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ESSENTIALS
OF
BACTERIOLOGY
Since the issue of the first volume of the
Saunders Question=Compends,
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of these unrivalled publications have been sold.
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SAUNDERS' QUESTION-COMPENDS, No. 20
ESSENTIALS
BACTERIOLOGY
CONCISE AND SYSTEMATIC INTRODUCTION
TO THE STUDY OF MICRO-ORGANISMS
/?
vCbv
M. V. BALL, M.D.
Formerly Resident Physician, German Hospital, Philadelphia ;
formerly Bacteriologist to St. Agnes' Hospital
FIFTH EDITION, THOROUGHLY REVISED
BY
KARL M, VOGEL, M.D.
Assistant in Pathology, College of Physicians and Surgeons,
Columbia University, New York City
With Ninety-Six Illustrations
some in Colors, and Six Plates
PHILADELPHIA AND LONDON
W. B. SAUNDERS & COMPANY
1905
Set up, electrotyped, printed, and copyrighted October, 1891. Reprinted October,
1892. Revised, reprinted, and recopyrighted May, 1893. Reprinted June,
1894. Revised, reprinted, and recopyrighted November, 1896. Reprinted
October, 1898. Revised, reprinted, and recopyrighted March,
1900. Reprinted May, 1903. Revised, reprinted,
and recopyrighted August, 1904.
Copyright, 1904, by W. B. Saunders & Company.
Reprinted October, 1905.
ELECTROTYPED BY
WESTCOTT & THOMSON, PHILAOA.
PRESS OF
W. B. SAUNDERS & COMPANY.
1165-
PREFACE TO THE FIFTH EDITION
The progress in bacteriology during the last few years
has involved more or less radical changes in many depart-
ments of the science. Recent work on such subjects as
immunity, tuberculosis, dysentery, yellow fever, the bu-
bonic plague, and other infectious diseases has rendered
obsolete many portions of any but the most modern books,
and in countless minor details the teaching of to-day differs
from that of even a few years ago.
In this revision the attempt has been made to reflect as
faithfully as possible the present status of bacteriology
without overstepping the limits set by the scope of a book
intended primarily as *an aid to students. Much assist-
ance has been derived from the " Manual of Bacteriol-
ogy" of Muir and Ritchie, and from F. C. Wood's
"Laboratory Guide to Clinical Pathology."
7
5506r()
PKEFAOE TO FIRST EDITION.
Feeling the need of a Compendium on the subject of
this work, it has been our aim to produce a concise treatise
upon the Practical Bacteriology of to-day, chiefly for the
medical student, which he may use in his laboratory.
It is the result of experience gained in the Laboratory
of the Hygienical Institute, Berlin, under the guidance of
Koch and Frankel ; and of information gathered from the
original works of other German, as well as of French,
bacteriologists.
Theory and obsolete methods have been slightly touched
upon. The scope of the work, and want of space, forbade
adequate consideration of them. The exact measurements
of bacteria have not been given. The same bacterium
varies often much in size, owing to differences in the media,
staining, etc.
We have received special help from the following books,
which we recommend to students for further reference : —
Mac6: Trait6 pratique de Bacteriologie.
Frankel: Grundriss der Bakterienkunde.
Eisenberg: Bakteriologische Diagnostik.
Crookschank, E. M.: Manual of Bacteriology.
Gunther: Einflihring in das Studium der Bacteriologie, etc.
Woodhead and Hare: Pathological Mycology.
Salmonsen: Bacteriological Technique (English translation).
M. V. BALL.
Buffalo, N. Y., October 1, 1891.
62 Delaware Avenue.
CONTENTS
PART I.
GENERAL CONSIDERATIONS AND TECHNIQUE.
PAGE
Introduction 15
Chapter I.— Classification, Structure, and Reproduction. 17
II.— Origin, Life, Growth, and Properties .... 23
" III.— Methods of Examination 26
" IV.— Staining of Bacteria 30
V— General Method of Staining Specimens ... 35
" VI.— Special Methods of Staining 39
" VII.— Methods of Culture 42
" VIIL— Nutrient Media 48
M IX.- -Solid Transparent Media 52
" X.— Inoculation of Gelatine and Agar 60
" XL— Growth and Appearances of Colonies ... 64
" XII. — Cultivation of Anaerobic Bacteria 67
" XIII.— Infection 70
" XIV.— Immunity 72
" XV.— Animal Experiments 76
PART II.
SPECIAL BACTERIOLOGY.
Chapter I.— Non-Pathogenic Bacteria 80
Bacillus Prodigiosus 80
Indicus 81
9
10 CONTENTS.
PAGE
Bacillus —
Mesentericus Vulgatus 81
Megateriuni 82
Ramosus 82
Bacterium Zopfi 83
Bacillus Subtilia 83
Spinosus 84.
Some Bacteria in Milk 84
Bacillus Acidi Lactici . . . 84
Butyricus 85
Amylobacter 85
Lactis Cyanogenus 86
Lactis Erythrogenes 86
Examination of Milk in Stained Specimens 87
Some Non-Pathogenic Bacteria of Water 87
Bacillus Violaceus * 87
Cceruleus 88
* Fluorescent Bacteria 88
Phosphorescent Bacteria 89
Leptothrix, Crenothrix, Cladothrix, and
Beggiatoa 90
Micro-organisms found in Urine 91
Spirillum 92
Rubrum; Concentricum 92
Sarcina 92
Lutea 92
Aurantica Flava, Rosea, and Alba 93
Ventriculi , 93
s-Oppler Bacillus 93
Chapter II. — Pathogenic Bacteria • . 94
Bacteria Pathogenic for Man and Other Animals ... 94
Bacillus Anthracis 94
• Tuberculosis 97
Lepra Bacillus 107
Syphilis Bacillus 108
Bacillus of Glanders 108
CONTENTS. 11
PAGE
Bacillus —
of Diphtheria HO
pseudo- 114
of Typhoid Fever 115
Paracolon or Paratyphoid . . . 121
Psittacosis 121
Coli Communis 122
Chapter III. — Pathogenic Bacteria — continued . . . . . 124
Spirillum Cholera 124
Bacteria Similar to Spirillum Cholerae ........ 127
Finkler-Prior . T\ .'. . ... r ..*... . . 127
Tyrogenum 128
Vibrio Metschnikovi 129
Bacteria of Pneumonia 130
Pneumobacillus of Friedlander ' . . 131
ofFrankel 132
Antitoxin of Pneumonia 134
Bacillus of Rhinoscleroma 135
Diplococcus Intracellularis Meningitidis 135
Micrococcus Tetragenus 135
Capsule Bacillus 136
Bacillus of Influenza 137
Micro-organisms of Suppuration , 137
Streptococcus Pyogenes 138
Staphylococcus Pyogenes Aureus 140
, Pyogenes Albus 141
Micrococcus Pyogenes Citreus ......... 141
Cereus Albus 141
Cereus Flavus 141
Pyogenes Tenuis 142
Bacillus Pyocyaneus 142
Micrococcus Gonorrhoeae 143
Microbes Similar to Gonorrhoea 145
Bacillus of Tetanus 147
12 CONTENTS.
PAGE
Bacillus —
(Edematis Maligni ............. 150
Spirillum of Relapsing Fever 152
Bacillus of Soft Chancre 153
Icteroides ........ 154
of Bubonic Plague 155
# of Dysentery 157
Aerogenes Capsulatus 158
Micrococcus Melitensis 159
Pathogenic Protozoa . 159
Malarial Parasite 159
Amoeba Dysenterise 163
Small-pox and Vaccinia . 163
Trypanosomes 163
Chapter IV. — Bacteria Pathogenic for Animals, but
not for Man 164
Bacillus of Symptomatic Anthrax 164
of Chicken Cholera . . . 165
Bacteria of Hemorrhagic Septicaemia, Swine Plague,
Duck Cholera, etc 166
Bacillus of Erysipelas of Swine 167
Murisepticus 168
Micrococcus of Mai de Pis 169
Bacillus Alvei 169
Micrococcus Amylovorus 170
Bacterium Termo 170
Proteus Vulgaris 170
Mirabilis 171
Zenkeri 0 ....... . 171
APPENDIX.
Yeasts 173
Oidiums 174
Moulds 175
CONTENTS. 13
PAGE
Cladothrices and Strep tothrices 177
Streptothrix, or Cladothrix Actinomyces (Ray Fungus) . . 177
Madura . . 178
Farcinica 179
Examination of Air 180
of Water 184
of Soil 187
Bacteria of Milk and Other Foods 188
Examination of the Organs and Cavities of the Human
Body 189
Tables of Chief Characteristics of the Principal Bacteria . . 194
Part I. Non-Pathogenic . 194
Part II. Pathogenic 218
INTRODUCTION
History. — The microscope was invented about the latter
part of the sixteenth century ; and soon after, by its aid,
minute organisms were found in decomposing substances.
Kircher, in 1646, suggested that diseases might be due to
similar organisms ; but the means at his disposal were in-
sufficient to enable him to prove his theories. Anthony
Van Leuwenhoeck, of Delft, Holland (1680 to 1723), so
improved the instrument that he was enabled thereby to
discover micro-organisms in vegetable infusion, saliva, fecal
matter, and scrapings from the teeth. He distinguished
several varieties, showed them to have the power of loco-
motion, and compared them in size with various grains of
definite measurement. It was a great service that this
"Dutch naturalist" rendered the world; and he can rightly
be called the " father of microscopy."
Various theories were then formulated by physicians to
connect the origin of different diseases with bacteria; but no
proofs of the connection could be obtained. Andry, in 1701,
called bacteria worms. Miiller, of Copenhagen, in 1786, made
a classification composed of two main divisions — monas and
vibrio ; and with the aid of the compound microscope was
better able to describe them. Ehrenberg, in 1833, with still
better instruments, divided bacteria into four orders: bac-
terium, vibrio, spirillum, and spirochete. It was not until
1863 that any positive advance was made in connecting
bacteria with disease. Rayer and Davaine had in 1850
15-
16
INTRODUCTION
already found a rod-shaped bacterium in the blood of ani.
mals suffering from splenic fever {sang de rate), but they
attached no special significance to their discovery until
Pasteur made public his grand researches in regard to fer-
mentation and the role bacteria played in the economy.
Then Davaine resumed his studies, and in 1863 established
by experiments the bacterial nature of splenic fever or an-
thrax.
But the first complete study of a contagious affection was
made by Pasteur in 1869, in the diseases affecting silk-worms
— pebrine and flacherie — which he showed to be due to micro-
organisms.
Then Koch, in 1875, described more fully the anthrax
bacillus, gave a description of its spores and the properties
of the same, and was enabled to cultivate the germ on arti-
ficial media ; and, to complete the chain of evidence, Pas-
teur and his pupils supplied the last link by reproducing the
same disease in animals by artificial inoculation from pure
cultures. The study of the bacterial nature of anthrax has
been the basis of our knowledge of all contagious maladies,
and most advances have been made first with the bacterium
of that disease.
Since then bacteriology has grown to huge proportions —
become a science in itself — and thousands of earnest workers
are adding yearly solid blocks of fact to the structure, which
structure it will be our aim to briefly describe in the pages
which are to follow.
ESSENTIALS OF BACTERIOLOGY.
PART I.
GENERAL CONSIDERATIONS.
CHAPTER I.
BACTERIA.
The bacteria occupy the lowest plane of plant life known to
us, though they are by no means as primitive in their biology
as was formerly supposed, and it is quite possible that still
simpler forms may be discovered.
The numerous unicellular vegetable organisms which form
the lower limit of plant life as we know it multiply by fission
and are hence called the Schizophyta, or splitting plants. This
group is subdivided into two classes — (a) the Schizophycese, or
fission alga?, and (b) the Schizomycetes, or fission fungi, or bac-
teria, as we usually call them.
Lately it has Become customary to subdivide the bacteria
themselves somewhat arbitrarily into two classes — the lower
bacteria and the higher bacteria.
The lower bacteria are unicellular masses of protoplasm of
microscopic size, multiplying by fission and existing without
chlorophyll. Three main types are found : (1) Globular forms,
called cocci ; (2) straight rod-shaped forms, called bacilli ; (3)
curved or spiral rods, called spirilla.
The higher bacteria show a tendency toward a more com-
plicated mode of organization in two ways : (1) They consist of
filaments made up of separate individuals, but which exhibit
enough independence to foreshadow the rudiments of a physi-
2 (17)
18 ESSENTIALS OF BACTERIOLOGY.
ological division of labor. (2) Certain elements may be differ-
entiated for the purpose of reproduction.
The Staphylococcus pyogenes, the anthrax bacillus, and the
spirillum of relapsing fever are typical forms of the lower bac-
teria, while the actinomyces, or ray-fungus, is the most impor-
tant pathogenic member of the higher bacteria.
Fig. 1.
-V
Micrococcus. Spirillum. Bacillus.
Structure. Bacteria are cells ; they appear as round or cylin-
drical of an average diameter or transverse section of 0.001 mm.
(=1 micromillimeter), written 1 /*. The cell, as other plant-
cells, is composed of a membranous cell- wall and cell-contents ;
" cell-nuclei " can in some cases be seen by the use of special
stains.
Cell- Wall. The cell-wall is composed either of plant cellu-
lose, or a form of albumin, since it is less permeable than cellu-
lose membrane. The membrane is firm, and can be brought
plainly into view by the action of iodin upon the cell-contents,
which contracts them.
Cell-Contents. The contents of the cell consist mainly of
protoplasm, usually homogeneous, but in some varieties, finely
granular, or holding pigment, chlorophyll, fat-droplets, and sul-
phur in its structure.
It is composed chiefly of mycoprotein.
Gelatinous Membrane. The outer layer of the cell-membrane
can absorb water and become gelatinoid, forming either a little
envelope or capsule around the bacterium or preventing the
separation of the newly-branched germs, forming chains and
bunches, as strepto- and staphylo-cocci. Long filaments are also
formed.
Zooglcea. When this gelatinous membrane is very thick, irre-
BACTERIA
19
gular masses of bacteria will be tunned, the whole growth being
in one jelly-like lump. This is termed a zooglcea {$<jwv, animal,
yloios, glue).
Locomotion. Many bacteria possess the faculty of self-move-
ment, carrying themselves in all manner of ways across the
microscopic field, some very quickly, others leisurely.
Vibratory Movements. Some bacteria vibrate in themselves,
appearing to move, but they do not change their place ; these
movements are denoted as molecular or " Broivnian" and are
due to purely physical causes.
Fig. 2.
Zoogloea.
Flagella. Little threads or lashes are found attached to many
of the motile bacteria, either at the poles or along the sides,
sometimes only one, and on some several, forming a tuft.
These flagella are in constant motion and can probably be
considered as the organs of locomotion ; they have not yet been
discovered upon all the motile bacteria, owing no doubt to our
imperfect methods of observation. They can be stained and
have been photographed. See Fig. 3. Flagella serve some-
times to increase food-supply, and have been found on some
species which are non-motile.
20
ESSENTIALS OF BACTERIOLOGY
Reproduction. Bacteria multiply through simple division or
fission as it is called. Spore formation is simply a resting stage
Fig. 3.
Flagella.
and not a means of multiplication. To accomplish division
the cell elongates, and at one portion, usually the middle, the
Fig. 4.
1 2
Division of a Micrococcus. (After Mace\)
-'TT^'N
Division of a Bacillus. (After Mace\)
cell- wall indents itself gradually, forming a septum and dividing
the cell into two equal parts, just as occurs in the higher plant
and animal cells. See Fig. 4.
BACTERIA.
21
Successive divisions take place, the new members either exist-
ing as separate cells or forming part of a community or group.
It has been computed that if division takes place every hour,
as it often does, one individual in twenty-four hours will have
17,000,000 descendants.
Spore Formations. Two forms of sporulation , Endosporous and
A rthrosporous. First, a small granule develops in the protoplasm
of a bacterium, this increases in size, or several little granules
coalesce to form an elongated, highly refractive, clearly defined
object, rapidly attaining its real size, and this is the spore. The
remainder of the cell-contents has now disappeared, leaving
the spore in a dark, very resistant, membrane or capsule, and
beyond this the weak cell-wall. The cell-wall dissolves gradu-
ally or stretches and allows the spore to be set free.
Each bacterium gives rise to but one spore. It may be at
either end or in the middle (Fig. 5). Some rods take on a pecu-
liar shape at the site of the spore, making the rod look like a
drum-stick or spindle, Clostridium (Fig. 6).
Fig. 6.
Sporulation. After De Bary.
a
Clostridium.
Spore Contents. What the real contents of spores are is not
known. In the mother cell at the site of the spore little gran-
ules have been found which stain differently from the rest of
the cell, and these are supposed to be the beginnings, the spwo-
22 ESSENTIALS OF BACTERIOLOGY.
genie bodies. The most important part of the spore is its cap-
sule; to this it owes its resisting properties. It consists of two
separate layers, a thin membrane around the cell, and a firm
outer gelatinous envelope.
Germination. AVhen brought into favorable conditions, the
spore begins to lose its shining appearance, the outer firm mem-
brane begins to swell, and it now assumes the shape and size
of the cell from which it sprang, the capsule having burst, so as
to allow the young bacillus to be set free.
Requisites for Spore Formation. It was formerly thought that
when the substratum could no longer maintain it, or had become
infiltrated with detrimental products, the bacterium-cell pro-
duced spores, or rather turned itself into a spore to escape anni-
hilation ; but we believe now that only when conditions are the
most favorable to the well-being of the cell, does it produce
fruit, just as with every other type of plant or animal life, a cer-
tain amount of oxygen and heat being necessary for good spore
formation. The question is still unsettled, however.
Asporogenic Bacteria. Bacteria can be so damaged that they
will remain sterile, not produce any spores. This condition can
be temporary only, or permanent.
Arthrosporous. All the above remarks relate to Endospores,
spores that arise within the cells.
In the other group called Arthrospores, individual members
of a colony or aggregation leave the same, and become the origi-
nators of new colonies, thus assuming toe character of spores.
The Micrococci furnish examples of this form.
Some authorities have denied the existence of the arthro-
sporous formation.
Resistance of Spores. Because of the very tenacious envelope,
the spore is not easily influenced by external measures. It is
said to be the most resisting object of the organic world.
Chemical and physical agents that easily destroy other life
have very little effect upon it.
Many spores require a temperature of 140° C. dry heat for
several hours to destroy them. The spores of a variety of potato-
bacillus (bacillus mesentericus) can withstand the application of
steam at 100° C. for four hours.
ORIGIN OP BACTERIA. 23
CHAPTER II.
ORIGIN OP BACTERIA AND THEIR DISTRIBUTION.
As Pasteur has shown, all bacteria develop from pre-existing
bacteria, or the spores of the same. They cannot arise de novo.
The wide and almost universal diffusion of bacteria is due to
the minuteness of the cells and the few requirements for their
existence. In a drop of water 1700 million cocci can find room.
Very few places are free from germs ; the air on the high seas,
and on the mountain tops, is said to be free from bacteria,
but it is questionable.
One kind of bacterium will not produce another kind.
A bacillus does not arise from a micrococcus or the typhoid
fever bacillus produce the bacillus of tetanus.
This subject has been long and well discussed, and it would
take many pages to state the " pros" and " cons," therefore, this
positive statement is made, it being the position now held by the
principal authorities.
Saprophytes and Parasites. {Saprophytes, gd-n-pSg, putrid. <j>vt6v,
plant. Parasites, napa, aside of, airoq, food.) Those bacteria
which live on the dead remains of organic life are known as
Saprophytic Bacteria, and those which choose the living bodies
of their fellow-creatures for their habitat are called Parasitic
Bacteria. Some, however, develop equally well as Saprophytes
and Parasites. They are called Facultative Parasites.
Conditions of Life and Growth of Bacteria. Influence of Tem-
perature.— In general, a temperature ranging from 10° C. to 40°
C. is necessary to their life and growth.
Saprophytes take the lower temperatures ; Parasites, the tem-
perature more approaching the animal heat of the warm-blooded.
Some forms require a nearly constant heat, growing within very
small limits, as the Bacillus of Tuberculosis.
Some forms can be arrested in their development by a warmer
or colder temperature, and then restored to activity by a return
to the natural heat.
24 ESSENTIALS OF BACTERIOLOGY.
A few varieties exist only at freezing point of water; and
others again will not live under a temperature of 60° C.
For the majority of Bacteria a temperature of 60° C. is de-
structive ; and several times freezing and thawing very fatal.
Influence of Oxygen. — Two varieties of bacteria in relation to
oxygen. The one terobie, growing in air; the other, anserobic,
living without air.
Obligate serobins, those which exist only when oxygen is present.
Facultative asrobins, those that live best when oxygen is present,
but can live without it.
Obligate or true anserobins, those which cannot exist where
oxygen is. Facultative anwrobins, those which exist better where
there is »^ oxygen, but can live in ito prcQcnce.'*"1^^^'-^
Some derive the oxygen which they require out of their nutri-
ment, so that a bacterium may be aerobic and yet not require
the presence of free oxygen.
iErobins may consume the free oxygen of a region and thus
allow the anserobins to develop. By improved methods of cul-
ture many varieties of anserobins have been discovered.
Influence of Light.— Sunlight is very destructive to bacteria.
A few hours' exposure to the sun has been fatal to anthrax
bacilli, and the cultures of bacillus tuberculosis. The sun's
rays, however, must come in direct contact with the germs, and
are usually only active on the surface-cultures. The rays at
the violet end of the spectrum are the most active. The
electric arc light has much the same effect as sunlight on bac-
teria.
Effects of Electricity. — Electricity arrests growth.
Effects of R'dntgen Rays. — Haye little or no effect on artificial
cultures, but in the living tissues a pronounced bactericidal
effect is produced, perhaps through the stimulation of the
body-cells.
Vital Actions of Microbes. Bacteria feeding upon organic com-
pounds produce chemical changes in them, not only by the with-
drawal of certain elements, but also by the excretion of these
elements changed by digestion. Sometimes such changes are
destructive to themselves, as when lactic and butyric acids are
formed in the media.
ORIGIN OF BACTERIA. 25
Oxidation and reduction are carried on by some bacteria. Am-
monia, hydrogen sulphide, and trimethylamin are a few of the
chemical products produced by bacteria. Nitrites in the soil
are reduced to ammonia.
Nitrification. — Albuminoids changed into indol, skatol, leucin,
etc. ; then these into ammonia. Ammonia into nitrites. Ni-
trites into nitrates.
Ptomaines. Brieger found a number of complex alkaloids,
closely resembling those found in ordinary plants, and which
he named ptomaines, from nrcbfia (corpse), because obtained
from putrefying objects.
Proteins. The components of the bacterial cell may cause
inflammation and fever.
Putrefaction. When fermentation is accompanied by devel-
opment of offensive gases a decomposition occurs, which is
called putrefaction, and this, in organic substances, is due
entirely to bacteria.
Producers of Disease. Various pathological processes are
caused by bacteria, the name given to such diseases being in-
fectious diseases, and the germs themselves called disease-pro-
ducing or pathogenic bacteria. Those which do not form any
pathological process are called non-pathogenic bacteria.
Ferments are diastatic, changing starch into sugar ; proteolytic,
transforming albumins into more soluble substances; gelatin
liquefaction is an example.
Inverting, changing a sugar from one that does not undergo
fermentation into one that does.
Coagulating, fat-splitting, hydrolytic ferments are some of the
other varieties.
Toxins and Toxalbumins are various albuminoids produced
in the animal organism and in culture-media which are very
poisonous, and are considered the prime cause of disease.
Pigmentation. Some bacteria are endowed with the property
of forming pigments either in themselves, or producing a thro-
mogenic body which, when set free, gives rise to the pigment.
In some cases the pigments have been isolated and many of the
properties of the aniline dyes discovered in them.
Phosphorescence. Many bacteria have the power to form
Zb ESSENTIALS OF BACTRIOLOGY.
light, giving to various objects which they inhabit a character-
istic glow or phosphorescence.
Fluorescence. An iridescence, or play of colors, develops in
some of the bacterial cultures.
Gas Formation. Many bacteria, anaerobic ones especially,
produce gases, noxious and odorless ; in the culture-media the
bubbles which arise soon displace the media.
Odors. Some germs form odors characteristic of them: some
are pleasant and even fragrant; others, foul and nauseous.
Effect of Age. With age, bacteria lose their strength and die.
CHAPTER III.
METHODS OF EXAMINATION.
We divide the further study of the general characteristics of
Bacteria into two portions : —
First. The examination of bacteria by aid of the microscope.
Second. The continued study through artificial cultivation.
They both go hand in hand ; the one incomplete without the
other.
Microscopical. The ordinary microscope will not suffice for
Bacteriologic research. Certain special appliances must first
be added. It is not so much required to have a picture very
large, as to have it sharp and clear.
Oil Immersion Lens. The penetration and clearness of a lens
are very much influenced by the absorption of the rays of light
emerging from the picture. In the ordinary dry system, many
of the light rays, being bent outward by the air which is be-
tween the object and the lens, do not enter the lens, and are
lost. By interposing an agent which has the same refractive
index as glass, cedar-oil, or clove-oil, for example, all the rays
of light from the object enter directly into the lens.
The " Homogeneous System/' or oil-immersion lens, consists
of a system of lenses which can be dipped into a drop of cedar-
oil placed upon the cover-glass, and which is then ready for use.
METHODS OF EXAMINATION.
27
Abbe's Condenser. The second necessary adjunct is a com-
bination of lenses placed underneath the stage, for bringing
wide rays of light directly under
the object. It serves to intensify Fig. 7.
the colored pictures by absorbing
or hiding the unstained structure.
This is very useful in searching
a specimen for bacteria, since it
clears the field of everything that
is not stained. It is called Abbe's
Condenser. Together With it is Abbe's Condenser.
usually found an instrument for
shutting off part of the light — a blender or diaphragm. When
the bacteria have been found, and their relation to the structure
is to be studied, the " Abbe* " is generally shut out by the iris
blender, and the structure comes more plainly into view. A
white light (daylight or a Welsbach burner) is best for bacterial
study : use the plane mirror for daylight and the concave
mirror for artificial light.
Iris Blender.
For all stained Bacteria the oil immersion lens and Abbe con-
denser, without the use of blender. For unstained specimens,
oil immersion and the narrowed blender.
When examining with low power objective, use a strong
ocular. When using high power objective use weak ocular. A
nose-piece will be found very useful, since it is sometimes neces-
28 ESSENTIALS OF BACTERIOLOGY.
sary to change the objective on the same field, and this insures
a great steadiness of the object.
Great cleanliness is needed in all bacteriological methods ; but
nowhere more so than in the microscopical examination.
The cover-glass should be very carefully washed in alcohol,
and dried with a soft linen rag. To remove the stains on the
cover-glasses that have been used, they should be soaked in
hydrochloric acid or placed in a 6 per cent, aqueous solution of
potassium bichromate with 6 per cent, of strong sulphuric acid,
washed in water, and kept in absolute alcohol.
Examination of Unstained Bacteria. As the coloring of bac-
Fig. 9.
Platinum Needles.
teria kills them and changes their shape to some extent, it is pre-
ferable to examine them when possible in their natural state.
We obtain the bacteria for examination, either from liquid or
solid media.
From Liquids. With a long platinum needle, the end of which
is bent into a loop, we obtain a small drop from the liquid con-
taining the bacteria, and place it on a cover-glass or slide ;
careful that no bubbles remain.
Sterilize Instruments. Right here we might say that it is
best to accustom one's self to passing all instruments, needles,
etc., through the flame before and after each procedure ; it in-
sures safety ; and once in the habit, it will be done automati-
cally.
From Solid Media. With a straight-pointed platinum needle,
a small speck of the medium is taken and rubbed upon a glass
METHODS OF EXAMINATION
29
slide, with a drop of sterilized water, or bouillon, and from this
a little is taken on cover-glass, as before.
The cover-glass with its drop is now placed on the glass slide,
carefully pressing out all bubbles. Then a drop of cedar-oil is
laid on top of the cover-glass, and the oil immersion lens dipped
gently down into it as close as possible to the cover-glass,
the narrow blender shutting off the Abbe condenser, for this
being an unstained specimen, we want but little light We
now apply the eye, and if not in focus, use the fine adjust-
ment or the coarse, but always away from the object — i. e. towards
us — since the distance between the specimen and the lens
is very slight, it does not require much turning to break the
cover-glass and ruin the specimen. Having found the bacte-
rium, we see whether it be bacillus, micrococcus, or spirillum ;
discover if it be motile, or not. That is about all we can ascer-
tain by this method.
Fig. 10.
Hanging Drop in Concave Glass Slide.
Hanging Drop. When the looped platinum needle is dipped
into a liquid, a very finely-formed globule will hang to it ; this
can be brought into a little cupped glass slide (an ordinary
microscopic glass slide with a circular depression in the centre)
in the following manner : The drop is first brought upon a
cover-glass ; the edges of the concavity on the glass slide are
smeared with vaseline, and the slide inverted over the drop;
the cover-glass sticks to the smeared slide, which, when turned
30 ESSENTIALS OF BACTERIOLOGY.
over, holds the drop in the depression covered by the cover-glass,
thus forming an air-tight cell; here the drop cannot evaporate.
Both slide and cover-glass should first be sterilized by heat.
Search for the bacteria with a weak lens ; having found them,
place a drop of cedar-oil upon the cover-glass, and bring the oil
immersion into place (here is where a nose-piece comes in very
usefully), careful not to press against the cell, for the cover-
glasses are very fragile in this position.
Search the edges of the drop rather than the middle ; the bac-
teria will usually be very thick in the centre and not so easily
distinguished.
Spores, automatic movements, fission, and cultivation in
general can be studied for several days. This moist chamber
can be placed in a brood-oven or on the ordinary warming
stages of the microscope.
Agglutination as observed in Widal's test is best seen in the
hanging drop.
CHAPTER IV.
STAINING OF BACTERIA.
Staining or coloring bacteria is done in order to make them
prominent, and to obtain permanent specimens. It is also
necessary to bring out the structure of the bacteria, and
serves in many instances as a means of diagnosis ; and lastly,
it would be well-nigh impossible to discover them in the tissues,
without staining.
Anilin Colors. Of the numerous dyes in the market, nearly
all have, at one time or other, been used in staining bacteria.
But now only a very few find general use, and with methyline
blue and fuchsin nearly every object can be accomplished.
Basic and Acid Dyes. Ehrlich was the first to divide the
anilin dyes into two groups, the basic colors to which belong —
Gentian violet, or pyoktanin, Basic fuchsin.
Methyl violet, or dahlia, Bismarck-brown,
Methylin blue {not methyl blue), Thionin,
Saffranin.
And the acid colors to which eosin and acid-fuchsin belong.
STAINING OF BACTERIA. 31
The basic dyes stain the bacteria and the nuclei of cells ; the
acidE dyes stain chiefly the tissue, leaving the bacteria almost
untouched. Carmine and Hematoxylin are also useful as con-
trast stains, affecting bacteria very slightly. The anilin dyes
are soluble in alcohol or water or a mixture of the two.
Staining Solutions. A saturated solution of the dye is made
with alcohol. This is called the stock or concentrated solution ;
1 part of this solution to about 100 parts of distilled water con-
stitutes the ordinary aqueous solution in use or weak solution.
It is readily made by adding to an ounce bottle of distilled
water enough of the strong solution until the fluid is still opaque
in the body of the bottle, but clear in the neck of the same.
These weak solutions should be renewed every three or four
weeks, otherwise the precipitates formed will interfere with the
staining.
Compound Solutions. By means of certain chemical agents,
the intensity of the aniline dyes can be greatly increased.
Mordants. Agents that t(p 6iie" into the specimen carrying
the stain with them, depositing it in the deeper layers, are
called mordants or etchers.
Various metallic salts and vegetable acids are used for such
purpose.
The mother liquid of the anilin dyes, anilin oil, a member of
the aromatic benzol group, has also this property.
Anilin Oil Water. Anilin oil is shaken up with water and
then filtered; the anilin water so obtained is mixed with the
dyes, forming the "anilin water gentian violet" or anilin water
fuchsin, etc.
Carbol Fuchsin. Carbolic acid can be used instead of anilin
oil, and forms one of the main ingredients of Ziehl's or Neelsen's
solution, used principally in staining bacillus tuberculosis.
Kuhne has a carbol- methylin blue made similar to the carbol
fuchsin.
Alkaline Stains. Alkalies have the same object as the above
agents ; namely, to intensify the picture. Potassium hydrate,
amnion, carbonate, and sodium hydrate are used.
Loffier's alkaline blue and Koch's weak alkaline blue have in
them potassium.
32 ESSENTIALS OP BACTERIOLOGY.
Heat. Warming or boiling the stains during the process of
staining increases their intensity.
Decolorizing Agents. The object is usually over-colored in
some part, and then decolorizing agents are employed. Water is
sufficient for many cases ; alcohol and strong mineral acids com-
bined are necessary in some.
Iodin as used in Gram's Method. Belonging to this group,
but used more in the sense of a protective, is tincture of 'iodin.
It picks out certain bacteria, which it coats; prevents them from
being decolorized, but allows all else to be faded. Then by
using one of the acid or tissue dyes, a contrast color or double
staining is obtained. Many of the more important bacteria are
not acted upon by the iodin, and it thus becomes a very useful
means of diagnosis.
Formulas of different Staining Solutions.
I. — Saturated Alcoholic Solution.
Place about 10 grammes of the powdered dye in a bottle and
add 40 grammes of alcohol. Shake well and allow to settle.
This can be used as the stock bottle.
II. — Weak Solutions.
Made best by adding about 1 part of number I. or stock solu-
tion to 10 of distilled water. This is the ordinary solution in use.
III. — Anilin Oil Water.
Aniline oil 5 parts.
Distilled water . . * . . 100 parts.— M.
Shake wTell and filter. To be made fresh each time.
IV.— Anilin Water Dyes.
Sat. alcoh. sol. of the dye . . 11 parts.
Aniline oil water .... 100 parts.
Abs. alcohol 10 parts. — M.
Can be kept 10 days.
STAINING OF BACTERIA. 33
V.— Alkaline Methylin Blue.
A. Loffler's.
Sat. ale. Sol. methylin blue . . 30
Sol. potass, hydrat. (1-10,000) . 100— M.
B. Koch's.
Sol. potass, hydrat. (10 per eent.) 0.2
Sat. ale. sol. methyl, blue . . 1.0
• Distilled water .... 200.0— M.
VI. — Carbolic Acid Solutions.
A. Ziehl-Neelsen.
Fuehsin (powd.) .... 1 part.
Alcohol 10 parts.
5 per eent. sol. acid, carbolic . 100 parts.— M.
Filter. The older the solution the better.
B. Kilhne.
Methylin blue . . . . 1.5
Alcohol 10.0
5 per cent. sol. ac. carbol. . . 100.0
Add the acid gradually. This solution loses strength with age.
VII. — Gram's Iodin Solution.
Iodine 1
Potass, iod 2
Aquae destillat 300.— M.
VIII.— Loffler's Mordant (for flagella).
Aq. sol. of tannin (20 per cent.) . 10 parts.
Aq. sol. ferri sulph. (5 per cent.) . 1 part.
Aqua? decoc. of logwood (1-8) . 4 parts. — M.
Keep in well-corked bottle.
IX. — Unna's Borax Methyl Blue.
Borax 1 part.
Methyl blue 1 part.
Water 100 parts.— M.
■6
34 ESSENTIALS OF BACTERIOLOGY.
X. — Gobbet's Acid Blue (rapid stain).
Methylin blue .... 2
25 per cent, sulphuric acid . . 100.— M.
XI. — Alkaline Anilin Water Solutions.
Sodium hydrat. (1 per cent.) . . 1
Anilin oil water .... 100. — M.
And add —
Fuchsin, or methyl-violet powd. . 4
Cork well. Filter before using.
XII. — Roux's Double Stain.
Dahlia or gentian violet ... 0.5 gramme.
Methyl green 1.5 "
Distilled water 200.0 grammes. — M.
Use as other stains, without acid.
XIII. — Neisser's Stain. (For
Diphtheria.)
Solution I.
Methylin blue ....
Alcohol (96 per cent.)
Dissolve and add water .
Glacial acetic acid .
1 gramme.
. 20 c.c.
. 950 c.c.
. 50 c.c— M.
Solution II.
Vesuvin
Water
2 grammes.
. 1000 c.c— M.
Stain cover-glasses (1) three seconds in Sol. I. ; (2) wash in
water; (3) three seconds in No. 2; (4) wash in water. Body of
bacillus, brown ; oval granules at each end, blue.
XIV. — Carbol-thionin. (Nicolle.)
Sat. sol. thionin in ale (90 per cent.) 10 c.c.
Aqueous sol. ac carbol. (1 per cent.) 100 c.c. — M.
Stain sections, one-half to one minute.
METHOD OF STAINING SPECIMENS. 35
XV. — Capsule Stain of Hiss.
Use the following, heated until it steams :
Sat. alcoholic solution of 1 K „ „
l . o c.c.
gentian violet or fuchsin j
Distilled water .... 95 c.c.
Wash in 20 per cent, solution of cupric sulphate crystals.
XVI. — Capsule Stain of Welch.
(1) Pour glacial acetic acid on film. After a few seconds
replace with anilin-water gentian violet without washing in
water. (2) Remove all acid by several additions of stain, and
allow it to act for three to four minutes. (3) Wash and ex-
amine in salt solution 0.8-2.0 per cent.
CHAPTER V.
GENERAL METHOD OF STAINING SPECIMENS.
Cover-Glass Preparations. The material is evenly spread in
as thin a layer as possible upon a cover-glass ; then, to spread
it still more finely, a second cover-glass is pressed down upon
the first and the two slid apart. This also secures two speci-
mens. Before they can be stained they must be perfectly dry,
otherwise deformities will arise in the structure.
Drying the Specimen.— The cover-glass can be set aside to dry,
or held in the fingers over the Bunsen burner (the fingers prevent-
ing too great a degree of heat). Since most of the specimens
contain a certain amount of albumenoid material, it is best in
all cases to "fix" it, t, e., to coagulate the albumen. This is
accomplished by passing the cover-glass (after the specimen is
dry) three times through the flame of the burner, about three
seconds being consumed in doing so, the glass being held in a
small forceps, smeared side up.
The best forceps for grasping cover-glasses is a bent one, bent
again upward, near the ends. (Fig. 11.) It prevents the flame
or staining-fluid from reaching the fingers.
36 ESSENTIALS OF BACTERIOLOGY.
The object is now ready for, staining.
Staining.— A few drops of the staining solution are placed
upon the cover-glass so that the whole specimen is covered,
and it is left on a few minutes, the time depending upon the
variety, the strength of stain, and the object desired. Instead
Fig. 11.
Author's Bent Forceps for Holding Cover-glass over Flame.
of placing the dye upon the object, the cover-glass can be im-
mersed in a small glass dish containing the solution ; or, if
heat is desired to intensify or hasten the process, a watch-
crystal holding the stain is placed over a Bunsen burner and
in it the cover-glass ; and, again, the cover-glass can be held
directly in the flame with the staining fluid upon it, which
must be constantly renewed until the process is completed,
or the cover-glass can be heated in a test-tube.
Removing Excess of Stain. The surplus stain is washed off
by dipping the glass in distilled water.
The water is removed by drying between filter paper or
simply allowed to run off by standing the cover-glass slant-
wise against an object. When the specimen is to be examined
in water (which is always best with the first preparation of
the specimen, as the Canada balsam destroys to some extent
the natural appearance of the bacteria), a small drop of ster-
ilized water is placed upon the glass slide, and the cover-glass
dropped gently down upon it, so that the cover-glass remains
adherent to the slide.
The dry system or the oil-immersion can now be used.
When the object has been sufficiently examined it can be per-
manently mounted by lifting the cover-glass off the slide (this
is facilitated by letting a little water flow under it, one end
METHOD OF STAINING SPECIMENS. 37
being slightly elevated). The water that still adheres is dried
off in the air or gently over the flame, and when perfectly dry
it is placed upon the drop of Canada balsam which has been put
upon the glass slide.
In placing the cover- glass in the staining solutions one must
be careful to remember which is the spread side.
By holding it between yourself and the window, and scraping
the sides carefully with the sharp point of the forceps, the side
having the specimen on it will show the marks of the instrument.
Little glass dishes, about one-half-dozen, should be at hand
for containing the various stains and decolorants.
Tissue Preparations. In order to obtain suitable specimens
for staining, very thin sections of the tissue must be made.
As with histological preparations, the tissue must be hardened
before it can be cut thin enough. Alcohol is the best agent for
this purpose.
Fig. 12.
I
' :
Spatula for Lifting Sections.
Pieces of the tissue one-quarter inch in size are covered with
alcohol for 24 to 48 hours.
When hardened it must be fixed upon or in some firm object.
A paste composed of—
Gelatine 1 part.
Glycerine 4 parts.
Water 2 parts.
will make it adhere firmly to a cork in about 2 hours, or it can
be imbedded in a small block of paraffine, and covered over with
melted paraffine. Celloidin may be used as an imbedding agent,
and formalin is useful to harden tissue quickly.
Cutting. The microtome should be able to cut sections j^B
inch in thickness ; this is the fineness usually required.
The sections are brought into alcohol as soon as cut unless
they have been imbedded in paraffine, when they are first washed
in chloroform to dissolve out the paraffine.
38
ESSENTIALS OF BACTERIOLOGY.
Staining. All the various solutions should be in readiness,
best placed in the little dishes in the order in which they are to
be used, as a short delay in one of the steps may spoil the speci-
men.
A very useful instrument for transferring the delicate sections
from one solution to another is a little metal spatula, the blade
being flexible.
A still better plan, especially when the tissue is "crumbling/
is to carry out the whole procedure on the glass-slide.
Fig. 13.
Section Microtome.
General Principles. The section is transferred from the alco-
hol in which it has been kept into water, which removes the
excess of alcohol, from here into —
Dish I, containing the stain; where it remains 5 to 15 minutes.
Then-
JJish II, containing 5 per cent, acetic acid (1 to 20); where it
remains ^ to 1 min. The acid removes the excess of stain.
Dish III, water to rinse off the acid. The section can now be
placed under the microscope covered with cover-glass to see if
the intensity of the stain is sufficient or too great. A second
STAINING AND MODIFICATIONS. 39
section is then taken, avoiding the errors, if any ; and having
reached this stage proceeded with as follows : —
Dish IV, alcohol, 2 to 3 seconds to remove the water in the
tissue.
V. A few drops of oil of cloves, just long enough to clear the
specimen to make it transparent (so that an object placed under
neath will shine through).
VI. Remove excess with filter-paper.
VII. Mount in Canada balsam (xylol balsam).
CHAPTER VI.
SPECIAL METHODS OF STAINING AND MODIFICATIONS.
Gram's Method of Double Staining. (For cover-glass speci-
mens.)— I. A hot solution of anil, water gentian violet 2 to 10
minutes.
II. Directly without washing, into Gram's solution of iod.
potass, iod. 1 to 3 min. (the cover-glass looks black).
III. Wash in alcohol 60 per cent, until only a light brown
shade remains (as if the glass were smeared with dried blood).
IV. Rinse off alcohol with water.
V. Contrast color with either eosin, picro-carmine, or bismark-
brown. The bacteria will appear deep blue, all else red or brown
on a very faint brown background.
The following bacteria do not retain their color with Gram's
method — are therefore not available for the stain : Bacillus of
typhoid ; spirillum of cholera ; bacillus of chicken cholera, of
hemorrhagic septicaemia, of malignant oedema, of pneumonia
(Friedlander), and of glanders; diplococcus of gonorrhoea ; spi-
rillum of relapsing fever.
Gram's Method for Tissues (modified by Giinther).
I. Stain in anil, water gent, violet . . 1 minute.
II. Dry between filter paper.
III. Iod. potass, iod. sol. .... 2 minutes.
IV. Alcohol £ minute.
V. 3 perct. sol. hydrochloric acid in alcohol 10 seconds.
VI. Alcohol, ol. of cloves, and Canada balsam.
40 ESSENTIALS OF BACTERIOLOGY.
To Stain Spores. Since spores have a very firm capsule,
which tends to keep out all external agents, a very intensive
stain is required to penetrate them, but once this object attained
it is equally as difficult to decolorize them.
A cover-glass prepared in the usual way, i. e., drying and
passing the specimen through the flame three times, is placed in
a watch-crystal containing Ziehl's carbol-fuchsin solution, and
the same placed upon a rack over a Bunsen burner, where it is
kept at boiling-point for one hour, careful to supply fresh solution
at short intervals lest it dry up.
The bacilli are now decolorized in alcohol, containing £ per
cent, hydrochloric acid. A contrast color, preferably methylin
blue, is added for a few minutes.
The spores will appear as little red beads in the blue bacteria,
and loose ones lying about.
Spore Stain (modified). — I. Carbol-fuchsin on cover-glass and
heated in the flame to boiling point 20 to 30 times.
II. 25 per cent, sulphuric acid, 2 seconds ; rinsed in water.
III. Methylin blue contrast.
Alex. Klein recommends the following spore method : mix a
little of the culture (potato) with 3 drops of physiologic salt
solution, and heat gently with an equal quantity of carbol-
fuchsin for a period of 6 minutes. Spread then on cover-glasses,
dry in the air, and fix by passing three times through Bunsen
burner flame. Decolorize in 1 per cent, sulphuric acid for 1 to
2 seconds ; contrast in weak methylin blue.
BowhilVs Orcein Stain.
Sat. alcoholic solution of orcein . . .15 c.c.
20 per cent, aqueous sol. tannin . . .10 c.c.
Distilled water 30 c.c. — M.
Filter.
Use orcein solution in watch-glass, float cover-glass in it and
heat gently, not boil, for 10 minutes. Wash in water. Dry and
mount in balsam.
Five per cent, chromic acid applied for 15 minutes has been
recommended in staining spores. This is followed by the
carbol-fuchsin stain as above.
STAINING AND MODIFICATIONS. 41
Flagella Stain, with Loffier's Mordant. — I. A few drops of the
mordant (No. viii. p. 33) are placed upon the spread cover-glass
and heated until it steams.
II. Washed with water until the cover-glass looks almost clean,
using a small piece of filter paper to rub off the crusts which have:
gathered around the edges.
III. Anilin water fuchsin (neutral) held in flame about 1£
minutes.
IV. Wash in water.
If the stain is properly made, the microbes are deeply colored
and the flagella seen as little dark lines attached to them.
Sporogenic bodies stain quite readily, and in order to distin-
guish them from spores Ernst uses alkaline methylin blue, slightly
warmed. Then rinse in water. Contrast with cold bfemark-
brown.
The spores are colored bright blue, the spore granules a dirty
blue, being mixed with the brown, which colors also the bacteria.
Kiihne's Method. — In sections, the alcohol used sometimes de-
colorizes too much. To obviate this Kulme mixes the alcohol
with the stain, so that while the section is being anhydrated it
is constantly supplied with fresh dye.
Weigert uses aniline oil to dehydrate instead of alcohol, and
here, too, it can be used mixed with the dye.
Unna's Method for Fungi (especially useful for epidermic;
scales). — Moisten horny scale or crust with acetic acid; mace-
rate between two glass slides ; dry in flame ; wash out fat with
ether and alcohol (equal parts) ; stain in borax methyl blue for
ten seconds (over flame) ; bleach with glycerine and aether (equal
parts) ; rinse in water, alcohol, dry, and mount.
Behavior of the More Important Bacteria to Gram's Stain.
Positive means that the bacteria retain the primary color, or
gentian violet.
Positive. Negative.
Tubercle Bacillus, Colon Bacillus,
Smegma Bacillus, Typhoid Bacillus,
Lepra Bacillus, Cholera Bacillus,
Anthrax Bacillus, Influenza Bacillus,
42 ESSENTIALS OF BACTERIOLOGY.
Positive. Negative.
Tetanus Bacillus, Friedlander's Bacillus,
Diphtheria Bacillus, Plague Bacillus,
Pneumococcus, Diplococcus intracellularis,
Streptococcus, Gonococcus,
Staphylococcus, Koch-Weeks' Bacillus,
Cocci of the urethra. Conjunctivitis Bacillus of Moras.
CHAPTER VII.
METHODS OF CULTURE.
Artificial Cultivation.— The objects of cultivation are to obtain
germs in pure culture, free from all foreign matter, isolated and
so developed as to be readily used either for microscopical ex-
amination or animal experimentation.
To properly develop bacteria we supply as near as possible
the conditions which hold for the especial germ in nature.
With the aid of solid nutrient media the bacteria can be easily
separated, and the methods are nearly perfect.
Sterilization. If we place our nutrient material in vessels
that have not been properly disinfected, we will obtain growths
of bacteria without having sown any.
If we have thoroughly cleaned our utensils, and then not taken
care to protect them from further exposure, the germs we have
sown will be effaced or contaminated by multitudes of others,
that are constantly about us. We therefore have two neces-
sary precautions to take : —
First. To thoroughly clean and sterilize every object that
enters into, or in any way comes in contact with, the culture.
Second. To maintain this degree of sterility throughout
the whole course of the growth, and prevent, by proper con-
tainers, the entrance of foreign germs.
Disinfectants. Corrosive sublimate (bichloride of mercury),
which is the most effective agent we possess, cannot be gene-
rally used because it renders the soil unproductive and therefore
METHODS OF CULTURE.
43
must only be employed in washing dishes, to destroy the old
cultures. Even after washing, a few drops of the solution may
remain and prevent growth, so that one must be careful to have
the glass-ware that comes in contact with the nutrient media
free from the sublimate.
Heat. Heat is the best agent we possess for general use.
Dry heat and moist heat are the two forms employed, but these
differ greatly in effectiveness. Thus Koch found that while
moist heat at 100° C. killed the spores of the anthrax bacillus
in one hour, it required three hours of dry heat at 140° C. to
produce death.
Fig. 14.
Hot Air Oven.
For obtaining dry heat— that is, a temperature of 150° C,
(about 300° F.)— a sheet-iron oven is used which can be heated
by a gas-burner. If it have double walls (air circulating be-
tween), the desired temperature is much more quickly obtained.
A small opening in the top to admit a thermometer is neces-
sary. These chests are usually about 1 foot high, 1^ foot wide,
and f foot deep. In them, glassware, cotton, and paper can be
sterilized. "When the cotton is turned slightly brown, it usually
44
ISSENTIALS OF BACTERIOLOGY,
denotes sufficient sterilization. All instruments, where practi-
cable, should be drawn through the flame of an alcohol lamp or
Bunsen burner. One hour in the oven at 170° C. usually steri-
lizes glass-ware, while the ordinary germs in liquids may be
Fig. 15.
Koch's Steam-chest.
killed by boiling for five minutes if no spores are present. The
boiling of any fluid at 100° C. for one and one-half hours nearly
always ensures sterilization.
Moist Heat. — Steam at 100° C. in circulation has been shown
to be a very effective application of heat.
METHODS OF CULTURE.
45
Koch's Steam-chest. Circulating steam is obtained by aid of
Koch's apparatus. This consists, of a cylindrical tin chest
.about 2£ feet high and about £ foofom diameter ; divided in its
interior by a perforated diaphragtiJja, an upper chamber for
the steam, c, and a lower one for ,, water, b. Two or more
gas-burners placed underneath the chest, which stands on a
Fig. 16.
Arnold's Steam-sterilizer.
tripod, supply the heat. In the cover is an opening for a ther-
mometer. The chest is usually covered with felt. When the
thermometer registers 100° C. the culture-medium or other sub-
stance to be sterilized is placed in the steam and kept there
from 10 to 15 minutes, or longer, as required.
Arnold's steam sterilizer will answer every purpose of the
Koch steam-chest. It is cheaper, also requiring less fuel to keep
it going. The steam does not escape, but is condensed in the
outer chamber, (rig. 16.)
46
ESSENTIALS OF BACTERIOLOGY
The autoclave of Chamberland allows a temperature of 120°
C. to be obtained, and is much used in Pasteur's laboratory.
Fig. 17.
Chamberland's Autoclave with pressure.
Instead of sterilizing for a long time at once, successive steri-
lization is practised with nutrient media, so that the albumen
will not be too strongly coagulated. Fifteen minutes each day
for three days in succession.
METHODS OF CULTURE,
47
Fractional Sterilization of Tyndall. Granted that so many
spores originally exist in the object to be sterilized, it is sub-
jected to 60° 0. for four hours, in which time a part at least of
those spores have developed into bacteria, and the bacteria
destroyed by the further application of the heat. The next day
more bacteria will have formed, and four hours' subjection to
60° heat will destroy them, and so at the end of a week, using
four hours' application each day, all the spores originally present
will have germinated and the bacteria destroyed.
Fig. 18.
Fig. 19.
i1 1
i'
!
i
1 1
i i
!
LJ
■I
!i
Wire-Cage.
Cotton plugged Test-Tubes.
Cotton Plugs or Corks. All the glass vessels (test-tubes, flasks,
etc.) must be closed with cotton plugs, the cotton being easily
sterilized and preventing the entrance of germs.
Tin-foil may be used to cover the cotton, or caps made of
india-rubber.
Test-tubes. New test-tubes are washed with hydrochloric
acid and water to neutralize the alkalinity often present in
fresh glass. They are then well washed and rubbed with a
48 ESSENTIALS OP BACTERIOLOGY.
brush, placed obliquely to drain, and when dry corked with
cotton plugs. Then put in the hot-air oven (little wire-cages
being used to contain them) for fifteen minutes, after which they
are ready to be filled with the nutrient media. (The cotton
should fit firmly in the tube and extend a short space beyond it.)
Test-tubes without flaring edges are more desirable since the
edges can easily be drawn out so as to seal the tube.
Instead of test-tubes, ordinary 3 oz. panel medicine bottles
can be used for retaining the nutrient media and cultures.
According to late investigations, the glass tubes become suffi-
ciently sterile in the steam-chest without the preliminary sterili-
zation in the dry oven.
CHAPTER VIII.
NUTRIENT MEDIA.
Of the many different media recommended and used since
bacteriology became a science, we can only describe the more
important ones now in use. Each investigator changes them
according to his taste.
Fluid Media.
Bouillon (according to Loftier). A cooked infusion of beef
made slightly alkaline with soda carbonate : 500 grammes of
finely-chopped raw lean beef are placed in a wide-mouthed jar
and covered with 1 litre of water ; this is left standing twelve
hours with occasional shaking. It is then strained through
cheese cloth, the white meat remaining being pressed until one
litre of the blood-red meat-water has been obtained. The meat-
water must now be cooked, but before doing this, in order to
prevent all the albumen from coagulating, 10 parts of peptone
powder and 5 parts of common salt are added to every 1000
parts meat-water. It is next placed in the steam-chest or
water-bath for three-quarters of an hour.
Neutralization. The majority of bacteria grow best on a
neutral or slightly alkaline soil, and the bouillon, as well as
NUTRIENT MEDIA. 49
other media, must be carefully neutralized with a sat. sol. of
carbonate of soda. Since too much alkalinity is nearly as bad
as none at all, the soda must be added drop by drop until red
litmus paper commences to turn blue. The bouillon is then
cooked another hour, and filtered when cold. The liquid thus
obtained must be clearly alkaline, and not clouded by further
cooking. If cloudiness occur, the white of an egg and further
boiling will clear the same. To make bouillon, beef-extract can
be used instead of fresh meat, 2 grammes to 1 litre of water.
This is boiled with 5 grammes of salt and 10 of peptone, neu-
tralized as above, and filtered when cold.
Schultz's Method of Neutralization. — A more accurate method of
obtaining the required reaction is to use an alcoholic solution
(£ per cent.) of phenolphthalein as an indicator ; a few drops of
this are mixed with 10 c.c. of the bouillon, and from a burette a
solution of caustic soda 0.4 per cent, is added drop by drop
until a faint red color appears. An average is taken from three
different samples, and the amount of soda needed for the entire
quantity of bouillon is calculated therefrom. Glucose broth,
which is a good medium for anaerobic organisms, consists of
bouillon to which 1 to 2 per cent, of grape-sugar has been
added. Glycerin broth is bouillon to which 6 to 8 per cent, of
glycerin has been added after filtration.
Sterilization of the Bouillon. Erlenmeyer flasks (little conical
glass bottles) or test-tubes plugged and properly sterilized are
filled one-third full with the bouillon, and placed with their
contents in the steam-chest. They are left in steam of 100° C.
one hour for three successive days, after which the tubes and
bouillon are ready for use.
Solid Media. The knowledge of bacteria and germs or moulds
settling and growing upon slices of potato exposed to the air, led
to the use of solid media for the artificial culture of the same.
It was also thus learned that each germ tends to form a separate
colony and remain isolated.
Potato-Cultures. A ripe potato with a smooth skin is the
best.
Several are brushed and scrubbed with water to get rid of the
dirt and the "eyes" are cut out.
4
50
ESSENTIALS OF BACTERIOLOGY.
Next placed in 1 to 500 solution of bichloride of mercury for
£ hour. Then in the steam-chest for £ hour.
In the meantime, a receptable is prepared for them. This is
called the moist chamber.
Fig. 20.
Moist chamber for potatoes.
The moist chamber consists of two large shallow dishes, one,
the larger, as a cover to the other.
These dishes are washed in warm distilled water.
Fig. 21.
Method of slicing potato. (After Woodhead and Hare.)
A layer of filter paper moistened with a 15 to 30 drops of 1 to
1000 bichloride is placed in the bottom of the glass dish.
The operator now prepares his own hands, rolling up his coat
sleeves and carefully washing his hands, then taking a potato
NUTRIENT MEDIA.
51
from the steam-oven and holding it between his thumb and
index finger in the short axis, he divides the potato in its
long axis with a knife that has been passed through the flame.
The two halves are kept in contact until they are lowered into
the moist chamber, when they of their own weight fall aside,
the cut surface uppermost. They are then ready for inoculation.
Esmarch's Cubes. The potato is first well cleaned and peeled.
It is then cut in cubes £ inch in size.
These are placed, each in a little glass dish or tray and then
in steam-chest for ^ hour, after which they are ready for inocu-
lation (the dishes first having been sterilized in hot-air oven).
Test-tube Potatoes. Cones are cut out of the peeled potato
and placed in test-tubes, which can then be plugged and easily
preserved.
Roux's test-tube (Fig. 22), specially designed Fig. 22.
for potato cultures, consists of a tube with a
small constricted portion at the bottom, in which
water may be kept to keep the potato moist.
Manner of Inoculation. With a platinum rod
or a spatula (sterilized) the material is spread
upon one of the slices, keeping free of the
edges. The growth on this first, or original,
potato will be quite luxuriant, and the individual
colonies often difficult to recognize; therefore
dilutions are made. (Fig. 23.)
From the original or first slice a small portion,
including some of the meat of the potato, is
spread upon the surface of a second slice,
which is first dilution. From this likewise a
small bit is taken and spread on a third slice, or
second dilution, and here usually the colonies
will be sparsely enough settled to study them in their indi-
viduality.
This is the principle carried on in all the cultivations. It is
a physical analysis.
Potato and Bread Mash. These pastes are used chiefly in the
culture of moulds and yeasts. Peeled potatoes are mashed with
distilled water until thick, and then sterilized in flasks f of an
hour for three successive days.
Tube for potato
culture.
52 ESSENTIALS OP BACTERIOLOGY.
Fig. 23.
Method of inoculation. (Woodhead and Hare.)
Bread Mush.— Bread devoid of crust, dried in an oven, and
then pulverized and mixed with water until thick and sterilized
as above.
CHAPTER IX.
SOLID TRANSPARENT MEDIA.
Solid Transparent Media are materials which can be used for
microscopical purposes and which can readily be converted
into liquids. Such are the gelatine and agar culture media.
Gelatine. Gelatine is obtained from bones and tendons, and
consists chiefly of chondrin and gluten.
The French golden medal brand is the one most in use, found
in long leaves with ribbed lines crossing them.
Koch-Loffler 10 per cent. Bouillon-Gelatine. To the meat-
water as made for the bouillon are added
100 grammes gelatine,
10 " peptone,
5 " salt,
to each 1000 grammes of the meat-water; or to the bouillon
made from beef-extract the gelatine is added ; this is placed in
a flask and gently heated until the gelatine is dissolved.
Neutralization with the soda and then cooking in water-bath
or carefully boiled over flame for 1 hour or more until the
SOLID TRANSPARENT MEDIA,
53
liquid seems clear, then add white of an egg and boil £ hour
longer ; the egg will produce
a clearer solution and save
much trouble. A small por-
tion, while hot, is now filtered
into a test-tube and tested for
alkalinity, and then re-heated
several times, watching if a
cloudy ppt. forms.
If the fluid remains clear
upon cooling, the remainder of
the material can be filtered.
It must be accomplished
while hot, else the gelatine
will coagulate and prevent
further filtration.
This can be carried on
either by keeping hot the so-
lution continually in water-
bath, and only filtering a small
quantity at a time through
the filter, or keeping the filter
itself hot, either with a hot
water filter or placing the
filter in steam chest. (Fig.
24.)
Clouding of Gelatine. If the gelatine does not come out clear,
or becomes turbid on cooling, it may be due to several things—
1. The filter-paper too thin or impure.
2. Too strongly alkaline.
3. Cooked too long or not long enough.
The addition of the white of an egg, as before mentioned, will
often clear it up ; if this avails not, re-filtering several times, and
attention to the few points mentioned.
Sterilizing the Gelatine. The gelatine is kept in little flasks
or poured at once into sterile test-tubes, careful not to wet the
neck where the cotton enters, lest when cool the cotton plug
stick to the tube.
The tubes are then placed in steam-chest for three successive
Hot-water
54 ESSENTIALS OP BACTERIOLOGY.
days, 15 minutes each day (or in water-bath 1 hour a day for
three days). Then set aside in a temperature of 15° to 20° C,
and if no germs develop and the gelatine remains clear, it can
be used for cultivation purposes.
Modifications. The amount of gelatine added to the meat-
water can be variously altered, and instead of making gelatine
bouillon milk, blood, serum, urine, and agar can be added.
Glycerine (4 to 6%) is a common addition, and sometimes
reducing agents to absorb the oxygen are mixed with it.
Agar- Agar. This agent, which is of vegetable origin, derived
from sea-plants gathered on the coasts of India and Japan, has
many of the properties of gelatine, retaining its solidity at a
much higher temperature; it becomes liquid at 90° C. and con-
geals again at 45° C. Gelatine will liquefy at 35° C.
It is not affected very much by the peptonizing action of
the bacteria — 38° C. is the temperature at which most patho-
genic germs grow best.
Preparation of Agar-Agar Bouillon or Nutrient Agar. The
ordinary bouillon is first made, and then the agar cut in small
pieces, added to the bouillon (15 grammes of agar to 1000
grammes bouillon. It is allowed to stand several minutes until
the agar swells, and then placed in water-bath or steam-chest
for six hours or more. It is then neutralized, very little of the
alkali being sufficient.
A white of an egg added, and boiled for several hours longer,
when, even if not perfectly clear, it is filtered.
The filtering process, very difficult because of the readiness
with which the agar solidifies, must be done in steam-chest or
with hot-water filter, and very small quantities passed through
at a time, changing the filter-paper often.
Cotton can be used instead of filter-paper, or filtering entirely
dispensed with, simply decanting.
A.s agar is seldom clear, a little more or less opaqueness will
not harm. The test-tubes are filled as with the gelatine, and
sterilized in the same manner. While cooling, some of the
tubes can be placed in a slanting position, so as to obtain a larger
surface to work upon.
Water of condensation will usually separate and settle at the
SOLID TRANSPARENT MEDIA. 55
bottom, or a little white sediment remain encysted in the centre ;
this cannot easily be avoided, nor does it form any serious obstacle.
The crude agar should first be rinsed in water, and then in
5% acetic acid and clear water again, to rid it of impurities.
If agar is boiled thoroughly over a hot flame or in an auto-
clave, it can be filtered much more readily. The main point
is to see that all the agar is dissolved.
It has been suggested to pour the hot agar into high cylin-
drical glass vessels and allow it to cool slowly in the steam
oven, the flame having been gradually lowered and then turned
out. After a time the cloudy portion will form a sediment at
the bottom ; the agar can then be shaken out as a long cylinder
and the cloudy portion cut off.
The Japanese Method. — Yokote prepares agar as follows : the
meat is cooked in water over a sand bath 1? hours. Filtered,
chopped agar is then added and the mixture cooked 1 hour
longer ; peptone and salt added next. Neutralization. After the
mixture has cooled to about 50° C. whites of 2 eggs are added
and the mixture shaken thoroughly.
Again the mixture is placed on the sand-bath and heated to
110° C. and over for lj to 2 hours, and then filtered through
ordinary filter-paper. Yokote claims that by this procedure
the agar can be filtered as easily as bouillon and without any
loss. (The water evaporated in boiling must be added before
filtering.)
Glycerine Agar. The addition of 4 to 6% of glycerine to
nutrient agar greatly enhances its value as a culture medium.
Gelatine-Agar. A mixture of 5 per cent, gelatine and 0.75
per cent, agar combines in it some of the virtues of both agents.
Blood Serum. Blood serum being rich in albumen coagulates
very easily at 70° C, and if this temperature is not exceeded,
a transparent, solid substance is obtained upon which the ma-
jority of bacteria develop, and some with preference.
Preparation of Nutrient Blood Serum. If the slaughter of
the animal can be supervised, it were best to have the site of
the wound and the knife sterilized, and sterile flasks to receive
the blood directly as it flows.
It is placed on ice forty-eight hours, and the serum is drawn
56
ESSENTIALS OF BACTERIOLOGY.
Fig. 25.
out with sterile pipettes into test-tubes; these are placed ob-
liquely in an oven where the temperature can be controlled
and maintained at a certain degree. See
Fig. 26.
Incubators or Brood-ovens. Incubators
or brood-ovens consist essentially of a double-
walled zinc or copper chest, the space between
the walls being filled with water.
The oven is covered with some imperme-
able material to prevent the action of the
surrounding atmosphere. (Fig, 27.) It is
supplied with a thermometer and a regu-
lator. The regulator is connected with a
Bunsen burner, and keeps the temperature
at a certain height.
There are several forms of regulators in
use, and new ones are invented continually.
The size of the flame in some is regulated
by the expansion of mercury, which, as it
rises, lessens the opening of the gas supply.
The mercury contracting on cooling allows more gas to enter
again. (Fig. 28.)
Koch has invented a safety burner, by which the gas supply is
shut oft* should the flame accidentally have gone out.
Coagulation of Blood Serum. The tubes of blood serum
having been placed in the oven, are kept at a tempera-
ture of 65° to 68° C, until coagulation occurs ; then removed
and sterilized.
Sterilization of Blood Serum. The tubes are placed 3 to 4
days in incubation at 58° C, and those tubes which show any
evidences of organic growth are discarded.
If now, at the end of a week, the serum remains sterile at the
ordinary temperature of the room, it can be used for experi-
mental purposes.
Perfectly prepared blood serum is transparent, of a gelatine-
like consistence, and straw-color. It will not liquefy by heat,
though bacteria can digest it. Water of condensation always
forms, which prevents the drying of the serum. Blood serum
Flask to receive blood
serum.
SOLID TRANSPARENT MEDIA.
57
may be prepared in a shorter way by coagulating the serum at
a temperature short of boiling-point. Sterilization is completed
in three days by exposing the tubes to a temperature of about
90° C. each day for five minutes. Tubes so prepared are opaque
and white.
Fro. 26.
Thermostat for blood serum.
Preservation of Blood Serum in Liquid State. Kirchner advises
the use of chloroform. To a quantity of serum in a well-stop-
pered flask a small amount of chloroform is added — enough to
form about a 2 mm. layer on the bottom. If the chloroform is
not allowed to evaporate, the serum remains sterile for a long
time. When needed for use test-tubes are filled and placed in
a water-bath at 50° C. until all chloroform has been driven off
(determined by absence of characteristic odor); the serum is
then solidified and sterilized as in the ordinary way.
58
ESSENTIALS OP BACTERIOLOGY.
Human blood serum derived from placenta, serum from as-
citic fluid and ovarian cysts, is prepared in a similar manner
to the above.
Blood Coagulum, suggested by the author, is the blood itself
(not the serum only) coagulated in test-tubes. It is dark brown
in color and allows some colonies of bacteria to be more visi-
ble. It requires less time to prepare, and is not so likely to
become contaminated as when the serum is used.
Fig. 27.
Fig. 28.
Babe's incubator. Thermo-regulators.
Loffler's Blood Serum Mixture (see p. 111).
Peptone Solution. (Dunham's.) Sodium chlorid, 0.5; pep-
tone, 1 ; water, 100. Boil, filter, and sterilize. Useful to detect
presence of indol.
Other Nutrient Media. Milk, urine, decoctions of various
fruits and plants, and lately for cultivating anaerobic bacteria,
eggs.
SOLID TRANSPARENT MEDIA. 59
Many combinations of the preceding are also in use, such as
glucose-agar, glucose gelatin, blood- or serum-agar; and litmus
is often added to media to show changes in reaction during
bacterial growth.
Dunham's Rosalie Acid Solution.
Peptone sol. (Dunham) .... 100 c.c.
2 per cent. sol. rosalic acid . . .0.5 gr.
Alcohol (80 per cent.) .... 100 c.c.
M. To detect acids and alkalies.
Eisner's Medium (for typhoid). (Iodo- potass. — Potato-gelatin.)
Five hundred grammes of (peeled and washed) potatoes are
mashed and pressed through a fine cloth. The juice is allowed
to settle, is filtered, and after 1 hour's cooking has added to it 10
per cent, gelatin; then 2\ c.c. ^ normal sodic hydrate solu-
tion, and finally 1 per cent, potassic iodid.
Typhoid Medium of Hiss. This consists of a slightly acid
mixture of gelatin and agar, beef-extract, sodium chloride, and
dextrose, used in different proportions for plate and tube cul-
tures. It is semi-solid in character and facilitates the identi-
fication of the motile typhoid bacilli which produce a uniform
clouding through the medium in tubes.
Urine Media (Gonococci).
Urine (sterile taken) . . . , .1 part.
2 per cent, agar solution . . . . 1 "
Fresh Egg Cultures, after Huppe. The eggs in the shell are
carefully cleaned, washed with sublimate, and dried with cotton.
The inoculation occurs through a very fine opening made in
the shell with a hot platinum needle ; after inoculation, the open-
ing is covered with a piece of sterilized paper, and collodion.
Boiled Eggs. Eggs boiled, shell removed over small portion,
and the coagulated albumen stroked with the material.
Guinea-pig Bouillon. The flesh of guinea-pigs as well as that
of other experiment-animals is used instead of beef in the prep-
aration of bouillon, for the growth of special germs.
The extracts of different organs have been added to the
various media for experimentation.
60
ESSENTIALS OF BACTERIOLOGY,
CHAPTER X.
INOCULATION OF GELATINE AND AGAR.
Glass Slide Cultures. Formerly the gelatine was poured on
little glass slides such as are used for microscopical purposes,
and after it had become hard, inoculated in separate spots as
with potatoes.
Fig. 29.
Manner of holding tubes for inoculation: a, tube with material; b, tube to be
inoculated ; c, cotton plugs. (After Woodhead and Hare.)
Test Tube Cultures. The gelatine, agar, or blood serum having
solidified in an oblique position, is smeared on the surface with
INOCULATION OF GELATINE AND AGAR. 61
the material and the growth occurs, or the medium is punctured
with a stab of the platinum rod containing the material. The
first is called a stroke or smear culture, the second a stab or thrust
culture. In removing the cotton plugs from the sterile tubes to
carry out the inoculation, the plugs should remain between the
lingers in such a way that the part which comes in contact with
the mouth of the tube will not touch anything.
After the needle has been withdrawn the plugs are re-inserted
and the tubes labelled with the kind and date of culture.
Plate Cultures. Several tubes of the culture medium are
made liquid by heating in water bath, and then inoculated with
the material as follows . A looped platinum needle is dipped
into the material and then shaken in the tube of liquid media,
(gelatine, agar, etc.).
This first tube is called original. From this three drops (taken
with the looped platinum rod) are placed in a second tube, the
rod being shaken somewhat in the gelatine or agar ; this is
labeled first dilution (a colored pencil is useful for such markings).
From the first dilution three drops are taken into a third tube,
which becomes the second dilution. (Fig. 29.)
The plugs of cotton must be replaced after each inoculation,
and during the same must be carefully protected from contami-
nation.
To hasten the procedure and lessen the danger of contamina-
tion, the tubes can be held in one hand aside of each other, each
Fig. 30.
Manner of holding plugs.
plug opposite its tube. They are now ready for spreading on
glass plates.
62
ESSENTIALS OF BACTERIOLOGY.
Glass Plates.
Fig. 31.
z^Ek?
The larger the surface over which the nutrient
medium is spread the more isolated will
the colonies be ; window glass cut in rec-
tangular plates Gx4 inches in size is used ;
about ten such plates are cleaned with dry
towel and placed in a small iron box or
wrapped in paper ; and sterilized in the
hot-air oven at a temperature of 150° C.
for ten minutes. (Fig. 31.) When the
plates have cooled they are placed upon
an apparatus designed to cool and so-
lidify the liquid media, which is now
poured upon the plates from the inocu-
lated test-tubes.
Nivellier Leveling and Cooling Apparatus. Ice and water
are placed in a shallow round glass tray ; on top of this a square
plate of glass, upon which the culture plate is placed, and cov-
ering this a bell-glass.
The whole is upon a low, wooden tripod, the feet of which
can be raised or lowered, and a little spirit-level used to adjust
it. (Fig. 32.) The glass plate taken out of the iron box is placed
under the bell-glass. The tube containing the gelatine is held
in the flame a second to singe the cotton plug to free it from dust,
and the plug removed, the edges of the tube again flamed, the bell-
glass lifted, and the inoculated gelatine carefully poured on the
plate, leaving about one-third inch margin from the borders ; the
Iron box for glass plates.
Fig. 32.
Nivellier leveling and cooling apparatus.
lips of the tube being sterile can be used to spread the media
INOCULATION OF GELATINE AND AGAR.
63
evenly. If the plate is at all cool, the fluid will solidify as it is
being spread. The glass cover is replaced until the gelatine or
agar is quite solid to prevent contamination.
Fig. 33.
Moist chamber with plates on benches.
When the gelatine is congealed, the plate is placed upon a
little glass bench or stand in the moist chamber.
The Moist Chamber Prepared Out of Two Glass Dishes, as for
the Potato- Cultures. • The glass benches are so arranged that
one stands upon the other. In order to avoid confusion, a slip
of paper with a number written on it is placed on the bench be-
neath each plate. As the original or first plate would have the
colonies developed in greatest profusion, it is placed the first
day on the topmost bench ; but, since the colonies would be
likely to overrun the plate and allow the gelatine to drop on the
lower plates, it is best, as soon as evidences of growth appear,
to place it below, and watch the third plate or second dilution
for the characteristic colonies, forgetting not all this time to
change the numbers accordingly.
The date of culture and the name can be written upon the
moist chamber.
Petri Saucers. Agar hardens very quickly, even without any
especial means for cooling, and it does
not adhere very well to the glass. There-
fore it is better to follow the method of
Petri and use little shallow glass dishes,
one covering the other. They are first
sterilized by dry heat, and then the in-
oculated gelatine or agar is poured into
the lower dish, covered by the larger one,
and placed in some cool place, different saucers being used for
each dilution.
Fig. 34.
Petri saucers.
64 ESSENTIALS OF BACTERIOLOGY.
This method is very useful for transportation ; the saucers
can be viewed under microscope similar to the glass plates, and
have almost entirely superseded them.
Esmarch's Tubes, or Rolled Cultures. This method, especially
used in the culture of anaerobic germs, consists in spreading the
inoculated gelatine upon the inner walls of the test tube in
which it is contained and allowing it to congeal. The colonies
then develop upon the sides of the tube without the aid of
other apparatus. The method is useful whenever a very quick
and easy way is required. The rolling of the tube is done under
ice-water or running water from the faucet. The tube is held
a little slanting, so as to avoid getting too much gelatine around
the cotton plug.
The tubes can be placed directly under the microscope for
further examination of the colonies.
It is almost impossible to separate certain organisms, such as
the tubercle bacillus and pneumococcus, from mixed cultures
by ordinary plate methods, and the plan of producing the dis-
ease in animals by inoculation, and then obtaining the organ-
ism in pure culture, has to be employed.
Spored organisms may also be separated from others by boil-
ing the mixture for a few minutes, when all the non-spored
forms will perish, and only the spores remain to germinate
subsequently.
CHAPTER XL
THE GROWTH AND APPEARANCES OF COLONIES.
Macroscopic. Depending greatly upon the temperature of
the room, which should be about 65° C, the colonies develop
so as to be visible to the naked eye in two to four days. Some
require ten to fourteen days, and others grow rapidly, covering
the third dilution in thirty-six hours. The plate should be
looked at each day.
The colonies present various appearances, from that of a
small dot, like a fly-speck, to that resembling a small leaf.
GROWTH AND APPEARANCE OF COLONIES.
65
Some are elevated, some depressed, and some, like cholera, cup-
shaped — umbilicated.
Fig. 35.
Staphylococcus pyogenes aureus : colony two days old, seen upon an agar-agar plate;
X 40 (Heim).
Then they are variously pigmented. Some liquefy the gela-
tine speedily, others not at all. The appearances of a few are
so characteristic as to be recognized at a glance.
Microscopic. We use a low-power lens, with the abbe nearly
shut out, that is the narrowest blender. The stage of the
microscope should be of such size as to carry a culture plate
easily upon it.
The second dilution or third plate is usually made use of, that
one containing the colonies sufficiently isolated.
These isolated ones should be sought for, and their appearances
well noticed.
There may be two or three forms from the same germ, the
difference due to the greater or less amount of oxygen that they
have received, or the greater or less amount of space that they
have had to develop in.
The microscopic picture varies greatly ; now it is like the
gnarled roots of a tree, and now like bits of frosted glass ; the
pictures are very characteristic, and the majority of bacteria
can be told thereby. (Fig. 32.)
66
ESSENTIALS OP BACTERIOLOGY.
Impression or "Klatsch" Preparations. In order to more
thoroughly study a certain colony and to make a permanent
specimen of the same, we press a clean cover-glass upon the
particular colony, and it adheres to the glass. It can then be
Microscopic appearances
of colonies.
Fig. 37.
Klatsch preparations.
stained or examined so. The Germans give the name oi
"Klatsch" to such preparations. Many beautiful pictures can
be so obtained.
Fishing. To obtain and examine the individual members oi
a particular colony the process of fishing, as it is called, is
resorted to.
The colony having been placed under the field of the micro-
scope, a long platinum needle, the point slightly bent, is passed
between the lens and the plate so as to be visible through the
microscope, then turned downward until the colony is seen to
be disturbed, and the needle is dipped into the colony. This
procedure must be carefully done, lest a different colony be
disturbed than the one looked at, and an unknown or unwanted
germ obtained.
After the needle has entered the particular colony, it is with-
drawn, and the material thus obtained is further examined by
staining and animal experimentation. The bacteria are then
again cultivated by inoculating fresh gelatine, making stab and
stroke cultures.
CULTIVATION OF ANAEROBIC BACTERIA
67
It is necessary to transfer the bacteria to fresh gelatine about
every six weeks, lest the products of growth and decay given
off by the organisms destroy them.
CHAPTER XII.
CULTIVATION OF ANAEROBIC BACTERIA.
Special methods are necessary for the culture of the anaerobic
variety of bacteria in order to procure a space devoid of oxygen.
Liborius's High Cultures. The tube is filled about
| full with gelatine, which is then steamed in a water
bath and allowed to cool to 40° C, when it is inocu-
lated by means of a long platinum rod with small
loop, the movement being a rotary vertical one, and
the rod going to the bottom of the tube.
The gelatine is next quickly solidified under ice ;
very little air is present. The anaerobic germs will
grow from the bottom upward, and any aerobins
present will develop first on top, this method being
one of isolation.
From the anaerobic germs grown in the lower part,
a stab culture is made into another tube containing
f gelatine, the material being obtained by breaking
test-tube with the culture.
Hesse's Method. A stab culture having been made
with anaerobic germs, gelatine in a semi-solid condi-
tion is poured into the tube until it is full, thus dis-
placing the air. (Fig. 39.)
Esmarch's Method. Having inoculated a tube with Libo™s's
the microbe, the gelatine is rolled out on the walls of
the tube, a " roll culture," and the rest of the interior filled with
gelatine, the tube being held in ice water. The colonies develop
upon the sides of the tube and can be examined microscopically.
Gases like Hydrogen to replace the Oxygen. Several ar-
rangements for passing a stream of hydrogen through the
culture: —
68
ESSENTIALS OP BACTERIOLOGY.
Frankel puts in the test tube, a rubber cork containing two
glass tubes, one reaching to the bottom and connected with a
hydrogen apparatus, the other very short, both bent at right
angles. When the hydrogen has passed through ten to thirty
minutes, the short tube is annealed and then the one in connec-
tion with the hydrogen bottle, and the gelatine rolled out upon
the walls of the tube. (Fig. 40.) Hiippe uses eggs as described
in Chapter IX.
Fig. 39. Fig. 40. Fig. 41.
Hesse's method.
Frankel's method.
Buchner's method.
Use of iErobic Bacteria to remove the Oxygen. Roux inocu-
lates an agar tube through a needle thrust after which semi-
solid gelatine is poured in on top. When the gelatine has solidi-
fied, the surface is inoculated with a small quantity of bacillus
subtilis or some other aerobic germ. The subtilis does not allow
the oxygen to pass by, appropriating it to itself.
CULTIVATION OF ANAEROBIC BACTERIA
69
Buchner's Method. The test tube containing the culture is
placed within a larger tube, the lower part of which contains
an alkaline solution of pyrogallic acid.
The tube is then closed with a rubber
stopper. (Fig. 41.)
Botkin's Method. Petri dishes, un-
covered, are placed on a rack under a
large bell-jar, into which hydrogen gas
is conducted. Alkaline pyrogallic acid
is placed in the upper and lower dishes
to absorb what oxygen remains.
Wright's Method. Applicable to
both fluid and solid media. After in-
oculating the test-tube, the plug, which
must be of absorbent cotton, is cut off
flush with the extremity of the tube
and pushed inward for a distance of 1
cm. It is then impregnated with 1 c.c.
of a watery solution of pyrogallic acid
and 1 c.c. of 5 per cent, sodium hydrate
solution. A tightly fitting rubber stop-
per is inserted, and the tube is then
ready for incubation.
Park's Method. An Erlenmeyer
flask containing the medium to be used
is boiled in a water-bath from ten to
fifteen minutes to drive off dissolved
oxygen, quickly cooled, and inoculated.
Hot melted paraffme is then poured
into the flask, which forms a layer over
the medium and on congealing pro-
vides an air-tight seal which does not
adhere to the glass so closely as to pre-
vent the escape of any gases formed by the bacterial growth.
Fin. 42.— Wright's method
for the cultivation of anae-
robes.
70 ESSENTIALS OF BACTERIOLOGY.
CHAPTER XIII.
INFECTION.
How Bacteria Cause Disease. Many theories have been
put forward to explain the action of bacteria in causing dis-
ease, but only a few of the more important ones can be taken
up here.
What are the Conditions Necessary to Produce Infection?
First. As to the Infective Agent. The organism must have the
power to produce disease. It must, in other words, be pathogenic. A
non-pathogenic bacterium under certain conditions may cause
disease, but this is not an infectious disease ; it is rather a tox-
emia, and is due to the absorption of poisons generated outside
of the body. It must be parasitic — have the power of growing
within the body of an animal.
Essentially an infectious disease is a toxemia, because it
depends upon poisons or toxins produced in the body. Para-
sitic or infectious bacteria cause disease by growing in the animal
organism and generating products therein which are toxic.
Saprophytic bacteria grow outside of the animal organism in dead
matter, decaying particles, etc., and they may give rise to prod-
ucts which also are toxic to the animal economy.
Second. The toxins or poisons elaborated must be present in
sufficient amount. Undoubtedly each animal organism is a law
unto itself in regard to the amount of poison it will tolerate
before disease is actually produced. The period of incubation
can be explained on the supposition that the germ requires so
much time to elaborate the amount of toxin necessary. This
time period varies with different organisms, some carrying the
toxin with them at the time of entry.
Third. The animal infected must be susceptible. Susceptibility
varies in different species of animals, in different members of
the same species, in the same individual at different times, and
in the same individual to the different forms of disease germs.
Susceptibility may be natural to the race, it may be acquired, it
may be inherited. Mice are naturally susceptible to anthrax.
Acquired susceptibility occurs upon exposure to conditions
INFECTION. 71
which lower vitality, as hunger, cold, advanced age, and sur-
gical shock. Inherited susceptibility is a less important factor
now than formerly. Many diseases were at one time considered
inherited which now are known to be acquired during the life-
time, of an individual. Still, certain physical characteristics,
such as narrow chest, mouth-breathing, etc. — clearly inheritable
characters — predispose to disease. Given a susceptible individ-
ual and an infective microorganism producing toxins in suffi-
cient amount, disease is certain to result.
Local Effects of Bacteria. By mechanical obstruction from
rapid growth, thrombosis, with its consequences, may occur.
Destruction of a part of the cells of a tissue with necrosis can
arise from irritation, as from a foreign body.
General Effects. Sapremia, when toxic products of local
suppuration are absorbed into the system. Septicemia, when the
infective agent itself enters the blood-stream and causes general
disturbance.
Suppurative bacteria are those which give rise to inflamma-
tion and suppuration locally at the point of entrance, and
secondarily through metastasis. Any organism may cause
suppuration, but a certain number are peculiarly inclined to
give rise to pus, and are known as pyogenic organisms.
Infective bacteria are as a rule specific, the particular toxin
having a specific action and causing a disease peculiar to the
microorganism. Thus typhoid fever is a disease distinctly
different from tuberculosis ; the infective organisms are distinct
and the poisons they produce have specific characteristics.
The Nature of Toxins. Very similar to the venom of ser-
pents; highly poisonous in minute doses (y^ gramme of
tetanus toxin will kill a horse weighing 600 kilos (1200 pounds,).
At first toxins were called ptomaines, or cadaveric alkaloids ; but
this term is applied now to such poisons as have a basic nature
and arise in decomposing meat, cheese, and cream as a result
of chemical change in the material, the bacteria causing the
change. Then they were called toxalbumins, and were supposed
to belong to an albumin series; but when the bacteria are grown
in non-albuminous media the toxins correspond more in their
chemical composition to a ferment, and therefore it is supposed
72 ESSENTIALS OF BACTERIOLOGY.
that the albumin part of the toxin is furnished by the blood or
albuminous media in which it is formed. The term toxin is to
be preferred in speaking of bacterial poisons.
Toxins may be of two sorts : (a) Chiefly within the bodies of
the bacteria, so that they are set free by the disintegration of
the organisms. This group comprises most of the pathogenic
bacteria and must be combatted by the use of antibacterial sera.
(b) The poisons seem to be excreted by the bacteria and are
found in the surrounding medium. Antitoxic sera are applica-
ble to this group, which includes the bacilli of diphtheria and
tetanus. Welch has suggested that even bacteria which do not
appear to form toxins in artificial cultures may do so in the
human body. In the effort to adapt themselves to their
environment and resist the hostile agencies of the body they
produce the poisons we call toxins. (For method of produc-
tion of an antitoxin, see article on Diphtheria.)
CHAPTER XIV.
IMMUNITY.
Immunity, as distinguished from susceptibility, is merely a rela-
tive term, as no animal is absolutely immune under all condi-
tions. It is merely less susceptible, and some animals are by
nature or can by artificial means be rendered so slightly sus-
ceptible that to all practical purposes they are immune— that is,
capable of resisting an attack of the particular disease against
which they are said to be immune.
Natural Immunity. The goat and dog are considered
naturally immune to tuberculosis. Algerian sheep are resistant
to anthrax, other varieties are susceptible.
The field mouse is susceptible to glanders, the white mouse
is ordinarily immune. House mice are susceptible to mouse
septicemia, field mice are immune.
Acquired Immunity. Immunity can be acquired in many
ways. Active and passive immunity are varieties.
Active immunity can be acquired from an attack of the disease;
IMMUNITY. 73
such infectious diseases as measles, scarlatina, and whooping-
cough usually confer immunity from future attacks. Some
diseases render the individual immune for only a short period.
Immunity from Inoculation with Attenuated or Weak-
ened Cultures of Bacteria. Vaccination is an example. Haff-
kine's cholera vaccines and Pasteur's vaccines of anthrax and
chicken cholera are likewise examples of this method.
Attenuation is produced as follows : Successive cultivation in
artificial media destroys the virulence of bacteria. Old cultures
are less virulent than fresh ones. Virulence is lessened by
passing the cultures through animals that are less susceptible
or entirely immune. The cautious use of chemicals and sun-
light lessens virulence. Heat is an effective agent. An anthrax-
culture exposed to a temperature of 42.6° C. for twenty days
will prove destructive only to animals no larger than mice.
Prolonged exposure to oxygen weakens the germs.
Immunity Through Inoculations of Small Doses of very
Virulent Microorganisms. A graduated resistance to the
disease is reached somewhat after nature's method. By succes-
sive inoculations with increased doses of the virus an immunity
is often reached sufficient to withstand ten times the lethal
dose. A poison-habit is thus acquired.
Increased Virulence is produced as follows : The cultures
may be greatly increased in virulence by successive cultivation
through animals, and gradually changing from smaller animals
to larger, until an amount of the culture that at the outset
would not destroy a guinea-pig becomes finally virulent for
chickens and dogs.
Immunity Through Injections of the Sterilized Products of
Bacteria. Cultures sterilized by heat or nitration through
germ-filters still contain the chemical products of bacteria, the
toxins ; and when these are injected in gradually increased doses
the same immunity is obtained as with the bacteria themselves.
Passive Immunity. The blood-serum and tissues generally
of animals rendered immune in the" ways described above, when
injected into susceptible animals render them immune against
the same infection. This has been called passive immunity, but
there is no strong reason why this term should be used. The
74 ESSENTIALS OP BACTERIOLOGY.
blood-serum of immune animals is simply another means for
immunization. It is less permanent than the other forms of
immunization, but it appears very soon after the injection, and
in a modified form has a curative action even when the symp-
toms of the infection are already present in the system.
Inherited Immunity. An immunity to disease acquired dur-
ing the lifetime of the parents is probably never transmitted
to the offspring, though the mother may transmit a temporary
immunity to the child in utero or the child itself may have
been subjected to the infection at the same time with its
mother. But this cannot be called inherited.
Theories of Immunity.
Several older theories need only to be mentioned, as they are
no longer tenable. They are the exhaustion theory of Pasteur,
the retention theory, and the humeral theory. At present
modifications of MetschnikofT's phagocytic theory and Ehrlich's
side-chain theory seem the most plausible.
Phagocytic or Cellular Theory. — Metschnikoff elaborated this
after his study on inflammation. Phagocytosis occurs in
animals when subjected to the action of an irritant. The leu-
cocytes are attracted to the injured spot and envelop the irri-
tating substance, be it bacteria or dead matter. The theory
given out at first was that if the leucocytes conquer the bacteria,
immunity results; if the bacteria eat up the leucocytes, disease
occurs.
Modified to suit other conditions, as, for instance, the germi-
cidal properties of serum freed from its cellular elements,
Metschnikoffnow states that at times phagolysis — that is, breaking
up or solution of the phagocytes — takes place, and the fluids in
which these cells are dissolved become charged with the powers
originally present in the phagocytes. Chemotaxis is the term
applied to the attraction of bacteria for the leucocytes, and is
supposed to be chemical in its nature. The phagocytic cells
comprise : (o) The polymorphonuclear leucocytes of the blood,
termed microphages, and (b) a group called macrophages which
includes all other cells having phagocytic properties, such as
leucocytes other than the polymorphonuclears, endothelial
cells, and connective-tissue corpuscles. When these cells are
IMMUNITY. 75
injured they set free their digestive ferments, known as micro-
cytases and macrocytases respectively, which correspond to the
alexins of Ehrlich.
EhrllcWs Side-chain Theory. This derives its name from the
fact that it presents an analogy to what happens in the benzol
ring of organic chemistry when its replaceable atoms of hydro-
gen are substituted by "side chains" of more or less complex
nature. The molecule of protoplasm is supposed to consist of
a central atom group provided with a large number of side
chains which subserve the vital processes of the molecule by
combining with other organic molecules. These side chains
are called receptors and are of many different kinds so as to fit
them for combination with many different varieties of extrane-
ous groups. Bacterial toxins contain two groups : (1) the
haptophores, by which the toxin molecule can become joined
to the cell, and (2) the toxophores, by virtue of which it can
attack the protoplasm after having been fixed to it by the hapto-
phore. If the attack on the molecule is not too severe, this is
stimulated into overactivity and throws out an abnormal num-
ber of receptors, some of which (the haptins) become detached
and are capable of uniting with free haptophores and prevent-
ing their combination with the protoplasm of the molecule.
In other words, they represent the antitoxin.
Bacteriolysis is the destruction of the bacterial cells by the
blood-serum, and is probably effected in a somewhat different
manner. Antibacterial sera are effective through the combined
activities of a destructive element, the "complement" (alexin
or cytase), and an "immune body" (amboceptor) which serves
the function of joining the complement to the bacterial mole-
cule. These two bodies differ markedly in their properties — for
example, the complement is destroyed at 60° C, while the
immune body is very resistant.
It is not stated what cells are the sources of these various
anti-bodies, but probably any cell capable of being attacked by
a toxin is also capable of responding by the production of anti-
substances.
Lysins. The substances producing destruction of bacteria
are called lysins. Normal blood-serum is bacteriolytic to a
76 ESSENTIALS OF BACTERIOLOGY.
slight degree, but during infection produces lysins specific for
the germ in question.
Agglutinins. These are bodies formed in the blood-serum in
response to the stimulation of certain bacteria, such as the
typhoid bacillus, Bacillus coli communis, Micrococcus meli-
tensis, the bacillus of dysentery, the cholera spirillum, etc.
When such a serum is added to cultures of the particular
organism concerned, the bacteria become clumped in motion-
less masses. A modified form of agglutination in which long
strings of bacteria are formed is known as the " thread " reac-
tion.
Precipitins. Animals immunized to certain bacteria or to
albumins of different sorts form bodies which cause the blood-
serum to give a precipitate when added to cultures of these
organisms or fluids containing the specific albumen. The
phenomenon has found forensic application in the identifica-
tion of blood-stains.
CHAPTER XV.
EXPERIMENTS UPON ANIMALS.
The smaller rodents and birds are the ones usually employed
for inoculation, as rabbits, Guinea-pigs, rats aud mice, and
pigeons, and chickens ; sometimes monkeys. These are pre-
ferred, because easily acted upon by the various bacteria, readily
obtained, and not expensive.
The white mouse is very prolific and easily kept, and is there-
fore a favorite animal for experiment. It lives well upon a little
moistened bread. A small box, perforated with holes, is filled
partly with sawdust, and in this ten to twelve mice can be kept.
When the female becomes pregnant she should be removed to
a glass jar until the young have opened their eyes, because the
males, which have not been raised together, are apt to attack-
each other.
Guinea-pigs. When Guinea-pigs have plenty of light and
air they multiply rapidly. Therefore it is best to have them in
EXPERIMENTS UPON ANIMALS. 77
some large stall or inclosure. They can be fed upon all sorts of
vegetables and grasses, and require but little attention.
Methods of Inoculation. L Inha lation.— Imitating the natural
infection, either by loading an atmosphere with the germs in
question or by administering them with a spray.
II. Tlirougk Skin or Mucous Membrane.
III. With the Food.
Method of Cutaneous Inoculation. The ear of mice is best
suited for this procedure. A small abrasion made with the
point of a lancet or needle, which has been dipped in the virus.
The animal is then separated from the rest and placed in a
glass jar, which is partly filled with sawdust and covered with
a piece of wire-gauze.
Subcutaneous. The root of the tail of mice is used for this
purpose. The hair around the root of the tail is clipped off,
and with a pair of scissors a very small pocket is made in the
subcutaneous connective tissue, not wounding the animal any
more than absolutely necessary, avoiding much blood. The
material is placed upon a platinum needle and introduced into
the pocket, solid bodies, with a forceps.
To hold the mouse still while the operation is going on a
little cone made of metal is used. The mouse just fits in here.
There is a slit along the top in which the tail can be fastened,
and thus the animal is secure and immobile.
Intravenous Injections. Rabbits are very easily injected
through the veins. Mice are too small.
The ear of the rabbit is usually taken. It is first washed with
1-2000 bichloride, which not only disinfects, but also makes the
vessels appear more distinct. The base of the ear is compressed
to swell the veins. Then a syringe, like the one used for the
injection of " tuberculine," a Koch syringe, which can be easily
sterilized, is filled with the desired amount of virus and slowly
injected into any one of the more prominent veins present.
(Fig. 43.)
Intra-peritoneal Injection. This is used with Guinea-pigs
mostly. The abdominal wall is pinched up through its entire
thickness, and the needle of the syringe thrust directly through,
so that it appears on the other side, then the fold let go, the
needle withdrawn just far enough so as to be within the cavity.
78
ESSENTIALS OF BACTERIOLOGY.
Inoculation in the Eye. The anterior chamber and the cornea
are the two places used. The rabbit is fixed upon a board;
the eyelids held apart and head held still by an assistant. A
small cut is made in the cornea, a few drops of cocaine having
first been introduced in the eye. The material is passed
through the opening with a small forceps, and with a few strokes
of a spoon it is pushed in the anterior chamber.
For the cornea a few scratches made in the corneal tissue
will suffice ; the material is then gently rubbed in.
Inoculation of the Cerebral Membranes. The skin and
Fig. 43.
Manner of making intravenous injections in the rabbit.
aponeurosis cut through where the skull is the thinnest. Then
the bone carefully trephined, and the dura exposed. In Babies
inoculation, the syringe containing the hydrophobic virus pierces
the dura and arachnoid, and the virus is discharged beneath the
latter.
Intra-Tracheal. The bacteria can be introduced directly into
the trachea, thus coming in contact with the lungs.
Intra-duodenal. — Cholera germs are injected into the intes-
EXPERIMENTS UPON ANIMALS. 79
tines after they have been exposed, by carefully opening the
abdomen. This is done in order to avoid the action of the
gastric juice.
Celloidin sacs of small size are sometimes used to introduce
living cultures of bacteria into the bodies of animals without
their coming into direct contact with the tissues.
Obtaining Material from Infected Animals. The animal
should be skinned, or the hairs plucked out, before it is washed,
at least the portion where the incision is to be made. Then the
entire body is washed in sublimate. Two sets of instruments
are required, one for coarser and one for finer work : the one
sterilized in the flame ; the other, to prevent being damaged,
heated in a hot air oven.
The animal, the mouse for example, is stretched upon a board,
a nail or pin through each leg, and the head fixed with a pin
through the nose. The skin is dissected awa}r from the belly
without exposing the intestines. Then the ribs being laid bare,
the sternum is lifted up, and the pericardium exposed. A pla-
tinum needle dipped into the heart after the pericardium has
been slit will give sufficient material for starting a culture. If
the other organs are to be examined, further dissection is made.
If the intestines were first to be looked at, they would be laid
bare first.
In this manner material is obtained, and the results of inocu-
lation noted.
Frequent sterilization of the instruments is desirable.
Koch's Rules in Regard to Bacterial Cause of Disease.
Before a microbe can be said to be the cause of a disease, it
must —
First. Be found in the tissue or secretions of the animal suf-
fering from, or dead with the disease.
Second. It must be cultivated outside of the body on artificial
media.
Third. A culture so obtained must produce the disease in
question when it is introduced into the body of a healthy
animal.
Fourth. The same germ must then again be found in the
animal so inoculated.
PART II.
SPECIAL BACTERIOLOGY.
CHAPTER I.
NON-PATHOGENIC BACTERIA.
Special Bacteriology. Under this head the chief character-
istics of individual bacteria will be detailed, pathogenic and non-
pathogenic being the main divisions. It is usual to describe the
non-pathogenic first.
Non-Pathogenic Bacteria. There are 300 varieties of non-
pathogenic bacteria, and the list is continually being added to.
Bacillus Prodigiosus. (Ehrenberg.) This bacillus, formerly
called a micrococcus, is very common, and one of the first
noticed, because of the lively red color it forms on vegetables
and starchy substances. "The bleeding host," miracles being
due to it.
Form. — Short rods, often in filaments, without spores.
Immobile. — Has no automatic movements.
Facultative anaerobic, that is, it can grow without air ; but
the pigment requires oxygen to show itself.
Growth. Gelatine. Liquefy rapidly.
Colonies. — At first white, round points with smooth edge
appearing brown under microscope, but soon changing to red.
Stab Cultures. — The pigment develops on the surface, the
growth occurring all along the line.
Potato is well suited to the growth, the pigment developing
after twelve hours. Agar and blood serum growths do well.
Temperature.— Grows best at 25° C.
Varieties. — By exposure to heat of brood-oven during several
generations the power to produce pigment can be temporarily
abolished.
(80)
NON-PATHOGENIC BACTERIA
81
Auto-
red
e Pigment. — A pigment-forming body is created by the
bacillus, and the action of oxygen upon it produces the color.
It is insoluble in water, slightly soluble in alcohol and ether ;
acids fade it, alkalies restore the color. The pigment resembles
fuchsin, presenting the same metallic lustre.
Gases. — A trimethylamin odor arises from all cultures.
Stain. — Takes all anilin dyes easily in the ordinary way.
Bacillus Indicus. (Koch.) Syn. Micrococcus Indicus.
Origin. — Found in the stomach of an Indian ape.
Form. — Short rods with rounded ends. No spores,
matic movements present ; faadtative ancerobin.
Growth. Gelatine. — Liquefy rapidly.
Colonies.— Round, or oval, granular margins ; brilliant
pigment.
Stab Cultures. — On the surface the pigment shows itself.
Grows well on other media.
Temperature.— Grows best at 35° C.
Action on Animals. — In very large quantities, if injected into
the blood, a severe and fatal gastro-
enteritis can be produced.
Stain.— Takes all dyes.
Bacillus Mesentericus Vulgatus.
The common potato bacillus of
Fliigge.
Habitat. — Surface of the soil, on
potatoes, and in milk.
Form. — Small thick rods with
rounded ends, often in pairs.
Properties. — Yery motile ; pro-
duce abundant spores ; liquefy
Fig. 44.
gelatine ; diastolic action.
Colony of Bacillus Mesentericus
Vulgatus.
Growth.— Rapid.
Plate Colonies.— Round, with transparent centre at first, then
becoming opaque. The border is ciliated ; little projections
evenly arranged.
Potato. — A white covering at first, which then changes to a
rough brown skin ; the skin can be detached in long threads.
Temperature.— Spores at ordinary temperatures.
6
82
ESSENTIALS OF BACTERIOLOGY.
Spores. — Are very resistant ; are colored in the manner de-
scribed in first part of the book for spores in general.
Bacillus Megaterium (de Bary).
Origin.— Found on cooked cabbage and garden soil.
Form. — Large rods, four times as long as they are broad,
2.5 fi. Thick rounded ends. Chains with ten or more members
often formed ; granular cell contents.
Properties. — Abundant spore formation ; very slow movement ;
slowly dissolves gelatine.
Growth.— Strongly aerobic ; grows quickly, and best, at a tem-
perature of 20° C.
Plate Colonies.— Small, round, yellow points in the depth of
the gelatine. Under microscope irregular masses.
Fig. 45.
Bacillus Megaterium, with spores.
Stab Culture. — Funnel-shaped from above downwards.
Potato. — Thick growth with abundance of spores.
Bacillus Ramosus.
Syn. Bac. Mycoides (Fliigge). Wurzel or root bacillus.
Origin.— In the upper layers of garden or farm grounds, and
in water.
Form.— Short rods, with rounded ends, about three times
as long as they are thick ; often in long threads and chains,,
NON-PATHOGENIC BACTERIA. 83
Properties. — Large, shining, oval spores ; a slight movement ;
liquefy gelatine.
Growth.— At ordinary temperatures, with plentiful supply of
air.
Plate Colonies. — Look like roots of an old tree gnarled together,
radiating from a common centre. On surface soon liquid.
Stab Culture.— Soon sl growth occurs along the needle track,
and the whole resembles a pine tree turned upside down. The
gelatine then becomes liquid, a thin skin floating on top, and
small flakes lying at the bottom.
Stroke Culture. — Feathery resemblance is produced.
Staining. — Spores stain readily with the ordinary spore stain.
Bacterium Zopfii. (Kurth.)
Origin. — Intestines of a fowl.
Form. — Short thick rods forming long threads coiled up,
which finally break up into spores, which were once thought to
be micrococci.
Properties. — Very motile ; does not dissolve or liquefy gela-
tine.
Growth. — In thirty hours abundant growth; cerobic; grows
best at 20° C.
Plates. — Small white points which form the centre of a very
fine netting. With high power this netting is found composed
of bacilli in coils, like braids of hair.
Excellent impress or "Klatsch" preparations are obtained
from these colonies.
Staining. — Ordinary dyes.
Bacillus Subtilis. (Hay Bacillus.) Ehrenberg.
Origin.— Hay infusions ; found also in air, water, soil, faeces,
and putrefying liquids. Very common, often contaminates
cultures.
Form. — Large rods, three times as long as broad ; slight
roundness of ends, transparent; seldom found singly; usually
in long threads. Flagella are found on the ends. Spores of
oval shape, strongly shining, very resistant.
Properties.— Yery motile ; dissolves gelatine.
Growth. — Rapid ; strongly eerobic.
Plate. — Round, gray colonies, with depressed white centre.
84 ESSENTIALS OF BACTERIOLOGY.
Under microscope the centre yellow ; the periphery like a wreath,
with tiny little rays projecting ; very characteristic.
Potato. — A thick moist skin forms in twenty-four hours.
Staining.— Rods, ordinary stain, spores, spore stain.
It is easily obtained by covering finely cut hay with distilled
water, and boiling a quarter of an hour. Set aside forty-eight
hours. A thick scum will show itself on the surface composed
of the subtilis bacilli, whose spores alone have survived the heat.
Bacillus Spinosus. (Liideritz.)
Called spinosus because small spine-like processes are formed
by the colonies.
Origin. — In the juices of the body of a mouse and guinea-pig
which were inoculated with garden earth.
Form.— Large rods, straight, some slightly bent, ends rounded ;
often in long threads.
Properties. — Large spores, the bacillus enlarging to allow the
spores to develop ; very motile ; gelatine slowly liquefied. A
gas is formed in the culture having an odor like Swiss cheese.
Growth. — The growth occurs at ordinary temperatures only
when the oxygen is excluded. Very strongly anaerobic. Glu-
cose added to the gelatine (1 to 2 per cent.) increases the nutri-
tive value.
Colonies in roll cultures and high stab cultures appear as little
spheres surrounded by a zone of liquefied gelatine. In the
deeper growths thorn-like projections or spines develop pro-
ceeding from a gray-colored centre.
Staining.— With ordinary methods. This bacillus, being
strongly anaerobic, must be cultured with the usual care taken
with anaerobins.
Some Bacteria found in Milk. Bacillus Acidi Lactici.
(Huppe.) Belongs to the same group as the Bacillus coli com-
munis.
Origin. — In sour milk.
Form.— Short thick rods, nearly as broad as they are long,
usually in pairs.
Properties.— Immotile. Spores large shining ones. Do not
liquefy gelatine. Breaks up the sugar of milk into lactic acid
and carbonic acid gas, the casein being thereby precipitated.
Growth.Siow ; is facultative anaerobic. Grows first at 10° C.
NON-PATHOGENIC BACTERIA. 85
Plate Colonies.— First small white points, which soon look like
porcelain, glistening. Under microscope the surface colonies
resemble leaves spread out.
Stab Culture. — A thick dry crust with cracks in it forms on the
surface after a couple of weeks.
Attenuation. — If cultured through successive generations, they
lose the power to produce fermentation. Several other bacteria
will give rise to lactic acid fermentation ; but this especial one
is almost constantly found, and is very wide spread.
In milk, it first produces acidity, then precipitation of casein,
and finally, formation of gases.
A bacillus described by Grotenfeldt, and called Bacterium
Acidi Lactici, forms alcohol in the milk. It was found in milk
in Bavaria.
Bacillus Butyricus. (Hiippe.)
This bacillus causes butyric acid fermentation.
Origin. — Found in milk.
Form.— Short and long thin rods wfth rounded ends; large
oval spores, seldom forming threads.
Properties. — Very motile ; liquefies gelatine rapidly ; produces
gases resembling butyric acid in odor. In milk it coagulates the
casein, decomposes it, forming peptones and ammonia, with a
bitter taste, and butyric acid fermentation. An alkaline reaction.
Growth. — Quickly, at 35° to 40° C, with oxygen. Spons very
resistant.
Colonies. Plate. — Small yellow points which soon run together,
becoming indistinguishable.
Stab Culture.— A small yellow skin formed on the surface with
delicate wrinkles ; cloudy masses in the liquefied portion.
Staining. — With ordinary stains.
Bacillus Amylobacter (Van Tiegham) ; or, Clostridium Buty-
ricum. (Prazmowsky.) (Vibrion butyrique of Pasteur.)
Origin. — Found in putrefying plant-infusions, in fossils, and
conifera of the coal period.
Form. — Large, thick rods, with rounded ends, often found in
chains. A large glancing spore at one end, the bacillus becoming
spindle-shape in order to allow the spore to grow ; hence the
name Clostridium.
86
ESSENTIALS OF BACTERIOLOGY.
Bacillus Amylobacter.
Fig. 46. Properties. — Yery motile ; gases arise
with butyric smell. In solutions of sugars,
lactates and cellulose-containing plants,
and vegetables, it gives rise to decomposi-
tions in which butyric acid is often formed.
Casein is also dissolved.
Like granulose, a watery solution of
iodine will color blue some portions of the
bacillus ; therefore it has been called amy-
lobacter.
Growth. — It is strongly anaerobic, and
fj\ 1 a # has not yet been satisfactorily cultivated.
Bacillus Lactis Cyanogenus. Bacterium
Syncyanum. (Hiippe.)
Origin.— Found in blue milk.
Form.— Small narrow rods about three times longer than they
are broad ; usually found in pairs. The ends are rounded.
Properties.— They are very motile ; do not liquefy gelatine ;
form spores usually in one end. A bluish-gray pigment is formed
outside of the cell, around the medium. The less alkaline the
media the deeper the color. It does not act upon the milk other-
wise than to color it blue.
Growth. — Grows rapidly, requiring oxygen. Colonies on plate.
Depressed centre surrounded by ring of porcelain-like bluish
growth. Dark brown appearance under microscope.
Stab Culture. — Grows mainly on surface ; a nail-like growth.
The surrounding gelatine becomes colored brown.
Potato. — The surface covered with a dirty blue scum.
Attenuation. — After prolonged artificial cultivation loses the
power to produce pigment.
Staining. — By ordinary methods.
Bacillus Lactis Erythrogenes. Bacillus of Bed Milk. (Hiippe
and Grotenfeldt.)
Origin. — Found in red milk, and in the faeces of a child.
Form. — Short rods, often in long filaments, without spores.
Properties. — Does not possess self-movement. Forms a nause-
ating odor ; liquefies gelatine. Produces a yellow pigment which
can be seen in the dark, and a red pigment in alkaline media,
NON-PATHOGENIC BACTERIA. 87
away from the light. In milk it produces the yellow cream on
top of the blood-red serum, or, fluid in the centre, and at the
bottom the precipitated casein.
Growth. — Grows rapidly in bouillon and on potatoes ; slower
on the other media; Plates. A cup-like depression in the centre
of the colony, with a pink coloration around it, the colony itself
being slightly yellow.
Stab Culture. — The growth mostly on surface. The gelatine
afterwards colored red and liquefied.
Potato. — A golden yellow pigment formed at 37° C, after six
days.
Examination of Milk in Stained Specimen. A drop of milk
diluted with a drop of distilled water is dried on the cover-glass
and fixed by heat. Chloroform methyl blue, prepared by mix-
ing 12 to 15 drops of saturated alcoholic solution of methyl blue
with 3 or 4 c.c. of chloroform, is used for staining. The chlo-
roform is then evaporated by exposing the specimen for a few
minutes to the air. Bacteria blue ; rest of field unstained.
Another method is to mix a drop of milk with two or three
drops of a 1 per cent, solution of sodium carbonate on a cover-
glass. Saponification of the fat occurs on heating the mixture
to evaporation. The preparation is then stained in the ordinary
manner.
Some Non-Pathogenic Bacteria found in Water. The bacteria
found here are very often given to producing pigments or phos-
phorescence, and are in great number. The more common ones
only will be described.
Bacillus Violaceus.
Origin. — Water.
Form.— A slender rod with rounded ends, three times as long
as it is broad, often in threads ; middle-sized spores.
Properties. — Very motile ; forms a violet-blue pigment, which
is soluble in alcohol, and depends upon oxygen for its growth.
Rapidly liquefies gelatine, but not agar.
Growth.— Grows fairly quick, is facultative anaerobic.
Cultures on Plate.— At first the colonies look like inclosed air-
bubbles. Low power shows irregular masses, with a centre
containing the pigment and a hairy-like periphery.
growth
—Terr fine little rods ; no
—Motile 5 fbnns
gelatine.
Rapid onhrafc ordinary
the periphery
the needle thrust ; the
MOX-PATHOOEfrlC BACTERIA, $$
Plate*.— Bound colonies, cup-shaped depressions, the solid
geb:ine that remains becoming colored witb greenish-yellow
Stab Culture.— ()n the surface, air-bobble depressions; toe
white colonies in the bottom of these depressions, and the solid
gelatine aroond the inoculation shining with the fluorescence.
Phosphorescent Bacteria, Six varieties of phosphorescent
bacteria bare been described ; they are found usually in sea*
water, or upon objects firing in the sea,
Bacillus Photphoreseens Indieus. (Fischer,)
Origin.— Tropical waters.
Form.— Thick rods, with rounded ends, sometimes forming
bag Uueada
Properties.— Very motile ; liquefying gelatine at a tempera*
tare of 2KP to Sf/"- C, with oxygen and a little moisture, and in
the dark, a peculiar electric-bine light develops a phosphores-
cence.
Growth.— Slowly ; must bare oxygen ; does not grow under
1CPC, ororerSCPC,
Plates,— Little round, gray points, which under low power
appear as green colonies with reddish tinge around them.
Cooked fish, when smeared upon the surface with a little of the
culture, show the phosphorescence most marked. Grows well
on potatoes aid Wood-serum.
Bacmusl^jsphcresceiis Indigents (Fischer,)
Origin.— Waters in the northern part of Germany, It differs
from the Indian bacillus, in that it grows at a temperature of
IP C, and does not develop upon potatoes or blood-serum,
BaciUiuPbospbcmseeiuGetidiiA (Fdrster,)
Origin — Surfaces of salt-water fish.
Form.— Short, thick rods, looking oral sometimes ; zoogkea
arc often formed.
Properties.— Motile ; does not liquefy gelatine ; a beautiful
phosphorescence from the surface of fish; it can be photographed
by its own light
Colonies.— Grows best between (P and 2<P C. ; grows slowly,
and mostly on the surface. The material must contain salt.
A bouillon made with sea-water, or 3 to 4 per cent, common
90 ESSENTIALS OF BACTERIOLOGY.
salt will suffice. The colonies appear as those of the Phospho-
rescens Indicus.
Fresh herring laid between two plates will often show phos-
phorescence in twent}r-four hours.
The other three varieties require glucose in the culture before
they give out any glow. They are Bacterium Pjlugeri, Bact.
Fischeri, and Bact. Baltlcum. They do not dissolve gelatine.
Several very indistinct species, found in waters from factories
and in some of the mineral waters, deserve yet to be men-
tioned. They have been given various names by observers ;
almost a new classification created. Such are the crenothrix,
cladothrix, and beggiatoa, which belong to the " higher bacteria."
Crenothrix Kuhniana. (Kabenhorst.) Long filaments joined
at one end ; little rod-like bodies form in the filaments ; and
these break up into spores.
Zoogloea are also formed by means of spores ; and these can
become so thick as to plug up pipes and carriers of water.
They are not injurious to health.
Cladothrix Dichotoma. (Cohn.) Very common in dirty
waters. The filaments branch out at acute angles, otherwise
resembling the crenothrix ; accumulations of ochre-colored
slime, consisting of filaments of this organism, are found in'
springs and streams.
Leptothrix Buccalis. In the mouth long filaments or threads
resembling bacteria are commonly found. At one end are seen
numerous cocci-like bodies, which some regard as spores. A
variety of this, or a nearly allied organism, is the most frequent
cause of noma or gangrenous stomatitis.
With iodin the leptothrix is colored yellow. At one time it
was considered the cause of "tartar" on the teeth, and often it
fills the crypts of the tonsils, forming there small masses which
are difficult to remove. Miller distinguishes three varieties —
Leptothrix buccalis innominata, maxima, and gigantea.
Beggiatoa Alba. (Vancher.) The most common of this
species. The distinction between this and the preceding species
lies in the presence of sulphur granules contained in the struc-
ture, and hence they are often found where sulphur or sulphides
exist ; but where the remains of organic life are decomposing
they can also be found.
NON-PATHOGENIC BACTERIA. 91
Several large spirilla and vibrio live in bog and rain-water,
but our space does not suffice to describe them.
Micro-organisms found in Urine. When freshly passed, urine
of a normal state contains no bacteria. By contact with the air
and the urinary passages exposed to air, a great number of yeast
moulds and bacteria soon accumulate in the fluid. Bacteria
also enter urine through the blood and during its secretion.
A number of bacteria have the property of converting urea
into carbonate of ammonia.
The urine should be centrifuged and the deposit then exam-
ined. The drying and fixing must proceed very slowly, since
otherwise crystals of salts will be precipitated and mar the
specimen.
Bacterium TJreae.
Origin. — Decomposed ammoniacal urine.
Form. — Thick, little rods, with round ends one-half as thick
as they are long.
Properties.— Does not dissolve gelatine ; changes urea into
carbonate of ammonia.
Growth.— At ordinary temperatures, very slowly. In two days
on gelatine very minute points, which in ten days have the size
of a cent. The colonies grow in concentric layers.
Micrococcus Urese. (Pasteur and Van Tiegham.)
Origin. — Decomposed urine and in the air.
Form. — Cocci, diplococci, and streptococci.
Properties.— Decomposes urea into carbonate of ammonia ;
does not liquefy gelatine.
Growth.— Grows rapidly, needing oxygen; can remain sta-
tionary below 0° C. ; growing again, when a higher temperature
\s reached.
Colonies on Plate.— On. the surface like a drop of wax.
Stab Cultures.— Looks like a very delicate thread along the
needle thrust.
Other bacteria are found in urine in various pathologic pro-
cesses, such as tubercle bacilli, typhoid bacilli, gonococci, and
other pyogenic organisms.
The Urobacillus liquefaciens, found by Schnitzler and Kro-
gius in cystitis, is supposed to stand in close relationship to this
disease.
92 ESSENTIALS OF BACTERIOLOGY.
Spirillum. Spirillum Rubrum. (Esmarch.)
Origin. — Body of a mouse dead with septicaemia.
Form. — Spirals of variable length, long joints, flagella on each
end ; no spores.
Properties. — Does not liquefy gelatine ; very motile ; produces
a wine-red pigment, which develops only by absence of oxygen.
Growth. — Can grow with oxygen, but is then colorless ; grows
very slowly ; ten to twelve days before any sign ; grows best at
37° C.
Gelatine Boll Cultures. —Small, round ; first gray, then wine-
red colonies.
.Stab Cultures. — A red-colored growth along the whole line ; it
is deepest below, getting paler as it approaches the surface.
Spirillum Concentricum. (Kitasato.)
Origin. — Decomposed blood.
Form. — Short spirals, two to three turns, with pointed ends ;
it has flagella on the ends.
Properties. — Very motile ; does not liquefy gelatine.
Growth. — Very slow ; mostly on the surface ; best at ordinary
temperatures.
Plates. — A growth of rings concentrically arranged, every
alternate one being transparent ; the furthest one from the
centre possessing small projections.
Stab Cultures. — Growth mostly on the surface.
Sarciua. Cocci in cubes or packets of colonies. A great
number have been isolated ; many producing very beautiful
pigments. The majority of them found in the air.
Sarcina Lutea. ( Seh roter. )
Origin. — Air.
Form. — Very large cocci in pairs ; tetrads and groups of
tetrads.
Properties. — Liquefies gelatine slowly ; produces sulphur-yel-
low pigment.
Growth. — Slowly ; at various temperatures ; strongly aerobic.
Plates. — Small, round, yellow colonies.
Stab Cultures.— -Grows more rapidly, the growth being nearly
all on the surface, a few separated colonies following the needle
NON-PATHOGENIC BACTERIA 93
thrust for a short distance. Agar, a very beautiful yellow,
along the stroked surface.
Sarcina Aurantica. — Flava, rosea, and alba are some of the
other varieties. Many are obtained from beer.
Sarcina Ventriculi. (Goodsir.) (Fig. 47.)
Origin. — Stomach of man and animals.
Form. — Colorless, oval cocci, in groups of eight and packets
of eight.
Fig. 47.
Sarcina ventriculi from stomach-contents ; X 530. (Van Valzah and Nisbet.)
Properties. —Does not liquefy gelatine ; shows the reaction of
cellulose to iodine.
Growth.— Rapid. At end of thirty-six hours, round, yellow
colonies, from which colorless cocci and cubes are obtained.
Habitat.— They are found in many diseases of the stomach,
especially when dilatation exists. Also normally; increased
when fermentation occurs.
Boas-Oppler Bacillus, also known as the Bacillus geniculatus.
Owing to the faculty possessed by this organism of growing in
the presence of amounts of lactic acid sufficient to check the
development of all other lactic-acid formers, it usually pre-
dominates in stomach-contents containing large amounts of
this substance. The parent type is composed of short rods,
but in the presence of considerable amounts of lactic acid these
94 ESSENTIALS OF BACTERIOLOGY.
change to a longer form which occurs singly or in long chains.
It is stained brown by Gram's iodine solution. The bacillus
affords confirmatory evidence of the presence of a new growth,
though it may occur in benign conditions.
CHAPTER II.
PATHOGENIC BACTERIA.
"We have divided this part into two portions.
I. Those bacteria which are pathogenic for man and other
animals.
II. Those bacteria which do not affect man, but are patho-
genic for*the lower animals.
Here again it will only be possible to give the more impor-
tant bacteria ; there are many diseases in which micro-organisms
have been found, but they have not yet been proven as causa-
tive of the disease, and have also been found in other diseases.
We cannot treat of them here.
Bacillus Anthracis. {Bayer and Davaine.) — Kayer and Da-
vaine, in 1850, first described this bacillus; but Pasteur, and
later Koch, gave it the importance it now has.
Synonyms — Bactericie du charbon (Fr.), Milzbrand bacillus
(German) ; bacillus of splenic fever, or malignant pustule.
Origin. — In blood of anthrax-suffering animals.
Form.— "Rods of variable length, nearly the size of a human
blood-corpuscle, broad cup-shaped ends; in bouillon cultures,
long threads are formed, with large oval spores.
Properties. — Liquefies gelatine ; immotile ; the spores are very
resisting, living twenty years, and resist boiling for five minutes.
Growth.— Grows rapidly, between 12° C. and 45° C, and re-
quires plenty of oxygen, but may be classed as a facultative
anaerobe ; grows well in all media.
Plates of Gelatine. — Colonies develop in two days, white shiny
spots, which appear under microscope as slightly yellowish
granular twisted balls, like a ball of yarn ; each separate string
PATHOGENIC BACTERIA. 95
or hair, if looked at under high power, being composed of bac-
teria in line.
Stab Cultures.— A white growth with thorn-like processes along
the needle-track ; later on, gelatine liquefied, and flaky masses
at the bottom.
Potato.— A dry creamy layer, and when placed in brood-oven,
rich in spores.
Varieties. Asporogenic.—'By cultivation in gelatine, contain-
ing 1 to 1000 ac. carbolic, a variety develop that cannot produce
spores. Also involution forms, differing from the usual type.
Fig. 48.
Anthrax bacilli in human blood (fuchsin staining), Zeiss 1-12 oil immersion.
No. 4 ocular taken from Vierordt.
Staining. — They readily take all the aniline dyes with the
ordinary methods. To bring out the cup-shaped concave ex-
tremities, a very weak watery solution of methylin blue is best.
Spores are stained by the usual method. When several bacilli
are joined together, the place of their joining looks like a spore
because of the hollowed ends. The double staining will develop
the difference.
Sections of tissue are stained according to the ordinary
methods, taking Gramas method very nicely.
Pathogenesis. — When mice are inoculated with anthrax mate-
96
ESSENTIALS OF BACTERIOLOGY.
rial through a wound in the skin, they die in twenty-four hours
from an active septicaemia, the point of inoculation remaining
unchanged, tfhe following appearances then present them-
selves : —
Peritoneum.— Covered with a gelatinous exudate.
Spleen. — Yery much swollen, dark red, and friable.
Liver. —Parenchymatous degeneration.
FiG. 49.
Fig. 50.
*&
Stab Cultures of Anthrax in Gelatine.
Blood. — Dark red. The bacilli are found wherever the capil-
laries are spread out, in the spleen, liver, intestinal villi, and
glomeruli of kidney, and in the blood itself. Only when the
capillaries burst are they found in the tubules of the kidney.
Mode of Entrance. — The bacilli can be infialed, and then a
pneumonia is caused, the pulmonary cells containing the bacilli ;
when the spores are inhaled, a general iufection occurs.
PATHOGENIC BACTERIA. 97
Feeding. — The cattle graze upon the meadows, where the
blood of anthrax animals has flowed and become dried, the
spores remaining, which then mix with the grass and so enter
the alimentary tract ; here they then cause the intestinal form
of the disease, ulcerating through the villi.
Local Infection. — In man usually only a local action occurs ; by
reason of his occupation — wool-sorter, cattle-driver, etc., he
obtains a small wound on the hand, and local gangrene and
necrosis set in.
Pneumonia by inhalation and intestinal infection also occurs
in man.
Susceptibility of Animals. — Dogs, birds, and cold-blooded ani-
mals affected the least ; while mice, sheep, and guinea-pigs
quickly and surely.
Products of Anthrax Bacilli. — A basic ptomaine has not been
found, but a toxalbumen or proteid, called anthraxin, has been
obtained. A certain amount of acid is produced by the virulent
form, alkali by the weak.
Attenuation and Immunity. — Cultures left several days at a
temperature between 40° and 42° C. soon become innocuous, and
when injected into animals protect them against the virulent
form.
The lymph obtained from lymph-sac of a frog destroys the
virulence of anthrax bacilli and spores temporarily.
Hankin obtained an alexin from the blood and spleen of rats,
they being naturally immune. It destroyed the anthrax bacilli
in vitro, and used by injection in susceptible animals made
them immune. It is insoluble in alcohol or water.
Protective Vaccination. — Animals have been rendered immune
by various ways — by inoculation of successive attenuated cul-
tures ; also with sterilized cultures — that is, cultures containing
no bacilli, and with cultures of other bacteria.
Habitat. — The anthrax disease seems confined to certain dis-
tricts in Siberia, Bavaria, and Auvergne, and mainly during the
summer months.
The bacillus has never been found free in nature.
Bacillus Tuberculosis. (Koch.)
This very important bacillus was first described, demonstrated,
98 ESSENTIALS OF BACTERIOLOGY.
and cultivated by Koch, who made his investigations public on
the 24th of March, before the Physiological Society of Berlin,
in the year 1882.
Origin. — In various tubercular products of man and other
animals.
Form. — Very slender rods, nearly straight, about one-quarter
Fig. 51.
/ >
2~ *
* » ^
Tubercle bacilli in sputum, carbol-fuchsin, and methylin blue. Zeiss 1:12 oil
immersion.
the size of a red corpuscle's diameter, their ends rounded, usu-
ally solitary, often, however, lying in pairs in such a manner as
to form an acute angle. Sometimes they are S -shaped. In
colored preparations little oval spaces are seen in the rod, which
resemble spores ; but the question of the existence of spores is
still undecided.
Properties. — Does not possess self-movement.
Growth.— Requires special media for its growth, and a temper-
ature varying but slightly from 37.5° C. It grows slowly, de-
veloping first after ten days, reaching its maximum in three
weeks. It is facultative anaerobic. On gelatine it does not
form a growth.
PATHOGENIC BACTERIA. 99
Colonies on Blood Serum. — Koch first used blood serum for
culture ground, and obtained thereon very good growths. Test-
tubes with stroke culture were placed in the brood oven at 37° C.
for ten to fourteen days, when small glistening white points ap-
peared which then coalesced to form a dry, white, scale-like
growth. Under microscope composed of many fine lines con-
taining the tubercle bacillus.
Glycerine Agar.— By adding four to six per cent, glycerine to
ordinary agar-peptone medium, Nocard and Koux obtained a-
culture ground upon which tubercle bacilli grew much better
than upon blood serum. This is now almost exclusively used.
Stroke cultures are here used as with blood serum. They are
placed in brood-oven after inoculation, and remain there about
ten days, at a temperature of 37° C. p10> 52
The cotton plugs of the tubes are
covered with rubber caps, the cotton
first having been passed through the
flame, and moistened with a few drops
of sublimate solution. The rubber
cap prevents the evaporation of the
water of condensation which always
forms, and keeps the culture from dry-
ing up.
The growth Which OCCUrs resembles Tubercle bacilli in human
the rusas of the stomach, and some- liver 500 X- (Frankei and
times looks like crumbs of bread moist-
ened. The impression or"Klatsch" preparation shows under
the microscope a thick curled-up centre around which threads
are wound in all directions. And these fine lines show the
bacilli in profusion.
Potato.— It can be cultivated on slices of potato which are
placed in air-tight test-tubes.
Bouillon. — Bouillon containing four per cent, glycerine is a
verv good nurture ground.
Varieties. — Branching and other aberrant forms are not rare,
and the tendency now is to class the organism with the " higher
bacteria." Other acid-fast bacilli exhibit similar types and it is
100
ESSENTIALS OF BACTERIOLOGY.
possible that the bacillary parasitic form is only one stage in
the life history of the organism.
Little granules arranged like streptococci, which take the
characteristic stain, and look as if the protoplasm had been
destroyed that enclosed them, are frequently found in sputum.
Fig. 53.
Klatsch preparation.
Bovine tubercle-bacilli are about one-third smaller than human
tubercle bacilli.
Staining.— The tubercle bacilli require special methods to
stain them, and a great number have been introduced. They
are stained with great difficulty : but once stained, they are
very resistant to decolorizing agents. Upon these facts all the
methods are founded.
PATHOGENIC BACTERIA. 101
It will only be necessary to describe those methods principally
in use ; and as the examination of sputum for bacilli is of so
frequent an occurrence, and so necessary, it is well to detail in
particular the method of staining.
Starting with the sputum, we search for little clumps or rolled-
up masses ; if these are not present, the most solid portions of the
mucus are brought with forceps upon a clean cover-glass ; very
little suffices. With another cover-glass it is pressed and spread
out evenly ; drawing one glass over the other, we obtain two
specimens, and these put aside or held high over the flame until
dry.
If we desire to examine the specimen quickly, or make a
hurried diagnosis, we use the rapid method, with hot solutions ;
Fig. 54.
Growth on Agar.
otherwise we let it stay, in cold solution until the next mornin<*
the advantages of which will be later on described.
Tlie Eapid Method.— (B. Frankel's method modified by Gab-
bet.) The principle is to combine with the contrast stain the
decolorizing agent ; but the preparations are not permanent ;
the method, however, is very useful.
Two solutions are required : one of Ziehl's carbol-fuchsin ;
the other Gabbet's acid methylin blue. (See No. X., on
page 34. )
The cover-glass containing the dried sputum is passed three
times through the flame, as described in the general directions.
It is then placed in the carbol-fuchsin solution five minutes (cold),
or two minutes in the hot, immediately then transferred to the
102 ESSENTIALS OP BACTERIOLOGY.
second solution, the acid blue, where it remains one minute,
then washing in water. The preparation is dried between
filter-paper, and mounted best first in water. Examined with
oil-immersion.
A somewhat longer, but preferable, method is to decolorize
the carbol-fuchsin with weaker acid. The smear is treated with
5 per cent, nitric or 10 per cent, sulphuric acid until, after
washing with water, a bright pink remains. The excess of
color is then washed out with 95 per cent, alcohol until no
further color is imparted to the alcohol and the smear is a
pinkish gray. The preparation is then washed writh water and
counterstained with aqueous methylin-blue for ten to thirty
seconds. A mechanical stage is of great assistance in the
search for the bacilli, as it permits every portion of the prepa-
ration to be inspected systematically.
In urine, owing to the almost inevitable contamination with
the smegma bacillus, special methods are necessary to avoid
error. The preparation may be left in 97 per cent, alcohol for
eight hours, when the smegma bacillus will have become de-
colorized, or Pappenheim's method may be used : (1) Smear
and fix as usual ; (2) stain with hot carbol-fuchsin for two
minutes, pour off the surplus dye without washing ; (3) counter-
stain and decolorize by pouring five times over the preparation
the following solution: A 1 per cent, alcoholic solution of
corallin is saturated with methylin-blue and 20 parts of gly-
cerine added. Wash in water, dry with blotting-paper, then in
the air, and examine. The tubercle bacilli are stained red,
smegma bacilli, blue.
The bacillus of leprosy resembles the tubercle bacillus in its
staining properties, but gives up the carbol-fuchsin more easily
and is usually decolorized by the acid and alcohol. It is colored
blue by Pappenheim's method.
Acid-fast bacilli have also been obtained from timothy grass,
butter, milk, manure, and the surfaces of animal bodies, but
differ from the tubercle bacillus in cultural characteristics.
Slow Method. — The stain may also be used without heating,
though in this case a much longer time is required before the
bacilli take up the stain. The preparation is left in a small
PATHOGENIC BACTERIA. 103
dish or beaker full of carbol-fuchsin for eight -to ten hours, and
then decolorized and counterstained in the usual way. The
method is less liable to produce artefacts than the quick
method, but is not much used on account of the time it takes.
BiederVs Method of Collecting Bacilli, when the bacilli are
very few in a great quantity of fluid, as urine, pus, abundant
mucus, etc., Biedert advises to mix 15 c.cm. of the fluid
with 75 to 100 c.cm. water and a few drops of potassium or
sodium hydrate, then boiling until the solution is quite thin. It
is placed in a conical glass for two days, and bacilli with other
morphological elements sink to the bottom of the glass ; when
the supernatant liquid is decanted, the residue can be easily
examined. In this way bacilli were found that had eluded
detection examined in the ordinary manner.
The centrifugal machine is used either in connection with
Biedert's sediment method or without, to obtain the solids sus-
pended in urine or serum.
When the bacilli are so few in number in sputum or urine as
to make their detection difficult, and also when doubt exists as
to the identity of acid-fast bacilli found, several guinea-pigs
should be injected in the groin and smears and sections made
from the enlarged glands resulting.
Carbolic Acid to Sediment Sputum. — Pure carbolic acid added
to sputum (about 1 part of the acid to 6 parts of sputum) will
in a few hours produce a coagulation and allow the sputum to
be spread evenly on the cover-glass, showing greater collections
of bacilli.
Without cover-glass. — Sputum can be spread and stained on
the glass slide without the use of a cover-glass, the oil of cedar
being placed directly on the stained sputum, and the oil immer-
sion lens dipping into it. It is a rapid and cheap way ; and
when a given case is to be studied daily the method is useful.
Pure Cultures from Sputum. — Kitasato recommends the tho-
rough washing, changing the water ten times, of the small masses
found in the sputum of tubercular persons. When such speci-
mens are examined they show tubercle bacilli alone, and when
inoculated in agar give rise to pure cultures.
Staining Bacillus Tuberculosis in Tissue (sections). — The general
104 ESSENTIALS OF BACTERIOLOGY.
method of Gram can be used, but the better way is to use the
following : —
Carbol-fuchsin, 15 to 30 minutes.
5 per cent, sulphuric acid, 1 minute.
Alcohol, until a light-red tinge appears.
Weak methylin blue, 3 to 5 minutes.
Alcohol, for a few seconds.
Oil of cloves, until cleared.
Canada balsam, to mount in.
Instead of carbol-fuchsin, alcoholic solution qffuchsin or aniline
water fuchsin can be used, but the sections must remain in the
stain over night.
Hardened sputum and sectioning. — Sputum can be hardened l»v
placing it in 98 per cent, alcohol. Thin sections can be obtained
by imbedding the hardened sputum in collodion. The sections
are then stained as ordinary tissue sections.
To preserve sputum. — Sputum can be preserved for future use
by placing it in alcohol, where it can be kept for months. Cover-
glass preparations can then be made by softening the coagula
with a small amount of liquor potassa.
The resisting action of the bacillus to acids is supposed to
be due to a peculiar arrangement of the albumen and cellulose
of the cell rather than to any particular capsule around it.
Pathogenesis. — When a guinea-pig has injected into its peri-
toneal cavity some of the diluted sputum containing tubercle
bacilli it perishes in about three weeks, and the following
picture presents itself at the autopsy : at the point of inoculation
a local tuberculosis shows itself, little tubercular nodules contain-
ing the characteristic bacilli. In the lungs and the lymphatics,
similar tubercles are found, a general tuberculosis.
If the animal lingers a few weeks longer, the tubercles become
necrosed in the centre and degeneration occurs, the periphery
still containing active bacilli, cavities having formed in the
centre.
Since the bacilli die in course of time, killed by their own pro-
ducts, their number forms no correct guide of the damage present.
Even their absence in the sputum does not preclude the ab-
sence of a tubercular process. It is their presence only that
PATHOGENIC BACTERIA. 105
warrants a positive declaration. The number of bacilli in a
given specimen is no indication of the severity of the disease.
They are found in the blood only when a vessel has come in
direct contact with a tubercular process through rupture or
otherwise. They have been found in other secretions, milk,
urine, etc.
Man is infected as follows : —
Through wounds. —Local tuberculosis.
Through nutrition.— Milk and meat of tuberculous animals.
Phthisical patients swallowing their own sputum and causing
an intestinal tuberculosis.
Inhalation. — This is the most usual way, probably constitu-
ting the cause in ^ of the cases, except in children.
The sputum of phthisical patients expectorated on the floors
of dwelling-houses in handkerchiefs, etc, dries, and the bacilli
set free are placed in motion by the wind or rising with the dust
are thus inhaled by those present. When the sputum is kept
from drying by expectoration in vessels containing water, this
great danger can be avoided.
Nearly all the cases of heredity can be explained in this man-
ner; the young children, possessing very little resistance, are
constantly exposed to the infection through inhalation and are
especially prone to intestinal infection through milk and other
foods.
Immunity. — No one can be said to be immune, though per-
sons who have been greatly weakened would offer less resistance
than healthy individuals.
Tuberculosis in animals. Tuberculosis is probably the most
widely disseminated disease among domestic animals, and
affects cattle, pigs, horses, dogs, cats, the smaller ruminants,
birds, and even turtles and fish. The conclusion of Koch,
made public in his address to the Tuberculosis Congress in
1901, that human and bovine tuberculosis are distinct and that
infection of human beings from cattle occurs so seldom that no
general regulations to restrict it are necessary, has found few
adherents. It is true that certain differences exist between
human and bovine tubercle bacilli, the latter appearing to be
more virulent to animals, and it is a fact that cattle are very
106 ESSENTIALS OF BACTERIOLOGY.
slightly susceptible to the human bacillus, but it is not likely
that the converse is so. Children are particularly liable to in-
fection through the gastrointestinal tract, and it has been
shown that the uninjured mucosa of the infant's intestine is
permeable to bacillus, so that the pulmonary disease in the
young may often be the result of tuberculous bronchial nodes
secondary to tuberculous glands of the mesentery.
Various observations on animals have shown that the bacillus
occurring in each species has acquired certain special charac-
teristics regarding growth and virulence. The bacilli causing
tuberculosis in the cold-blooded animals have departed farthest
from the human type, those of birds to a less degree, and those
of cattle least of all.
Products of Tubercle Bacilli. The true nature of the tuber-
cle toxin is not yet clear. It is not unlikely that several toxic
bodies differing from one another in their properties are pro-
duced. Koch's tuberculin (1890) was obtained by filtering,
through unglazed porcelain, concentrated glycerine bouillon
cultures of tubercle bacilli. It was speedily shown to be devoid
of curative power, and is now used mainly for diagnosing the
disease in cattle. In healthy animals little or no reaction is
produced by the injection of 30 to 40 eg. of tuberculin, but if
tubercular, the temperature rises 2° or 3° F. in eight to twelve
hours, and remains elevated for a like period of time. In man
the use of tuberculin as a diagnostic measure is falling into dis-
favor, as it is both dangerous and unreliable.
Tuber culocidin. — This is an albuminoid obtained from the
original tuberculin by precipitation with alcohol. Klebs used
it as a cure for tuberculosis.
Tuberculin R. is an extract made from dried and powdered
living bacilli, and was recommended by Koch in place of the
original tuberculin, but it has likewise proved useless.
Agglutination. Arloing and Courmont have described an
agglutination reaction for the tubercle bacillus similar to the
Widal reaction of typhoid fever. It is very unreliable, how-
ever, and but little importance is attached to it.
Antituberculous Serum. The attempts to produce an effec-
tive serum have so far been unsuccessful. Marmorek, by grow-
PATHOGENIC BACTERIA. 107
ing the bacillus on a special serum obtained by injecting calves
with the leucocytes of guinea-pigs, has secured a toxin which
he used to immunize horses, and the serum so obtained has
been tried with encouraging results, but its value is still doubt-
ful.
Examination of Milk for Tubercle Bacilli. Place a drop of
the sample on a cover-glass and mix it with 2 drops of a 1 per
cent, solution of sodium carbonate. The cover-glass is then
gently warmed until evaporation is complete. The saponified
fat is then stained as the ordinary cover-glass preparation.
Only a ver}r few persons have succeeded in discovering the
bacillus in milk.
Lepra Bacillus. (Hansen.)
Origin. — In 1880 Armauer Hansen declared, as the result ot
many years' investigation, that he found a bacillus in all leprous
processes.
Form.— Small slender rods somewhat shorter than tubercle
bacilli, otherwise very similar in appearance.
In the interior of the cell two to three oval spaces are usually
seen, not known if spores or otherwise.
Properties.— They are immotile, do not liquefy the nutrient
media.
Growth.— Bordoni-Uftreduzzi have obtained growths upon
blood serum to which peptone and glycerine had been added,
but the accuracy of this observation is very doubtful.
Staining. — They resist the decolorizing action of acids as the
tubercle bacilli, but they are easily stained, requiring but a
few minutes with the ordinary watery solutions. They take
Gram's stain readily.
Pathogenesis.— Arning has inoculated prisoners with tissue
obtained from leprous patients, and produced true leprosy.
Rabbits which had been infected through the anterior chamber
of the eye showed the lepra nodules (containing the lepra
bacilli) diffused through various organs, but here again the
results are not wholly satisfactory.
In man the skin and peripheral nerves are principally affected,
but the lymphatic glands, liver, and spleen can also become the
seat of the lepra nodules. The lepra cells which compose these
108 ESSENTIALS OF BACTERIOLOGY.
nodules contain the bacilli in large numbers. By applying a
vesicant to the leprous skin the serum thereby obtained will con-
tain great numbers of bacilli. This is a simple diagnostic test.
Method of Infection. — Not yet determined ; the air, soil, water,
and food of leprous districts have been carefully examined with-
out result. The nasal secretion is very infectious.
Syphilis Bacillus of Lustgarten (Smegma Bacillus of Alvarey
and Tavel). Lustgarten in 1885, through a certain staining
process, found peculiar bacilli in syphilitic tissues which he
thought had a direct connection with the disease.
Van Niessen, Joseph and Piorkowski, DeLisle and Jullien all
describe other organisms that they have found in syphilitic
lesions, and Schuller mentions a protozoon-like body he has
seen in many cases, but these results still lack confirmation.
Metschnikoff, Roux, and Lassar have lately succeeded in inocu-
lating chimpanzees with what appears to be true syphilis.
The question yet remains an open one, what relation the
syphilis or the smegma bacillus bears to syphilis, and will
remain so until the bacillus can be cultivated, which so far has
not been accomplished.
Bacillus of Glanders. {Bacillus Mallei, Loffler-Shutz.) Botz
bacillus.
Origin. — In the " farcy buds" or little nodules of the disease,
by Loffler and Shiitz in 1882.
Form. — Small slender rods, about the size of the tubercle
bacillus. The ends rounded. Never appearing in large collec-
tions, usually singly. Spores are said to exist, but this is
doubtful.
Properties. — The rods are very resistant, living in a dried state
for three months and longer without any spores present. They
are not motile ; possess, however, great molecular vibration.
Growth. — The growth occurs between 25° and 40° C, best
at 37° C. ; it is very sparse upon gelatine, but on glycerine-agar
or blood serum a very abundant growth occurs.
Colonies. — On agar or glycerine-agar there appear in two to
three days small white glistening drops, which under microscope
seem as round granular masses with an even periphery.
Stroke Cultures. — On glvcerine-asrar and blood serum small
PATHOGENIC BACTERIA
109
transparent drops of whitish or grayish color, which soon
coalesce to form a broad band.
Potato. — An amber-colored honey-like growth which gradually
turns red, then brown, and greenish-brown around it. Weakly
acid potatoes are a good medium and give the most typical
growth.
Fig. 55.
Bacillus of Glanders.
Staining. — Since the bacillus is very easily decolorized, some
special methods have been recommended.
Lbffler's.— (For cover-glass preparations.)
1. Alkaline methylin blue (Loffler's). 5 minutes.
2. Acetic acid with a few drops of tropaeolin. 1 second.
3. Washed in water.
For Sections. — Instead of tropseolin acetic acid, the following
mixture is used : —
$.— Oxalic acid 5 per cent. . . . gtt. j.
Cone, sulphuric, acid gtt. ij.
Aq. destill. 3ij. — M.
The sections are kept in this 5 seconds.
Kuhne^s method. Coverglass.
1. Warm carbol-blue 2 min.
2. Decolorized in weak sol. of muriatic acid (10 parts to 500).
3. Washed in water.
110 ESSENTIALS OF BACTERIOLOGY.
Sections of Tissue.
1. Carbol-blue, £ hour.
2. Decolorized in £ per cent, muriatic acid.
3. Washed in distilled water.
4. Dehydrated in alcohol 1 second.
5. Aniline oil with 6 gtt. of turpentine. 5 min.
6. Turpentine, xylol, Canada balsam.
If contrast stain, add 5 gtt. of safranin (Bismark-brown) to
turpentine, and use it after the xylol.
Pathogenesis. — If horses, field mice, or guinea-pigs be inocu-
lated subcutaneously, with but a very small quantity of culture,
a local affection results, followed some time after by a general
disturbance ; ulcers form at the point of inoculation ; little
nodules, which then caseate, leaving scars and involving the
lymphatics ; metastatic abscesses then occur in the spleen and
lungs, and death arises from exhaustion. Cattle, pigs, and rab-
bits are not easily affected ; man is readily attacked. The ba-
cilli gain entrance to the blood and urine. Nasal glanders
occurs whatever the mode of inoculation.
Manner of Infection.— Glanders being a highly contagious dis-
ease, it requires but a slight wound to allow it to gain entrance.
In horses the primary sore seems to be at the nasal mucous
membrane. In man it is usually on the fingers. Boiling water
or 1-10,000 sublimate solution will quickly destroy the virulence
of this bacillus.
Mallein. A substance called mattein has been obtained from
the cultures grown in glycerin bouillon. It gives a reaction
when injected into cattle suffering from glanders, and is said
to be useful in diagnosing the disease.
Bacillus of Diphtheria. (Klebs-Loffler.)
Origin. — Klebs found it in membrane in 1883; it was isolated
by Loffler in 1884.
Form. — Small, slightly curved rods about as long as tubercle
bacilli and twice as broad; the ends are at times swollen; spores
have not been found. Their form is, however, very variable —
sometimes much longer than usual, one end often greatly
knobbed. Normal bacilli are found only in membrane.
Stained forms are characteristic, since the ends are more easily
PATHOGENIC BACTERIA. Ill
colored than the centre, and usually the bacillus stains in seg-
ments, so that it seems to be made up of very short sections.
At first sight it appears like a chain of cocci.
Properties.— They do not possess any movement; do not
liquefy gelatine. They are not very resistant, being destroyed
by a temperature of 50° C, but they have lived on blood-serum
five months.
Growth.— Grow readily on all media, but best on blood-serum
mixtures, between temperatures of 20° and 40° C. They are fac-
ultative anaerobic ; they grow quite rapidly and profusely. Egg
cultures (Hueppe's method) give good growths. Passing cur-
rents of air increase the growth.
Colonies on Gelatine Plates.— At 24° C. little round colonies,
white under low-power, granular centre; irregular borders.
Stab Cultures.— Small, white drops along the needle track. In
glycerine-agar a somewhat profuse growth.
Potato.— On alkaline surface, a grayish layer in 48 hours.
Blood-Serum (after Loftier). — Blood serum 3 parts, and bouil-
lon 1 part ; the bouillon contains peptone, 1 per cent., chloride
of sodium, £ per cent., and dextrin (or glucose), 1 per cent.
In a few hours (eight to sixteen) on the white opaque surface
a slight moisture is noticeable, which, if examined, is composed
of bacilli. In twenty -four hours small round colonies are found
which seem to arrange themselves concentrically. The growth
becomes more abundant, and the individual colonies larger and
yellowish. On blood-coagulum the growth is usually gray and
the margins of the culture crenated. Often a diagnosis can be
made in four hours if the serum tubes are kept in a brood oven.
Serum- Agar. — Joos finds serum-agar better than Loffler's
serum : 300 c.c. blood-serum mixed with 50 c.c. normal soda
solution and 150 c.c. water, heated in water bath for 2 to 3 hours
at 60° to 70° C, then raised to 100° O, or in steam chest I hour.
Then 500 c.c. peptone bouillon (slightly alkaline) and 20 gm.
agar. When the agar is dissolved by heat, avoiding prolonged
boiling, the mixture is filtered and sterilized \ hour at 100° to
HO" C. in autoclave ; then poured into petri dishes. Strepto-
cocci do not grow on this medium, whereas diphtheria bacilli
will grow in from 6 to 12 hours.
112
ESSENTIALS OP BACTERIOLOGY.
Bouillon. — In bouillon an abundant growth takes place, and
this medium is used to obtain the toxins.
.f<;
Fig. 50.
W
i
«nv <^*
/&%<
V
ttrM it* :
Bacillus diphtheria?, from a pure culture
Fig. 57.
&
Sis" V
i
«'
Bacillus diphtherise, from a culture upon blood-serum ; X 1000 (Fraukel and Pfeiffer).
Staining. — Is not colored by Gram's method. Stained best
with Loffler's alkaline methylin-blue.
PATHOGENIC BACTERIA. 113
Pathogenesis. — By inoculation, animals, which naturally are
not suhject to diphtheria, have had diphtheritic processes de-
velop at the site of infection ; hemorrhagic oedema then follows,
and death.
In rahhits paralyses develop, and when the inoculation occurs
upon the trachea, all the prominent symptoms of diphtheria
show themselves.
Manner of Infection in Man. — The exact way is not yet known.
It is supposed that the mucous membrane altered in some man-
ner, the diphtheria bacillus, then gains entrance and the disease
develops. The bacilli may be found in healthy individuals who
may act as a source of infection to susceptible individuals with-
out themselves becoming infected.
Products. — But it is not the mere presence of the bacillus that
gives rise to all trouble ; certain products which they generate
get into the system and produce the severe constitutional symp-
toms.
Roux and Yersin, in 1888, discovered that the injection of the
filtered culture bouillon (that is, freed of all diphtheria bacilli)
gave rise to the same palsies as when the bacilli themselves were
introduced.
Toxins of Diphtheria. — Brieger and Frankel filter the bouillon
culture, evaporate (in vacuo at 27° C.) to i volume, then treat
with 10 volumes of alcohol and acetic acid, the precipitate re-
dissolved in water and reprecipitated with the acidulated alco-
hol until a clear aqueous solution is obtained ; this is then
dialyzed for 72 hours, and again precipitated with alcohol, and
dried ; a white amorphous body results, giving all the reactions
of an albumen, and called by them toxalbumen.
The toxin of diphtheria, first demonstrated by Roux and
Yersin, is not an albumen. It is obtained by growing virulent
bacilli in bouillon for three or four weeks at 37° C. After a
sufficient alkalinity has been produced the cultures are fil-
tered, and the filtrate should have a toxicity that will destroy
a 500-gramme guinea-pig in twenty-four hours when 0.1 c. cm.
of the toxin is injected.
Antitoxin. Behring found that animals rendered immune
8
114 ESSENTIALS OF BACTERIOLOGY.
had a principle in their blood that was antagonistic to the
development of the toxin.
Immunity. — Brieger and Frankel, by injecting 10 to 20 c.cm.
of a three weeks' old culture of diphtheria bacilli, which had
been heated at 70° C. for one hour, produced an immunity in
guinea-pigs against the virulent form.
This important discovery has been utilized in a practical Avay.
Horses are made immune by gradually increased doses of the
toxin until 300 c. cm. can be borne without bad effect. This
may require several months' time. The serum of such an im-
munized animal is now possessed of antitoxic properties.
Behring has standardized the strength of antitoxic serum, so
that we say a serum has an immunizing strength of 60 units
or 100 units, which means that 0.1 c. cm. of the serum would
protect against 1 c. cm. of the toxin when injected together into
guinea-pigs. 1 cubic centimetre of this is the unit. The strength
commonly employed in human beings is 1500 units in 10 c. cm.
If this amount is injected into a child suffering from diphtheria
in the earlier stages (second to third day), the disease is often
arrested. The membrane begins to disappear, and in two or
three days has vanished. The constitutional symptoms are
likewise greatly influenced by the injection. If a smaller dose
is injected into persons who have been exposed to contagion,
the disease is prevented from appearing.
The antitoxin has no influence on the bacteria themselves;
their virulence and length of residence in the body is not
lessened.
The toxin generated by the germ is supposed to be neutral-
ized by the antitoxin and prevented from injuring the body
tissues.
Pseudo-diphtheria bacilli, so called, differ from the true
organism in certain cultural and morphological characteristics,
and do not produce a toxin, but their true status is still uncer-
tain.
Site of Bacilli. — Bacilli are usually found in the older portions
of the pseudo-membrane very near to the surface. The secre-
tions of the throat of a diphtheritic child produced bacilli three
weeks after the temperature was down to normal.
PATHOGENIC BACTERIA.
115
Streptococcus in Diphtheria. Streptococci have been found
quite constant in diphtheria, but they resemble the strepto-
coccus pyogenes, and have no specific action.
Fig. 58.
Bacillus typhi, from an agar-agar culture six hours old, showing the flagella stained
by Loffler's method ; X 1000.(Frankel and Pl'eifier.)
Pseudo-diphtheritic bacillus is probably a weakened or a vir-
ulent form of the true bacillus.
Bacillus of Typhoid or Enteric Fever. (Eberth-Gaffky.)
Origin. — Eberth found this bacillus in the spleen and lym-
phatic glands in the year 1880, and Gaff ky isolated and cultivated
the same four years later.
Form. — Rods with rounded ends about three times as Ions as
they are broad. Usually solitary in tissue-sections, but in arti-
ficial cultures found in long threads. Flagella on the side.
Properties. — They are very motile ; they take the aniline dyes
less deeply than some similar bacilli. Spores have not yet been
found ; they do not liquefy gelatin.
Growth. — They are facultative anaerobic; grow best at 37°
C, but can also develop at ordinary room temperature. All
116 ESSENTIALS OF BACTERIOLOGY.
nutrient media can be used as culture ground. They develop
chiefly on the surface, and very slowly. Repeated freezing and
thawing do not affect the vitality of the germ, and carbolic acid
in 1 to 2 per cent, solution has no effect on it. A ten-minute
exposure to 60° C. is invariably fatal.
Colonies on Gelatine Plates. — Two forms ; the ones near the
surface spread out like a leaf, transparent with bluish fluor-
escence. The deeper ones resemble whetstone crystals of uric
acid, with the same yellowish tinge.
In five days they attain to 3 millimetres in diameter.
On Potato Gelatine. — The colonies do not have the yellow
color, they are transparent, later on they become dark brown
with green iridescence.
Stab Cultures.— Mainly on the surface a pearly layer.
Stroke Cultures. — A transparent thick layer.
Potato. — The growth here is quite characteristic. At 37° C.
Fig. 59. Fig. 60.
Typhoid fever bacillus in pure cul- Colonies of typhoid bacilli 3 days
ture. 650 diameters. old 100 X. (Frankel and Pfeiffer.)
in 48 hours a moist transparent film is formed over the whole
surface, but so transparent that it can hardly be seen without
close observation. If a small portion of this is placed under a
microscope, it will be seen swarming with bacilli.
The growth never becomes more prominent ; the potato must
have a neutral or acid reaction.
Milk. — The bacteria grow very well in milk, producing a
slightly acid reaction, but no coagulation.
PATHOGENIC BACTERIA. 117
Carbolized Gelatine. — Gelatine which has added to it ^ per
cent, carbolic acid will allow the typhoid bacillus to develop,
other similar bacilli being destroyed.
Glucose Gelatine. — In glucose gelatine there is no gas-produc-
tion. Indol is likewise not generated by the typhoid bacillus,
whereas it is by the colon bacillus. On Eisner's potato-gelatine
the colon bacillus and the typhoid bacillus grow readily. The
medium of Hiss is of great assistance in isolating the germ.
Fig. 61.
The Widal agglutination reaction (Slater and Spitta).
The Gruber- Widal blood-serum test, or, as otherwise known, the
agglutination-phenomenon (Fig. 61), has the following history:
About 1889, Charrin and Roger observed in the serum of im-
munized animals that the B. pyocyaneus arranged itself in little
clumps. Other investigators reported the same thing for other
bacteria, and Metschnikoff added that motility was destroyed.
In 1895, Bordet showed that the serum of cholera-immunized
animals, when mixed with bouillon cultures of cholera spirilla,
affected their motility and caused them to form masses, or
"Klumpen," as the Germans call it-
R. Pfeiffer, in the same year, showed that the introduction of
immune serum at the same time with virulent cholera spirilla
118 ESSENTIALS OF BACTERIOLOGY.
into the peritoneum of guinea-pigs, prevented infection from
taking place, and the spirilla were transformed into granular
masses. He likewise showed this reaction to be specific, the
serum of cholera-immune animals acting only on cholera
vibrio; and hence he suggested using the serum as a means
of diagnosis for the cholera vibrio and typhoid bacillus. Gru-
ber about the same time made some studies upon the use of
this serum property in differentiating bacteria, but it was con-
sidered as yet a property connected in some way with immu-
nity.
In 1896, Widal and Griinbaum, working separately, developed
what is now spoken of as the " Widal serum-test," or "Widal
reaction." It consists in testing a drop of blood of a patient
suspected of having typhoid fever, by mixing a dilution of it
with a drop of a fresh bouillon culture of typhoid bacilli, and
examining the mixture in a hanging drop under the micro-
scope. Within fifteen minutes to an hour the motility of the
bacilli will cease, and they will have arranged themselves into
clusters, as if stuck or glued together. If this reaction occurs
within an hour, and with the proper dilution of the serum, the
case is one of typhoid. Widal first used the serum of the blood ;
this has been modified so that even a drop of dried blood is suf-
ficient. The method as applied in city laboratories is as follows :
The physician is told to clean the finger of the patient with
water (no germicides), and with a needle draw a drop of blood
on to a piece of ordinary note-paper. This is then sent to the
laboratory ; the paper with the dried blood is soaked for a few
minutes in a watch-glass containing 4 drops of clean water, thus
obtaining a dilution of 1 : 5. One drop of this is then mixed with
one drop of a bouillon culture of typhoid bacilli of about 24
hours' growth, and examined under the microscope in the
hanging drop. Weaker dilutions of the serum have been rec-
ommended (1 : 50), and this should be used in cases of doubt
So far, about 95 per cent, of the cases examined, and which clin«
ically were considered typhoid fever, have given a positive reac-
tion. It is not often present until the fifth day of the fever, and
disappears usually within a year, though in some individuals it
has been found ten years after an attack of the disease.
PATHOGENIC BACTERIA. 119
The agglutinating properties have been found in nearly all
the secretions of the body — tears, urine, milk, pleuritic effusions,
serous fluid from blisters, etc.
There is no relation between the reaction and the bactericidal
power of the serum ; the agglutination is not a destruction. The
agglutinating power is active, though the blood be dried and
sealed up for months. It seems to have no direct relation with
the question of immunity, since it occurs at the height of the
disease, and intense agglutinating serum may be had in severe
cases and in cases with relapses. A negative result does not
exclude typhoid.
The test is quantitative — i. e., it depends upon the dilution of
the blood-serum, since the serum of healthy persons in strong
dilution will cause agglutination and loss of mobility.
The test must occur within a certain limit of time to be of
value, since agglutination is liable to appear of itself with non-
typhoid sera after a period of an hour.
As a clinical test of the disease it has considerable value,
although operative at a time when other symptoms have devel-
oped sufficiently to determine the diagnosis.
Staining. — Colored with the ordinary aniline dyes, when they
are warmed; since they are easily decolorized, acids should be
avoided.
Gram's method is not applicable. Tissue sections stained as
follows : —
Alkaline methylin-blue . .1 hour.
Alcohol 5 seconds.
Aniline oil 5 minutes.
Turpentine oil 1 minute.
Xylol and Canada bals.
Such a specimen should first be examined with low power, to
focus little colored masses, then examined with immersion lens ;
these masses will be found composed of bacilli.
Similar Bacteria. The Neapolitanus bacillus of Emmerich, or
fxces bacillus of Brieger, resembles the typhoid bacillus in many
ways, the colonies being the same and its structure similar.
But the growth on potato is very different; a thick, yellow,
pasty layer is formed thereon.
120 ESSENTIALS OF BACTERIOLOGY.
The colon bacillus not only resembles the typhoid germ in
form, but also in some of the pathologic processes produced.
For points of resemblance and difference, see Bacillus coli com-
munis.
In Water. Bacilli have been found which also resemble ty-
phoid bacilli, and one must be very careful not to make any
positive statement.
Examination of Water for Typhoid Bacilli. — When a water is
supposed to contain typhoid bacilli, 500 c.cm. of the same is
mixed with 20 gtt. of |-per cent, carbolic acid, which destroys
many of the saprophytes.
Plates are then made as described under Water Analysis.
Those colonies which then form and have a tendency to liquefy,
are touched on second day with permanganate of potassium,
and when so colored, destroyed with bichloride of mercury.
Those that now develop are transferred by inoculation to fresh
plates. At the end of eight days they are examined under
microscope ; every colony not possessing motile bacilli is dis-
carded. The motile bacilli are tested with Gram's method of
staining ; those that do not take the stain are alone retained.
Cultures are made from these upon potatoes, and, if the char-
acteristic growth occurs, then only can they be called typhoid
bacilli with any certainty.
Pathogenesis. — Lower animals have npt yet been given enteric
fever, though their death has been caused by injection of the
bacilli into the veins of the ear.
In man it has been found in the urine, blood, sputum, milk,
intestinal discharges, roseolar spots, and in various organs, as
spleen, liver, lymphatic glands, and intestinal villi.
It is found in secretions several days after the attack has sub-
sided. It is found only in this disease, and regularly.
Way of Infection. — The bacilli in the dejecta of the diseased
person find their way into drinking water, milk, or dirty clothes,
and so into the alimentary tract of a person predisposed to the
disease. They enter the blood through the lymphatics, and so
become lodged in various organs. They are quite resistant, liv-
ing for some time in the soil and water, and are not affected as
PATHOGENIC BACTERIA. 121
other organisms by carbolic acid. An epidemic has been traced
to the eating of oysters taken from contaminated water.
Persistence in Water. — Franckland kept bacilli alive in water,
sterilized by heat, 75 days ; in filtered water at 19° C, 5 days ;
at 6° C, 12 days. In ordinary water they are likely to be de-
stroyed in a few days by the overgrowth of other bacteria.
Products. — Brieger found a ptomaine in the cultures which he
named typhotoxin with the formula C9HnN02. It has no
specific action. A toxalbumen insoluble in water has also been
isolated, but, as experiment animals are immune to the disease,
no definite actions have yet been determined.
The cultures, when old, show an acid reaction.
Paracolon or paratyphoid bacilli are members of the colon
group recently described by Widal, Gwyn, Schottmuller, and
others. They are of importance, since they produce fevers
clinically resembling a mild form of typhoid, and which are
rarely fatal. They may be the sole cause of the disease, and
probably also occur together with the typhoid bacillus in mixed
and secondary infections. Morphologically they resemble the
typhoid bacillus, but differ from it culturally and give their
own serum reactions with the blood of affected patients. They
ferment glucose, but not lactose or saccharose ; litmus milk at
first becomes acid, but later grows alkaline and is not coagu-
lated. On potato a slight visible growth occurs ; indol is usually
not formed. Typhoid sera do not agglutinate paracolon bacilli,
and vice versa; also different paracolon infections may not
agglutinate each other. The Bacillus enteritidis of Gartner is
a related form.
Bacillus psittacosis is an allied form occurring in parrots,
and producing hemorrhagic septicemia in them and experi-
ment animals. The disease is readily communicated to man
from the affected birds, and causes, after ten days' incubation,
a disease, the chief symptoms of which are fever, delirium,
vomiting, diarrhoea, and albuminuria, about a third of the
cases ending fatally. The organism is agglutinated by strong
dilutions of typhoid serum, but the clumping is incomplete and
the bacillus differs further from the typhoid bacillus in its
growth on potato and in the nature of the infection produced.
122 ESSENTIALS OF BACTERIOLOGY.
Bacillus Coli Communis. (Escherich.)
Found in human feces, intestinal canal of most animals, in
pus and water.
Form. — Short rods with very slow movement, often associated
in little masses resembling the typhoid germ, flagellated, does
not form spores.
Fig. 62.
Bacillus coli communis, from an agar-agar culture; X 1°00 (Itzerott and Niemann).
Properties.— Does not liquefy gelatine, causes fermentation in
saccharine solutions in the absence of oxygen, produces acid
fermentation in milk.
Growth.— On potato a thick, moist, yellow-colored growth.
Very soon after inoculation on gelatine a growth similar to
typhoid. It can also develop in carbolized gelatine, and with-
stands a temperature of 45° C. without its growth being de-
stroyed.
Pathogenesis.— Inoculated into rabbits or guinea-pigs, death
follows in from one to three days, the symptoms being those of
diarrhoea and coma ; after death tumefactions of Peyer's patches
and other parts of the intestine ; perforations into peritoneal
cavity, the blood containing a large number of germs.
With the blood of immunized animals a serum reaction
PATHOGENIC BACTERIA.
123
similar to that of typhoid fever may be obtained with cultures
of colon bacilli. The colon bacillus is held responsible for most
of the complications of typhoid fever, such as peritonitis,
cholangitis, etc., by many writers.
Staining. — Ordinary stains; do not take Gram.
Site.— The bacillus has been found very constant in acute
peritonitis and in cholera nostras. Its presence in water would
indicate fecal contamination, as it is normally present in the
intestine.
Points of Resemblance between Bacillus Typhi and Bacillus Coli
Communis. — 1. Microscopic appearance; 2. Agar and gelatine
cultures ; 3. Sometimes growth on potato the same ; 4. Stain-
ing peculiarities ; 5. Resistance to carbolic acid.
Points of Difference :
Colon Bacillus.
Less motile,
Gelatine colonies develop more
rapidly,
Produces gas on dextrose or
lactose media,
Coagulates milk,
Produces indol,
Growth on potato visible,
Changes neutral red to yellow.
Typhoid Bacillus.
Actively motile,
Develop more slowly,
Does not,
Does not,
Does not,
Invisible,
Does not reduce neutral red.
Differences are also noted in the growth on special media,
such as those of Hiss and Eisner.
Varieties. — By some bacteriologists the following bacilli are all
considered forms of the colon bacillus : B. lactis aerogenes of
Escherich, B. cavicida of Brieger, B. neapolitanus of Emmerich,
B. enteritidis of Gartner, and, together with some other allied
organisms, they are spoken of as the " colon group."
124 ESSENTIALS OF BACTERIOLOGY
CHAPTER III.
PATHOGENIC BACTERIA — CONTINUED.
Spirillum Cholerse. (Koch.) Comma bacillus of cholera.
Origin. — Koch, as a member of the German expedition sent
to India, in 1883, to study cholera, found this micro-organism
in the intestinal contents of cholera
Fig. 63. patients, and by further experiments
identified it with the disease.
Form.— The microbe as seen ordi-
narily appears as a short, arc-like body,
about half the size of a tubercle bacillus,
but when seen in large groups, spirals
are formed, each little arc appearing
then as but a segment, a vibrio ; each
arc is about three times as long as it
Comma bacillus, pure cul- is broad, and possesses a flagellum at
ture. 600 diameters. one or more rarely both ends.
Properties. — They are very motile ; liquefy gelatine. They are
easily affected by heat and dryness. Spores have not been
found, though some (Hiippe) claim arthrospores, but these
bodies represent only degenerative changes.
Growth. — Develops at ordinary temperatures on all nutrient
media that have an alkaline or neutral reaction. They are
facultative anaerobic.
Colonies, gelatine. — After 24 hours, small white points which
gradually come to the surface, the gelatine being slowly lique-
fied, a funnel-shaped cavity formed holding the colony in its
narrow part, at the bottom, and on the fifth day all the gelatine
is liquid. If the colonies of three days' growth are placed under
microscope they appear as if composed of small bits of frosted
glass with sharp irregular points.
9W
PATHOGENIC BACTERIA
125
Stab Culture.— After 30 hours a growth can be distinguished
along the needle track, and on the surface a little cavity has
been formed, filled up by a bubble of air, and this liquefaction
proceeds until on the sixth day it has reached the sides of the
tube, tapering, funnel-shaped to the bottom of the tube. After
several weeks the spirilla
are found in little collec- FlGJ- 64-
tions at the bottom of the
fluid gelatine. In eight
weeks the bacilli have
perished.
Agar. — Stroke cultures.
A shiny white layer lasts
many months.
Potato. — A yellow honey-
like transparent layer, if
the potato is kept at ani-
mal heat.
Bouillon. — A wrinkled
scum is soon formed in
bouillon. They live wel]
and grow in sterilized mill*
and sterilized water, re^
maining virulent in the
latter for many months.
In ordinary water, the bacteria present are destructive to the
comma bacillus, and they die in a few days.
Dunham's Peptone Solution. — Useful for the development of
nitrites and the indol reaction.
WidaVs serum test, as used in typhoid, is applicable in the
diagnosis of cholera, using cholera cultures in place of the
typhoid.
Staining. — They are colored well with watery aniline solu-
tions. The flagella can be well seen by staining according to the
flagella stain.
Pathogenesis. — Experiment animals are not subject to cholera
Asiatica, but by overcoming two obstacles Koch has produced
choleraic symptoms in guinea-pigs. Nieati and Rietsch pre-
vented peristalsis and avoided the acidity of the stomach juices
Cholera colonies after 30 hours 100 X . (Frankel
and Pfeiffer.)
126 ESSENTIALS OF BACTERIOLOGY.
by direct injection into the duodenum, after tying the gall-duct.
Koch alkalinizes the gastric juice with 5 c.cm. of 5 per cent,
sol. of sodii carbonas, and then injecting 2 grams of opium tinc-
ture for every 300 grams of weight into the peritoneal cavity
paralyzes peristalsis. The cholera culture then introduced
through a stomach-tube, the animals die in forty-eight hours,
presenting the same symptoms in the appearance of the intes-
tines as in cholera patients, the serous effusion containing great
numbers of spirilla.
Manner of Infection in Man.— Usually through the alimen-
tary tract, with the food or drink, the intestinal discharges of
cholera patients having found entrance into the source of drink-
ing water. Soiled clothes to fingers, fingers to the mouth, etc. ;
torpid catarrhal affection of the digestive tract predisposing.
The microbe is not found in the blood or any organ other than
the intestines, the tissue of the small intestines. It is also
found in the vomit and the intestinal contents.
Fig. 65.
Comma bacillus in mucus, from a case of Asiatic cholera.
Products. — " Cholera red." When chemically pure nitric or
sulphuric acid is added to nutrient peptone cultures of the
PATHOGENIC BACTERIA. 127
cholera bacillus a rose-red color is produced. This will not take
place with other bacilli unless nitrous acid is present. The cholera
bacillus forms nitrites from the nitrates present in the media,
and also indol. The mineral acid splits the nitrites, setting free
nitrous acid, which, with the indol, forms the red reaction.
This pigment has been isolated and extracted and called
" cholera red." A ptomaine, identical with cadaverin, and sev-
eral other alkaloids have been obtained from the cultures. A
toxalbumen and a toxicpeptone have lately been isolated, but
no special actions ascribed to them.
Detection of Cholera Organisms in Drinking-water. — When a few
bacteria are supposed to be present in fecal matter or drinking-
water it is best to add a large quantity of the material (200
c. cm. of drinking-water) to about 10 c. cm. of bouillon, and
place the mixture for twenty-four hours in an incubator,
which will cause rapid reproduction, and then the organisms
can be readily discovered.
HafFkine has obtained a great reduction in mortality in
cholera regions by the use of anti-cholera vaccines as pro-
tective and curative measures.
Cholera Immunity of Pfeiffer. — Intraperitoneal, subcutaneous,
and intravenous injections of living or dead cholera bacteria
cause a disease in animals similar to the cold stage of cholera.
Death is the result of toxemia. If the animal lives, the blood
has protective properties of a specific nature; it has bacteri-
cidal properties against cholera vibrio, and by the injection of
this serum into non-immune animals it renders them immune.
The blood-serum of convalescents and cholera-vaccinated indi-
viduals contains the same bactericidal substances.
Bacteria Similar to the Spirillum of Cholera.
Finkler-Prior Vibrio, or Spirillum Finkleri.
Origin. — Found in the intestinal contents of a patient suffer-
ing from cholera Asiatica in 1884, by Finkler and Prior, who
thought it identical with the spirillum of cholera; it differs
from it, however, in many ways, and has been found in healthy
persons.
Form. — Somewhat thicker than the cholera vibrio: but forms
the long spirilla less often. Has flagella.
128
ESSENTIALS OF BACTERIOLOGY.
Liquefies gelatine in a short
It
Fig. 67.
Properties. — It is very motile
time.
Growth. — It grows quickly at ordinary room temperature.
is facultative aerobic.
Colonies on Gelatine Plates. — Round, finely granular colonfes,
which in twenty-four hours are ten times as large as the cholera
colonies, and in forty-eight hours the whole plate is liquefied,
it being then impossible to distinguish any separate colonies.
The microscopic appearances in no way
resemble the cholera colony.
Stab Cultures. — The gelatine is lique-
fied from above downwards, like a stock-
ing in appearance, and in three days is
completely liquid.
Potato. — At ordinary temperature a
thick gray layer covering the whole sur-
face.
Water. — It soon perishes in water.
Staining. — Ordinary aniline dyes.
Pathogenesis. — For man it has no spe-
Fig. 66.
Spirillum Finkleri. 700 diameters. (Fl
!ugge.)
Stab Culture. (Finkler-
Prior.)
cific action. If it is injected into Guinea pigs, prepared as
described under the cholera bacillus, they die, the intestines
having a foul odor, and the bacilli then found in great numbers.
Spirillum Tyrogenum. (Deneke.)
Origin.— In 1885 Deneke found in old cheese a spirillum very
similar in appearance to the cholera spirillum.
Form. — The same as the cholera vibrio.
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PATHOGENIC BACTERIA. 129
Properties. — Very motile, liquefy gelatine.
Growth. — They grow quicker than the cholera, and slower
than the Finkler; they are also facultative aerobic.
Colonies. — At first resemble cholera colonies ; have, however,
a yellow-green iridescence, and are more irregular; also grow
more rapidly.
Stab Cultures. — A thick line along the needle-track and yellow
colonies forming at the bottom, on the surface a bubble of air
similar to the cholera. The gelatine is liquid in two weeks.
Potato. — At brood-heat a thin yellow membrane, but not
always constant. Staining, as cholera bacillus.
Pathogenesis. — When injected into animals prepared as for the
cholera bacillus, a certain number die.
Vibrio Metschnikovi. (Gamaleia.)
Origin. — In the intestines of fowls suffering from a gastro-
enteritis, common in Kussia. Gamaleia found a spirillum which
bears so close a resemblance to the cholera bacillus, both in form
and growth, that it cannot be distinguished by these character-
istics alone.
Form. — As cholera bacillus.
Growth. — Two kinds are found on the gelatine plate— one that
is identical in appearance with the cholera colony, the other more
liquefying, resembling the Finkler spirillum. If now a second
plate be inoculated from either one of these forms, both kinds
again are found grown, so that it is not a mixture of two bacilli.
Stab Culture. — Similar to the cholera growth, a trifle faster in
growing. Staining, as cholera.
Pathogenesis.— To differentiate it from cholera, these bacilli,
when injected into animals, prove very fatal, and no especial
precautions need be taken to make the animal susceptible. In
the pigeon, guinea-pig, and chicken it produces a hemorrhagic
oedema, and a septicaemia which has been called " Vibrion
septicaemia." The blood and organs contain the spirilla in
great numbers.
Products.— The nitrites are formed just as in cholera bacillus,
and the red reaction given when mineral acids added to gelatine
cultures. Certain products also which, when injected, give
130 ESSENTIALS OP BACTERIOLOGY.
immunity. The cultures are first heated for one half hour at
100° C, which destroys the germs, and then this sterilized pro-
duct injected. (5 c.cm. of a five days' old sterilized culture.)
In a couple of weeks 1 to 2 c.cm. of the infected blood can be
injected without causing any fatal result.
A great many more spirilla resembling the spirillum of chol-
era have been isolated from drinking-waters in the past few
years, and some bacteriologists are inclined to consider them
as varieties of the true cholera spirillum which require only
certain conditions to make them pathogenic. Among these,
besides those already described, are Spirillum Berolinesis, S.
Dunbar, S. Danubicus, S. Wernicke, S. BonhofF, S. Weibeli, S.«
Schuylkiliensis, S. Milleri, S. Aquatilis. The last two are non-
pathogenic for experiment animals.
Bacteria of Pneumonia. Two forms of bacteria have been
found in this disease, and thought at different times to be the
cause of the same.
Neither one of them is constant in pneumonia ; and since
many other pathological processes have shown them they can
hardly be set down as the sole cause of pneumonia.
Klebs in 1875 called attention to the presence of bacteria in
pneumonia, and in 1882 Friedlander developed a bacillus from
the lung tissue of a pneumonic person, which he thought was a
coccus, and called it pneumococcus.
In 1886 A. Frankel and Weichselbaum proved that this
microbe was not constant, in fact was rare.
A. Frankel obtained in the majority of cases of pneumonia a
microbe that he had described in 1884 under the name of
sputum-septicaemia micrococcus.
Weichselbaum called it " Diplococcus pneumonias," and be-
lieved it to be the real cause of pneumonia. It has been found
in many other serous inflammations, and also in the mouths of
healthy persons. It is the generally accepted -organism of the
disease, and can be isolated from nearly all cases of acute
croupous pneumonia. It is found in about three-quarters of
all cases of pneumonia.
Streptococcus pyogenes and staphylococcus pyogenes aureus have
been found in some cases.
PATHOGENIC BACTERIA
131
Pneumo-bacillus (Pneumococcus). (Friedlander.)
Origin. — In the lung of a croupous-pneumonia person, by
Friedlander, in 1882.
Fig. 68.
Bacillus pneumoniae of Friedlander, from the expectoration of a pneumonia patient ;
X 1000 (Frankel and Pfeitterj.
Form.— Small, almost oval-shaped rods, nearly as wide as
they are long ; often in pairs, they were at first believed to be
cocci. In bouillon cultures the rod-form becomes more visible.
In tissues each bacillus is surrounded by a faint capsule ; but
not around those developed in artificial cultures. Spores have
not been found.
Properties.— They are immobile ; do not liquefy gelatine. A
gas is produced in gelatine cultures.
Growth.— Grows rapidly on all media at ordinary temperature ;
is facultative aerobic.
Colonies.— On gelatine plates. Small white round colonies,
reaching the surface in the course of three or four days ; appear-
ing then as little buttons, with a porcelain-like shimmer, the
edges smooth.
132
ESSENTIALS OF BACTERIOLOGY.
Pig. 69.
Stab Culture. — A growth along the needle-track, but on the
surface a button-like projection, which gives to the growth the
appearance of a nail driven into the gelatine,
its head resting on the surface ; therefore
such cultures are called "Nail cultures.''''
See Fig. 69. Old cultures are colored brown,
and contain bubbles of gas.
Potato. — A yellow, moist layer in a few
days at brood-heat. Gas bubbles develop.
Staining.— The ordinary aniline stains.
The sections do not take Gram's method ;
are therefore not suited for double staining.
Capsule.— Stained as follows :—
Cover glasses.
1. Acetic acid, two minutes.
2. Allow acetic acid to dry by blowing air
upon it through a glass tube.
3. Saturated, aniline water. Gent, violet,
ten seconds.
4. Rinse in water. Mount in Canada balsam.
For Sections.
Bacillus of Pneumo- f cone, ale. gent, violet, 50.0
nia. stab Culture. 1. Stain in warm ] aqua, 100.0
LNaii culture.) I acetic acid, 10.
M. for 24 hours.
2. Rinse in one per cent, acetic acid.
3. Alcohol to dehydrate. Mount in balsam.
The capsule will be found stained a light blue, the bacillus a
deep blue. (See also the capsule stain of Hiss, p. 35.)
Pathogenesis. — Animals are not affected unless the culture is
injected intrapleura.
Pneumobacillus of Frankel. (A. Frankel and Weichselbaum.)
Synonyms. — Pneumococcus ; Diplococcus of Pneumonia ; Mi-
crococcus of sputum septicaemia ; Micrococcus Pasteuri ; Diplo-
coccus lanceolatus.
Origin. — A. Frankel found it in the sputum of pneumonic
PATHOGENIC BACTERIA.
133
patients, thinking it at first to be the micrococcus of sputum
septicemia ; later he believed it to be the cause of pneumonia.
Form. — Oval cocci they were at first called, but they are now
known to be rod-shaped, being somewhat longer than broad ;
varying, however, much in size and shape. Usually found in
pairs, sometimes in filaments of three and four elements. In
the material from the body a capsule surrounds each rod. In
the artificial cultures this is not found.
Fig. 70.
Bacillus of pneumonia in blood of rabbit 1000 X. (Frankel and Pfeifler.)
Properties. — They are without self-movement; do not liquefy
gelatine. There are no spores.
Growth.— Grow only at high temperature, 35° C. ; are facul-
tative anaerobic. The culture media must be slightly alkaline ;
the growth is slow.
Colonies on Gelatine Plates. — Since the temperature must be
somewhat elevated, the gelatine media need to be thicker than
usual (15 per cent, gelatine), in order to keep it solid, and a
temperature of 24° C. used. Little round white colonies, some-
what granular in the centre, growing very slowly.
Stab Cultures. — Along the needle-track small separate white
granules, one above the other, like a string of beads.
Stroke Culture. — On agar, transparent, almost invisible little
drops resembling dew moisture.
134 ESSENTIALS OF BACTERIOLOGY.
Bouillon. — They grow better here than in the other media,
remaining alive a longer period of time.
Staining.— Takes Gram's method and the other aniline stains
very readily. The capsule stained the same way as that of the
Friedlander bacillus.
Pathogenesis. — Rabbits and guinea-pigs, if subcutaneously in-
jected, die in the course of a couple of days with septicaemia.
(0.1 com. of a fresh bouillon culture suffices.)
Autopsy shows greatly enlarged spleen and myriads of bacilli
in the blood and viscera, the lungs not especially affected. If
injected per trachea, a pneumonia occurs. In man in 90 per
cent, of croupous pneumonia they are found and usually only
during the existence of the rusty sputum, i. e., the first stage.
Fig. 71.
1&
**
Micrococcus tetragenus in sputum (tubercle bacillus also).
They have also been found in pleuritis, peritonitis, pericarditis,
meningitis, and endocarditis. They stand in some intimate re-
lation with all infectious inflammations of the body. Their
presence in healthy mouth secretion does not speak against
this, it requiring some slight injury to allow this ever-present
germ to develop into disease.
Anti-toxin of Pneumonia. (Klemperer.)
The injection of very diluted cultures of the virulent bacilli in-
travenously has produced an immunity in rabbits and guinea-
pigs. The serum of such artificially immune animals when filtered
PATHOGENIC BACTERIA. 135
through a Chamberland filter and injected into a rabbit suffer-
ing with pneumonia, cured the same; or when injected into a
susceptible animal produced in it immunity very quickly. This
principle is ascribed to an anti-toxin formed in the tissues by
the diluted proteids, and this anti-toxin neutralizes the toxicity
of the strong virus.
Bacillus of Rhinoscleroma. (Frisch. 1882.) It was found in
the tissue of a rhinoscleroma, but resembles the Friedlander
bacillus in nearly every respect, and as the disease rhinoscleroma
was not reproduced by the inoculation of the bacillus in animals,
it can be considered identical. The growth, cultures, and pro-
perties are the same as the pneumobacillus of Friedlander.
Diplococcus Intracellularis Meningitidis. Weichselbaum
claims to have found a special diplococcus in epidemic cerebro-
spinal meningitis, which differs in a few respects from the pneu-
mococcus of Frankel : growth most abundant on blood-serum —
round, white, shining colonies in twenty-four hours. It does
not take Gram's stain ; does not affect animals when injected
eubcutaneously. Inoculated into the meninges of the dog and
goat, a meningitis has been produced, and when found in the
exudate of the meninges lies in the protoplasm and nuclei of
the leucocytes. The organism has many points in common
with the gonococcus, but differs from it in the ease of cultivation.
Micrococcus Tetragenus. (Koch. Gaffky).
Origin. — Koch found this microbe in the cavity of a tuber-
culous lung. Gaffky, in 1883, studied its pathogenic actions
and gave it the name it now bears.
Form. — Cocci which are gathered in the tissues in groups of
four, forming a square, a tetrad. See Fig. 71. In artificial
culture, sometimes found in pairs. A capsule of light gelat-
inous consistence surrounds each tetrad.
Properties. — They are immobile ; do not liquefy gelatine.
Growth. — They grow well on all nutrient media at ordinary and
brood temperatures ; are facultative aerobic. They grow slowly.
Colonies in gelatine plates. In two days, little white spots,
which when on the surface form little elevations of a porce-
lain-like appearance; under low power they are seen very
finely granulated.
136
ESSENTIALS OF BACTERIOLOGY.
Fig. 72.
v\
Stab Culture. — Small round separated colonies along the
needle-track, and on the surface a button-like elevation, a
form of " nail culture." See Fig. 72.
Potato. — A thick slimy layer which can be
loosened in long shreds.
Staining.— Colored with the ordinary ani-
line stains. Gram's method also applicable.
Pathogenesis.— White mice and guinea-pigs
die in a few days of septicaemia when injected
with the tetragenus cultures, and the micro-
coccus is then found in large numbers in the
blood and viscera. Field mice are immune.
In the cavities of tubercular lungs, in the
sputum of phthisical and healthy patients, it
is often found, but what action it has upon
man has not yet been determined.
Capsule Bacillus. (Pfeiffer.)
Origin. — Stringy exudate and blood of a
dead guinea-pig.
Form. — Thick little rods, sometimes in long
threads. Large oval capsules in the stained
preparations.
Properties. — Immotile, not liquefying, an
odorless gas in gelatine cultures.
Growth. — At ordinary temperatures, rap-
idly; facultative anaerobin.
Gelatine Plates. — Oval points, and like a por-
celain button on the surface.
Stab Cultures. — Like the pneumonia bacillus
of Friedlander.
Potatoes. — Abundant growth, yellow color
and moist, coming off in strings.
Staining. — Hot fuchsin colors the capsule intensely ; carefully
decolorizing with acetic acid, the capsules are red or light violet
around the deeply-tinged bacillus. Gram's method not applic-
able.
Pathogenesis. — Subcutaneously injected in mice, they die in
48 hours. Rabbits die when a large quantity is injected into
Stab Culture.
Micrococcus tetra-
genus.
PATHOGENIC BACTERIA. 137
the circulation. The blood and juices have a peculiar stringy
fibrinous consistence.
Bacillus of Influenza. (Pfeiffer, 1892.)
A small bacillus about one-half the size of the bacillus of
mouse septicaemia, and arranged in chain-form. It develops
Fig. 73.
» . • * <«
Bacillus influenzae, from a gelatin culture; X 1000 (Itzerott and Niemann).
upon blood-serum agar. It is aerobic. Without movement;
does not take the Gram stain. It is best stained with diluted
carbol-fuchsin, the contrast-stain being Loffler's methylene-
blue. Upon glycerine-agar, over which a drop of blood has been
spread, in an incubator at the end of twenty-four hours a very
delicate growth occurs, which resembles condensed moisture.
It is found in the sputum and in the bronchial nasal secretions
and blood of influenza patients, but cannot as yet be said to be
the cause of influenza.
Micro-Organisms of Suppuration. The suppuration of wounds
is due to the presence of germs. The knowledge of this fact is
the basis of the antiseptic treatment in surgery ; for when the
microbes can be destroyed or their entrance prevented, the
wounds are made clean and kept without suppurating. Vari-
ous forms of bacteria have been found in septic processes, and
138
ESSENTIALS OF BACTERIOLOGY-.
the formation of pus cannot be ascribed to any particular one
alone ; some, more common than others, are found in nearly all
forms of suppuration ; others give rise to special types.
Wounds are often irritated by for-
FlG# 74, eign bodies and chemicals, and a dis-
charge occurs in them even when
every aseptic and antiseptic precau-
tion has been taken ; but such a dis-
charge is free from bacteria, and no
more like pus than a benign growth
is like a malignant one.
Fig. 75.
Streptococcus pyogenes: cult-
ure upon agar-agar two days old
(Frankel and Pfeiffer).
Streptococcus pyogenes (Jakob).
Streptococcus Pyogenes. (Rosenbach.) Streptococcus erysipe-
latis. (Fehleisen.)
Origin. — Fehleisen discovered this microbe in the lymphatics
of the skin in erysipelas, and he thought it the cause of the
same. Under the name streptococcus pyogenes, Rosenbach
described an identical coccus which has been found in nearly
all suppurative conditions.
Form. — Small cocci singly and in chain-like groups. Spores
PATHOGENIC BACTERIA. 139
have not been found, though it is supposed because of their
permanency that spores are present.
Properties. — They are immotile, do not liquefy gelatine.
Growth. — They grow slowly, usually on the surface, and best
at higher temperatures.
Colonies. — In three days a very small grayish speck, which
hardly ever becomes much larger than a pin-head ; under micro-
scope, looking yellowish, finely granular, the edges quite defined.
Stub Cultures. — Along the needle-track little separated colonies
like strings of beads, which after a time become one solid white
string.
Stroke Culture. — Little drops, never coalescing, having a bluish
tint.
Potato. — No apparent growth.
Bouillon.— At 37° C. clouds are formed in the bouillon, which
then sink to the bottom, and long chains of cocci found in this
growth.
Staining. — Easily colored with the ordinary stains. Gram's
method is also applicable.
Pathogenesis. — Inoculated subcutaneously in the ear of a
rabbit, an erysipelatous condition develops in a few days,
rapidly spreading from point of infection.
In man, inoculations have been made to produce an effect
upon carcinomatous growths. Erysipelas was always produced
thereby. When it occurs upon the valves of the heart, endo-
carditis results. Puerperal fever is caused by the microbe in-
fecting the endometrium, the Streptococcus puerperalis of Frankel
being the same germ.
In scarlatina, variola, yellow fever, cerebro-spinal meningitis,
and many similar diseases, the microbe has been an almost con-
stant attendant. It is often associated with the diphtheria
bacillus in true diphtheria, and is the cause of many of the
diphtheritic affections of the throat in which the diphtheria
bacillus is absent.
An antistreptococcic serum has been used as a curative
agent in puerperal fever, scarlatina, and other diseases sup-
posed to be due to this germ.
A mixture of a culture of Pyogenes and Prodigiosus has been
140 ESSENTIALS OF BACTERIOLOGY.
used as an injection, with apparent benefit, in inoperable cases
of sarcoma.
Staphylococcus Pyogenes Aureus. (Rosenbach.)
Fig. 76.
Staphylococcus pyogenes albus (Jakob).
Origin. — Found commonly in pus (80 per cent, of all suppura-
tions), in air, water, and earth ; also in sputum of healthy persons.
Form. — Micrococci in clusters like bunched grapes, hence the
name staphylo, which means grape. They never form chains.
Spores have not been found, though the cocci are very resistant.
Properties. — Without movement ; liquefying gelatine. It gives
rise to an orange-yellow pigment in the various cultures.
Growth. — It grows moderately fast at ordinary temperature,
and can live without air, a facultative serobin and anterobin.
Colonies on Gelatine. — On second day small dots on the surface,
containing in their centre an orange-yellow spot. The gelatine
all around the colony is liquefied ; the size is never much greater
than that attained the second day.
Colonies on Agar. — The pigment remains a long time.
Stab Culture. — At first, gray growth along the track, which,
after three days, has settled at the bottom of the tube in a yel-
low granular mass, the gelatine being all liquid.
Stroke Culture on Agar. — The pigment diffused over the sur-
face where the growth is, in moist masses.
PATHOGENIC BACTERIA.
141
Fig.
Potato. — A thin white layer which gradually becomes yellow
and gives out a doughy smell.
Staining.— Very readily colored with ordinary stains; also
with Gram's method.
Pathogenesis.— When rabbits are injected with cultures of this
microbe into the knee-joint or pleura, they die in a day. If
injected subcutaneously, only a local action occurs, namely,
abscesses.
If directly into circulation, a general phleg-
monous condition arises, the capillaries be-
come plugged with masses of cocci, infarct
occur in kidney and liver, and metastatic ab-
scesses form in viscera and joints. Garre\ by
rubbing the culture on his forearm, caused
carbuncles to appear.
Several varieties of the pyogenic staphylo-
cocci are recognized according to their color-
producing properties and slight variations of
growth. Of these, the staphylococcus pyog-
enes aureus is the most virulent, and is con-
sidered the type of the group. They are
always present on the surface of the body,
beneath the nails, in the nose and mouth, in
the dust of streets, and on the floor of houses.
Staphylococcus pyogenes albus differs
from the preceding only in the absence of Stab culture. Micro-
pigment and in its slight virulence. Welch coccus Py°genes
° aureus.
describes a variety constantly found both on
the skin and in its deeper layers, which he calls the staphylo-
coccus epidermidis albus.
Micrococcus Pyogenes Citreus. (Passett.) This liquefies gel-
atine less rapidly than the pyogenes aureus, and forms a citron-
yellow pigment instead of the orange-yellow of the aureus.
Micrococcus Cereus Albus. (Passet.) Differs from the pyo-
genes albus in the form of colony. A white shiny growth like
drops of wax; hence the name cereus.
Micrococcus Cereus Flavus. (Passet.) A lemon-yellow
colored growth after a short time, otherwise not differing from
cereus albus.
142 ESSENTIALS OF BACTERIOLOGY.
Micrococcus Pyogenes Tenuis. (Rosenbach.)
Origin.— Found in the pus of large inclosed abscesses.
Form. — Cocci, without any especial arrangement.
Properties. — Not much studied.
Growth. — Cultivated on agar, it forms clear thin colonies; along
the needle-track an opaque streak, looking as if varnished over.
Bacillus Pyocyaneus. (Gessard.)
Synonyms. — Bacterium seruginosum, bacillus fluorescens.
(Schroter.) The bacillus of bluish-green pus.
Origin. — Found in 1882 in the green pus in pyocyeemia.
Form. — Small slender rods with rounded ends, easily mistaken
for cocci. Often in groups of four and six, without spores.
Properties. — Very motile ; liquefy gelatine rapidly ; a peculiar
sweetish odor is produced in the cultures, and a blue pigment.
Fig. 78.
*«*N*«-
Bacillus pyocyaneus, from an agar-agar culture ; X 1000 (Itzerott and Niemann).
Growth. — Develops readily at ordinary temperature, growing
quickly and mostly on the" surface ; it is aerobic. Colonies on gela-
tine plate, in two or three days a greenish iridescence appears
over the whole plate, the colonies having a funnel-shaped lique-
faction, and appearing under low power when still young, as
yellowish green, the periphery being granulated.
Stab Cultures. — Mainly in upper strata, the liquefaction funnel-
shaped, the growth gradually settling at the bottom, a rich green
PATHOGENIC BACTERIA. 143
shimmer forming on the surface, and the gelatine having a deep
fluorescence.
Potato. — The potato is soaked with the pigment, a deep fold
of green occurring on the surface.
Staining. — With ordinary aniline dyes.
Pathogenesis. — When animals are injected with fresh cultures
in the peritoneal cavities or cellular tissues, a rapidly spreading
oedema with general suppuration develops. The bacilli are
found in the viscera and blood.
If a small quantity is injected, a local suppuration occurs, and
if the animal does not die it then can withstand large quanti-
ties. It is immune.
The Pigment. Pyocyanin. — When the pus, bandages, and
dressings containing the bacillus pyocyaneus are washed in
chloroform, the pigment is dissolved and crystallizes from the
chloroform in long needles. It is soluble in acidulated water,
which is turned red thereby, and when neutralized the blue color
returns. It has no pathogenic action. It is an aromatic com-
pound. The bacillus has no especial action on the wound, and
is found sometimes in perspiration of healthy persons.
Bacillus Pyocyaneus. |3. (Ernst.) A bacillus found in gray-
ish pus-colored bandages.
The only especial difference between this and the above is the
formation of brownish-yellow pigment instead of pyocyanin. The
form and appearance of cultures otherwise the same.
Micrococcus Gonorrhoeae. Gonococcus. (Neisser.) In 1879
Neisser demonstrated the presence of this
germ in the secretion of specific urethritis. Fi®. 79.
Form. — Cocci, somewhat triangular in
form, found nearly always in pairs, the base
of one coccus facing the base of the other,
and giving the appearance of a Vienna roll,
hence the German name Semmel (roll)-form.
:■%.
•v»- '
Immotile.
Culture. — On gelatine-agar or potato they
do not grow, and only upon human-blood Gonococci in gon-
serum have they given any semblance of a nne^methyi' *toi£
growth. The temperature must be between (650 diameters.)
Four to twelve such pairs are often found
together. r
144
ESSENTIALS OF BACTERIOLOGY.
83° and 37° C, and the growth occurs very slowly and
sparsely.
Method of Cultivation (Wertheim).— Gonorrheal pus is mixed
in a test-tube with liquid human blood serum of 40° C. temper-
ature, and two dilutions are made with blood of the same tem-
perature. An equal quantity of 2 per cent, agar solution is
now poured into each tube, and three glass dishes are covered
at once with this mixture. After being in the brood oven for
twenty-four hours colonies can be discovered.
Fig. 80.
Gonococcus in urethral pus; X 1000 (Frankel and Pfeiffer).
In three days a very thin, almost invisible, moist 3rellowish
growth, seemingly composed of little drops.
Under low power small processes are seen shooting out from
the smooth border.
It requires to be then transferred to fresh media, as it quickly
perishes.
Cultivation has also occurred on acid gelatine, gelatine con-
taining acid urine, in acid urine itself, and albuminous urine
with agar.
Staining. — Colored easily with all ordinary aniline stains.
PATHOGENIC BACTERIA. 145
Gram's method is not applicable, this being one of its main
diagnostic features.
The following method is recommended by Neisser.
The cover-glasses, with some of the urethral discharge smeared
upon them, are covered with a few drops of alcoholic solution of
eosin and heated for a few minutes over the flame. The excess
of the dye is removed with filter paper, then the cover-glass
placed in concentrated methylin blue (alcoholic solution) for 15
seconds, and rinsed in water.
The gonococci are dark blue, the protoplasm of the cell pink,
and the nucleus a light blue, the gonococci lying in the proto-
plasm next to the nucleus.
Other bacteria are similar to the gonococci in form ; they are
distinguished from the gonococcus, in that they are colored with
Gram's method, whereas the micrococcus of gonorrhoea is not.
The points on which the diagnosis is to be made are the char-
acteristic biscuit shape, the intracellular position of the organ-
ism, and its failure to stain with Gram.
Pathogenesis. — The attempts to infect the experiment ani-
mals with gonorrhoea have so far been without success. In man,
upon a healthy urethra, a specific urethritis was produced with
even the 20th generation of the culture. Gonorrhoeal ophthalmia
contains the cocci in great numbers, and endocarditis and gon-
orrhceal rheumatism are said to be caused by the cocci.
The microbes have been found long after the acute attack,
when only a very slight oozing remained, and the same were
very virulent.
The specific inflammations of the generative organs of the
female are due to this microbe, having gained entrance through
the vagina, extending its influence. It is found chiefly in the
superficial layers of the mucous membrane.
A temperature of 40° C. for 12 hours destroys the gonococci.
Gonotoxin.—A toxin has been isolated which causes fever, loss
of weight, and finally death. The urethra is not immunized by
repeated injections. In man the toxin causes painful indura-
tions lasting several days.
Similar Microbes found in the Urethra and Vagina.
Micrococcus Citreus Conglomerata. (Bumra.) Similar to
10
116 ESSENTIALS OP BACTERIOLOGY.
the gonococci in form, they are, however, easily cultivated, and
form yellow colonies which dissolve the gelatine and grow rap-
idly; the surface of the gelatine is at first moist and shiny, but
later on wrinkled. Colored with Gram's method, and have no spe-
cial pathological action. Found in the air and gonorrhoeal pus.
Diplococcus Albicans Amplus. (Bumm. ) In vaginal secretion.
The diplococci are much larger than the gonococci, but similar
in form. They are also cultivated upon gelatine plates, grayish-
white colonies, which slowly liquefy gelatine. They grow mode-
rately rapid. Stained with Gram's method, and have no
pathogenic action.
Diplococcus Albicans Tardissimus. (Bumm.)
Origin. — In urethral pus. Form, like gonococci. Properties,
immotile ; do not liquefy gelatine. Growth, very slow at ordi-
nary temperature, but more rapid at brood-heat. The colonies
are small white points, which under low power appear brown
and opaque.
Agar Stroke Culture. — Grayish-white growth, which after two
months is like a skin upon the surface.
Staining. — Takes Gram's method.
Pathogenesis. — None known.
Micrococcus Subflavus. (Bumm.)
Origin.— In lochial discharges, in vagina and urethra of
healthy persons.
Form. — As gonococci.
Properties. — Not motile ; liquefy gelatine slowly ; a yellow-
brownish pigment.
Growth. — Grows slowly on all media, forming on gelatine,
after two weeks, a moist yellowish surface growth.
Potato. — Small half-moon-shaped colonies which, after three
weeks, become light-brown in color, and covering the surface as
a skin.
Staining. — Colored ivith Gram.
Pathogenesis. — Not acting upon the mucous membrane, but
when injected in cellular connective tissue, an abscess results
which contains myriads of diplococci.
The gonococcus is distinguished from all these similar micro-
cocci by being found usually within the cell protoplasm.
PATHOGENIC BACTERIA. 147
Secondly.— Not stained with Gram's method.
Thirdly. — Refusing to grow readily upon gelatine.
All the similar bacteria being easily cultivated.
These characteristics, taken in toto, form sufficient features for
its ready recognition, and as it is often a serious question to
decide, not so much because of the patient's health as because
of his character, we should be very careful not to pronounce a
verdict until we have tested the micro-organism as above. When
the germ so tested is found, the process can be called specific
without a doubt.
Fig. 81.
/
* :^v
Bacillus of Tetanus with spores.
Bacillus of Tetanus. (Nicolaier-Kitasato.)
Oi*igin.— Nicolaier found this bacillus in the pus of a wound
in one who had died of tetanus, describing it in 1884.
Kitasato has since then been able to isolate and cultivate this
germ. (1889.)
Form.— A very delicate, slender rod, somewhat longer than
the bacillus of mouse septicaemia, which is the smallest bacillus.
When the spores form, a small swelling occurs at the end
where the spore lies, giving it a drum-stick shape.
148
ESSENTIALS OF BACTERIOLOGY.
Properties.- JSot very motile, though distinctly so ; liquefies
gelatine slowly. The cultures give rise to a foul-smelling gas.
Growth.— Develops very slowly, best at brood-heat (36° to 38°
Fig. 82.
Fig. 83.
tf
Racillus tetani : culture four days
old in glucose-gelatine (Frankel and
Pfeiffer).
Six days' culture of bacillus
of tetanus in gelatine (deep
stab). (Frankel and Pfeiffer.)
C), and only when all oxygen is excluded, an obligatory ancero-
bin. In an atmosphere of carbon dioxide gas it cannot grow,
but in hydrogen it nourishes.
PATHOGENIC BACTERIA. 149
Colonies on gelatine plates in an atmosphere of hydrogen.
Small colonies. After four days a thick centre and radiating
wreath-like periphery, like the colonies of bacillus subtilis.
Hujh Stab-Culture. — (The gelatine having 2 per cent, glucose
added and filling the tube.) Along the lower portion of the needle-
track, a thorny-like growth, little needle-like points shooting
out from a straight line. The whole tube becomes clouded as
the gelatine liquefies, and then the growth settles at the bottom
of the tube.
Agar. — At brood-heat, on agar, the growth is quite rapid, and
at the end of forty-eight hours gas bubbles have formed and the
growth nearly reached the surface.
Bouillon. — Adding glucose to the bouillon gives a medium in
which an abundant growth occurs.
Cultivation from Spores. — Kitasato, by exposing a portion of sus-
pected material to a temperature of 80° C. for one hour, killed off
all the spores save those of tetanus, which were then cultivated.
Staining. — All the ordinary stains, Gram's method also; the
spores being colored in the usual way.
Pathogenesis. — A small amount of the pure culture injected
under the skin of experiment animals will cause, in two to three
days, death from true tetanus, the tetanic condition starting
from the point of infection. At the autopsy nothing characteristic
or abnormal is found, and the bacilli have disappeared, except
near the point of entrance. This fact is explained as follows :
Several toxic products have been obtained from the cultures,
and they are produced in the body, and give rise to the morbid
symptoms. These have been isolated, and when injected singly
cause some of the tetanic symptoms. The virus enters the
circulation, but does not remain in the tissues. The spores are
very resistant to heat, drying, and chemicals.
Four toxins (among them tetanin, tetanotoxin, and spasmo-
toxin) have been found. The blood and the urine contain the
toxin and are fatal to animals.
Immunity. — Kitasato, by inoculation of sterilized cultures, has
caused immunity to the effects of virulent bacilli.
An anti-toxin obtained by Tizzoni and Cattani from the serum
of animals made immune by sterilized cultures has been used
150
ESSENTIALS OF BACTERIOLOGY.
with curative effects in several cases of tetanus in man. It is
a globulin, but differs from the anthrax anti-toxin, and it is
found exclusively in the serum. By precipitation with alcohol
and drying in vacuo the anti-toxin is obtained in a solid state.
The aqueous solution is used for injection subcutaneously or
subdurally through a trephine opening. Its injection into the
spinal canal by lumbar puncture has also been recommended.
Anti-toxin is more beneficial in chronic cases than in acute.
Habitat. — The bacillus is present in garden earth, in manure ;
and it has been isolated even from mortar.
The earth of special districts seems to contain the bacilli in
greater quantities.
Fig. 84.
Bacillus of malignant oedema, from the body-juice of a guinea-pig inoculated with
garden earth ; X 1000 (Frankel and Pfeiffer).
Bacillus (Edematis Maligni. (Koch, 1881.) Vibrion Septique.
(Pasteur, 1875.)
Origin. — In garden earth, found lately also in man, in severe
wounds when gangrene with oedema had developed. Identical
with the bacillus found in Pasteur's septicaemia.
Form. — Rods somewhat smaller than the anthrax bacilli, the
PATHOGENIC BACTERIA.
151
ends rounded very sharply. Long threads are formed. Very
large spores which cause the rods to become spindle-shaped.
Fig. 85.
Fig. 86.
ESfr-wl
Cultures in agar of malignant
(Edema, after 24 hours, at 37° C.
(Fr&nkel and Pfeiffer.)
Bacillus of malignant oedema
growing in glucose-gelatine
(Frankel and Pfeiffer).
Properties. — Very motile ; liquefy gelatine ; do not produce
any foul gaseous products in the body.
152 ESSENTIALS OF BACTERIOLOGY.
Growth. — Grows rapidly, but only when the air is excluded,
and best at brood or body heat.
Roll Cultures. — (After Esmarch's method.) Small, round colo-
nies with fluid contents, under low power, a mass of motile
threads in the centre, and at the edges a wreath-like border.
High Stab- Culture. — With glucose gelatine, the growth at first
seen in the bottom of the tube, with a general liquefaction of
the gelatine, gases develop and a somewhat unpleasant odor.
Agar. — The gases develop more strongly in this medium, and
the odor is more prominent.
Guinea-Tig Bouillon. — In an atmosphere of hydrogen cloud-
ing of the entire culture medium without any flocculent pre-
cipitate until third day.
Staining.— Are stained with the ordinary dyes, but Gram's
method is not applicable.
Pathogenesis. — When experiment animals, mice or guinea-
pigs, are injected with a pure culture under the skin they die in
8 to 15 hours, and the following picture presents itself at the
autopsy : In guinea-pigs from the point of infection, spreading
over a large area, an oedema of the subcutaneous tissues and
muscles, which are saturated with a clear red serous exudate
free from smell, containing great quantities of bacilli.
The spleen is enlarged, especially in mice. The bacilli are
not found in the viscera, but are present in great numbers on
the surface, i. e., in the serous coverings of the different orgfcns ;
though when any length of time has elapsed between the death
of the animal and the examination, they can be found in the
inner portions of the organs, for they grow well upon the dead
body. In man they have been found in rapidly spreading gan-
grene. They are present in the soil, in putrefactions of various
kinds, and in dirty water.
Immunity. — Is produced by injection of the sterilized cul-
tures, and also the filtered bloody serum of animals dead with
the disease.
Spirillum of Relapsing Fever. (Obermeier.)
Syn. — Spirochseta Obermeieri.
Origin. — Found in the blood of recurrent fever patients,
described in 1873.
PATHOGENIC BACTERIA. 153
Form.— Long, wavy threads (16 to 40 fx long), a true spiril-
lum ; flagella are present.
Properties. — Very motile. Has not been cultivated.
Staining. — Ordinary aniline stains. Bismark brown best for
tissue sections.
Pathogenesis. —Found in the organs and blood of recurrent
fever. Man and monkeys inoculated with blood from one suf-
fering from this disease become attacked with the fever, and
in their blood the spirillum is again found. It is found in the
blood, only in the relapses (during the fever). After the attack
the spirilla gather in the spleen and gradually die there. It
has been found in the brain, spleen, liver, and kidneys. In the
secretions it has not been discovered.
©°©«
Fio. 87.
.©
<&Pod& °
Spirochseta Obermeieti in the blood (von Jaksch).
Bacillus of Soft Chancre. (Ducrey-Unna.)
A diplobacillus which is specific has been described by Ducrey
as obtained from the secretion and in the depth and margins
of the chancroid. Unna's bacillus is narrower and unbroken
in the center.
Cultivation. — Unsuccessful.
Staining.— With borax, methylen-blue, decolorized with weak
acetic acid.
Pathogenesis. — Probably a mixed infection occurs in most
chancroids, especially if buboes result. The bacillus of Ducrey
154
ESSENTIALS OF BACTERIOLOGY.
is not found in unopened buboes, though often contaminating
the ulcerated ones.
Bacillus Icteroides. (Sanarelli, 1897.)
Fig. 88.
Considered the cause of yellow fever by Sanarelli, but Stern-
berg and Novy regard this as not determined. It is not
identical, as was once supposed, with the Bacillus X of Stern-
berg, which is a variety of the Bacillus Coli.
Origin. — In the tissues and blood of yellow-fever patients.
Form. — A small bacillus with rounded ends, often arranged in
pairs, sometimes in threads, with lateral flagella.
Properties. — Motile, readily stained, decolorized by Gram's
method. Does not liquefy gelatine -nor produce glucose fermen-
tation. Aerobic. No acid reaction in milk.
Growth. — Gelatine plates ; white kidney -shaped colonies, with
a central darker portion or nucleus.
Agar. — Colonies look like drops of paraffin, with margins
raised above the surface. Kept alternately at 22° C. and 37° C,
the colonies take on a characteristic appearance, as if an im-
pression had been made in soft wax.
PATHOGENIC BACTERIA. 155
Potato. — Creamy pale growth, turning brown in a week.
Pathogenesis. — Dogs and rabbits, when inoculated with pure
cultures, are affected with symptoms exactly similar to those
seen in yellow-fever patients— a hemorrhagic gastro-enteritis,
steatosis of the liver, and albuminuria.
Two theories exist as to the etiology of yellow fever : (a) That
it is due to the Bacillus icteroides. The results of Sanarelli
have not been universally accepted, (b) That the causative
agent is so small as to be microscopically invisible, and that it
is transmitted from man to man through the bite of a mos-
quito, the Stegomyia fasciata, which acts as intermediate host.
Individuals who have allowed themselves to be bitten by in-
fected mosquitoes have contracted the disease, while others
exposed to infection in all ways but this have remained well.
Bacillus of Bubonic Plague. (Yersin and Kitasato, 1894. )
Bubonic plague or pest is an extremely infectious disease
more or less common in China and the East, and is believed
to have its origin in man from rats and other rodents. It
spreads with great rapidity, especially among those living under
unsanitary conditions.
Nearly at the same time Yersin and Kitasato, working inde-
pendently, discovered in the bubonic swellings and blood of
affected persons a distinctive bacillus which has conformed to
all the conditions necessary to make it the cause of the disease.
Origin. — In the tissues and all the body fluids and secretions
of affected individuals.
Form. — Short, thick rods with an indistinct capsule, rounded
ends. Growing in chains in fluid media.
Properties. — Immotile. Stains readily. No spores. Culti-
vated best in oxygen, but is facultative anaerobic. Stains
stronger at the ends, producing bipolar appearance. Gelatine
not liquefied. Easily destroyed by sunlight and drying.
Growth.— Best at 37° C.
Gelatine. — At 22° C, in 24 hours white, point-like colonies on
the plates, with broad and flat surface, turning gray and then
brown.
Stab. — Snow-white, spreading out on the surface to the edge,
and fluorescent.
156
ESSENTIALS OF BACTERIOLOGY.
Bouillon. — Granular precipitate, with clear fluid above.
Agar and Blood-serum. — Glass-like colonies like drops of dew
at first, then growing larger with iridescent edges.
Potato. — At 37° C. small white mass.
No gas formation in glucose media.
Staining readily with all basic dyes.
Pathogenesis. — After subcutaneous injection in rats death fol-
lows in 40 to 60 hours, with symptoms of severe toxaemia and
convulsions. The point of infection shows a local oedema and
inflammation of the lymphatics. All the organs congested and
surrounded by a bloody exudate. The characteristic bacilli in
all the tissues and secretions. Nearly all the domestic animals
Fig. 89.
I
,1s Ij
** »? '■ *
\ * '*. * ' <#
V'~svx<irK? ■■•'■■■ 7r
i f^r J * ■»■' ' £ * S * ■ *■■
Bacillus of bubonic plague (Yersin).
are susceptible. Mosquitoes and pigeons, however, are im-
mune; flies are not.
Products. — A toxin has been obtained and immunity has been
effected; the serum of immune animals has protective prop-
erties. The serum likewise shows agglutinating powers, as with
typhoid and cholera serums.
Habitat.— Not found in water, but most likely spreads from the
soil in damp and darkened areas. Rats become affected first,
PATHOGENIC BACTERIA. 157
and then through bites and scratches affect man and other ani-
mals. Clothing, vomit, and the excretions generally, likewise,
act as carriers of the infection. In man three forms of the dis-
ease are recognized according to the mode of infection and
course of the disease — viz., bubonic, pulmonic, septicemic.
Bacillus Dysenteriae. (Shiga, 1898.)
The term dysentery is applied to an intestinal disease dis-
playing more or less constancy in its clinical manifestations,
but having, as is now known, a variety of causative agents. It
is fairly certain that one type is the result of infection with an
amoeba, while non-amoebic forms can probably be produced by
several bacteria. Chief among these is the bacillus first de-
scribed by Shiga in Japan, and since then found by Kruse in
Germany, by Flexner, Strong, and Harvie in the Philippine
Islands, and by Vedder and Duval in the United States.
Although it is not absolutely proved that it is the cause of the
disease, still the feet that it is constantly present in the faeces in
one type of dysentery, that such cases give a positive agglu-
tination reaction, the production of a curative serum by the
immunization of animals with pure cultures, and the results
on experiment animals, leave little doubt as to the specificity
of the organism.
Origin. — The dejecta of dysenteric patients.
Form. — A plump bacillus with rounded ends, resembling the
typhoid and colon bacilli.
Properties. — Motility doubtful, but numerous flagella have
been demonstrated. Does not form spores.
Staining. — Stains readily, negative to Gram, facultative an-
aerobe.
Growth.— Best at 37° C. Killed by ten minutes' exposure to
55° C.
Gelatine. — A white line of growth along puncture; super-
ficial growth slight.
Bouillon. — Uniform clouding. Indol usually not produced;
milk not coagulated.
Agar. — Resembles typhoid bacillus.
Potato. — Thin whitish layer, turning light brown.
No gas-formation in glucose or lactose media.
158 ESSENTIALS OP BACTERIOLOGY.
Pathogenesis. — Mice and guinea-pigs die in one or two days
after intraperitoneal inoculation. Rabbits usually recover,
though lesions analogous to those of human dysentery have
been produced. Dogs die in five or six days, with well-marked
diarrhoea.
Products. — The patient's blood-serum agglutinates the bacillus
in cases in which it can be cultivated from the stools. The re-
action is absent from other cases. Shiga has reduced the mor-
tality from 34.7 to 9 per cent, by means of a serum obtained
from immunized horses.
Habitat. — Found in the stools and in shreds of mucous mem-
brane from the intestinal walls.
Bacillus Aerogenes Capsulatus. (Welch, 1891.)
Origin. — The intestine of man and animals, soil, sewage, and
water.
Form. — A thick bacillus, 3 to 6 \i in length, frequently capsu-
lated.
Properties. — Not motile, anaerobic, forms spores chiefly in cul-
tures on blood-serum.
Growth.— Best at 37° C.
Gelatine. — Liquefied slowly or not at all.
Bouillon. — Forms gas.
Milk. — Coagulated and becomes acid.
Potato. — Thin, grayish-white growth with gas-production.
Forms gas in abundance on dextrose, lactose, or saccharose
media.
Pathogenesis. — Is not usually pathogenic for rabbits and mice,
though in guinea-pigs and birds it produces "gas phlegmons."
It is sometimes found in autopsies on human subjects, produc-
ing bubbles or cavities in the viscera (Schaumorgane), but this
is probably due to postmortem migration of the germ from the
intestine. It has been recovered from the blood during life,
however, and. is the most frequent cause of emphysematous
gangrene. Various foreign observers have described organisms
having similar properties and have given them such names as
Bacillus perfringens, Bacillus enteritidis, Granulobacillus immo-
bilis, etc., but they were probably dealing with the Bacillus
aerogenes capsulatus.
PATHOGENIC BACTERIA. 159
Micrococcus Melitensis. (Bruce, 1887.)
Malta fever, also known as Mediterranean fever, occurs in the
region from which it derives its name, but has been observed in
India, the Philippine Islands, and Porto Rico. Bruce culti-
vated a micrococcus from the spleen and proved its specificity.
Origin. — Is found most abundantly in the spleen.
Form. — Rounded or oval, 5 // in diameter, singly, in pairs, or
short chains.
Properties. — Non-motile, though flagella said to be present;
grows slowly, best at body-temperature.
Gelatine. — Not liquefied ; growth very slow.
Bouillon. — Turbid, with sediment.
Agar. — Pearly white growths.
Potato. — Slight invisible growth.
Stained by ordinary aniline dyes.
The disease may be produced in monkeys by even small
amounts of pure culture. In man a chronic, remittent febrile
disease is produced, with sweating and arthritis. The mortality
is 2 per cent. A serum reaction can be obtained and is diag-
nostic.
Micro-organisms have been found by various observers in
measles, scarlatina, mumps, and whooping-cough, but their
specificity is still in doubt.
PATHOGENIC PROTOZOA.
Certain diseases are produced by animal parasites belonging
to the protozoa, and although not pertaining to the realm of
bacteriology, still the fact that they were long considered bac-
terial in nature and require somewhat similar methods for their
study renders it proper to include a brief mention of them.
The Malarial Parasite. It has been definitely proved that
malarial fever is the result of the presence in the blood of a
protozoon which in the vast majority of cases gains entrance
to the body through the bite of a particular genus of mosquito
(Anopheles). Three varieties of the organism are recognized
in man, though possibly more exist, and each produces a char-
acteristic clinical picture. 1. The Hazmamazba vivax, the para-
site of tertian fever. 2. The Hmmamazba malarias, the parasite
160 ESSENTIALS OP BACTERIOLOGY.
of quartan fever. 3. The Hcemomenas prsecox, the parasite of
sestivo-autumnal fever.
According to its situation, the parasite exhibits two distinct
phases of existence : in the human blood it passes through an
asexual reproductive cycle, while in the body of the mosquito
it undergoes an entirely different series of sexually reproductive
changes. It is simpler first to describe the life history of the
organism in general, pointing out the differences shown by the
three varieties later.
1. The Asexual Cycle in Man. — An infected mosquito conveys
the parasites into the blood as minute hyaline bodies which
enter the blood-cells. At first they are small, round, colorless
bodies, exhibiting more or less active amoeboid motion in the
fresh blood. Sometimes, particularly in the sestivo-autumnal
form, a ring shape is assumed. Their size gradually increases
and pigment granules appear, while in stained specimens a
nucleus containing chromatin granules is visible. As the para-
site approaches maturity the chromatin becomes scattered,
and finally the protoplasm divides into six to twenty spores
(merozoites), each containing a portion of the chromatin. The
number of spores formed and their arrangement before seg-
mentation takes place differ in the three varieties and will be
noted below. The spores burst through the envelop of. the red
corpuscle and become free in the blood, but speedily enter fresh
corpuscles and pass through the same series of changes. The
febrile stage is synchronous with sporulation and liberation of
the young forms.
Certain of the parasites do not, however, go on to segmenta-
tion, but, after reaching maturity, remain quiescent and form
the so-called gametes or sexual types. In the tertian and quartan
varieties these are not very different from the mature organ-
isms, but the sestivo-autumnal gametes are crescentic in shape
and very characteristic.
2. The Sexual Cycle in the Mosquito. — If, now, the blood is shed,
certain of the gametes (the male forms or microgametocytes) ex-
trude long protoplasmic processes containing a central core of
chromatin, and which represent the male fertilizing element
(microgametes). These become detached, and, entering a female
Plate III.
Various Forms of Malarial Parasites (Thayer and Hewetson). Figs. 1-10
inclusive, tertian organisms ; Figs. 11-17 inclusive, quartan organisms ; Figs.
18-27 inclusive, estivo-autumnal organisms.
Fig. 1.— Young hyaline form; 2, hyaline form with beginning pigmenta-
tion ; 3, pigmented form ; 4, full-grown pigmented form ; 5, 6, 7, 8, segmenting
forms ; 9, mature pigmented form ; 10, flagellate form.
Fig. 11.— Young hyaline form ; 12, 13, pigmented forms ; 14, fully developed
form ; 15, 16, segmenting forms ; 17, flagellate form.
Figs. 18, 19, 20.— Ring-like and cross-like hyaline forms: 21, 22, pigmented
forms ; 23, 24, segmenting forms ; 25, 26, '27, crescents.
f
PLATE III.
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8
9
10
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is
18
19
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21
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27
PATHOGENIC BACTERIA. 161
gamete (macrogamete), a true sexual fertilizing process takes
place. In the alimentary canal of the mosquito these fertilized
cells penetrate the stomach-walls and form cysts filled with a
large number of filiform spores, which are extruded into the
body cavity of the insect, and some of which reach the salivary
glands, whence they are ejected when the mosquito bites.
This cycle of development takes seven or eight days.
Differential points of the three forms :
1. The Tertian Form. — The adult forms are large, not very re-
fractile, and their outline is somewhat indistinct. There is an
abundance of fine pigment-granules, and the ameboid motion
is vigorous. Segmenting forms divide into 15 to 20 merozoites ;
the sexual forms or gametes are large. The red cell containing
the organism is swollen and pale. Sporulation and, therefore,
the malarial paroxysm occur every forty-eight hours.
2. The Quartan Form. — The organism is smaller, is more re-
fractile, and its outline is more distinct. The pigment is coarse
and situated at the periphery of the organism, while the proto-
plasmic motion is sluggish. Segmentation forms only 6 to 12
spores, and has the regular " daisy-head " appearance ; the
gametes are small. The red cells become dark in color, and
the cycle requires seventy-two hours.
3. JEstivo-autumnal Form. — The adult forms are found mainly
in the spleen and other viscera, and do not very often occur in
the peripheral blood ; their outline is sharp, and they are highly
refractile. The pigment is scanty and fine; the motion is
active. A variable number of merozoites is formed — usually 6
to 12. The gametes are characteristic, being crescentic in
shape and very resistant to quinine. The red cell becomes
shrivelled and yellowish. The cycle usually takes forty-eight
hours, though it is somewhat variable.
Mixed infections with the different organisms or with two or
more broods of the same organism may occur, so that quotidian
and irregular paroxysms may be produced.
Methods of Examination.
1. Fresh preparations are made by placing a small drop of
blood on a slide and a cover-glass over it, so that only a thin
film is formed. A ring of vaseline is smeared over the edges
11
162 ESSENTIALS OF BACTERIOLOGY.
of the cover-glass to prevent evaporation. This is the best
method for studying flagellation and fertilization, but is less
satisfactory for routine clinical work than —
2. Stained Smears. — These are made by spreading a drop of
blood in a thin film over one slide with the edge of another,
drying in the air, and staining. Many stains have been devised
for the malarial organism, but the following are sufficient for
ordinary use :
(1) Marchoux's Thionin Stain.— Add 20 c.c. of saturated solu-
tion of thionin in 50 per cent, alcohol to 100 c.c. of 2 per cent,
carbolic acid. Fix the smears and stain for fifteen to twenty
seconds. The malarial organisms are stained a deep purple,
strongly contrasting with the faint green of the red cells, so
that they are readily recognized.
(2) Jenner's Stain. — This is excellent for routine work, as no
preparatory fixation is required. Equal parts of a 1.2 per cent,
aqueous solution of Griibler's water-soluble eosin and a 1 per
cent, aqueous solution of Griibler's medicinal methylin-blue
are mixed and the resulting precipitate allowed to stand for
twenty-four hours, washed, and dried. Half a gram of this is
dissolved in 100 c.c. of pure methyl-alcohol. The smears are
dropped into this stain for one to three minutes, without pre-
vious fixation, and at once rinsed in distilled water. The
malarial parasites are stained blue, the cell-bodies a reddish
brown.
(3) Wright's Chromatin Stain. — This is the best of the chroma-
tin stains. For its preparation, which is quite complicated, see
Wright, Journal of Medical Research, vol. vii., 1902. It is used
as follows :
1. The stain is poured over the film and allowed to remain
for one minute to secure fixation.
2. Add distilled water drop by drop until a metallic scum is
formed on the surface. The staining now takes place and re-
quires two to three minutes. Wash in distilled water until a
pinkish tint appears in the thin portions of the smear. The
body of the malarial parasite is stained blue, and its chromatin
a lilac to red color. The red cells are orange pink.
PATHOGENIC BACTERIA. 163
If possible, examinations for malarial organisms should
always be made before quinine is administered.
Amoeba Bysenteriae. Found in the intestinal ulcers, fasces,
and secondary liver abscesses in certain cases of dysentery. A
non-pathogenic form, Amoeba coli, also occurs. The Amoeba
dysenteries is a unicellular animal organism measuring 25 to
35 fi in diameter, though larger and smaller forms occur. There
are a nucleus and a nucleolus ; the protoplasm of the cell-body
is vacuolated and often contains red blood-cells and bacteria.
In fresh, warm stools active ameboid motion may be observed.
The non-pathogenic form is smaller and never contains red
blood-cells.
Small-pox and Vaccinia. The exciting agent of small-pox is
still unknown, but numerous bacteria and protozoon-like bodies
have been described and given etiological significance by vari-
ous authors. There is some evidence in favor of Funck's belief
that vaccinia is caused by a protozoon, the Sporidium vaccinale.
Animals inoculated with this organism developed both vaccinia
and variola.
Trypanosomes. These are protozoa belonging to the order
flagettaJta, and have been found in the blood in certain diseases
of man and animals. Surra, a fatal tropical disease of horses
and mules, the tse-tse fly disease of South Africa, and the
sleeping sickness or negro lethargy of the Guinea coast, are due
to organisms of this group. It is probable that insects of vari-
ous species are the intermediary or definitive hosts of the
trypanosome and convey the infection by their bites.
Texas cattle-fever or bovine malaria is due to an endoglobular
parasite, the Pi/rosoma bigwninum, not unlike the malarial
organism, and is transmitted through the larvae of the cattle-
tick. A similar organism has lately been found to be the cause
of the Rocky Mountain fever of man. The infection here also
seems to be through the mediation of a tick.
164 ESSENTIALS OF BACTERIOLOGY.
CHAPTER IV.
BACTERIA PATHOGENIC FOR ANIMALS BUT NOT FOR MAN.
Bacillus of Symptomatic Anthrax. (Bollinger and Feser.)
(Charbon symptomatique. Arloing, Cornevin, and Thomas.;
Origin. — This bacillus, described already in 1879, has only
lately been isolated, and by animal inoculation shown to be the
cause of the " black-leg" or " quarter evil" disease of cattle.
Form. — Large slender rods, which swell up at one end or in
the middle for the spore. (See Plate IV., Fig. 1.)
Properties. — They are motile, and liquefy gelatine quite
rapidly.
A rancid odor is developed in the cultures.
Cultures.— The growth occurs slowly, and only in an atmo-
sphere of hydrogen, being very easily destroyed by oxygen and
carbon dioxide ; grows best at blood heat ; under 15° C. no
growth.
Glucose-gelatine.— In a few days little round colonies develop,
which, under low power, show hairy processes around a compact
centre.
Stab Cultures in full test tubes. — The first growth in the lower
portion of the tube not very characteristic. Gases develop
after a few days, and the gelatine becomes liquid.
Agar at brood temperature, in 24 to 48 hours, an abundant
growth with a sour odor and abundant gas formation.
Staining.— Ordinary methods. Gram's method is not appli-
cable to the rods ; but the spores can be colored by the regular
double stain for spores.
Pathogenesis. — If a small amount of the culture be injected
under the skin of a guinea-pig, in twenty hours a rise of tempera-
ture, pain at the site of injection, and in a few hours more
death. At the autopsy, the tissues blackened in color and
soaked with a bloody serous fluid ; in the connective tissue large
collections of gas, but only in the neighborhood of the point
of infection. The bacilli are found in great numbers in the
BACTERIA PATHOGENIC FOR ANIMALS
165
serum, but only appear in the viscera some time after death,
when spores have developed.
The animals are usually infected through wounds on the
extremities ; the stalls or meadows having been dirtied by the
spore-containing blood of animals previously dead of the dis-
ease. " Baaachbrand" is the German name; " Cliarbon symp-
tomatique," the French, from the resemblance in its symptoms
to anthrax.
Immunity.— Rabbits, dogs, pigs, and fowls are immune by
nature, but if the bacilli are placed in a 20 per cent, solution of
lactic acid, and the mixture injected, the disease develops in
them. The lactic acid is supposed to destroy some of the
natural resistance of the animal's cells.
When a bouillon culture is allowed to stand a few days, the
bacilli therein lose their virulence, and animals are no longer af-
fected by them.
But if they are placed in 20 per cent, lactic acid and the mix-
ture injected, their virulence returns.
Immunity is produced by the injections of these weakened
cultures, and also by some of the products which have been ob-
tained from the cultures.
Bacillus of Chicken Cholera. (Pasteur.)
Syn.— Micrococcus cholera gallinarum. Microbe en huit. Ba-
cillus avicidus. Bacillus of fowl septicemia.
Origin. — In 1879 Perroncito observed this cocci-like bacillus
in diseases of chickens, and Pasteur, in 1880, isolated and
reproduced the disease with the microbe in question.
Form. — At first it was thought to be a micro-
coccus, but it has been seen to be a short rod
about twice as long as it is broad, the ends
slightly rounded. The centre is very slightly
influenced by the aniline colors, the poles
easily, so that in stained specimens the bacil-
lus looks like a dumb-bell or a figure-of-eight.
(Microbe en huit. )
Properties. — They do not possess self-move-
ment ; do not liquefy gelatine.
Growth. — Occurs at ordinary temperature, requiring oxygen
for development. It grows very slowly.
Fig
Chicken cholera
in blood 1000 X-
(Frankel and
Pfeiffer.)
166 ESSENTIALS OP BACTERIOLOGY.
Gelatine Plates. — In the course of three days little round,
white colonies, which seldom increase in size, having a rough
border and very finely granulated.
Stab Culture. — A very delicate gray line along the needle-
track, which does not become much larger.
Agar Stroke Culture.— A moist, grayish-colored skin, more
appreciable at brood heat.
Potato.— At brood heat after several days a very thin, trans-
parent growth.
Staining. — Methylin blue gives the best picture. Gram's
method is not applicable. As the bacillus is easily decolorized,
aniline oil is used for dehydrating tissue sections, instead of
alcohol.
Method :
Loffler's methylin blue . £ hour.
Alcohol 5 seconds.
Aniline oil 5 minutes.
Turpentine 1 minute.
Xylol and Canada balsam.
Pathogenesis. — Feeding the fowls or injecting under the skin
will cause their death in from 12 to 24 hours, the symptoms pre-
ceding death being those of a heavy septicaemia.
The bacillus is then found in the blood and viscera, and the
intestinal discharges, the intestines presenting a hemorrhagic
inflammation.
Guinea-pigs and sheep do not react. Mice and rabbits -are
affected in the same manner as the fowls.
Immunity. — Pasteur, by injecting different-aged cultures into
fowls, produced in them only a local inflammation, and they
were then immune. But as the strength of these cultures could
not be estimated, many fowls died and the healthy ones were
endangered from the intestinal excretions, which is the chief
manner of infection naturally ; the freces becoming mixed with
the food.
Bacteria of Hemorrhagic Septicaemia. (Hueppe.)
Under this heading Hueppe has gathered a number of bac-
teria very similar to the bacillus of chicken cholera, differing
BACTERIA PATHOGENIC FOR ANIMALS. 167
from it and each other but very little. They have been described
by various observers and found in different diseases.
(1) The bacteria of this group color themselves strongly at
the poles, giving rise to tbe dumb-bell shape. They do not take
the Oram stain. They are without spores,
(2) And do not liquefy gelatine.
They have been placed in three general divisions : —
f Wild Plague. (Hueppe.)
I German Swine Plague. (Loffler, Schtitz.)
1st division. -{ Rabbit Septicaemia.
I Ox Plague. (Oresti-Armanni.)
[ Steer Plague. (Kitt.)
The bacteria of the first division are not motile, do not grow-
on potato, and are found scattered through the bloodvessels.
A local reaction is uncommon.
f American Swine Plague. (Billings.)
J French Swine Plague. (CornilandChantemesse.)
2d division. { CaUle piague Texas Je(jer> (Billings.)
{ Frog Plague. (E berth.)
Here the bacteria are motile. They grow on potatoes and
are similar to the typhoid bacillus in gelatine. They form
small embolic processes in the capillaries. They cause only
a local disturbance in rabbits when subcutnneously injected.
An acid fermentation is produced in milk.
,. . . f Hog Cholera. (Salmon.)
1 Swedish Swine Plague. (Lelander.)
The bacteria of this third division are very motile. The hog-
cholera bacilli lie in the spleen and other organs in small masses
like the typhoid bacillus.
Rabbits die in four to eight days without any local disturb-
ance. The growth on potato is strong.
The Swedish swine-plague bacillus occupies a position be-
tween that of Hog Cholera and Bacillus Coli Communis.
The various swine-plague bacilli are but little active in fowls,
differing thus widely from the chicken cholera bacillus.
Bacillus of Erysipelas of Swine. (Loffler, Schiitz.) Schweine-
roilaufbacillus (German). Bouget du pore (French).
168 ESSENTIALS OF BACTERIOLOGY.
Origin. — Found in the spleen of an erysipelatous swine by
Loffler in 1885.
Form.— One of the smallest forms of bacilli known ; very thin,
seldom longer than 1 p, looking at first like little needle-like
crystals. Spores have not been found.
Properties. — They are motile ; do not liquefy gelatine.
Growth in culture at ordinary temperature, very slowly, and
the less oxygen the better the growth.
Gelatine Plate. — On third day little silver-gray specks, seen
best with a dark background, coalescing after awhile, pro-
ducing a clouding of the entire plate.
Stab Cultures. — In a few days a very light, silvery -like clouding,
which gradually involves the entire gelatine ; held up against
a dark object, it comes plainly into view.
Staining. — All ordinary dyes and Gram's method also.
Tissue sections stained by Gram's method show the bacilli in
the cells, capillaries, and arterioles in great numbers.
Pathogenesis. —Swine, mice, rabbits, and pigeons are sus-
ceptible ; guinea-pigs and chickens, immune.
When swine are infected through food or by injection a tor-
pidity develops with diarrhoea and fever, and on the belly and
breast red spots occur which coalesce, but do not give rise to
any pain or swelling. The animal dies from exhaustion in 24 to
48 hours. In mice the lids are glued together with pus.
At the autopsy the liver, spleen, and glands are enlarged and
congested, little hemorrhages occurring in the intestinal mucous
membrane and that of the stomach.
Bacilli are found in the blood and all the viscera.
One attack, if withstood, protects against succeeding ones.
Immunity. — Has also been attained b}T injecting vaccines of
two separate strengths.
Bacillus Murisepticus. (Koch.) Mouse septicaemia.
Origin.— Found in the body of a mouse which had died from
injection of putrid blood, and described by Koch in 1878.
Form.— Differs in no particular from the bacillus of swine
erysipelas, excepting that it is a very little shorter, making it
the smallest known bacillus. Spores have been found, the cul-
tures exactly similar to those of swine erysipelas.
BACTERIA PATHOGENIC FOR ANIMALS. 169
The pathological actions are also similar. Field mice are
immune ; whereas for house and white mice the bacillus is fatal
in two to three days.
Micrococcus of Mai de Pis. (Nocard.) Gangrenous mastitis
of sheep.
Origin.— In the milk and serum of a sheep sick with the
umai depis."
Form. — Yery small cocci seldom in chains.
Properties, immotile ; liquefying gelatine.
Growth.— Growth occurs best between 20° and 37° C, is very
rapid, and irrespective of oxygen.
Plates of Gelatine.— White round colonies, some on the surface
and some in the deeper strata, with low power, appearing brown
surrounded by a transparent areola.
Stab Culture. — Very profuse along the needle-track, in the
form of a cone after two days, the colonies having gathered at
the apex.
Potato. — A dirty gray, not very abundant, layer somewhat
viscid.
Staining, with ordinary methods ; also Gram's method.
Pathogenesis.— If a pure culture is injected into the mammary
gland of sheep, a u mal de pis" is produced which causes the
death of the animal in 24 to 48 hours. The breast is found
oedematous, likewise the thighs and perineum ; the mammae
very much enlarged, and at the nipples a blue-violet coloration.
The spleen is small and black ; other animals are less susceptible.
In rabbits abscesses at the point of infection, but no general
affection.
Bacillus Alvei. (Cheshire and Cheyne.) Bacillus melittoph-
tharus. (Cohn.)
Origin.— In foul-brood of bees.
Form. — Slender rods, with round and conical-pointed ends ;
very large oval spores, the rod becoming spindle-shaped when
they appear.
Properties. — Motile, liquefying gelatine rapidly.
Grotvth.— Grows best between 20° C. and 37° C, very slowly ;
aerobic.
Gelatine Plates.— Small grooves are slowly formed, which unite
170 ESSENTIALS OF BACTERIOLOGY.
so as to form a circle or pear-shaped growth, from which linear
grooves again start.
Stab Culture.— Grows first on surface, then gradually along
the needle-track, long processes shooting out from the same,
clouding the gelatine. Later, air-buhbles form like the cholera
culture, and in two weeks the whole gelatine liquefied.
Staining. —Do not take aniline dyes very well. Gram's method
is, however, applicable.
Pathogenesis. — If a pure culture is spread over the honey-
comb containing bee larvae, or if bees are fed upon infected
material, foul-brood disease will occur. Mice, if injected, die in a
few hours. (Edema around the point of infection, and many
bacilli contained in the cedematous fluid, otherwise no changes.
Micrococcus Amylovorus (Burrill.)
Origin.— In the disease called "Blight," which affects pear-
trees and other plants.
Form.— Small oval cells, never in chains, more the form of a
bacillus.
Pathogenesis. — Introduced into small incisions in the bark of
pear-trees the trees perished from the " blight." The starch of
the plant cell was converted into carbon dioxide, hydrogen,
and butyric acid.
Bacterium Termo. (Cohn.)
This was a name given to a form of micro-organism found in
decomposing albuminous material, and was supposed to be one
specific germ. Hauser, in 1885, found three different distinct
microbes which he grouped under the common name of Proteus,
which have the putrefying properties ascribed to B. Termo.
Proteus Vulgaris.
Origin. — In putrid animal matter, in the feces, and in water.
Form. — Small rods, slightly curved, of varying lengths, often
in twisted chains, having long cilia or flagella.
Properties.— Very motile, and very soon liquefying gelatine ;
forms hydrogen sulphide gas ; causes putrefaction in meat.
Growth. —Growth very rapid, best at 24° C, is facultative
aerobic.
Gelatine Plates. — Yellowish-brown, irregular colonies, with
prolongations in every direction, forming all sorts of figures ; an
BACTERIA PATHOGENIC FOR ANIMALS. 171
impression preparation shows these spider-leg processes to con-
sist of bacilli in regular order.
Stab Culture. — The gelatine soon liquid, a gray layer on the
surface, but the chief part of the culture in small crumbs at the
bottom.
Pathogenesis.— Rabbits and guinea-pigs injected subcutane-
ously die quickly, a form of toxaemia, hemorrhagic condition of
lungs and intestines present. When neurin is injected previ-
ously the animals do not die. This ptomaine is supposed to be
generated by the proteus vulgaris.
Proteus Mirabilis. (Hauser.)
Differs from P. vulgaris in that the gelatine is less rapidly
liquefied. Found also in putrid material.
Proteus Zenkeri. (Hauser.)
Does not liquefy gelatine ; otherwise similar to the other two.
We have now considered some of the characteristics of the
more important bacteria. The scope of this work does not allow
a more extended study than we have made, which, as we are
aware, has been very superficial. The larger works must be
referred to, if a deeper interest is taken in the subject.
APPENDIX
YEASTS AND MOULDS.
In works on bacteria, these true fungi, yeasts and moulds, are
usually considered. They are so closely related to bacteria, and
so often contaminate the culture media, and are so similar in
many respects, that a description is almost a necessity.
But there are several thousand varieties, and we cannot
attempt to describe even all of the more important ones. It
will answer our purpose to detail a few of the more common
kinds, and give the principal features of the different orders.
' Saccharomycetes or Yeasts increase through budding; the
spores are attached to the mother cell like a tuber on a potato.
Yeasts are the cause of alcoholic fermentation in the saccha-
roses. A description of the most common ones will suffice.
Saccharomyces Cerevisise. (Torula Cerevisice.) This is the
ordinary beer yeast.
Form. — Bound and oval cells ; a thin membrane inclosing a
granular mass, in which usually can be seen three or four irre-
gular-shaped spores. When these become full grown they pass
through the cell wall and form a daughter cell. Sometimes long
chains are produced by the attached daughter cells.
Growth.— They can be cultivated as bacteria in bouillon, but
they grow best in beer.
There are several varieties of beer yeast, each one giving a
characteristic taste to the beer. Brewers, by paying special
attention to the nutrient media, cultivate yeasts which give to
their beers individual flavors.
Mixed yeast gives rise to a poor quality of beer.
Saccharomyces Rosaceus. S. Niger and S. Albicans. These
yeasts are found in the air ; and instead of producing alcoholic
(173)
174 APPENDIX.
fermentation they give rise to a pigment in the culture media.
They grow upon gelatine which they do not liquefy.
Saccharomyces Mycoderma. This yeast forms a mould-like
growth, a skin, on the surface of fermented liquids, but does not
cause any fermentation itself. It forms the common u mould"
on wine, preserves, and " sour krout."
Pathogenic Yeasts. In recent years a number of workers
have interested themselves in experiments with yeasts in their
relation to disease; and under the name of Blastomycetes, San-
felice has grouped yeasts that produce tumors resembling epi-
theliomata ; and he has tried to prove that the so-called animal
parasites found in malignant growths, and variously known as
coccidia and sporozoa, are yeasts. The whole subject is still
under discussion.
Oidium. A form which seems to be the bridge between the
yeast and the moulds is the oidium. Sometimes it resembles
the yeasts, sometimes the moulds, and often both forms are
found in the same culture. Several are pathogenic for man.
Oidium Lactis.
Origin. — In sour milk and butter.
Form.— The branches or hyphens break up into short rod-like
spores. No sporangium, as in moulds.
Growth.— In milk it appears as a white mould.
Artificially cultured on gelatine plates, or milk gelatine plates,
it forms satin-like, star-shaped colonies, which slowly liquefy.
Under microscope the form of the fungus is well seen.
Agar Stroke Culture.— The little stars, very nicely seen at first ;
then the culture becomes covered with them, causing a smeared
layer to appear over the whole surface, with a sour odor.
Properties. — The milk is not changed in any special way. It
is not pathogenic for man or animals. It is found when the
milk begins to sour.
Oidium Albicans. {Soor.) Thrush Fungus.
Origin. — Mucous membrane of the mouth, especially of infants.
Form. — Taken from the surface of the culture, a form like
yeasts ; but in the deeper layers, mycelia with hyphens occur.
Growth. — Not liquefying; snow-white colonies on gelatine
plates.
YEASTS AND MOULDS. 17o
Stab Culture.— Radiating yellow or white processes spring from
the line made by the needle, those near the surface having oval
ends.
Potatoes.— The yeast form, develops as thick white colonies.
Bread Mash. — Snow-white veil over the surface.
Pathogenesis. — In man the parasitic thrush, or "white mouth, n
is caused by this fungus. In the white patches the spores and
filaments of this microbe can be found. Rabbits receiving an
intravenous injection perish in twenty-four to forty-eight hours,
the viscera being filled with mycelia.
True Moulds. Fliigge has made five distinct divisions of
moulds. It will, however, serve our purpose to classify those
to be described under three headings : Penicillium, Mucor, and
Aspergillus.
Penicillium Glaucum.
Origin.— The most widely distributed of all moulds, found
wherever moulds can exist.
Form. — From the mycelium, hypha? spring which divide into
basidia (branches), from which tiny filaments arise (sterigmata ),
arranged like a brush or tuft. On each sterigma a little bead
or conidium forms, which is the spore. In this particular fungus
the spores in mass appear green.
Growth. — It develops only at ordinary temperatures, forming
thick grayish-green moulds on bread-mash. At first these ap-
pear white, but as soon as the spores form, the green predomi-
nates. Gelatine is liquefied by it.
Mucor Mucedo. Next to the penicillium glaucum, this is the
most common mould. Found in horse dung, in nuts, and
apples, in bread and potatoes as a white mould.
Form. — The mycelium sends out several branches, on one of
which a pointed stem is formed which enlarges to form a globu-
'ar head, a spore-bulb, or Sporangium. The spore-bulb is par-
ilioned off into cells in which large oval spores lie. When the
spores are ripe a cap forms around the bulb, the walls break
down and the wind scatters the spores, leaving the cap or
M columella"11 behind.
Growth. — Takes place at higher temperatures on acid media.
It is not Pathogenic.
176
APPENDIX
Achorion Schbnleinii.
Trichophyton Tonsurans.
Microsporon Furfur.
These three forms are similar to each other in nearly every
particular and resemble in some respects the oidium lactis, in
other ways the mucors. The first one, Achorion Schbnleinii, was
discovered by Schonlein in 1839, in Favus, and is now known as
the direct cause of this skin disease.
Fig. 91.
Achorion Schbnleinii (after Kaposi).
Origin, — Found in the scaly crusts of favus.
Form. — Similar to oidium lactis.
Growth. — Is very sparse. On gelatine round white masses
inclosed by a zone of liquefied gelatine.
In milk it is destroyed.
Pathogenesis. — Causes favus in man.
Trichophyton Tonsurans. Found, in 1854, by Bazin, in Tinea.
Form. — Similar to the achorion or favus fungus.
Growth. — Somewhat more rapid than the favus, and the gela-
tine quickly liquefied, Old cultures are of an orange-yellow
color. Colonies have a star-shaped form.
Pathogenesis. — Herpes tonsurans and the various tineee are
produced by this fungus.
Microsporon Furfur. Found in tinea versicolor, almost iden-
YEASTS AND MOULDS. 177
tical with the above, forms dry yellow spots, usually on the chest
in persons suffering from wasting diseases.
Aspergillus Glaucus.
Origin. — In saccharine fruits.
Form. — The hypha has formed upon its further end a bulb,
from which pear-shaped sterigmata arise and bear upon their
ends the conidia or spores.
Growth.— Best upon fruit juices. Non-pathogen fc. The mould
is green. Aspergillus flams has the tufts and spores of a yellow
color.
A. Fumigatus. Is pathogenic for rabbits when injected into
them. At the autopsy their viscera are found filled with the
mould.
Examination of Yeasts and Moulds. Yeasts and moulds are
best examined in the unstained condition. A small portion of
the colony rubbed up with a mixture of alcohol and a few drops
of liquor ammonia ; of this, a little is brought upon the glass-
slide covered with a drop of glycerine and the cover-glass pressed
upon it. If the preparation is to be saved, the cover-glass is
secured by ringing around the edges. Yeasts take methylin-blue
stain very well.
Cladothrices and Streptothrices. The streptothrix and
cladothrix groups are classed with the higher bacteria, but
their exact status is still undetermined. They may be consid-
ered as representing the transition from the bacteria to the
lower fungi.
Streptothrix, or Cladothrix Actinomyces (ray fungus).
Actinomycosis is a disease caused in man and cattle by this
organism, which is commonly found in grain, particularly
barley. It is probable that several varieties of the parasite can
produce the characteristic lesions. It has been discovered in
all countries and in various organs of the body, although its
place of election is about the lower jaw, where it tends to form
hard ulcerating abscesses, affecting other organs secondarily.
Form. — In the granular masses of an abscess cylindrical fila-
ments are matted together, and radiating outward from this
zone are club-shaped branches, as the petals of an aster. In the
center of the granule are numerous cocci-like bodies, and some
12
178 APPENDIX.
of the ovoid or club-shaped hyphae lie detached from the
clusters. Through cultivation it was found that the ovules give
rise to filaments, and they then form the ovules again.
Cultivation. — At 38° C. on glycerine-agar in a period of one
to two weeks, pointed scales about the size of a millet-seed,
center dry and prominent, margins hyaline, composed only of
filaments, short and long, massed together, but no clubbed
forms.
By some the clubs are considered the spore organs ; by others
they are thought to be encapsulated or thickened filaments.
Pathogenesis. — When a portion of the growth obtained in
eggs was injected into the abdominal cavity of a rabbit, actinomy-
cotic processes developed upon the peritoneum.
It usually gains access to the living body through a wound in
the gum or some caries of the teeth. A new growth is formed,
ulceration being first set up.
The new tissue, composed of round cells, then undergoes soft-
ening, purulent collections form and the normal structure is
destroyed.
The usual seat is in the maxillary bones, but the fungus has
been found in the lungs, tonsils, intestines, and various other
organs in man and cattle.
Examination. — Well seen in the unstained condition. From
the pus or scraping a small portion is taken and squeezed upon
the glass slide ; if calcareous matter is present, a drop of nitric
acid will dissolve the same.
Glycerine will preserve the preparation.
Staining.— Cover-glass specimens stained best with Gram's
method. Tissue sections should be stained as follows : —
Ziehl's carbol-fuchsin, ten minutes. Kinse in water.
Cone, alcohol. sol. of picric acid, five minutes. Rinse in water.
Alcohol, 50 per cent., fifteen minutes. Alcohol absolute, clove
oil, balsam.
The rays stained red, the tissue yellow.
Streptothrix Madurae. (Vincent.)
Origin. — Found in the disease known as Madura foot, or
Mycetoma, an ulceration affecting the feet, especially of indi-
YEASTS AND MOULDS
179
viduals living in the tropics. Two varieties, the pale and the
black, have been described.
Form. — Branched filaments resembling the actinomyces strep-
tothrix in the mycelia. Spores are seen.
Cultivation. — In liquid media containing vegetable infusions
growth occurs best. Temper-
ature of 37° C. most suited. Fig. 92.
The colonies near the surface
become colored red.
Agar. — Glazed colonies, at
first colorless, then rose-col-
ored, about the size of a pea,
with the central part umbili-
cated and pale. Gradually the
rose color fades.
Acid Potato. — A slow and
meager growth.
Pathogenesis. — Only local re-
action has been caused by in-
oculation in animals. In man
the disease usually follows a
slight injury and attacks the leg
or foot, slowly forming a nodu-
lar growth, which in the course
of months or a year begins to
soften and ulcerate, and with
the sero-pus are discharged
numerous little granules, some
black, some pink, containing
mycelia. The limb becomes
much deformed, the tissue
■WP<
•
M
Streptothrix Madura; in a secti
eased tissue(Vincent)
dis-
vascularized, and the degenerated area. filled with the strep-
tothrix filaments.^
Staining. — The organism itself stained with ordinary stains.
Gram's method for the tissue.
Streptothrix Farcinica. (Nocard.) Bovine Farcy, Farcin du
Boeuf.
180 APPENDIX.
Origin. — A disease affecting cattle and giving rise to tubercle-
like lesions in the lungs, liver, and spleen. Common in France.
Form. — Small interwoven mass of threads arranged in tufts
found in the centers of the tubercles.
Culture. — At body-temperature in various media.
Bouillon. — Colorless masses irregular in size and shape.
Agar and Gelatine. — Small, rounded, opaque colonies, thicker
at the periphery.
Potato. — Rapid growth of pale yellow dry scales, consisting of
many spores.
Pathogenesis. — Pure cultures introduced into the peritoneum
of guinea-pigs give rise in 9 to 20 days to tubercle-like lesions.
Subcutaneous injections cause abscesses with secondary in-
volvement of the lymphatics, ending in recovery. Dogs,
horses, and rabbits are immune.
Staining. — Wright's double stain for tissues; also Gram's.
Examination of Air, Soil, and Water.
Air. — Many germs are constantly found in the atmosphere
about us. Bacteria unaided do not rise into the air and fly
about ; they usually become mixed with small particles of dirt
or dust and are moved with the wind. The more dust the more
bacteria, and therefore the air in summer contains a greater
number than the air in winter, and all the other differences can
be attributed to the greater or less quantity of dust and wind.
Methods of Examination. The simplest method is to ex-
pose a glass or dish covered with gelatine in a dust-laden
atmosphere or in the place to be examined. In the course of
24 to 48 hours colonies will be seen formed wherever a germ has
fallen. But this method will not give any accurate results in
regard to the number of bacteria in a given space ; for such a
purpose somewhat more complicated methods are needed, so
that a certain amount of air can come in contact with the
culture media at a certain regulated rate of speed.
Hesse's Method. This is the most useful of the various
methods in vogue.
A glass cylinder, 70 centimetres long and 3.5 centimetres in
diameter, is covered at one end, by two rubber caps, the inner
AIR, SOIL, AND WATER.
181
one having a hole in its centre 10 millimetres in diameter ; and
at the end B a rubber cork fits in the cylinder; through this
cork a glass tube 10 mm. in diameter passes, which is plugged
at both ends with cotton. The cylinder and fittings are first
washed in alcohol and sublimate and then placed for one hour
in the steam chamber.
Removing the cork of the cylinder, 50 cubic centimetres of
sterile gelatine in a fluid condition are introduced and rolled
out on the sides of the tube, after the manner of Esmarch,
leaving a somewhat thicker coating along the under side of the
Fig. 93.
cylinder. The aeroscope, as the cylinder and its fittings are
called, is placed upon an ordinary photographer's tripod and
the glass tube, which passes through the rubber cork, connected
with an aspirator, the cotton having first been removed from its
82
APPENDIX.
Fig. 94.
outer end. The aspirator consists of two ordinary wash-bottles
connected with each other by a rubber tube, 0. They are at-
tached to the tripod with a small hook one above the other, the
upper one half filled with water and slightly tilted.
When the apparatus is wanted, the outer rubber
cap at the end A of the aeroscope is removed, the
air can then pass through the small hole in the
other cap, and the germs fall upon the gelatine in
the tube, the cotton in the small glass tube at the
other end preventing the germs from getting out.
The aspirator is set in use by tilting the upper
bottle so that the water flows into the lower, this
creates suction and draws the air through the
aeroscope.
The amount entering estimated by the capacity
of the wash-bottle. The rate at which it enters
depending upon the rate of the flow of water,
which can be regulated.
Hesse advises for rooms and closed spaces 1 to 5
litres, at the rate of 2 minutes a litre, and for open
spaces, 10 to 20 litres at 4 minutes a litre. Plate
cultures can be made from the colonies which de-
velop in 8 to 10 days in the cylinder.
Petri's Method. The air pumped or sucked
through sand filters, and the sand then mixed with
gelatine.
Sand is sterilized by heating to redness, and
while still warm placed in test tubes which are
Sand filter then plugged. (Sand which has been passed
a ter e n. through a sieve with meshes 0.25 millimetre wide
is the kind required.) A glass tube 9 centimetres long is pro-
vided with two portions of sand each 3 cm. long and £ cm. apart,
little plates of brass gauze keeping the portions in position.
The tube and its contents now sterilized in hot air oven at
150° C, the ends having first been plugged with cotton.
One end of the tube is then fitted with a rubber cork through
which passes a glass tube, which is connected with an aspirator
(a hand-pump with a known capacity).
AIR, SOIL, AND WATER.
183
If a hundred litres of air pass through the tube in fifteen min-
utes the germs should all be arrested in the first sand filter.
And when the filters are removed and thoroughly mixed with
gelatine, each filter for itself, there should be no colonies de-
veloped from the second filter, i. c, the one nearest the aspirator.
Sedgwick-Tucker Method. A special form of tube is used,
called an aerobioscope. It consists of a neck 2.5 cm. in length,
Fig. 95.
Sedgwick-Tucker aerobioscope.
an expanded portion 15 cm. long, and a long narrow tube of 15
cm. After sterilization the tube is partly filled with granulated
sugar, which is the filtering material. By means of a vacuum
gauge and an air-pump, or ordinary aspirating bottles, the vol-
ume of air passing through the apparatus can be determined.
After the air has been passed through, the sugar is gently
shaken from the narrow tube into the expanded portion, and
20 c. c. of liquefied gelatine is poured in. The sugar dissolves,
and the mixture is then rolled on the inner side of the glass
as an Esmarch tube. This part of the apparatus is divided into
squares to make the counting of colonies easy. The aerobio-
scope is very highly recommended.
Varieties Found in Air. The only pathogenic bacteria found
with any constancy are the staphylococcus aureus and citreus;
but any bacterium can be, through accident, lifted into the atmo-
sphere, and in certain places may be always found — the bacillus
tuberculosis, for example, in rooms where many consumptives
are living.
Non-Pathogenic. The micrococci predominate. Sarcinse,
yeasts, and moulds constantly contaminate cultures.
In the ordinary habitations the average number of germs to
the litre of air does not exceed five.
Around water-closets, where one would imagine a great num-
ber to exist, owing to the undisturbed condition of the air, but
few will be found.
184 APPENDIX.
Examination of Water. The bacteriological examination of
water is to-day of as much importance as the chemical analy-
sis, and must go hand in hand with it.
At the start we must say that a water containing thousands
of germs to the cubic centimeter is far less dangerous than one
containing but two germs, if one of these two be a typhoid ba-
cillus. It is not the number that proves dangerous, it is the
kind.
If a natural water contains more than 500 germs to the cubic
centimeter, it were well to examine its source.
Bacteriology performs the greatest service in testing the devices
which are intended to render water fit for drinking.
As a diagnostic aid the examination is of but little use. An
epidemic of typhoid fever occurs, the water is suspected, an ex-
amination is undertaken ; but the days of incubation and the
days passed before the water is analyzed have given the typhoid
germs, if any had been present, ample time to disappear, since
in water that contains other bacteria they live a very short time
only. Again, the water tested one day may be entirely free and
the next day contain a great number, and before the typhoid
germ can be proven to be present in that particular water, the
epidemic may be past.
Purity of Waters. The purest water we have is the natural
spring water— water that has slowly filtered its way through
various layers of gravel and sand and comes finally clear and
sparkling from the ground. It is without germs ; but let such
a water stand walled up in cisterns or wells, it becomes as
surface water, open to all sorts of impurities, and the bacterial
nature of it changes every moment.
Artesian or Driven Well. The driven well will secure to a cer-
tain extent a pure water. It is the only form of well or cistern
that will insure this, since the water does not become stagnant
in it ; but it may connect with an outhouse, the soil being very
loose, allowing the products of germs of refuse water to find their
way into the well. If a chemical examination shows increased
amounts of chloride of sodium, a contamination can be mooted.
Filtered Water. Dangerous as surface water is, the greater
quantity used, is such : the inhabitants of larger towns and cities
AIR, SOIL, AND WATER. 185
using chiefly the rivers and other large waters which course
near them for drinking purposes. A purification or filtration
can in a certain measure render these waters harmless.
Filtration is often carried on on a large scale in the water-
works of cities and towns.
Bacteriological examination is here of great service to deter-
mine if a water, which has been filtered and may have a very
clear appearance, and give no harmful chemical reaction, yet
be entirely free, or nearly so, from germs ; in other words, if
the filter is a germ filter or not.
Charcoal Sponge and Asbestos, the materials formerly in use,
are objectionable because germs readily develop on them and
clog them, so that they require frequent renewal. In very
large filters, sand and gravel give the best results ; the number
of germs in a cubic centimetre is reduced to forty or fifty and
kept at that number. This is a very pure water for a city water,
though, as we stated before, not a safe one, for among those
forty germs very dangerous ones may be found. It is then
necessary for the users to refilter the water before drinking it,
through a material which will not allow any germs to pass.
Pasteur-Chamberland Filter. This very perfect filter, which
is now in almost universal use, consists of a piece of polished
porcelain in the form of a cylinder closed at one end and pointed
at the other. It is placed in another cylinder of glass or rubber
and the pointed portion connected with a bottle containing the
water, or directly with faucet of the water-pipe. The water
courses through the porcelain very slowly and comes out entirely
free from germs ; pipe-clay, bisque, infusorial earth, and kaolin
are also perfect filters. The only disadvantage is the long time
it takes for the water to pass through. Pressure is used to
accelerate the passage in the form of an aspirator or air-pump.
The force of the hydrant water is also sufficient to produce a
steady, small stream.
These porcelain cylinders can easily be sterilized and the
pores washed out.
All the cylinders or bougies are not germ proof, so that they
must be tested, and most of them must be cleaned every fourth
day, or they will allow germs to pass through.
186 APPENDIX.
Boiling as a means of purifying. When such a filter cannot
be obtained, the only alternative is to boil all the water to be
used for drinking ; and this should especially be done in times
of typhoid and cholera epidemics.
Methods of Examination. Since the germs rapidly multiply
in stagnant water, an examination must not be delayed longer
than an hour after the water has been collected. Every pre-
caution must be taken in the way of cleanliness to prevent con-
tamination ; sterilized flasks, pipettes, and plugs must be at
hand, and the gelatine tubes best inoculated on the spot. If
this cannot be done, the sample should be packed in ice until
it arrives at the laboratory. The sample is placed in a steri-
lized glass flask, and the flask then closed with a sterile cotton
plug. A sterilized pipette is then dipped into the flask and
1 c.c. of the water withdrawn and added to a tube of gelatine,
the gelatine being in a fluid condition. To a second tube,
£ c.c. is added. The tubes are then shaken so as to thor-
oughly mix the water with the gelatine, and then poured
upon wide glass plates, one plate for each tube ; the plates are
then placed in the moist chamber, and in two or three days
examined. A temperature of 18° to 20° C. is best. Many water-
bacteria are hindered by higher degrees of heat. If the germs
are equally divided, there should be one-half the number on
one plate that there is on the other; thus the ? c.c. serves as
control.
Water that is very rich in germs requires dilution with ster-
ilized water 50 to 100 times. Fewer colonies will be found on
agar than on gelatine even at the same temperature.
To count the colonies which develop upon the plates, a spe-
cial apparatus has been designed, known as
Wolfhiigel's Apparatus. A glass plate divided into squares,
each a centimeter large, and some of these subdivided. This
plate is placed above the gelatine plate with the colonies, and
the number in several quadrants taken, a lens being used to
see the smaller ones.
The petri saucers can be used instead of plates, and an appa-
ratus on the Wolf hiigel plan can be obtained to count the colo-
nies. It is best to count all the colonies on the plate or dish.
AIR, SOIL, AND WATER. 187
Agar and bouillon are used in qualitative analyses. A large
quantity of the water is taken (about 100 c. cm.) and mixed
with 25 c.c. of bouillon ; the mixture is then placed in an in-
cubator. The ordinary water-bacteria do not bear the higher
temperatures very well, and therefore pathogenic organisms—
as cholera, for instance — will be found almost in pure cultures.
The growth of intestinal bacteria is also favored by glucose
bouillon (2 per cent.), and fermentation ensues. If a fermenta
tion-tube (Smith's) is used, the gas collects at one end, and the
bacteria can be further cultivated and studied.
Varieties Found. The usual kinds found are non-patho-
genic, but, as is well known, typhoid and cholera are princi-
pally spread through drinking water, and many other germs
may find their way into the water. Some of the common
varieties give rise to fluorescence, or produce pigment.
Eisenberg gives 100 different varieties as ordinarily found.
Other intestinal diseases also are supposed to be water borne, and
the presence of large numbers of the Bacillus coli communis is
strongly suggestive of sewage contamination. Ice supplies
require the same supervision as water supplies, for many bac-
teria, like the typhoid bacillus, retain their vitality for weeks
after freezing.
The Examination of the Soil. The upper layers of the soil
contain a great many bacteria, but because of the difficulty in
analyzing the same, the results are neither accurate nor con-
stant. The principal trouble lies in the mixing of the earth
with the nutrient medium ; little particles of ground will cling
to the walls of the tube, or be imbedded in the gelatine, and
may contain within them myriads of bacteria. As with water,
the soil must be examined immediately or very soon after it is
collected, the bacteria rapidly multiplying in it.
When the deeper layers are to be examined, some precautions
must be taken to avoid contamination with the other portions of
the soil. One method, very laborious and not often practical, is
to dig a hole near the spot to be examined and take the earth
from the sides of this excavation.
Frankel's Borer. Frankel has devised a small apparatus in
the form of a borer, which contains near its lower end a small
188 APPENDIX.
cavity, which can be closed up by turning the handle, or opened
by turning in the opposite direction.
It is introduced with the cavity closed, and when it is at the
desired depth, the handle is turned, the earth enters the cavity,
the handle again turned, incloses it completely, and the borer is
then withdrawn.
The earth can then be mixed with the gelatine in a tube, and
this gelatine then rolled on the walls of the tube after the man-
ner of Esmarch, or it can be poured upon a glass plate, and the
colonies developed so.
Another method is to wash the earth with sterilized water,
and the water then mixed with the gelatine, as many of the
germs are taken up b}r the water.
The roll-cultures of Esmarch give the best results, many of
the varieties usually found being anaerobic.
Animals inoculated with the soil around Berlin die almost
always of malignant oedema, and with that of some other towns
invariably of tetanus. Many of the germs found are nitrogen
formers and play a great role in the economy of the soil.
Nitrifying organisms are found in the superficial layers of the
earth. Organic matters found in sewage and in the faecal evac-
uations of animals form the basis for their activity, whereby
nitrates, ammonias, and nitric acid result. The nitrogen neces-
sary for the growing plant is thus produced. The nitro-monas
of Winogradsky belongs to this group.
The Bacteria of Milk and Other Foods. Milk as secreted is
sterile, but at every step in its passage from the cow to the con-
sumer it is liable to contamination. Even the lower portion of
the teat is a source of infection, owing to the presence of stagnated
milk from the former milking, and, as consumed, milk usually
contains thousands to millions of bacteria to the cubic centimetre.
Sterilization or Pasteurization and supervision of the dairies
should always be carried out on milk used for infant feeding.
Foods eaten after little or no cooking, such as fruits, salads,
and the like, and also oysters, are possible sources of bacterial
diseases, and the not infrequent so-called ptomaine poisoning
observed after the consumption of ice-cream, sausage, canned
meats, etc., is the result of the action of bacteria or their
products
EXAMINATION OF THE HUMAN BODY. ] go,
BACTERIOLOGIC EXAMINATION OF THE ORGANS AND
CAVITIES OF THE HUMAN BODY.
The body, on account of its constant contact with the sur-
rounding air, is necessarily exposed to infection, and we would
be likely to find on the skin and in the oral, anal, and nasal
cavities the varieties of micro-organisms commonly around us.
Through the water and food the body is also contaminated;
but some organisms by predilection inhabit the mouth, intes-
tine, and other cavities, and form there a flora distinctly their
own.
The Skin. The majority of micro-organisms met with on the
skin are non-pathogenic, although underneath the nails and in
the hair, pus-forming micro-organisms often occur, producing
sometimes serious abscesses.
In the sweat-glands and the sebaceous glands various organ-
isms have been found. The Staphylococcus epidermidis albus
of Welch is present normally.
In foul-smelling perspiration of the feet Rosenbach found
Saprogenes No. II., which is pathogenic for rabbits.
Micrococcus cereus albns and flavus, Diplococcus liquefa-
ciens albus and flavus, Staphylococcus pyogenes aureus, and
Streptococcus pyogenes are found underneath the nails.
In eczema, Diplococcus albicans tardus, D. citreus liquefa-
ciens, D. flavus liquefaciens, and Ascobacillus citreus.
In colored sweat, Micrococcus hsematodes, Bacillus pyocya-
neus.
A diplococcus is found in acute pemphigus.
The lepra bacillus, the tubercle bacillus in lupus, and the
typhoid bacillus in the eruption of typhoid fever are a few of
the specific germs found on the skin.
The Conjunctiva. The micrococcus of trachoma, the Koch-
Weeks bacillus, considered to be the specific cause of acute
catarrhal conjunctivitis, or " pink eye," and the Bacillus xero-
sis, are special germs found on the conjunctiva; the other va-
rieties of air- and water-organisms, and those usually present
on the skin, are also found.
190 APPENDIX.
The Mouth. The mouth is a favorite seat for the development
of bacteria. The alkaline saliva, the particles of food left in
the teeth, the decayed teeth themselves, all furnish suitable
soil for their growth.
Quite a number of germs have been isolated and their prop-
erties partly studied. Many have some connection with the
production of caries of the teeth, as Miller has well shown in
his careful studies. The Leptothrix buccalis, found in nearly
all mouths, is a long chain or filamentous bacillus which stains
blue with iodin. It was formerly considered the cause of tartar
on the teeth.
The Spirillum sputigenum, Spirochaeta dentium, Micrococcus
gingivae pyogenes, Bacillus dentalis viridans, B. pulpae pyogenes,
Microccocus of sputum-septicaemia, and M. salivarus septicus
are a few of the germs cultivated by Miller and Biondi from
the mouth. Besides these, the pneumo-bacteria, diphtheria
bacillus, and tubercle bacillus are often met with, the first two
in the mouths of healthy persons. The expired air in quiet
respiration is free from bacteria, but in coughing, sneezing, etc.,
large numbers of organisms are violently ejected and the
atmosphere about tubercular patients is always saturated with
tubercle bacilli.
Ear. In the middle ear of new-born infants no pathogenic
organisms were found, but quite a number of non-pathogenic
ones. In affections of the ear the pneumo-bacillus and the
Staphylococcus pyogenes are most frequent.
Nasal Cavity. The nasal secretion, containing as it does
dead cells and being alkaline in reaction, forms a good soil for
the growth of germs.
Diplococcus coryzae, Micrococcus nasalis, Bacillus fcetidus
ozaenae, B. striatus albus et flavus, B. capsulatus mucosus, and
Vibrio nasalis are some of the organisms described by various
observers.
Stomach and Intestine. The secretion of the stomach is in
its normal state not a favorable soil for the development of
bacteria, yet some germs resist the action of the gastric juice
and flourish in it. When the acids of the stomach are dimin-
ished in quantity or absent altogether, the conditions for the
EXAMINATION OF THE HUMAN BODY. 191
growth of bacteria are more favorable. The alimentary canal
of the newly born infant is sterile, but in a few hours micro-
organisms begin to appear.
Some gastric bacteria normally present are Sarcina ventric-
uli, Bacterium lactis aerogenes, Bacillus subtilis, B. amylo-
bacter, B. megaterium.
The intestinal organisms are more numerous, and the mu-
cous lining of the intestines and the secretions there present
are favorable to germ-growth.
Bacillus geniculates, Boas considers a sign of carcinoma of
the stomach, and is always present, he claims, when the con-
tents contain lactic acid.
Some investigators consider digestion dependent on microbic
activity, but experiments with animals have recently shown
that life and digestion can proceed in a perfectly sterile condi-
tion. Food and air sterilized will not develop bacteria in the
faeces.
In the faeces of the young a great many bacteria have been
found that are supposed to stand in close relation with the
intestinal disorders common to nurslings. The majority of
bacteria usually present in the intestines are non-pathogenic.
The following varieties may be met with in the feces : Micro-
coccus aerogenes, Bacillus subtilis, B. butyricus, B. putrificus
coli, B. lactis aerogenes, B. coli commune, B. subtiliformis, and
the bacteria of cholera, dysentery, and typhoid, besides many
yeast-cells.
Genito-urinary Passages. In vaginal secretion Bumm has
been able to find a number of organisms, some of which
closely resemble the gonococcus; thus, there is the Diplococ-
cus subflavus, Micrococcus lacteus faviformis, Diplococcus albi-
cans amplus, and the vaginal bacillus.
In the urethra of healthy persons bacteria are sometimes
found, usually having entered from the air.
In the normal secretions around the prepuce a bacillus called
the smegma bacillus has been discovered, and it is considered
identical with the so-called syphilis bacillus of Lustgarten.
In urethral pus a number of diplococci other than the gono-
cocci have been isolated.
192 APPENDIX.
From the urine itself a great number of bacteria have been
obtained, but mostly derived from the air, finding in the urine
a suitable soil. A description of uro-bacteria will be found on
page 91.
Micro-organisms of the Blood. Many of the bacteria de-
scribed in the body of this book are found in the blood of the
animal they infect ; thus, anthrax bacilli are always found in
the blood, whereas tubercle bacilli seldom, if ever, enter this
secretion.
When animals are subcutaneously injected with pneumo-
cocci they are found in large quantities in the blood. The dis-
eases of a hemorrhagic nature affecting fowls and swine usually
show the presence of bacteria in the vascular system.
Bacteria may be recovered from the blood in all forms of
septic infection, such as general sepsis, malignant endocarditis,
and puerperal sepsis.
Method of Examination. — A drop of blood can be spread on
a cover-glass and stained with the ordinary dyes, as sputum,
pus, or serum ; but in order to eliminate the coloring matter
of the red corpuscles and bring the stained bacteria more
prominently into view, Gunther recommends that the blood,
after drying and fixing, should be rinsed in a dilute solution
of acetic acid (1 to 5 per cent.). The haemoglobin is thereby
extracted, and the corpuscles appear then only as faint out-
lines.
Instead of " fixing " by heat, Canon employs alcohol for five
minutes, especially in staining for influenza bacilli, which have
been detected in the blood.
This method, however, requires the presence of enormous
numbers of bacteria in order to succeed, and the plan com-
monly employed consists in making " blood cultures." As
large a quantity of blood as possible — never less than 10 c.c. —
is taken from a superficial vein, the median basilic, for ex-
ample, by means of a sterile antitoxin syringe, a small incision
being made through the skin over the vein in order to avoid
skin infection. The blood so obtained is immediately trans-
ferred to culture tubes, which are then studied in the custom-
arv manner.
PLATE IV.
-' < h ! y
i .
' \>
H I
o
BACILLI OF SYMPTOMATIC ANTHRAX, WITH SPORES iooo*
(Frankel and Pfeiffer.)
DIPHTHERIA BACILLUS PURE CULTURE ioooX-
(Frankel and Pfeiffer.)
PLATE V.
PFEIFFER'S CAPSULE BACILLUS IN BLOOD ioooX-
'Frankel and Pfeiffer.)
YEAST-CELUS 500 X-
(FrSnkel and Pfeiffer.)
PLATE VI.
PENICILLIUM GLAUCUM 500 X.
(Frankel and Pfeitter.)
ASPERGILLUS FUMIGATUS 500 X-
(Frankel and Pfeiffer.)
INDEX
Abbe's condenser, 27
Achorion Schonleinii, 176
Actinomyces, 177
Actinomycosis, 177
Aerobin, 24
.Kstivo-autumnal form of malarial
parasite, 161
Agar-agar, 54
bouillon, 54
glycerine, 55
Agglutination reaction for tubercle
bacillus, 106
Agglutinins, 7G
Air, examination of, 180
Alexin, 75
Amboceptor, 75
Amoeba dysenteriae, 163
Anaerobin, 24
Anilin dyes, 30
oil, 31
oil water, 31
Animals for experiment, 76
tuberculosis in, 105
Anopheles, 159
Anthrax, 94
Anthraxin, 97
Antitoxin of diphtheria, 113
of pneumonia, 134
of tetanus, 149
Antituberculous serum, 106
Arnold's sterilizer, 45
Arthrospores, 22
Asexual cycle in man, 160
Aspergillus fumigatus, 177
glaucus, 177
Autoclave of Chamberland, 46
Bacillus acidilactici, 84
aerogenes capsulatus, 158
Bacillus, alvei, 169
amylobacter, 85
anthracis, 94
avicidis, 165
Boas-Oppler, 93
butyricus, 85
capsule, 136
coeruleus, 88
coli communis, 122
comma, 124
erythrosporus, 88
fluorescens, 142
fluorescens liquefaciens, 88
geniculatus, 93
icteroides, 154
indicus, 81
Klebs-Loffler, 110
lactis cyanogenus, 86
erythrogenes, 86
lepra, 107
mallei, 108
megaterium, 82
melittophtharus, 169
mesentericus vulgatus, 81
Milzbrand, 94
murisepticus, 168
mycoides, 82
cedematis maligni, 150
of American swine plague, 167
of anthrax, 94
of bluish-green pus, 142
of bubonic plague, 155
of cattle plague, 167
of chicken cholera, 165
of diphtheria, 110
pseudo-, 114
of dysentery, 157
of French swine plague, 167
of glanders, 108
237
238
INDEX.
Bacillus of hog cholera, 167
of influenza, 137
of malignant oedema, 150
of mouse septicaemia, 1 68
of ox plague, 167
of rabbit septicaemia, 167
of soft chancre, 153
of steer plague, 167
of Swedish swine plague, 167
of swine erysipelas, 167
of symptomatic anthrax, 164
of syphilis, 108
of tetanus, 147
of typhoid fever, 115
paracolon, 121
paratyphoid, 121
phosphorescens gelidus, 89
indicus, 89
indigenus, 89
pneumo-, 131
potato, 81
prodigiosus, 80
pseudo-diphtheria, 114
psittacosis, 121
pyocyaneus, 142
P-, 143
ramosus, 82
smegma, 108
spinosus, 84
subtilis, 83
tuberculosis, 97
products of, 106
violaceus, 87
Bacteria, 17
action in causing disease, 70
asporogenic, 22
effect on body, 71
fluorescent, 88
higher, 17
in air, 183
in milk, 84
in urine, 91
in water, 87, 184
infective, 71
life of, 23
lower, 17
non-pathogenic, 80, 183
of food, 188
of hemorrhagic septicaemia, 166
Bacteria of milk, 188
of pneumonia, 130
origin of, 23
pathogenic, 25, 70, 94, 183
persistence in water, 121
phosphorescent, 89
pyogenic, 71
similar to cholera, 127
staining of, 30
structure of, 18
suppurative, 71
tables of, 194-235
toxic, 70
unstained, 27
vital actions of, 24
Bactericie du charbon, 94
Bacteriolysis, 75
Bacterium acidi lactici, 84
aeruginosum, 142
Balticum, 90
Fischeri, 90
Pflugeri, 90
syncyanum, 86
termo-, 170
ureae, 91
zopfi, 83
Beef extract, 48
Beggiatoa alba, 90
Behavior of bacteria to Gram'
stain, 41
Benches for glass plates, 63
Biedert's method. 103
Black-leg, 164
Blight, 170
Blood cultures, 192
Blood-coagulum, 58
Blood-serum as media, 55
Boas-Oppler bacillus, 93
Botkin's method, 69
Bouillon, 48
gelatine, 52
guinea-pig, 59
preparation of, 48
Bovine tuberculosis, 105
Bowhill's orcein stain, 40
Bread mash, 51
Brood-oven, 56
Brownian movements, 19
Buchner's method, 69
INDEX.
239
CArsuLE stain of Hiss, 35
of Welch, 35
Carbol-thionin solution, 34
Cattle plague, 167
Cell contents, 18
wall, 18
Celloidin sacs, 79
Cellular theory, 74
Charbon symptomatique, 164
Charcoal niter, 185
Chemotaxis, 74
Cholera, 124
Pfeitfer's immunity, 127
Cladothrices, 177
Cladothrix actinomyces, 177
dichotoma, 90
Clostridium, 21
- butyricum, 85
Complement, 75
Cotton plugs, 47
Cover-glass preparations, 35
Crenothrix, 90
Kulmiana, 90
Cultivation, 42
artificial, 42
methods of, 42
of anaerobins, 67
Cultures, appearances of, 64
egg, 59
glass-plate, 62
glass-slide, 60
potato, 49
rolled, 64
test-tube, 60
Cytase, 75
Dkcolorants, 32
Diphtheria, 110
Diplococcus albicans ampins, 146
tardissimus, 146
lanceolatus, 132
of meningitis, 135
of pneumonia, 130
Disinfectants, 42
Drying specimens, 35
Dunham's rosalic acid solution,
59
peptone solution, 125
Dysentery, 157
Effect of age, 26
Ehrlich's side-chain theory, 75
Eisner's medium, 59
Endospores, 21
Enteric fever, 115
Esmarch's method, 67 •
tubes, 64
Examination of human body,
189
Experiments on animals, 76
Favus, 176
Ferments, 25, 71
Filters, 185
hot-water, 53
Pasteur-Chamberland, 185
sand, 182
Fishing, 66
Fission, 20
Fission-fungi, 17
Flagella, 19
staining of, 41
Fluorescence, 26
P'ood, bacteria of, 188
Foul-brood, 169
Franker s borer, 187
method for anaerobins, 68
stain for tubercle bacillus, 101
Frog plague, 167
Fuchsin, carbol-, 31, 33
Fungi, 173
Fungus, rav, 177
thrush, 174
Gabbett's stain, 34, 101
Gametes, 160
Gas formation, 26
Gelatine, 52
bouillon, 52
carbolized, 117
paste, 37
plates, 170
Gelatinous membrane, 18
Germination, 22
Glucose broth, 49
Glycerin broth, 49
Gonococcus, 143
Gonorrhoea, 143
Gonotoxin, 145
240
INDEX
Gram's stain, 33
Gruber-Widal blood-serum test, 117
HjEMAMCEBA malarise, 159
vivax, 159
Hsemomenas prcecox, 160 ,
Hanging-drop, 29
Haptins, 75
Haptophores, 75
Heat as disinfectant, 43
dry, 43
moist, 44
Hemorrhagic septicaemia, 166
Herpes tonsurans, 176
Hesse's method for air, 180
for anaerobins, 67
Hiss' capsule stain, 35
typhoid medium, 59
Hog cholera, 167
Homogeneous lens, 26
Hot-air oven, 43
Hot- water filter, 53
Hiippe's method, 68
Immersion lens, 26
Immune body, 75
Immunity, 72
theories of, 74
Incubators, 56
Infection, 70
conditions necessary to produce,
70 >
Inoculation of animals, 76
Iodin, 32
Iris blender, 27
Iron box for plates, 62
Japanese method, 55
Jenner's stain, 162
Klatsch preparations, 66
Koch's rules, 79
stain, 33
steam-chest, 44
Kiihne's method, 41
stain, 33
Leprosy, 107
Leptothrix buccalis, 90
Liborius's method for anaerobins,
67
Locomotion, 19
Loftier' s alkaline stain, 33
blood serum, 111
mordant, 33
Lysins, 75
Macrocytases, 75
Macrogamete, 161
Macrophages, 74
Malarial parasite, 159
Malignant oedema, 150
Mallein, 110
Marchoux's thionin stain, 162
Marmorek's serum, 106
Material from animals, 79
Media, nutrient, 48
solid, 49
transparent, 52
Merozoites, 160
Metchnikoff ' s theory, 74
Microbe en huit, 165
Micrococci similar to gonococcus,
145
Micrococcus amylovorus, 170
cereus albus, 141
flavus, 141
cholera gallinarum, 165
citreus conglomeratus, 145
indicus, 81
melitensis, 159
of gonorrhoea, 143
of mal de pis, 169
of sputum septicaemia, 132
Pasteuri, 132
pyogenes aureus, 141
citreus, 141
tenuis, 142
subflavus, 146
tetragenus, 135
ureae, 91
Microcytases, 75
Microgametes, 160
Microgametocytes, 160
Micro-organisms of suppuration,
137
Microphages, 74
Microscope, 26
NDEX.
241
Microsporon furfur, 176
Milzbrand, 94
Moist chamber, 50
Mordants, 31
Mosquito, sexual cycle in, 160
Moulds, 175
examination of, 177
Mouse septicaemia, 168
Movements, vibratory, 19
Mucor mucedo, 175
Mycoprotein, 18
Nail culture, 132
Neisser's stain, 34
Nicolle's solution, 34
Nitrification, 25
Nitromonas, 188
Nivellier apparatus, 62
Nutrient media, 48
Odors in cultures, 25
Oi'dium, 174
albicans, 174
lactis, 174
Oil immersion, 26
Orcein stain, 40
Oxidation, 25
Parasites, 23
malarial, 159
Park's method, 69
Pasteur filter, 185
Pathogenic yeasts, 174
Penicillium glaucum, 175
Peptone solution, 58
Dunham's, 125
Petri's sand Altera, 182
saucer, 63, 186
Pfeiffer's immunity from cholera,
127
Phagocytic theory, 74
Phosphorescence, 25
Pigmentation, 25
Platinum needles, 28
Pneumo-bacillus, 131, 132
Potato cubes, 51
cultures, 49
inoculation of, 51
in test-tubes, 51
16
Potato mash, 51
Precipitins, 76
Products of tubercle bacilli, 106
Proteins, 25
Proteus, 170
mirabilis, 171
vulgaris, 170
Zenkeri, 171
Protozoa, pathogenic, 159
Pseudo-diphtheria bacilli, 114
Ptomaines, 25, 71
Putrefaction, 25
Pyocyanin, 143
Pyrosoma bigeminum, 163
Quartan forms of malarial para-
site, 161
Rabbit septicaemia, 167
Rauschbrand, 165
Ray fungus, 177
Receptors, 75
Reduction, 25
Relapsing fever, 152
Removing excess of stain, 36
Reproduction, 20
Rosalie acid solution, 59
Rotz, 108
Rouget du pore, 167
Roux's stain, 34
test-tube, 51
Saccharomyces albicans, 173
cerevisiae, 173
mycoderma, 174
niger, 173
rosaceus, 173
Sapremia, 71
Saprophytes, 23
Sarcina, 92
alba, 93
aurantica, 93
flava, 93
lutea, 92
rosea, 93
ventriculi, 93
Schizomycetes, 17
Sehizophyceae, 17
Schizophyta, 17
242
INDEX.
Schultz's method, 49
Schweinerotlauf, 167
Sedge wick-Tucker method, 183
Septicemia, 71
Serum, antituberculous, 106
test, 125
Serum-agar, 111
Sexual cycle in mosquito, 160
Slides, concave, 29
Small-pox, 163
Soil, examination of, 187
Solutions, composite, 31
formulae of, 32
saturated, 32
stock, 31, 32
weak, 31, 32
Soor, 174
Spasmotoxin, 149
Specimens, cover-glass, 35
cutting of, 37
drying of, 35
Klatsch, 66
Spirillum, 18
cholera?, 124
concentricum, 92
Finkleri, 127
of relapsing fever, 152
rubrum, 92
tyrogenum, 128
Spirochete Obermeieri, 152
Spores, arthro-, 21, 22
contents of, 21
endo-, 21, 22
formation of, 21, 22
requisites for, 22
resistance of, 22
staining of, 40
Sporidium vaccinale, 163
Sputum, hardened, 104
Stain, alkaline, 31, 33
aniline-water, 32
chloroform methyl-blue, 87
Gabbett's, 34
Gram's, 33
Hiss', 35
Koch's, 33
Kiihne's, 33
Loffler's, 33
Neisser's, 34
Stain, Roux's, 34
Unna's, 33
Welch's, 35
Ziehl-Neelsen, 33
Staining, Ernst's method of, 41
general method of, 35
Gram's method of, 39
Jenner's method of, 162
Kiihne's method of, 41, 109
Loffler's method of, 109
Marchoux's method of, 162
of capsule of bacillus of pneu-
monia, 132
of flagella, 41
of malarial parasite, 162
of milk, 87
of spores, 40
of sporogenic bodies, 41
of tissue sections, 37, 39
rapid method of, for bacillis tu-
berculosis, 101
slow method of, for bacillus tu-
berculosis, 102
solutions, 31
special methods of, 39
Weigert's method of, 41
Wright's method of, 162
Staphylococcus, 18
pyogenes albus, 141
aureus, 140
epidermidis albus, 141
Sterilization, 42
fractional, 47
Streptococcus, 18
erysipelatis, 138
puerperalis, 139
pyogenes, 138
Streptothrix, 177
farcinica, 179
Madura, 178
Suppuration, 137
Swine erysipelas, 167
plague, 167
Tertian form of malarial para-
site, 161
Test-tubes, 47
Tetanin, 119
Tetanotoxin, 149
INDEX
243
Tetanus, 147
Thermo-regulator, 58
Thread reaction, 76
Thrush, 174
Tinea, 176
Tissue preparations, 37
Toxalbumins, 25, 71
Toxins, 25, 71
nature of, 71
Toxophores, 75
Trichophyton tonsurans, 176
Trypanosomes, 163
Tuberculin, 106
Tuberculin E, 106
Tuberculocidin, 106
Tuberculosis, 97
in animals, 105
Typhoid fever, 115
medium of Hiss, 59
Typhotoxin, 121
Urine media, 59
Vaccinia, 163
Vibrio, Finkler-Prior, 127
Metschnikovi, 129
Vibrion septique, 150
Water, bacteria in, 87, 127, 180
examination of, 184
persistence of bacilli in, 121
Weigert's method of staining, 41
Welch's capsule stain, 35
Widal's serum test, 125
Wild plague, 167
Wire cages, 47
Wolf hiigel's apparatus, 186
Wright's chromatin stain, 162
method, 69
Yeasts, 173
examination of, 177
pathogenic, 174
Yellow fever, etiology, 155
Ziehl's solution, 33
Zoogloea, 18
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Sh^w on Nervous Diseases and Insanity
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Essentials of Nervous Diseases and Insanity : their Symptoms and
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System, Long Island College Hospital, New York. 121110 of 204 pages,
illustrated. Cloth, $1.00 net. In Saunders" Question- Compend Series.
Powell's Diseases of Children 3d Edition, Revised
Essentials of the Diseases of Children. By William M.
Powell, M. D. Revised by Alfred Hand, Jr., A. B., M. D., Dis-
pensary Physician and Pathologist to the Children's Hospital, Philadel-
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1. ESSENTIALS OF PHYSIOLOGY. 2d edition. By Sidney
P. BUDGETT, M.D.
2. ESSENTIALS OF SURGERY. 7th ed. 90 illustrations. By
Edward Martin, M.D.
3. ESSENTIALS OF ANATOMY. 7* ed. 151 illustrations. By
C. B. Nancrede, M.D.
4. ESSENTIALS OF MEDICAL CHEMISTRY. 6th ed. By Law-
rence Wolff, M.D. Revised by A. Ferree Witmer,
Ph.D.
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W. Easterly Ashton, M.D.
6. ESSENTIALS OF PATHOLOGY AND MORBID ANATOMY.
By H. Harlow Brooks, M.D. A new work. Preparing.
7. ESSENTIALS OF MATERIA MEDICA, THERAPEUTICS,
AND PRESCRIPTION-WRITING. 7th ed. By Henry
Morris, M.D. Revised by W. A. Bastedo, Ph.G., M.D.
8,9. ESSENTIALS OF PRACTICE OF MEDICINE. By W. R.
Williams, M.D. (Double number, $1.75 net.)
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10. ESSENTIALS OF GYNECOLOGY. 6th ed. With 57 illustrations.
By Edwin B. Cragin, M.D. Revised by Frank S. Mathews,
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1 1. ESSENTIALS OF DISEASES OF THE SKIN. 6th edition.
61 illustrations. By H. W. Stelwagon, M.D.
12. ESSENTIALS OF MINOR SURGERY, BANDAGING, AND
VENEREAL DISEASES. 2d ed. 78 illustrations. By
Edward Martin, M.D.
13. ESSENTIALS OF GENITO-URINARY AND VENEREAL DIS-
EASES. By S. S. Wilcox, M.D. A new work, fully illus-
trated. Just Ready.
14. ESSENTIALS OF DISEASES OF THE EYE.
trated. By Edward Jackson, M.D.
15. ESSENTIALS OF DISEASES OF CHILDREN.
Wm. M. Powell, M.D.
17. ESSENTIALS OF DIAGNOSIS. 2d ed.
M.D., and A. A. Eshner, M.D.
19. ESSENTIALS OF NOSE AND TH
By E. B. Gleason, M.D.
20. ESSENTIALS OF BACTERI
and 6 plates. By M. V
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21. ESSENTIALS OF
4th ed. 53 ill
by Smith F
24. ESSENTIA! illus-
trate
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