act like rennet in coagulating milk. Other
bacteria are capable of forming sugar from starch. Others
have the power of changing cane-sugar into glucose.

1 Consult Buxton, " Mycotic Enzymes," American Medicine, July 25,
1903-

PRODUCTS OF THE GROWTH OF BACTERIA. 129

Bacteria which are able to decompose cellulose are found
in the stomachs of ruminant animals. Although it is doubt-
ful whether the products of cellulose decomposition have
any nutritive value, the process is useful in effecting a sub-
division of the coarse food, consisting of grass, hay, and
the like.

Some bacteria have the power of decomposing neutral
fats into fatty acids and glycerin, after the manner of the
fat-splitting ferment of the pancreatic juice.

The end-products which result from the growth of bac-
teria upon albuminous nutrient media are very numerous.
They are complicated and not well understood. Among
these end-products may be mentioned peptone, indol, skatol,
phenol, leucin and tyrosin. Nearly related are the toxins,
which play an important part in the production of disease
by pathogenic bacteria. In the decomposition of urine by
bacteria the urea is converted into ammonium carbonate.

The formation of indol in cultures is an important pecu-
liarity of certain bacteria, which may be tested as follows :
The bacteria are cultivated in Dunham's peptone solution or
in dextrose-free bouillon; after twenty-four to forty-eight
hours the test may be made. Add ten drops of concentrated
sulphuric acid; the development of a rose-color indicates
the presence of both indol and nitrites. If no rose-color
forms, to another tube add, first i c.c. of a o.oi per cent,
solution of sodium nitrite, and then the sulphuric acid. The
development of a rose-color indicates the formation of indol
but not of nitrites. If there is no rose-color, no indol has
been formed. The color appears usually in a few minutes,
but it may only develop after a somewhat longer time. Con-
trol tests must be made upon tubes of the same peptone
solution but which have not been inoculated. The reaction
may be hastened by warming slightly. The value of this
reaction will be understood when, to give one illustration.

130 MANUAL OF BACTERIOLOGY.

it is remembered that the bacillus coli communis produces
indol and the bacillus of typhoid fever usually does not.
The reaction depends upon the liberation of nitrous acid,
which, with indol, forms a red color.

The change of organic substances into more stable ones
does not cease with the compounds mentioned above. Cer-
tain bacteria of the soil which will be discussed further on
are able to complete the conversion of ammonia into nitrous
acid (leading to the formation of nitrites) ; and others still
that of nitrites into nitric acid, which at once forms nitrates,

Formation of Acids. — In the course of their growth
many bacteria produce acids, especially from substances
containing sugar. The power of developing lactic acid is
possessed by a large number of species. Acetic acid is
another common by-product. Besides these, butyric acid,
formic acid, propionic acid and many more are formed by
different bacteria.

Development of Gas. — The evolution of gas from bac-
terial growths is of frequent occurrence. Carbon dioxide,
hydrogen sulphide and nitrogen are among the better
known gases that may be formed. The odors that arise
from cultures and that are so characteristic of putrefactive
processes depend upon the development of gases, or a mix-
ture of gases, of considerable complexity. The bacillus
aerogenes capsulatus leads sometimes to the formation of
gas in the organs of the human cadaver within a short time
after death.

T. Smith considers the formation of gases in media con-
taining sugar of importance in discriminating between dif-
ferent species. Bouillon containing i per cent, of dextrose
(or lactose, etc.) is the culture-medium advised. The
test is best conducted in a U-shaped tube, closed at one end,
and at the other end provided with a bulb (Fig. 46). The
tube is stoppered with cotton, sterilized by dry heat, after-

PRODUCTS OF THE GROWTH OF BACTERIA. 13!

ward filled with the bouillon, and sterilized by steam in the
usual manner. After the last sterilization it should be tilted
until the closed end is completely filled with the medium.
After it has been inoculated with the species under con-
sideration, any development of gas will be indicated by the
collection of the gas at the closed end. The amount of gas
formed may be estimated and its FlG

quality tested. To accomplish the
latter fill the bulb with 2 per cent,
solution of sodium hydroxide,
close the outlet, and tilt the tube
to allow the mixture to come in
contact with the gas. After
shaking, this causes the absorp-
tion of the carbon dioxide and
diminution in the quantity of
gas. The portions which remain
may be mixed with air and
ignited, when the presence of
hydrogen and some of its com-
pounds will be indicated by an
explosion. (See The Detection
of Bacillus coli communis in

,T. Fermentation-tube.

\Vater, Part II., Chapter III.)

The development of gas may readily be tested by inocu-
lating the bacteria by a deep puncture into agar containing
i per cent, of dextrose or other sugars. The development
of gas causes bubbles to form in the agar, often to the extent
of splitting it, and sometimes forcing out the cotton plug
(see Fig. 68).

The activities of bacteria which have just been enumer-
ated are fundamental to the phenomena which go by the
names of fermentation and putrefaction. These words
have been defined differently at different times and by dif-
ferent writers, but in general both are used as names for

132 MANUAL OF BACTERIOLOGY.

the breaking up of complex organic compounds by micro-
organisms with the formation of simpler compounds. Fer-
mentation refers especially to the formation of useful prod-
ucts like alcohol. The term putrefaction is employed chiefly
for the breaking up of nitrogenous compounds with the
development of foul-smelling gases. The term fermentation
is also applied to the decomposition of complex substances
through the influence of unorganized ferments or enzymes.

The work of bacteria in fermentation and putrefaction is
indispensable to the existence of the organic world as we
find it. Green plants convert the stable compounds of nitro-
gen, the carbon dioxide of the atmosphere, and water into
the complex and unstable albumins and carbohydrates which
serve as food for animals. Animals, on the other hand, con-
vert these unstable and complex compounds back into simpler
forms. The work of changing them back into the simple
and stable condition, in which they serve as the food for
plants, is performed by animal life in part only, and its com-
pletion is left to the activities of bacteria. It is the work of
bacteria in this direction which we call fermentation and
putrefaction. Without that work, as we understand it, the
existence of life upon the earth would soon come to an end,
and the dead and undecomposed bodies of living things and
their products of all kinds would lie about unchanged, as
they had fallen.

Bacterium tcnno is the name formerly given to a sup-
posed species of bacteria which was credited with being
the producer of putrefaction. The individuals were rep-
resented as being short rods, mostly going in pairs, and
actively motile. The term has been abandoned since it
appears to have included a number of different species.

DISTRIBUTION OF BACTERIA. 133

CHAPTER III.

DISTRIBUTION OF BACTERIA.

The Bacteria of the Soil. — Bacteria are present in the
soil in enormous numbers — 100,000 or more in i c.c. of
virgin soil, according to Fliigge. The depths to which
they penetrate will depend upon the character of the soil
and the character of the life upon it, and whether or not it
has been artificially disturbed, as by cultivation. In gen-
eral, at a depth of 1.25 meters (about four feet) the num-
ber will have become very small, and a little deeper the soil
will be entirely sterile.

The bacilli of tetanus and malignant edema, and bacillus
aerogenes capsulatus are present in the soil of many locali-
ties. According to Woodhead, certain savage tribes of
Africa and the East Indies use as an arrow-poison soil that
is capable of producing tetanus. The bacillus of anthrax
may be found in soil which has been infected with this
organism.

Most of the bacteria of the soil are harmless or useful
saprophytes.1 The nitrifying bacteria described by Wino-
gradsky and by Jordan and Richards belong to the latter
class. There occur in soil organisms which have the power
of converting ammonia into nitrous acid which forms
nitrites, and others which complete the change of nitrites
into nitrates. Both varieties are widely distributed. These
organisms will not grow on ordinary culture-media, and
their cultivation presents great difficulties. Probably a good
many bacteria have similar properties to some extent. The

1 See Conn, " Agricultural Bacteriology."

134 MANUAL OF BACTERIOLOGY.

work done by nitrifying bacteria in making nitrates from
sewage, manure and the like is indispensable to most plant
life. Bacteria have also been credited with the assimilation
of free, atmospheric nitrogen, resulting in the addition of
a valuable proportion of nitrogen compounds to the soil.
This is spoken of as nitrogen -fixation. Inasmuch as a large
part of the excrementitious products of animals containing
nitrogen are not retained in the soil, where they may be
employed as food by plants, but are washed directly or in-
directly into the sea by means of sewage and the rivers, it
will be seen that the supply of nitrogen compounds might
be in a way to suffer gradual exhaustion. Furthermore, it
has already been noticed (page 130) that one of the products
of decomposition by bacteria is nitrogen, which is not avail-
able to animals and most plants as food. These facts have
met with practical recognition by agriculturists in the adop-
tion of various methods of fertilizing the soil. It appears
that the roots of peas, beans, clover, alfalfa and some other
plants frequently present minute tubercles. These tubercles
are pathological growths, caused by the development of
microorganisms related to the bacteria. The organisms
appear to have the power of assimilating atmospheric nitro-
gen and of converting it into nitrogen compounds. Experi-
ments show that these observations may be destined to be of
great value to the farmer.1

The bacteria of the soil may easily be studied in plate-
cultures made from small portions of soil collected with the
necessary precautions to avoid contamination, or plate-cul-
tures may be made from sterilized water with which a por-
tion of the soil has been properly mixed. Anaerobic bac-
teria must be cultivated by the special methods adapted to
them.

1 For simple experiments to illustrate these phenomena see Buxton,
Journal of Applied Microscopy, September, 1902.

DISTRIBUTION OF BACTERIA. 135

Bacteria of the Air.— The bacteria of the air will be
found for the most part clinging to solid particles in suspen-
sion in the shape of dust. As has already been stated,
bacteria will not rise from moist surfaces unless forcibly
removed, as by agitation or currents of air. Conditions of
dryness and wind tend to increase the number of micro-
organisms in the air. They are fewer after a fall of rain
or snow, and the number is smaller in winter than in sum-
mer. The air of cities contains more germs than that of
the country. The atmosphere over the sea and at the tops
of high mountains is nearly or wholly free from germs.
The bacteria which do occur in the air will seldom be
pathogenic. Their character will depend upon the character
of the dust. It is obvious that dust which consists in part
of the dried, pulverized expectoration of cases of pulmonary
tuberculosis may contain tubercle bacilli. Anthrax of the
lungs sometimes arises in men who handle the wool of
sheep that were infected with anthrax (Wool-sorter's dis-
ease) , and is due to the inhalation of anthrax spores attached
to the wool. It is likely that the atmosphere in the imme-
diate vicinity of cases of the exanthematous fevers may
contain the organisms, whatever they may be, that cause
these diseases.

In a rough way one may obtain some knowledge of the
character of the organisms in the air of a given locality by
removing the cover of a Petri dish containing sterilized
gelatin or agar for a few minutes, replacing it, and allow-
ing the organisms to develop. In most cases a large pro-
portion of the growths that appear will be moulds. Yeasts
are also common, and among the bacteria the micrococci
are abundant. Chromogenic varieties are likely to be
present.

A few studies of this character will show that the num-
ber of organisms that are present depends chiefly upon

136 MANUAL OF BACTERIOLOGY.

whether the air is quiet or has recently been disturbed by
draughts, gusts of wind, or sweeping. These facts are of
fundamental importance in laboratory work, where plate-
cultures are being studied, if we wish to avoid contamina-
tion of the plates. Among various devices that have been
proposed for the accurate study of the organisms of the
air, the Sedgwick-Tucker aerobioscope is the simplest and
most accurate. It consists of a glass tube, one end of
which is drawn out so as to be smaller than the other.
The small end contains a quantity of fine granulated sugar ;
both ends are plugged with cotton, and the instrument is

FIG. 47-

Sedgwick-Tucker aerobioscope.

sterilized. A definite quantity of air is to be aspirated
through the large end, after removing the cotton, which
may be done by means of a suction-pump applied to the
other end, or by siphoning water out of a bottle the upper
part of which is connected with the end of the aerobioscope
by means of a rubber tube. The sugar acts as a filter and
sifts out of the air the microorganisms which are contained
in it. Liquefied gelatin or agar may be introduced into
the large end of the instrument by means of a bent funnel ;
and, after replacing the cotton, it may mix with the sugar
which dissolves. The culture-medium may be spread
around the inside of the larger portion of the tube after the
manner of an Esmarch roll-tube. The bacteria which
were filtered out by the sugar will develop as so many
colonies upon the solidified medium.

Bacteria of Water and of Ice. — The water of rivers,
lakes and the ocean always contains bacteria. The num-

DISTRIBUTION OF BACTERIA. 137

her of organisms varies greatly in different places and
under different conditions. The number of different spe-
cies found in water is also very large. Ground-water1
contains few or no bacteria under normal conditions, and
is therefore suitable for a source of water-supply, when a
sufficient amount is available. The possibility of contami-
nation of the ground- water from unusual or abnormal con-
ditions should always be eliminated before it is taken for
drinking-water. Numerous epidemics of typhoid fever have
been traced to contamination of wells. The location of wells
with reference to privy-vaults and other possible sources of
contamination should be chosen with the greatest care.

The ordinary bacteria of water are harmless, as far as
is known.2 Bad odors and tastes in drinking water that is
not polluted with putrid material are usually due to minute
green plants (algae).3 The diseases most commonly dis-
seminated by water are typhoid fever and Asiatic cholera,
and probably also dysentery. The spirillum of cholera will
usually die in natural water (not sterilized water) inside
of two or three weeks ; the bacillus of typhoid fever will usu-
ally die in two or three weeks. Under exceptional circum-
stances these organisms may perhaps maintain their vitality
for a longer period. They appear, however, to be less
hardy than the ordinary water bacteria. As wre now un-
derstand these diseases, the organisms causing them will
be present only in a water-supply which has been contami-
nated by the excreta from a case of the disease. Notwith-
standing the rapid death of these organisms in water, they

1 Ground-water is the water which — originally derived from rain or
snow — sinks through superficial porous strata, like gravel, and collects
on some underlying, impervious bed of clay or rock.

2 See Fuller and Johnson, " The Classification of Water Bacteria,"
Journal of Experimental Medicine, Vol. IV., p. 609.

1 " Contamination of Water Supplies by Algae." G. T. Moore in
Yearbook U. S. Department of Agriculture, 1902.

138 MANUAL OF BACTERIOLOGY.

may exist long enough to infect individuals habitually
drinking the water. Many epidemics of cholera and typhoid
fever have been traced to water polluted with the discharges
from cases of these diseases.

By self-purification of water is meant the removal through
natural processes of contaminating organisms such as might
occur from the discharge of sewage into it. It depends
upon the sedimentation of the contaminating material, in
the form of mud, upon the growth of the ordinary water-
plants and protozoa, upon the exhaustion of the food
supply by the growth of bacteria themselves, upon the
destructive influence of direct sunlight, and the dilution of
the matter added with a large volume of water.1 It is not
usually to be relied upon as a means of freeing the water-
supply from pathogenic bacteria.

Storage of water. AYhen water is kept in large reser-
voirs, the solid particles in it, including bacteria, tend to fall
to the bottom. The number of bacteria in a water-supply
may be considerably reduced in this way.

Filtration. — Filtration on a large scale has been more
commonly in use in the cities of Europe than elsewhere, until
lately. Similar filtration-plants now exist in several cities
of the United States.

Slow Sand Filtration. — The filter consists of successive
layers of stones, coarse and fine gravel. The uppermost
layers are of fine sand. The whole filter is from I to 2
meters thick. The sand should be 60 cm. in thickness. The
upper layers may be removed from time to time, the re-
mainder not becoming less than 30 cm. in thickness. The
first water coming from the filter is discarded. The actual
filtration is done largely by the slimy sediment which collects
on the surface of the layer of fine sand. The filter-beds may
be several acres in extent, and are protected by arches of
brick or stone. They require renewal occasionally. This

1 See Jordan, Journal of Experimental Medicine, Vol. V., p. 271.

DISTRIBUTION OF BACTERIA. 139

kind of filtration has come largely into use since the cholera
epidemic of 1892-93, and it appears to be very effective.

Mechanical Filtration. — This method of filtration is also
called the American system. It is more rapid than the pre-
ceding method and does not require a large area for filter
beds. Although sand is required also, filtration is accom-
plished by a jelly-like layer of aluminum hydroxide. This
product is formed by adding to the water a small quantity
of alum. The carbonates in the water decompose the alum
and produce aluminum hydroxide. It precipitates as a
white, flocculent deposit, entangling solid particles, includ-
ing bacteria, as coffee is cleared with white of egg. Only a
trace of alum should appear in the water. This method of
filtration has not been tested so extensively as slow sand
filtration, but seems likely to prove efficient. With water
poor in carbonates, these may have to be added.

Various methods for the purification of water by means
of chemicals have been proposed. The use of ozone for this
purpose has met with considerable favor.1

The filtration of water on a small scale, as is ordinarily
done for domestic purposes, is generally entirely useless.
The so-called Pasteur filter of unglazed porcelain is effec-
tive if it is properly constructed and if the filter-tubes are
sterilized by heat frequently (every few days) — conditions
which are seldom complied with. Distillation of water, or
thorough boiling will usually be the most practical method
for sterilizing drinking-water.

Collection of Samples. — Samples from the water-supply
of a city may be drawn from the faucet, but the water
should first be allowed to run for half an hour or longer.
From other sources the supply should be collected in ster-
ilized tubes or bottles, taking care to avoid contamination.
Sternberg bulbs (see Fig. 38) will be found useful for

1 Consult " Disinfection and Disinfectants/' Rosenau, 1902.

J4O MANUAL OF BACTERIOLOGY.

small samples. These samples should be examined as
quickly as possible, for the water bacteria increase rapidly
in number after the samples have been collected. When
transportation to some distance is unavoidable the samples
should be packed in ice.

The number of bacteria may be determined by making'
plates of a definite quantity of the water with gelatin or
agar. The amount examined ordinarily is I c.c. When
the number of bacteria is very large, a smaller quantity
must be taken, and it may be necessary to dilute the sample
ten times or more with sterilized water. The amount should
be measured with a sterilized, graduated pipette. The water
is to be mixed with liquefied gelatin or agar in a tube which
has been allowed to cool after melting. After thorough
mixing, remove the plug, burn the edge of the tube in the
flame, hold in a nearly horizontal position until cool, and
pour into a sterilized Petri dish. The number of colonies
may be counted on the third or fourth day; the later the
better, as some forms develop slowly and may not present
visible colonies for several days; but the plates are often
spoiled after three or four days by the profuse surface
growths of certain forms or by the rapid liquefaction of
gelatin, if that be used, by other forms. The number of
colonies that develop is supposed to represent the number
of individual bacteria contained in the quantity measured.
That will probably not always be the case, however, as
colonies may develop from a clump of bacteria which have
not been separated from one another by the mixing process.
Abbott has shown that the number of colonies is usually
larger on gelatin plates than upon agar plates, and at the
room temperature than in the incubator. This observation
illustrates the fact that there are doubtless many kinds of
bacteria that do not find favorable conditions for develop-
ment on ordinary culture-media. The reaction of the

DISTRIBUTION OF BACTERIA. I^-T

medium has an important influence upon the development of
these water bacteria in plate-cultures.

When the number of colonies is small, there is no diffi-
culty in counting them as they appear in the ordinary
Petri dish. When the number is large some kind of me-
chanical device may be used to assist counting. The
Wolffhiigel plate is a large square of glass resting in a
wooden frame painted black. The glass plate is ruled
in squares. It was designed particularly with reference
to the form of plate-cultures first made by Koch. The
Petri dish, however, may be placed upon the glass plate
and the cross lines be used to assist in counting. Lafar,
Pakes and Jeffer recommend a surface painted black, ruled
with white lines which represent the radii of a circle, which
may be still further subdivided by other lines. Many find
counting easier when a black surface divided into squares
is employed. An ordinary card with a smooth black sur-
face divided into squares by white lines may be placed under
a Petri dish and will be found to serve very well. For the
mere examination of the colonies no better surface can be
devised than the ferrotype plate used by photographers.
The examination of the colonies will be easier if a small
hand-lens be used. Care must be taken not to mistake air-
bubbles or particles of dirt for colonies of bacteria.

In any case, if possible, all the colonies in the plate
should be counted. The number contained within several
squares may be counted and the average taken; knowing
the size of the squares and the area of the plate, the num-
ber contained in the whole plate may be calculated. Such
estimations, however, are likely to give results very wide
of the truth.

The plating may be done by rolling the medium after the
manner of Esmarch. When the number of colonies is not
large this may serve very well. Counting may be assisted by

142 MANUAL OF BACTERIOLOGY.

drawing lines with ink on the outer surface of the test-tube.

It has been said that a water-supply containing no more
than 500 bacteria per cubic centimeter is to be regarded as
safe, one having between 500 and 1000 is to be looked upon
with suspicion, and that where there are more than 1000
to the cubic centimeter the water is unfit for drinking pur-
poses. It is obvious, however, that the character of the
bacteria is of prime importance; that pathogenic organ-
isms may occasionally be present, even when the number
of bacteria to the cubic centimeter is small. But knowing
the number usually found in a good water-supply, any
sudden variation above that number is to be looked upon
with suspicion. An increase is to be expected when the
water has been subjected to unusual agitation from \vinds
or currents.

The detection of pathogenic bacteria in water1 involves
great difficulties, and our knowledge in this direction is very
meagre. Koch and several others have reported finding
the spirillum of Asiatic cholera in water. The examination
of water-supplies for this organism has disclosed the fact
that bacteria resembling the organism of cholera in many
respects are not uncommon in water. This adds to the diffi-
culty of detecting the cholera germ in water.

The bacillus of typhoid fever has many times been de-
scribed as occurring in water-supplies suspected of being
contaminated with the excreta of cases of the disease. The
interpretation of these observations is at present doubtful.
It is now known that several forms of bacteria exist which
closely resemble the bacillus of typhoid fever, and which
would make its recognition in an unknown specimen very
difficult.

It will at once be appreciated that the number of cholera
and typhoid organisms necessary to contaminate a consid-

1 See also articles in Part IV. on the bacillus of typhoid fever, bacillus
coli communis and spirillum of cholera.

DISTRIBUTION OF BACTERIA. 143

erable body of water, and sufficient to cause an outbreak of
the disease among some of the people drinking the water,
may still be so small that many different cubic centimeters
of the water might be studied before a single one of the
specific organisms would be encountered. Anyone who
has examined plates made from samples of water will recog-
nize the difficulty of detecting one or a few colonies of the
bacteria of cholera or typhoid fever among a hundred or
more colonies of ordinary water-bacteria. The existence of
contamination with animal excreta might, however, be indi-
cated by finding the bacillus coli communis, whose detection
offers a greater prospect of success. It is not certain just
how much importance is to be attributed to the presence of
small numbers of the colon bacillus in water.1 Until our
knowledge is more complete any suspicious water should be
discarded.

Certain devices have been adopted to hasten the develop-
ment of the desired bacteria and to retard that of the ordi-
nary water-bacteria. Among these may be mentioned the
influence of the heat of the incubator, which will hasten the
growth of organisms derived from the human body, and
which retards the growth of water-bacteria. Another is
the addition of a solution of peptone to a large quantity of
the water to be examined with a view to assisting the
development of the desired bacteria by furnishing them
suitable food for growth. In another method (Parietti's)
small quantities of carbolic acid are added to bouillon and
mixed with the water, with a view to retarding the develop-
ment of all except typhoid and colon bacilli. Suspected bac-
teria may be tested by inoculation into animals ; the pos-
session of pathogenic properties creates a probability in
favor of their having come from some contamination with
animal excreta.

Jordan, Journal of Hygiene, Vol. I., 1901. Savage, Journal of Hy-
giene, Vol. II., 1902. Winslow & Hunnewell, Journal Medical Research,
Vol. VIII., 1902.

144 MANUAL OF BACTERIOLOGY.

Detection of Bacillus coli communis in Water. — To each of a number
of fermentation-tubes containing i per cent, dextrose-bouillon add some
of the suspected water (.1 to I c.c.). Place in the incubator. Each day
mark the amount of gas that has formed in the closed arm. In three
days B. coli communis should render the bouillon strongly acid and
produce 50 per cent, of gas (or about that amount). From tubes show-
ing these characters, plates may be made and the usual tests for the
colon bacillus applied.1 (See Part IV.)

Ice. — The bacteriological examination of ice differs in
no respect from that of water. Although development may
be arrested, the vitality of bacteria is not necessarily im-
paired by freezing. Prudden found the bacillus of typhoid
fever alive in ice after more than one hundred days. How-
ever, Sedgwick and Winslow have stated that when typhoid
bacilli are frozen in water the majority of them are de-
stroyed. Nevertheless, it is safest to have the source from
which ice is taken as carefully scrutinized as that of the
water-supply, especially in view of the universal habit of
cooling water in the summer time by adding ice directly to
the water. It is better to cool water and articles of food by
surrounding the vessels containing them with ice.2

Bacteria of Milk and Other Foods."— Of the different
food substances, milk is probably the most important from
a bacteriological point of view. In the first place, most
other foods are cooked before eating. Furthermore, cow's
milk constitutes a large part of the food of many young
infants who are highly susceptible to certain bacteria, or to
substances in the milk itself, after it has undergone certain
alterations due to bacteria. The milk of the healthy cow
as it- is secreted in the mammary gland is sterile; how-
ever, after milking the cow a little milk generally remains

1 T. Smith, American Journal Medical Sciences, Vol. no, 1895.

3 Clark, " Bacterial Purification of Water by Freezing," Reports Amer-
ican Public Health Association, Vol. XXVII. , 1901.

3 See Conn, " Bacteria in Milk and its Products," 1903. Russell,
" Dairy Bacteriology."

DISTRIBUTION OF BACTERIA. 145

in the milk-ducts and in the lower part of the teat in which
numerous bacteria will have developed before the next
milking-time.1 The first milk obtained at a milking should
therefore be discarded, as it may contain an excessive
number of bacteria.

Contamination with bacteria may occur from the outer
surface of the udder of the cow, the hands of the milker or
dirty pails, or through agitation of the air of the stable, and
in other ways readily conceived of. Bacillus coli commu-
nis is often found in milk. Excluding the tubercle bacillus,
the organisms which contaminate milk will be pathogenic
only in exceptional cases. Occasionally typhoid fever,
cholera, and possibly scarlet fever, diphtheria and other
diseases are disseminated by means of contaminated milk.
In the case of typhoid fever, it is probable that the milk cans
have been washed with polluted water; after the cans were
filled, a few typhoid bacilli left in drops of water in the
cans, might multiply enormously. Streptococci have been
found quite frequently in the milk sold in cities.2 The
mixture with the milk of non-pathogenic organisms from
the air, and their growth, may induce changes in it which
render it unfit for consumption, and even poisonous. These
alterations may be evident to the senses, as the ordinary
lactic acid fermentation (souring of milk), or they may not.
The character of the alterations doubtless varies much with
the temperature and with the character of the contaminating
bacteria. Summer temperatures of course favor decom-
position and fermentation. Specialists in children's diseases
attribute to alterations in milk with the formation of poison-
ous substances a preeminent influence in the production of
the intestinal disorders of infancy so common in the summer.

1 See Harrison and dimming, " The Bacterial Flora of Freshly
Drawn Milk," Journal of Applied Microscopy, November, 1902.

2 See Reed and Ward, " The Significance of the Presence of Strepto-
cocci in Market Milk," American Medicine, February 14, 1903.

146 MANUAL OF BACTERIOLOGY.

Poisoning with milk, ice-cream or cheese is not rare, as
is well known. There are many records of whole compa-
nies of individuals having been taken violently ill after
having eaten one of these foods from the same source of
supply. The symptoms in such cases resemble those pro-
duced by irritant mineral poisons such as arsenic : nausea
and vomiting, vertigo, dryness of the mouth, sense of burn-
ing and constriction in the throat, difficulty in swallowing,
cramps and griping pain in the bowels, constipation or
diarrhea, general prostration or even collapse. Vaughan
isolated from poisonous cheese a ptomaine which he called
tyrotoxicon. It appears, however, that other toxins may
be present in cheese, and that tyrotoxicon is a somewhat
rare poison. Vaughan believes that bacteria of the colon
group play an important part in producing poisons in milk
and cheese.

To prevent the alteration by bacteria of milk intended
to be the food of infants, the practice of sterilizing milk
has been largely in vogue. Unfortunately, during steriliza-
tion the milk undergoes some kind of alteration which
makes it disagree with certain infants. Furthermore,
among the organisms which would be likely to contam-
inate milk the bacilli of hay and potato, whose spores are
so excessively resistant, would be prominent, and they are
not killed by any process to which the milk intended for
an infant's consumption could possibly be subjected in the
household. Least of all can sterilization be expected to
purify milk in which bacterial poisons are already formed.

The process called pasteurization is designed, not to ster-
ilize the milk completely, but to destroy the vegetative
forms of bacteria, and to destroy the ordinary pathogenic
bacteria with which the milk might possibly be contami-
nated.1 The milk is subjected to a temperature of only

1 See Journal of Experimental Medicine, Vol. IV., p. 217. "The Ther-
mal Death-point of Tubercle Bacilli in Milk," etc., by T. Smith.

DISTRIBUTION OF BACTERIA. 147

about 70° to 75° C. This temperature is less likely to pro-
duce alteration in the milk than sterilization by steam at
100° C. According to Freeman, milk which had been pas-
teurized at 75° C. and distributed among the poor people of
New York City, whose homes were not supplied with ice,
usually kept very well even in the summer time (see p. 67).

The number of bacteria in milk may be reduced con-
siderably by the use of the centrifuge (separator).

It has been undertaken recently to do away as far as
possible with the contamination usually inevitable in the
barnyard and stable by the use of extraordinary measures
to keep the cows, and especially their udders, clean; also
the hands of the milker and the milk-pails ; and by sprink-
ling the floor of the milk-room to prevent dust.1 The milk
is to be transported to the city on ice. Milk which has
been collected in this manner is furnished in several cities
in the United States. The cattle from which the milk is
derived are regularly inspected by veterinary surgeons as
well as subjected periodically to the tuberculin test. The
surroundings and drainage of the stables are investigated
by physicians and sanitary engineers. The milk is also
regularly analyzed by a chemist. It has been found possi-
ble to reduce the number of bacteria in milk very notice-
ably. This milk is of course- sold at a considerably higher
price than ordinary milk.

The number of bacteria which occur in samples of milk
varies greatly. In ordinary milk as furnished by milkmen
the number of bacteria to the cubic centimeter is usually
many thousands to millions; grocer's milk may contain
hundreds of thousands to millions of bacteria to the cubic
centimeter; frequently figures are reached which are be-
yond computation.

Human milk often contains the staphylococcus epicler-

3 See W. H. Park, Journal of Hygiene, Vol. I., 1901.
13

148 MANUAL OF BACTERIOLOGY.

midis albus, and not seldom the staphylococcus pyogenes
aureus, under normal conditions.

Of the different pathogenic bacteria liable to furnish a
source of danger in milk, the most important is the bacillus
tuberculosis. Tuberculosis is a disease to which cattle are
exceedingly prone. There is good reason to believe that
infants acquire tuberculosis through taking as food the milk
of tuberculous cows, although the danger from this source
has probably been overestimated. The milk of tubercu-
lous cows may contain tubercle bacilli when there is no
tuberculous disease of the udder.1 The frequency of tuber-
culosis among milch cows sometimes becomes as high as 25
per cent., or even more. Butter derived from the milk of
such cows may contain tubercle bacilli. The proper man-
ner for the States to deal with this problem, for it is one
that doubtless will fall to the individual States, has not yet
been determined. The cost of killing such a large number
of valuable cows would be very great. Furthermore, it is
by no means certain that this procedure would eradicate
the disease. The flesh of cattle also is capable of trans-
mitting tuberculosis, but is a smaller source of danger on
account of the universal practice in the United States of
thoroughly cooking beef.

" Ripening " of cream and cheese is due to the growth of
bacteria which produce agreeable flavors in the butter and
cheese. Molds are also important in the ripening of some
kinds of cheese.2

In examining milk for bacteria the number may be esti-
mated by precisely the same technique as is used for the
estimation of bacteria in water, except that the milk must
be diluted; otherwise the plates are rendered opaque by

1 See Mohler, " Infectiveness of Milk of Cows which have Reacted
to the Tuber nlin Test," U. S. Dept. Agriculture, Bureau Animal In-
dustry, Bull. No. 44, 1903.

2 Conn, " Agricultural Bacteriology."

DISTRIBUTION OF BACTERIA. 149

the fat. It may be diluted one hundred times with steril-
ized water; when the number of bacteria is very great a
second dilution may be required. Estimations based upon
such high dilutions can only be approximate. The quan-
tity taken for examination may be o.i to i c.c. Plates
should be made immediately upon collection of the sample.
If the milk stands for a few hours at the room temperature
in the laboratory, the number of bacteria will become
enormously increased.

The detection of a particular kind of pathogenic bacterfa
in milk or butter involves very great difficulties. Staining
of bacteria in milk may be done by the usual methods, but
the results are rendered unsatisfactory by the oil in the milk.
The demonstration of tubercle bacilli by staining methods is
likely to involve many difficulties. In this connection it is
necessary to remember the group of bacilli which resemble
the tubercle bacillus in resisting decolor ization with acids
after staining. (See p. 44.) The procedure of injecting
milk into guinea-pigs has been resorted to largely, but the
results are only obtained after the lapse of weeks, when the
development of tuberculosis in the guinea-pigs would indi-
cate that the milk was tuberculous, provided that control
guinea-pigs remained healthy. Furthermore, the other acid-
proof bacilli which may occur in milk or butter are capable
of producing nodules resembling tubercles.1 (See Bacillus
tuberculosis, Part IV.) The most satisfactory plan will be
to apply the tuberculin test to the cow from which the milk
was derived.

Among the other articles of food, those are to be most
carefullv scrutinized which are to be eaten after little or no
cooking, particularly salads, green vegetables, fruits, and
the like. Under exceptional circumstances they may be-
come agents for conveying infectious diseases. Conn

1 Rabinowitsch, Zeitschrift f. Hygiene, Bd. XXXVII. , p. 439.

15O MANUAL OF BACTERIOLOGY.

showed that there was good reason for attributing an epi-
demic of typhoid fever among students at Middletown,
Connecticut, to raw oysters. After having been collected
from the oyster-beds, these oysters were placed in a small
stream to fatten, where they were exposed to contamination
from a sewer. Into this sewer the discharges of a case of
typhoid fever were found to have been running at the time
when the oysters were fattening. An epidemic at Atlantic
City, New Jersey, in 1902, was traced to nearly similar
causes and conditions.1

The ordinary processes for curing and salting meat can-
not be relied upon to destroy pathogenic bacteria.

Cases of poisoning by eating oysters, fish, meat in the
form of sausage or canned meat, and other articles of food
are not rare. These cases belong to the same class as
those poisoned by milk and cheese already mentioned. They
are due to products of bacterial decomposition. Such affec-
tions are quite commonly called " ptomaine poisoning/' al-
though the poisons are not ptomaines in most cases. Prob-
ably a number of bacteria exist which are capable of affecting
changes in meat and other foods either before or after
ingestion. Among these are an anaerobic bacillus described
by Van Ermengem (B. botulinus), bacillus enteridis (Gaert-
ner) and members of the groups of which B. coli communis
and B. proteus are types.2

1 Philadelphia Medical Journal, November I, 1902.

2 See Vaughan and Novy, " The Cellular Toxins," 1902. Ohlraacher,
" Food-Intoxication from Oatmeal," Journal of Medical Research, Vol.
VII., p. 420. Galeotti and Zardo, Centralblatt f. Bakteriologic, Vol.
XX.vL, 1902, Orig. p. 593.

THE BACTERIA OF THE NORMAL HUMAN BODY.

CHAPTER IV.

THE BACTERIA OF THE NORMAL HUMAN BODY.

THE numerous solid tissues and organs of the human
body, the fluids circulating in the interior like the blood
and lymph, and the cavities that have no connection with
the outer world, are entirely free from bacteria.1 So also
the maxillary, ethmoidal and frontal sinuses, middle ear,2
urinary bladder, uterus and Fallopian tubes, and to a less
extent the lungs and gall-bladder,3 although having external
connections, are usually sterile when in a healthy condition.
When bacteria do enter the tissues from any of the surfaces
their progress is checked by means of the activities of the
cells or fluids of the body, and if they succeed in penetrating
to any considerable distance their advance is usually arrested
by the nearest group of lymph-nodes, which appear to be
important safeguards for preventing the dissemination of
bacteria throughout the body. As a rule, the secretions of
the mucous membranes are inimical to bacteria.

The skin, as might be expected, is liable to have upon
it numerous bacteria, especially micrococci, and moulds.

1 This view is not upheld by the experiments of Ford, who found
small numbers of bacteria in the normal organs of rabbits, cats and
dogs in the majority of those examined. The species of bacteria
obtained were mostly common saprophytes, and to some extent constant
in the same kind of animal. Journal of Hygiene, Vol. I., 1901.

2 Calamida and Bertarelli, Centralblatt f. Bakteriologie, Vol. XXXII.,
1902, Orig. p. 428. Torne, Ibidem, XXXIII., 1903, p. 250. Hasslauer,
Ibidem, Ref. XXXII., p. 174. An examination of these articles will
show that investigators disagree somewhat, with regard to the sterility
of these cavities.

3 See Review on the " Bacteriology of the Gall-Bladder and its Ducts,"
American Journal Medical Sciences, Vol. 123, p. 372.

152 MANUAL OF BACTERIOLOGY.

The staphylococcus pyogenes aureus, the streptococcus
pyogenes, the bacillus pyocyaneus and the bacillus coli
communis sometimes occur on the skin. According to
Welch, it always contains the staphylococcus epidermidis
albus, which may be a form of the staphylococcus pyogenes
albus. This organism is of some importance to surgeons
on account of its relation to the cleansing of the skin before
operations. It seems impossible, by any amount of clean-
ing, to dislodge all of the germs in the skin, especially
those under the nails.

The bacteria of the exposed mucous membranes like the
conjunctiva and the nasal cavity1 and the mouth cavity will
naturally be very fluctuating both in quantity and quality;
they will be, in fact, those which happen to fall upon the
surface or to be drawn in from the external air.

In the mouth, however, there is a certain group of organ-
isms more or less characteristic of it, many of which have
not been successfully cultivated. These have been thor-
oughly studied by Miller, to whose works students are re-
ferred.2

Several species of spirilla have been discovered in the
mouth and are found along the margins of the gums. The
leptothrix buccalis, and related organisms which have a
long, ribbon-like form, also occur in the mouth. The mi-
crococcus lanceolatus (or pneumococcus) appears to be
present in many human mouths. In 15 to 20 per cent, of
human mouths this organism is sufficiently virulent to pro-
duce fatal septicemia when inoculated into susceptible ani-
mals. Pyogenic bacteria, especially streptococci, occur fre-
quently, although not regularly, in the mouth. Putrefactive

1 Hasslauer, " Die Bakterien flora der gesunden und kranken Nasen-
schleimhaut," L'cntralb'.utt f. Bakteriologic, Vol. XXXIII., 1902, Orig.
p. 47.

- Miller, " Microorganisms of the Mouth." For a recent review on
the bacteria of the month, see Madzar, Ccntralblatt f. Bakteriologic, Vol.
XXXi, 1902, Ref. p. 489; Vol. XXXII., p. 609.

THE BACTERIA OF THE NORMAL HUMAN BODY. 153

bacteria acting on particles of food about the teeth produce
the bad odor from the mouths of persons of careless habits.
According to Miller, bacteria play an important part in the
production of dental caries. Certain of the bacteria of the
mouth produce fermentation in the vicinity of the teeth with
the formation of acids, which dissolve the calcium salts of
the teeth. The softening and destruction of the decalci-
fied matrix is then accomplished by other and liquefying
forms.

The expired air coming from the mouth and nose, con-
trary to the popular notion, is free from bacteria, excepting
those which become forcibly detached, as by efforts of
sneezing and coughing.

Among the other exposed mucous surfaces, the urinary
meatus and the vagina may be included. The urinary
meatus and at least part of the urethra will be found to con-
tain bacteria, which, in health, should be non-pathogenic,
although interest attaches to the fact that diplococci have
been described which behaved with stains in the same man-
ner as the gonococcus (pseudo-gonococci).

There has been much dispute as to whether or not the
pyogenic bacteria occur in the vagina normally. It is prob-
able that the healthy vagina is in most cases free from the
pyogenic bacteria ; although bacteria of some sort are always
present, and the pyogenic bacteria may exceptionally be
found there in health. The normal secretion of the vagina
has a bactericidal influence which may be attributed in part
to its acidity. The upper part of the normal cervix uteri is
sterile, while bacteria are present in the lower part.

According to Doderlein the properties of the vaginal secretion are
due to bacilli which very commonly occur in it. The secretion is most
abundant and important during pregnancy.1

TJ. W. Williams, "Obstetrics, A Text-Book, etc.," 1903, pp. 34, 773-
7/5. Wadsworth, American Journal of Obstetrics, Vol. XLIIL, 1901.

154 MANUAL OF BACTERIOLOGY.

The smegma of the external genitals contains numerous
bacteria, among which are frequently found bacilli which
retain their color after treatment with acids in the Gabbett
method for staining tubercle bacilli. It is uncertain whether
these, bacilli form a special group of organisms by them-
selves, having as one of their properties the power of re-
taining the stain after acids, or whether they are bacilli of
no particular sort, which resist acids after staining owing
to the oily material with which they have been impregnated
in this peculiar secretion. These organisms must be taken
into account in staining for tubercle bacilli, urine or other
secretions which might accidentally contain particles of
smegma. Usually the employment of alcohol after the acid
will remove the color from the smegma bacilli (Hueppe).
Sometimes smegma bacilli are as resistant as tubercle bacilli
to decolorizing agents (Welch) ; see page 44. Similar acid-
proof bacilli occur about the genitals of the domestic ani-
mals.1

The bacteria of the stomach and intestines are of great
interest and importance. The alimentary tract of new-born
infants and the meconium are sterile. In from four to
eighteen hours organisms begin to appear. They may
enter either from the mouth or the anus. There seems to
be no constancy in the nature of the forms which are found
at first, but their character depends upon the surroundings.

The bacterial inhabitants of the stomach are less constant
than we shall find those of the intestines to be. Under
normal circumstances they seem to be those introduced
from the mouth. Different investigators, at all events,
have met with quite different species. It appears that the
hydrochloric acid (about 2 parts per thousand) present in
the gastric juice at the height of digestion possesses decided
germicidal properties. This germicidal power exercises a
restraining influence upon fermentation due to bacteria, and
1 Cowie, Journal Experimental Medicine, Vol. V., p. 205.

THE BACTERIA OF THE NORMAL HUMAN BODY. 155

probably serves as a safeguard against the introduction of
pathogenic germs into the intestines. That is particularly
important in the case of the spirillum of cholera, which is
excessively sensitive to the action of acids. Nevertheless
many bacteria are able to reacrTthe intestines uninjured, as
the acidity of the gastric juice does not reach its height
until some hours after eating. Such bacteria will be those
which are most resistant and those which form spores. In
the intervals when hydrochloric acid is absent from the
stomach, lactic acid appears. It is formed from carbohy-
drates by a large number of species of bacteria. In con-
ditions of fermentation, sarcina ventriculi and yeasts may
be present in large numbers; in the healthy stomach they
occur in much smaller numbers.

The intestine of the infant in whom feeding has become
well established was found by Escherich to contain two
principal species of bacteria — in the lower part of the in-
testine the bacillus coli communis, in the upper part the
bacillus lactis aerogenes. More recently it has been shown
that the stools of milk-fed infants, and to a less extent of
adults, contain large numbers of anaerobic bacilli, which
stain by Gram's method (bacillus bifidus — Tissier, bacillus
acidophilus — Moro). These bacteria have not been fully
studied.1

The number of bacteria in a milligram of human fecal
matter has been estimated at from seventy thousand to
thirty-three million. It is estimated that about one-third of
the fecal matter of adults consists of bacteria. The small
intestine of adults has been found by different observers to
contain very different species. The majority of these ap-
pear to have been introduced from the mouth in food or
water. The bacillus coli communis, however, occurs in-
variably in health not only in the intestine of man, but also

1 Metchnikoff, " Les Microbes Intestinaux," Bulletin dc I'Institut Pas-
teur, May 15 and 30, 1903.

156 MANUAL OF BACTERIOLOGY.

in that of many animals, especially in the lower part.1 The
pyogenic micrococci very often occur in the intestine.

In the case of ruminant animals like the cow and sheep,
the decomposition of cellulose, which forms so large a part
of their food, appears to be effected by bacteria. Bacteria
having this power are constantly found in the stomachs of
ruminants. The best known species is that called bacillus
amylobacter. It is questionable whether the products of
the decomposition of cellulose have any nutritive value.

Pasteur some years ago expressed the opinion that if animals could
be placed in such surroundings that bacteria could be excluded from the
alimentary canal and the food, life would be impossible. This view
has excited much controversy, and was apparently disproved by the
experiments of Nuttall and Thierfelder. These investigators succeeded
in removing guinea-pigs from the mother by Caesarean section, and in
keeping them alive in sterile surroundings, upon sterile food, so that the
contents of the alimentary canal remained sterile. Schottelius, who
worked with chickens, obtained contrary results, however, so that this
interesting question is still undecided.

1 Moore and Wright, " Bacillus coli communis from Certain Species
of Domesticated Animals," American Medicine, March, 1902.

BACTERIA IN DISEASE. 157

CHAPTER V.

BACTERIA IN DISEASE.

To the physician and the student of medicine the study of
bacteriology is interesting chiefly on account of the great
importance attributed to bacteria in producing disease.
The presence in an organism of one or a number of organ-
isms of another species, which flourish as parasites upon
the first, is a phenomenon of very wide occurrence in na-
ture. It is, in fact, nearly universal. It may be observed
among plants as well as animals, for example in the familiar
galls seen on some of the higher plants, and mostly caused
by the larvae of insects harbored by the plant. We also
find animals, such as tape-worms and the trichina spiralis,
living as parasites upon other animals. The functions of
the bacteria make them peculiarly suited to leading a para-
sitic existence. The fact that they possess no chlorophyll,
and that they are therefore unable to form carbon com-
pounds from the carbon dioxide of the atmosphere, makes
it necessary for them to secure such compounds from pre-
existing organic matter. Many of them, furthermore,
flourish better when they are able to obtain nitrogenous
food from organic matter rather than from inorganic salts
containing nitrogen. Most bacteria find the necessary nutri-
ment in the dead bodies of other animals and plants ; they
constitute what are known as saprophytes. But some of
them flourish upon the living bodies of other plants and
animals in whom they may produce disease.

The phenomena of disease, we shall find, are very largely
due to the numerous waste products of the activities of
bacteria, which act as poisons to the host.

158 MANUAL OF BACTERIOLOGY.

The diseases of plants known to be caused by bacteria
are not very numerous. Among them may be mentioned
pear-blight, due to micrococcus amylovorus.1 Among lower
animals bacteria very frequently produce diseases — for
example, chicken-cholera, symptomatic anthrax, erysipelas
of swine, hog-cholera, tuberculosis, anthrax and glanders.

Koch formulated certain rules which he considered must
be complied with in order to p.ove that any microorganism
was the cause of a particular disease :

First. That the organism should always be found micro-
scopically in the bodies of animals having the disease; that
it should be found in that disease and no other; that it
should occur in such numbers and be distributed in such a
manner as to explain the lesions of the disease.

Second. That the organism should be obtained from the
diseased animal and propagated in pure culture outside of
the body.

Third. That the inoculation of these germs in pure cul-
tures, which had been freed by successive transplantations
from the smallest particle of matter taken from the original
animal, should produce the same disease in a susceptible
animal.

Fourth. That the organism should be found in the lesions
thus produced in the animal.

An infectious disease is a disease which is caused by a
microorganism growing in the body of the animal having
the disease. Such microorganisms are usually bacteria,
but not always; for example, malaria is produced by a
minute animal organism.

A contagious disease is one which is acquired from an
individual having the disease. Most contagious diseases
are infectious, but infectious diseases are not necessarily
contagious. The words are often used very loosely, and

1 See E. Smith in Ccntralblatt f. Baktcriologic, etc., Z \\eite Abtheilung,
TU1. V., p. 271 ; Bd. VII., p. 88.

BACTERIA IN DISEASE. 159

it is no longer possible or very desirable to draw the line
sharply between them. Fomites are the materials on which
the contagious element is conveyed.

A miasmatic disease is a variety of infection in which
the microorganisms are not received from another case of
the disease, but are derived from the external world, par-
ticularly through foul air.

The following is a list of the most important diseases of
man caused by bacteria. The proof as required by the
rules of Koch is not complete for all of them :

Tuberculosis, Gonorrhea,

Leprosy, Chancroid or soft chancre,

Glanders, Lobar pneumonia,

Anthrax, Influenza,

Tetanus, Diphtheria,

Malignant edema, Typhoid fever,

Bubonic plague, Dysentery (not amcebic dys-

Malta Fever, entery),

Suppuration and certain Asiatic cholera,
inflammatory conditions Relapsing fever,

allied to it, Rhinoscleroma (?),

Erysipelas, Actinomycosis.

Malaria and amoebic dysentery are caused by microscopic
animal organisms (protozoa). It has been claimed that
small-pox is caused by protozoa; this view has acquired
added interest from the recent researches of Councilman.
Recent work indicates that the " sleeping sickness " (of
Africa) and yellow fever may be caused by protozoa (see
appendix). Thrush and certain parasitic skin diseases are
caused by fungi of more highly organized structure than
bacteria.

In each of the following diseases there is good reason to
think that the cause is some kind of microorganism, but it
has not yet been discovered :

l6o MANUAL OF BACTERIOLOGY.

Syphilis, Mumps,

Chicken-pox, Whooping-cough,

Measles, Yellow fever,

Scarlet fever, Typhus fever,

German measles, Hydrophobia,

Dengue.

Rheumatic fever and Beri-beri would be placed in this
list by many writers.

The causes of these diseases have been very carefully
sought for by ordinary bacteriological methods with inde-
cisive results. Some of them no doubt are due to bacteria.
In recent years numerous observers have described a diplo-
coccus or short streptococcus as the cause of rheumatic fever
or acute rheumatism. In the case of yellow fever Sanarelli
described an organism (bacillus icteroides) as its cause, but
his view is not upheld by most of those who have worked
on yellow fever.1 The bacillus described by a number of
observers as having been found in cases of whooping-cough
may also be the cause of that disease.2 Bacilli have also
been described in cases of measles on several occasions.
Lustgarten has described a bacillus found in the lesions of
syphilis which resembles tubercle and smegma bacilli. More
recently Joseph and Piorkowsky3 have cultivated another
bacillus from cases of syphilis ; how much importance should
be attached to it cannot as yet be stated. It is likely that
for some of the diseases mentioned other procedures than
the usual methods of research will have to be devised in
order that the cause may be discovered. The protozoa may
play a part in the etiology of some of them. Roux believes

1 Sanarelli, La Semainc Medicare, April 4, 1900. Reed and Carroll,
Journal Experimental Medicine, Vol. V.

2 See Czaplewski, Ccniralblatt f. Baktcnologic, Bd. XXIV., 1898, p.
865.

3Ccutralb!att f. Bakteriologie, Vol. XXXI., 1902, Orig. p. 445. Ber-
liner klin. Wochcnschrift, 1902, Nos. 13 and 14.

BACTERIA IN DISEASE. l6l

that contagious pleuro-pneumonia of cattle is due to a
microbe so minute that it is barely visible with the highest
powers of the microscope, so that its outlines and its mor-
phology can not be studied. The virus of this disease re-
mains virulent after being passed through a Pasteur filter,
showing that it is small enough to go through its pores.
Similar experiments have succeeded with a number of other
affections of animals (of which the best known is foot and
mouth disease) . The virus may pass through a Pasteur or
Berkenfeld filter of a certain coarseness, but is restrained
by one sufficiently fine. The most important of the diseases
in this class is yellow fever. Reed and Carroll found that
the infective agent of yellow fever is in the blood, and that
the serum could produce yellow fever in a non-immune
person after filtration through a Berkenfeld filter.1 These
facts suggest the possibility that failure to find the causes
of some other diseases may lie in the fact that their organ-
isms are so small as to be nearly or entirely invisible to the
microscope.

Modes of Introduction. — There are various avenues by
which bacteria may enter the body to produce disease.
Infection of the embryo through the ovum or semen seems
to be of rare occurrence. Syphilis (which may not be due
to bacteria) is transmitted in this manner. The embryo
may be infected through the placenta, although not com-
monly. The bacilli of typhoid fever and the pus-forming
bacteria have been known to be conveyed through it.
Tuberculosis may also be transmitted through the placenta,
how frequently is still- uncertain. The comparatively com-
mon occurrence of endocarditis on the right side of the
heart in the fetus may be due to placental infection. Oc-

1 See Reed and Carroll, American Medicine, February 22, 1902. For
an admirable review of this subject see Roux, " Sur les Microbes dits
' Invisibles,' " Bulletin de I'Institut Pasteur, Vol. I., Nos. I and 2.

1 62 MANUAL OF BACTERIOLOGY.

casionally the exanthematous fevers are transmitted from
the mother to the fetus.

The surfaces covered with thick stratified epithelium are
not likely to be penetrated by bacteria excepting by direct
introduction through some wound or other lesion. This,
for instance, is true of the skin, the mouth, the vagina and
bladder. The infection of bubonic plague appears to be
introduced most often by means of wounds in the skin.
Bacteria more easily penetrate surfaces having a thin
columnar epithelium such as occurs in the intestines, the
middle ear, bronchi and bronchial tubes, uterus and Fallo-
pian tubes.

The thin, flat epithelial cells of the air-vesicles of the
lungs, as would be expected, seem to be passed with com-
parative ease. On epithelial surfaces covered with cilia,
as in the bronchi and bronchial tubes, the Eustachian tubes,
the uterus and Fallopian tubes, the current toward the ex-
terior created by the cilia acts beneficially in removing
bacteria.

The tonsils and lymph-follicles of the intestines, espe-
cially the lymphoid tissue of the ileum and the vermiform
appendix, are points where bacterial invasion frequently
begins. The lymphoid tissue of the appendix may have
some influence in predisposing to infection at that point
and to appendicitis. On the other hand, it is certain that
the progress of many infections is checked by the lymph-
nodes. That is repeatedly seen in the ordinary post-mor-
tem wound where the spread of the inflammation along the
arm is checked suddenly at the elbow or axilla. The par-
ticipation of the lymphoid structures in most infections is
well known. How far this is a conservative process it is
impossible to say.

In most cases of infectious disease a point of entrance
for the bacteria may be discovered. As a rule, the invading

BACTERIA IN DISEASE. 163

microbes produce a lesion at the point where they are in-
troduced, as in the familiar cases of boils and carbuncles
when pyogenic bacteria enter the skin, or of the tubercles
found in the lungs when the bacilli lodge in the respiratory
tract. However, there are cases of septicemia and pyemia
in which the most careful search fails to reveal the place at
which the bacteria entered. The bacilli of plague usually
produce no reaction at the point of entrance.

It is probable that tubercle bacilli may pass through thin
epithelial surfaces and lodge in the deeper structures un-
derneath, where they produce definite lesions. For exam-
ple, they may pass by the lungs and enter the bronchial
glands, and form tubercles in that situation.

Experiments on animals have shown that bacteria may
be very rapidly disseminated after their introduction. The
inoculation of mice, for instance, with anthrax bacilli has
been known to prove fatal, although the wound was washed
immediately with the strongest antiseptic solutions or the
part amputated within a few minutes.

The manner in which infectious agents reach human
beings varies considerably. Generally speaking, the most
important element will be found to be direct or indirect
connection with another case of the same disease. W. H.
Park was able to cultivate diphtheria bacilli from bed cloth-
ing soiled by the expectoration of diphtheria cases. Bald-
win has shown that tubercle bacilli may be found on the
hands of patients having pulmonary tuberculosis, especially
those who expectorate on handkerchiefs.

Excepting under certain special conditions, the Air will
not contain the germs of disease. The dried pulverized
sputum of cases of pulmonary tuberculosis may float in the
atmosphere as dust which will contain tubercle bacilli.
Fliigge states that powerful expiratory efforts like coughing
and sneezing may carry tubercle bacilli with small particles

164 MANUAL OF BACTERIOLOGY.

of secretion into the air, and that they may remain in sus-
pension some time. The pus-producing bacteria may be
present in dust. Infectious elements which happen to be
present in the air will usually be attached to particles of
dust. Wool-sorter's disease is a name sometimes applied
to anthrax in man when acquired by those engaged in the
work of handling wool, and in which the anthrax bacilli
or spores may be conveyed to the lungs in dust.

The atmosphere in the vicinity of cases of the exanthem-
atous fevers at times probably contains the germs of these
diseases.

Water is the usual medium for the transmission of the in-
fection in typhoid fever, and Asiatic cholera, and probably
all forms of dysentery.

Milk from tuberculous cows may carry the bacilli of
tuberculosis; it is of most importance in the case of young
infants. Typhoid fever and cholera, and probably scarlet
fever and diphtheria are sometimes conveyed through the
medium of milk. Bacteria may reach the intestines in
uncooked food, fruit and vegetables.

The Soil is of importance in connection with tetanus and
malignant edema, whose bacteria are frequently found in
soil. Bacillus aerogenes capsulatus may occur in the soil,
and may infect dirty wounds. The spores of anthrax bacilli
are present in the soil of certain localities, and may produce
anthrax in cattle.

Flies and other insects and related animals are capable
of carrying the bacteria of disease. Under suitable condi-
tions flies play an important part in transporting the bac-
teria of cholera and typhoid fever from the excreta of these
diseases to food substances, which they may contaminate.
To what extent diseases are disseminated by fleas, bed-
bugs and similar creatures is still uncertain.1

'Nnttall, "Role of Insects, etc., in Disease," Johns Hopkins Hospit.-il
Reports, Vol. VIII., 1900.

BACTERIA IN DISEASE. 165

In this connection it is proper to refer to certain diseases
due to animal microorganisms. Malaria is conveyed from
man to man by mosquitoes of the genus Anopheles, and
is probably transmitted exclusively in this manner. The
parasite of malaria undergoes part of its cycle of develop-
ment in man, and another part in the mosquito. Similarly,
in Texas Fever, a disease of cattle, it has been shown by
T. Smith that the parasite (a protozoon, Piroplasma) passes
from cow to cow through the cattle-tick (Boophilus annul-
atus or bovis).1 In surra, a disease chiefly affecting horses,
and in the tsetse-fly disease of animals the parasite (a proto-
zoon, Trypanosoma ) , is transmitted by the bites of flies.2
It has recently been shown that the infectious agent of
yellow fever may be introduced into man by mosquitoes of
the genus Stegomyia. Under the administration of the
United States Army yellow fever was suppressed in Havana
chiefly by measures intended to prevent the disease from
being carried by mosquitoes.3

Auto-Infection. — It is possible for the bacteria of disease
to be derived from the individual's own body — auto-infec-
tion. The microbes of lobar pneumonia, for instance,
flourish in the mouths of a large number of people. The
bacillus coli communis, which constantly inhabits the in-
testines, may invade other organs and exhibit pathogenic
properties when the way is opened up for it by other dis-
ease processes.

Bodily Conditions that Dispose to Infection. — The de-
velopment of an infectious disease may be favored by cer-
tain bodily conditions. Hunger, cold and exhaustion make
the body more liable to the inroads of pathogenic bacteria ;
so also do anemia and chronic diseases. Those suffering
from diabetes, as is well known, are especially liable to

1 See V. A. Moore, " Infectious Diseases of Animals," 1902.

2 Report on Surra, U. S. Bureau Animal Industry, 1902.

3 Carroll, Journal American Medical Association, May 23, 1903.

1 66 MANUAL OF BACTERIOLOGY.

infection by the pus-producing bacteria and the bacillus
tuberculosis. Dr. Roswell Park believes that prolonged
anesthesia makes patients who have undergone operations
more liable to surgical infections, and that absorption of
bacterial poisons and auto-intoxication due to the products of
disordered metabolism of the patient's own cells, predis-
pose to infection. Some of the above-mentioned conditions
can be imitated in laboratory experiments. Hens in a nor-
mal condition are not susceptible to the anthrax bacillus, but
Pasteur succeeded in making them contract anthrax by
artificially cooling them. Frogs, on the other hand, which
also are resistant to anthrax, may be made susceptible by
keeping them at an abnormally high temperature. Rats
were made more susceptible to anthrax by physical ex-
haustion produced by making them run a treadmill, and
pigeons by starvation.

Abbott found " that the normal vital resistance of rabbits
to infection by streptococcus pyogenes is markedly dimin-
ished through the influence of alcohol, when given daily to
the stage of acute intoxication." It was less noticeable for
bacillus coli communis, and not observed for staphylococcus
pyogenes aureus. Pigeons and other animals have been
made susceptible to anthrax by intoxicating doses of alcohol.

Climate and altitude appear to influence the liability to
infection with the tubercle bacillus, which occurs less com-
monly in Colorado and some other elevated regions than
in lower and more densely populated districts.

Age. — In general, infants are more susceptible to infec-
tions than adults, though apparently nearly exempt from
the exanthematous fevers during the early weeks of life.
Osteomyelitis is commoner in infants than in adults, as also
is tuberculous meningitis.

How much influence is to be ascribed to individual pre-
disposition in contracting or warding off infection is unccr-

BACTERIA IN DISEASE. 167

tain. Welch says, " the fact that some individuals are
attacked, and others, apparently equally exposed to the
danger of infection, escape, is not always due to any espe-
cial predisposition on the part of the former. It may be
that the germs hit the one and miss the other, and we
would have no more right to say that the former are espe-
cially predisposed than to say that those who fall in battle
are predisposed to bullets and those who escape are bullet-
proof." It is probable that the importance of an hereditary
tendency to certain infections, notably tuberculosis, has been
overrated.

Race. — The influence of racial predisposition is undeni-
able. For example, it is known that the negro race is much
less susceptible to yellow fever than the white race.

Local conditions often have a most important influence
in determining the occurrence of infections. In endocar-
ditis the lesion usually occurs along the line of closure of
the heart- valves, indicating that the point subjected to the
greatest friction is the part of the endocardium most liable
to infection. Regions where there is passive hyperemia
are more vulnerable, as is seen in hypostatic pneumonia.
Localities which have suffered from previous inflammation
or irritation are rendered more liable to subsequent infec-
tion, as when the bladder or pelvis of the kidney contain-
ing a calculus becomes the seat of a suppurative cystitis or
pyelitis.

Local conditions become of great importance in surgery.
The surgeon can seldom be certain of dealing with a per-
fectly aseptic wound, and must rely to a large extent upon
the power inherent in the fluids and tissues to prevent the
development of bacteria. It is important, therefore, to keep
the resisting power of the tissues at the highest possible
point. Injury of the tissues disposes the part to infection;
so do strangulation and necrosis. In operating, it is to be

1 68 MANUAL OF BACTERIOLOGY.

remembered that hyperemic and edematous parts are more
likely to become infected; so also are anemic regions. An
infarct of the lung which was originally sterile may be in-
fected with bacteria through inhalation, and undergo sup-
puration or gangrene. The presence of foreign bodies in
the tissues disposes to infection. Injection of the staphy-
lococcus pyogenes aureus into a rabbit's tissues is not always
followed by suppuration, but if a foreign body, like a piece
of sterilized potato, be inserted at the same time, infection
is much more likely to occur. When lesions are produced in
the internal viscera of animals by cauterization or crushing
and bacteria then injected subcutaneously or into the blood,
the bacteria lodge in the lesions and multiply.1

Amount of Infectious Material. — A large number of
bacteria introduced into the body simultaneously will be
more likely to produce infection than a small number. This
factor is of less importance with organisms whose virulence
is very constant than with those of more variable virulence.

Variability in the Virulence of Bacteria. — The occur-
rence of an infectious disease depends very largely upon
the virulence of the bacteria. Any species of pathogenic
bacteria may vary in virulence at different times. In some
cases the virulence is not easily lost, as with the anthrax
bacillus; in others the virulence is maintained in cultures
only with difficulty, as in the case of the micrococcus lan-
ceolatus (of pneumonia) and the streptococcus pyogenes.
As a rule, the virulence is likely to be diminished in old
cultures. It may sometimes be preserved better in the ice-
chest than at the room temperature. The virulence of the
anthrax bacillus becomes diminished if it is cultivated at
42° C. Exposure to light and to oxygen tends to weaken
the virulence; and also cultivation upon unfavorable media,
such as those containing a small proportion of carbolic acid
or certain other chemical germicides.

1 Cheesman and Meltzer, Journal Experimental Medicine, Vol. III.

BACTERIA IN DISEASE. 169

In laboratory work the virulence is usually maintained
best by inoculating the bacteria from time to time into sus-
ceptible animals. Bacteria coming freshly from infected
animals are likely to be highly virulent. The virulence
may be increased by beginning with an especially sensitive
animal like a very young guinea-pig, and progressively in-
oculating into less sensitive animals. The infection of rela-
tively insusceptible animals may sometimes be produced by
the injection of a very large dose of the bacteria. The
addition of the toxic products of the bacteria, which may
be obtained by using large doses of cultures in bouillon,
makes infection more likely. Cultivation on a particular
medium may maintain or increase the virulence.

Finally, the combination of two or more kinds of bac-
teria may produce infection when neither one would do so
alone. On the other hand, it is said that the fatal effects
of an inoculation of virulent anthrax bacilli into a sus-
ceptible animal may be averted if the animal be inoculated
with a culture of bacillus pyocyaneus shortly afterward.

Mixed Infection. — It is not uncommon in disease to find
two kinds of bacteria associated together, producing a
mixed infection. In diphtheria, very frequently, the ba-
cillus of diphtheria is found to be accompanied in the mem-
brane by the streptococcus pyogenes. The course of the
diphtheria may be modified in this manner. The term
secondary infection is rather loosely used. It is sometimes
employed to designate an infection occurring in an indi-
vidual, the resisting power of whose tissues has been weak-
ened by some chronic organic disease. Such an infection
is often called a terminal infection. Terminal infections
are very common in cases of carcinoma, chronic nephritis,
arteriosclerosis, and in many other diseases.

Concerning terminal infections Osier says : " It may
seem paradoxical, but there is truth in the statement that

MANUAL OF BACTERIOLOGY.

persons rarely die of the disease with which they suffer.
Secondary infections, or, as we are apt to call them in
hospital work, terminal infections, carry off many of the
incurable cases in the wards."

The term secondary infection is also used for the modifi-
cation of an infectious process which has been in existence
for some time, by infection with a second variety of bacte-
ria. That takes place, for instance, in pulmonary tubercu-
losis, when the invasion of the already tuberculous lungs by
the pyogenic micrococci assists in the formation of cavities.
In this sense it will be seen that the term secondary infec-
tion is used as a name for a variety of mixed infection. In
the secondary, mixed and terminal infections, the bacteria
which enter secondarily are likely to be of the pus-producing
varieties, especially the streptococcus pyogenes.

As to the mechanism which bacteria make use of in
order to produce disease, according to our present knowl-
edge, they work chiefly through the poisonous substances
formed by them and deposited in the bodies of the persons
suffering from the disease. The theory that bacteria have
an important influence through the destruction of substances
taken by them from the body of the patient for food, is no
longer entitled to much weight; neither are we able in most
cases to account for the phenomena of disease by any
mechanical action on the part of the bodies of bacteria.
That such action does occasionally take place may be seen
in experimental anthrax in mice, where the blood-capil-
laries of the liver and kidneys may be completely plugged
with masses of anthrax bacilli. The diseases in which
the circulating blood is swarming with bacteria are much
commoner in the lower animals than in man.

Toxemia. — By toxemia is meant the absorption of poison-
ous bacterial products from a localized point of invasion,
and their dissemination throughout the body by means of

BACTERIA IN DISEASE.

the circulation. We see typical toxemias in diphtheria and
tetanus. In surgery the term sapremia is used to cover a
similar condition of affairs when the absorption proceeds
from a wound or denuded surface, as may happen in the
puerpural uterus.

Septicemia. — In septicemia there is not only absorption
of the bacterial poisons, but the bacteria have invaded the
living tissues. Bacteriologists usually employ the word
septicemia to describe the wide dissemination of bacteria
through the body and the presence of a large number of
them in the circulating blood. In this sense septicemias
are commoner in lower animals than in man; anthrax and
infection with micrococcus lanceolatus would be examples.
Typical septicemias in man are found in relapsing fever and
certain cases of bubonic plague. For pyemia, see the article
on Suppuration, Part IV.

The principal agencies in effecting recovery from in-
fectious diseases are the presence or formation in the body
of substances which destroy bacteria (lysins), the develop-
ment of new substances which also neutralize their action
(antitoxins), and their destruction by the cells of the body
(phagocytosis). These phenomena are discussed in the
chapter on immunity. A factor of less importance is the
elimination of bacteria by the excretory organs. Experi-
ments on animals indicate that bacteria which have been
injected into the body do not appear in the urine until they
have damaged the structure of the kidney. In typhoid fever,
the bacilli of typhoid may occur in the urine in great num-
bers; the condition of the kidney in the generality of such
cases has not thus far been determined. The extent to
which the excretory organs act in eliminating bacterial
toxins is not yet known. Some bacteria, as has already
been stated, may, in the end, produce substances that are
inimical to their own growth.
'5

172 MANUAL OF BACTERIOLOGY.

CHAPTER VI.

TOXINS.1

IT is now generally believed that in most, if not all of
the infectious diseases, the principal symptoms and lesions
are to be attributed to the action of poisonous substances
formed by the bacteria. The part that bacteria play can
be understood best by recalling the work of the saprophytes
in producing fermentation and putrefaction. It has already
been shown that the poisoning that comes from eating de-
composed meat, fish, or cheese results from poisons which
bacteria have elaborated in the course of their growth.
In infectious diseases we suppose the bacteria to grow in-
side of the body and to form their poisons in it; not before
their introduction into it, as in these cases of poisoning with
spoiled food. If it were possible for the cells of ordinary
yeast to grow in the living human body and to produce
alcohol from the grape-sugar of the body-fluids, the person
so infected might be expected to suffer from alcoholic in-
toxication as long as the infection lasted. This impossible
illustration although not entirely accurate may help to make
clear what does happen in an infectious disease due to bac-
teria, where poisons formed in a manner analogous to the for-
mation of alcohol produce intoxications analogous to alco-
holic intoxication. Certain infectious diseases exhibit the
element of poisoning by bacterial products in an extremely
marked manner. In tetanus the local wound may be trifling,
and may seem utterly incapable of having given rise to the

1 For a full consideration of this subject see Vaughan and Novy,
" The Cellular Toxins," 1902.

TOXINS. 1/3

violent muscular spasms from which the patient suffers. In
diphtheria, although the condition in the throat may be one
of severe inflammation, it is, of itself, insufficient to explain
the profound prostration and other symptoms of general
poisoning which the case manifests.

The first bacterial poisons to be studied thoroughly were
those called ptomaines. Observing the poisonous effects
which follow the injection into animals of certain ptomaines
derived from bacterial cultures, it was suggested that similar
ptomaines, formed by the action of bacteria in the living
body, might account for the symptoms of many of the in-
fectious diseases. The ptomaines were most readily studied
because of the comparative facility with which they could
be isolated in a condition of purity, where their exact
chemical nature could be determined.

" A Ptomaine is an organic chemical compound, basic in
character, formed by the action of bacteria on nitrogenous
matter." (VAUGHAN and Now.)

The peculiar coloring which distinguishes cultures of the
bacillus pyocyaneus is due to a ptomaine called pyocyanin
and its derivatives.

A group of substances of a similar nature called leuco-
maines has been discovered, which are formed within the
body and not by the action of the bacteria. Leucomaincs
may then be defined as " basic substances which result from
tissue metabolism in the body." (VAUGHAN and Now.)

Further study has demonstrated, however, that the char-
acteristic features of the infectious diseases are not due to
ptomaines. Some of the poisons formed by bacteria have
been described as albumens and have given rise to the
name toxalbumen. It appears, however, that bacterial
poisons are not necessarily of an albuminous nature either,
and at the present time it seems best to call the bacterial
poisons whose chemical nature is uncertain simply to.ruis.

174 MANUAL OF BACTERIOLOGY.

Substances which produce effects in animals similar to the
bacterial poisons may be extracted from certain plants,
notably abrin, which is derived from the jequirity bean, and
ricin, which comes from the castor-oil bean. The venom
of poisonous snakes, which is elaborated by the epithelial
cells of certain glands, also acts in much the same manner.

Bacterial poisons are of two principal sorts : ( i ) Some-
times they appear to be formed as excretions from the bac-
teria. They then occur in solution in liquid cultures, and
they may be separated from the bacteria by filtration.
The toxins of diphtheria and tetanus are typical examples.
(2) The poisons of most bacteria occur chiefly in the bodies
of the bacterial cells. Such toxins are called intracellular.
Those of typhoid fever and cholera are examples. Precisely
how intracellular toxins are set free in the body of an in-
fected animal is not clearly understood. It is, however,
known that many of the bacteria in an infected individual
are dead and disintegrated. The theories concerning toxins
are also considered in the next chapter.

Sometimes the results of the injection of excessively
small doses of a toxin are so tremendous as to have given
rise to the suggestion that the toxins may be allied to the
ferments, like pepsin and trypsin, in their nature.

Owing to the instability of the toxins it has not been
possible to isolate them in a state of purity so as to deter-
mine their exact chemical character. They have, never-
theless, been obtained in some cases in an extremely con-
centrated form. Brieger and Conn obtained a toxin from
tetanus bacilli of which .00000005 gram killed a mouse
weighing 15 grams. Roux and Yersin obtained a toxin
from diphtheria bacilli of which .00005 gram was capable of
killing a guinea-pig. These figures indicate a capability
for poisoning that is simply inconceivable. Such proper-
ties permit bacteria growing in a comparatively limited area
to manifest their evil effects at remote parts of the body.

TOXINS. 175

A curious and unexplained effect of some toxins is the
production of minute areas of necrosis in certain viscera, as
the liver. Such " focal necroses " have been observed to
be formed by the poisons of the bacilli of diphtheria, of
typhoid fever, and of the micrococcus lanceolatus (of pneu-
monia), and following the injection of abrin and ricin.

Besides the poisonous substances produced by the bacilli
of diphtheria and of tetanus, toxic substances have been
obtained from the spirillum of cholera, the bacillus of ty-
phoid fever, the bacillus coli communis, the bacillus of
bubonic plague, and from the bacilli of tuberculosis and
glanders. The extract from cultures of tubercle bacilli, called
tuberculin, and that from glanders bacilli, called mallein,
contain toxins produced by these germs, and will be spoken
of in connection with the bacteria themselves. Vaughan1
has succeeded in cultivating anthrax bacilli, colon bacilli,
and other bacteria on large surfaces of solid media, so as to
secure quantities of the bacterial cells sufficient for extensive
chemical tests. The toxin of the colon bacillus proved to be
a very stable substance, and resistant to heat. Most toxins
become inactive at comparatively low temperatures. (60°
to 70° C.)

Prudden and Hodenpyl found that the injection of dead
tubercle bacilli was followed by the development of lesions
resembling tubercles, which, of course, did not increase in
number or become disseminated.

There is good reason on both clinical and experimental
grounds to believe that toxic substances are formed by the
micrococcus lanceolatus (of pneumonia). The symptoms
of a disease as it occurs in man cannot be imitated in any
other case as accurately as happens after the injection of the
toxins of tetanus and diphtheria in the lower animals.

1 American Medicine, May 18, 1901. Journal American Medical Asso-
ciation, March 28, 190.3.

176 MANUAL OF BACTERIOLOGY.

CHAPTER VII.

IMMUNITY.

UNDER the title of immunity a number of nearly related
subjects may be discussed. It has already been shown that
the body is constantly liable to the attacks of pathogenic
microbes, which are endeavoring to effect an entrance. Some
of the defences of the body have been described. But it has
been found that there are other more subtle and at the same
time far more powerful weapons, which usually succeed in
repelling the invasion. This will answer the question often
asked, Why do we not all constantly have infectious dis-
eases ? In case bacteria do gain a foothold in the body and
produce what we call an infectious disease, we have to con-
sider the means by which the intruders are overcome. The
exemption from a second visitation which often follows one
attack of an infectious disease also needs to be accounted
for.

Certain facts concerning immunity, some of which were
observed many years ago, are extremely interesting, but
very difficult of explanation. Even in the light of recent
bacteriological researches their interpretation is by no means
clear. The immunity which an individual who has suffered
from an attack of measles or scarlet fever possesses from a
second attack of the same disease is well known ; so also is
the immunity from small-pox which is conferred by vacci-
nation. Such an immunity is called " acquired." There is
also a " natural " immunity. Field-mice are susceptible to
glanders and house-mice are not. House-mice are suscep-
tible to mouse-septicemia and field-mice are not. Although

IMMUNITY. 177

sheep, as a rule, are easily infected with anthrax, this disease
seldom occurs in sheep of the Algerian variety or race. The
immunity which belongs to a race, but not to a whole species,
is sometimes called " racial."

The occurrence of immunity from a second attack of an
infectious disease has given rise to numerous hypotheses in
the past. One theory supposed that after an attack of the
disease certain bacterial products are retained within the
body which prevent a second invasion. Another theory
supposed that the attack of the disease exhausts the supply
of some substance necessary for the growth of the microbes,
as plants sometimes exhaust the soil they grow in.

It will be best to deal first with the results of some of the
experimental attempts to produce immunity. The more
recent theories will then be considered.

Small-pox and Vaccination. — The origin of vaccination
against small-pox with the virus of cow-pox has been de-
scribed in the historical sketch (p. 21). The nature of the
protection furnished by this virus has been the subject of
much controversy. The opinion of the present day inclines
to regarding vaccinia as small-pox which has been modified
by passage through a relatively insusceptible animal. Cer-
tainly there are many analogies between the protection
against small-pox afforded by vaccination and the other
examples of artificial immunity mentioned below.

This question cannot be settled with certainty until the
organisms causing small-pox and vaccinia have been isolated
in pure culture. Their identity and mode of action may then
be determined.

Small-pox has been inoculated into calves and passed
through other calves in succession, producing finally an
eruption indistinguishable from cow-pox. Lymph taken
from such calves has been used successfully to vaccinate
children. Not only does cow-pox protect against small-pox,

178 MANUAL OF BACTERIOLOGY.

but it has been shown that small-pox protects against cow-
pox.

Immunity Produced by Inoculation with Bacteria of
Diminished Virulence. — Pasteur conceived the idea of
attenuating the virulence of the bacilli of fowl-cholera by
prolonged exposure to the air. He made use of the atten-
uated virus as a vaccine against the disease.

A nearly similar principle was shortly afterward applied
by him to the preparation of a vaccine against anthrax.
When anthrax bacilli were cultivated at a temperature of
43 °C., Pasteur obtained bacilli of very slight virulence.
Such bacilli did not produce death when inoculated into
animals that were ordinarily susceptible. Yet animals that
were vaccinated with this virus were able afterward to
resist inoculation with fully virulent anthrax bacilli. (See
Bacillus anthracis, Part IV.)

In the case of erysipelas of swine (French rouget; Ger-
man, Schweinerothlauf) Pasteur secured bacilli of dimin-
ished virulence by injecting virulent bacilli into relatively
insusceptible animals. The animal used was the rabbit.
The bacilli were passed through several rabbits in succes-
sion. Cultures taken from the last of the series produced
a milder form of the disease and an amount of immunity,
the value of which is in dispute.

In still another disease, black leg of cattle or symptomatic
anthrax ( French, charbon sytnptomatique ; German, Rausch-
brand), an attenuated virus is secured by the use of heat.
The pulp from the infected muscle of a diseased animal,
containing the bacilli, is squeezed from it and heated to a
temperature of 95° to 99° C. for six hours. The dried
material mixed with water constitutes the vaccine. The
Department of Agriculture of the United States now fur-
nishes this vaccine free to farmers. The results of this
method are said to be very gratifying.1

1 See Recent Annual Reports, Bureau of Animal Industry, U. S.
Department of Agriculture.

IMMUNITY. 179

In the human disease, bubonic plague, a nearly similar
procedure has been proposed by Haffkine. To protect
against plague, cultures of plague bacilli, previously steril-
ized by heat and carbolic acid are injected. (See article on
Bubonic Plague, Part IV.)

Inoculation Against Rabies or Hydrophobia. — The im-
munity produced in this case probably depends upon prin-
ciples similar to those underlying the examples related on
the preceding pages. But this question cannot be regarded
as settled until the organism of rabies has been isolated and
cultivated. Attempts to discover this organism have, as yet,
been futile. Pasteur discovered that rabbits are susceptible
to rabies when portions of the medulla oblongata of a dog
which has died of the disease are inserted beneath the dura
mater of the rabbit. Spinal cords taken from rabbits thus
injected are placed in a desiccating chamber. Under these
circumstances the unknown virus undergoes a diminution
in virulence. Emulsions are made from spinal cords desic-
cated in this manner. First the patient is injected with part
of an emulsion from a cord which has been desiccated a
longer time, and in which the virulence of the poison has
been much reduced. Injections are then made at intervals
from cords that have been subjected to desiccation for
shorter and shorter periods, and therefore of greater and
greater virulence. At the end of about the twenty-fifth day
the patient is supposed to be immune from rabies. The
period of incubation in rabies is longer than in most infec-
tious diseases, being usually one to two months. Al-
though the injections take place after he has been bitten by
a rabid dog, it is hoped the patient may be rendered immune
before the period of incubation has ended.1

Reports of cases managed according to this method have
been conflicting in the past. However, there seems no longer

1 F. Cabot, " The Dilution Method of Immunization from Rabies,"
Journal Experimental Medicine, Vol. IV., p. 181.
16

I SO MANUAL OF BACTERIOLOGY.

to be any question as to its efficiency. Laboratories where
Pasteur's method of treatment is used now exist in all parts
of the world. According to statistics collected by Ravenel,
based on many thousands of cases, the mortality from rabies
in those so treated is less than one per cent.1

Antitoxins. — The first efforts to point out the way along
which antitoxins might be secured were made by Salmon
and Smith in 1886. In their experiments pigeons were in-
jected with filtrates from cultures containing the products
resulting from growth of the hog-cholera bacillus. Such
pigeons were found to be immune to this bacillus, which
is pathogenic to ordinary pigeons.

As was stated in the last chapter, bacterial poisons may be
of twro sorts. In one group the poisons occur chiefly within
the bodies of the bacteria. This group seems to contain the
majority of the pathogenic bacteria. Methods of protection
against infections caused by them will be considered here-
after. In the other group, the poisons do not, for the most
part, remain in the bodies of the bacteria, but are readily
diffused from them into their surroundings. It is for the
bacteria of the latter group that antitoxins have been made.
Its most important members are the bacilli of diphtheria and
tetanus. Their poisons may be found in the culture-media
in which they have grown. The principle employed in pre-
paring antitoxins was established by Behring. The bacilli

lln cases of dog-bite there is often some doubt as to whether or not
the dog was rabid. The dog should be allowed to die under observa-
tion. A large ganglion, as that of the pneumogastric nerve, should be
removed and placed in absolute alcohol or ten per cent, fcrmaldehyde
solution. Microscopic changes have been observed in the ganglia which
seem to be quite constant in rabies. See Ravenel and McCarthy, Uni-
versity of Pennsylvania Medical Bulletin, June, 1901 ; also editorial in
Philadelphia Medical Journal, March 14, 1903. The diagnosis may be
made by inoculating a rabbit as above described, but the results are not
obtained quickly enough to be of value. See V. A. Moore, ''Infectious
Diseases of Animals."

IMMUNITY. l8l

are cultivated in bouillon. The cultures are freed from all
living bacilli by filtration. The liquid filtrate contains the
toxin. This filtrate is injected into healthy animals, usually
horses, in increasing doses. Eventually enormous doses of
toxin are given, and the animal acquires a high degree of
immunity. The blood of the animal is withdrawn, taking
care to avoid contamination. The serum of the blood is
collected and constitutes the antitoxin. Such antitoxins
have produced brilliant results in the treatment of diph-
theria, and have given partial success with tetanus. " (See
the articles on the bacteria of these diseases.)

Ehrlich discovered that the vegetable toxins, abrin and
ricin, behave in a manner very similar to soluble bacterial
poisons when injected into animals, and that by their injec-
tion an immunity for the same poisons may be secured.
There is an analogy between the tolerance acquired in this
manner from bacterial and other toxins and that which vic-
tims of the morphine and cocaine habits have for immense
doses of those drugs. Ehrlich also found that the milk of
animals which had been immunized against abrin and ricin
might confer immunity upon young animals. In most cases
we look to the blood-serum for the immunizing agent.

Active and Passive Immunity. — The kind of immunity
which results from the injection of substances from im-
munized animals is called " passive immunity." Diphtheria
and tetanus antitoxins produce passive immunity. " Active
immunity " may be brought about in several ways :

(i) By an ordinary attack of an infectious disease; (2)
by an attack excited artificially through inoculation with
small doses of virulent cultures, or (3) large doses of atten-
uated cultures; (4) or by the injection of bacterial products
(toxins) freed from the bacteria themselves. Pasteur's
methods of protective inoculation for anthrax, etc., and
Haffkine's injections for bubonic plague produce active

1 82 MANUAL OF BACTERIOLOGY.

immunity. Active immunity is usually more enduring than
passive immunity. Passive immunity, established through
the direct introduction of antitoxins, may be brought about
more quickly than would be possible for an active immunity.

THEORIES OF IMMUNITY.

Phagocytosis.1 — Metchnikoff described under the name
" phagocytosis " a phenomenon which, he maintained, could
explain immunity and recovery from bacterial invasion.
This theory is based on the well-known fact that certain cells
of the body have the power of surrounding and ingesting
foreign substances. The cells in question are chiefly poly-
nuclear leucocytes, but to some extent other leucocytes, and
enclothelial and other cells. There are many examples of
this process. The leucocytes of the lungs constantly take up
small bits of carbon inhaled with the air. Particles of car-
mine injected into the tissues will later be found within
leucocytes. After a hemorrhage, phagocytic cells may be
found containing red blood-corpuscles or particles of blood
pigment. The presumption is that phagocytic cells serve to
remove irritating and foreign bodies to less sensitive parts.
Metchnikoff showed that phagocytes also absorb bits of de-
generating or useless tissue. Such particles disintegrate
and their identity is lost. They are digested and become a
part of the protoplasm of the phagocytes. This process is
seen when the tail of the tadpole shortens. The superfluous
part is absorbed, at least in part, by phagocytic leucocytes.
MetchnikofFs observations were made largely on the in-
vertebrates, whose transparent bodies may be studied while
living. One illustration was furnished by a small crustacean
(Daphnia or water-flea), which was often infected with a
fungus. Some infected individuals died, others recovered.
Metchnikoff found that the cells of the fungus might be

1 Greek, (bayelv, to eat ; nvroq , a cell.

IMMUNITY. 183

ingested and destroyed by the leucocytes of the Daphnia.
He described the history of this disease as a contest between
the parasitic cells and the phagocytes, in which either might
succeed. Similarly, when anthrax bacilli were introduced
into frogs, which are immune from anthrax, the bacilli were
ingested by the frog's leucocytes. MetchnikofF contended
that this function of leucocytes and other phagocytic cells
constituted the principal defence of the body against bac-
teria.

Other investigators also have seen bacteria enclosed
within the bodies of leucocytes. It has been urged by some
that the bacteria are already dead when the leucocytes de-
vour them. In some cases, as with the gonococcus which is
commonly found enclosed within leucocytes, it is possible
that the bacteria retain their full vigor after being ingested.

It is well known that a suppurating part contains large
numbers of leucocytes, and one of the most characteristic
events in the inflammatory process is the migration of
leucocytes to the point of irritation. This indicates a posi-
tive chemotaxis for leucocytes on the part of substances in
the inflamed area. Metchnikoff believed that the function
of these leucocytes is to destroy the bacteria and to arrest
their further progress. On this theory bacteria have often
been likened to an invading army and the leucocytes or
phagocytes to a force designed to repel their attacks.

It is certain that in some infectious diseases the number
of leucocytes, chiefly of the polynuclear neutrophilic variety,
in the circulating blood is increased (leucocytosis). This is
the case usually in lobar pneumonia and acute suppurative
infections. In other infectious diseases there is no leucocy-
tosis ; for example, tuberculosis, typhoid fever and malaria.
It is interesting to observe that in trichinosis, and more
rarely in infection with other animal parasites, the eosino-

1 Metchnikoff, " Comparative Pathology of Inflammation," trans.,
Starling, 1893.

184 MANUAL OF BACTERIOLOGY.

philic leucocytes become much more numerous in the blood
than normally. There is every reason for believing that the
leucocytes and other body cells play an important part in
combating bacteria. However, the doctrine of phagocytosis,
as primarily stated, is insufficient to account for many facts.
Metchnikoff himself has modified his original views, to con-
form with more recent studies.

Ehrlich's Side-Chain Theory of Immunity.1 — This the-
ory was first proposed to explain the resistance of the
body to soluble bacterial poisons, as those of diphtheria and
tetanus. In these cases what is known as antitoxic immunity
is produced. With the infections in which the poisons occur
in the bodies of the bacteria the protective mechanism is
different, giving rise to the term bacteriolytic immunity.

Antitoxic Immunity. — Ehrlich first endeavored to ex-
plain from a chemical standpoint the action of toxins on
cellular protoplasm and the formation of antitoxins. To
begin with, the molecules of the protoplasm are to be re-
garded as being endowed with chemical groups, present in
the form of lateral appendages to the molecule, called side-
chains. They can be illustrated by the analogies presented
by the graphically written formulae of some complex mole-
cules. It is necessary to conceive of molecules made of an
immense number of atoms, and bristling with projecting
side-chains. The function of the side-chains is to become
attached to other organic molecules with which thev have
affinities. In this manner they aid in absorbing the sub-
stances essential for the nutrition of the protoplasm of cells.

1The literature of this subject is very extensive. An exhaustive
review is that by L. Aschoff, " Ehrlich's Seitenkettentheorie," ZeitscJmft
f. aUgcmcinc Physiologic, 1902.

The following are also of a general character: H. C. Ernst, " Modern
Theories of ^pcternl Immunity," 1903; Prudden, Medical Record,
February i 1. 1003; RilrlrV. Journal of Hygiene, Vol. II., 1902; Bergey,
American Medicine, October n, 1902.

IMMUNITY. 155

The side-chains are therefore preferably called " receptors"
The numerous receptors which a molecule has are of many
kinds, with affinities for other molecules of different kinds.
Each kind of receptor will then have an affinity for a mole-
cule of a particular kind, which it may be said to " fit," as a
key fits in a lock, although this expression must not be taken
in a literal sense. A receptor to which tetanus toxin might
become attached would not " fit " diphtheria toxin. In order
that toxins may be able to combine with the receptors their
structure must be nearly like that of the food molecules
which the receptors are adapted to receive.

FIG. 48.

\— Antitoxin
Body cell > Antitoxin

Diagram to Illustrate Side-chain Theory (modified from Aschoff).

Secondly, soluble toxins are to be looked upon as definite
chemical bodies excreted by bacteria, and containing two
essential groups. One group is the haptophorc, by means of
which the toxin may be linked with the receptors of the
molecules of the cell. The other group is the ioxophore,
which is capable of destroying the protoplasmic molecule,
after being attached to the receptor of the latter by the
haptophore.

These relations may be represented schematically though
in a very crude manner. In Fig. 48 a portion of a cell is
shown, with receptors. Molecules of toxin, with hapto-
phores and toxophores are near by or attached. At the

1 86 MANUAL OF BACTERIOLOGY.

right we see a free receptor (antitoxin) and one which has
united with toxin to neutralize it.

COOH

/ \
H/ \H

/ \

1 1/ \OH

Hlrr
\ /•**

\ /

\/
II

Benzol.

Phenol.

Salicylic Acid.

The chemical nature of this conception may be illustrated
by recalling the formula of a molecule of benzol. Here one
or more H atoms may be replaced with equivalent groups
making phenol, salicylic acid and other derivatives.

As the side-chains or receptors of the protoplasm are
essential to its existence, their combination with the toxin,
through its haptophore, might result in destruction of the
molecule. But if the damage be not too serious, the proto-
plasm may be stimulated to produce numerous similar side-
chain groups. Not all of these are necessary for the per-
formance of its functions, and the superfluous ones are
thrown off into the surrounding serum. It is well known
that many cells of the body exhibit analogous heightened
activities under stimulating influences. Such free side-
chains or receptors may combine with the haptophorous
groups of the toxin, when it will no longer be able to com-
bine with the protoplasm. Thus they act as a kind of buffer
in protecting the protoplasm from the attacks of the toxins.
They constitute the antitoxic part of the serum.

Numerous experiments have been made which illustrate
the probable chemical nature of antitoxic action. A fatal
dose of diphtheria or tetanus toxin may be neutralized out-
side of the body by mixing it with its appropriate antitoxin.
Injection of the mixture shows it to be innocuous to animals.

The manner in which toxins combine with protoplasm has

IMMUNITY. 187

been shown in the case of tetanus toxin. The filtrate from
cultures of tetanus bacilli will kill guinea-pigs, presumably
by damage to the central nervous system. The same filtrate
rubbed up with brain or spinal cord has been found to have
lost its toxic proporties. It may be assumed that the poison
has combined with the protoplasm of the cells.

Bacteriolysis. — Although the hypothesis of Ehrlich just
stated may seem very complicated, more recent studies along
similar lines have led to conceptions so intricate, that they
can be followed only with the greatest difficulty. The
nomenclature of this subject has also become extremely
involved, as different words have often been coined to con-
vey a similar idea. In other cases a single word has been
used in different senses.

The problems we have still to consider relate to the bac-
teria which do not produce soluble toxins. In infections
with these organisms, attempts to make protective or cura-
tive substances analogous to antitoxins have met with little
success. The outlook for better results in the future is
promising.

The agencies which lead to the destruction of bacteria
during the progress of an infectious disease have been
studied chiefly in experimental infections in the lower ani-
mals. There are numerous bacteria, such as the spirillum
of cholera, bacillus of typhoid and bacillus coli communis,
injection of which may produce a fatal septicemia in ani-
mals. It also usually is possible to render animals
immune to them by injections of the dead bacteria, or
small doses of living bacteria, or the two in succession.
When an animal has been immunized in this manner, in-
jection of living cultures of the same kind is followed by
destruction of the bacteria injected. This phenomenon
appears to depend on the combined action of two substances.
Neither of these is effective without the other. The actual

1 88 MANUAL OF BACTERIOLOGY.

destruction of bacteria (bacteriolysis) is performed by the
complement, an unstable body, destroyed at quite low tem-
peratures (about 60° C.). The union of the complement
with the bacterial cell requires the presence of an inter-
mediary body, which is more stable, and more resistant to
heat.

Some of the synonyms for these terms are :

Complement, Intermediary Body,

Addiment, Amboceptor,

Cytase, Immune Body,

Alexin, Substance Sensibilitrice.

The nature of complements and intermediary bodies has
in a large measure been worked out by the study of im-
munity to substances not directly related with bacteriology.
Among these the blood1 of various animals and snake
venom2 are notable examples.

The substances developed or increased by immunization
are the intermediary bodies, which have, therefore, also been
called immune bodies. Their ultimate source must have
been in the cells of the host. They may be supposed to have
been receptors originally, which were cast off, after the
manner of antitoxins. They are specific for the particular
kind of organism used in developing immunity. Whether
or not various kinds of complements exist is still in doubt.

Metchnikoff holds that while intermediary bodies may
occur in blood-serum, the complement exists within leuco-
cytes and other forms of cells. Union of intermediary
bodies with the molecules of a bacterial cell, permits de-
struction of the bacteria by the complement in the phagocyte.

1 Prtidden, Medical Record, February 14, 1903.

2 Flexner and Noguchi, " Snake Venom in Relation to Hemolysis,
etc.," Journal Experimental M'edicine. Vol. VI., 1902, p. 277. Univer-
sity of Pennsylvania Medical Bulletin, November; 1902.

IMMUNITY. 189

Disintegration of leucocytes may give rise to the presence
of complement in the serum.

An example of the destruction of bacteria in immunized
animals is seen in an experiment performed by Pfeiffer. A
guinea-pig is given repeated injections of the spirilla of
cholera, which have been killed by chloroform or heat.
When living spirilla are introduced into the peritoneal
cavity of such an immunized guinea-pig they rapidly
undergo disintegration. Since no such disintegration takes
place when other bacteria than the spirilla of cholera are in-
jected into the animal made immune from this organism, it
has been suggested by Pfeiffer that this reaction could be
made use of in the diagnosis of that disease. If the serum
of the immune animal be introduced along with the cholera
spirilla into the peritoneal cavity of an animal not immune,
the same disintegration takes place. Furthermore the serum
of the immune animal may be heated to 70° C. and will still
cause disintegration of the organisms in the peritoneum
of a non-immune guinea-pig. The heated serum alone is
found inactive. The explanation of this phenomenon ap-
pears to be that the serum of the normal animal contains
the complement only. The serum of the immunized animal
has developed in it the intermediary or immune body, besides
the normal complement. The immune body resists heat,
while the complement is destroyed by heat. The previously
heated serum from the immunized animal, mixed with
organisms, finds the necessary complement in the normal
animal. The organisms then become disintegrated.

Such disintegration of bacteria is called bacteriolysis.
The substances which effect it are called lysins. It is prob-
able that the development of lysins is one of the most im-
portant factors in checking infections and in promoting
recovery. The preparation of specific bacteriolytic blood-
serum from immunized animals has been attempted for the

190 MANUAL OF BACTERIOLOGY.

treatment of various diseases. The partial or complete fail-
ure of such efforts may be ascribed to the fact that the
serum prepared does not contain both the complement and
the intermediary or immune body in proper kinds and pro-
portions to be effective. There is likely to be a deficiency
of complement. Investigators are at present very hopeful
of being able to make up for this deficiency. The outlook
is encouraging at the present time particularly with regard
to typhoid fever and dysentery.

Welch's Hypothesis. — When bacteria do not form sol-
uble toxins, we are obliged to suppose that large numbers
of them must disintegrate in the body of the host. Thus
their intracellular toxins may be liberated and produce the
symptoms of intoxication manifested in infections of this
character. Welch has also suggested that production of
toxins may occur in infection of the living body even when
test-tube cultures of the same bacteria contain no soluble
toxins or only small amounts. Bacteria growing as para-
sites in the living body may adapt themselves to the condi-
tions found there. The existence of the bacteria requires
them to have some means of combating the substances
elaborated by the host for his protection. Bacteria may
also be able to form similar substances for their own protec-
tion. Such substances, from the standpoint of the host,
would constitute toxins.1

Nuttall made the important discovery that the serum of
the blood deprived of all cells possesses the power of destroy-
ing pathogenic bacteria.

The ingredients of the blood-serum that exert the bacteri-
cidal influence have been named alexins,2 they have also
been called defensive proteids. The nature of these sub-
stances has not been determined with certainty. Vaughan

1 Welch, Bulletin Johns Hopkins Hospital, December, 1902.

2 This word has unfortunately been used partly in another sense, sec
page 1 88.

IMMUNITY.

has shown that they may be in part nucleins. He has found
that blood-serum contains nuclein and that nuclein has
germicidal power. Such substances apparently serve as
safeguards to the body against all kinds of bacterial in-
vasion. They have not necessarily a specific action as
regards any particular kind of infection.

It now appears that the bactericidal power of blood-serum
also depends in a large measure upon the joint operation of
complements and intermediary bodies.

The amount of intermediary body appears to be more
constant than that of the complement. Flexner1 found
that the blood-serum from cases of chronic diseases had
diminished bactericidal power for the staphylococcus pyo-
genes aureus. His observations were regarded as explain-
ing the liability of these patients to terminal infections.
(See page 169.) Longcope2 has since shown that in such
affections as chronic nephritis, cirrhosis of the liver and
diabetes the complement, active in destroying bacteria, is
diminished. Abbott and Bergey3 proved that the comple-
ment active in disintegrating the blood-corpuscles of another
species is reduced in quantity by alcoholic poisoning. Their
conclusions are important taken in connection with Abbott's
previous work on the effect of alcohol in lowering the re-
sisting power to infection (see page 166).

Agglutinins.4 — It has been shown that the blood-serum
of patients having typhoid fever contains a substance which,
when mixed with living typhoid bacilli, causes them to
gather into groups or clumps and at the same time to lose
their motility. In the great majority of cases no such
clumping occurs when the blood of typhoid cases is mixed

1 Journal of Experimental Medicine, Vol. L, 1896, p. 21.

2 University of Pennsylvania Medical Bulletin, November, 1902, p. 331.

3 University of Pennsylvania Medical Bulletin, Aug.-Sept., 1902, p. 186.

4 Sailer, University of Pennsylvania, Medical Bulletin, Aug.-Sept.,
1902.

IQ2 MANUAL OF BACTERIOLOGY.

with other bacteria than the bacilli of typhoid fever. The
nature of this agglutinating substance, as it is called, is not
known nor is its significance understood. It has been
applied to the diagnosis of typhoid fever, where it is called
the " serum-reaction," and will be discussed in connection
with the bacilli of typhoid fever. Similar agglutinating
bodies form in many other infections. Bacteria that are not
motile may nevertheless be agglutinated. Among the in-
fections where such a reaction occurs, the following are
noteworthy — with the cholera spirillum, bacillus pyocya-
neus, bacillus proteus, bacillus coli communis, micrococcus
melitensis, bacillus of glanders, bacillus tuberculosis, diplo-
coccus of pneumonia, bacillus of bubonic plague, and the
bacillus of dysentery (Shiga). The protozoon, Try-
panosoma (see appendix), is said to become agglutinated
in the blood of infected rats. Not all of these have been
studied sufficiently to be available for diagnostic purposes.
It has not yet been shown that agglutinins are concerned in
producing immunity or recovery from infectious diseases.
These substances are relatively resistant to heat, not being
destroyed by temperatures below 70° C.

Under conditions similar to those under which agglutina-
tion occurs, bacteria have been observed to form in long
filaments. This is the so-called " thread-reaction." Its sig-
nificance is uncertain.

Precipitins. —After repeated injections of albumins for-
eign to it, an animal's fluids undergo still another form of
adaptation. Substances are now developed in the blood-
serum, which cause precipitates to form where it is mixed
with the foreign albumin.1 Thus a rabbit may be immu-

1 These reactions have been most fully studied in connection with the
precipitation of blood-serum of a particular animal by the serum of
another species which has been immunized to the serum of the first
animal. The principle is of some importance in medico-legal work
for the identification of human blood, as in stains on clothing. See
Nuttall, Journal of Hygiene, Vol. I., 1901.

193

IMMUNITY.

nized or adapted to hen's egg-albumen. The rabbit's serum
will then precipitate hen's egg-albumen, but no other form
of albumin. It may, however, imperfectly precipitate al-
bumen from the egg of a species closely allied to the henr
It has not been shown that this property plays any important
part in bacteriology, though that is not improbable.

194 MANUAL OF BACTERIOLOGY.

CHAPTER VIII.

DISINFECTANTS AND ANTISEPTICS.1

A disinfectant or germicide is a substance capable of
killing bacteria. The latter term is of more recent develop-
ment than the former, and apparently needed on account
of the loose application of the term disinfectant.

An antiseptic is a substance capable of preventing the
growth and reproduction of bacteria. It differs from a
disinfectant or germicide in that it simply prevents devel-
opment without actually killing.

A deodorizer is a substance capable of so changing a
noxious odor that it is less unpleasant to the sense of smell.
At the present time the term is usually and properly re-
stricted to those substances which, without disinfectant ac-
tion, simply replace or destroy an odor.

TESTING ANTISEPTICS AND DISINFECTANTS.

The determination of the antiseptic value of a material
is a comparatively simple matter. A virulent culture of
the organism used as a test is inoculated into sterile bouil-
lon containing a known quantity of the antiseptic. The
process is repeated with varying strengths of the material
until the smallest quantity of it capable of preventing
growth is determined. This dilution may be considered
the antiseptic value of the material in question for the organ-
ism used, under the conditions of the test.

1 By Thomas B. Carpenter, M.D., Assistant City Bacteriologist,
Buffalo, N. Y.

DISINFECTANTS AND ANTISEPTICS. IQ5

Determination of the disinfectant power of a substance
is a problem of much greater magnitude, and the method
used must be altered more or less to suit the properties of
the substance tested. It is obvious that some of the sub-
stance tested remains in contact with the organisms in the
method given for determining the antiseptic value, and
that we do not know whether the bacteria are alive and
merely inhibited in growth, or actually killed.

Sternbergs Method. — To a measured quantity of a
virulent bouillon-culture of the test-organism is added a
known quantity of the substance to be tested. After vary-
ing lengths of time inoculations are made from this mixture
into culture-media, preferably bouillon, and growth watched
for under suitable conditions as to temperature and the like.
The shortest exposure of the test-organism to the smallest
quantity of the substance is taken as the germicidal value
of that substance for the particular organism used.

Koch's Method. — Usually employed for spore-bearing
bacteria like the bacillus of anthrax. The hay bacillus is
convenient to use when experiments are being made by
large classes of students. Small pieces of sterile silk or
cotton thread are soaked for some hours in a bouillon-cul-
ture of the test-organisms. They are removed, partially
dried, and then placed in a solution of known strength of
the substance being tested, and exposed for a definite
length of time. The thread is removed from the solution,
• washed carefully in sterile water, planted in bouillon, and
growth is watched for. As in other methods, the greatest
dilution of the germicide that will kill the test-organism in
the shortest time is taken as the germicidal value of that
substance for the organism used.

Hill's Method. — The test organism is dried upon the end
of a sterile glass rod contained in a sterile test-tube, the end
of the rod projecting through a cotton plug. The end of
17

196 MANUAL OF BACTERIOLOGY.

the rod is inoculated with a small amount of a fluid culture
of the test organism and allowed to dry. It is then ready
to test by exposure to any disinfectant, either liquid or
gaseous.

All of these methods are open to serious sources of error,
particularly in the testing of powerful germicides. In
Sternberg's method, small quantities of the substances
tested may be carried over with the organisms, and, if a
powerful germicide, in sufficient amount to prevent growth,
and thus give erroneous results. In Koch's or Hill's
method this factor may be partially obviated by washing in
sterile water after exposure to the germicide. This does not
remove another source of error, namely, the chemical action
that may take place between the substance and the proto-
plasmic contents of the bacterial cell. This action may ex-
tend deeply enough to restrain the growth of an organism
for a very long time without actually killing it. When
placed under suitable conditions, such union may be broken
up and the organism regain its power to develop. It has
been suggested that, to remove errors in the above methods,
test-cultures containing bacteria supposed to be killed by
the smallest quantity of germicide be inoculated into sus-
ceptible animals; but Sternberg's experiments in this direc-
tion have shown that bacteria may become so altered in
virulence by the action of germicides insufficient to kill, that
animal inoculation experiments are worthless.

Geppert suggested a valuable modification of these
methods while determining the germicidal value of bichlo-
ride of mercury. After exposing his test-organism to
bichloride of mercury, and before inoculating into bouillon
to determine death of the organism, he treated with a dilute
ammonium sulphide solution, thus effectually neutralizing
any mercury-salt remaining.

Sedgwick developed this method still further, and to him

DISINFECTANTS AND ANTISEPTICS. 197

we are indebted for demonstrating its accuracy and "practi-
cability.

Method. — To 15 c.c. of sterile water in a 60 c.c. Erlen-
meyer flask add 2 c.c. of a virulent culture of the test-
organism. Then add a solution of the substance under in-
vestigation in the proportion necessary to give the dilution
wished. Mix thoroughly, and allow this " action-flask " to
stand as long as it is desired to have the germicide in con-
tact with the test-organism (action-period). Transfer 0.5
c.c. from the action-flask to a flask containing 200 c.c. of a
solution of some chemical capable of decomposing the sub-
stance being tested with the formation of inert or insoluble
compounds. In this " inhibition-flask " the strength of the
solution should be such that molecular proportions of the
chemical are present in sufficient quantity to combine with
all the germicide carried over. The inhibition-flask is
shaken for 30 seconds, and i c.c. transferred from it to 100
c.c. of sterile water in another, the " dilution-flask." After
two minutes, three agar tubes are inoculated with i c.c. each
from the dilution-flask, plated, and growth watched for.

Control-experiments should be performed to determine
that the dilution of the test-culture is not too great when
carried through the three flasks. It likewise should be de-
termined that the inhibiting chemical has no effect on the
bacteria.

What the inhibiting chemical shall be must be deter-
mined for each individual case. For salts of the heavy
metals ammonium sulphide answers well ; for mercury salts,
stannous chloride may be used; for formaldehyde, ammo-
nium hydrate; for carbolic acid, sodium sulphate.

The testing of gaseous disinfectants, such as sulphur
dioxide and formaldehyde, must be conducted under condi-
tions as nearly parallel to actual practice as possible. The
test-organisms may be exposed on threads or glass rods, and

198 MANUAL OF BACTERIOLOGY.

acted upon by a known volume strength of disinfectant for
a known length of time. Subsequent treatment of the
organisms with a suitable inhibitor is necessary when pos-
sible, and should growth occur in the cultures following, the
test-organism should be recognized in order that possible
contamination by extraneous organisms may be excluded.

In determining the value of germicides for sterilizing
ligatures, the students can apply methods based on the fore-
going principles. Great care and ingenuity are necessary
to arrive at correct conclusions, particularly in the case of
animal tendons. In many instances quite stable compounds
are formed between tendon and germicide, and living or-
ganisms may be so imbedded in such a substance that sub-
sequent growth in a test-culture is impossible. The use of
a suitable inhibitor, and, prior to final culture-tests, a pro-
longed soaking in sterile water, will promote the accuracy
of the results.

CHEMICAL DISINFECTION. 1

Heat properly applied is the simplest and at the same
time the surest disinfectant (see Part I., Chapter II.) ; but
for many purposes it cannot be used, and we have recourse
to those chemicals that practice and investigation have
shown to be of value. The efficiency of chemical disin-
fectants as ordinarily used is over-rated. An immense
number of substances possess germicidal properties, but un-
fortunately, the majority are objectionable in that they are
expensive, intensely poisonous, or so corrosive that damage
may be done to articles of value with which they may come
in contact.

In the following pages only those substances which are
in common use or seem to be of special value will be con-
sidered.

1 For fuller details on this subject consult Rosenau, "Disinfection and
Disinfectants," 1902.

DISINFECTANTS AND ANTISEPTICS.

Mercuric Chloride or Bichloride of Mercury. — This
substance is probably more commonly used than any other
one disinfectant. In the strength of i-iooo it will some-
times kill the spores of anthrax within a few minutes (see
Bacillus anthracis, Part IV.). It is claimed that its affinity
for albuminous bodies, and the readiness with which it
combines with such substances, detracts from its value for
some purposes. On the other hand, many observers claim
that the albuminous combinations formed under such cir-
cumstances are soluble in an excess of albuminous fluid,
and that its value as a germicide is not affected thereby.
To obviate this possible difficulty it is customary in prac-
tice to combine the bichloride of mercury with some sub-
stance that will prevent the precipitation of the mercury
salt by albumin. For this purpose 5 parts of any one of
the following substances to i part of bichloride of mercury
may be used — hydrochloric acid, tartaric acid, sodium
chloride, potassium chloride or ammonium chloride. A
very practical stock-solution for laboratory purposes has
the following composition :

Hydrochloric acid 100 c.c.

Bichloride of mercury 20 grams.

5 c.c. in a liter of water makes a solution of about i-iooo strength.

Mercuric Iodide. — An extremely high antiseptic value
has been placed on this substance by Miquel, who claims
that the most resistant spores are prevented from develop-
ing in a culture-medium containing 1-40,000. In combi-
nation, as potassio-mercuric iodide, it has been used in
soaps (McClintock) with very favorable results. The sub-
stance is not extensively employed, and further investiga-
tion is necessary to determine its true value.

Attempts are being made to manufacture combinations
of mercury and other powerful metallic germicides with
organic acid and basic bodies, the purpose being to utilize

2OO MANUAL OF BACTERIOLOGY.

the metallic base in greater strength without injury to the
living tissues. Such compounds are exemplified by uicr-
curol, said to be a combination of mercury with nucleinic
acid, and to possess active germicidal properties, great
penetrating power and no injurious effect on living tissue.
It is also said to have a particularly destructive action upon
the gonococcus.

Silver Nitrate. — This salt probably occupies the next
position to the bichloride of mercury in disinfectant power.
Behring claims it to be superior to bichloride of mercury
in albuminous fluids. The anthrax bacillus is killed by a
solution of 1-20,000 after two hours' exposure. At least
forty-eight hours' exposure to a i-i 0,000 solution is re-
quired to kill the spores of anthrax. It is very irritating,
and possesses strong affinities for chlorides, forming with
them, insoluble chloride of silver, a salt without germicidal
value. For these reasons the use of silver nitrate is lim-
ited. In the solutions usually employed for douching the
cavities of the body, the available silver nitrate is imme-
diately converted into the insoluble chloride, and little if
any germicidal action takes place. To this fact may be
ascribed the varying clinical results reported.

Many semi-proprietary silver compounds are on the mar-
ket, introduced to replace the nitrate and its objection-
able features. The most important are argentamin, ar-
gonin, protargol and argyrol, all organic silver combina-
tions. They do not combine with chlorides, are less irritat-
ing than the nitrate, and, not coagulating albumin, they pos-
sess greater penetrating power. Clinical reports and in-
vestigations have been so contradictory thus far that their
value cannot be readily estimated.

Carbolic Acid. — One of the most important and most
widely-used disinfectants. It is usually employed in
strengths of from i to 5 per cent. A 3 per cent, solution

DISINFECTANTS AND ANTISEPTICS. 2OI

will sometimes kill the spores of anthrax after two days'
exposure (see Bacillus anthracis, Part IV.)- In the ab-
sence of spores the anthrax bacillus is destroyed by a I per
cent, solution in one hour. The less resistant pus cocci are
destroyed rapidly by a 2 per cent, solution. Combination
with an equal proportion of hydrochloric acid enhances the
efficacy of carbolic acid to a marked extent. This is due
to the prevention of albuminous combinations, thus allow-
ing- greater penetration of the disinfectant.

Many other substances closely related to carbolic acid
are used and possess marked germicidal properties.
Among them may be mentioned creolin, cresol and lysol.
They are all slightly superior to carbolic acid in actual
germicidal value.

Aniline Dyes. — Many of these substances possess germ-
icidal properties, notably pyoktanin (methyl-violet). A
solution of 1-5000 will kill the anthrax bacillus in two
hours. A much stronger solution, 1-150, is required to
kill the typhoid bacillus in the same time. Malachite-
green is said to possess even greater germicidal value than
pyoktanin. Methylene-blue also possesses considerable
germicidal power.'

Formaldehyde. — A gaseous substance placed on the
market in a 40 per cent, aqueous solution. Remarkable
claims have been made for this substance, and numerous
investigations have shown it to possess, both in the liquid
and gaseous forms, wonderful disinfecting power under
certain conditions. It is a noticeable fact that the more
recent the investigation the lower the value placed upon it.
In solutions of i-iooo an exposure of twenty-four hours is
necessary to destroy the staphylococcus pyogenes aureus,
while 1-5000 is sufficient to restrain its growth (Slater
and Rideal). Its use in a gaseous form as a house-disin-

202 MANUAL OF BACTERIOLOGY.

fectant is by far the most important application at the
present time.

Harrington's investigations have shown that an atmosphere produced
by vaporizing 435 c.c. of formalin (40 per cent, aqueous solution of
the gas) in 1000 cubic feet of air space, equivalent to i quart to a room
15 feet square and 10 feet high, will destroy all exposed organisms in
half an hour ; when protected by one fold of cotton-cloth, an exposure
of one and one-half hours is necessary. In a perfectly dry atmosphere
the gas penetrates slightly, and will disinfect through one layer of
cotton-cloth; in a moist atmosphere no penetration can be obtained.

In vaporizing the gas many methods have been em-
ployed. Simple evaporation of solutions without heat
cannot be relied upon, for the solid, polymerized para-
formaldehyde is easily formed under these circumstances.
Better results can be obtained with the aid of heat, although
polymerization is apt to occur unless evaporation is rapid.
To produce the best results it has been found necessary to
use special forms of lamps or generators for its production,
a few of which are mentioned below.

Sanitary Construction Company's Lamp. — This lamp
consists of a tank to hold the formaldehyde solution, and a
spiral tube by which the solution is slowly conducted through
a flame and vaporized. The necessary amount of solution
is placed in the tank and the apparatus started, outside
the room, the gas being conducted through the keyhole by
a suitable tube.

Trillat Autoclave. — A small silver-lined pressure-boiler,
fitted with lamp, safety-valve, pressure-gauge, thermome-
ter and escapement-tube. The necessary amount of form-
aldehyde solution is placed within the apparatus, together
with an equal amount of 20 per cent, solution of calcium
chloride ; the addition of the latter salt is to prevent forma-
tion of the solid polymeric modification, the so-called para-
form. The autoclave is closed and heated from below to
a temperature of 135° C. The escapement-valve is then
opened carefully and the gas allowed to enter the room

DISINFECTANTS AND ANTISEPTICS. 203

slowly through the escapement-tube, which has meanwhile
been passed through the keyhole. About thirty minutes
are required to discharge all the gas from 500 c.c. of solu-
tion. If the temperature has not been allowed to go above
135° C. the gas will contain but little moisture and possess
its maximum efficiency.

Schering Lamp. — This lamp is intended to utilize para-
form or para-formaldehyde, a polymeric modification of
formaldehye, occurring as a white salt. It is decompos-
able by heat, yielding formaldehyde gas. It is placed on
the market in the form of tablets, each one of which yields
a definite amount of gas. The lamp consists of a small
iron tray for the reception of tablets, and so arranged above
the heating-apparatus that sufficient draught is created to
carry off the gas as rapidly as formed. In operating, a
sufficient number of tablets are placed on the tray, the lamp
lighted and placed in the room to be disinfected.

Methyl-Alcohol Lamps. — Several of these lamps are on
the market, all operating on the well-known principle of
the oxidation of wood-alcohol to formaldehyde when the
alcohol is vaporized by projection against a heated, plati-
nized, asbestos disk. In operating such an apparatus, the
alcohol is lighted until the asbestos disk becomes hot. The
flame is then extinguished; the heat from the disk is suffi-
cient to vaporize the alcohol, which undergoes oxidation
and keeps the disk at a red heat. When the apparatus is
operating in a satisfactory manner the room is closed and
disinfection allowed to proceed. It must be said, however,
that it is difficult to estimate or control the amount of form-
aldehyde evolved in generators of this type.

Formaldehyde Candles. — Mixtures of para-formaldehyde

and paraffin or other combustibles, which may be moulded

into candles, each enclosed in a tin case, make a convenient

apparatus to generate formaldehyde gas for room disinfec-

204 MANUAL OF BACTERIOLOGY.

tion. The candle is placed in a suitable fire-proof dish,
it is then ignited, and generation of the gas is allowed to
proceed in the tightly closed room.

Sulphur Dioxide. — This substance is used extensively
for house disinfection, and is usually prepared by burning
sulphur. Much difference of opinion exists regarding the
value of it as a disinfectant. The spores of anthrax are
not killed by several days' exposure to the liquefied gas.
Anthrax and other bacilli are destroyed in thirty minutes
when exposed on moist threads in an atmosphere contain-
ing one volume per centum of the gas. An exposure of
twenty-four hours in an atmosphere containing four vol-
umes per centum of the gas will destroy the organisms of
typhoid fever, diphtheria, cholera and tuberculosis. The
presence of moisture greatly enhances the activity of the
disinfectant, owing to the formation of the more energetic
sulphurous acid.

For the destruction of insects, such as mosquitoes, this
agent is superior to formaldehyde. Its application for this
purpose is important in preventing the spread of yellow
fever and malaria.

In practice, at least 3 pounds of sulphur per 1000 cubic
feet should be used, and moisture must be present. This
latter requirement can be fulfilled by evaporating several
quarts of water within the tightly closed room just prior to
generating the gas. In using powdered or flowers of sul-
phur, the necessary amount is placed on a bed of sand or
ashes in an iron pot, which should rest on a couple of
bricks in a pan or other vessel containing an inch or two
of water. The sulphur is ignited by means of some glow-
ing coals, or by moistening with alcohol and applying a
a match. Difficulty is often experienced in keeping the sul-
phur burning, and for this reason it is surer and more con-
venient to use the so-called sulphur candles now on the

DISINFECTANTS AND ANTISEPTICS. 2C«5

market. In operating with these, a sufficient number are
placed on bricks in a pan of water and the wicks lighted.
Liquefied sulphur dioxide may be used, and can now be
obtained in convenient tin receptacles containing a suffi-
cient quantity for the disinfection of an ordinary room.
The can is opened by cutting through a soft metal tube
projecting from the top. The fluid vaporizes at the room
temperature, and it is simply necessary to place the can in a
convenient porcelain dish and allow the fluid to evaporate.

Sulphur dioxide is objectionable on account of its lack
of power when dry, and on account of its corrosive action
on metal and its bleaching effect on hangings and draperies
in the presence of moisture; it is, therefore, preferable to
use formaldehyde when possible.

Chlorine. — A very active gaseous disinfectant, particu-
larly in the presence of moisture. An atmosphere con-
taining i per cent, of the dry gas is fatal to anthrax spores
in three hours. The anthrax bacillus is killed in twenty-
four hours by exposure to a moist atmosphere containing
the gas in the proportion of 1-2500. The bacillus of tuber-
culosis is killed by an exposure of one hour to a moist atmos-
phere containing the gas in the proportion of 1-200. Ex-
tremely minute quantities in solution will prevent the
development of putrefactive organisms. The substance
has been used for house and ship disinfection, but is now
seldom employed on account of its extremely irritating
properties and the difficulty of handling it.

Bromine. — Used in the gaseous and liquid form. The
dry vapor possesses but little disinfectant power; when
moist it is much more efficient. In saturated aqueous so-
lution it will kill the anthrax bacillus in twenty-four hours.

Calcium Hypochlorite, usually known as Chloride of
Lime. — This is a most practical and valuable disinfectant,
depending for its efficiency on the available chlorine con-

206 MANUAL OF BACTERIOLOGY.

tained in it. Its alkalinity favors penetration, and for
many purposes it cannot be excelled. A I per cent, solu-
tion will destroy anthrax spores in one hour. A solution
of the same strength will disinfect typhoid stools in ten
minutes.

Lime. — The addition of o.i per cent, of unslaked lime
to fluid-cultures of the typhoid bacillus and cholera spiril-
lum will render them sterile in four or five hours. Ty-
phoid dejecta are sterilized in six hours by the addition of
3 per cent, of slaked lime; the addition of 6 per cent, will
accomplish the same result in two hours. A convenient
form for practical use is an aqueous mixture containing 20
per cent, of lime — so-called milk of lime. Typhoid and
cholera dejecta are sterilized in one hour after the addition
of 20 per cent, of this mixture. In practice it is safer to
use a considerable excess of lime. From the foregoing facts
it would seem probable that lime or whitewash as ordi-
narily applied would possess disinfectant properties. Ex-
perimental work has demonstrated this to be a fact. The
organisms of anthrax, glanders and the pus cocci were
destroyed within twenty-four hours by one application.
For spore-forming organisms and the bacillus of tubercu-
losis the power is not so great, the latter organism not
being destroyed by three applications of the whitewash.
This is due, perhaps, to the large amount of fatty matter
in the bacillus of tuberculosis, and suggests the possibility
of enhancing the efficacy of the lime by the addition of a
small proportion of caustic alkali.

Hydrogen Peroxide. — This substance is placed on the
market in solutions varying in strength from 10 to 30 vol-
umes; the mode of expression indicating that correspond-
ing solutions will liberate ten to thirty times their volume
of oxygen when appropriately treated. It possesses the
property of rapidly oxidizing purulent secretions, and on

DISINFECTANTS AND ANTISEPTICS. 2OJ

this account is much used for cleansing infected wounds.
It deteriorates in strength so rapidly that only fresh solu-
tions of known strength should be used.

Potassium Permanganate. — Koch asserts that a 3 per
cent, solution will destroy anthrax spores in twenty-four
hours, but that a i per cent, solution cannot be depended
upon to kill pathogenic organisms. Its disinfectant value in
practice is very low on account of its ready decomposition
by inert material. In the dilute solutions usually used for
medicinal injections and irrigations no disinfectant action
occurs.

lodoform. — This substance possesses little if any disin-
fectant power. It is mildly antiseptic in moist wounds,
due to the gradual liberation of small quantities of iodine.

Boric Acid. — This material possesses practically no dis-
infectant power. It is a mild antiseptic when applied as
an undiluted powder to wounds. A saturated aqueous
solution is much used, and is weakly antiseptic.

Essential Oils. — Many of these bodies possess germi-
cidal value, notably the oils of cinnamon and cloves. The
oil of mustard is also a valuable disinfectant, but so irri-
tating that the pure oil cannot be used. The use of pow-
dered mustard in the autopsy-room will remove the foul
odor from the hands more rapidly and completely than any
other means.

Coal Oil or Petroleum. — While the disinfectant value of
this substance is slight, its use in destroying the larvae of
insects, such as the mosquito, has given it an important
position in preventing the spread of malaria and yellow
fever. A small amount poured on a stagnant pool rapidly
spreads over the surface, and effectually destroys such
larvae.

Ferrous Sulphate (Copperas). — This salt has been much
used, but possesses only feeble disinfectant powers. A 3

208 MANUAL OF BACTERIOLOGY.

per cent, solution requires three days to kill the bacillus of
typhoid fever. On account of its affinity for ammonia and
sulphides it is an efficient deodorizer for temporary use, but
cannot be relied upon to kill the bacteria producing the
noxious gases.

Cupric Sulphate (Blue Vitriol}. — This salt is quite an
efficient disinfectant. In a solution of 1-3000 the spirillum
of cholera is destroyed in ten minutes. A 5 per cent, solu-
tion will kill the typhoid bacillus in ten minutes. A solu-
tion of from 2 to 3 per cent, in strength can be relied upon
to destroy all pathogenic organisms that do not form spores.

Zinc Sulphate. — This salt is a very feeble disinfectant.
Pus cocci are not destroyed in two hours by a 20 per cent,
solution. As a deodorizer it has about the same value and
acts in the same way as ferrous sulphate.

Zinc Chloride. — A 2 per cent, solution will kill pus cocci
after an exposure of two hours. It is therefore a much
more powerful disinfectant than the sulphate.

Disinfection of Dejecta and Urine. — A 4 per cent, solu-
tion of calcic hypochlorite (chloride of lime) is most effi-
cient and rapid for this purpose. A convenient solution
contains 6 ounces of the salt to i gallon of water. The
excreta should be received in a suitable vessel and imme-
diately mixed with an equal bulk of the disinfectant. The
contents of the vessel should be allowed to stand for one
hour before emptying. A 20 per cent, milk of lime is just
as efficient, and possesses the advantage of cleanliness and
lack of odor. It should be used in the same quantity and
allowed to act for the same length of time. A 5 per cent,
solution of carbolic acid may be used, but should be allowed
to act for at least four hours.

Disinfection of Sputum. — The chemical disinfection of
tuberculous sputum is somewhat difficult on account of the
large amount of albumin in it and the fatty matter associated

DISINFECTANTS AND ANTISEPTICS. 2(X)

with the bacillus of tuberculosis. Dilute solutions of bi-
chloride of mercury are apt to be decomposed and rendered
inert by the albumin. Carbolic acid is open to the same
objection, but its combination with hydrochloric acid can
be used successfully in a strength of 5 per cent. each.
Milk of lime cannot be relied upon for this purpose. A
4 per cent, solution of calcic hypochlorite (chloride of lime)
is the best for general use, and the spit-cup should be kept
nearly full of this solution. Sputum may also be disinfected
by exposure to the action of steam in the steam sterilizer
or by boiling for 15 minutes. If napkins or old pieces of
cloth are used for the reception of sputum they may be
immediately destroyed in a fire.

Disinfection after Postmortems. — After autopsies on
infectious cases it is necessary to disinfect the table and
fluid products coming from it prior to emptying into the
sewer. The table may be successfully disinfected by a
liberal sprinkling with 4 per cent, calcic hypochlorite solu-
tion. All fluids should be treated with an equal quantity
of the same solution. The table should not be cleaned for
at least one hour after application of the disinfectant. The
same rule applies to the disinfection of the fluids — an ex-
posure of at least one hour to the disinfectant before final
disposition.

The Cadaver in Contagions Diseases. — In cases of death
from a contagious disease all the orifices of the body should
be packed with cotton soaked in a strong solution ( i to 500)
of bichloride of mercury, the skin washed with a i to 1000
solution, and the cadaver wrapped in a sheet wet with the
same. The funeral should be private and the body dis-
posed of within twenty-four hours, preferably by cremation.

House Disinfection. — After infectious disease it is essen-
tial that the house or the apartment in which the patient
has been confined should be disinfected. It is rarely neces-

2IO MANUAL OF BACTERIOLOGY.

sary to carry out the process in more than two rooms ; but
should it be so, the process can be applied to the whole
house.

After thorough bathing of the patient, preferably with
an antiseptic soap, the individual should be wrapped in a
clean sheet and removed to a clean room. All articles or
materials that are of little value should be destroyed. All
bedding, towels and the like should be placed in wooden
tubs and covered with a i-iooo solution of bichloride of
mercury. The room should then be made as nearly air-
tight as possible ; this can be accomplished by pasting strips
of paper over registers, cracks, spaces between window-
sashes and the like. Formaldehyde gas is then passed
through the keyhole into the room (or it may be generated
by formaldehyde candles) in sufficient quantity to destroy
the infectious element. The room should be sealed for at
least twelve hours, after which time it may be opened and
aired. The process is completed by washing all exposed
surfaces in the room with i-iooo bichloride of mercury.
This latter requirement is not essential if the gaseous disin-
fection has been complete, but since we have no absolute
knowledge on this point, the secondary washing should be
carried out. This method can be considered reliable for
surface disinfection, but for the interior of mattresses and
stuffed furniture-cushions it is not certain. In all cases
where absolute disinfection is demanded, such articles must
be ripped apart and loosely exposed to the gas. They may
be disposed of by fire or sterilized by steam under pressure.
The latter method must necessarily be a matter of municipal
control, and can only be carried out by means of suitable
apparatus in the hands of a municipal disinfecting corps.
Instead of formaldehyde, sulphur dioxide may be used for
room disinfection, but in the light of present knowledge the
formaldehyde method is superior.

PREPARATION OF INSTRUMENTS, ETC. 211

CHAPTER IX.

THE PREPARATION OF INSTRUMENTS, LIGATURES, DRESS-
INGS, ETC., FOR SURGICAL PURPOSES.1

THE purpose of this chapter is to explain the application
of the principles set forth on the preceding pages to sur-
gical technique. It has been shown that all objects about
us may have bacteria on them, and that bacteria are pres-
ent on all the surfaces of our bodies that come in contact
with the air. All the care that is needed in working with
bacteria in the laboratory, and more, must be exercised in
surgical operations. Everything that has not been steril-
ized must be regarded as having the possibilities of infec-
tion in it. After the hands have been cleansed, if they touch
the clothing or furniture, they must be cleansed again.
If a sterilized instrument falls on the floor, it must be ster-
ilized again. The same applies to dressings, sponges, liga-
tures, or anything which is to be used about the wound.

The value of chemical germicides has probably been over-
rated in the past. They are used only to destroy the bac-
teria on living tissues and on articles that would be damaged
by heat. They give less reliable results than boiling.
Wherever boiling or steam sterilization is permissible, it
should be used. With materials that may contain a small
quantity of substance in which bacteria can grow, the frac-
tional method of sterilization should be used (see page 63).
With glass and metallic objects, obviously a single boiling
can accomplish as much as boiling on three consecutive days.

1 By Marshall Clinton, M.D., Instructor in Clinical Surgery, Medical
Department, University of Buffalo.

212 MANUAL OF BACTERIOLOGY.

The failures in the practice of aseptic surgery are gen-
erally due to the hands of the operator and assistants and
the skin of the patient.

The following formulae have been selected from the many
published as they are successfully used by many surgeons,
and meet the theoretical grounds of bacteriology as far as
is possible with our present knowledge.

Sterilization of Hands. — There is no known method for
perfect sterilization of the human skin. A close approach
to sterility is reached by any one of the methods that has
as its basis mechanical cleanliness.

Park's method : ( i ) Hands and forearms are thoroughly
rubbed with a mixture of green soap and cornmeal, which
serves to remove all the loose dirt and epithelium. Rinse
carefully until hands and forearms are clean. (2) A paste
of mustard flour and cold water is rubbed into the hands
and forearms until they begin to sting. (3) Rinse in run-
ning sterile water; then soak in a hot i-iooo bichloride of
mercury solution for a few minutes, the fluid being well
rubbed into the skin.

FUrbringer's method : ( i ) Thorough scrubbing of the
hand and forearms with soft soap, water and a nail-brush
for at least three minutes, especial attention being paid to the
nails. (2) Removal of all fat and debris by rubbing hands
and forearms while immersed in 95 per cent, alcohol. (3)
Rinsing of hands and forearms in a i-iooo bichloride of
mercury solution, rubbing the fluid well into the skin.

Schatz's method : ( i ) Hands and forearms are cleansed
by brisk scrubbing with soft soap and a clean brush for
from three to five minutes. (2) Soaking in saturated solution
of permanganate of potassium at a temperature of 110° F.
until the hands and forearms are a deep mahogany brown.
(3) Immersion in a saturated solution of oxalic acid, tem-
perature of 110° F. until the skin is entirely decolorized.

PREPARATION OF INSTRUMENTS,, ETC. 213

(4) Rinsing with sterile lime water to rid of excess of acid.

(5) Washing in i-iooo bichloride of mercury solution for
one minute.

Weir's method : ( i ) Hands and forearms are scrubbed
as in other methods. (2) A scant tablespoonful of chlori-
nated lime is moistened with enough warm water to make a
thick paste. This is carefully rubbed into hands and fore-
arms. (3) A piece of carbonate of soda one inch square and
one-half inch thick is crushed and rubbed into the paste.
From three to five minutes are thus employed. (4) Rinsing
in sterile water and washing in a solution of £ of I per cent,
of ammonia, removes the odor of chlorine.

E. R. McGuire1 states that prolonged scrubbing with frequent changes
of brushes in running sterile water will give the nearest approach
to sterility. The use of antiseptics on the hands is not to be relied
upon, for their precipitation by chemicals or normal tissue fluids may
break up their combination with bacteria that were considered inactive
or dead, but are not so in reality. He suggests the use of hot-air, by
cabinet-bath or Kelly hot-air apparatus, to " sweat " out of the glands
in the skin as much as possible of their contents before the skin is
cleansed.

Prolonged soaking of the skin in a soap poultice or strong
antiseptic may damage and irritate the tissues, so that it is
not advisable to prepare the field of operation more than
twelve or twenty-four hours before the time set for an
operation.

Maylard2 recommends the sterilization of the skin by
inunctions of oleate of mercury. The method employed is
as follows : ( i ) Cleanse the skin in the usual way with soap
and water. (2) Anoint freely and widely with hydrated
lanolin-oleate of mercury, 20 per cent., and rub in; smear
a piece of gauze with the same and leave until a second
inunction is performed twelve hours later. Every case

1 American Medicine, February 28, 1903.

2 Annals of Surgery, January, 1902.

214 MANUAL OF BACTERIOLOGY.

should be treated for at least twenty-four hours before opera-
tion; preferably forty-eight hours should be given, with at
least two separate periods of " rubbing in " for about ten
minutes on each occasion. (3) On the operating table the
piece of gauze is removed, and the superfluous ointment
rubbed off with a piece of sterile gauze.

To Prepare the Field of Operation. — Wash with green
soap and water, scrubbing thoroughly and carefully, paying
particular attention not to scrub hard enough to render the
skin tender or to make abrasions. Shave parts with clean
razor. Wash with ether and alcohol, to remove debris and
epithelium, and cover with a sterile towel. If the skin of the
patient is thick a soap poultice may be left on, care being
taken to see that the skin does not become macerated. After
the patient is anesthetized the field is briskly scrubbed with
sterile brushes, soap, and water, washed with i-iooo
bichloride of mercury solution and covered with sterile
towels.

It is important to remember that during an operation
patient, operator and assistants, may perspire and that in this
way fresh masses of bacteria from the deeper parts of the
glands may be brought to the surface of the skin. Careful
attention must be paid to maintaining cleanliness during an
operation. The patient's skin is kept covered with sterile
towels, changed as often as they become soiled. For the
surgeon's and assistant's hands rubber gloves do this per-
fectly. If an operator or assistant finds that the hands per-
spire during an operation the use of rubber gloves becomes
essential. Rubber gloves may be sterilized by boiling.

Instruments are best sterilized by contact with super-
heated steam, or steam under pressure for ten minutes, or
by boiling in a I per cent, carbonate of soda solution. If
soda is unavailable use water that is actively boiling, as this
avoids spotting and rusting of the instruments. Imme-

PREPARATION OF INSTRUMENTS, ETC. 215

diately after use instruments should be thoroughly scrubbed
with a brush and washed with soap and hot water and
boiled, before being replaced in the instrument case.

The practice of passing an instrument through a flame
a few times cannot be relied on to destroy the bacteria that
may be present.

Aspirating syringes, needles, trocars, drainage tubes and
glass nozzles are best sterilized by boiling for ten minutes.
If syringes have leather washers (which should be avoided)
they may be cleansed with hot water and soap, rinsed with
alcohol, filled and refilled with boiling water ten or more
successive times, and placed in a 1-40 carbolic acid solution.

Instrument trays, ligature dishes, basins for sponges, etc.,
are to be sterilized by boiling for ten minutes, and protected
from dust with sterile towels.

Catgut is made from the intestines of sheep, and sheep are
subject to anthrax infection, while tetanus bacilli may occur
in the intestine. Therefore, catgut must be sterilized by
some method that will kill the spores of these organisms if
present (see Bacilli of anthrax and tetanus, Part IV.).
There are many methods devised for the preparation of
sterile catgut that have as a basis an incorporation within
the catgut of some antiseptic. They are open to the objec-
tion that any antiseptic introduced into the tissues acts as an
irritant, aside from the fact that organisms may be liberated
from partially absorbed catgut. This is seen in cases of
late suppuration — ten to fifteen days after an operation.

Catgut comes in sizes from double zero up to No. 8. The
sizes mostly used are o to 4. Catgut when ready for use
should be smooth, soft, pliable, and very strong ; wiry catgut
is apt to cut through tissues.

Cumol method.1 The catgut is rolled on glass spools, and
these put in a glass beaker. The bottom of the beaker is

1 Clark and Miller, Bulletin Johns Hopkins Hospital, Vol. XL, Sep-
tember, 1900.

2l6 MANUAL OF BACTERIOLOGY.

covered with a layer of cotton on which the catgut rests.
The beaker is heated with a Bunsen burner over a sand-
bath. The top of the beaker is covered with a piece of
cardboard. Through a hole in the center of the cardboard
a thermometer passes. Heat is now applied to the sand-
bath, and the temperature of the catgut slowly raised to
80° C. In this manner all moisture is driven out of the
catgut. This degree of heat is maintained for one hour.
Cumol1 at a temperature of 100° C. is now added to the
beaker, completely covering the catgut. The beaker should
be covered with copper-wire netting to prevent ignition of
the cumol, which is very inflammable. The temperature is
then increased to 165° C., and kept at this point for one
hour. The fluid is now poured off, and the catgut allowed
to dry in the beaker on the sand-bath at a temperature of
1 00° C. for two hours. It is then transferred to sterile jars
or test-tubes until needed, or it may be preserved in sterile
alcohol.

Formaldehyde catgut.2 Three-quarter-inch glass spools
are notched on each flange. The catgut is wound upon the
spool tightly in one layer, and evenly, the ends passing over
the flange of the spool in the notch; the longer end, after
passing through the notch, goes through the barrel of the
spool and is securely tied to the shorter end which has
passed over the other notched flange. By thus winding the
gut there will be enough for one or two ligatures or sutures
of good length. Gut prepared by this process tends to
contract forcibly, and on account of this strain must be
held securely or it will shrink and be useless. The object
of winding in a single layer, evenly, is to prevent over-
lapping or crossing of one strand over another. If in

1 Cumol is a fluid hydrocarbon, with a boiling point somewhat above
165° C. It dissolves the fat in catgut. After boiling it has a brown
color.

2Frederick, American Journal Obstetrics, Vol. 89, 1899.

PREPARATION OF INSTRUMENTS, ETC.

the process of soaking in formaldehyde and the consequent
shrinking, one strand crosses another, the one next to
the glass will be so pressed upon as to prevent hardening
at that point. When the gut is boiled later in water,
that point will gelatinize and break at the least strain.
Formaldehyde comes in a 40 per cent, solution. We use a
3 per cent, solution, pouring one part of the 40 per cent,
formaldehyde solution and thirteen parts of water into a
wide-mouthed bottle. Immerse the spools in this solution
for periods of time varying with the size of the gut. No. o
is left in one hour. Nos. i, 2 and 3 are given three, five,
and seven hours respectively. If left too long in the solu-
tion the gut will become too hard, too brittle, and the
strength will be impaired. Wash in running water for a
longer time than it was in the formaldehyde solution. Up
to this time the gut has not been sterilized. It has under-
gone a chemical change whereby it may be boiled without
spoiling it. The sterilization of the gut consists in boiling
for fifteen minutes, with the receptacles in which it is to be
kept. With sterile forceps place the spools, each size by
itself, in wide-mouthed-ground-glass-stoppered bottles or in
rubber-sealing fruit jars, sterilized by boiling. Pour over
the gut clean 95 per cent, alcohol with 8 to 10 per cent, of
glycerine. To sterilize the glycerine it should be placed in
a bottle in water and raised to the temperature of boiling
water for half an hour.

To make chromicizcd catgut wind the spools as before.
Place the spools in a solution of : bichromate of potassium,
1.5 grammes; glycerine and carbolic acid each 10 c.c. ; water
I liter. Allow them to remain in this solution for twenty-
four hours. Take out and drain, allowing them to dry for a
few hours. Then place in the formaldehyde solution and
put through the same process as with formaldehyde catgut.

Kangaroo tendon, owing to its slow absorption, is used as

2l8 MANUAL OF BACTERIOLOGY.

a heavy retaining suture, and is prepared by washing the
strands in ether to free from fat. Soak in a 4 per cent, solu-
tion of chromic acid for twenty-four hours. Then sterilize
by the cumol method.

Silk may be sterilized by the fractional method (see p. 63)
as this does not impair the strength as does boiling.

Silkworm gut is prepared by steam sterilization by the
fractional method or by boiling in plain water for one half
hour. It should not be boiled in soda solution as this spoils
the gut.

Horsehair strands are cut into two-foot lengths, washed
with soap and water and sterilized with steam by the frac-
tional method. They make a very fine suture and are
used where an inconspicuous scar is particularly desirable,
as on the face. Only the finer grades are used for this
purpose.

Silver Ti'/'/T.1 This material has the advantage over other
suture materials of having a germicidal or at least a restrain-
ing influence on bacteria. If we remember that absolute
sterilization of the skin is not possible by any means, we
must see that in silver wire as a skin suture we have a safe
and valuable material. Recent annealing by heating to a
dull red increases the flexibility of the wire but almost
totally destroys its germicidal property. This will reappear
in a month and is not disturbed by boiling. Therefore pre-
pare it by boiling for ten minutes in the I per cent, soda
solution.

Sponges. The best absorbents to use in surgical work are
those whose sterility is undoubted. Pads of gauze are easily
sterilized by steam as for dressings. Sea sponges2 may be
prepared by beating with a wooden mallet to remove sand
and dirt. Soak in a 1-64 solution of hydrochloric acid for

1 Bolton, Transactions Association American Physicians, 1894.
" McBurney, "International Text-Book of Surgery/' June, 1900, p.
284.

PREPARATION OF INSTRUMENTS, ETC. 219

twelve hours to remove lime deposits. Wash in running
warm water. Soak for fifteen minutes in a saturated solu-
tion of permanganate of potassium, then place in a satu-
rated solution of oxalic acid until they are perfectly bleached.
After immersion for half an hour in this solution rinse
thoroughly in sterile water and put in a i-iooo bichloride
of mercury solution for twenty-four hours. Remove and
place in 1-20 carbolic acid solution until required for use.
At operation remove from solution, rinse out in normal salt
solution, and place in receptacle filled with salt solution. If
sea sponges are used on a septic case they should be thrown
away and no attempt made to resterilize them. If used on
clean cases they may be resterilized as above.

Dressings. The two materials universally used to dress
wounds are " gauze " or cheese-cloth and absorbent cotton.
If they are properly sterilized, the impregnation of gauze
or cotton with antiseptics does not* add to their value.
Gauze is usually cut in portions one yard square and folded
in pieces called compresses. A number of compresses are
wrapped with a piece of cotton cloth and the edges stitched
loosely into a closed bundle. After sterilization by the frac-
tional method, the bundles can be placed in sterile jars or
receptacles and each bundle removed as needed. Ripping
open the stitches gives untouched sterile bundles of com-
presses, convenient and handy for using. Cotton is sterilized
by the fractional method in rolls or bundles as for gauze.
These dressings should be warmed before being placed in
the steam sterilizer or they will be unnecessarily wet when
removed.

Irrigating Solutions. Chemical germicides, such as bichlo-
ride of mercury when in solution, cause necrosis of tissue.
Plain sterile water causes maceration of epithelium. Nor-
mal salt solution is the least irritating to the tissues and is
the one most generally employed for irrigating purposes. It

22O MANUAL OF BACTERIOLOGY.

is .6 per cent, sodium chloride, prepared roughly by adding
a teaspoonful of salt to the pint of water. This solution
may be sterilized by boiling for half an hour on three con-
secutive days. It does not injure tissue, and may be freely
used in operations for irrigating. It has no germicidal or
antiseptic properties.

Accident wounds are generally lacerated or contused and may con-
tain pathogenic bacteria. They should be promptly and carefully
cleansed with sterile salt solution, wiped with sterile gauze and if
necessary scrubbed vigorously with sterile soap and brush to remove
all infectious dirt. When there is any doubt of this being accomplished
it is better to dress such wounds wide open, filled with sterile gauze,
for forty-eight hours 'or more. Retained blood clots form a good
medium for the development of bacteria so that drainage for a day or
two is safer in doubtful cases.

Infected wounds. There is no known method for promptly sterilizing
infected wounds without destroying tissue. An infected wound, if
the infection be not too deep, may be sterilized by cauterizing with pure
carbolic acid.

Care must be exercised in the application of antiseptic solutions in
infected wounds for the antiseptic rarely penetrates as deeply into the
tissues as the bacteria are found, therefore, further necrosis of tissue
and mechanical cleanliness are about all they accomplish.

After-treatment of wounds. Close attention to details is important in
the technique of a first dressing after an operation. All instruments,
irrigating fluids, bowls, basins, etc., are to be sterilized. When the
dressing is removed the skin surrounding the wound should be cleansed
by washing with salt solution or peroxide of hydrogen. The sutures
or drainage should be removed with sterile forceps, and fresh sterile
dressings applied. If a wound is found infected, all accumulations of
blood-clot, pus, etc., should be gently and carefully washed out, and
free drainage provided. In the care of infected wounds careful attention
must be paid to maintaining mechanical cleanliness and avoiding
infection with some organism which may not be already present.

It should be borne in mind that anything that tends to depress a
patient's resisting powers encourages infection ; such as prolonged
exposure to cold during an operation, loss of blood and infliction of a
great degree of surgical shock.

PART III.

NON-PATHOGENIC BACTERIA.

THE number of varieties of non-pathogenic bacteria is
very large. Eisenberg1 describes 376 species of bacteria,
mostly non-pathogenic. Sternberg2 enumerates 489 species,
including the pathogenic varieties, but the majority, of
course, are non-pathogenic. Fliigge3 considers about 500
species of bacteria. Migula4 recognizes nearly 1,300, and
Chester5 about 800 species. Probably some of the bacteria
which have been described as distinct species are in reality
not different; but, on the other hand, it is also probable
that a still larger number of species have not been described
at all ; how many it is impossible to say. In a work of this
character it is feasible to mention only a few of the com-
monest and best-known species of non-pathogenic bacteria.

Micrococcus agilis. — Found in water; coccus about i p
in diameter, usually appearing as diplococci, sometimes as
streptococci and tetrads ; liquefies gelatin slowly ; grows at
room temperature, on ordinary culture-media, forming a
rose-red pigment on agar and potato. This micrococcus is
remarkable in being actively motile ; it possesses a flagellum.
It is stained by Gram's method.

Micrococcus urese. — Found in decomposed, ammoniacal
urine and in the air ; coccus .8 to I [*• in diameter, occurring

" Bakteriologische Diagnostik," 1891.

" Manual of Bacteriology," 1893.
'"Die Mikro organism en" 1896.
1 " System der Bakterien," 1900.
5 " Manual of Determinative Bacteriology," 1901.

222 MANUAL OF BACTERIOLOGY.

singly or in various combinations ; does not liquefy gelatin ;
facultative anaerobic; grows rapidly, best at 30° to 33° C. ;
grows on ordinary gelatin, but best on special media; it
decomposes urea, producing ammonia and carbon dioxide,
which form ammonium carbonate.

Sarcinae. — There is a large number of species of sarcinse.
They are common organisms in the air. They frequently
contaminate plate-cultures. Many of the sarcinae of the
air present, in cultures, growths having brilliant colors,
from which some of them are named ; thus there are orange,
yellow, rose-colored and white sarcinae, and others.

Sarcina pulmonum. — Found in the air passages of man;
I to 1.5 /J. in diameter, occurring in tetrads or cubes of eight
cells; aerobic; does not liquefy gelatin; grows slowly, best
at ordinary temperatures, preferably upon gelatin. It de-
composes urine with the formation of ammonia. It is said
to form endogenous spores which are extremely resistant
to heat.

Sarcina ventriculi. — Found in the stomachs of man and
of animals; 2.5^ in diameter, occurring in cubes of eight
cells or more; it does not liquefy gelatin; aerobic; grows
on ordinary culture-media; the growths tend to become
yellow. Small numbers of sarcinre may occur in the normal
human stomach ; the presence of large numbers indicates the
existence of abnormal fermentative processes.

Bacillus fluorescens liquefaciens. — Found in water and
putrid fluids; very common; appears as a small rod, actively
motile; aerobic, but somewhat variably; liquefies gelatin;
grows rapidly at ordinary temperatures upon the usual cul-
ture-media. It forms a pigment having a beautiful greenish-
yellow fluorescence, best seen in transparent media ; the
growth on potato has a brown color. Does not stain by
Gram's method and does not form spores.

Bacillus fluorescens putidus. — Found in water; a short
rod with rounded ends; actively motile; does not liquefy

NON-PATHOGENIC BACTERIA. 223

gelatin; aerobic; does not form spores; grows rapidly at
l lie ordinary temperatures upon the common media. Gel-
atin cultures give off a powerful, foul odor of trimethyl-
amin. It produces a greenish, fluorescent pigment, best
seen in transparent media; on potato the growths form a
thin, gray to brown, slimy layer.

There are several other fluorescing bacilli, mostly found
in water.

Bacillus Indicus. — Found by Koch in the stomach-con-
tents of an ape in India ; a fine short bacillus with rounded
ends; motile; does not form spores; facultative anaerobic;
liquefies gelatin; grows rapidly, best at 35° C. upon the
ordinary media ; produces a brick-red pigment. Very large
doses injected into rabbits caused death in three to twenty-
four hours.

Bacillus prodigiosus. — \Yiclely disseminated in the at-
mosphere of certain places ; a short bacillus with rounded
ends, in form often nearly like the micrococci ; facultative
anaerobic; not motile, as a rule; does not form spores;
liquefies gelatin rapidly; grows rapidly, best at 25° C. on
the ordinary culture-media; milk is coagulated; gas forms
in sugar-media : cultures on potatoes give off a foul odor
of trimethylamin. A brilliant red color, which only de-
velops in the presence of oxygen, appears in cultures. The
pigment appears as granules outside of the bacteria.

Bacillus violaceus (of Berlin). — Found in water; a slim
rod with rounded ends which may form threads; actively
motile; facultative anaerobic; liquefies gelatin rapidly;
forms endogenous spores placed near the centers of the ba-
cilli ; grows rapidly, and not at high temperatures, upon
ordinary media, forming a deep, violet-colored pigment.
There are several bacilli related to this one.

Bacillus amylobacter (Clostridium butyricum, Bacillus
butyricus, Prazmowski). — Found widely distributed in na-

224 MANUAL OF BACTERIOLOGY.

ture in decomposing vegetable material and in the stomachs
of ruminant animals; a large, thick rod with round ends,
often arranged in chains; actively motile; anaerobic; forms
spores, which are located in the center of the bacillus and
give it a spindle-shaped form, or at one end when it has the
outline of a tadpole; has not been cultivated satisfactorily
on ordinary media; grows best at 35° to 40° C. ; decom-
poses carbohydrates with the formation of butyric acid ;
decomposes cellulose. Organisms of similar form have
been found as fossils belonging to the carboniferous period.

Bacillus butyricus (Hueppe). — Found in milk; appears
as a small, irregular rod, also forming threads; very actively
motile ; aerobic ; rapidly liquefies gelatin ; forms centrally
located spores; grows best at 35° to 40° C. ; grows rapidly
on ordinary media; coagulates milk, redissolving the coag-
ulum, producing also butyric acid. A large number of
bacteria, both aerobic and anaerobic, produce butyric acid
fermentation.

Bacillus megatherium. — Obtained by DeBary from
cooked cabbage-leaves ; common on plants and earth ; a large
bacillus with rounded ends, often forming chains; motile;
slowly liquefies gelatin ; aerobic ; forms spores, especially in
potato cultures; grows rapidly at room temperature on the
ordinary media.

Bacillus mesentericus vulgatus (Potato bacillus).—
Found on potatoes; common in earth; a large, long rod
with rounded ends, often forming long chains ; motile ; it
is stained by Gram's method ; liquefies gelatin ; aerobic ;
forms spores; grows rapidly, best at about 20° C. ; grows
on ordinary media, forming on potato a thin, wrinkled
membrane which spreads rapidly over the surface. It
coagulates milk, redissolving the coagulmn. It possesses
numerous flagella. The spores are extremely resistant to
heat.

NON-PATHOGENIC BACTERIA.

Bacillus phosphorescens Indicus. — Obtained from sea-
water; a small, thick, rod-shaped bacillus with rounded
ends, also forming threads; actively motile; not stained
by Gram's method; liquefies gelatin; aerobic. It grows
slowly, best between 20° and 30° C, upon the usual media;
except milk and potato. Its cultures, when old, especially
when on animal- nutrient-media and in the presence of
certain sodium salts, are phosphorescent in the dark.

There are various other bacilli which produce phospho-
rescence, some of which do not liquefy gelatin.

Bacillus mycoides (Bacillus ramosus, Wurzelbacillus).
Found in the earth and in water, very common; a large,
short bacillus with rounded ends, often forming chains and
threads ; slightly motile ; liquefies gelatin ; aerobic ; forms
centrally located, oval spores; gro\vs rapidly at room and
incubator temperatures upon the usual media. It is said
to rapidly decompose albumin with the formation of
ammonia.

Bacillus subtilis (Hay bacillus). — Found on hay, in the
air, water, ground and decomposing fluids; very common;
a large bacillus somewhat resembling the anthrax bacillus
in form, with rounded ends, often forming chains or long
filaments; motile; possessing flagella; liquefies gelatin;
aerobic; it is stained by Gram's method. It may have
large, centrally located spores, which form best on potato
at about 30° C. The spores are extremely resistant to heat
and to chemical germicides. It grows best at about 30° C.
upon the ordinary culture-media; milk is peptonized.
Bacillus subtilis may easily be isolated in pure culture by
adding finely-cut hay to bouillon ; place in the steam ster-
ilizer for five or ten minutes; then let the tubes develop in
the incubator. Plates made from the bouillon will probably
show colonies of the bacillus subtilis only, as the steam
may be expected to have destroyed all organisms except

226

MANUAL OF BACTERIOLOGY.

its very resistant spores. The hay bacillus is entirely with-
out pathogenic properties.

FIG. 49.

Bacillus subtilis. (X 1000.)

Bacillus erythrosporus. — Found in decomposing fluids
and water; a slim bacillus with rounded ends; motile; does
not liquefy gelatin; facultative anaerobic; forms oval, red-
colored spores, two to eight in each filament ; grows rapidly,
only at ordinary temperatures; produces a greenish-yellow
fluorescent pigment. On potato it forms a limited, reddish
growth, becoming nut-brown.

Bacillus cyanogenus (Bacterium syncyanum, Bacillus
lactis cyanogenus, Bacillus of blue milk). — A bacillus of
variable size, with rounded ends; motile; spore formation
doubtful; is aerobic; not stained by Gram's method; grows
rapidly at ordinary but not so well as incubator tempera-
tures on the usual culture-media ; does not liquefy gelatin ;
produces a grayish-blue pigment, brighter in acid media,
at ordinary temperatures; milk is not coagulated, or ren-
dered acid.

NON-PATHOGENIC BACTERIA. 22/

Bacillus acidi lactici (Hueppe). — Found in sour milk; a
short, plump rod; not motile; does not liquefy gelatin;
facultative anaerobic; grows on the ordinary media; in
milk causes development of lactic acid with precipitation
of casein and production of gas and alcohol. It belongs in
the same group as B. coli communis and B. lactis aerogenes
(see Part IV.).

There are numerous other bacteria, such as the bacte-
rium acidi lactici, which cause the formation of lactic acid
in milk.

Bacterium ureae. — A short, thick bacillus with rounded
ends; net motile; aerobic; found in ammoniacal urine;
grows slowly at room temperature upon gelatin, which is
not liquefied ; decomposes urea, forms ammonium carbonate.

Bacterium Zopfii. — Found in the intestines of hens, in
water and in fecal matter; a bacillus .75 to i JJL broad and 2
to 5 /j. long ; may form threads. Actively motile ; does not
liquefy gelatin; aerobic; involution forms are often seen
and they have been described as spores; grows rapidly,
best at 20° C. upon gelatin; forms branching zoogloese.
It is a member of the same group as B. proteus (see Part
IV.)-

Spirillum rubrutn. — Found by Esmarch in the putrefy-
ing cadaver of a mouse; short spirals twice the breadth of
the cholera spirillum, usually with one to three turns; in
bouillon growing into long spirals ; motile with flagella ;
spore formation doubtful ; facultative anaerobic ; does not
liquefy gelatin; grows slowly, best at about 37° C. on the
ordinary media; produces a wine-red pigment only when
the air is excluded.

Spirillum or Spirochaeta dentium. — Found in the mouths
of healthy persons, on the margins of the gums when they
are covered with a dirty deposit; long spirals with several
windings uneven in thickness ; has not been cultivated.

^28 MANUAL OF BACTERIOLOGY.

Spirillum sputigenum. — Found in the human mouth in
healthy persons at the margin of the gums; curved rods or
short spirals which resemble the spirillum of cholera in
form ; has not been cultivated.

Spirillum rugula (Vibrio rugula). — Found in swamp
water, in fecal matter, and in the tartar of the teeth ; a
curved rod .5 to 2.5^ broad and 6 to 8/2 long, having one
flat spiral winding; motile, with flagella at the ends; prob-
ably anaerobic; forms spores located at the ends.

FIG. 50.

^ X

. <\

Spirilla from Swamp Water. (X about 500.)

Spirillum volutans. — Found in swamp water; very long
spirals with several turns; 1.5 to 2 /* broad and 25 to 30 //
long; motile, with a flagellum at each extremity. The
protoplasm is granular.

Spirillum undula. — Found in putrefying infusions con-
taining organic matter ; a rather short spiral form with three
turns or less, about i [* thick and 8 to 12^ long; actively
motile, with a tuft of flagella at each extremity; has been
cultivated on agar.

NON-PATHOGENIC BACTERIA. 229

Spirillum or Spirochaeta plicatile. — Found in swamp
water; spiral forms of various lengths; sometimes 100 to
200 /•* long; actively motile.

The spirilla (vibrios or comma-shaped forms), closely
resembling the spirillum of cholera, will be considered in
connection with that organism. A form of chronic pseudo-

FIG. 51.

Spirilla from Swamp Water, showing Flagella, Loffler Stain. (X 1000.)

membranous inflammation of the pharynx has been attrib-
uted to an organism called the fusiform bacillus or spirillum
of Vincent.1

Higher Bacteria. — Certain organisms (beggiatoa, thio-
thrix, leptothrix, cladothrix, actinomyces or streptothrix)
of more complicated structure than most bacteria, but re-
sembling them in many respects, are called " higher bac-
teria." They consist of definite filaments which are usually
made up of rod-shaped elements, but the relation between
these elements is very intimate. Some of them (beggiatoa,

1 Mayer, American Journal Medical Sciences, Vol. 123, 1902, p. 187.

230 MANUAL OF BACTERIOLOGY.

thiothrix) contain sulphur granules. Many of them occur
in water. There are forms among them which are found
attached to some object by one end of the filament (thio-
thrix). Some of them (actinomyces or streptothrix) have
branching filaments, which are rarely seen among the lower
bacteria (see page 119). Often one end of the filament be-
comes specialized for the purposes of reproduction. The
fungus of actinomycosis is the best known of this group.
There are many other members, however, both pathogenic
and non-pathogenic. Most of them require still further
study. The tubercle bacillus and other acid-proof bacilli
which resemble it, have some points of resemblance with
actinomyces (see B. tuberculosis, Part IV.).

Leptothrix buccalis. — Found in the mouth cavity. This
name has been applied to large, twisted, thread-like organ-
isms, in which segments can be demonstrated with diffi-
culty or not at all. Apparently, different organisms have
been described under this name. Vignal claims to have
cultivated a leptothrix buccalis. Miller recognizes two
principal species, neither of which could be cultivated,—
leptothrix innominata, which shows no transverse divisions,
and which is stained faintly yellow by iodine; and bacillus
buccalis maximus, in which the transverse divisions are
distinct, and which is stained brownish-violet by iodine.
Miller's leptothrix maxima buccalis is similar to the last
except in lacking the iodine reaction.

A variety of leptothrix, or a nearly related organism, ap-
pears to be the most frequent cause of the form of gangren-
ous inflammation of the mouth and genitals called noma.
It stains faintly by Gram's method. It does not grow on
ordinary media.1 Another organism of this group has been

1Blumer and MacFarlane, American Journal Medical Sciences,
November, 1901.

YEASTS AND MOULDS.

231

described which is pathogenic to a number of domestic
animals.1

Yeasts and Moulds. — In the course of bacteriological
work one constantly encounters yeasts and moulds, which,
although not bacteria, must nevertheless be understood and
recognized to avoid error. Accidental contamination of
tubes or plates is likely to be the result of the growth of some

FIG. 52.

Yeast Cells, stained with Fuchsin. (X 1000.)

of these forms. The yeasts generally go by the name of
sac char omyces, of which there are several species. The
saccharomyccs cerevisia is the ordinary yeast of alcoholic
fermentation. Some of the yeasts present colored growths
—red, white and black. They consist of large, oval cells,
which readily stain with the aniline dyes. They multiply by

1 It has also been called " Necrosis bacillus," and " Streptothrix
cuniculi." Pearce, University of Pennsylvania Medical Bulletin, Novem-
ber, 1902.

232

MANUAL OF BACTERIOLOGY.
FIG. S3-

a. Penicillium glaucum. b. Oidium lactis. c. Aspergillus glaucus. d. The
same more highly magnified. c. Mucor mucedo (Baumgarten).

YEASTS AND MOULDS. 233

the protrusion of a little bud from the cell, which develops
into a new cell. In an actively germinating growth of yeast
these budding cells are readily distinguished (Fig. 52).

Yeasts have been found that were pathogenic to animals.
They have also been supposed to be the cause of some
malignant tumors, but this view has been, for the most part,
abandoned.

Among the moulds the varieties most commonly en-
countered are the mucor, the penicillium, the aspergillus
and the oidlum. There are various species of each of
them. They consist of cells arranged end to end, making
a thread-like body called a hypha. The threads are matted
together and form a mycelium. Certain threads project
upward from the mycelium, and on them are borne spores,
or conidia. The arrangement of the spores is characteristic
in each variety of mould (Fig. 53). A group of organisms
exist which have affinities both with yeasts and mould-
fungi. Some of them are pathogenic. The form of infec-
tion of the mouth called thrush, is due to a fungus of this
class, which is generally considered an oidium. A chronic
inflammatory affection of the skin (blastomycetic der-
matitis) is due to related organisms.1 The Sporotricha of
Schenck2 which produces chronic subcutaneous abscesses,
may be mentioned here, provisionally. A number of skin
affections, such as Tinea favosa and Tinea trichophytina,
are due to fungi, which have some similarity with those
above mentioned.

Among the mould fungi, several species of aspergillus and
of mucor are pathogenic. Man, as well as the lower ani-
mals, may be affected. In man the lungs may be involved
in a broncho-pneumonia (pneumonomycosis), usually due to

'Ricketts, Journal of Medical Research, Vol. VI., 1901; Hyde and
Montgomery, Journal American Medical Association, June 7, 1902.
2Hektoen, Journal Experimental Medicine, Vol. V.

234 MANUAL OF BACTERIOLOGY.

aspergillus, and often secondary to some preexisting disease
of the lung. Mould fungi, especially aspergillus, may grow
in the external ear (otomycosis). The growth is usually
superficial. These fungi rarely produce lesions in other
organs.

PART IV.

PATHOGENIC BACTERIA.

Suppuration and Allied Conditions. — The occurrence of
suppuration is characterized by certain appearances which
we are accustomed to describe under the name of inflamma-
tion. The study of inflammation belongs to pathology, and
cannot be considered here. However, certain evidences
which are characteristic of the suppurative variety of inflam-
mation need to be outlined on account of their relation to the
action of the pyogenic bacteria.

In a suppurating area, as is well known, the blood-ves-
sels are dilated, and the lymph-spaces become filled with
serum. Leucocytes are attracted to the neighborhood in
large numbers, we may suppose by a positive chemotaxis,
and crowd the small veins and capillaries. The leucocytes,
by reason of their amoeboid movement, pass through the
walls of the vessels at little openings filled with cement-
substance, situated between the lining endothelial cells. Ac-
cording to the theory of phagocytosis, they are bent on find-
ing the irritant which has led to the inflammation, and upon
isolating it and rendering it harmless. At the point which
appears to be the center of the inflammatory area there is
usually, but not always, a necrosis of the cells of the tissue;
this constitutes the central slough or the familiar core of
some boils. The necrosis is to be attributed to poisons
formed by the micrococci. In sections cut through such an
abscess the nuclei of the central necrotic cells fail to take
the nuclear stain ; the necrotic mass does not stain, or takes

236 MANUAL OF BACTERIOLOGY.

the dye diffusely and irregularly, and it exhibits many fine
granules.

We find the cells of the tissues surrounding the necrotic
area mingled with large numbers of polynuclear leucocytes,
which enclose the area of irritation.

The nuclei of the cells near the center of the abscess are
frequently broken up into a number of small parts (frag-
mentation), which indicates the commencement of their
destruction. In sections through small abscesses it is pos-
sible, by means of a double stain of carmine followed
with gentian-violet, according to Gram's method, to bring
out the histological character of the tissue, and at the same
time to stain the common pyogenic bacteria, which are
usually found near the center of the abscess in large num-
bers, even making masses visible with a low power of the
microscope. Preparations, most convincing and of great
beauty, may be secured in this manner. It is often pos-
sible to demonstrate masses of micrococci filling up the
lumina of capillaries in which they are lodged as emboli.

The production of pus in the center of the abscess is due
to the liquefaction of the necrotic tissue, which apparently
results from the action of some peptonizing ferment. In
the liquid thus formed, immense numbers of the polynu-
clear leucocytes are found floating, and they constitute the
greater part of the so-called pus-cells. The nuclei of
these cells are obscured by clouds of extremely fine granules.
The granules are of an albuminoid nature, and are dissolved
by acetic acid, when the nuclei become visible. The nuclei
generally consist of three, four, five or more portions.
The presence of the fine albuminoid granules in the pus-
cells is to be counted as a degenerative change. Although
it is possible to produce suppuration in laboratory experi-
ments by the introduction of sterilized irritants, such as
croton oil, in the vast majority of cases suppuration is due
to the action of pyogenic bacteria.

PATHOGENIC BACTERIA. 237

Specimens of pus will nearly always be found to contain
bacteria, which can be demonstrated by cultivation, and,
as a rule, also in smears made and stained upon cover-
glasses. The bacteria are generally found outside the pus-
cells. In the case of the gonococcus and the diplococcus
intracellularis meningitidis they are characteristically found
in pairs, inside. of, or at least attached to the pus-cells.
The character of the suppuration differs somewhat with the
different species of pyogenic bacteria. The kind of abscess
above described — localized and having a central slough,
usually rather slow in progress — is typical for the staphylo-
coccus pyogenes aureus, which is prone to produce circum-
scribed areas of suppuration. The streptococcus pyogenes,
on the other hand, oftener leads to suppuration of a more
diffused character, such as we see in cellulitis and erysipelas;
but either organism may, at times, produce the effects
usually characteristic of the other. Pus having a blue or
green tinge generally owes the color to the presence of the
bacillus pyocyaneus. The commonest pus-producing organ-
ism is then the Staphylococcus pyogenes aurcus, and next to
that the streptococcus pyogenes. Among the other pyogenic
bacteria the following may be named :

Staphylococcus pyogenes albus, including staphylococ-
cus epidermidis albus; streptococcus of erysipelas (prob-
ably identical with streptococcus pyogenes); gonococcus;
diplococcus intracellularis meningitidis ; Staphylococcus
pyogenes ciireus; micrococcus tetragenus ; micrococcus
pyogenes tennis, which may be the same as the micrococcus
lanceolatus; Staphylococcus cereus albus and flavus.

Pus-formation may also be due to micrococcus lanceola-
tus, bacillus pyocyaneus, bacillus proteus, bacillus coli
communis, bacillus pyogenes fetidus, bacillus pneumonias
(of Friedlander), bacillus aerogenes capsulatus, the ray
fungus of actinomycosis, and possibly the bacillus of bu-

238 MANUAL OF BACTERIOLOGY.

bonic plague. Besides these organisms, there are others
whose effects are usually more marked in a specific way
which sometimes form pus, as the bacilli of diphtheria,
tuberculosis, glanders and typhoid fever.

Frequently two or more species of pyogenic bacteria will
be found associated.

The table on page 239, quoted from Dowd, shows the fre-
quency of the occurrence of various pyogenic bacteria in
135 cases of different types of suppuration.

The condition of the animal's tissues is of great impor-
tance in determining whether or not suppuration is to oc-
cur. It will be seen that we are repeatedly subjected to
infection with pyogenic bacteria, but that in most cases
suppuration nevertheless does not occur. The local condi-
tions have an important influence in determining infection.
Regions of hyperemia, edema, anemia or necrosis are
especially liable to suppuration, as are tissues which have
been bruised, lacerated, strangulated or otherwise dam-
aged. Furthermore, the general condition of the patient
is of great importance. Chronic diseases and conditions
of exhaustion or depression dispose to suppuration, and
the depraved condition of the tissues in diabetes renders
the sufferer from this disease especially liable to it. These
facts have already been enumerated in a previous chapter
(page 165). In the lower animals we find that it is often
very difficult to produce suppuration artificially with the
ordinary pyogenic bacteria. In rabbits the subcutaneous
introduction of staphylococcus pyogenes aureus frequently
fails to produce an abscess. Suppuration is likely to result,
however, if an irritant body like a piece of sterilized potato
or sterilized glass be introduced along with the bacteria.

Pyogenic bacteria are most frequently introduced into
the body through the agency of injuries and wounds of
various sorts. They are very widely disseminated in

PATHOGENIC BACTERIA.

239

nature, and are always liable to be clinging to external
objects, especially in cities and around dwellings. The
infection of a wound in this manner, when the suppuration
is of a spreading character, such as is most character-
istic of streptococcus infection, is known in every-day Ian-

in 0i

•U «

•

is 3

^ i

c™

ft!

Streptococcus pyo^enes alone

Streptococcus pyogenes predominant. . . .

9
23

Streptococcus pyogenes relatively few..
Staphylococcus pyogenes aureus alone. .

3
ii

I
I

6
i

i
6

Staphylococcus pyogenes aureus pre-

dominant

Staphylococcus pyogenes aureus rela-

tively few

T ?

Staphylococcus pyogenes or epidermidis

albus alone

Staphylococcus pyogenes or epidermidis

albus predominant

Staphylococcus pyogenes or epidermidis

albus relatively few

Staphylococcus cereus albus

•j

Staphylococcus citreus

No growths on agar

1 1

Very few growths on agar

Bacillus pyocyaneus ...

Bacillus coli communis

Overgrown

Few undetermined colonies

guage as " blood-poisoning." It is possible for infec-
tion to take place around hair-follicles through the un-
broken skin. In such instances the suppurative inflamma-
tion first shows itself in a minute red pimple with a hair in
the center. The pimple presently becomes a pustule. The
process may cease at this point, or it may be only the com-
mencement of a large carbuncle with a central slough. Such
infection has been produced experimentally on the human

24O MANUAL OF BACTERIOLOGY.

skin by rubbing in cultures of staphylococcus pyogenes
aureus. It is, furthermore, the constant experience of post-
mortem examiners that infection may occur around the hair-
follicles when no wound of the skin has been inflicted.

In many instances, infection with the pyogenic bacteria
follows upon some preexisting infection; this happens, for
instance, in tuberculosis, when tuberculous lungs become
infected with streptococcus pyogenes, leading to the forma-
tion of a cavity. It is a common occurrence in gonorrhea,
after the acute stage of the disease has passed, when we
find the gonococcus in the pus, mingled with other pyo-
genic micrococci. Secondary infection with pyogenic bac-
teria is frequently due to the streptococcus pyogenes, often
also to the micrococcus lanceolatus.

Sometimes we are obliged to admit that the manner in
which the pyogenic bacteria enter the body is unknown.

The severe general symptoms, familiar to every physi-
cian, often accompanying acute suppuration, indicate the
formation of toxic bacterial products and their absorption.
Experimental evidence of the formation of such toxic prod-
ucts is not so clear, however, for the pyogenic organisms
as for some of the other bacteria. It has been shown that
cultures of staphylococcus pyogenes aureus, in which the
bacteria have been killed, are capable of producing suppura-
tion in the lower animals.

The pyogenic bacteria play a somewhat different part in
producing disease, which is fully as important as the typ-
ical suppuration seen in an abscess. This happens when
the suppurative condition is mixed with other phenomena,
or when there is inflammation of another variety without
suppuration at all ; or there may be lesions not inflamma-
tory in a strict sense. These differences in their action
depend largely upon the organ affected. One such condi-
tion is osteomyelitis, which is, usually, suppuration occur-

PATHOGENIC BACTERIA. 24!

ring in bone, but which does not present the ordinary
picture of pus-formation owing to the hard and unyielding
character of the tissue. Other conditions of very great
importance are meningitis, pericarditis, pleuritis, pneumo-
nia (croupous and broncho-), peritonitis and endocarditis.
It will be observed that these affections are, for the most
part, inflammations of the serous membranes. Such in-
flammations, when they are produced by pyogenic bac-
teria, are likely to be of great severity, accompanied by
the formation of fibrinous exudates ; pus-formation may or
may not be present. We find that the cause at times is
the staphylococcus pyogenes aureus; this is often the case
in malignant endocarditis. Generally speaking, in such in-
flammations the streptococcus pyogenes, the staphylococcus
pyogenes aureus, and the pneumococcus occur most com-
monly, although they are by no means the only organisms
found. Many cases of peritonitis show the presence of B.
coli communis, either in combination with other bacteria,
or alone.1 This is explained by the proximity of the intes-
tine, and especially by the frequent occurrence of peritonitis
after perforation of the intestine.

In inflammations of mucous membranes the common
pyogenic organisms play the most important though not an
exclusive part. In acute bronchitis, pneumococci and strep-
tococci were found by Ritchie to be the commonest causes.

In inflammations of the middle ear the principal causes are
the pneumococcus, the streptococcus, and the staphylococcus
aureus and albus.3

In 25 cases of acute cystitis in women Brown4 found
B. coli communis, 15 times; S. pyogenes albus, 5 times; S.

1 Flexner, " Etiology, etc., of Peritonitis," Philadelphia Medical Jour-
nal, November 12, 1898.

2 Ritchie, Journal Pathology and Bacteriology, Vol. VII., December,
1900.

3Hasslauer, Ccntralblatt f. Baktcriologic, XXXII., Ref., 1902, p. 174.
Compare Ibidem, pp. 240 and 246.

4 Johns Hopkins Hospital Reports, Vol. X., 1902.

242 MANUAL OF BACTERIOLOGY.

pyogenes aureus, 2 times; B. typhosus, I time; B. pyocy-
aneus, i time ; B. proteus vulgaris, i time.

A number of investigators have recovered from cases of
acute articular rheumatism organisms resembling the pyo-
genic cocci. Most frequently a diplococcus or short strepto-
coccus has been found, which has sometimes produced
arthritis and endocarditis when inoculated into rabbits.

Staphylococcus pyogenes aureus in Pus, stained by Gram's Method.
(X 1000.)

From a point where there is suppuration or other local-
ized infection, pyogenic bacteria may enter the circulation
and become widely disseminated throughout the body.
That happens very commonly in malignant endocarditis. In
this manner secondary or metastatic abscesses may be pro-
duced in the most diverse organs.

The term pyemia is used to describe the dissemination of
pyogenic bacteria in the circulating blood, with the forma-
tion of metastatic abscesses.

PATHOGENIC BACTERIA.

243

Staphylococcus pyogenes aureus. — A micrococcus of
variable size, arranged in irregular clumps, sometimes in
pairs; about .8 to .9^ in diameter; not motile (Fig. 55). It
stains by Gram's method; it is facultative anaerobic; grows
rapidly, best at 30° to 37° C. It liquefies gelatin. Upon
gelatin plates small colonies appear at the end of about two
days. It grows well upon all the culture-media. Milk is
coagulated. It does not lead to fermentation with the pro-
duction of gas but produces various acids.

FIG. 55.

Staphylococcus pyogenes aureus, Pure Culture. (X 1000.)

The growths in the first place are pale, subsequently
becoming golden-yellow in color, but only in the presence
of oxygen. This color appears well on all media, and is
especially distinct on potato. Sometimes the color is slow
in developing.

In a fresh, moist condition the organism is killed by ten
minutes' exposure to 58° C. ; in a desiccated condition it
requires a temperature of 90° to 100° C. to destroy it. It

244 MANUAL OF BACTERIOLOGY.

resists drying in a considerable degree. In thq same speci-
men the micrococci may have quite different resisting powers
to chemical germicides. Some of them are destroyed by
i-iooo solution of bichloride of mercury in five minutes;
others survive exposure to the same for from ten to thirty
minutes. (Abbott.)

Sterilized cultures introduced into animals may produce
local suppuration. The toxic substances occur in the bac-
terial cells.1

As has already been mentioned, the staphylococcus pyo-
genes aureus is the commonest of the pyogenic bacteria
in man. It has been obtained from a great variety of
sources, and appears to be able to exist as a saprophyte.
It has been found on the skin, in the mouth, in the nasal
and pharyngeal mucus, and also in the alimentary canal.
It has furthermore been detected in the air and in dust. It
appears to find the conditions necessary for its existence
in the vicinity of human habitations.

Cultures of the staphylococcus pyogenes aureus vary
considerably in virulence. These variations are sometimes
to be explained through cultivation on unfavorable media
or repeated transplantation from one medium to another;
but at times the diminished virulence is due to unknown
causes. The lower animals used for experiments are not
as readily infected as man. The local introduction in rab-
bits or guinea-pigs of a part of a culture of staphylococcus
pyogenes aureus may be entirely without effect. The use
of a very large dose, or the addition at the same time of
some kind of irritant, may produce an abscess. Large
amounts of cultures in bouillon may often be injected into
the peritoneal cavity of the dog without effect, when the
simultaneous addition of a piece of sterile potato or an
injury to the gut may lead to fatal peritonitis. Introduc-

1 See also Morse, Journal Experimental Medicine, Vol. I., p. 613.

PATHOGENIC BACTERIA.

245

FIG. 56.

tion of fluid cultures into the venous circulation of the rabbit
generally produces metastatic abscesses in the kidneys, the
heart-muscle and the voluntary muscles, and causes death.

In man this organism produces sup-
puration of a localized character, such
as we are familiar with in boils and
carbuncles. It has been shown to be
the usual cause of infectious osteo-
myelitis. Osteomyelitis has been pro-
duced experimentally in rabbits by the
injection of the staphylococcus pyo-
genes aureus, both with and without
previous injury to the bone of the ani-
mal. Ulcerative endocarditis has on
numerous occasions been shown to be
due to this organism. It has been
found possible to produce ulcerative
endocarditis experimentally in animals
by the injection of the staphylococcus
pyogenes aureus when the valves of the
heart have first been mechanically in-
jured. The staphylococcus pyogenes
aureus has also been found in acute
abscesses of the lymph-nodes, tonsils,
parotid gland, and mammary gland,

. . , ~r 1 Staphylococcus py-

m suppurating joint affections and em- ogenes aureus, Geia-
pyema. It appears, furthermore, in tin Culture, i Week
acute inflammation of the serous mem-
branes,— pleuritis, pericarditis, peritonitis, — although less
frequently than the streptococcus pyogenes.

Staphylococcus pyogenes albus. — In form and manner
of growth this organism behaves like the staphylococcus
pyogenes aureus, with the exception that it produces no
colored growths and its cultures appear white. Its patho-
genic properties are less marked, and it is a less frequent

246 MANUAL OF BACTERIOLOGY.

cause of suppuration than the staphylococcus pyogenes
aureus. It has, however, been found in acute abscesses on
numerous occasions.

Staphylococcus epidermidis albus. — According to Welch,
the epidermis of man contains with great regularity the
organism to which he gave the above name, and which he
considers to be a variety of staphylococcus pyogenes albus.
It grows, liquefies gelatin, and coagulates milk more slowly
than the ordinary staphylococcus pyogenes albus. It is,
furthermore, possessed of less marked pus-producing ten-
dencies. Welch found it impossible to sterilize the skin so
as to remove this micrococcus from it. The organism is
usually innocuous. It has been found in healthy wounds
on numerous occasions. It is capable of causing trouble
in wounds when necrotic or strangulated tissues are present,
or where a foreign body like a drainage-tube has been left
in the wound. It is a common cause of stitch abscesses.

Streptococcus pyogenes. — Appears as micrococci ar-
ranged in chains, often in pairs, when the adjacent cocci
may be flattened. Sometimes the chains are very long.
The diameters of the cocci vary from .4 to i //. Attempts
have been made to create varieties of streptococci accord-
ing to the length of the chains. On that basis a strepto-
coccus brevis and a streptococcus longus have been de-
scribed.

The streptococcus pyogenes is not motile. It stains by
Gram's method. By the method of Hiss (page 57) capsules
may sometimes be demonstrated. It is facultative anaero-
bic; grows best in the incubator; more slowly at room tem-
perature, and does not liquefy gelatin. In gelatin plates it
produces small, round, white, punctiform colonies which are
slow of development, and are only visible after about three
days. It grows on the ordinary media; with the exception
of potato, according to some authors. Milk may or may not

PATHOGENIC BACTERIA. 247

be coagulated. The growths are never very luxuriant, and
may die out entirely after a few transplantations.

It is killed by exposure to 52° to 54° C, in ten minutes.
The streptococcus pyogenes occurs frequently on the mu-
cous surfaces of the healthy body. It is often found in
pus, especially pus of spreading inflammations of the kind

FIG. 57.

Streptococcus pyogenes, from a Pure Culture. (X 1000.)

known as cellulitis. This organism is the commonest infec-
tious agent in puerperal fever, metritis and peritonitis. It
occurs commonly in inflammations of the serous membranes
— pleuritis, pericarditis and peritonitis. It has been dis-
covered many times in ulcerative endocarditis, and in
broncho-pneumonia. It is frequently present in the false
membrane found in genuine diphtheria. It is also the cause
of many of the pseudo-membranous or so-called " diph-
theritic " affections of the throat where the Klebs-Loffler
bacillus of diphtheria is wanting. These cases may be in-
distinguishable clinically from genuine diphtheria, and their

248 MANUAL OF BACTERIOLOGY.

nature will only be revealed on bacteriological examination.
They are, however, as a rule, milder than genuine diph-
theria. The pseudo-membranous affections of the throat
which occur in scarlet fever and measles are generally caused

FIG. 58.

•

Streptococcus pyogenes in Pus, Gram's Stain. (X 1000.)

by the streptococcus pyogenes, although those diseases may
be complicated by genuine diphtheria. Streptococci are very
commonly present in the throat in scarlet fever,1 and some-
times occur in the blood. Some observers believe that scarlet
fever is caused by streptococci. Streptococci are very often
found in the pustules of small-pox, and may also appear in
the blood.

The streptococcus pyogenes is pathogenic for mice and
rabbits, but the virulence is very variable. That may some-
times be increased by passing through a number of animals
in succession, but is rapidly lost in artificial cultures. It is
said that the virulence is best maintained when cultures on
gelatin, after forty-eight hours' growth, are kept in a cool

' Weaver, American Medicine, April 18, 1903.

PATHOGENIC BACTERIA.

249

FIG. 59.

place, as in the ice-chest. Marmorek undertakes to main-
tain or increase the virulence by growing it first in a mix-
ture of human blood-serum (or
that of the ass or the horse) with
bouillon, and then inoculating it
into the body of a rabbit, alter-
nating these procedures, to obtain
a culture of very high virulence.
A serum of uncertain value de-
rived from an immunized horse
or ass and intended to cure strep-
tococcus infection, has been pre-
pared by Marmorek.

A number of other sera have
been prepared to combat strepto-
coccus infection. These have
been used for human cases, in-
cluding also scarlet fever. The
results appear somewhat encour-
aging, although still uncertain.

It is said that streptococci may
be agglutinated by serum from
animals immunized to the strepto-
coccus.

Coley has recommended a
bouillon culture of streptococcus
pyogenes (or of erysipelas), in
which the bacillus prodigiosus was
afterward grown, to be adminis-
tered by injection, after steriliza-

Streptococcus pyogenes, cul-

ture on agar (slightly en-

tion of the cultures by heat, in lar ed)
cases of inoperable sarcomatous

tumors. These injections appear in some cases to have ac-
complished remarkable and wholly unexplainable cures.

250 MANUAL OF BACTERIOLOGY.

Streptococcus of Erysipelas. — A streptococcus has been
derived from cases of erysipelas which in all essential re-
spects, in its morphology, its growth on culture-media, its
behavior with stains, and its pathogenic properties, is simi-
lar to the streptococcus pyogenes. It is probable that these
organisms are identical.

Micrococcus tetragenus. — Found in the cavities in the
lungs of pulmonary tuberculosis, in sputum and in pus.

FIG. 60.

Micrococcus tetragenus in pus from a large abscess on the arm ; showing
capsule, Gram's stain and eosin. (X 1000.)

The micrococci are enclosed in a transparent capsule, best
seen in preparations from the tissues of inoculated animals,
and are arranged in pairs or in fours ; about I p. in diameter ;
not motile; stain by Gram's method. It grows well at the
room temperature, but rather slowly; is facultative anaero-
bic; does not liquefy gelatin. Gelatin plates show little,
white, punctiform colonies, which, with the low power, are
finely granular, and have a peculiar glassy shimmer ; and in
stab-cultures the growths appear as little colonies along

PATHOGENIC BACTERIA. 25!

the line of puncture. On agar, round white colonies form,
not spreading. It produces a thick, slimy film on potato and
a broad, white, moist growth on blood-serum. This organ-
ism is only occasionally found in pus. It is pathogenic to
white mice and guinea-pigs, not to gray mice and rabbits.
It may produce a septicemia or only a localized suppuration
in guinea-pigs. In white mice a general septicemia results,
when the micrococcus tetragenus is found in the blood and
in the great viscera. White mice usually die in from two
to six days; guinea-pigs in from four to eight days.

Micrococcus lanceolatus (Micrococcus pneumoniae crou-
posse, Micrococcus Pasteuri, Diplococcus pneumonise, Micro-
coccus of sputum septicemia, Streptococcus lanceolatus
Pasteuri, and Pneumococcus of Frankel). — This organism
was discovered by Sternberg in his saliva in 1880, and after-
ward demonstrated to be the cause of lobar pneumonia by
Frankel and Weichselbaum. The micrococci usually occur in
pairs. The pair of micrococci, in its most typical form, ap-
pears like a couple of curved triangles with their bases close
to each other. The outline is usually described as being lan-
cet-shaped. The micrococci are frequently oval or round;
they often form chains. When it is most characteristic, each
pair of micrococci is surrounded with a capsule, which is
best shown in preparations made from the blood of infected
animals or from pneumonic sputum; the capsule is not
usually seen in preparations made from cultures. For
methods of demonstrating the capsule see page 57. The
pneumococcus is not motile. It stains by Gram's method,
which also is useful in demonstrating the capsule. It is
facultative anaerobic. It grows only at elevated tempera-
tures, preferably about 35° to 37° C. Gelatin is not lique-
fied. It grows well upon agar, upon blood-serum and upon
Guarnieri's medium (p. 81). It does not grow upon potato.
Milk usually becomes acid, and may or may not be coagu-

252 MANUAL OF BACTERIOLOGY.

lated. The colonies are seen in their characteristic form
upon agar, and are developed after about forty-eight hours,
appearing as minute, whitish, translucent, circular growths.

It is killed by an exposure to 52° C. for ten minutes.

It is best cultivated from the blood of an animal which
has been infected with the sputum of a case of lobar pneu-

FIG. 61.

Pneumococcus of Frankel in sputum of pneumonia, Gram's stain and
eosin. (X 1000.)

monia. Cultures need to be transplanted every few days;
they cannot usually be propagated more than a couple of
months.

The virulence of the organism for animals diminishes
rapidly in cultures. In cultures it frequently grows as a
streptococcus. When virulent, it is pathogenic to mice and
rabbits, less so to guinea-pigs. In these animals it is likely
to lead to inflammations, and to rapidly fatal septicemia
(twenty-four to forty-eight hours). The blood may con-
tain great numbers of the diplococci. It may be introduced

r ,

PATHOGENIC BACTERIA. 253

subcutaneously or into the peritoneum, or by intravenous
injection when liquid cultures are used. Its virulence is
very variable. In the sputum of a case of lobar pneumonia,
early in the disease, it is likely to be virulent. The virulence
is best maintained by repeated inoculations into mice or

rabbits.

FIG. 62.

Pneumococcus showing capsule, from pleuritic fluid of infected rabbit,
stained by second method of Hiss. (X 1000.)

This organism is detected very frequently in the human
mouth. When taken from the mouth it is not, however,
pathogenic to animals in many instances, being found
virulent in only from 15 to 20 per cent, of human mouths.
It is the specific cause of croupous or lobar pneumonia in
man. In that disease the characteristic lesion consists of an
inflammation of the lung, involving large areas, usually one
or several lobes. An exudate is poured into the air-vesicles,
which in the early part of the disease contains red blood-
cells, imparting the rusty color to the sputum. The principal
element in the exudate is fibrin. The formation of fibrin
produces the liver-like consolidation or " hepatization."

254 MANUAL OF BACTERIOLOGY.

The diplococci can readily be demonstrated in sections of
pneumonic lung, which are best stained by carmine and
gentian-violet, by the Gram method. Although the exudate
at first contains many red blood-cells and the solid lung
appears red, subsequently it becomes decolorized and pre-
sents a gray color. Many leucocytes will now be found to
have migrated into the air-vesicles, and the lung will have
become relatively anemic, instead of hyperemic. Finally,
the fibrinous exudate and the cells entangled in it become
softened and liquefied. Some of this liquefied exudate is
absorbed into the lymphatics in the walls of the air-vesicles ;
part of it is expectorated.

The micrococcus lanceolatus can be detected in large
numbers, sometimes almost unmixed with other bacteria,
in the rusty sputum of lobar pneumonia, often showing the
peculiar unstained capsule. On account of its liability to
be mixed with other forms of bacteria, its presence in the
sputum of cases suspected of being pneumonia is not of
very great value in differential diagnosis, especially con-
sidering that it is so commonly present in the normal mouth.
In a suspicious case its appearance in sputum in nearly pure
culture may be significant.

Cultures from the blood of cases of pneumonia, where a
large amount of blood is taken, have shown the presence
of the pneumococcus in a considerable proportion of the
cases, especially when severe or fatal.

The micrococcus lanceolatus is often also the cause of
broncho-pneumonia and of meningitis. It produces inflam-
mations in other situations as well, the most important be-
ing pleuritis, pericarditis, endocarditis and arthritis. The
micrococcus lanceolatus may produce pseudomembranous
inflammation1 and also ordinary suppuration, although not
very commonly.

1 Gary and Lyon, American Journal Medical Sciences, Vol. 122, 1901.

PATHOGENIC BACTERIA. 255

G. and F. Klemperer claim to have obtained toxins from
cultures of the pneumococcus, and to have established im-
munity in animals with the development in the blood of
antitoxic substances. Similar attempts have been made by
Washbourn and others, but the interpretation of them at
the present time is not clear. An agglutination reaction
has been described as occurring with the pneumococcus, but
it does not yet appear to have any practical value in diag-
nosis.

Organisms related to the pneumococcus have been de-
scribed under the names of pseudopneumococcus1 and strep-
tococcus mucosus.2

The organism named by Rosenbach, micrococcus pyo-
gcncs tennis, is probably only a variety of the pneumococcus.

Micrococcus melitensis. — A micrococcus found by Bruce
in cases of Malta fever. It is a round or slightly oval
organism, about .5 IJL in diameter, occurring singly, in pairs
or in short chains. It is usually said to be non-motile,
though flagella have been described. It is stained by ordi-
nary aniline dyes, but not by Gram's method. It grows
slowly, even in the incubator, and more slowly at ordinary
temperatures. In gelatin the growth is feeble; there is no
liquefaction. On agar pearly white growths appear after
three or four days. Bouillon becomes turbid, with a sedi-
ment later. On potato there may be slight invisible growth.

Malta fever occurs chiefly about the Mediterranean. It
has been observed in India, in the Philippine Islands and in
Porto Rico.

It is a chronic febrile disease, accompanied by pains in the
joints and perspiration, and not very fatal. At autopsies
the organisms may best be recovered from the enlarged
spleen. Accidental infection in man has occurred from pure

1 Richardson, Journal Boston Society of Medical Sciences, Vol. V.,
1901.

2 Howard, Journal Medical Research, Vol. VI., 1901.

256 MANUAL OF BACTERIOLOGY.

cultures on a number of occasions. The disease may be
reproduced in monkeys by inoculation with pure cultures.
An agglutination reaction occurs in this disease. The
diagnosis is best made by applying this test to the blood-
serum of the patient, with a known pure culture of micro-
coccus melitensis.1 A suspension of an agar culture is
made in normal salt solution. The diluted serum is added
so as to secure a dilution of about i to 50, but the dilutions
used have varied widely. Precipitation quickly occurs.
According to Craig the test may be made on a slide, examin-
ing with the microscope as for the typhoid bacillus (see
Serum-test for typhoid fever).

Diplococcus intracellularis meningitidis.2 — Found in the
exudate of cerebro-spinal meningitis by Weichselbaum ; a
micrococcus about the size of the common pyogenic cocci ;
grows in pairs or fours, more often in pairs consisting of
two hemispheres separated by an interval which does not
stain ; usually found within the pus-cells, in which respect it
resembles the gonococcus. It is stained by ordinary methods
with the aniline dyes, and is decolorized by Gram's method.
It does not grow at the room temperature but only in the
incubator; gelatin is not available. There is no growth on
potato and scanty growth on agar or in bouillon. The
development is most abundant upon Loffler's blood-serum,
when round, white, shining, viscid-looking colonies with
sharp outlines may be seen in twenty-four hours. The
serum is not liquefied. Upon agar, or better upon glycerin-
agar, the colonies are flat, round, translucent, viscid-look-

1 Mtisser and Sailer, PhiladelpJiia Medical Journal, December 31, 1898,
July 8, 1899; Strong and Musgrove, Ibid., November 24, 1900; Curry,
Journal Medical Research, Vol. VI., 1901.

2 The writer is indebted for the brief statement, which it is possible to
give here, chiefly to the exhaustive Report to the Massachusetts Board
of Health by Councilman, Mallory and Wright, 1898. The photograph
was made from a preparation kindly furnished by Dr. Mallory.

PATHOGENIC BACTERIA. 257

ing, under the low power having a yellowish-brown color.
The organism should be transplanted to fresh media fre-
quently, as it rapidly loses its power of reproduction. Many
of the tubes inoculated with the original material or with
pure cultures show no growth.

It is moderately pathogenic for guinea-pigs and rabbits
when inoculated into the pleura or peritoneum. Menin-
gitis and encephalitis have been produced in the dog and
goat by inoculation in the meninges.

FIG. 63.

Diplococcus intracellularis meningitidis and pus-cells. (X 1000.)

This organism appears to be the principal if not the only
cause of epidemic cerebro-spinal meningitis. The lesion
consists of a purulent inflammation of the pia and arach-
noid, extending into the brain substance, over the cord,
and along the nerves. General invasion of the tissues of
the body seems not to occur, but focal areas of pneumonia
may be present. Spinal puncture in the lumbar region is
recommended as a means of diagnosis. The puncture
should be made early, and the fluid should be examined
with the microscope and by cultures.

258 MANUAL OF BACTERIOLOGY.

Micrococcus gonorrheas (Gonococcus of Neisser).—
Found in pus in cases of gonorrhea. The micrococci gen-
erally are in pairs, occasionally in groups of four. The
cocci are flattened, the flattened sides facing each other, and
they are often compared to a pair of biscuits. The long
diameter of the pair of biscuit-shaped elements is about
1.25 /*. The organisms are usually found attached to the
epithelial cells or inside of the pus-cells; they are also
found in smaller numbers floating free in the fluid. They
stain with ordinary aniline dyes, for example Loffier's
methylene-blue, but not by Gram's method.

The occurrence, (i) inside of the pus-cells, (2) of pairs
of biscuit-shaped micrococci (3) which are not stained by
Gram's method, will serve to distinguish the gonococcus
from all the other ordinary pus-forming bacteria. There
are other diplococci (pseudo-gonococci), probably non-
pathogenic, which have rarely been found in the vulvo-
vaginal tract and in the urethra, which, it is said, are also
decolorized by Gram's method. Such organisms are not
likely to present all the points mentioned as characteristic
of the gonococcus. The recognition of the gonococcus in
the discharges of a case of acute gonorrhea is usually a
matter of the greatest possible ease. It must be admitted,
however, that in cases having chronic discharges, when its
detection is most to be desired, the diagnosis may become
very difficult and is frequently impossible, except by culture-
methods, owing to secondary infection with the ordinary
pus-forming or other bacteria, which may be present in
larger numbers than the gonococci themselves.

The gonococcus grows only in the incubator, and cannot
therefore be cultivated upon gelatin. Its cultivation is in
fact a matter of some difficulty. The medium usually
selected is a mixture of agar with human blood-serum. The
blood-serum from the placental blood or pleuritic or peri-
toneal transudates, or hvdrocele fluid, has been taken. The

PATHOGENIC BACTERIA.

259

addition of human urine, -sterilized by filtration through
porcelain, to the mixture of blood-serum and agar im-
proves its character according to some writers. A con-
venient medium is one consisting of one part of human
serum derived from a pleuritic effusion, added to two parts
of a 2 per cent, nutrient agar. The agar has previously

FIG. 64.

Gonococci and pus-cells. (X 1000.)

been sterilized; the two are mixed in tubes while fluid;
they are cooled while in an inclined position, and are
sterilized between 65° and 70° C. by the fractional method
on six consecutive days. They are afterward tested in the
incubator for two days.

The colonies of the gonococcus are very small, grayish-
white, circular, translucent; appearing after from twenty-
four to forty-eight hours. They may attain a diameter of
i to 2 mm. The gonococcus will occasionally develop on
ordinary glycerin-agar or Loffler's blood-serum medium,
but the growth is likely to be feeble and cannot be relied

260 MANUAL OF BACTERIOLOGY.

on. The cultures live for a considerable time if kept from
drying. The gonococcus is not known to produce ure-
thritis or conjunctivitis in any of the lower animals. In
the peritoneum it may cause suppurative inflammation in
mice and guinea-pigs. Reproduction of the disease in
man has been effected by experimental inoculation with pure
cultures. Besides being the cause of gonorrheal urethritis
and infection of the cervix uteri, the gonococcus has been
isolated from cases of vaginitis in little girls, and from
gonorrheal conjunctivitis. It has been found to be the cause
of many cases of pyosalpinx, as well as of gonorrheal proc-
titis, arthritis, myocarditis and endocarditis; these condi-
tions complicating gonorrhea may also be secondary or
mixed infections.

Bacillus of Soft Chancre (of Ducrey). — A small, oval
bacillus, usually occurring in chains. It stains with ordinary
aniline dyes, but not by Gram's method. It has been culti-
vated on human blood-agar (also rabbit blood-agar; the
medium deteriorates in a few weeks, Davis). It is culti-
vated with difficulty. It is found in the pus of soft chancre
or chancroid, usually mixed with other organisms. It has
been demonstrated in sections of the ulcers. There seems to
be uncertainty with respect to its occurrence in buboes.
Ducrey was able to secure it in pure culture by successive
inoculations on the human skin. Although this bacillus has
not yet been sufficiently studied there seems little doubt that
it is the cause of soft chancre.1

Bacillus pneumonise (of Friedlander) , or Bacillus nin-
cosus capsulatus.2 — A short bacillus with rounded ends,
sometimes growing out to a greater length; sometimes
occurring in pairs; surrounded by a capsule which is only
seen in preparations made from the tissues of infected ani-

1 Davis, Journal Medical Research, Vol. IX., 1903.

2 Howard, Philadelphia Medical Journal, February 19, 1898; Curry,
Howard, Perkins, Journal Experimental Medicine, Vols. IV. and V.

PATHOGENIC BACTERIA. 26l

mals, and is not demonstrated in cultures. This bacillus is
not motile. It does not form spores. It stains with the ordi-
nary aniline dyes, but does not stain by Gram's method. It
is aerobic and facultative anaerobic. It may be cultivated
at ordinary temperatures, but grows better in the incubator.
It does not liquefy gelatin. Stick-cultures in gelatin develop
especially at the point where the puncture enters the surface
of the gelatin, making what is called a " nail-shaped "
growth; the growth in gelatin is white; in old cultures the
gelatin acquires a brown color. It develops also on the other
media. Dextrose and lactose are fermented by it ; in cultures
on potato, gas is formed; milk is not coagulated. It does
not produce indol.

The thermal death-point is about 56° C. It is patho-
genic for mice, less so for guinea-pigs and rabbits. This
bacillus is sometimes found in the healthy mouth and nose.
It has been known to cause inflammation, especially in the
vicinity of the mouth, nose and ear, broncho-pneumonia,
and more rarely empyema and meningitis. It was described
by Friedlander as the specific cause of lobar pneumonia.
Subsequent investigations indicate that it is comparatively
seldom found in pneumonia.

There are various capsulated bacilli (capsule bacilli of
R. Pfeiffer and others) which closely resemble the bacillus
of Friedlander, and at least belong to the same group. The
bacillus of ozaena, which has often been found in that dis-
ease is very similar. B. lactis aerogenes and B. coli com-
munis also have many points in common with the Fried-
lander bacillus.

Bacillus of Rhinoscleroma. — A short bacillus with
rounded ends, often united in pairs, also growing to a
greater length ; surrounded by a capsule ; not motile ; stained
by the ordinary aniline dyes. It is much like the bacillus
of Friedlander, but some writers have said that it is not so

262 MANUAL OF BACTERIOLOGY.

easily decolorized by Gram's method; this may be doubted,
however. The organism has been cultivated. It is faculta-
tive anaerobic. It grows rapidly, best in the incubator. It
does not liquefy gelatin ; its growth in gelatin stick-cultures
resembles the bacillus of Friedlander. It grows on the
ordinary media. Gas may be developed upon potato.

It is pathogenic for mice and guinea-pigs, less so for
rabbits. Its virulence is less than that of Friedlander's
bacillus.

It has been obtained from the tissues of cases of rhino-
scleroma. Rhinoscleroma is a disease characterized by a
chronic tubercular thickening and swelling of the skin
around the nose and similar swelling of the nasal mucous
membrane, sometimes followed by ulceration. It is com-
monest in Austria and Italy. It has been seen in America
only with the greatest rarity.

The organisms may be stained in the diseased tissues,
but their detection is a matter of considerable difficulty,
and they are not always found. It is not yet certain that
they are the cause of rhinoscleroma.

Bacillus pyocyaneus. — A slim bacillus with rounded
ends. It is motile. It does not form spores. At 56° C.
it is killed in ten minutes. It is decolorized by Gram's
method. It is aerobic; grows well at ordinary tempera-
tures; liquefies gelatin, and grows on the ordinary culture-
media. Cultures present a blue or green color, especially
in transparent media. This color is not confined to the
growth itself, but a blue or green fluorescence spreads
over the whole medium. In old agar-cultures the color
may become very dark. The pigment forms in the pres-
ence of oxygen, and is due, at least in part, to the pto-
maine, pyocyanin. On potato the growth is usually brown,
which may be tinged with green. Milk is coagulated and
peptonized and an acid reaction is developed. Indol is

PATHOGENIC BACTERIA. 263

formed in Dunham's peptone solution. Blood-serum is
liquefied.

The bacillus pyocyaneus seems to be rather widely dis-
tributed in nature; it has been found on the skin, in nor-
mal feces, also in diarrheal discharges and in dysentery. It
is the cause of the color in blue or green pus. It has fre-

FIG. 65.

M'/,

' l/^x---

% » , I V >

\ * '
C. * 'I

* * / I

.. \*t '

Bacillus pyocyaneus, pure culture. (X 1000.)

quently been demonstrated in pus, but oftenest perhaps, in
mixed infections. It has been found in various abscesses, in
otitis media, peritonitis, appendicitis and broncho-pneu-
monia. It has been known to produce general septicemia.1
It is pathogenic for guinea-pigs and rabbits, in whom it may
produce septicemia. In animals it may lead only to local
suppuration, from which they may recover, being made im-

1Lartigau, Philadelphia Medical Journal, September 17, 1898; Journal
Experimental Medicine, Vol. III., 1898; Perkins, Journal Medical Re-
search, Vol. VI., 1901.

264 MANUAL OF BACTERIOLOGY.

mune to subsequent infection with this organism. It ap-
pears that an antagonism exists between the products of
the bacillus pyocyaneus and the anthrax bacillus. Rabbits
which have been inoculated with cultures of the anthrax
bacillus may recover if they are injected shortly after with
a culture of the bacillus pyocyaneus.

Bacillus proteus. — A bacillus with rounded ends, vary-
ing much in length, breadth .4 to .6 ^ ; frequently appear-
ing as short ovals like micrococci; sometimes growing out
into long filaments, so that it is said to be pleomorphic.
Rounded involution forms occur. It is not stained by
Gram's method. It is motile. Spore formation has not
been observed. It is aerobic and facultative anaerobic. It
grows rapidly at ordinary temperatures. This organism
was originally described by Hauser as three different
species — proteus mdgaris, which was said to liquefy gel-
atin rapidly, proteus mirabilis, which liquefied gelatin
slowly, and proteus Zenkcri, which did not liquefy gelatin.
It seems probable that these organisms were, in fact,
varieties of the same species, now called bacillus proteus.
Upon gelatin-plates the colonies present a characteristic
phenomenon, when seen under the low power, in the pro-
jection of processes which subsequently change their form
and position, and which may become entirely detached
from the original colony, so that the surface of the gelatin
may become covered with so-called " swarming islands."

The proteus grows on the usual media tending to pro-
duce foul odor, decomposition and alkaline reaction. In
urine it converts urea into ammonium carbonate.

This organism is one of those which were formerly de-
scribed under the name of bacterium termo. It is among
the most common and widely-distributed bacteria. It has
been found in decomposing animal and vegetable sub-
stances, in the feces, in the urine in cystitis, and in the dis-

PATHOGENIC BACTERIA. 265

charges of children having cholera infantum. It appears
that this organism may occasionally be pathogenic to man,
causing pus-formation, peritonitis, and even general infec-
tion.1 Cultures injected in considerable amounts may be
pathogenic to animals.

Bacillus of Bubonic Plague. — An oval or short rod-
shaped bacillus, with rounded ends, sometimes possessing
a capsule. It is not motile. It does not form spores. With
the aniline dyes the ends stain more deeply than the middle,

FIG. 66.

X. %% *<r

^*> » *

• * ju i

Bacillus of Bubonic Plague. (Yersin.)

called polar staining; by Gram's method it is decolorized.
It is aerobic. It grows at ordinary temperatures, but better
in the incubator. It grows on most media. The growths
are grayish-white. Gelatin and blood-serum are not lique-
fied. In bouillon, the medium remains clear, while a granu-
lar deposit forms on the sides and bottom of the tube. In
bouillon to which a few minute drops of sterile oil, as
cocoanut oil, have been added, a growth takes place from
the under side of the oil drops. Such growths extend down,
and are called stalactite growths. The stalactites break off,
with the slightest disturbance.
1 Ware, Annals of Surgery, Vol. XXXVL, 1902.

266 MANUAL OF BACTERIOLOGY.

On agar containing 3 per cent, of common salt remark-
able involution forms appear. The stalactite growths and
the forms occurring on salt-agar are considered the most
characteristic cultural tests.1

It is sometimes sensitive to drying, but may survive pro-
longed drying. It is killed in three to four hours by direct
sunlight, when spread in thin layers; in a few minutes by
steam at 100° C., and in one hour by I per cent, carbolic
acid.2 It is pathogenic to rats, mice, guinea-pigs, rabbits,
and a number of other animals.

In man it appears usually to enter through wounds of
the skin. Other possible avenues of infection are the air
passages, the mouth, and the gastrointestinal tract. Plague
is usually regarded as having three different possible forms,
—the bubonic, the pneumonic and the septicemic. The
bubonic form is commonest. The point in the skin at which
the inoculation takes place seems generally to exhibit no
inflammatory reaction. The lymph-nodes are generally
swollen, especially the deep inguinal and axillary nodes.
The swollen lymph-nodes may suppurate. The suppurating
nodes often are infected simultaneously with micrococci.
The bacilli are numerous in the enlarged lymph-nodes, but
may be detected in the other organs of the body and in the
blood. Fluid drawn from the buboes with a hypodermic
needle may be examined microscopically, by cultures and by
inoculation into rats or guinea-pigs. In the pneumonic or
pulmonary form the bacilli occur in the sputum, and may
be tested in the same manner. This type of the disease is
said to be very fatal. In the septicemic form no primary
bubo is found, or a bubonic case may become septicemic.
This form is very fatal.

1 Wilson, Journal Medical Research, Vol. VI., 1901.

2 See Viability of Bacillus pestis, Rosenau, Marine Hospital Service,
Hygienic Lab'y, Bull. No. 4, 1901.

PATHOGENIC BACTERIA. 267

During epidemics of plague it has been noted that rats
may die in large numbers, and plague bacilli have often been
recovered from the bodies of such rats. The systematic
destruction by health departments of all the rats possible is
important where an epidemic is present or is feared. The
same applies to mice. The agency of fleas as carriers of
the bacilli has been suggested, but has not yet been proved ;
this is equally true as to flies.

The greatest care must be used in working with the
bacillus of plague. A number of fatal results have occurred
through it in laboratory investigators.

Haffkine has invented a method of protective inoculation
against plague by the injection of cultures of plague bacilli
which have been sterilized by heat, and a little carbolic acid
added. An active immunity, w-hich is quite lasting, it is
maintained, may be secured in some days. The injection
is sometimes followed by considerable constitutional dis-
turbance. This method seems likely to be of considerable
value.

Yersin and others have prepared protective sera on the
same general principles used in making other sera for effect-
ing passive immunity. It is hoped they may be useful in
producing quickly a temporary immunity; and the outlook
for their employment in the treatment of the disease is
very encouraging.

An agglutination reaction has been described; it is not
likely to be cf great value in diagnosis.

The period of incubation in this disease is from two to
seven days. It has occasionally appeared in civilized
countries during recent times though not to a very seri-
ous extent. Among the localities of importance to us it has
recently visited the Philippine Islands, California and
Mexico. It has ravaged the southeastern part of Asia
within a few years. In the Middle Ages, and in succeeding
23

268

MANUAL OF BACTERIOLOGY.

centuries, it devastated many of the countries of Europe,
where it was one of the most important of the pestilences
that went in those days by the name of the " Plague." It
appears to have been the disease known in English history
as the " Black Death."1

Bacillus aerogenes capsulatus. — A thick bacillus, 3 to 6
fj- in length, frequently capsulated, discovered by Welch and
Nuttall. The capsules may be found in preparations from

FIG. 67.

Bacillus aerogenes capsulatus, Smear-preparation from Rabbit's Liver.

(X 1000.)

animal tissues, but rarely in cultures. It sometimes forms
spores chiefly in cultures on blood-serum. The vegetative
forms are destroyed at 58° C. moist heat in ten minutes,
but the spores withstand boiling nearly 8 minutes. It is not
motile. It stains by Gram's method. It is anaerobic, and is

1 For further details concerning plague consult articles by Barker,
Novy and Flexner, Trans. Association American Physicians, 1902;
Calvert, American Medicine, January 24, 1903.

PATHOGENIC BACTERIA.

269

readily cultivated by Buchner's
method for anaerobes. It grows
best at the body temperature, but
will grow at the room tempera-
ture. It may liquefy gelatin
slowly or not at all. The growths
are whitish. In media containing
lactose, dextrose, or saccharose it
produces an abundance of gas ; but
it is also, according to Welch,
able to form gas from proteids.
Milk is coagulated, and the re-
action becomes acid. Gas forms
upon potato, where the growth
is thin and grayish-white.

It occurs in the intestine of
man and various other animals,
in soil, sewage and water. It is
not usually pathogenic to rabbits
and mice. In guinea-pigs, spar-
rows and pigeons it may produce
" gas phlegmons." It has been
found on numerous occasions in
the organs of human cadavers
in which a development of gas
had taken place, producing bub-
bles or cavities in the tissues, im-
parting to them a peculiar spongy
character (German, Schaumor-
gane). Probably this is as a rule
a post-mortem invasion, but there
is reason to believe that in some
cases it enters the circulation dur-
ing life. It has been found in

FIG. 68.

cases of emphysematous gangrene gas-bubbles.

Bacillus aerogenes capsulatus,
culture in dextrose-agar showing

270 MANUAL OF BACTERIOLOGY.

or cellulitis, in various uterine infections, including physo-
metra and emphysema of the uterine wall, in pneumothorax
and pneumoperitonitis, and in other pathological conditions
where gas occurs in the tissues. Exceptionally it may cause
pus-formation.1 This bacillus, or the gas formed by it in
the organs of human cadavers, appears to have furnished
the basis for some of the cases in which death has been
ascribed to the entrance of air into the veins during life. It
is the same as the organism described by E. Frankel as
bacillus phlegmones emphysematosae.

Bacillus edematis maligni (French, ribrion scptiquc).
—A bacillus about I n in breadth, 2 to 10 ,« in length, which
may form threads, having rounded ends when occurring
singly. It is motile, having flagella at the sides and ends.
It forms spores, and may bulge at the center in consequence
of the spores lorn^ed there. It is decolorized by Gram's
method. It is a strict anaerobe and is best cultivated under
hydrogen. It grows at ordinary temperatures, but better
in the incubator. It liquefies gelatin and blood-serum. The
colonies in gelatin are spherical and appear like little
bubbles. It grows well upon agar. Gas may be produced
in these media.

It is found in garden earth, street dirt, and in putrefying
organic material. It is pathogenic to rabbits, guinea-pigs,
mice, pigeons and various other animals, including man.
Inoculation results in the production of swelling and edema,
spreading from the point of inoculation. Gas may be pro-
duced in the tissue. It may lead to widespread septicemia.

Bacillus tetani. — A slim, straight bacillus, with rounded
ends, which may form in threads. It is slightly motile.
Spores form in culture-media at the end of thirty hours in
the incubator. The spores are located at one end, which
is swollen, so that in this stage the organism has the shape

1 Welch, Philadelphia Medical Journal, August 4, 1900.

PATHOGENIC BACTERIA. 27 1

of a drum-stick. The spores are extremely resistant, and
in the dry condition can exist for years. They are killed
by moist heat at 100° C. in five minutes; by 5 per cent,
carbolic acid in fifteen hours ; by bichloride of mercury,
i-iooo, in three hours. The tetanus bacillus stains by
Gram's method. It is a strict anaerobe; it grows in an
atmosphere of hydrogen, but not of carbon dioxide. It may
sometimes be made to grow very well by Buchner's method.

FIG. 69.

Tetanus bacilli, showing spores. (X 1000.)

It may be cultivated at the room temperature, but better in
the incubator. It grows upon ordinary culture-media, pref-
erably those containing dextrose. Gelatin is liquefied
slowly; the colonies in gelatin present characteristic radi-
ating filaments and look like a thistle. It grows on the
other culture-media. Gas formation is not pronounced.

This organism appears to be widely spread in external
nature, especially in the soil. It is often found in garden
earth, and in the feces of herbivorous animals. McFarland

272 MANUAL OF BACTERIOLOGY.

believes that it may occur in vaccine virus when that is care-
lessly prepared, which would explain the rare occurrence of
tetanus after vaccination.1 Tetanus bacilli have been found
in gelatin, and it is stated that the tetanus has followed the
injection of gelatin as a hemostatic. The infection appears
almost always, if not always, to be introduced through
some wound.2 Clinically, persons having the disease suffer
from spasms of the muscles about the neck and the lower
jaw (lock jaw). The spasms finally become general.

Inoculation with a pure culture produces tetanus in mice ;
also in rats, guinea-pigs and rabbits. The tetanic spasms
begin in the vicinity of the point of inoculation and after-
ward become general. The bacilli are not widely scattered
through the body ; they occur only in the immediate vicinity
of the original lesion, and there are no important macro-
scopic alterations in the internal viscera.

Tetanus is the type of the purely toxic disease. Its
symptoms may be produced in animals by the injection of
liquid cultures which have been deprived of their bacteria
by filtration. The toxic substance appears not to be a pto-
maine, as was at first supposed, and its exact nature is not
determined.

The poison is tremendously powerful (see page 174). It
acts as an excitant to the motor cells of the central nervous
system, especially the spinal cord. Bolton and Fisch have
pointed out the possibility that horses used for the prepara-
tion of diphtheria antitoxin may be infected with tetanus,
and have tetanus toxin in the blood. ::

The activity of the poison is destroyed by heat, and by
direct sunlight ; various chemicals diminish its intensity.

Antitoxins for tetanus have been prepared according to
the principles employed for antitoxins in general. They

1 Journal Medical Research, Vol. VII., 1902.

2 Wells, "Fourth of July Tetanus,'' American Medicine, June 13, 1903.

3 Trans. Association A^ncrican Physicians, 1902.

PATHOGENIC BACTERIA. 2/3

have not proved very markedly successful. Unfortunately
the disease is seldom suspected until a relatively large
amount of toxin has formed and begun to manifest its action
in the patient's body.1

Bacillus anthracis. — This is the largest of the patho-
genic bacteria with the exception of the spirillum of relaps-
ing fever, which is longer but more slender. The bacillus
of anthrax is 1.25 // broad, and from 3 to 10 n long. Ba-

FIG. 70.

Anthrax bacilli, from a pure culture.2 (X 1000.)

cillus aerogenes capsulatus is of about the same size. It
often forms long threads. A capsule is sometimes present.
It is not motile. It forms spores, which are placed in the
centers of the bacilli. The spores form only in the pres-
ence of oxygen; they do not appear in the body of an in-
fected animal during life. Anthrax spores are the most

1 See also Moschkowitz, Studies Department Pathology, College Physi-
cians and Surgeons, New York, Vol. VII., 1899-1900. Annals of
Surgery, 1900, p. 442.

2 The culture was derived from a case of malignant pustule in a
tanner. The lesion was excised promptly, and the patient recovered.

274 MANUAL OF BACTERIOLOGY.

resistant of all pathogenic bacteria; they have been known
to withstand boiling for twelve minutes,1 5 per cent, carbolic
acid for forty days, and i-iooo bichloride of mercury for
nearly three days. The anthrax bacillus is aerobic, although
not strictly so. It stains by Gram's method. It grows at
the room temperature, but better in the incubator. It
liquefies gelatin and blood-serum. Colonies in gelatin seen

FIG. 71.

Anthrax bacilli, showing spores. (X 1000.)

under a low power display numerous, irregular, fine, hair-
like projections; stab-cultures in gelatin also present fine
projections passing from the needle-puncture into the solid
gelatin. It grows on the ordinary culture-media; the
growths are usually whitish. Cultures on potato kept in
the incubator are particularly favorable to the development
of spores. Milk is coagulated and later peptonized.

1 More than half an hour. V. A. Moore. "Infections Disease of
Animals," 1902.

PATHOGENIC BACTERIA.

275

FlG

It is pathogenic to mice and guinea-pigs, less so to rab-
bits; it is also pathogenic to sheep and cattle. Rats and
pigeons are quite resistant but not entirely immune; cats,
dogs and frogs are not sus-
ceptible, or but slightly so.

Anthrax is a disease
which occurs chiefly in cat-
tle and sheep. It is com-
moner on the continent of
Europe and in Siberia than
in America. In susceptible
animals inoculated with
virulent cultures of the an-
thrax bacillus septicemia is
produced. Large numbers

Of the bacilli are found in Colony of Anthrax bacilli (low power).

FIG. 73.

Bacillus of anthrax. Stick-culture in gelatin. (Giinther.)

the blood, and may be crowded together in the capillaries
of the liver and kidneys. Men are occasionally affected,
24

276 MANUAL OF BACTERIOLOGY.

especially those whose occupations bring them in contact
with cattle or with the hides and wool of animals that die
of the disease. The infection may enter through wounds
of the skin, where it usually produces a localized inflam-
mation known as malignant pustule. Anthrax of the lungs
may be acquired by inhalation of material containing the
spores of the bacilli (" Wool-sorter's disease "). Infection
FlG by way of the intestine oc-

curs occasionally but is less
common. Laboratory
workers engaged in study-
ing the anthrax bacillus
have been accidentally in-
fected in a number of in-
stances.

The anthrax bacillus,
owing to its large size,
was the first of the patho-
genic bacteria to be recog-
nized, and its study has

Anthrax bacilli with square or furnished the basis for
slightly concave ends sometimes seen ; , r

much of our knowledge

fuchsin stain. (X 1000.) . &

concerning the infectious

diseases. It was for anthrax that Pasteur developed the
idea of making a protective vaccine, shortly after he had
invented a similar vaccine for chicken-cholera. There is
some danger attending its use.

In order to obtain material free from spores the blood of
an animal which has recently died of anthrax is taken,
because anthrax spores do not form in the living body.
Cultures made in bouillon are kept at a temperature of
from 42° to 43° C. At this temperature spores do not
form, while the virulence of the anthrax bacillus becomes
gradually diminished. In time the virulence is so far
diminished that rabbits will survive inoculation, and even-

PATHOGENIC BACTERIA. 277

tually also mice and guinea-pigs, which are extremely sus-
ceptible to anthrax. Small doses of a culture of extremely
weak virulence are given to the animals which it is desired
to protect, like cattle and sheep (never human beings), and
subsequently a somewhat more virulent culture is employed.1

FI<;. 75

Anthrax bacilli in the capillaries of the liver of a mouse, sketched from
a section stained with fuchdn.

Bacillus influenzas. — A small bacillus, .2 to .3 j*. by .5 IJL
with rounded ends. It does not form spores, is not motile,
and is decolorized by Gram's method. It is aerobic, grows
only in the incubator, and upon media containing hemo-
globin. The medium is prepared by smearing sterile blood
over the surface of a tube of agar. Fresh, uncoagulated
blood may, with care, be mixed with melted agar sufficiently
cooled; the mixture may be poured into tubes and slanted;

1 For details as to the results of this method, see V. A. Moore, " In-
fectious Diseases of Animals," 1902. For other and unique researches on
immunity for anthrax see Emmerich, Centralblatt f. Bakteriologie, Orig.
Bd. XXXII, p. 821.

2/8 MANUAL OF BACTERIOLOGY.

the tubes should be tested in the incubator before using.
The blood of some animals, as the pigeon and rabbit, may
be used instead or human blood.1 The colonies are small
and transparent, looking like little drops of water, not be-
coming confluent.

Of a large number of bacilli, the majority are destroyed
in twenty-four hours or less by drying. They die out in
a similar manner in water. Experiments upon animals
appear up to this time not to have been very convincing.
In diagnosis, the sputum should be carefully collected in a
sterile bottle. If the particles of sputum are likely to have
become contaminated, rinse in sterile water. Inoculate on
agar and on blood-agar. The influenza bacillus should grow
only on blood-agar and have the other characters above
mentioned. As far as is known, this organism grows only
in man, and not outside of the human body. In cases of
influenza it is found in the mucous discharges, and in the
bronchi and lungs. It is the predominating organism in
some cases of bronchitis.2 According to Canon, the bacilli
may sometimes be found in the blood.

Bacillus diphtherias (Klebs-Loffler). — A straight or
slightly-curved bacillus, usually 1.2 to 2.5 ft in length, with
rounded or slightly pointed ends, remarkable for showing-
irregularities of form, sometimes being club-shaped or
spindle-shaped; branching forms have been found.3 It is
not motile, and does not form spores. It retains its color
after Gram's method, but it is best stained with watery
solutions of the aniline dyes, especially LofHer's alkaline
methylene-blue. Very characteristic pictures are obtained
by the method of Neisser :

».

1 Ccntralblatt f. Baktcriologic, Bel. XXXII., Orig. p. 667.

2 See Lord, Hoston Medical and Surgical Journal, December 8, 1902.
;: Hill, Journal Medical Research, Vol. VII, 1902.

PATHOGENIC BACTERIA. 279!

SOLUTION No. i.

Methylene-blue I

Alcohol (96 per cent. ) 20

Distilled water 95°

Glacial acetic acid 50

SOLUTION No. 2.

Bismarck brown I

Boiling distilled water 500

Stain the cover-glass preparation which has been fixed
in the flame in No. i one to three seconds ; wash in water ;
stain in No. 2 three to five seconds; wash in water; mount

FIG. 76.

* » -*j -*

[ r O, ' \ v.

Bacillus of diphtheria. (X 1000.)

as usual. The body of the bacillus is stained pale brown,
with dark blue spots, especially at the ends. (Fig. 77.)

The diphtheria bacillus is peculiar in staining irregularly ;
certain spots stain more sharply than other portions, and
darkly-stained spots are likely to occur at the ends. It is
facultative anaerobic. It grows most rapidly in the incuba-
tor, and slowly, or not at all, below 20° C. Gelatin is not
liquefied. It may be cultivated on various alkaline culture-

28O MANUAL OF BACTERIOLOGY.

media, but grows best on Loffler's blood-serum mixture.
On this medium the growth consists of small white or cream-
colored, slightly elevated colonies, which may become con-
fluent. The morphology of the bacillus is most character-

Bacillus of diphtheria stained by Xeisser's method. (X 1000.)

istic when it is cultivated on blood-serum. It also grows
upon glycerine-agar. On potato it produces an invisible
growth (see Bacillus of Typhoid Fever). In alkaline
bouillon containing dextrose (or muscle-sugar) the reaction
becomes acid in forty-eight hours. The reaction of the
bouillon subsequently becomes alkaline. The growth may
form a pellicle over the surface of the bouillon. It has also
been successfully cultivated on various media to which egg-
albumen has been added.

It is killed by a temperature of 58° C. in ten minutes.
It resists desiccation well.

Bacteriological diagnosis of Diphtheria. — In many large
cities the bacteriological diagnosis of diphtheria is under-
taken bv boards of health. The methods used differ some-

PATHOGENIC BACTERIA.

28l

FIG. 78.

what in detail, but are similar in the main, and are based
upon the procedure devised by Biggs and Park for the
Board of Health of New York City. Two tubes are fur-
nished in a box. The tubes are like ordinary test-tubes,
about three inches in length, rather
heavy, and without a flange. Both are
plugged with cotton. One contains slant-
ed and sterilized Loffler's blood-serum
mixture; the other contains a steel rod,
around the lower end of which a pledget
of absorbent cotton has been wound and
the tube afterward sterilized. The swab
is wiped over the suspected region in
the throat, taking care that it touches
nothing else, and is then rubbed over the
surface of the blood-serum mixture. The
swab is returned to its test-tube and the
cotton plugs are returned to their respec-
tive tubes. The plugs, of course, are held
in the fingers during the operation, and
care must be taken that the portion of the
plug thatgoes into the tube touches neither tube usf;.n ,th.c

nosis of diphtheria.

the finger nor any other object. The prin-
ciples, in fact, are the same as those laid down in general for
the inoculation of culture-tubes with bacteria (see page 84).
In board of health work these tubes are returned to the
office. When it is desirable, a second tube may be inoculated
from the swab. The tubes are placed in the incubator,
where they remain for from twelve to twenty-four hours,
and a microscopical examination is then made of smear pre-
parations stained with Loffler's methylene-blue. On Loffler's
blood-serum kept in the incubator the bacillus of diphtheria
grows more rapidly than the other organisms which are ordi-
narily encountered in the throat, a property which to a cer-
tain extent sifts it out, as it were, from them, and makes its

Swab and culture-

282

MANUAL OF BACTERIOLOGY.

recognition with the microscope easy in most cases. The
growth, furthermore, is quite characteristic, and its nature

can be predicted with consider-
able accuracy, even without mi-
croscopical examination, by one
who has had much practice.
Colonies of streptococci fre-
quently look very like those of
the bacillus of diphtheria, but
these two are easily distin-
guished from each other with
the microscope. The diagnosis
of the diphtheria bacillus, then,
is made from the character of
the growth upon blood-serum
and the microscopical exami-
nation, taking into account the
size and shape of the bacilli,
with the frequent occurrence
of irregular forms and the pe-
culiar irregularities in stain-
ing. In doubtful cases a sec-
ond culture should be made
from the throat.

The very large number of
examinations that have been
made by various boards of
health, have shown that pseudo-
membranous inflammations of
the throat are sometimes caused
by streptococci alone, or by
They have also shown that the
diphtheria bacillus may persist in the throat for a long

1 Bissell. .Ifedictil NeiVS, May 31, 1902; American Journal Medical
Sciences, February, 1903, Review of Work of Massachusetts Boards of
Health.

Bacillus of diphtheria, culture
on glycerine-agar.

other pyogenic bacteria.1

.PATHOGENIC BACTERIA.

time, occasionally several weeks after the patient has
apparently recovered; also that diphtheria bacilli are
occasionally found in the throat when there is an in-
flammatory condition without any pseudo-membrane,
and that they sometimes appear in an apparently healthy
throat, especially in children who have been associated with
cases of diphtheria. It has been found that bacilli some-
times occur in the throat which have all the morphological
and cultural properties of the diphtheria bacillus, but which
are devoid of virulence when tested upon animals, i. e.t
do not produce diphtheria toxin. Such diphtheria bacilli
have frequently been called pseudo-diphtheria bacilli. A
bacillus closely resembling the diphtheria bacillus, but with-
out virulence, has been found in xerosis of the conjunctiva.
It is called the xcrosis bacillus. If not a transformed diph-
theria bacillus, it is at least closely related. The diphtheria
bacillus is subject to wide variations in morphology, so that,
in dealing with unknown cultures where the forms of the
bacilli are not characteristic and injection into animals is
without result, it may be difficult to decide whether or not
the organisms are diphtheria bacilli. Consequently another
view with regard to pseudo-diphtheria bacilli has arisen.
While recognizing that avirulent diphtheria bacilli occur,
it is also claimed that a distinct pseudo-diphtheria bacillus
exists, different from the diphtheria bacillus, though re-
sembling it. It is shorter, stains more evenly, shows no
polar granules by Neissers method of staining, does not
produce acid in dextrose-bouillon, and is not pathogenic to
animals. It is found occasionally in the nose and throat,
and has no connection with diphtheria, according to this
view.1

1The different sides of this question will be found fully discussed
by the following: Wesbrook, Wilson and McDaniel, Trans. Association.
American Physicians, 1900; Gorham, Journal Medical Research, Vol.
VI., 1900; A. Williams, Ibid., Vol. VIII., 1902; Denny, Ibid., Vol. IX.,
1903.

284 MANUAL OF BACTERIOLOGY.

The diphtheria bacillus is pathogenic to animals. When
it is injected into them it produces a toxemia. In the
guinea-pig, which is especially susceptible, local inflamma-
tion results, and death occurs usually in two or three days.
The bacilli are found to be confined to the vicinity of the
wound, and not usually to be disseminated throughout the
whole body. The death of the animal, therefore, is due to
the poisons elaborated by the diphtheria bacilli — either
poisons introduced at the original injection, or substances
produced by the bacilli which may have multiplied in the
animal's body. The internal viscera, especially the liver,
often exhibit small areas consisting of necrotic cells; a
transudation of serum takes place in the great serous cavi-
ties, and the lymph-nodes are swollen. A genuine diph-
theritic membrane may be produced on the trachea of a
young kitten by rubbing into it a part of a culture of the
diphtheria bacillus.

As is well known, the pseudo-membranous affection pro-
duced by the diphtheria bacillus in man is generally seen in
the larynx and pharynx. Membranous rhinitis is also
caused by the diphtheria bacillus. On the other hand,
pseudo-membranous affections of the larynx and pharynx
may be produced by streptococci. Pseudo-membranes
occurring in the throat during scarlet fever and measles
may be due to the diphtheria bacillus, but are more
often caused by streptococci. The affection known as mem-
branous croup is usually diphtheria of the larynx, produced
by the diphtheria bacillus. The diphtheria bacillus is a rare
cause of puerperal fever. Although the uninjured skin is
not attacked by the diphtheria bacillus, it may be present in
pseudo-membranes on wounded surfaces, usually in con-
nection with diphtheria in the throat. Most pseudo-mem-
branes formed upon wounds of the skin are produced by
other bacteria than the diphtheria bacillus, as is also the case

PATHOGENIC BACTERIA. 285

with the pseudo-membranous inflammations of the intestines
and bladder. Although such inflammations are often called
" diphtheritic," it must be remembered that the expression
is used in an anatomical sense, meaning that a fibrinous
pseudo-membrane has formed, extending deeply into the
tissues, which is not necessarily caused by the diphtheria
bacillus.

In cases of diphtheria in man,1 the diphtheria bacillus is
generally found limited to the vicinity of the pseudo-mem-
brane, and at autopsies it is not usually found in the in-
ternal viscera, excepting in the lungs, where diphtheria
bacilli may or may not be present when diphtheria is com-
plicated with broncho-pneumonia. The general symptoms
of the disease, including the paralysis which sometimes
follows it, are due to the toxins produced by the bacilli in
the throat.

Diphtheria antitoxin. It is necessary first to obtain the toxin pro-
duced by diphtheria bacilli in a concentrated form. Virulent diphtheria
bacilli are cultivated in alkaline bouillon, in flasks plugged with cotton,
exposing a large surface to the air. The cultures are grown in the
incubator. After five to ten days they are ready, and are filtered through
porcelain. The filtrate contains the toxin. The animal usually em-
ployed is the horse, which should be healthy; the presence of tubercu-
losis and glanders should have been excluded, testing with tuberculin
and mallein ; tetanus should also be considered, see page 272. The toxin
is injected into the horse in small doses — about i c.c. of the filtrate from
the bouillon culture. The dose depends on the strength of the toxin.

The injection is repeated at intervals of about one week, using larger
and larger doses, until the animal is able to tolerate a very large dose
indeed — as much as 300 c.c., or even more. If the treatment is suc-
cessful the general condition of the animal should not suffer. The

1 For a full study of the lesions of diphtheria see the Monograph of
Councilman, Mallory and Pearce, Boston, 1901.

2 See articles by Park, A. Williams, Atkinson and T. Smith, Journal of
Experimental Medicine, Vol. I., p. 164; Vol. III., p. 513; Vol. IV., pp.
373 and 649; Journal Medical Research, Vol. IX., p. 173.

3 W. H. Park adds 10 per cent, of a 5 per cent, solution of carbolic acid
to kill the bacilli, and filters through paper on the following day; after
adding carbolic acid the Berkenfeld filter may be used with advantage
instead of filter-paper.

286 MANUAL OF BACTERIOLOGY.

injections last over a long period — usually about two or three months.
The general condition of the animal remaining good, the toleration of
these large doses of toxin is presumed to indicate the existence of a con-
centrated antitoxic substance in the blood. Small quantities of blood
may be withdrawn from time to time, and the serum tested for its
antitoxic strength. When a satisfactory serum has been attained, the
animal may be bled and the serum saved for therapeutic purposes.
Through an incision in the skin a trochar is inserted into the jugular
vein. The blood is conducted into sterilized flasks with every precau-
tion to insure sterility. The blood is allowed to coagulate and is placed
for a time in the ice-chest. The serum is then withdrawn with steril-
ized pipettes. Small amounts of chemical germicides, as carbolic acid
or chloroform, are sometimes added to assist in preserving it. This
serum is the so-called antitoxin used in medical practice.

A standard to express the potency of the serum, called an immunity
unit, has been devised by Behring and modified by Ehrlich. Such an
immunity or antitoxic unit is the amount of antitoxic principle con-
tained in that quantity of serum, which, when mixed and injected with
one hundred times the fatal dose of toxin will preserve the life of a
guinea-pig weighing 250 grains for four days.

It has been proposed to modify this test so as to lessen certain
sources of error. According to the new method, one hundred fatal
doses of toxin would not be used, but the amount of toxin, which,
when mixed with one unit of some special and previously standardized1
antitoxin and injected into a guinea-pig weighing 250 grams, will kill
the animal in four days. This procedure would partly do away with
inaccuracies, unavoidable where one hundred fatal doses of toxin are
used as a standard, resulting from changes that occur in the toxin
(development of toxoids, presence of IOXOIK-S, etc, Ehrlich).

It has been found possible to prepare antitoxin of a high
degree of concentration, so that 500 to 1,500 units may be
contained in a quantity of serum which it is practicable to
give at a single hypodermic injection. The large volume of
statistics that have been collected from hospitals, and from
physicians in private practice indicates that the use of this
serum has effected a very great reduction in the mortality
from diphtheria.

'The standard largely used in this country is an antitoxin prepared
for this purpose by the lustitut fiir cxpcrimcntcllc Tlicrapic, Frankfort
a Main, (iermany. Prof. Ehrlich, Director.

PATHOGENIC BACTERIA. 287

Bacillus tuberculosis. — A slim bacillus 1.5 to 4 ^ in
length, which very frequently presents a beaded appear-
ance, owing to its being dotted with bright, shining spots.
Branching forms have been described. The tubercle bacillus
is considered by some to be a member of the actinomyces
group. It is not motile. It has not been proved that spores
are formed; nevertheless certain structures, like caseous
lymph-nodes, have been shown to be capable of infecting
guinea-pigs with tuberculosis, although tubercle bacilli could

FIG. 80.

Bacillus tuberculosis, from a pure culture. (X 1000.)

not be demonstrated in them with the microscope. This
makes it seem possible that the organisms were present as
spores which eluded the microscopical examination. The
tubercle bacilli stain with the ordinary aniline clyes and by
Gram's method. As has already been stated, when stained
with aniline-water dyes or carbol-fuchsin they are not
readily decolorized by acids and alcohol, which fact distin-
guishes them from all other known bacteria excepting the
leprosy bacillus, the bacillus of smegma, possibly the bacillus

. \A x

288 MANUAL OF BACTERIOLOGY.

of syphilis (Lustgarten), and certain bacilli found in milk,
butter and cow-dung and on various grasses. All of these
may resist decolorization by acids or alcohol, and some
resist both. They must always be kept in mind in making
a diagnosis of tuberculosis. (See pages 44 and 295.) In
FIG. 8r. examining sputum it is particularly

important to bear in mind that acid-
proof bacilli, resembling tubercle
. bacilli, have rarely been found in

r cases of gangrene of the lung.

They are likely to be longer than
> tubercle bacilli, and branch more
/ often, besides being less resistant

to decolorization.1 The tubercle
Branching form of bacilli appear to owe their peculiar

tubercle bacillus from a . . . f ,

staining properties to fatty sub-
culture. (X 1000.) l J

stances contained in the bodies of the

bacilli. In stained preparations the bacillus usually appears
very distinctly beaded, owing to the presence of stained areas
which alternate with unstained areas; these unstained areas
have been considered by some to be spores.

The bacillus tuberculosis is aerobic. It is cultivated with
considerable difficulty, best at about 38° C. It does not
grow at a temperature below 29° C., and cannot therefore
be cultivated upon gelatin. It grows best upon blood-serum,
where the growth becomes visible in from ten to fourteen
days in the incubator. It forms a dry, mealy, scaly mass,
elevated above the surface, of a grayish-brown color. It also
grows upon glycerin-agar ; or glycerin-bouillon, on which
it forms a pellicle; upon potato; upon milk containing i per
cent, of agar and upon coagulated egg (see page 81). It
is important to have the medium moist. It can be cultivated

1 Ophiils, Journal Medical Research, Vol. VIII., 1902; Ohlmacher,
Journal ^linerican Medical Association, 1901.

PATHOGENIC BACTERIA. 280

from tuberculous sputum only with very great difficulty. It
is best to obtain it from the tissues of an animal that has
died of tuberculosis, where the tubercle bacilli may be found
unmixed with other bacteria. Pieces of tissue should be
taken with the precautions necessary to avoid contamina-
tion, and should be broken up and rubbed over the surface
of the medium. The tubes must be closed with sealing-wax,
paraffin or rubber stoppers, or covered with rubber caps,

FIG. 82.

Bacillus tuberculosis in sputum, stained with fuchsin and methylene blue.
Photomicrograph in two colors. (X 1000.)

to prevent drying in the incubator. If rubber caps are used
they should first be left in i-iooo bichloride of mercury for
an hour, and the cotton plug should be burned before putting
on the rubber cap. A number of tubes should be inoculated,
'using rather large particles of the tuberculous material.
Among the tubes inoculated, many will fail to present any
growth. After the organism has once been grown upon
a culture-medium it may be propagated with less difficulty.
It is best cultivated the first time upon blood-serum.

290 MANUAL OF BACTERIOLOGY.

It is killed by 5 per cent, solution of carbolic acid in a
few minutes. In sputum it is destroyed in twenty-four
hours by a three per cent, solution of carbolic acid. It
resists desiccation for months, but is killed in some hours
by direct sunlight. It is destroyed in a few minutes by
boiling..

It is not known to grow outside of the animal body. It
is the cause of tuberculosis in man. It produces tubercu-
losis in apes, cows, sheep, horses, rabbits, guinea-pigs, cats,
field-mice, and occasionally in other animals. Guinea-
pigs and rabbits are extremely susceptible. A guinea-pig in-
oculated with tuberculous sputum (provided it does not die
of septicemia, due to the pyogenic micrococci which are fre-
quently present in sputum) will present a swelling of the
neighboring lymph-nodes in the course of two to four weeks,
and will die as a rule in from four to eight weeks, although
the time may be longer.

Tuberculosis in cattle ((lerman, J'crJsuc/if} is characterized by large.
nodular lesions, \\itb a marked tendency to become fibrous, caseous
and calcified. Tbe tubercle bacilli of cattle differ somewhat from those
of human tuberculosis, as was noted by T. Smith.1 \Yhether or not
men could be infected with bovine tubercle bacilli, has been a question
that has been warmly debated in recent years. It seems to have been
shown that such infection is possible: also that it is possible that cattle
may be infected with human tubercle bacilli. Bovine tubercle bacilli
are more virulent for some animals, as rabbits, than human tubercle
bacilli.2 It seems possible that the danger of infection from cattle has
been somewhat overrated.

The lesion produced by the tubercle bacilli in the tissues
of men and the lower animals is called a tubercle, which
in the beginning is a grayish-white area about the size of a

^Journal Experimental Medicine, Vol. TIL, p. 451.

2 T. Smith, Medical Xe-^'s. February 22, 1902 ; Salmon, Bureau of
Animal Industry. Bui. \«>. .^ : Adami, Philadelphia Medical Journal,
February _>2, iuo_>: Ravenel, Unirersity of Pennsylvania Medical
bulletin. May, 1902; Larti.uau. Journal Medical Research. Vol. VI., 1901.

PATHOGENIC BACTERIA. 29!

millet-seed. In sections of the tissue young tubercles are
found to present several different structures. Near the
center, one or more very large cells called giant-cells occur.
They contain several or many nuclei which are frequently
arranged in a crescentic manner at one side of the cell.
Tubercle bacilli can sometimes be demonstrated inside of the
giant-cell. Except possibly in the very youngest tubercles,
a small area of necrotic tissue will always be found at the
center of the tubercle.

Around the giant-cells and the necrotic area are seen
large cells with distinct nuclei which resemble epithelial
cells, and are often called epithelioid cells ; they are also
often termed granulation cells, and represent an attempt
at the formation of granulation tissue. But no new-formed
blood-vessels, such as are found in granulation tissue as a
rule, occur in the tubercle. Tubercle bacilli may also be
found among the epithelioid cells. Outside of these epithe-
lioid cells is another layer of small cells called lymphoid
cells which represent leucocytes that have appeared in this
situation as a part of the inflammatory reaction excited by
the presence of the tubercles. The zone of lymphoid cells
may be very indistinct or wanting. Frequently it may be
very difficult to make out that the cells are arranged in
distinct zones at all. The cells are imbedded in a matrix
consisting of the connective tissue originally belonging to
the part, to which some fibrin may be added. In addition
to the fact that no new blood-vessels are formed to main-
tain the nutrition of these newly-formed cells, the small
vessels included in the tubercle and around it suffer from
inflammatory changes. Owing to these causes and to a
toxic substance formed by or in the tubercle bacilli, de-
generative changes and necrosis take place at the central
part of the tubercle. As a result of these degenerative
changes the center of the tubercle becomes converted into
26

2Q2 MANUAL OF BACTERIOLOGY.

a dry, yellowish-white, friable mass, resembling dry cream-
cheese. Such material is said to be caseous, and the pro-
cess is called cascation. Prudden and Hodenpyl found that
the injection of dead tubercle bacilli into animals produced
lesions having the histological characters of tubercles, but
caseation did not take place.

The small tubercles first formed are called gray or mili-
ary tubercles. As they become larger they also frequently
become confluent. The larger, confluent, caseous tubercles
are often called yellow tubercles. Swollen tuberculous
lymph-nodes of the neck are among the manifestations of
the condition formerly known as scrofula.
""Masses of caseous tubercles sometimes undergo soften-
ing. In the lungs the discharge of the softened material
results in the formation of a cavity. This formation of a
cavity in the lungs is frequently, if not usually, accom-
panied by secondary infection with pyogenic micrococci.
Caseous tuberculous masses may become partly calcified.
Very often they may be encapsulated by new formed fibrous
or scar tissue. It is possible for tuberculosis to become
cured for all practical purposes by means of this process.
Autopsies on human subjects have shown that such cures
not rarely take place, especially in tuberculosis of the lungs
occurring over a localized area. The statistics of autopsies
vary widely as to the number of persons that at some time
of life suffer from tuberculosis (25 or 30 per cent, and
upwards). When a tuberculous area has become caseous
and encapsulated and apparently quiescent, it is possible for
it to be excited to renewed activity under suitable conditions,
and, owing to the softening and the discharge of infected
material into one of the vessels or cavities of the body, a
wide-spreading and rapidly fatal tuberculosis may follow.

Tuberculosis may become disseminated throughout the
body from a small focus as a starting-point. The tubercle

PATHOGENIC BACTERIA. 293

bacilli may travel through the lymph-spaces and affect adja-
cent tissues, some of them reaching the nearest group of
lymph-nodes. In tuberculosis of the lungs it is usual also to
find tubercles in the bronchial lymph-nodes, and in tuber-
culosis of the intestines there is also tuberculosis of the
mesenteric lymph-nodes. The disease may travel along the
serous surfaces and become widely scattered throughout a
cavity like that of the pleura or peritoneum. The bacilli
may be expelled on some mucous surface and be carried
along it to infect some point farther on, as happens when
the larynx becomes infected in tuberculosis of the lungs,
and when in the same disease tuberculous sputum is swal-
lowed and leads to infection of the intestines. Finally, the
infectious material may enter the blood-vessels, especially
the veins, and be swept along with the blood-current to
become scattered generally throughout the body. In such
cases we are likely to have general or acute miliary tubercu-
losis. Almost every organ of the human body may be in-
fected by tuberculosis. Among the most common may be
mentioned the lungs, the lymph-nodes, the bones, the intes-
tines, the skin, the meninges, and the serous membranes.

Infection, as far as we know, is always to be attributed
directly or indirectly to some preexisting case of tubercu-
losis in man or the lower animals. The entrance into the
body is most commonly by way of the lungs, where also
tuberculous disease is commonest in man, going by the
name of consumption. This is doubtless due to the prev-
alent habit of expectorating in public places. Out of fifty-
six samples of sputum collected in street cars by Dr. W. G.
Bissell, City Bacteriologist in Buffalo, four were tubercu-
lous. In forty-eight samples taken from the floors of a
public building by Dr. C. R. Orr, of the pathological labor-
atory of the University of Buffalo, tubercle bacilli were
found three times. According to the researches of Nuttall,

294 MANUAL OF BACTERIOLOGY.

a case of tuberculosis may expectorate many millions of
tubercle bacilli in the course of twenty-four hours. Cough-
ing and similar efforts may serve to disseminate the bacilli
(see page 163).

Concerning the occurrence of tubercle bacilli in cow's
milk and butter, see pages 148 and 149.

Cases have been recorded in which the disease was trans-
mitted from the mother to the child in the uterus ; how fre-
quently this happens is uncertain. It is usual to attribute
greater importance to an inherited tendency to tuberculosis
than to the inheritance of the tubercle bacilli themselves.

Agglutination of the tubercle bacillus is said to occur with
the serum of cases of tuberculosis under certain circum-
stances. The reaction does not seem likely to be of practical
value.

Tuberculin is made by concentrating a culture of tuber-
cle bacilli grown in glycerin-bouillon to one-tenth of its
original volume, over a water-bath, and filtering through an
unglazed porcelain filter. It therefore represents the pro-
ducts of tubercle bacilli. It was proposed by Koch as a
remedy for tuberculosis, but it has not met with great suc-
cess, and is little used as a therapeutic agent. It has been
found, however, of great value in the diagnosis of tubercu-
losis, especially in cattle. When tuberculin is injected into
a tuberculous animal there results considerable general dis-
turbance, of which the most noticeable evidence is a sud-
den rise in temperature, while hyperemia is excited around
the tuberculous area. In a healthy subject the injection
produces no reaction. There is danger attending its use,
so that its application in diagnosis is practically confined to
cattle.1 As a diagnostic measure in cattle it has been found
accurate in the great majority of cases. Concerning tuber-

1 For details as to its use in cattle see V. A. Mi tore, "Infectious

Diseases <>f Animals," i<)O_\ p. 151.

PATHOGENIC BACTERIA. 295

culosis in cows, see page 148. Supposing that some cura-
tive principle exists in the bodies of the tubercle bacilli
themselves which could not be procured from cultures de-
prived of their bacilli by nitration through porcelain, Koch
has recently proposed a new form of tuberculin called
" tuberculin R," which consists of an extract made from
dried and pulverized living tubercle bacilli. The value of
this new tuberculin as a remedy is at least doubtful, and
physicians are disposed to regard it with a great deal of
caution.

Immunity from tuberculosis has been attained experimentally to a
certain degree. In very old cultures the virulence of tubercle bacilli
sometimes becomes greatly diminished. Animals which survive injec-
tions of such bacilli may afterwards withstand large doses of virulent
bacilli.1

Acid-proof bacilli resembling tubercle bacilli have been alluded to
a number of times (pages 149, 154, and 288). A number of such bacilli
have been cultivated, such as those of butter and grass. Injected into
animals they may produce nodules more or less like tubercles. In these
nodules they sometimes assume forms resembling the fungus of actino-
mycosis. The tubercle bacillus rarely shows similar forms. All the
bacilli of this class, including the tubercle bacillus, sometimes show
branching. It is probable that the bacilli of this group are related to
the fungus of actinomycosis.2 Similar organisms have been found in
fishes, in whom they produce nodules resembling tubercles; it is quite
possible that the latter organisms are tubercle bacilli, which have been
modified by an altered environment. Another acid-proof bacillus has
been found which is pathogenic to rats, producing lesions of the skin
with nodules ; the disease appears in wild rats in certain localities.

Tuberculosis of Birds. — Fowls, ducks and other birds sometimes
suffer from tuberculosis due to a bacillus closely resembling the tubercle
bacillus of mammals. It has similar staining properties. It sometimes
grows in long, branching forms. It differs somewhat from the tubercle
bacillus of mammals in its cultural properties. The liver is the organ
most often affected. Guinea-pigs are much less susceptible to it than to

'Truder.ti, Neiv York Medical Journal, July 18, 1903. Salmon, Phila-
delphia Medical Journal, June 13, 1903.

2 Abbott and Gildersleeve, University Pennsylvania Medical Bulletin,
June, 1902.

296 MANUAL OF BACTERIOLOGY.

mammalian tuberculosis. Rabbits are somewhat susceptible, though less
so than to mammalian tuberculosis.

Pseudo-tuberculosis. — Guinea-pigs and other rodents sometimes pre-
sent lesions macroscopically very similar to those of tuberculosis, in
which, however, the tubercle bacilli cannot be found. These affections
appear not to be tuberculosis at all, and their nature is not well under-
stood. Several organisms have been found in them, all of which are
entirely unlike the tubercle bacillus.

Bacillus leprae (of leprosy). — A slim bacillus about 4/^
in length. It is probably not motile. It is uncertain whether
or not it forms spores. It stains by the Gram and the
Weigert fibrin method, and it is also colored by the methods
used for staining the tubercle bacillus. It takes the dye,
however, more readily than the tubercle bacillus. In stained
preparations it appears very similar to the tubercle bacillus,
and resembles it in having alternate colored and unstained
spots. Although several observers have reported success
in attempts to cultivate the bacillus of leprosy, their claims
have been disputed. The results of inoculation into man
and the lower animals of material coming from cases of
leprosy have also been uncertain. The bacillus of leprosy
has been found so constantly in the tissues of those having
the disease that it is generally admitted to be the specific
cause. The skin and the peripheral nerves are the parts
most affected, although other tissues and the internal viscera
may be involved. A granulation tissue, forming nodules
and thickenings, appears in the affected parts. The bacilli
are found in large numbers in the nodules, partly outside of
the cells, but mostly within the cells. It is still uncertain
whether or not the disease can be transmitted directly from
one individual to another, in extra -uterine life, or whether
it can be inherited from the parents. However, no explana-
tion can be given for the appearance of the infection in any
patient, except communication with some other case.
Transmission by contact seems at any rate not to take place
easily.

PATHOGENIC BACTERIA. 297

Bacillus mallei (of glanders). — A slim bacillus with
round or pointed ends, which often shows alternate light
and dark spots in stained preparations. Branching forms
have been described. It is not motile. It probably does
not form spores. It is decolorized by Gram's method.
After staining with the ordinary aniline dyes it is easily de-
colorized, and on that account it is difficult to demonstrate
in sections of tissues. It is facultative anaerobic. It grows
at the room temperature, but better in the incubator. It
grows slowly on gelatin, and does not liquefy it, or only
after a long time. On agar it produces a moist, white
growth, on blood-serum a yellowish or brownish growth;
blood-serum is not liquefied. Milk is coagulated slowly,
and the reaction becomes acid. On potato the growth is
characteristic in one or two days in the incubator, becom-
ing translucent amber-yellow, later a reddish-brown, while
the surface of the potato becomes discolored.

It is killed in five minutes by a 5 per cent, solution of
carbolic acid, in two minutes by 1-5000 bichloride of mer-
cury. It may survive drying for a number of weeks.

In the horse and ass it produces the disease known as
glanders, which affects the mucous membrane of the nasal
cavity. When the skin is involved the disease goes by the
name of farcy. In the nose, nodules appear in the mucous
membrane which become necrotic, forming ulcers. They
may become confluent, and may extend along the adjacent
surfaces as far as the lungs. There is a profuse discharge
from the nose. The neighboring lymph-nodes become in-
volved and are swollen, and nodules may be present in the
internal viscera. In the skin the nodes lying underneath
the skin are called farcy-buds. Histologically the nodules
consist of a granulation tissue, but they tend to break down
rapidly, and the process in some respects is very like ordi-
nary suppuration.

298 MANUAL OF BACTERIOLOGY.

This bacillus is pathogenic1 to guinea-pigs, field-mice and
cats; rabbits, sheep and dogs are less susceptible or only
slightly so, also white and house-mice, and hogs ; cattle are
immune. Men are occasionally affected, especially those
who have come in contact with horses. The mucous mem-
brane of the nasal cavity may be the part involved, or the
skin, or the internal viscera. In a number of instances,
workers in the laboratory have been accidentally infected.

The diagnosis of the disease is best effected by the in-
oculation of a male guinea-pig with the material from a
case suspected of being glanders, introducing it into the
peritoneal cavity (Method of Straus). In about two to
three days there appears a characteristic swelling of the
testicle indicating the beginning of suppuration, which
presently takes place ; the animal usually dies after two or
more weeks. At least two guinea-pigs should be inocu-
lated: and the test may sometimes fail, when it should be
repeated on other guinea-pigs.2

Mallcin is a product obtained from an old glycerin-
bouillon culture of the bacillus mallei. The cultures are
placed in a steam sterilizer for several hours, and are fil-
tered through unglazed porcelain. The filtrate contains
the products of the growth of the bacillus mallei and is of
much the same character as tuberculin. Injected into ani-
mals suspected of having glanders, if it produces a local
and febrile reaction, the existence of glanders is indicated.
It is usually successful in the diagnosis of the disease in
lower animals, especially in horses, where it has been
largely employed. An agglutination reaction has been de-
scribed for the bacillus of glanders.

1The statements of different writers differ considerably \\ilh regard
to some of these animals.
2 Frothingham, Journal Medical Research. Vol. VI., 1901.

PATHOGENIC BACTERIA.

299

Actinomyces bovis1 ( Streptothrix actinomyces, Ray- fun-
gus of actinomycosis). — The morphology of this organism
is quite different from that of most of the bacteria. It is
sometimes considered to be a bacterium of a higher type.
The organism appears in the form of threads which show
genuine branching. These threads make radiating, inter-
lacing masses. Their external ends are swollen and bulbous
under certain conditions. Colonies formed in this manner,
seen under moderate mag-
nification, have a radiat-
ing appearance which has
given rise to the name,
ray-fungus. The club-
shaped external ends are
readily distinguished and
the growth possesses a
very distinctive form.
This is the shape which
the organism presents as
it grows in the animal
body. The club-shaped
ends are generally re-

1 i i Ray-fungus of Actinomycosis. Fresh,

garded as a degener-

0 ° unstained preparation from a case of

ative Or involution form. lump.jaw in a cow. Diagrammatic.

Transverse divisions may

sometimes be distinguished upon the threads. Spherical
forms resembling micrococci may appear which may pos-
sibly be spores. In some members of this group spores
form in cultures on the ends of the filaments (conidia).
The organism stains with the ordinary aniline dyes, by
Gram's method or the Weigert fibrin stain.

The fungus may be cultivated upon the usual culture-
media, though not easily. It is facultative anaerobic. It

1 Hektoen, Philadelphia Monthly Medical Journal, November, 1899;
Ewing, Bulletin Johns Hopkins Hospital, November, 1902.

300

MANUAL OF BACTERIOLOGY.

grows both at ordinary temperatures and in the incubator.
The growth is not rapid. The colonies are fine, dry,
elevated, irregular in form, becoming opaque. Bulbous
ends upon the threads do not usually appear in cultures.
The results of the injection of these cultures into the lower
animals are as yet uncertain.

The disease produced by the ray-fungus is called actino-
mycosis. It occurs in cattle chiefly, seldom in swine and

FIG. 84.

Actinomyces bovis, smear preparation from a pure culture, stained by
Gram's method. (X 1000.)

horses, and occasionally in man. Infection appears to be
carried by grain or particles of vegetable fiber which pene-
trate the tissue. The presence of such foreign particles as
well as the organism appears to favor infection. The infec-
tious material frequently enters through the mouth, espe-
cially in the vicinity of the teeth, but it may also occur
through the skin or the mucous membranes. It leads to the

PATHOGENIC BACTERIA. 3OI

formation of inflammatory, tumor-like nodules, hence the
name " lump-jaw " given to the disease in cattle. Necrosis
of the tissue takes place with the formation of an abscess.
The pus is peculiar in containing small whitish particles
which consist of little colonies of the ray-fungus, and which
readily permit the disease to be diagnosed by the micro-
scope. The material may be examined in the perfectly
fresh condition without any staining. The jaw or its neigh-
borhood is very frequently affected, or the disease may be
present in other situations about the head and neck, and
may involve the lungs, the intestines, and the vertebrae, ribs,
and other bones. The disease is usually localized, but a
number of areas may be affected simultaneously.

Besides the common actinomyces, there are numerous other ray-fungi,
more or less closely related, and whose pathogenic properties are not
fully determined. Generally speaking they appear to be saprophytes
naturally, which occasionally become parasitic and pathogenic under
especially favorable conditions. A number of species have been found
in air, dust, etc., some of them chromogenic. Wolff and Israel described
an anaerobic species, pathogenic to man and animals. Madura disease,
Madura foot, or mycetoma is a disease occurring in India (rarely else-
where), affecting one of the extremities, characterized by swellings,
nodular deposits and abscesses. Some cases are certainly due to a mem-
ber of the actinomyces group.1

Other branching organisms, some of them acid-proof, have been de-
scribed chiefly under the name of streptothrix. In man they have been
found in a variety of suppurative and necrotic lesions, in particular,
broncho-pneumonias.2

Bacillus iyphosus (Bacillus of Eberth). — A bacillus
with rounded ends, varying in length, sometimes making
very short, oval forms, sometimes growing out into long
threads. It is very actively motile, and possesses numerous

1 Compare Wright, Journal Experimental Medicine, Vol. III., p. 421.

2 Norris and Larkin, Journal Experimental Medicine, Vol. V., p. 155;
Musser, Philadelphia Medical Journal, September 7, 1901; Flexner,
Journal Experimental Medicine, Vol. III.; MacCallum, Centralblatt f.
Bakteriologie, Orig. Bd. XXXI., 1902.

3O2 MANUAL OF BACTERIOLOGY.

flagella which arise from all parts of the surface. It does
not form spores. It is not stained by Gram's method, but
it may be colored with the ordinary aniline dyes, when the
stain will frequently be somewhat irregular. It may be
stained in sections of tissues from cases of typhoid fever,
with the aniline dyes, such as Loffler's alkaline methylene-
blue. It is facultative anaerobic. It grows at ordinary
temperatures, better in the incubator, but grows rather more
slowly than B. coli commnnis. Gelatin is not liquefied.

- ^ / I

•"* '-*.

'"•

>•/»• \
' *N\ 1%

Bacillus of typhoid fever. (X n

surface colonies in gelatin appear whitish, with
irregular borders and more or less wrinkled surfaces, when
slightly magnified. It grows on the ordinary media, and the
growths are whitish. Bouillon is clouded. Milk becomes
slightly acid, but is not coagulated. In media containing
dextrose, acid is formed but no gas. In lactose-bouillon
neither acid nor gas is formed, although when grown in
milk the typhoid bacilli produce an acid reaction. The lac-

PATHOGENIC BACTERIA.

303

tose-litmus-gelatin or -agar of VVurtz makes use of the blue
tinge possessed by colonies of the typhoid bacillus on this
medium to distinguish them from colonies of the colon
bacillus and other bacteria which form acids from lactose.
Neutral red has been used in the same manner, as it is said
not to be altered by the typhoid bacillus, but to be changed
by the colon bacillus to a yellow color. (To neutral, plain
agar add i per cent, of a saturated aqueous solution of
neutral red, and some also add .3 per cent, dextrose.)

FIG. 86.

•

-K&-

i '

•~f-

Bacillus of typhoid fever, stained by Loffler's method to show flagella.

(X 1000.)

Ill Dunham's peptone solution indol is not formed, as a
rule. On potato it usually forms what is called an invisible
growth; that is, although no development is apparent to the
eye, numerous bacilli may be shown under the microscope
in smear preparations made from the surface of potato
inoculated about forty-eight hours previously. Occasionally
a slight visible growth is seen on potato.

3O4 MANUAL OF BACTERIOLOGY.

The typhoid bacillus is killed at 60° C. in ten minutes.
It resists drying well. It can survive in soil and sewage a
long time.

For a comparison of the properties of the typhoid bacillus
and the colon bacillus see the latter.

Concerning the detection of the typhoid bacillus in
water see page 142.

A new medium has been suggested by Hiss1 for the isolation of the
typhoid bacillus. It consists of gelatin and agar, beef-extract, sodium
chloride and dextrose, and is given a slightly acid reaction. These sub-
stances are used in different proportions for plate- and for tube-cul-
tures. This medium is of a semi-solid character, and makes use of the
great motility of the typhoid bacillus in producing a uniform clouding
of the medium in tubes, with the absence of a gas formation; while
in plate-cultures the colonies exhibit peculiar filamentous outgrowths.
It is claimed that it can be determined whether organisms are typhoid
bacilli or not after thirty-six hours in the incubator.

Other special media for the identification of the typhoid bacillus have
been devised by Eisner, Stoddart, by Capaldi and Proskauer, and bj
Piorkowski.2 The medium of Stoddart is based upon principles similar
to those applied in the medium of Hiss.

M. W. Richardson has devised an application of the serum-test to
plate-colonies suspected of containing typhoid bacilli. If a typhoid colony
be torn with a needle, under moderate magnification " a seething motion
resembling much the appearance of a swarm of bees " may be seen.
This appearance is due to the motility of the bacteria. If such a colony
be touched with a small quantity of blood-serum from a case of typhoid
fever, the motion is said to cease instantly and almost absolutely.
Colonies of other motile bacteria do not undergo a corresponding loss
of motility.

THE SERUM-TEST FOR TYPHOID FEVER.3
When a small quantity of a culture of typhoid bacilli is
mixed with a little blood-serum derived from a case of ty-

1 Journal Medical Research, Vol. VIII., 1002.

"Eisner, Zcitschrift f. Hygiene, Bd. XXL, 1895, p. 25; Stoddart.
Journal of Pathology and Bacteriology, Vol. IV., p. 429, 1897; Capaldi
and Proskauer, Zcitschrift fiir Hygiene, etc., Bd. XXIII. , p. 452, 1896;
Piorkowski, Berliner klinischc irochcnschrift, 1899, p. 145.

3 This test is often known as the " Widal reaction." For a history and
general discussion of the subject see Durham, Journal of Experimental
Medicine, Vol. V., p. 353.

PATHOGENIC BACTERIA.

305

phoid fever, within a few minutes the motility of the typhoid
bacilli is abolished and they become agglutinated into
clumps or masses. Occasionally the bacilli may eventually
undergo disintegration into granular material. This re-
action does not take place with the blood-serum of healthy
persons or of those suffering with other diseases, nor when
the blood-serum of a typhoid fever case is mixed with motile
bacteria other than typhoid bacilli. It has been observed
in the blood-serum of an infant born while the mother was
convalescing from typhoid fever.

FIG. 87.

Application of the serum-reaction to typhoid bacilli. A shows the dis-
tribution of the bacilli before the reaction. It is to be remembered that
they are motile and their positions may change continually. B shows
clumping of the motionless bacilli after mixture with the serum of a case
of typhoid fever. Diagrammatic.

The agglutinating substance has been found in blister-
serum and in the milk of typhoid cases, in fluids from the
serous cavities and inflammatory and edematous areas in
variable amounts, and occasionally in urine, bile and tears.

The reaction may be obtained by adding blood-serum to
a young bouillon-culture of typhoid bacilli kept in the in-
cubator, when the occurrence of agglutination becomes

306 MANUAL OF BACTERIOLOGY.

manifest by the collection of the bacteria into visible masses
or flocculi, which form a sediment. Most investigators
prefer to watch the results under the microscope, using an
ordinary slide, or, better, the hanging-drop. Young cultures
—less than twenty-four hours old — in bouillon, and kept in
the incubator, may be used, or better cultures kept at room-
temperature for twenty-four hours. Johnston and Mc-
Taggart recommend that the bouillon cultures be freshly
made each time from stock cultures on agar, which need only
occasionally be transplanted. Certain stocks of typhoid
bacilli seem especially suited to this reaction, and such a
stock should be secured.

Blood-serum, blister-serum, fresh blood and dried blood
have all been tried with success. Blood dried on unglazed
paper or cover-glasses as proposed by Wyatt Johnston is ex-
tremely convenient. To perform the test, it is mixed with
sterilized distilled water, bouillon, or normal salt solution;
the objection to it lies in the difficulty of securing an accu-
rate dilution. An approximate knowledge of the degree of
dilution may be acquired by mixing drops of dried blood
of known volume with definite amounts of water, and ob-
serving the tints. These should be kept in mind as stand-
ards. The dilution may be measured with the hemoglo-
1)inometer or with the pipette of the hemacytometer. The
Xe\v York Board of Health have found blister-serum satis-
factory and easy to obtain. A little of the diluted serum is
mixed on the cover-glass with a definite amount of the fresh
bouillon-culture, and is examined as a hanging-drop. In
a short time the characteristic clumping and loss of motility
occur. At the same time a drop of the culture alone, and
a drop of the culture mixed with normal scrum-, similarly
diluted, should be examined as controls. The dilutions
used vary from I part of serum in 30 to I in 50. The
higher dilutions are more accurate. The time within which

PATHOGENIC BACTERIA. 307

the reaction occurs varies from a few minutes to one or two
hours. With little dilution the time should be short; with
greater dilution it may be longer. Both clumping and
paralysis of motility should take place. In a positive case
the reaction should be distinct. Normal blood sometimes
exhibits agglutinative properties in some degree. If the
reaction in any case is not satisfactory it should be tried
with a higher dilution, i to 50, and the result should be
positive if the case is a genuine case of typhoid fever.

The reaction usually appears between the seventh day
and the end of the third week of the disease; it may be
seen earlier; it is often delayed and appears late. The
test frequently has to be repeated when the first result is
doubtful or negative. Reports indicate that the method is
a great aid in the diagnosis of typhoid fever, though not
infallible.

Considerable experience is necessary to acquire the
judgment needed in using this test.

The agglutinating power becomes lessened after recovery,
and usually is wanting at the end of a year. Rarely it may
be present for a longer time, a fact that is to be borne in
mind in diagnosis.

Typhoid bacilli have frequently been obtained from the
stools of cases of the disease, but they are isolated only with
considerable difficulty. At autopsies they are best culti-
vated from the spleen, in which, however, it is to be remem-
bered, the bacillus coli communis may also be present.
Puncture of the spleen with a sterilized hypodermic needle,
during life, has also been resorted to as a means of diag-
nosis. The drop of fluid withdrawn may be examined by
culture-methods for typhoid bacilli. There is probably some
clanger to the patient attending this procedure. Cultures
made from the blood, where several c.c. are taken show that
a few bacilli occur in the blood in a large proportion of cases
27

308 MANUAL OF BACTERIOLOGY.

of the disease, and probably in a majority. Typhoid bacilli
frequently appear in the urine (in about twenty per cent,
of all cases) and the examination of urine for them has
been used in diagnosis. The bacilli often occur in the
gall-bladder. They have been found associated with gall-
stones, and have been supposed to be one of the causes
for the formation of gall-stones.1 They may remain pres-
ent in the gall-bladder or in the urine2 long after convales-
cence from the disease. They have been demonstrated in
the " rose spots " on the abdomen. They may be present
in the lesions of the pneumonia, which frequently compli-
cates typhoid fever, and may appear in the sputum.

Inoculation experiments in animals have not been very
satisfactory. With a few exceptions, possibly, anatomical
lesions resembling those of typhoid fever have not been
produced by the inoculation of typhoid bacilli into animals.
The injection of cultures into animals may produce death,
but it can usually be shown to have resulted from the poisons
contained in the cultures.

Typhoid fever is rare during the first two years of life.
It frequently attacks young and robust men. The causes
that bring about susceptibility to infection are not known.

The principal lesion in typhoid fever lies in the Peyer's
patches of the lower part of the small intestines; the mes-
enteric lymph-nodes and spleen also are swollen. The
typhoid bacillus may be demonstrated in sections of the
walls of the diseased portions of the intestine. Cases are
recorded in which no lesions were found in the intestines
but where the typhoid bacilli were widely spread through
the organs of the body, and which therefore represented
typhoid septicemia.

Periostitis and osteomyelitis, which are not uncommon
sequelae of typhoid fever, may be caused by typhoid bacilli.

1 Pratt, American Journal Medical Sciences, Vol. 122, 1901.

2 M. \Y. Richardson, Journal Experimental Medicine, Vol. IV., 1899.

PATHOGENIC BACTERIA. 309

Ordinary suppuration may be produced by the typhoid
bacillus, but most suppurative affections during or following
typhoid fever are mixed infections, or are due to the ordi-
nary pyogenic bacteria.

Typhoid fever is transmitted chiefly through the medium
of water. It is sometimes conveyed by milk, green vege-
tables and oysters. Infection through the medium of dust,
and by the hands and clothing probably occurs, but not com-
monly. Under certain circumstances the bacilli may be
carried by flies.1 In caring for cases of typhoid fever the
stools, urine, sputum and linen should be disinfected. Per-
sons handling the patient should wash and disinfect their
hands.

The injection of typhoid bacilli which have been killed by heat has
been advised (by Wright) as a preventive measure. The results appear
to have been partially successful, but the method is still being actively
studied.

Bacillus coli communis (Bacterium coli commune of
Escherich, probably the same as Bacillus Neapolitanus of
Emmerich, often called simply the colon bacillus. Passet
described an organism under the name of Bacillus pyo-
genes fcetidus, from .foul pus and mixed infections, which
is probably the same as B. coli communis). — A bacillus with
rounded ends, frequently of a short, oval form, when it
may be difficult to distinguish from micrococci ; often
longer; often forming threads. It is slightly motile, hav-
ing several flagella. It does not form spores. It stains
with the ordinary aniline dyes and is decolorized by Gram's
method. It is facultative anaerobic. It grows well at the
room temperature, but more rapidly in the incubator. It
does not liquefy gelatin. In gelatin plates the surface
colonies are of a bluish-white color; the centers are denser

1 Vaughan, Philadelphia Medical Journal, June 9, 1900.

3IO MANUAL OF BACTERIOLOGY.

than the borders, which are translucent. It usually grows
more rapidly in gelatin than the bacillus of typhoid fever.
Its growths in other media are mostly whitish. Bouillon
becomes clouded. Nitrates are reduced to nitrites. In
peptone solution it forms indol. On potato it forms an
abundant visible growth from cream-color to pale brown.

FIG. 88.

v> P*

I* ' ~ "

% \ * 0 »'.\ ^ !» -

Bacillus coli comraunis. (X 1000.)

Milk becomes acid and is usually, but not always, coagu-
lated slowly. It causes the development of gas and acid
in media containing dextrose or lactose. In media contain-
ing neutral red it is stated that the colon bacillus produces a
yellow color with a green fluorescence. Differential points
between the bacillus of typhoid fever and the bacillus coli
communis are as follows :

1st. The typhoid bacillus is actively motile; the • colon
bacillus less actively, or slightly motile.

2d. The typhoid bacillus has numerous flagella which
rise from all parts of the surface; the colon bacillus has a
smaller number of flagella.

PATHOGENIC BACTERIA. 311

3d. In both, spore formation is absent.
4th. Both are decolorized by Gram's method.
5th. The colonies of the typhoid bacillus in gelatin de-
velop more slowly than those of the colon bacillus.

6th. The appearance of superficial colonies in gelatin

plates.

FIG. 89.

Bacillus coli communis with flagella, stained by Van Ermengem's
method. (X 1000.)

7th. In media containing dextrose or lactose, the typhoid
bacillus does not produce fermentation with gas and the
colon bacillus does produce gas.

8th. The typhoid bacillus produces an acid reaction with-
out coagulation in milk, and the colon bacillus produces an
acid reaction and coagulation.

9th. In peptone solution the typhoid bacillus, as a rule,
produces no indol , and the colon bacillus produces indol.

loth. The typhoid bacillus usually produces an invisible
growth on potato, the colon bacillus a visible growth.

nth. The typhoid bacillus is said not to reduce neutral

312 MANUAL OF BACTERIOLOGY.

red in media, and the colon bacillus to change it to a yellow
color.

To these may be added the growth of the two organisms
on special media like those of Wurtz, of Eisner and of
Hiss, and the application of the serum-reaction.

Injections of cultures of the bacillus coli communis into
animals produce variable and uncertain results. Subcuta-
neous injection may lead to pus-formation ; in rabbits and
guinea-pigs injections may produce death apparently from
poisons introduced. With the blood of immunized animals
a serum-reaction, similar to that described for typhoid fever,
may be demonstrated.

Concerning the occurrence of the bacillus coli communis
in the intestine of man, see page 155.*

At autopsies on human subjects the great viscera are
often found to have been infected by the colon bacillus,
usually when some lesion of the intestine existed simul-
taneously, but in most cases without having produced much
apparent damage to the organs invaded. The bacillus coli
communis frequently occurs in mixed infections, as in
wounds, inflammations and abscesses. It is often found in
the peritoneum in peritonitis, in the pus in appendicitis,
and in the urine in cystitis; it frequently occurs in the in-
terior of gall-stones with whose formation it may be con-
nected.2

There is a large number of more or less closely-related
organisms which go by the name of the "colon group."
The limits of the colon group are extremely ill-defined.

Paracolon or paratyphoid bacilli are the names applied to certain
members of the colon group which have recently been shown to be
pathogenic to man. They may produce clinical symptoms resembling
typhoid fever of a mild and atypical form. The affection is rarely

1 See also Moore and \Yright, " Bacillus coli communis in the Do-
niesticnu-d Animals." American Medicine, March .'<). K;O-'.

2 Lartigau, Journal American Medical Association, April 12, 1902.

PATHOGENIC BACTERIA. 313

fatal. Probably they may occur with typhoid fever in mixed and second-
ary infections. Characteristic lesions have not yet been observed. The
bacilli have been found in the blood, spleen, liver, gall-bladder and
urine. Like typhoid and colon bacilli they are motile, have flagella, are
not stained by Gram's method and do not liquefy gelatin. They ferment
dextrose and maltose, producing acid and gas. They do not ferment
lactose. Milk at first becomes acid, later it becomes alkaline, and is not
coagulated. On potato a slight visible growth occurs. Media contain-
ing neutral red become yellow, as with B. coli communis, but more
slowly, and the red color sometimes returns. In respect to the fermen-
tation of saccharose and the formation of indol reports differ; both are
usually negative. The blood of the patient agglutinates the bacilli. But,
as among the closely related members of this group mutual reactions are
sometimes seen, this test is not to be considered invariable.1 Several
bacilli allied to the above are known. The bacillus enteridis of Gaertner
is a related form which has been found in cases of meat-poisoning.

Bacillus lactis aerogenes (Bacillus aerogenes). — A ba-
cillus having a form similar to that of the colon bacillus,
described as being larger and plumper. In the main its
properties are similar to those of the colon bacillus. Its
colonies are more circumscribed and elevated. It is also
non-motile. It coagulates milk more rapidly than the
colon bacillus. It produces gas upon potato more rapidly
than the colon bacillus, and more abundantly. It was de-
scribed by Escherich, who also described the colon bacillus,
assigning the bacillus lactis aerogenes rather to the upper
part of the small intestine, and the colon bacillus to the
lower portion. According to Kruse, the bacillus lactis aero-
genes and its relatives differ from the bacillus coli com-
munis chiefly in lacking motility. Like the colon bacillus
it has been found many times in the urine in cystitis. See
also B. acidi lactici, page 227.

Bacillus dysenteriae (Shiga). — A bacillus with rounded
ends, of the size and shape of typhoid and colon bacilli,

'Gushing, Bulletin Jo/ins Hopkins Hospital, July-August, 1900;
Strong, Ibid., May, 1902; Johnstone, Hewlett, Longcope, American
Journal Medical Sciences. August, 1902; Libman, Buxton, Journal
Medical Researcli. Vol. VIII., 1902.

314 MANUAL OF BACTERIOLOGY.

seldom forming threads. Most observers have found it non-
motile. Vedder and Duval have demonstrated flagella. The
bacillus does not form spores. It may be stained with the
ordinary aniline dyes; it does not stain by Gram's method.
It is facultative anaerobic. It grows at ordinary tempera-
tures, but better in the incubator. It grows on the usual
culture-media, but more slowly than B. coli communis.
The growths are whitish. Colonies on gelatin plates re-
semble those of the typhoid bacillus. Bouillon is diffusely
clouded ; a precipitate may form, but no pellicle. Indol is
not produced. Milk becomes acid and is not coagulated.
On potato a thin pale layer forms which may become light
brown. No gas is formed in media containing glucose or
lactose.

Xeutral-red agar is not changed. From the feces the
bacillus is best cultivated on agar plates, in the incubator.
Colonies of B. coli communis are often more numerous
than those of the dysentery bacillus. The colonies which
develop in twenty-four hours are likely to be colonies of
B. coli communis. Their position may be marked on the
glass with a pencil. Those which appear later are to be
planted in dextrose-agar. If gas develops they are not the
bacillus of dysentery; otherwise they are to be studied and
identified by the cultural and other tests mentioned above,
and by the agglutination reaction.

The bacilli are destroyed in a few minutes by boiling, and
at 58° C. in half an hour. They appear not to be particularly
resistant to the influences that are harmful to bacteria in
general.

They have been found in the intestine and the discharges
of acute and epidemic dysentery in various climates and
countries, including the United States. Thus far their dis-
semination in the blood and distant organs has not been
demonstrated. The lesion of this form of dysentery con-

PATHOGENIC BACTERIA. 315

sists of a severe acute inflammation of the colon, frequently
with necrosis of the surface and the formation of pseudo-
membrane. Ulceration may occur, but is usually superficial.
Duval and Bassett found the bacillus of dysentery in the
stools of infants having summer diarrhoea.

The introduction of pure cultures into animals by way of
the alimentary canal has sometimes been followed by a cer-
tain amount of diarrhoea, but it does not appear that dysen-
tery, as it occurs in man, has been reproduced. Most labor-
atory animals are, however, very sensitive to the injection
into the tissues or veins of cultures, living or dead. They
show the lesions produced by many toxins.

The bacillus is agglutinated by the patient's blood, but
often only late in the disease and apparently not in all cases.
This test seems to have only a limited value in clinical diag-
nosis. Many prefer to secure the reaction in a test-tube.
The dilutions used vary greatly (from i in 20 to I in 100).
Immunized animals develop the agglutinins in the blood.
The outlook for a curative serum is encouraging.

It now seems that the bacillus of Shiga has numerous
close allies, constituting with it a " group." To what extent
the others of the group may be concerned in the causation
of diarrhoeal diseases, or may occur in the normal intestine
is uncertain. According to W. H. Park some of these form
indol and develop acid from mannit, which the bacillus of
Shiga does not; they also differ from it in their agglutina-
tion reactions.1

Spirillum cholerae (Comma bacillus of cholera). — A rod-
or staff-shaped organism, somewhat curved, and with

1 Shiga, Ccntralblatt f. Baktcriologic, Bd. XXIV., 1898; Flexner,
Philadelphia Medical Journal, September I, 1900; Vedder and Duval,
Journal Experimental Medicine, Vol. VI.; Gay, University of Pennsyl-
vania Medical Bulletin. November, 1902; Duval and Bassett, American
Medicine, 1902, Vol. IV., p. 417; Park and Carey, Journal Medical Re-
search, Vol. IX., 1003; Strong and Mtisgrove, Journal American Medical
Association, Vol. XXXV., 1900, •). 498.

3i6

MANUAL OF BACTERIOLOGY.

pointed ends, hence the name " comma " bacillus. The
curved forms, placed end to end, may produce an S-shaped
body. The length is from .8 to 2 n and the breadth from

&$*!§(&

fea>i ^r^/rC?
SiGS&:£.V?-

P. f'*fr

s*>

Spirillum of cholera. (X 1000.)

.3 to .4//. In cultures, genuine spirilla may be seen. In the
whitish particles found in the stools of cases of cholera the
organisms may be present in very large numbers. In these
particles they may exhibit a very curious arrangement, lying
parallel with one another, and, as remarked by Koch, they
resemble a school of fish moving1 up stream. Involution
forms, irregular in outline and staining poorly, are often
seen in old cultures. The organism is motile, having a
rlagellnm at one end. It does not form spores. It stains
with the ordinary aniline dyes, but not by Gram's method.
It is aerobic. It grows at the room temperature, but better
in the incubator. On the ordinary media the growths are
whitish. It grows best on neutral or alkaline media, and is
very sensitive to a small amount of acid. It liquefies gelatin.
The colonies on gelatin plates have a very characteristic

PATHOGENIC BACTERIA. 317

appearance. They are nearly round at first, and granular
as seen under the low power of the microscope; but at the
end of about twenty-four hours the outline is slightly irregu-
lar, and the surface looks as though it were covered with
finely-broken glass. The outline later becomes still more
irregular or scalloped. As liquefaction of the gelatin takes
place a funnel-shaped depression is formed, into which the
colony sinks. The plates should be kept at a temperature of

FIG. 91.

Involution forms of the spirillum of cholera. (Van

from 20° to 22° C. In stab-cultures in gelatin a white
growth forms around the stab, and at the end of about
thirty-six to forty-eight hours a funnel-shaped depression
occurs at the surface, owing to the liquefaction of the
gelatin. This depression increases in size, and the surface
of the liquefied gelatin seems to be surmounted by an air-
bubble, which appears to have taken the place of the part
of the fluid gelatin which has evaporated. In the deeper
portion of the stab liquefaction is less noticeable. The
growths on agar are not characteristic. In bouillon a pellicle
forms on the surface. On potato in the incubator the
growth is whitish or brownish, not conspicuously elevated.
After growing it in Dunham's peptone solution in the incu-

MANUAL OF BACTERIOLOGY.

bator, the addition of sulphuric acid develops a red color,
owing to the formation of indol and nitrites, the so-called
" cholera red " reaction.

The spirillum of cholera is said to be very sensitive to
drying, and, provided the drying be complete, is usually
killed within twenty-four hours. It is killed in five minutes
at a temperature of 65° C. and in one hour at 55° C. It

FIG. 92.

a b c

Spirillum of cholera, colonies on gelatin plates, X 100 to 150. (a) Twenty-
four hours old. (b) Thirty hours old. (c) Forty-eight hours old.
(Frankel and Pfeiffer.)

may retain its vitality in water for a long time; observa-
tions vary widely in respect to determining how long. In
the ordinary food-substances it may survive long enough to
allow them to act as carriers of the infection if eaten raw.
The important fact is that the cholera spirillum is not a
strict parasite, but under favorable conditions it may main-
tain its vitality for some time outside of the human body.

PATHOGENIC BACTERIA. 319

The animals ordinarily used for laboratory experiments
are, in their normal condition, not susceptible to infection
with the spirillum of cholera through the alimentary canal,
and no animal is known which suffers from cholera except-
ing man, though a disease resembling cholera can be re-
produced in animals when certain conditions
are complied with. In particular it is
necessary to avoid the influence of the acid
gastric juice.

The following plan was adopted by
Koch : The gastric juice was neutralized
with a solution of sodium carbonate;
the movements of the intestines were quieted
by the injection of i c.c. of tincture of
opium for each 200 grams of the body-
weight; and a portion of a pure culture
of the cholera spirillum was introduced into
the stomach. When guinea-pigs were
treated in this manner, in most cases a
condition closely simulating cholera was SpirnTum of
produced. The animal died with symptoms cholera, stab-cui-
of collapse. The small intestine contained ture in gelatin, two
a watery, flocculent fluid in which the spi- d*ys °l* <!™k-

* el and Pfeiffer.)

nlla of cholera were numerous. The mucous
membrane of the intestine was swollen and reddened.

When mice or guinea-pigs receive an intra-peritoneal in-
jection from a pure culture, death usually results, appar-
ently from the toxic substances contained in the culture.
Pfeiffer has shown that by repeated doses, insufficient to
kill the animal, of cultures whose vitality has been de-
stroyed by heat or otherwise, the animal may be made im-
mune. He has also shown that when living comma bacilli
are introduced in the peritoneum of an immune animal they
are rapidly destroyed and disintegrated (see page 189). He

32O MANUAL OF BACTERIOLOGY.

has advised the use of this reaction as a means of diagnosis,
inasmuch as the spirilla which apparently resemble the
spirillum of cholera, but are in reality different from it, do
not become disintegrated when they are introduced in the
peritoneum of an animal made immune to the spirillum of
cholera. It has been shown also that blood from animals
made immune to cholera has an agglutinating action upon
the spirillum of cholera like that seen when the blood-serum
of cases of typhoid fever is mixed with living typhoid
bacilli.

The outlook is encouraging for the production of a safe method for
immunizing healthy persons from cholera during an epidemic.1

Although a positive demonstration that the spirillum of
Koch is the cause of cholera is lacking, as far as the exact
reproduction of the disease in animals is concerned, the
necessary proof has been supplied by the accidental or
intentional infection of laboratory investigators who were
working with cholera, which has happened on several occa-
sions.

Bacteriological investigations of the victims of cholera
have shown that the spirilla of cholera are present in very
large numbers in the watery contents of the intestine, espe-
cially early in the disease. They appear in the lumina of
the glands, and they may be seen underneath the epithelial
cells. They may occur in the matters vomited. They
usually are not found widely spread through the organs of
the body. It is probable that the symptoms of the disease
result from poisonous substances produced by the spirilla
or contained in them.

The infectious element in cholera is usually transmitted
through water, and numerous epidemics have been studied
where the infection was traced to drinking-water, and the
origin of the contamination was discovered. The organ-

1 Strong, American Medicine, Aug. 15, 19x53.

. PATHOGENIC BACTERIA. 321

isms may, however, be carried by other articles of food,
and may be conveyed occasionally through contaminated
clothing and bedding, and probably by flies. The excreta
and bedding should be thoroughly sterilized, the hands of
the attendants must be carefully disinfected. Although
commoner in the summer-time, epidemics of cholera have
been known to occur in the winter.

Bacteriological Diagnosis of Cholera. — When cases sus-
pected of being cholera appear in a community, it becomes
a matter of the utmost importance to determine the exact
nature of the disease in order that it may not become
epidemic. One of the first occasions when bacteriological
methods were put into practice in the diagnosis of cholera
was at the time of the appearance of that disease in the
Port of New York in 1887.

According to Koch, the diagnosis may be made in twenty-
four hours or less. It is important to obtain the discharges
from the intestines as early in the course of the disease as
possible, and while they are perfectly fresh. It may be
necessary, however, to examine the moist dejecta on the
linen or clothing, when no other material is available.

In the first place, one of the small, partly-solid particles
which may be found in the discharges from the intestines
should be smeared upon a cover-glass, fixed in the usual
manner, stained with one of the aniline dyes, and examined
with the microscope. If taken early in the disease, the
comma bacilli may be present in large numbers, and they
are likely to be arranged in more or less parallel groups
(see above). If comma-shaped bacilli are thus found, a
strong probability is created that the disease is Asiatic
cholera. The motility of the organisms can be determined
by examination in the hanging-drop. It is to be remem-
bered that spirilla of various forms are common in the
normal mouth, and may appear in the stools (see pages
152 and 228).

322 MANUAL OF BACTERIOLOGY.

The diagnosis should be confirmed by the use of culture-
methods. Using the small, semi-solid particles from the
intestinal discharges, gelatin plates in the usual three dilu-
tions (see page 97) should be made and kept at a tempera-
ture of 20° to 22° C. At the end of twenty-four hours or
less the colonies of the spirillum of cholera should have
been developed and should present the picture characteristic
for these colonies in gelatin plates (Fig. 92), which enables
them to be differentiated from colonies of other bacteria.
From one of these colonies, preparations may be made for
microscopic examination, and a set of tubes may be inocu-
lated. The most characteristic growth will be from stick-
cultures in gelatin. The growth in Dunham's peptone
solution may be tested for the development of indol and
nitrites.

At the time that the first smear preparations and gelatin
plates are prepared, tubes of peptone solution should be
inoculated directly from the intestinal contents, and kept
in the incubator (Schottelius). After development has oc-
curred, the production of indol may be tested by the addi-
tion of sulphuric acid. These tubes are especially valuable
when unfavorable material or when material containing
small numbers of the spirilla is used. In the incubator the
spirilla may be expected to multiply in the peptone solution
rapidly, and to appear upon the surface of the liquid in
large numbers, even forming a visible film in six hours.
Smears may be made from the surface part of these tubes,
stained, and examined with a microscope. From the same
material gelatin plates should be prepared, and examined
as soon as the colonies develop.

\Yhen cultures are obtained, their effects may be tested
upon guinea-pigs by injecting them into the peritoneum.
The reaction described by Pfeiffer as resulting from the
injection of cholera spirilla into the peritoneum of immune

PATHOGENIC BACTERIA. 323

animals has been recommended as an additional means of
diagnosis between the cholera spirillum and related forms.
The agglutinating power which the blood of animals im-
munized to cholera has for the cholera spirillum may be
employed in the same way.

In the examination of water for the spirillum of cholera,
to i liter, or more, of water, add enough of a strong pep-
tone solution to make it contain i per cent, peptone and
.5 per cent, sodium chloride. (The strong peptone solution
contains 20 per cent, peptone and 10 per cent, sodium
chloride; is alkaline and sterile.) The water, with the
peptone in it, is divided among a number of sterilized
flasks. After twelve hours in the incubator, any vibrios in
it are likely to have multiplied and to have formed a scum
on the surface, which may be investigated for the char-
acteristics of the spirillum of cholera according to the
methods given above. See also page 142.

Since Koch's discovery of the cholera spirillum in
1883-84 a considerable number of bacteria have been
described which resemble the cholera spirillum more or
less closely, and which have to be taken into account in
making examinations of material of any sort for it. This
is particularly necessary in the investigation of water, in
which such spirilla seem to occur quite frequently.

Vibrio Metchnikovi. — A comma-shaped organism,
which though somewhat shorter and thicker may be very
similar to the comma bacillus of cholera in form, and which,
like it, may sometimes form genuine spirilla. It is motile
and has a flagellum at one end. It does not form spores.
It is aerobic. It stains with the aniline dyes, and is not
stained by Gram's method. It grows at the room tempera-
ture. It liquefies gelatin somewhat more rapidly than the
spirillum of cholera. The colonies on gelatin plates are
not all alike; some of them resemble those of vibrio pro-
tens, and others are extremely like those of the spirillum of
28

324 MANUAL OF BACTERIOLOGY.

cholera. It grows upon the usual media. Blood-serum is
liquefied by it. The growth on agar is grayish to yellowish,
and abundant. It forms a pellicle on bouillon. In milk
an acid reaction is developed with coagulation. In peptone
solution it produces indol and nitrites like the spirillum of
cholera. It is said to lead to the production of indol more
intensely than the spirillum of cholera.

It is killed by a temperature of 50° C. in five minutes.
It was discovered in chickens suffering from gastro-ente-

FIG. 94.

ritis. It is pathogenic to chickens, pigeons and guinea-
pigs, less so to mice and to rabbits. The comma-shaped
organisms are found in the blood in guinea-pigs, pigeons
and young chickens.

Vibrio proteus (of Finkler and Prior). — A comma-shaped
organism somewhat larger than the spirillum of cholera,
sometimes exhibiting genuine spiral forms, and also, at

1The magnification is a little greater than in the other photomicro-
graphs.

PATHOGENIC BACTERIA. 325

times, involution forms. It is motile and has a flagellum
at one end. It liquefies gelatin much more rapidly than
the spirillum of cholera, and the colonies in gelatin develop
more rapidly. At the end of twenty-four hours the colo-
nies are uniformly circular, larger than those of the spiril-
lum of cholera, and uniformly granular, when slightly
magnified. On the other culture-media the growths are
usually whitish. On potato it produces an abundant, moist,
grayish-yellow deposit, and grows at the room temperature.
It liquefies blood-serum; milk becomes acid. In peptone
solution it does not form indol. It is less pathogenic to
animals than the spirillum of cholera. It was supposed by
its discoverers to be the cause of cholera nostras, but it ap-
pears to have no relation to that disease.

Spirillum Milleri. — A comma-shaped organism resem-
bling vibrio proteus in many respects, and probably ident-
ical with it. In gelatin it grows more rapidly, and produces
liquefaction more rapidly than the spirillum of cholera.
On gelatin plates, at the end of twenty-four hours, the
colonies are uniformly circular and granular, lying in little
depressions resulting from the liquefaction of the gelatin.
Its growths in the other media are not characteristic. It
liquefies blood-serum. It does not produce indol. It is
less toxic to animals than the spirillum of cholera. It was
isolated by Miller from a carious tooth.

See also Spirillum sputigenum, Part III.

Spirillum tyrogenum (of Deneke). — A comma-shaped
organism not so large as the spirillum of cholera. It is
motile, having a flagellum at one end. It does not form
spores. In cultures, genuine spirilla may develop. Gelatin
is liquefied more rapidly than by the spirillum of cholera,
and the colonies develop more rapidly. The circumference
of the colony is round, the surface may appear somewhat
granular, and it has a greenish-brown color, seen under

326 MANUAL OF BACTERIOLOGY.

the low power. The colonies differ noticeably from the
colonies of the cholera spirillum, in the more rapid lique-
faction of gelatin. Milk containing litmus becomes acid,
is subsequently decolorized, and is also coagulated. It
liquefies blood-serum. It does not form indol in Dunham's
peptone solution. No pellicle forms in cultures upon bouil-
lon. It is less toxic to animals than the spirillum of cholera.
It was isolated originally from old cheese.

Vibrio Berolinensis. — A comma-shaped organism resem-
bling the spirillum of cholera in form and in the position
of its flagellum. It does not stain by Gram's method. It
grows at the room temperature, but more rapidly in the
incubator. The colonies upon gelatin, one or two days old,
when magnified, are decidedly more finely granular and
more transparent than those of the spirillum of cholera, and
the margin is almost absolutely smooth and circular. As
the colonies become older they assume a more irregular
and lobulated appearance, but are still more finely granular
than the colonies of the cholera spirillum. Gelatin is very
slowly liquefied. Its growth on the other culture-media is
not remarkable. It forms indol in peptone solution, and it
increases in the upper layers of the fluid. When guinea-
pigs are inoculated in the peritoneal cavity, death occurs in
one to two days. This organism was discovered in the
water-supply of Berlin.

Other spirilla have been isolated from water by Gunther
(vibrio aquatilis in Spree water) ; by Dunbar from the Elbe
River; by Russell from the Gulf of Naples; by Heider
from the water of the Danube Canal ; and in America, by
Abbott, from the water of the Schuylkill (vibrio Schuyl-
kiliensis) ; and many others have been described to which
the limits of this work will not permit of further allusion.

The Spirillum or Spirochaeta Obermeieri (of relapsing
fever). — A slim spirillum with numerous turns, 16 to 40 /*

PATHOGENIC BACTERIA. 327

in length. The ends are pointed. It is actively motile.
The spirillum is not stained by Gram's method but may be
colored by the ordinary aniline dyes. The organism has
never been cultivated. It is found abundantly in the blood

FIG. 95.

Spirillum of Relapsing Fever in the Blood. Sketched from a Stained

Specimen.

and in the spleen during the attack of fever. The spleen
is enlarged. The disease has been produced in apes by
inoculating them with blood taken from men having the
disease.

It is asserted that the spirillum is transferred by bed-
bugs from one person to another.1

1 Karlinski, Centralblatt f. Baktcriologie, Bd. XXXI., Orig., 1902.

328 MANUAL OF BACTERIOLOGY.

APPENDIX.
PATHOGENIC PROTOZOA.

PROTOZOA are unicellular animal organisms. As they are
studied by methods that have much in common with those
used for the bacteria they may be considered here briefly.
Protozoa are numerous in pond and ditch water, and these
species seem to be harmless. However, many diseases of
the lower animals are caused by protozoa, such as surra,
Texas fever, and coccidium disease of rabbits. Birds,1 rep-
tiles and frogs2 may show organisms in the blood, resem-
bling the parasites of malaria. Until recently it has been
doubtful whether any pathogenic protozoon has ever been
propagated in pure culture outside of the body of the host.
This has recently been accomplished by Novy and McXcal
for a parasite (Trypanosoma) from the blood of the rat
on rabbit-blood-agar.

Amoeba Dysenterise (Amoeba Coli). — Associated with
annt'bic dysentery and believed to be its causative agent is
the (juurba d \scntcr ice, more often named amcrba coli.
These organisms are found in the intestinal ulcers, the feces,
the secondary liver abscesses and the sputum (in the latter
only when an amoebic liver abscess has perforated into the
lung). The lesion in the colon is a severe inflammation
accompanied by necrosis chiefly of the submucous layer, and
leading to extensive ulceration.3 According to Strong, at
least two distinct species of amoebae have been found in the

1 Opie. MacCallum, Journal Experimental Medicine, Vol. III.
•"Lnnirniann. AYu' Y< rk Medical Journal, January 7, 1899.
3 See Councilman and Laflcur. Johns Hopkins Hospital Reports, Vol.
II.; soo also Harris, American Journal Medical Sciences, Vol. 115, 1898.

APPENDIX. 329

feces in man, only one of which is pathogenic and the cause
of dysentery. Unfortunately the designation, amoeba coli,
has been applied to both species. The amoeba of dysentery
should be designated amoeba dysenteries, limiting the term
amoeba coli to the non-pathogenic form or forms.

The amoeba dysenteric is a unicellular organism, 20-50 /*
in diameter when at rest, consisting of a clear, homogeneous
ectosarc and a granular endosarc, with an eccentrically
placed nucleus. The endosarc contains a number of vacu-
oles of variable size and very frequently red blood-cor-
puscles, as well as other foreign bodies such as bacteria,
pigment granules, etc. Many red blood-corpuscles may be
seen crowded together in a single amoeba. The organism is
actively amoeboid, extending its substance into processes
or pseudopodia of varying forms. This amoeboid motion
assists in making easy the recognition of the parasites under
the microscope and in distinguishing them from large,
swollen cells found in the feces. The stool should be ex-
amined while fresh and still warm.

The non-pathogenic amoeba (amoeba coli), also occasion-
ally found in the intestinal tract of man, differs from the
pathogenic dysenteric organism chiefly in its much smaller
size ( 10-24 /;) and the invariable absence of red corpuscles
from its interior. The protoplasmic granules are also, as a
rule, smaller and are difficult to recognize. The amoeba
dysenteric produces experimentally definite ulceration of
the gut of cats, whereas the amoeba coli is harmless. Both
varieties of amoebae may be stained by a special stain de-
vised by Mallory.2

1 Strong, Circulars on Tropical Diseases, No. I, Chief Surgeon's
Office, Headquarters, Division of the Philippines, Manila, P. I., Feb-
ruary, 1901. Ibid., No. IT, April, 1901. (Both reports may be obtained
from the United States Government, Washington.)

2 Mallory, Journal of Experimental Medicine, September, 1897, Vol.
II., p. 529.

33° MANUAL OF BACTERIOLOGY.

The Malarial Parasite.1 (Plasmodium or Hematozoon
Malaria). — The organisms of malaria consist of at least
three different species, each associated with one of the three
types of malarial fever: The tertian parasite with benign
tertian malarial fever, the parasite reaching maturity in
forty-eight hours; the quartan parasite with benign quar-
tan malarial fever, the cycle of development requiring
seventy-two hours; and the &stivo-autumnal parasite with
malignant, aestivo-autumnal fever, developing to maturity
in a variable period of from twenty-four to forty-eight
hours. The parasites are studied to best advantage in a
drop of fresh, fluid blood placed between a cover-glass and
slide and examined with an oil-immersion objective. For
method of making and staining dry preparations, see pages
55 and 108.

Tertian Parasite. — This appears in its youngest form as
a small, round, colorless, hyaline body within the red cor-
puscle, seen during and just after the chill of the disease.
This body may be actively amu'boid. suddenly changing its
contour into various forms. Its size gradually increases,
and fine, dark, actively-motile, dancing pigment granules
begin to appear at its periphery.

The red corpuscle harboring the parasite, with the growth
of the latter, becomes gradually paler and expands in size.
The parasite as it grows loses its earlier amoeboid movement
and the pigment granules, still actively motile, accumulate.
Xear the end of forty-eight hours, the organism finally fills
the red corpuscle, only a faint rim indicating the latter. The
ripe parasite now divides it into from fifteen to twenty-live
small, round, hyaline spores which are arranged somewhat
radially about the pigment granules which have lost their
motility and become concentrated in a clump at the center of

1 Thaver and llewetson, "The Malarial Fevers of Kaltimore." Johns
Hopkins Hospital Reports, iS<>5. Vol. V., and reprinted; Thayer, "Lec-
tures on the Alalarial Fevers," Xe\v York,

APPENDIX.

331

the spore- forming organism. The spores finally break apart
and scatter, each destined to invade a red corpuscle and
start anew the cycle of development. This cycle may be
repeated over and over again, producing a corresponding
number of malarial paroxysms.

FIGS. 96-99.

Malarial Parasites in Various Stages. (X 1000.) 96, 97 and 98 are
tertian parasites ; 98 shows the completion of segmentation. 99 is the
crescentic form of the festivo-autumnal parasite.

Certain full-grown parasites do not complete the cycle of development
by sportilation, as described, but, breaking loose from the corpuscle,
remain as "extra-cellular" bodies. These are seen chiefly after the
paroxysm as large, round, pale bodies containing numerous dancing
pigment granules scattered through their substance. They ultimately
degenerate and disappear. Some of these extra-cellular forms may be
seen to develop long slender processes (flagella), having a very active
whip-like motion. Flagella are never observed in perfectly fresh blood
but develop only after the blood has been drawn some time, usually
fifteen or twenty minutes.

332 MANUAL OF BACTERIOLOGY.

The extra-cellular forms of the parasite (gametes), incapable of fur-
ther development in their human intermediate host, can continue their
life cycle only when, by chance, they happen to be sucked into the body of
a mosquito of the genus Anopheles (definite host), in which they un-
dergo a second complete (sexual) cycle of development with the ultimate
production of spores (sporozoids). When in turn the spores chance to
be inoculated into the blood of man by the bite of an infected Anopheles,
the man becomes infected, and the cycle of development in the red
corpuscle, already outlined, commences. The second or sexual cycle of
the parasite in the mosquito, here described for the tertian organism,
applies as well to the other varieties of the malarial organism, namely
the quartan and the aestivo-autumnal forms, in the case of each starting
from the extra-cellular mature forms of the organism found in the
blood of the human host.1

Quartan Parasite. — This resembles quite closely the ter-
tian parasite, but differs from it in certain respects. The
young, hyaline, intra-corpuscular parasite is more highly
refractive, its amoeboid motion is less marked and more
sluggish, and the pigment granules are darker, much coarser,
and have very slight motility. The infected red corpuscles
are usually somewhat contracted instead of swollen and
their color is apt to be darker, assuming a bronzed hue. The
full-grown parasite is much smaller than the corresponding
form of the tertian, approximating the size of a normal red
corpuscle. As segmentation begins, a characteristic appear-
ance develops which distinguishes the quartan organism,
namely, the coarse pigment granules are drawn toward the
center of the parasite in certain converging straight paths,
giving a stellate arrangement to the pigment, until finally it
becomes clumped entirely at the center in a solid mass. The
segmenting forms of the quartan parasite thus present a
more symmetrical arrangement of the spores, which often
resemble the petals of a " marguerite." These spores are
oval and number only from six to twelve, being fewer than
those of the tertian segmenting parasite. The quartan

1 Lyon, "The Inoculation of Malaria by the Mosquito: A Review of
the Literature," Medical Record, February 17, 1900.

APPENDIX. 333

extra-cellular forms are smaller than those of the tertian,
being about the size of a red corpuscle, and contain coarse
pigment granules in active motility until degeneration
occurs. Flagella may develop from certain extra-cellular
forms. The entire development of the quartan parasite
occupies about seventy-two hours.

/Estwo-autumnal Parasite. — This parasite develops to
maturity in from twenty-four to forty-eight hours and is
usually regarded as representing a single species, though
certain observers claim to distinguish two distinct varieties.
The usual description of a single variety is here adopted.
The youngest forms (hyaline bodies) resemble those of the
tertian and quartan organisms, but are distinctly smaller and
more highly refractive. They often present a ring-like
appearance. They are amoeboid. Pigment granules later
appear at their periphery, but are exceedingly minute and
scanty, seldom more than one or two being seen. These
granules have little or no motility and in fact are with diffi-
culty made out. The hyaline bodies remain small, seldom
exceeding one third the diameter of a red corpuscle. The
infected corpuscle is apt to be crenated, shrunken and dark.
These are the forms seen in the circulating blood in early
infections; the mature forms, with the exception of the
extra-cellular forms, developing in the spleen and bone-
marrow, rarely reach the general circulation. Blood from
the spleen shows the full-grown forms in abundance. The
segmenting forms resemble those of the tertian parasite
both in the numbers of the segments and in their arrange-
ment, but are much smaller in the aggregate, as well as in
the individual segments.

After the fever has lasted about one week, extra-cellular
forms make their appearance in the circulating blood. These
are crescentic, ovoid or small round bodies, containing coarse
pigment granules at their center, generally arranged in a

334 MANUAL OF BACTERIOLOGY.

ring. The crescents and ovoid bodies are highly refractive
and are in length about equal to the diameter of a red cor-
puscle, sometimes larger. The round forms are smaller than
a red corpuscle, with the pigment arranged centrally in a
ring. They may become flagellated after the blood has
remained outside the body for some minutes. Any of the
extra-cellular bodies may show remnants of the red cor-
puscle attached to its side, like a bib. The extra-cellular
forms are concerned in the cycle of development of the
organism in the mosquito, and are sterile in the human
body. They are exceedingly resistant to quinine and may
continue in the blood for long periods of time.

Melaniferous leucocytes (phagocytes) are seen in the
blood, being especially abundant after the paroxysm in all
forms of malarial infection.1

Small-pox and Vaccinia. — Micrococci of various sorts
have been found in the pustules of small-pox and vaccinia,
but indicate only a secondary infection. Other microorgan-
isms have been described. The most important are certain
bodies often considered protozoa. In both small-pox and
vaccinia small round homogeneous bodies, 2 to 4 /* in
diameter, have been found in the epithelial cells of the
vesicles. Inoculation of vaccine lymph into the rabbit's
cornea leads to the production of similar bodies in the
epithelial cells of the cornea. W. Reed2 found small amoe-
boid bodies in the blood in cases of small-pox and vaccinia.
Vaccine virus that has been filtered through the Chamber-
land or Berkenfeld filter is no longer active. From this it
may be presumed that the organism causing it is not too
small to be seen with the microscope.

Councilman, Magratli and Brickerhoff,3 as a result of

'See also E\vin.u\ Journal Experimental Medicine, Yols. V. and VI.

'Journal Experimental Medicine, Vol. 11.. 515; sec also, Anna Wil-
liams and Flournoy, and W. H. Park, N. Y. Univ. Bull Medical
Sciences, Tl., October, 1902.

3 Journal Medical Research. Vol. IX., May, 1903.

APPENDIX. 335

recent studies, believe that the bodies above mentioned are
protozoa. Segmentation of the bodies is described, result-
ing in the formation of spore-like bodies. The spore-like
bodies undergo a further or second cycle of development
within the nucleus. The second cycle also ends in segmenta-
tion. The two cycles were seen in small-pox; in vaccinia,
only the first or extranuclear bodies were observed.

YELLOW FEVER.

It has already been indicated (page 160) that the study of cases of
yellow fever has failed to prove that this disease is caused by bacteria.
On the other hand, evidence that it is transmitted by the mosquito,
Stegomyia, has been increasing.

As malaria and some other diseases transmitted by mosquitoes are due
to protozoa, careful search has been made for protozoa in yellow fever.
Examinations of the blood of individuals having yellow fever have been
without result. Recently Parker, Beyer and Pothier have studied speci-
mens of Stegomyia fed on such blood. They claim to have found that
these mosquitoes became infected with a protozoon parasite, a portion
of whose cycle was worked out.1 Ordinary mosquitoes did not contain
the parasite. Blood-serum from a case of yellow fever was filtered
through a Berkenfeld filter and injected into two healthy subjects; one
subject developed yellow fever and the other did not. The interpreta-
tion of these results in connection \vith the experiments of Reed and
Carroll (page 161) is not at present clear.

Trypanosomes.— A number of species of Trypanosoma have been de-
scribed, which produce diseases in the lower animals; recently one has
been stated to be the cause of disease in man.2 The trypanosoma is a
protozoon belonging to the flagellata. It is of an elongated spindle-
shaped form, with a nucleus, and has a flagellum at one end, which
extends along a thin edge, called the undulating membrane. It is ac-
tively motile. It occurs in the blood, between, but not in, the blood-
corpuscles. Its length is two to several times the diameter of a red
corpuscle. Members of this genus are the cause of surra (a fatal dis-

1 Marine Hospital Service. Yellow Fever Institute, Bulletin No. 13,
March, 1903.

2 For a full description of the life history and classification of Trypan-
osoma see Salmon and Stiles, Emergency Report on Surra. U. S.
Bureau Animal Industry, Bulletin No. 42, 1902. See also Francis,
Marine Hospital Service, Hygienic Laboratory, Bulletin No. n, 1903.

336 MANUAL OF BACTERIOLOGY.

ease of horses and mules occurring in India and the Philippine islands)
and of the tsetse-fly disease of South Africa; while others are found
in rats, birds, amphibia and fishes. In the horse the infection is trans-
mitted by the bites of flies. Novy and McNeil have succeeded in culti-
vating the trypanosoma of rats on rabbit blood-agar.1

Several cases were reported during 1902 where trypanosomes were
found in the b ood of individuals from tropical Africa, showing that
this group of parasites may occur in man.2 The symptomatology of
these infections requires further study. Still more recently it has been
claimed by Castellani that a trypanosoma is the cause of " sleeping sick-
ness," a disease of the natives of Africa. He states that the parasites
may be demonstrated in the cerebro-spinal fluid obtained by lumbar
puncture and, with greater difficulty, in the blood, during life. Many
cases also show at autopsy streptococcus infection, which is believed to
be a secondary invasion.3

1 Novy and McNeil, in Contributions to Medical Research dedicated
to Victor C. Vaughan, 1903.

2 British Medical Journal, May 30, 1903.

3Britisli Medical Journal and Lancet, June 20, 1903.

Surface Divided in Square Centimeters for Counting Colonies,

O/ 6

Plate for Counting Colonies of Bacteria in Petri Dishes.

INDEX.

ABBE condenser, 31
Abrin, 174, 181
Abscesses, 235, 245

metastatic, 242
Absorbent cotton, 82, 92, 219
Acetic acid, 40, 52, 130
Accidental infection of laboratory

workers, 115
Acid, acetic, 40, 52, 130

alcohol, 42, 45

aniline dyes, 39

boric, 207

butyric, 130, 224

carbolic, 200, 129, 197, 220

formic, 130

fuchsin, 39

hydrochloric, 154, 199, 201

lactic, 130, 145, 227

oxalic, 212

picric, 39

propionic, 130

pyrogallic in cultivating anae-
robes, 92

rosolic, 79
Acid-proof bacilli, 43, 46, 149, 154,

287, 295, 301

Acids, formation by bacteria, 130
Actinomyces, 299, 229, 230, 287, 295
Actinomycosis, 300
Acquired immunity, 176
Active immunity, 181
Acute miliaiy tuberculosis, 293
Addiment, 188

Aerobic bacteria, definition, 126
Aerobioscope, 136
Agar-agar, 76

Age, relation to infections, 166
Agglutinating substances in blood-
serum, 191, 305
Agglutinins, 191, 305
Air, bacteria of, 135

bacteria conveyed by, 163
Albumen, culture-media containing,
81

Albumen, fixative, 50
Alcohol, acid, 42, 45

fixation of tissues by, 48

relation to infection, 166, 191
A.exins, 188, 190

Alimentary canal, bacteria of, 154
Alum filter, 139
Amboceptor, 188
American filtration system, 139
American Public Health Ass'n, di-
rections for preparing media, 73
Amoeba coli, 328, 329

of dysentery, 328
Anaerobic bacteria, cultivation, 91

definition, 126
Aniline dyes, 39

alcoholic solutions, 39
as germicides, 201
watery solutions, 39

oil, 41, 53

-water solutions, 41, 46
Animals, autopsies on, 105

care of, 103, 104

inoculation of, 96, 103
Anopheles, 165, 332
Anthrax bacillus (see also Bacillus
of anthrax), 273

protective inoculation, 178,276

symptomatic, 178
Antiseptic, definition, 194
Antitoxic unit, 286
Antitoxins, 180, 184

for diphtheria, 285, 180, 186,

272

tetanus, 180, 186, 272
Argentamin, 200
Argonin, 200
Argyrol, 200

Arnold steam sterilizer, 64
Arrow-poisons, bacteria in, 16, 133
Arthritis, 242, 254, 260
Arthrospore, 123
Asiatic cholera (see Cholera)
Aspergillus glaucus, 233

337

338

INDEX.

Autoclave, 68

Auto-infection, 165

Autopsies, on animals, 105

bacteriological examinations at,

105, no

disinfection at, 105, 106, 209
on human subjects, no

Avian tuberculosis, 295

•pABES-ERNST bodies, 121
\j Bacilli, branching forms, 119
Bacilli, acid-proof, 43, 46, 149, 154,

287, 295, 301

Bacillus, definition, 14, 118
acidi lactici, Hueppe, 227
acidophilus, 155
aerogenes, 313, 155, 261
capsulatus, 268, 133
amylobacter, 223, 156
anthracis, 273, 25, 133, 135, 163,

164, 166, 183, 264
bifidus, 155
botulinus, 150
buccalis maximus, 230
butyricus, Hueppe, 224

Prazmowski, 223
capsule, of Pfeiffer, 261
coli communis, 309, 145, 146,
150, 156, 175, 192, 261,
3i3
comparison with typhoid

bacillus, 310
in water, 143, 144
comma of cholera (see Spiril-
lum of cholera)
cyanogenus, 226
diphtheriae, 278, 115, 163, 184
dysenteric, 313, 137
edematis maligni, 270, 133
enteridis, Gartner, 150, 313
erythrosporus, 226
fluorescens liquefaciens, 222

putidus, 222
icteroides, 160
Indicus, 223
influenzas, 277

Klebs-Loffler (see Bacillus diph-
theria)
lactis aerogenes, 313, 155, 261

cyanogenus, 226
leprae, 296, 287
mallei, 297, 192
megatherium, 224

Bacillus, mesentericus vulgatus (see

also Potato Bacillus), 224
mucosus capsulatus, 260
mycoides, 225
Neapolitanus, 309
of anthrax, 273, 25, 133, 135,

163, 164, 166, 183, 264
of blue milk, 226
of bubonic plague, 265
of chancroid, 260
of diphtheria, 278, 115, 163, 184
of Ducrey, 260
of dysentery, 313, 137
of Eberth, 301
of Emmerich, 309
of Escherich, 309
of Friedlander, 260
of glanders, 297, 192
of influenza, 277
of leprosy, 296, 287
of malignant edema, 270, 133
of ozaena, 261
of rhinoscleroma, 261
of Shiga, 313, 137
of smegma, 44, 154
of soft chancre, 260
of syphilis, Lustgarten, 160

Joseph and Piorkowsky, 160
of tetanus, 270, 96, 133
of typhoid fever, 301, 137, 142,

M4. 145

of Vincent, 229
of Xerosis, 283
paracolon, 312
paratyphoid, 312
pest is buboniae, 265
phlegmones emphysematosae, 270
phosphorescens Indicus, 225
pneumoniae, Friedlander, 260
prodigiosus, 223
proteus, 264, 150, 192
pyocyaneus, 262, 192
pyogenes fetidus, 309
ramosus, 225
subtilis (see also Hay bacillus),

225

tetani, 270, 96, 133
tuberculosis, 287, 96, 161, 163
in milk, 148, 149
staining of, 43, 54, 149,

287
typhi abdominalis (B. typho-

sus), 301, 137, 142, 144, 145

INDEX.

339

Bacillus, vaginalis, 153

violaceus, 223

Bacteria, acid-proof, 43, 46, 149,
154, 287, 295, 301

aerobic, 126

anaerobic, 126

cultivation of, 91

chlorophyll, relation to, n, 125,
132, iS7_

chromogenic, 128

cultivation of, 84

classification, 117

definition, 13

diseases caused by, 159

distribution, 14, 133

examination with the micro-
scope, 29

ferments formed by, 128

fluorescent, 128, 222, 262

forms of, 117, 118

higher, 229

in disease, 157

influence of electricity, 127
of oxygen, 126
of sunlight, 126

microscopic examination, 29

motility, 124

multiplication, 122

non-pathogenic, 120, 221

number of species, 221

nutrition of, 125

of air, 135, 163

of the alimentary canal, 154

of foods, 144, 164

of the cranial sinuses, 151

of the gall-bladder, 151, 308,
312

of the intestines, 154, 155

of ice, 136, 144

of milk, 144, 164

of the mouth, 152, 227, 228, 230,
253, ^61

of the nasal cavity, 152, 261

of the normal human body,

151

of the skin, 151
of soil, 133, 164
of the stomach, 154
of the urethra, 153
of the vagina, 153
of water, 136, 164
pathogenic, 121, 235
phosphorescent, 128
29

Bacteria, products of growth, 128,

146, 150, 172
pyogenic, 237
size, 14, 121
staining, 39, 40

in tissues, 48, 51
transmission of specimens by

mail, no

vegetative forms, 122
Bacterial products, 128, 146, 150,

172

Bacteriolysis, 184, 187, 189
Bacterium, definition, 119

coli commune, 309, 145, 146,

150, 156, 175, 192, 261, 313
syncyanum, 226
termo, 132, 264
urese, 227
Zopfii, 227

Balsam, Canada, 38, 40, 52
Basic aniline dyes, 39
Basophilic granules, 51, 53
Beef-tea, 71
Beggiatoa, 229
Beri-beri, 160
Berkenfeld filter, 69
Bichloride of mercury (see Mercury,

bichloride)
Biedert's method for examining

sputum, 47

Birds, tuberculosis of, 295
Bismarck brown, 39, 42
Black death, 268

leg, 178

Blastomycetic dermatitis, 233
Blood-agar, 81, 277
Blood, cultures from, 109

of another species, immunity

for, 1 88, 192
-poisoning, 239
specimens of, 108
-serum-agar, 80

germicidal power, 190

Loffler, 80

Marmorek, 81, 249

preparation, 79

sterilization, 67, 79

-test for typhoid fever, 304,

191

staining of, 55
Blue milk, bacillus of, 226
pus, 263
vitriol, 208

340

INDEX.

Bodily conditions disposing to in-
fection, 165

Boiling, sterilization by, 63, 139,
21 1, 214

Boils, 245

Bodphilus, 165

Boric acid, 207

Bouillon, 71

sugar-free, 74

Bovine tuberculosis, 148, 290, 294

Branching forms of bacilli, 119

Bread-paste, 81

Bromine, as a germicide, 205

Bronchitis, 241, 278

Brownian movement, 35

Bubonic plague, bacillus, 265

Buchner's method for cultivating
anaerobes, 91

Butter, tubercle bacilli in, 148, 149,

44
Butyric acid, 130, 224

/CADAVER, care of, in contag-
\_^ ious diseases, 209

Calcium compounds as germicides,
205, 206

hypochlorite, 205
Canada balsam, 38, 40, 52
Capaldi's culture-medium, 304
Capsule bacillus of PfeitTer, 261
Capsules of bacteria, 121, 57

staining of, 57
Carbol-fuchsin, 45
Carbolic acid, 200, 129, 197, 220
Carbon dioxide, 130
Carbuncles, 245
Carmine, 54, 55
Caries of the teeth, 153

ition, 292

Catgut, surgical preparation, 215
( 'edar-wood oil, 31
Celloidin imbedding, 48
Cells, epithelioid, 291

giant, 291

pus, 236

Cellulitis, _'47, 270
Cellulose, decomposition by bac-
teria, 129, 156

Centrifuge for milk separator, 147
Cerebro-spinal meningitis, _>5;
Chancroid, bacillus of, 260
Charbon (see Anthrax)
Cliarbon sytnptomatiqiic, 178

Cheese-poisoning, 146
Chemotaxis, 124
Chicken-pox, 160
Chloride of lime, 205
Chlorine, as a germicide, 205
Chloroform, as a preservative, 80
Chlorophyll, relation to bacteria, n,

125. 132, 157
Cholera, diagnosis, 321

infantum, 265, 315

nostras, 325

-red reaction, 318

spirillum (see also Spirillum of

cholera), 315
Chromicized catgut, 217
Chromogenic bacteria, 128
Cladothrix, 229
Classes in bacteriology, hints for

teaching, 112, 113, 114, 115
Classification of bacteria, 117
Cleaning fluid, 36
Climate, influence on infections, 166
Clostridium, definition, i_>4

butyricum, 223
Coal-oil, 207

Coccus, definition, 14, 118
Collodion, 48

capsules, 106
Colon bacillus (see also Bacillus

coli communis), 309
contrasted with typhoid

bacillus, 310
Colon group, 312
Colonies of bacteria, 97, 99, 101
Comma bacillus of cholera (see also

Spirillum of cholera), 315
Comma-shaped bacteria, 118, 120
Complement, 188
Condenser, Abbe, 31
Conidia. -233, 299
Conjunctivitis, gonorrheal, 260
Consumption, 293
Contagious disease, definition, 158

disinfection after, 210
Contrast-stains, 39, 42, 43, 47, 54
Copperas, 207
I opper sulphate, 208
Cornet forceps, 36, 37
Corrosive sublimate, see Mercury

bichloride
Cotton plugs for tubes, etc., 23, 82,

Cotton, absorbent, 82, 92, 219

INDEX.

341

Cover-glasses, 36
Cover-glass forceps, 36, 37

preparations, 36, 37, 39
Cow-pox, 21, 177
Cranial sinuses, bacteria of, 151
Cream, ripening, 148
Creolin, 201
Cresol, 201

Croup, membranous, 284
Cultivation of anaerobic bacteria, QI

of bacteria, 84
Culture-media, definition, 17

neutralization, 71, 72, 73
preparation, 71
reaction of, 71, 74, 126
sterilization, 63, 71, 74, 79,

83
-tubes, 82

inoculation of, 84
sterilization of, 82
Cultures at autopsies, 105, no
from blood, 1 09
destruction of, 99, 115
sealing of, 91
Cumol, 216

Cutting of sections, 50
Cupric sulphate, 208
Cystitis, 241, 264, 308, 312, 313
Cytase, 188

DELAFIED'S hematoxylin, 54
Deneke's spirillum, 325
Dengue, 160
Dental caries, 153
Deodorizers, 194
Dermatitis, blastomycetic, 233
Dextrose, 74

-agar, 77

-bouillon, 74

media for anaerobes, 91
Diagnosis of actinomycosis, 301

of bubonic plague, 266

of cholera, 321

of diphtheria, 280, 247

of dysentery, 315, 329

of glanders, 298

of gonorrhea, 258

of influenza, 278

of malaria, 330

of Malta fever, 256

of meningitis, cerebro-spinal,
257

of pneumonia, 254

Diagnosis of tuberculosis, 287, 44,

294, 295

of typhoid fever, 304, 307, 308
Dilution-cultures, 99
Diphtheria, 284, 247

antitoxin, 285, 180, 186, 272
bacillus, 278, 115, 163, 184
diagnosis, 280, 247
toxin, 174, 186, 284, 285
Diphtheritic inflammation (see also
Pseudamembranous inflamma-

tion), 247, 282, 284
Diplococcus, definition, 118

intracellularis meningitidis, 256
of gonorrhea, 258, 183
of pneumonia (see also Micro-
coccus lanceolatus), 251
Disease, bacteria in, 157
Diseases caused by bacteria, 159

by protozoa, 328
probably due to microorgan-
isms, 160
infectious, recovery from, 171,

176, 189

Disinfectant, 194
Disinfection at autopsies, 105, 106,

209

of cultures, 99, 115
of dejecta, 208
of hands, 212

of houses, 209, 201, 204, 206
of sputum, 45, 208
of stools, 208
of test-tubes, 99, 115
of urine, 208
surgical, 211, 220
Distribution of bacteria, 133
Dorset's egg-medium, 81
Dressings, surgical preparation, 219
Drinking water, sterilization of, 139
Drying, influence on bacteria, 123,

125

Ducrey's bacillus, 260
Dunham's peptone solution, 79
Dyes, aniline, 39

as germicides, 201
Dysentery, 263, 314
amoebic, 328
bacillus, 313

EAR, middle, bacteria of, 151, 241
Eberth's bacillus (see also Ba-
cillus of typhoid fever), 301

342

INDEX.

Edema, malignant, bacillus, 270, 133
Egg-albumen as a culture-medium,

Egg-medium of Dorset, 81
Eggs, in cultivating anaerobes, 81, 95
Ehrlich's side-chain theory, 184
Electricity, influence on bacteria,

127

Eisner's culture-medium, 304
Emmerich's bacillus, 309
Emphysematous gangrene, 269
Endocarditis, 241, 245, 247, 254, 260
Endogenous spores, 123
Enzymes, 128, 174
Eosin, 39, 42
Epithelioid cells, 291
Erysipelas, 250
Escherich's bacillus, 309
Esmarch's method for anaerobes, 95

roll-tubes, 99

Essential oils as germicides, 207
Eye-piece, 29, 30

FALLOPIAN tube, bacteria of,
151

tarcy-buds, 297

Fat in culture media, 81

Fats, decomposition by bacteria, 129

Feces, bacillus of tetanus in, 271

bacteria of, 155

disinfection, 208

typhoid bacilli, examination for,

304, 307
Fermentation, 22, 131

-tube, 130

Ferments, development by bacteria,
128

and toxins, 1 74
Ferrous sulphate, 207
Fibrin, Weigert's stain, 53
Film-preparations, 37, 38, 39
Filter, alum, 139

American, 139

Berkenfeld, 69

infusorial earth, 69

Kitasato, 69

mechanical, 139

Pasteur-Chamberland, 69, 139

sand, 138

unglazed porcelain, 69, 139
Filtration, sterilization by, 69, 139

of water, 138
Finkler and Prior spirillum, 324

Fishing from colonies, 101
Fission of bacteria, 13
Fixation of cover-glass preparations,
37, 39

of slide-preparations, 38, 39

of tissues, 48
Flagella, 124

staining, 58
Flies, bacteria carried by, 164, 309,

321

Fluid for cleaning, 36
Fluorescence of bacteria, 128, 222,

262

Focusing the microscope, 32, 35
FomI es, definition, 159
Foods, bacteria of, 144, 149

poisoning by, 146, 150
Food used by oacteria, 125
Foot and mouth disease, 161
Forceps, Cornet, 36, 37

cover-glass, 36, 37

for slides, Kirkbride, 38

Stewart, 36, 38

Formaldehyde as a germicide, 201,
197, 210

catgut, 216

disinfection of rooms, 210

fixation of tissues with, 48
Formalin (see Formaldehyde)
Formic acid, 130
Fowl-cholera, protective inoculation,

178

Fowls, tuberculosis of, 295
Fractional sterilization, 63
Frankel's method for anaerobes, 93

pneumococcus (see also Micro-
coccus lanceolatus), 251
Freeman's pail for pasteurizing, 67,

Freezing, influence on bacteria. 144
Friedlander's bacillus of pneumonia,

260
Fuchsin, 39, 40

acid, 39

Fiirbringer's method for disinfec-
ting hands, 212
Fusiform bacillus of Vincent, 229

/~* ABBETT'S method for staining
VJJ" tubercle bacilli, 46, 154
Gall-bladder, bacteria of, 151, 308.

312
Gangrene, emphysematous, 269

INDEX.

343

Gas-burner, Koch's, 90

formation by bacteria, 130

-phlegmons, 269

-regulator, 89

Gastric juice, germicidal power, 154
Gauze, sterilization of, 219
Gelatin, 74

liquefaction, 128

tetanus bacilli in, 272
Gelose, see agar-agar, 76
Gentian-violet, 39, 40
Geppert's test for germicides, 196
Germicidal power of blood-serum,

190

Germicide, definition, 194
Germicides, tests for, 195
Germ, use of the word, 13
German measles, 160
Giant-cell, 291
Glanders, bacillus, 297, 192

Straus' method for diagnosing,

298

Glass plates, 100

Glassware, sterilization of, 61, 62
Gloves, rubber, 214
Glucose (see also Dextrose), 74
Glycerin-agar, 77

-albumen, 50
Glycerin-bouillon, 74
Gonococcus of Neisser, 258, 183
Gonorrhea, 258, 240, 260

diagnosis, 258
Gram's method, 41, 52

bacteria stained by, 42
not stained by, 43
Gram-Giinther method, 42
Gram-Weigert method, 53
Gray tubercle, 292
Green pus, 263
Ground- water, 137
Groups of bacteria, 118
Guarnieri's medium, 81
Gun-cotton, 48

Giinther's modification of Gram's
method, 42

HAFFKINE'S inoculations for
plague, 267, 179

Hair-follicles, infection around, 239
Hands, disinfection, 212
Hanging-block, 36
Hanging-drop, 34
Haptophore, 185

Hardening of tissues, 48

Hay bacillus, 225, 113, 123, 146,

i95
Heat, effect on growth of bacteria,

125

Heat, sterilization by, 61, 211
Hematoxylin, 54
Hematozoon of malaria, 330
Higher bacteria, 229
Hill's test for germicides, 195
Hiss, medium of, 304
Hiss, stain for capsules, 57
Historical sketch of bacteriology, 18
Hog cholera, 180
Holmes, O. W., 22
Honing of knives, 51
Horse-hair, surgical preparation, 218
Hot-air sterilizer, 62
Houses, disinfection, 209, 201, 204,

206

Hueppe's method for anaerobes, 95
Hydrochloric acid, 154, 199, 201
Hydrogen, cultivation of anaerobes
under, 93

peroxide, 206

sulphide, 130
Hydrophobia, 179, 160

preventive inoculation, 179
Hypha, 233

Hypochlorite of calcium, 205
Hypodermic inoculation of animals,
104, 105

ICE, bacteria of, 136, 144
Ice-cream poisoning, 146
Illumination for the microscope, 32
Imbedding, 48
Immune-body, 188
Immunity, 176, 26

acquired, 176

active, 181

antitoxic, 184

bacteriolytic, 184

natural, 176

passive, 181

racial, 177

side-chain theory, 184

theories of, 182

unit, 286

Impression-preparation, 37
Incubator, 87, 88
Indol, 129

test for, 129

344

INDEX.

Infected wounds, 220
Infection, bodily conditions favor-
ing, 165

local conditions favoring, 167
of investigators with pathogenic

bacteria, 115
of wounds, 167, 169
mixed, 169

secondary, 169, 170, 240
terminal, 169, 191
Infectious disease, definition, 158
Inflammation, 235, 240

diphtheritic, see also Pseu-
domembranous inflammation,
247, 282, 284
Influenza bacillus, 277
Infusorial earth in filters, 69
Inoculation of animals, 103

in isolating bacteria, 96
of tube-cultures, 84
Inoculations, preventive, 177
for anthrax, 178, 276
for blackleg of cattle, 178
for bubonic plague, 267, 179
for cholera, 320
for erysipelas of swine, 178
fowl-cholera, 178
for hydrophobia, 179
for small-pox, 20, 177
for tuberculosis, 295
for typhoid fever, 309
Insects, infections spread by, 164,

165, 309, 321, 327, 332, 335
Insects, destruction of, 204, 207
Instruments, surgical preparation,

21 i, 214

Intermediary body, 188
Intermittent sterilization, 63
Intestine, bacteria of, 154, 155
Intravenous inoculation, 104
Invisible growth on potato, 303

microbes, 121, 161
Involution forms of bacteria, 121
Iodide of mercury, 199
Iodine solution, 42
lodoform, 207
Iris diaphragm, 29
Itch, 25

ENNER, 21

Journals of bacteriology, 18

KANGAROO tendon, surgical
preparation of, 217
Kerosene, 207

Kirkbride forceps for slides, 38
Kitasato filter, 69
Khitschpreparat, 37
Klebs-Loffler bacillus (see also B.

diphtheria), 278
Knives, sharpening of, 51
Koch, 26
Koch's gas-burner, 90

method for anaerobes, 94

plate-cultures, 26, 96,

100

rules, 158

steam sterilizer, 66, 67
tests for germicides, 195

T ACTIC ACID, 130, 145
J_/ Lactose, 74
Leeuwenhoek, 20
Leprosy bacillus, 296, 287
Leptothrix, 229, 230

buccalis, 230, 152

innominate, 230

maxima buccalis, 230
Leucin, 129
Leucocytosis, 183
Leucomaines, 1 73
Ligatures, surgical preparation, 215,

217, 218, 198

Light, influence on bacteria, 126
Lime as a germicide, 206
Liquefaction of gelatin, 128
Lister, 24

Lithium-carmine, 55
Litmus-agar, 77

-milk, 79

Lockjaw (see Tetanus)
Loffler's bacillus of diphtheria, 278,
115, 163, 184

blood-serum, 80

methylene-blue, 41

stain for flagella, 58
Lump-jaw, 301
Lungs, bacteria of the, 151
Lustgarten's bacillus of syphilis,

160

Lymphoid tissues, relation of bac-
teria to, 151, 162
Lysins, 189
Lysol, 201

INDEX.

345

MADURA disease, Madura foot,
301

Magnifying power of objectives, 30
Mails, transmission of specimens of

bacteria in, no

Malachite-green as a germicide, 201
Malaria, 165, 204, 207

parasite of, 330
Malarial parasite, staining of, 55,

109
Malignant edema, bacillus, 270, 133

pustule, 276
Mallein, 298

Malta-fever, micrococcus of, 255
Marmorek's anti-streptococcus

serum, 249

serum-medium, 81, 249
Massachusetts steam sterilizer, 66
Mastzellen, 51

Mayer's glycerin-albumen, 50
Measles, 160, 248, 284
Meat, tubercle bacilli in, 148
Mechanical filter, 139
Medium, culture- (see Culture-me-
dium)
Membranous croup, 284

rhinitis, 284
Meningitis, 241, 254, 257, 261

cerebro-spinal, 257
Mercuric chloride (see Mercury bi-
chloride)
iodide, 199
Mercurol, 200
Mercury bichloride, 199, 196, 197

stock solution, 199
Metachromatic granules of bacteria,

121

Metastatic abscesses, 242
Metchnikoff, theory of phagocytosis,

182, 188
vibrio of, 323

Methyl alcohol lamp in formalde-
hyde disinfection, 203
Methylene-blue, 39, 40, 41

as a germicide, 201
Loffler's, 41

Methyl-violet as a germicide, 201
Miasmatic disease, definition, 159
Microbe, use of the word, 13
Micrococcus, definition, 14, 118
agilis, 221
amylovorus, 158

Micrococcus, gonorrheas, 258

lanceolatus, 251

melitensis, 255

of sputum septicemia, 251

Pasteuri, 251

pneumonia crouposae, 251

pyogenes tenuis, 255

tetragenus, 250

ureae, 221

Micromillimeter, 32
Micron, u, 32
Microscope, 29

Microscopical examination of bac-
teria, 29
Microtome, 50
Miliary tubercle, 292

tuberculosis, 293
Milk as a culture-medium, 78

bacteria of, 144, 164

number of bacteria in, 147, 148

pasteurization, 67, 146

pathogenic bacteria in, 145

-poisoning, 146

samples of, 108

staining bacteria in, 149

sterilization in infant feeding,
146

tubercle bacilli in, 148, 149

of lime, 206
Miller's spirillum, 325
Milzbrand (see Anthrax)
Mixed infection, 169
Moisture, effect on growth of bac-
teria, 125
Mosquitoes as carriers of infectious

disease, 165, 332, 335
Mosquitoes, destruction of, 204, 207
Motility of bacteria, 35, 124
Moulds, 231, 114, 135, 233

cultivation, 81
Mouth, bacteria, 152, 227, 228, 230,

253, 261

Movement, Brownian, 35
Mucor mucedo, 233
Mucous membranes, bacteria of, 151

152

Multiplication of bacteria, 122
Mumps, 1 60

Mustard as a deodorizer, 207
Mycelium, 233
Mycetoma, 301

346

INDEX.

NASAL cavity, bacteria of, 152,
361

Natural immunity, 176
Necrosis bacillus, 231
Neisser's gonococcus, 258, 183

stain for diphtheria bacilli, 278,

279
Neutral red in culture-media, 77,

303, 310, 313, 3i4
Neutralization of culture-media, 71,

72, 73

Nitrate of silver, 200
Nitrifying bacteria, 130, 133
Nitrogen fixation by bacteria, 134

liberation by bacteria, 130
Nitroso-indol reaction, 130
Noma, 230
Non-pathogenic bacteria, definition,

120

bacteria, 221
Normal solutions, 73
Nose-piece, 29

Novy's method for anaerobes, 94
Nucleins, 191

Number of bacteria in feces, 155
milk, 147, 148
soil, 133
water, 140

species of bacteria, 221
Nutrient agar-agar, 76
bouillon, 71
gelatin, 74
Nut.ition of bacteria, 125

OBERMEIER'S spirillum, 326
Objectives, 29
Ocular, 29
Odors developed by bacteria, 130

from water, 137
Oese, 33

Oidium lactis, 233
Oil, aniline, 41, 53

cedar-wood, 31

culture-media containing, 81,
265

-immersion objective, 30, 31
Oil, kerosene, 207
Oils, essential, as germicides, 207
Osteomyelitis, 240, 245, 308
Otomycosis, 234

Ovum, bacteria conveyed in, 161
Oxalic acid, _M j
Oxygen, relation of bacteria to, 126

Oysters, typhoid fever conveyed by,

150

Ozaena, bacillus, 261
Ozone in purifying water, 139

PARACOLON bacillus, 312
Paraffin imbedding, 49
Faraform or paraformaldehyde, 202
Parasite, definition, 120
Paratyphoid bacillus, 312
Park, Roswell, method for disinfect-
ing hands, 212
Park, W. H., method for cultivating

anaerobes, 94
Parietti's method for examination of

water, 143

Passive immunity, 181
Pasteur, 23, 26

Pasteur-Chamberland filter, 69, 1 39
Pasteurization, 67, 146
Pathogenic bacteria, definition, IJT
descriptions of species, 235
Pear-blight, 16, 158
Penicillium glaucum, 233
Peptone, 71, 129

solution concentrated, 323
Dunham, 79
Peptonizing ferments formed by

bacteria, 128

Pericarditis, 241. 245. 247, 254
Peritonitis, 241. 245, 247, 263, 265,

312

I'crlsucht, 290

Permanganate of potassium, 207, 212
Peroxide of hydrogen, 206
Petri dishes, 98
Petroleum for destroying insects,

207
Pfeiffer's capsule bacillus, 261

reaction for cholera spirillum
(Pfeiffer's phenomenon), 189,
319

Phagocytosis, 182, 188, 235. 334
t'henol (see also Carbolic acid), 129
Phenolthalein, 72
Phosphorescence of bacteria, 128,

225

Picric acid, 39

Piorkowski's culture-medium, 304
Piroplasma, 165
Placenta, bacteria transmitted

through, 161
Plague, bubonic, bacillus of, 265

INDEX.

347

Plants, diseases of, 16, 158
Plasmodium of malaria, 330
staining of, 55, 109
Plasmolysis, 121
Plate-cultures, 96
Platinum wire, 33

rules for use, 33, 85
Pleuritis, 241, 245, 247, 254
Pleuro-pneumonia of cattle, 161
Plugs, cotton, for tubes, etc., 82, 92
Pneumococcus of Frankel (see also

Micrococcus lanceolatus), 251
Pneumonia, broncho-, 241, 247, 254,

261, 263, 285, 301, 308
croupous, 241, 253, 261
diagnosis, 254, 255
Pneumonomycosis, 233
Poisoning by food, 146, 150
Porcelain filter, 69
Post-mortems, disinfection at, 105,

1 06, 209

Post-office rules for mailing speci-
mens of bacteria, no
Potassium permanganate, 207, 212
Potato as a culture-medium, 78
invisible growth on, 303
bacillus, 224, 95, 113, 123, 146
Precipitins, 192

Predisposition to infection, 166
Products, bacterial, 128, 146, 150,

172

Propionic acid, 130
Protargol, 200
Protective inoculation, 177

for anthrax, 178, 276
for blackleg of cattle, 178
for bubonic plague, 267, 179
for Asiatic cholera, 320
for erysipelas of swine, 178
for fowl-cholera, 178
for hvdrophobia or rabies,

179

for small-pox, 20, 177
for tuberculosis, 295
for typhoid fever, 309
Proteus mirabilis, 264
vulgaris, 264
Zenkeri, 264

Protozoa, pathogenic, 328, 27, 165
Pseudo-gonococcus, 258

-diphtheria bacillus, 283
-membranous inflammations,
247, 254, 282, 284, 315

Pseudo-pneumococcus, 255

-tuberculosis, 296
Ptomaine poisoning, 150
Ptomaines, 173
Puerperal fever, 21, 247, 284
Pure cultures, 25, 96, 101
Pus, blue, 263

-cells, 236

formation, 236

green, 263

samples of, 108, no
Putrefaction, 23, 131
Pyemia, 242
Pyocyanin, 173, 262
Pyogenic bacteria, 237
Pyoktanin, 201
Pyosalpinx, 260

Pyrogallic acid for cultivating anae-
robes, 92
Pyroxylin, 48

QUARANTINE, 19

RABIES, 179, 160
Racial immunity, 177

predisposition to infec-
tion, 167
Rats, acid-proof bacilli of, 295

relation to bubonic plague, 267
Ranschbrand, 178
Ray-fungus of actinomycosis, 299
Reactions of culture-media, 71, 74,

126

Receptor, 185
Recovery from infectious disease,

171, 176, 189

Reichert's gas-regulator, 89
Relapsing fever, spirillum, 326
Rheumatic fever, 160, 242
Rheumatism, 160, 242
Rhinoscleroma, bacillus, 261
Ricin, 174, 181
Ripening of cream, 148
Roll-tubes of Esmarch, 99
Rooms, disinfection, 209, 201, 204,

206

Root-tubercle organisms, 134
Rosolic acid, 79
Rougct, 178
Rubber caps for culture-tubes, 86,

9i
gloves, 214

348

INDEX.

Rubber stoppers for culture-tubes,

86, 91, 92
Rules for students, 115, 98, 99, 105,

1 06

of Koch, 158
of post-office, no

SABOURAUD'S culture-medium,
82

Saccharomyces cerevisiae, 231
Saccharose, 74
Salt-agar, 266
Sanarelli's bacillus of yellow fever,

160

Sand filter, 138
Sapremia, 171

Saprophyte, definition, 120, 157
Sarcina, 118, 222
pulmonum, 222
ventriculi, 222, 155
Sarcoma, toxins of streptococcus

for, 249

Scarlet fever, 160, 248, 249, 284
Schatz's method for disinfecting

hands, 212

Schizomycetes, definition, 13
Schultz's method for neutralizing

culture-media, 72
Scrofula, 292
Sclr^'cinerothlauf, 178
Sealing culture-tubes, 91
Secondary infection, 169, 170, 240
Section-cutting, 50
Sections, staining bacteria in, 51
carmine, 55
Gram's method, 52
hematoxylin, 54
tubercle bacilli, 54
Weigert method, 53
Sedgwick's test for germicides, 197

-Tucker aerobioscope, 136
Self-purification of water, 138
Semen, transmission bacteria by,

161

Semmelweis, 21
Separator for milk, 147
Septicemia, 171
Serum (see Blood-serum)

-test for typhoid fever, 304,

191
Shiga's bacillus of dysentery, 313,

137
Side-chain theory of immunity, 184

Silk threads in testing germicides,
195

surgical preparation, 218
Silkworm gut, surgical preparation,

218
Silver, germicidal power of, 218

nitrate, 200

wire in surgery, 218
Sinuses, cranial, bacteria of, 151
Size of bacteria, 14, 121
Skatol, 129
Skin, bacteria of, 151

disinfection, 152, 212, 213, 214
Sleeping sickness, 336
Slides, forceps for, 38

glass, 38
Small-pox, 334, 177, 248

inoculation of, 20
Smear-culture, 86

preparations, 37, 38, 39
Smegma bacilli, 44, 154
Snake- venom, 174, 188
Sodium hydroxide, 71, 73. 92
Soft-chancre, bacillus of, 260
Soil, bacteria of, 133, 164
Solutions, normal, 73
Species of bacteria, 117
Spirilla in the mouth, 152, 227, 325

in water, 114, 142, 228, 326
Spirillum, definition, 14, 118, 120

dentium, 227

of Asiatic cholera, 315, 137,
142, 145, 155, 189

of Deneke, 325

of Finkler and Prior, 324

of Metchnikoff, 323

of Miller, 325

of Obermeier, 326

of Vincent, 229

plicatile, 229

relapsing fever, 326

rubrum, 227

rugula, 228

sputigenum, 228

tyrogenum, 325

undula, 228

volutans, 228
Spirochaeta, definition, 120

dentium, 227

Obermeieri, 326

plicatile, 229
Splenic fever (see Anthrax)

puncture in typhoid fever, 307

INDEX.

349

Sponges, surgical preparation of, 218
Spontaneous generation, 13, 23
Spores, 13, 24, 122

arthro-, 123

endogenous, 123
Spores of the malarial parasite, 330,

332
Spores of moulds, 233

resistance to heat, etc., 123

staining, 56

Sporotricha or sporothrix, 233
Sputum, collection, 44, 108

disinfection, 45, 208

staining, 44, 108, 287
Stab-culture, 86
Staining, 39, 40

bacteria in tissues, 48, 51

blood, 55

capsules, 57

diphtheria bacillus, 278

flagella, 58

gonococcus, 258

Gram's method, 41, 52

malarial parasite, 55, 109

sections, 51

spores, 56

tubercle bacillus, 43

in milk, 44, 149

sputum, 44, 45,

1 08

tissue, 54
Stalactite growth of plague bacillus,

265, 266
Staphylococcus, definition, 118

cereus albus, 237
flavus, 237

epidermidis albus, 246, 147, 152

pyogenes albus, 245

aureus (see also Suppura-
tion), 243, 148

pyogenet, citreus, 237
Steam sterilization, 63, 211
Stegomyia, 165, 335
Sterilization, 63, 211

after autopsies, 105, 106, 209

by the autoclave, 68

by boiling, 63, 139, 211, 214

by filtration, 69, 139

by steam, 63, 211

by the naked flame, 61

fractional, 63

hot-air, 61

Sterilization, intermittent, 63

of blood-serum, 67, 79

of culture-media, 63, 71, 74,
79, 83

of cultures, 99, 115

of dressings, 219

of glassware, 61, 62

of gloves, rubber, 214

of hands, 212

of instruments, 214

of ligatures, 215, 217, 218

of milk in infant feeding, 146

of test-tubes, 82

of water, 139

steam, 63, 211
Sterilizer, Arnold, 64

hot-air, 62

Koch, 66, 67

Massachusetts, 66

steam, 64
Stern berg's bulbs, no

determination thermal death-
point of bacteria, 125

tests for germicides, 195
Stewart's forceps, 36, 38
Stick-culture (see Stab-culture)
Stitch-abscesses, 246
Stoddart's culture-medium, 304
Stomach, bacteria of, 154
Stools, disinfection, 208
Storage of water, 138
Straus' method for diagnosis of

glanders, 298

Streptococcus, definition, 118
brevis, 246

lanceolatus, 251

longus, 246

mucosus, 255

of erysipelas, 250

pyogenes (see also Suppura-
tion), 246, 145, 170

serum, 249
Streptothrix, 229, 230, 301

actinomyces, 299

cuniculi, 231
Stropping knives, 51
Substance sensibilitrice, 188
Sugar-free bouillon, 74
Sugars in culture-media, 74, 76, 77
Sulphur, use in disinfection, 204,

210

Sunlight, influence on bacteria, 126
Suppuration, 235

350

INDEX.

Surgical disinfection, 211, 220

infection, 167, 238
Surra, 335

Swarming islands, 264
Swine erysipelas, 178
Symptomatic anthrax, 178
Syphilis, 160, 161

Systematic study of species of bac-
teria, 112

''T^EACHING bacteriology, sugges-

JL tions for, 112, 113, 114, 115

Tendons, animal, as ligatures, 198,

215
Test-tubes, 82

inoculation of, 84
manner of holding, 84
plugs for, 82, 92
sealing of, 91
sterilization, 82
Teeth, bacteria of, 153

caries of, 153

Terminal infections, 169, 191
Tetanus antitoxin, 180, 186, 2~ 2
bacillus, 270, 96, 133
toxin, 174, 1 86, 187, 2-2
Tetrad, definition, 118
Texas fever, 165

Thermal death-point of bacteria, de-
termination, 125
Thermophilic bacteria, i J5
Thermostat (see Gas-regulator)
Thiothrix, 229, 230
Thread-reaction of bacteria, 192
Thrush, 233
Thymol, 108
Tinea favosa, 233
Tinea trichophytina. j.$3
Tissues, fixation and hardening, 48

staining bacteria in, 48, 51
Titration of culture-media, 72, ;.}
Toxalbumens, 173
Toxemia, 170
Toxin, definition, 173
Toxins, \-j2, -2, uo, 157, 180, 181,

185
of diphtheria, 174, 186, 284,

285

of tetanus, 174, 186, 187, 272
Vrxophore, 185
Trichophyton, cultivation, 82

rypanosome, 335, 192
Tsetse-fly disease, 336, 165

Tubercle, gray, miliary, yellow, 292
structure, 290, 291
bacillus, 287, 96, 161, 163
in butter, 44, 148, 149
in meat, 148
in milk, 44, 148, 149
staining, 43, 54, 149, 289
in milk, 44, 149
in sputum, 44, 45, 108

287
in sections of tissue;,

54
Tuberculin, 294

R., 295
Tuberculosis, 292

acute miliary, 293
bovine, 148, 290, 294
diagnosis, 287, 44, 294, 295
frequency, 148, 292
immunity, 295
of birds, 295
organs affected by, 293
pseudo-, 296

spread of, in the body, 292, 293
Typhoid fever, bacillus, 301, 137,

142, 144, 145

contrasted with colon ba-
cillus, 310
fever diagnosis, 304, 307, 308

serum-test, 304, 191
Typhus fever, 160
Tyrosin, 129
Tyrotoxicon, 146

TTLTRA-MICROSCOPIC organ-
\^j isms, i _> i , 161
Unit, immunity, 286

decomposition by bacteria, 129
Urethra, bacteria. 153
Urethritis. gonorrheal, 260
Urinary bladder, bacteria of (see

also cystitis), 151
Trine, disinfection, 208

samples, 108

-serum-agar, 259

typhoid bacilli in, 308
Uterus, bacteria of, 151

VACCIXATIOX. 21, 177
and tetanus, j;_-
Vaccinia, parasites in, 334
Vagina, bacteria of, 153
Vaginitis, gonorrheal, 260

INDEX.

351

Van Ermengem's method for stain-
ing flagella, 59

Vegetative forms of bacteria, 122
Venom of snakes, 174, 188
Vibrio, definition, 120

aquatilis, 326

Berolinensis, 326

Metchnikovi, 323

proteus, 324

rugula, 228

Schuylkiliensis, 326
Vibrion septique, 270
Villemin, 22
Vincent, bacillus of, 229
Vinegar, bacteria in, 15
Violet, gentian-, 39, 40

methyl, 201
Virulence of bacteria, 126, 168

WARMTH, effect on growth of
bacteria, 125
Water, bacillus coli communis in,

143, 144
bacteria of, 136

conveyed by, 137, 164
filtration, 138
ground-, 137

infections carried by, 137
number of bacteria in, 140
pathogenic bacteria in, 137, 142
purification by ozone, 139
samples of, 108, 139
self-purification, 138
spirilla in, 114, 228, 326
sterilization of, 139
storage of, 138
Watery solutions of aniline dyes, 39

Weir's method for disinfecting

hands, 213
Welch's stain for capsules, 57

hypothesis, 190
Whooping-cough, 160
Weigert's stain for fibrin and bac-
teria, 53
Widal's serum-test for typhoid fever,

304
Wire baskets, 83

platinum, 33

silver, 218

Wolffhiigel plate, 141
Wounds, infected, 220

infection of, 167, 238

irrigation of, 220
Wool-sorters' disease, 135, 276
Wright's stain for blood, 55

method for anaerobes, 92
Wurtz's culture-medium, 303
Wurzcl bacillus, 225

X-RAYS, 127
Xerosis bacillus, 283
Xylol, 49, 52

YEASTS, 231, 114, 135, 155
Yellow fever, 335, 160, 161,

165, 204, 207
Yellow tubercle, 292
Yersin's serum for plague, 267

ZIEHL'S carbol-fuchsin, 45
Zinc chloride, 208

sulphate, 208
Zoogloea, 122

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Disease, Official and Practical Pharmacy, and Minute Direc-
tions for Prescription Writing, etc. Including over 650
Prescriptions and Formulae. By SAMUEL O. L. POTTER,
M.A., M.D., M.R.C.P. (Lond.), formerly Professor of the
Principles and Practice of Medicine, Cooper Medical Col-
lege, San Francisco ; Major and Brigade Surgeon, U. S.
Vol. Ninth Edition, Revised and Enlarged. 8vo.
With Thumb Index in each copy.

Cloth, $5.00 ; Leather, $6.00

*^.*This is the most complete and trustworthy book
for the use of students and physicians. Students who pur-
chase it will find it to contain a vast deal of information not
in the usual text-books arranged in the-most practical man-
ner for facilitating study and reference. It cannot be sur-
passed as a physician's working book.

WHITE AND WILCOX. Materia Medica,
Pharmacy, Pharmacology, and Thera-
peutics* Fifth Edition.

A Handbook for Students. By W. HALE WHITE, M.D.,
F.R.C.P., etc., Physician to, and Lecturer on Materia
Medica and Therapeutics at, Guy's Hospital, etc. Fifth
American Edition, Revised by REYNOLD W. WILCOX,
M A , M.D. , LL.D. , Professor of Clinical Medicine and Thera-
peutics at the New York Post-Graduate Medical School and
Hospital ; Visiting Physician, St. Mark's Hospital ; Assist-
ant Visiting Physician, Bellevue Hospital. I2mo.

Cloth, ^3.00; Leather, $3. 50

SUBJECT INDEX.

Gould's Medical Dictionaries, - Pages 12, 13
Morris' Anatomy, New Edition, - - Page 4
Compends for Students, - Page 27

SUBJECT. PAOB

Alimentary Canal (see Sur-
gery) 24

Anatomy 7

Anesthetics 18, 19

Autopsies (see Pathology) 20

Bacteriology 8

Bandaging (see Surgery) . . 24

Blood, Examination of . . . 8

Brain 8

Chemistry. Physics 9

Children, Diseases of 11

Climatology 19

Clinical Charts 25

Compends 27

Consumption (see Lungs). 16

Cyclopedia of Medicine. . . 13

Dentistry 11

Diabetes (see Urin. Organs) 25

Diagnosis 11

Diagrams (see Anatomy) . 8

Dictionaries, Cyclopedias. 12

Diet and Food 13

Disinfection 16

Dissectors 7

Ear 14

Electricity 14

Embryology 7

Emergencies 24

Eye 14

Fevers 15

Food 13

Formularies 21

Gynecology 15

Hay Fever 25

Heart 15

Histology 15

Hydrotherapy 19

Hygiene 16

Hypnotism 8

Insanity 8

Intestines 23

Latin, Medical (see Phar-
macy) 21

Life Insurance 19

Lungs 16

Massage 17

Materia Medica 17

Mechanotherapy 17

Medical Jurisprudence. ... 18

SUBJECT. PAOK

Mental Therapeutics 8

Microscopy 18

Milk 8,10

Miscellaneous 18

Nervous Diseases 19

Nose 25

Nursing 20

Obstetrics 20

Ophthalmology 14

Organotherapy 18

Osteology (see Anatomy). 7

Pathology 20

Pharmacy 21

Physical Diagnosis 11

Physical Training 17

Physiology 22

Pneumotherapy 19

Poisons (see Toxicology) . . 18

Practice of Medicine 22

Prescription Books (Phar-
macy) 21

Refraction (see Eye) 14

Rest 19

Sanitary Science 16

Serum-Therapy 17

Skin 23

Spectacles (see Eye) 14

Spine (see Nervous Dis-
eases) 19

Stomach 23

Students' Compends 27

Surgery and Surgical Dis-
eases 24

Technological Books 9

Temperature Charts 25

Therapeutics 17

Throat 25

Toxicology 18

Tumors (see Surgery) .... 24

U. S. Pharmacopoeia 22

Urinary Organs 25

Urine 25

Venereal Diseases 26

Veterinary Medicine. ..... 26

Visiting Lists, Physicians'.
(Send Jar Special Circu-
lar.)

Water Analysis 16

Women, Diseases of 15

Self-Examination tor Medical Students. 3500 Questions on
Medical Subjects, with References to Standard Works in which
the correct replies will be found. Together with Questions
from State Examining Boards. 3d Ed. Paper Cover, 10 oi».

SUBJECT CATALOGUE OF MEDICAL BOOKS. 7

SPECIAL NOTE. — The prices given in this catalogue are
net ; no discount can be allowed retail purchasers under any con-
sideration. This rule has been established in order that everyone
will be treated alike, a general reduction in former prices having
been made to meet previous retail discounts. Upon receipt of
the advertised price any book will be forwarded by mail or
express, all charges prepaid.

ANATOMY. EMBRYOLOGY.

MORRIS. Text-Book of Anatomy. Third Revised and Enlarged
Edition. 846 Illustrations, 267 of which are printed in colors.
Thumb Index in Each Copy. Cloth, $6.00 ; Leather, $7.00

"The ever-growing popularity of the book with teachers and
students is an index of its value." — Medical Record, New York.

BROOMELL. Anatomy and Histology of the Human Mouth
and Teeth. 2d Edition, Enlarged. 337 Illus. Cloth, $4.50

DAVISSON. Mammalian Anatomy. With Special Reference
to the Cat. 110 Illustrations. In Press.

DEAVER. Surgical Anatomy. A Treatise on Anatomy in its
Application to Medicine and Surgery. With 499 very hand-
some full-page Illustrations Engraved from Original Drawings
made by special Artists from dissections prepared for the pur-
pose. Three vols. By Subscription only.

Half Morocco or Sheep, $24.00; Half Russia, $27.00

GORDINIER. Anatomy of the Central Nervous System. With
271 Illustrations, many of which are original. Cloth, $6.00
HEATH. Practical Anatomy. 9th Edition. 321 Illus. $4.25
HOLDEN. Anatomy. A Manual of Dissections. Revised by A.
HEWSON, M.D., Demonstrator of Anatomy, Jefferson Medical
College, Philadelphia. 320 handsome Illustrations. 7th Ed.
In two compact 12mo volumes. 850 pages. Large New Type.
Vol. I. Scalp— Face— Orbit— Neck— Throat— Thorax— Up-
per Extremity. $1.50
Vol. II. Abdomen — Perineum — Lower Extremity — Brain —
Eye — Ear — Mammary Gland — Scrotum — Testes.

$1.60

HOLDEN. Human Osteology. Comprising a Description of the
Bones, with Colored Delineations of the Attachments of the
Muscles. The General and Microscopical Structure of Bone
and its Development. With Lithographic Plates and numer-
ous Illustrations. 8th Edition. $5.25
HOLDEN. Landmarks, Medical and Surgical. 4th Ed. .75

HUGHES AND KEITH. Dissections. With 527 Colored Plates
and other Illustrations. In three parts.

I, Upper and Lower Extremity. $3.00

II, Abdomen— Thorax. $3.00

III, Head— Neck— Central Nervous System. $3.00

LAZARUS-BARLOW. Pathological Anatomy. 21 Plates and

171 other Illustrations. Just Ready. $6.50

McMURRICH. Embryology. The Development of the Human

Body. 276 Illustrations. $3.00

8 SUBJECT CATALOGUE.

MARSHALL. Physiological Diagrams. Eleven Life-Size
Colored Diagrams (each seven feet by three feet seven inches).
Designed for Demonstration before the Class.

In Sheets, Unmounted, $40.00; Backed with Muslin and
Mounted on Rollers, $60.00; Ditto, Spring Rollers, in hand-
some Walnut Wall Map Case, $100.00; Single Plates— Sheets,
$5.00; Mounted, $7.50. Explanatory Key, .50. Purchaser
mutt pay freight charge*.

MINOT. Laboratory Text-Book of Embryology. 218 Illustra-
tions. Just Ready, $4.50

POTTER. Compend of Anatomy, Including Visceral Anatomy.
7th Edition, Revised and Enlarged. 16 Plates and 138 other
Illustrations. Just Ready. .80; Interleaved, $1.00

WILSON. Anatomy, llth Edition. 429 Illus., 26 Plates. $5.00

YUTZY. Guide to the Dissection of the Human Body. Based
on Morris' Anatomy. Paper Cover, .25

BACTERIOLOGY.

CONN. Agricultural Bacteriology. Including the Study of
Bacteria as relating to Agriculture, Soil, Dairy and Food
Products, Sewage, Domestic Animals, etc. Illustrated. $2.50

CONN. Bacteria in Milk and Its Products. Designed for
Students of Dairying, Boards of Health, Bacteriologists, etc.
Illustrated. $1.25

EMERY. Bacteriological Diagnosis. 2 Colored Plates and 32
other Illustrations. $1.50

HEWLETT. Manual of Bacteriology. 75 Illustrations. Second
Edition, Revised and Enlarged. $4.00

SMITH. Laboratory Exercises in Bacteriology. A Handbook
for Students. Illustrated. $1.50

WILLIAMS. Bacteriology. A Manual for Students. 88 Illus-
trations. 3d Edition. Revised. Just Ready. $1.50

BLOOD, Examination of.

DA COSTA. Clinical Hematology. A Practical Guide to the
Examination of the Blood, with Reference to Diagnosis. Six
Colored Plates and 48 other Illus. Cloth. $5.00 ; Sheep, $6.00

BRAIN AND INSANITY (see also
Nervous Diseases.)

BLACKBURN. A Manual of Autopsies. Designed for the Use

of Hospitals for the Insane and other Public Institutions. Ten

full-page Plates and other Illustrations. $1.25

CHASE. General Paresis. Illustrated. $1.75

DERCUM. Mental Therapeutics, Rest, Suggestion. Set Cohen,

Physiologic Therapeutic*, page 17.

GORDINIER. The Gross and Minute Anatomy of the Central

Nervous System. With full -page and other Illus. $6.00

IRELAND. The Mental Affections of Children. 2d Ed. $4.00

LEWIS (SEVAN). Mental Diseases. A Text-Book having

Special Reference to the Pathological Aspects of Insanity. 26

Lithographic Plates and other Illustrations. 2d Ed. $7.00

MEDICAL BOOKS.

MANN. Manual of Psychological Medicine. $3.00

PERSHING. Diagnosis of Nervous and Mental Disease. Illus-
trated. $1-25
REGIS. Mental Medicine. Authorized Translation by H. M.
BANNISTER. M.D. $2.00

STEARNS. Mental Diseases. With a Digest of Laws Relating
to Care of Insane. Illustrated. Cloth, $2.75; Sheep, $3.25

TUKE. Dictionary of Psychological Medicine. Giving the
Definition, Etymology, and Symptoms of the Terms used in
Medical Psychology, with the Symptom?, Pathology, and
Treatment of the Recognized Forms of Mental Disorders.
Two volumes. $10.00

WOOD, H. C. Brain and Overwork. .40

CHEMISTRY AND TECHNOLOGY.

Special Catalogue of Chemical Books tent free upon application.

ALLEN. Commercial Organic Analysis. A Treatise on the
Modes of Assaying the Various Organic Chemicals and Prod-
ucts Employed in the Arts, Manufactures, Medicine, etc.,
with Concise Methods for the Detection of Impurities, Adul-
terations, etc. 8vo.

Vol. I. Alcohols, Neutral Alcoholic Derivatives, etc., Ethers,
Vegetable Acids, Starch, Sugars, etc. 3d Edition. $4.50
Vol. II, Part I. Fixed Oils and Fats, Glycerol, Explosives,
etc. 3d Edition. $3.50

Vol. II, Part II. Hydrocarbons, Mineral Oils, Lubricants,
Benzenes, Naphthalenes and Derivatives, Creosote, Phenols,
etc. 3d Edition. $3.50

Vol. II, Part III. Terpenes, Essential Oils, Resins, Camphors,
etc. 3d Edition. Preparing.

Vol. Ill, Part I. Tannins, Dyes, and Coloring Matters. 3d
Edition, Enlarged and Rewritten. Illustrated. $4.50
Vol. Ill, Part II. The Amines, Hydrazines and Derivatives,
Pyridine Bases. The Antipyretics, etc. Vegetable Alka-
loids, Tea, Coffee, Cocoa, etc. 8vo. 2d Edition. $4.50
Vol. Ill, Part III. Vegetable Alkaloids, Non-Basic Vegetable
Bitter Principles. Animal Bases, Animal Acids, Cyanogen
Compounds, etc. 2d Edition, 8vo. $4.50

Vol. IV. The Proteids and Albuminou* Principles. 2d
Edition. $4.50

BAILEY AND CADY. Qualitative Chemical Analysis. $1.25
BARTLEY. Medical and Pharmaceutical Chemistry. A Text-
Book for Medical, Dental, and Pharmaceutical Students. With
Illustrations, Glossary, and Complete Index. 5th Ed. $3.00

BARTLEY. Clinical Chemistry. The Examination of Feces,
Saliva, Gastric Juice, Milk, and Urine. $1.00

BLOXAM. Chemistry, Inorganic and Organic. With Experi-
ments. 9th Ed., Revised. 284 Engravings. $6.00

BUNGE. Physiologic and Pathologic Chemistry. From the
Fourth German Enlarged Edition. $3.00

CALDWELL. Elements of Qualitative and Quantitative Chem-
ical Analysis. 3d Edition, Revised. $1.00

10 SUBJECT CATALOGUE.

CAMERON. Soap and Candles. 54 Illustrations. $2.00

CLOWES AND COLEMAN. Quantitative Analysis. 6th Edi-
tion. 125 Illustrations. $3.50

COBLENTZ. Volumetric Analysis. Illustrated. $1.25

CONGDON. Laboratory Instructions in Chemistry. With
Numerous Tables and 56 Illustrations. $1.00

GARDNER. The Brewer, Distiller, and Wine Manufacturer.
Illustrated. $1.50

GRAY. Physics. Volume I. Dynamics and Properties of
Matter. 350 Illustrations. $4.50

GROVES AND THORP. Chemical Technology. The Applica-
tion of Chemistry to the Arts and Manufactures.
Vol. I. Fuel and its Applications. 607 Illustrations and 4
Plates. Cloth, $5.00; * Mor., $6.50

Vol.11. Lighting. Illustrated. Cloth, $4.00; i Mor., $5.50
Vol. III. Gas Lighting. Cloth, $3.50; * Mor., $4.50

Vol. IV. Electric Lighting. Photometry.

Cloth, $3.50; * Mor., $4.50

HEUSLER. The Chemistry of the Terpenes. $4.00

HOLLAND. The Urine, the Gastric Contents, the Common
Poisons, and the Milk. Memoranda, Chemical and Micro-
scopical, for Laboratory Use. 6th Ed. Illustrated. $1.00

LEFFMANN. Compend of Medical Chemistry, Inorganic and
Organic. 4th Edition, Revised. .80; Interleaved, $1.00

LEFFMANN. Analysis of Milk and Milk Products. 2d Edition,
Enlarged. Illustrated. $1.25

LEFFMANN. Water Analysis. For Sanitary and Technic Pur-
poses. Illustrated. 4th Edition. $1.25

LEFFMANN. Structural Formulae. Including 180 Structural
and Stereo-Chemical Formuhe. 12mo. Interleaved. $1.00

LEFFMANN AND BEAM. Select Methods in Food Analysis.
Illustrated. $2.50

MUTER. Practical and Analytical Chemistry. 3d American
from the Ninth English Edition. Revised to meet the re-
quirements of American Students. 56 Illustrations. $1.25

OETTEL. Exercises in Electro-Chemistry. Illustrated. .75

OETTEL. Electro-Chemical Experiments. Illustrated. .75

RICHTER. Inorganic Chemistry. 5th American from 10th
German Edition. Authorized translation by EDGAR F. SMITH,
M.A , PH.D. 89 Illustrations and a Colored" Plate. $1.75

RICHTER. Organic Chemistry. 3d American Edition, trans-
lated from the 8th German by EDQAR F. SMITH, lllus. 2 vols.
Vol. 1. Aliphatic Series. 625 pages. $3.00

Vol. II. Carbocyclic Series. 671 pages. $3.00

ROCKWOOD. Chemical Analysis for Students of Medicine,
Dentistry, and Pharmacy. Illustrated. $1.50

SMITH. Electro-Chemical Analysis. 3d Ed. 39 lllus. $1.50

SMITH AND KELLER. Experiments. Arranged for Students
in General Chemistry. 4th Edition. Illustrated. .60

SUTTON. Volumetric Analysis. A Systematic Handbook for
the Quantitative Estimation of Chemical Substances by
Measure, Applied to Liquids, Solids, and Gases. 8th Edition,
Revised. 112 Illustrations. $5.00

SYMONDS. Manual of Chemistry. 2d Edition. $2.00

TRAUBE. Physico-Chemical Methods. 97 Illustration*. $1.50

MEDICAL BOOKS. 11

THRESH. WaWr and Water Supplies. 3d Edition. $2.00

ULZER AND FRAENKEL. Chemical Technical Analysis.

Translated by Fleck. Illustrated. $1.25

WOODY. Essentials of Chemistry and Urinalysis. 4th Edition.

Illustrated. $1.50

*** Special Catalogue of Books on Chemistry free upon application.

CHILDREN.

HATFIELD. Compend of Diseases of Children. With a
Colored Plate. 3d Ed. Just Ready. .80; Interleaved, $1.00
IRELAND. The Mental Affections of Children. Idiocy, Im-
becility, Insanity, etc. 2d Edition. $4.00
POWER. Surgical Diseases of Children and their Treatment
by Modern Methods. Illustrated. $2.50
STARR. The Digestive Organs in Childhood. The Diseases of
the Digestive Organs in Infancy and Childhood. 3d Edition,
Rewritten and Enlarged. Illustrated. $3.00
STARR. Hygiene of the Nursery. Including the General Regi-
men and Feeding of Infants and Children, and the Domestic
Management of the Ordinary Emergencies of Early Life,
Massage, etc. 6th Edition. 25 Illustrations. $1.00
SMITH. Wasting Diseases of Children. 6th Edition. $2.00
TAYLOR AND WELLS. The Diseases of Children. 2d Edition,
Revised and Enlarged. Illustrated. 8vo. $4.50
"It is well worthy the careful study of both student and prac-
titioner, and can not fail to prove of great value to both. We
do not hesitate to recommend it." — Boston Medical and Surgical
Journal.

DIAGNOSIS.

BROWN. Medical Diagnosis. A Manual of Clinical Methods.
4th Edition. 112 Illustrations. Cloth, $2.25

DA COSTA. Clinical Hematology. A Practical Guide to Exam-
ination of Blood, with Reference to Diagnosis. 6 Colored
Plates, 48 other Illustrations. Cloth, $5.00 ; Sheep, $6.00

DOUGLAS. Surgical Diseases of Abdomen, with Reference to
Diagnosis. 20 Full-Page Plates. Just Ready.

Cloth, $7.00 ; Sheep, $8.00

EMERY. Bacteriological Diagnosis. 2 Colored Plates and 32
other Illustrations. $1.50

MEMMINGER. Diagnosis by the Urine. 2d Ed. 24 Illus. $1.00

PERSHING. Diagnosis of Nervous and Mental Diseases.
Illustrated. $1.25

STEELL. Physical Signs of Pulmonary Disease. $1.25

TYSON. Handbook of Physical Diagnosis. For Students and
Physicians. By the Professor of Clinical Medicine in the Uni-
versity of Pennsylvania. Illus. 4th Ed., Improved and En-
larged. With 2 Colored and 55 other Illustrations. $1.50

DENTISTRY.

Special Catalogue of Dental Books tent free upon application.
BARRETT. Dental Surgery for General Practitioners and
Students of Medicine and Dentistry. Extraction of Teeth,
ete. 3d Edition. Illustrated. $1.00

12 SUBJECT CATALOGUE.

BROOMELL. Anatomy and Histology of the Human Mouth
and Teeth. Second Edition, Revised and Enlarged. 337
handsome Illustrations. Cloth, $4.50; Leather, $5.50

FILLEBROWN. Operative Dentistry. Illustrated. $2.25

GORGAS. Dental Medicine. A Manual of Materia Medica and
Therapeutics. 7th Edition. Cloth, $4.00; Sheep,$5.00

GORGAS. Questions and Answers for the Dental Student.
Embracing all the subjects in the Curriculum of the Dental
Student. Octavo. $6.00

HARRIS. Principles and Practice of Dentistry. Including
Anatomy, Physiology, Pathology, Therapeutics, Dental Sur-
gery, and Mechanism. 13th Edition. Revised by F. J. S.
GOROAS, M.D., D.D.S. 1250 Illus. Cloth, $6.00 ; Leather, $7.00

HARRIS. Dictionary of Dentistry. Including Definitions of Such
Words and Phrases of the Collateral Sciences as Pertain to the
Art and Practice of Dentistry. 6th Edition, Revised and
Enlarged by FERDINAND J. S. GOROAS, M.D., D.D.S.

Cloth, $5.00 ; Leather, $6.00

RICHARDSON. Mechanical Dentistry. 7th Edition. Thor-
oughly Revised and Enlarged by DR. GEO. W. WARREN. 691
Illustrations. Cloth, $5.00; Leather, $6.00

SMITH. Dental Metallurgy. 2d Edition. Illustrated. $2.00

TAFT. Index of Dental Periodical Literature. $2.00

TOMES. Dental Anatomy. 263 Illustrations. 5th Ed. $4.00

TOMES. Dental Surgery. 4th Edition. 289 Illus. $4.00

WARREN. Compend of Dental Pathology and Dental Medicine.
With a Chapter on Emergencies. 3d Edition. Illustrated.

.80; Interleaved, $1.00

WARREN. Dental Prosthesis and Metallurgy. 129 Illus. $1.25
WHITE. The Mouth and Teeth. Illustrated. .40

DICTIONARIES. CYCLOPEDIAS.

GOULD. The Illustrated Dictionary of Medicine, Biology, and
Allied Sciences. Being an Exhaustive Lexicon of Medicine and
those Sciences Collateral to it: Biology (Zoology and Botany),
Chemistry, Dentistry, Pharmacology, Microscopy, etc., with
many useful Tables and numerous fine Illustrations. 1633
pages. Fifth Edition.

Sheep or Half Morocco, $10.00; with Thumb Index, $11.00
Half Russia, Thumb Index, $12.00

GOULD. The Medical Student's Dictionary, nth Edition. Il-
lustrated. Including those Words and Phrases generally used
in Medicine, with their Proper Pronunciation and Definition,
Based on Recent Medical Literature. With Table of Epq-
nymic Terms and Tests and Tables of the Bacilli, Micrococci,
Mineral Springs, etc., of the Arteries, Muscles, Nerves, Ganglia,
Plexuses, etc. Eleventh Edition. Enlarged and illustrated
with a large number of Engravings. 840 pages.

Half Morocco, $2.50: with Thumb Index, $3.00
Flexible Leather, Burnished Edges, Thumb Index, 3.50

MEDICAL BOOKS. 13

GOULD. The Pocket Pronouncing Medical Lexicon. 4th Edi-
tion. (30,000 Medical Words Pronounced and Denned.) Con-
taining all the Words, their Definition and Pronunciation,
that the Medical, Dental, or Pharmaceutical Student Gener-
ally Cornea in Contact with; also Elaborate Tables of Epo-
nymic Terms, Arteries, Muscles, Nerves, Bacilli, etc., etc., a
Dose List in both English and Metric Systems, etc., Arranged
in a Most Convenient Form for Reference and Memorizing.
Fourth Edition, Revised and Enlarged. 838 pages.
Full Limp Leather, Gilt Edges, $1.00; Thumb Index, $1.25
145,000 Copies of Gould's Dictionaries have been sold.

GOULD AND PYLE. Cyclopedia of Practical Medicine and
Surgery. Seventy-two Special Contributors. Illustrated. One
Volume. A Concise Reference Handbook of Medicine, Sur-
gery, Obstetrics, Materia Medica, Therapeutics, and the Vari-
ous Specialties, with Particular Reference to Diagnosis and
Treatment. Compiled under the Editorial Supervision of
GEORGE M. GOULD, M.D., Author of "An Illustrated Dictionary
of Medicine," etc.; and WALTER L. PTLB, M.D., Assistant
Surgeon Wills Eye Hospital; formerly Editor "International
Medical Magazine," etc., and Seventy-two Special Contribu-
tors. With many Illustrations. Large Square 8vo, to corre-
spond with Gould's "Illustrated Dictionary."
Full Sheep or Half Mor., $10.00; with Thumb Index, $11.00
Half Russia, Thumb Index, $12.00 net.

GOULD AND PYLE. Pocket Cyclopedia of Medicine and Sur-
gery. Based upon above book and uniform in size with
"Gould's Pocket Dictionary."

Full Limp Leather, Gilt Edges, $1.00
With Thumb Index, $1.25

HARRIS. Dictionary of Dentistry. Including Definitions of
Such Words and Phrases of the Collateral Sciences as Pertain
to the Art and Practice of Dentistry. 6th Edition, Revised
and Enlarged by FERDINAND J. S. GORGAS, M.D., D.D.S.

Cloth, $5.00; Leather, $6.00

LONGLEY. Pocket Medical Dictionary. Cloth, .75

TREVES AND LANG. German-English Medical Dictionary.

Half Calf, $3.25

DIET AND FOOD.

ALLEN. Proteids and Albuminous Principles. An analytical
Study of Food Products. 2d Edition. $4.50

BURNETT. Foods and Dietaries. A Manual of Clinical Diet-
etics, with Diet Lists for Various Diseases, etc. 2dEd. $1.50

DAVIS. Dietptherapy. Food in Health and Disease. With
Tables of Dietaries, Relative Value of Foods, etc. Set Cohen,
Physiologic Therapeutics, page 17.

GREENISH. Microscopical Examination of Foods and Drugs.
Illustrated. Just Ready. $3.50

HAIG. Diet and Food. Considered in Relation to Strength and
Power of Endurance. 4th Edition. $1.00

LEFFMANN. Select Methods in Food Analysis. Illua. $2.50

14 SUBJECT CATALOGUE.

EAR (see also Throat and Nose).

BURNETT. Hearing and How to Keep It. Illustrated. .40

HOVELL. Diseases of the Ear and Naso-Pharynx. Including

Anatomy and Physiology of the Organ, together with the

Treatment of the Affections of the Nose and Pharynx which

Conduce to Aural Disease. 128 Illustrations. 2d Ed. $5.50

PRITCHARD. Diseases of the Ear. 4th Edition, Enlarged.

Many Illustrations arid Formulae. In Press.

ELECTRICITY.

BIGELOW. Plain Talks on Medical Electricity and Batteries.

With a Therapeutic Index and a Glossary. 43 Illustrations.

2d Edition. $1.00

HEDLEY. Therapeutic Electricity and Practical Muscle Testing.

99 Illustrations. $2.50

JACOB Y. Electrotherapy. 2 volumes. Illustrated. See Cohen,

Physiologic Therapeutics, page 17.
JONES. Medical Electricity. 3d Edition. 117 Illus. $3.00

EYE.

A Special Circular of Books on the Eye sent free upon application.

DARIER. Ocular Therapeutics. Just Ready. $3.00

DONDERS. The Nature and Consequences of Anomalies of
Refraction. With Portrait and lllua. Half Morocco, $1.25

PICK. Diseases of the Eye and Ophthalmoscopy. Translated
by A. B. HALE, M.D. 157 llius. Cloth, $4.50; Sheep, $5.50

GOULD AND PYLE. Compend of Diseases of the Eye and Re-
fraction. Including Treatment and Operations, and a Section
on Local Therapeutics. With Formulae, Useful Tables, a
Glossary, and 111 Illus., several of which are in colors. 2d
Edition, Revised. Cloth, .80; Interleaved, $1.00

GREEFF. The Microscopic Examination of the Eye. Illus-
trated. $1.25

HARLAN. Eyesight, and How to Care for It. Illus. .40

HARTRIDGE. On the Ophthalmoscope. 4th Edition. With
4 Colored Plates and 68 Wood-cuts. $1.50

HARTRIDGE. Refraction. 104 Illustrations and Test Types.
12th Edition, Enlarged. $1.50

HANSELL AND SWEET. Treatise on Diseases of the Eye.
With many Illus. drawn by special artists, etc. In Press.

HANSELL AND REBER. Muscular Anomalies of the Eye.
Illustrated. $1.50

HANSELL AND BELL. Clinical Ophthalmology. Colored
Plate of Normal Fundus and 120 Illustrations. $1.50

HENDERSON. Notes on the Eye. 3d Ed. Just Ready. $1.50

JENNINGS. Manual of Ophthaimoscopy. 95 Illustrations and
1 Colored Plate. $1.50

MORTON. Refraction of the Eye. Its Diagnosis and the Cor-
rection of its Errors. 6th Edition. $1.00

OHLEMANN. Ocular Therapeutics. Authorized Translation,
and Edited by DR. CHARLEB A. OLIVER. $1.75

PARSONS. Elementary Ophthalmic Optics. With Diagram-
matic Illustrations. $2.00

MEDICAL BOOKS. 15

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MEDICAL BOOKS. 21

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MEDICAL BOOKS. 28

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DA COSTA

Clinical Hematology

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*.£* A new, thorough, systematic, and comprehensive
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OERTEL

Medical Microscopy

JUST READY

A GUIDE TO DIAGNOSIS, ELEMEN-
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AND MICROSCOPIC TECHNIC

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Professor of Pathology and Clinical Microscopy, Medical Depart-
ment, University of Georgia.

WITH 131 ILLUSTRATIONS. 121110. Cloth, $2.00

The Pocket Cyclopedia, of
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Uniform ivith "Gould's Pocket Dictionary"

A concise practical volume of nearly 600
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By Drs. Gould and Pyle

Based upon their large "Cyclopedia of
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*^* This is a new book which will prove of the greatest
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in shape for quick reference. In no other book can be
found so much exact detailed knowledge so conveniently
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It is Multum in Parvo. Sample Pages Free.
29

A NEW EDITION

CROCKER ON THE SKIN

The Diseases of the Skin. Their Description, Pathology,
Diagnosis, and Treatment, with Special Reference to the
Skin Eruptions of Children. By H. RADCLIFFE CROCKER,
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versity College Hospital, London. With new Illustrations.

Third Edition, Rewritten and Enlarged

OCTAVO. JUST READY; CLOTH, $5.00

*#* This new edition will easily hold the high position
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logical Societies, and a recognized authority the world over.

STURGIS— MANUAL OF
VENEREAL DISEASES

By F. R. STURGIS, M.D., Sometime Clinical Professor of
Venereal Diseases in the Medical Department of the Uni-
versity of the City of New York. Seventh Edition, Revised
and in Part Rewritten by the Author and FOLLEN CABOT,
M.D., Instructor in Genito-Urinary and Venereal Diseases
in the Cornell University Medical College. I2mo. 216
pages. Cloth, $1.25

*x* This manual was originally written for students'
use, and is as concise and as practical as possible. It pre-
sents a careful, condensed description of the commoner
forms of venereal diseases which occur in the practice of
the general physician, together with the most approved
remedies.

FOR THE^ DISSECTING ROOM

Holden's Anatomy — Seventh Edition
320 Illustrations

A Manual of the Dissections of the Human Body. By JOHN
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Demonstrator of Anatomy, Jefferson Medical College, Phila-
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Oil Cloth, $1.50

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445 Pages. 167 Illustrations.

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Hughes a.i\d Keith — Dissections
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A Manual of Dissections by ALFRED W. HUGHES, M.B.,
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KEITH, M.D., Joint Lecturer on Anatomy, London Hospital
Medical College, etc. In three parts. With 527 Colored
and other Illustrations.

I. Upper and Lower Extremity. 38 Plates, 116 other
Illustrations. Cloth, $3.00

II. Abdomen. Thorax. 4 Plates, 149 other Illus-
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Each volume sold separately.

*.£* The student will find it of great advantage to have
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anatomy. These books meet all requirements, and as they
can be purchased in parts as wanted, the outlay is small.
31

EDGAR'S

OBSTETRICS

A NEW TEXT -BOOK
I 22 i Illustrations

Edgar's Obstetrics excels all
other works on this subject
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formity and consistency in
arrangement, thoroughness
and clearness in handling
details, and in the number
and usefulness of its illus-
trations.

OCTAVO. CM.oni, 56.oo; SIIKKP, <7.oo

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