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SAUNDERS' QUESTION COMPENDS, NO. 20.

ESSENTIALS OF BACTERIOLOGY:

BEING A

CONCISE AND SYSTEMATIC INTRODUCTION TO THE
STUDY OF MICRO-ORGANISMS

FOR THE USE OF

STUDENTS AND PRACTITIONERS.

BY

M. V. BALL, M.D.,

LATE RESIDENT PHYSICIAN GERMAN HOSPITAL, PHILADELPHIA; ASSISTANT IN
MICROSCOPY, NIAGARA UNIVERSITY, BUFFALO, NEW YORK, ETC.

WITH SEVENTY-SEVEN ILLUSTRATIONS, SOME IN COLORS,

PHILADELPHIA:

W. B. S AU N DE ES,

913 WALNUT STKEET.
1891.

COPYRIGHT, 1891.
BY W. B. SAUNDERS,

COLLINS PRINTING HOUSE,
705 JAYNE STREET.

oo

PREFACE.

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 : —

MAC£: TraitS pratique de Bacteriologie.
FRANKEL: Grundriss der Bakterienkunde.
EISENBERG: Bakteriologische Diagnostik.
CROOKSCHANK, E. M.: Manual of Bacteriology.
GUNTHER: Einflihriug 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.

(v)

CONTENTS.

PART I.
GENERAL CONSIDERATIONS AND TECHNIQUE.

PAGE

Introduction • • • . ix

CHAPTER I.— CLASSIFICATION, STRUCTURE, AND RE-
PRODUCTION . . .... 17

" II.— ORIGIN, LIFE, GROWTH, AND PROPERTIES 23

" III.— METHODS OF EXAMINATION . . . 26

" IV.— STAINING OF BACTERIA .... 30

" V.— GENERAL METHOD OF STAINING SPECI-
MENS 34

" VI.— SPECIAL METHODS OF STAINING . .37

" VII.— METHODS OF CULTURE .... 39

" VIII. — NUTRIENT MEDIA 44

" IX.— SOLID TRANSPARENT MEDIA ... 47

" X.— INOCULATION OF GELATINE AND AGAR . 53

" XI.— GROWTH AND APPEARANCES OF COLONIES 58

" XII.— CULTIVATION OF ANAEROBIC BACTERIA . 60

" XIII.— MANNER IN WHICH BACTERIA ACT UPON

BODY 62

" XIV.— IMMUNITY 66

« XV. — ANIMAL EXPERIMENTS .... 68

(vii)

viii CONTENTS.

PART II.
SPECIAL BACTERIOLOGY.

PAGE

CHAPTER I.— NON-PATHOGENIC BACTERIA . . .72

Bacillus Prodigiosus 72

Indicus .72

Mesentericus Yulgatus 7.']

Megatherium 74

Ramosus 74

Bacterium Zopfi 75

Bacillus Subtilis 75

Spinosus 7(3

Some Bacteria in Milk 76

Bacillus Acidi Lactici 76

Butyricus 77

Amylobacter 77

Lactis Cyanogenus .... 78

Lactis Erythogenes .... 78

Some Noil-Pathogenic Bacteria of Water ... 79

Bacillus Violaceus ; Coeruleus ... 79

Fluorescent Bacteria 80

Phosphorescent Bacteria . . . .80

Crenothrix, Cladothrix, and Beggiatoa . 81

Bacterium Urese 82

Spirillum 83

Rubrum ; Concentricum 83

Sarcina 83

Lutea 83

Aurantica Flava, Rosea, and Alba ; Ventriculi . 84

CHAPTER II.— PATHOGENIC BACTERIA . * . . .84

Bacteria Pathogenic for Man and other Animals . 84

Bacillus Anthracis 84

Tuberculosis 88

Lepra Bacillus 96

Syphilis Bacillus 97

CONTENTS. IX

PAGE

Bacteria —

Bacillus of Glanders . . . . . .98

of Diphtheria 100

of Typhoid Fever' 101

Neapolitanus 104

CHAPTER III.— PATHOGENIC BACTERIA— CONTINUED . 105

Spirillum Choleras 105

Bacteria Similar to Spirillum Cholerse . . . 108

Finkler-Prior 108

Tyrogenum 109

Vibrio Metschnikoff 110

Bacteria of Pneumonia 110

Pneumo-bacilltis of Friedlander . . . .111

of Frankel . . . . .112

Bacillus of Khinoscleroma „ . . . . 115

Micrococcus Tetragenus 115

Capsule Bacillus . . . . . . . ' . 116

Micro-Organisms of Suppuration .... 116

Steptococcus Pyogenes 117

Staphylococcus Pyogenes Aureus .... 118

Micrococcus Pyogenes Albus, Citreus, Tenuis . 120

Cereus Albus, Flavus . . .120

Bacillus Pyocyaneus 120

Micrococcus Gonorrhoea .... . 121

Microbes Similar to Gonorrhoea .... 123

Bacillus of Tetanus 124

(Edematis Maligni 127

Spirillum of Relapsing Fever .... 129

Bacillus Malarise 130

Hsematozoa of Malaria 130

CHAPTER I V.— BACTERIA PATHOGENIC FOR ANIMALS,

BUT NOT FOR MAN . . . . . 133

Bacillus of Symptomatic Anthrax .... 133

of Chicken Cholera . . ...... .134

Bacteria of Hemorrhagic Septicaemia, Swine Plagues,

Duck Cholera, etc 136

CONTENTS.

PAGE

Bacillus of Rabbit Septicaemia . . . . . 136

of Erysipelas of Swine 130

Murisepticus 138

Micrococcus of Mai de Pis 138

Bacillus Alvei 139

Micrococcus Ainylivorus 139

Bacterium Termo 139

APPENDIX.

Yeasts 141

Oidiums 142

Moulds 143

Actinomyces or Ray Fungus 144

Examination of Air 146

of Water 149

of Soil 152

CONCLUSION . . . • . . 153

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 spirochsete. It was not until
1863 that any positive advance was made in connecting
bacteria with disease. Rayer and Davaine had in 1850

(xi)

Xll 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 of 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.

BACTERIA (daxtypiov, little staff) is the name given to a group
of the lowest form of plants, very closely following the algae.
They were called Fission- Fungi or Schizomycetes (o^t^w, to cleave,
/uor^j, fungus), because it was thought that, as the Fungi, they
lived without the chlorophyll. The word fission was supplied
to distinguish them from moulds and yeasts, it denoting the
manner of reproduction. Since several bacteria have been
found to possess chlorophyll, and as a great many increase in
other ways than by simple fission— the name of Schizomycetes
can no longer be applied, though the word Bacteria leaves much
to be desired.

Classification. Ferdinand Cohn, in the middle of the present
century, was the first to demonstrate bacteria to be of vegetable
origin, they being placed previous to that among the infusoria.
He arranged them according to their form under four divisions.

Cohn's System. I. Spherobacteria (globules).

II. Microbacteria (short rods). \ ^
III. Desmobacteria (long rods). /
IY. Spirobacteria (spirals).
As expressed at the present time, Micrococcus, Bacillus, and
Spirillum. This classification is very superficial, but because a
better one has not been found it is most in use to-day.

2 (17)

18 ESSENTIALS OF BACTERIOLOGY.

De Bary's System. De Bary divides bacteria into two groups,
those arising from or giving rise to endospores and those devel-
oped from arthrospores. This division has a more scientific
value than the first.

FIG. 1.

Jo^Tv ^

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" have not yet been observed, but the latest re-
searches point to their presence.

Cell- Wall. The cell-wall is composed of plant cellulose, which
can be demonstrated in some cases by the tests for cellulose.
The membrane is firm and can be brought plainly into view by
the action of iodine 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, granulose, and sul-
phur in its structure.

It is composed chiefly of myco^yrotcin.

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.

Zoogloea. "When this gelatinous membrane is very thick, irre-
gular masses of bacteria will be formed, the whole growth being
in one jelly-like lump. This is termed a zoogl<wv (£ww, animal,

. .jlue).
Locomotion, Many bacteria possess the faculty of self-move-

BACTERIA. 19

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 riot change their place ; these
movements are denoted as molecular or " Brownian."

FIG. 2.

Zooglcea.

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.

Reproduction. Bacteria multiply either through simple divi-
sion or through fructification by means of small round or oval
bodies called spores from spora-seed. In the first case, division,
the cell elongates, and at one portion, usually the middle, the
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.

20

ESSENTIALS OF BACTERIOLOGY.

FIG. 3.

Flagella.

Successive divisions take place, the new members either exist-
ing as separate cells or forming part of a community or group.

FJG. 4.

/-;

1 Z 3

Division of a Micrococcus. (After Mac6.)

*._.. — .-*

/«

^

Division of a Bacillus. (After Mace".)

Spore Formations. Two forms of sporulation, Endosporous
and Artlirosporous. A small granule develops in the protoplasm

BACTERIA.

21

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. 5.

FIG. 6.

Sporulation. After De Bary.

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 sporo-
genic 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. When brought into favorable conditions, the
spore begins to lose its shining appearance, the outer firm mem-

22 ESSENTIALS OF BACTERIOLOGY.

brane begiris 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 know now that only when the 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.

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 the 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 OF BACTERIA. 23

CHAPTER II.

ORIGIN OF BACTERIA AND THEIR DISTRIBUTION.

As Pasteur has shown, all bacteria develop from pre-existing
bacteria, or the spores of the same. They cannot, do not arise
de novo.

Their wide and almost universal diffusion is due to the minute-
ness of the cells and the few requirements for their existence.

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, $a?tpo?, putrid, $vtov,
plant. Parasites, rtapa, aside of 6iro$, 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 that will not live under a temperature of 00° C.

For the majority of Bacteria a temperature of 00° 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 cerofo'c, growing in air ; the other anccrobic, living
without air.

Obligate azrobins, those which exist only when oxygen is present.

Facultative a>robins, those that live best when oxygen is present,
but can live without it.

Obliijate or true anccrobins, those which cannot exist where
oxygen is.

facultative ancerobins, those which exist better where there is
no oxygen, but can live in its presence.

Some derive the oxygen which they require out of their nutri-
ment, so that a bacterium may be terobic and yet not require
the presence of free oxygen.

JErobins may consume the free oxygen of a region and thus
allow the anrerobius to develop. By improved methods of cul-
ture many varieties of ancerobins 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 have been killed
by a few days' standing in daylight.

Effects of Electricity. — Electricity arrests growth.

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.

Oxidation and reducivm are carried on by some bacteria. Am-
monia, hydrogen sulphide, and trimethylamin are a few of the
chemical products produced by bacteria.

Ptomaines, Brieger found a number of complex alkaloids,
closely resembling those found in ordinary plants, and which

ORIGIN OF BACTERIA. 25

he named ptomaines, from rt^^a (corpse), because obtained
from putrefying objects.

Fermentation, This form of "splitting up"— fermentation,
as it is called — is due to the direct action of vegetable organisms.
Many bacteria have the power of ferments.

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.

Liquefaction of Solid Gelatine. Some varieties of bacteria
digest the nutrient gelatine, and so dissolve it; others excrete
a ferment which liquefies the gelatine.

Producers of Disease. Various pathological processes are
caused by bacteria, the name given to such diseases being
infectious diseases and the germs themselves called disease-pro-
ducing pathogenic bacteria. Those which do not form any
pathological process are called n'.n-pathogenic bacteria.

Pigmentation. Some of the bacteria are endowed with the
property of forming pigments either in themselves, or producing
a chromogenic body which, when set free, gives rise to the pig-
ment. In many 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
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 displacing the media.

Odors. Some germs form odors characteristic for them ; some
a sweet aromatic one, and others a very foul, disagreeable smell ;
some give a sour or rancid exhalation.

Effect of Age. With age, bacteria lose their strength and die.

Bacteria thus carry on all the functions of higher organized
life.

They breathe, eat, digest, excrete, and multiply ; and they are
very busy workers.

26 ESSENTIALS OF BACTERIOLOGY.

CHAPTER III.

METHODS OF EXAMINATION.

WE divide the further study of the general characteristics of
Bacteria into two portions : —

First the examination of the same 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
Bacteriological 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," as this lens is called, dips into
a drop of cedar-oil placed upon the cover-glass, and is then ready
for use.

Abbe's Condenser. The second necessary adjunct is a com-
bination of mirrors for bringing
FlG- 7' wide rays of light directly under

the object. It serves to intensify
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

Abbe's condenser. Condenser. Together with it is

usually found an instrument for

METHODS OF EXAMINATION. 27

shutting off part of the light— a Blender. When the bacteria
have been found, and their relation to the structure is then
wished to be studied, the " Abbe" is generally shut out by the
Iris blender, and the structure comes more plainly into view.

FIG. 8.

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-
sary to change the objective on the same field, and that 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.

Examination of Unstained Bacteria. As the coloring of bac-

28 ESSENTIALS OF BACTERIOLOGY.

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.

Right here we might say that it is best to accustom one's self
to passing all instruments, needles, etc., through the flame, be-
fore and after each procedure ; it insures safety ; and once in the
habit, it will be done automatically.

From Solid Media. With a straight-pointed platinum needle,
a small pinch of the medium is taken and rubbed upon a glass
slide, with a drop of sterilized water, or bouillon, and from this
a little taken on cover-glass, as before.

FIG. 9.

Platinum Needles.

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 nn unstained specimen, we want but little light. We now
apply the eye, and if not in focus, use the fine adjustment, or,
using the coarse, but always away from the object that is to-
wnrds us, since the distance between the specimen and the lens

METHODS OF EXAMINATION. 29

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
over, holds the drop in the depression covered by the cover-
glass, thus forming an air-tight cell ; here the drop cannot
evaporate.

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 ; it will
usually be very thick in the centre and not so easily distin-
guished.

30 ESSENTIALS OF BACTERIOLOGY.

Spores, automatic movements, fission, and cultivation in
general can be studied for several days. This moixt chmnlur
can be placed in a brood-oven or on the ordinary warming
stages of the microscope.

CHAPTER IY.

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.

Only since the aniline colors bave come into active use, has
the technology of staining become developed.

Aniline 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
aniline dyes into two groups, the basic colors to which belong—
Gentian violet,
Methyl violet,
Methylin blue,
Fuchsin,
Bismark-brown,
And the acid colors to which eosin belongs.

The basic dyes stain the bacteria and the nuclei of cells ; the
acid dyes stain chiefly the tissue, leaving the bacteria almost
untouched. Carmine and Hcematoxylin are also useful as con-
trast stains, affecting bacteria very slightly. The aniline 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 ;

STAINING OF BACTERIA. 31

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 just
opaque in the body of the bottle, but still 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 "frrte" 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 aniline dyes, aniline oi7, a member
of the aromatic benzol group, has also this property.

Aniline Oil Water. Aniline oil is shaken up with water and
then filtered ; the aniline water so obtained is mixed with the
dyes forming the "aniline water gentian violet" or aniline
water fuchsin, etc.

Carbol Fuchsin. Carbolic acid can be used instead of aniline
oil, and forms one of the main ingredients of Ziehl's or Neelsen's
solution, used principally in staining bacillus tuberculosis.
Kiihne 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,
ammon. carbonate, and sodium hydrate are used.

Loffler's alkaline blue and Koch's weak alkaline blue make
use of potassium.

Heat. Warming or boiling the stains during the process of
staining increases their intensity.

Decolorizing Agent's. 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.

32 ESSENTIALS OF BACTERIOLOGY.

Iodine as used in Gram's Method. Belonging to this group,
but used more in the sense of a protective, is tr. iodine. It picks
out certain bacteria, which it coats ; prevents tliem 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 iodine, 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.— Aniline Oil Water.

Aniline oil 5 parts.

Distilled water .... 100 parts.— M.
Shake well and filter. To be made fresh each time.

IV. — Aniline 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.

V.— Alkaline Methylin Jllue.

A. J30fer*«.

Sat. ale. sol. methylin blue . . 30
Sol. potass, hydrat. (1-10,000) . 100— M.

B. KodSs.

Sol. potass, hydrat. (10 per cent.) 0.2

Sat. ale. sol. methyl, blue . . 1.0
Distilled water 200.0— M.

STAINING OF BACTERIA. 33

VI. — Carbolic Acid Solutions.

A. Ziehl-Neelsen.

Fuchsia (powd.) .... 1 part.

Alcohol 10 parts.

5 per cent. sol. acid, carbolic . 100 parts.— M.
Filter. The older the solution the better.

B. Kuhne.

Methylin blue .... 1.5

Alcohol 10.0

5 per cent. sol. ac. carbol. . . 100.0
Add the acid gradually. Tins solution loses strength with age.

VII. — Gram's Iodine Solution.
Iodine ...... 1

Potass, iod 2

Aquae destillat. . . . . 300.— M.

VIII.— Lqffler's Mordant (for flagella).
Aq. sol. of tannin (20 per cent.) . 10 parts.
Aq. sol. ferri sulph. (5 per cent.) . 1 part.
Aqure decoc. of logwood (1-8) . 4 parts. — M.
Keep in well-corked bottle.

IX. — Picro-carmine ( Ranvier).

Carmine 1

Water 10

Sol. atnmon. ..... 3

Sat. sol. picric acid . . . 200.— M.

X. — Gobbet's Acid Blue (rapid stain).
Methylin blue .... 2
25 per cent, sulphuric acid . . 100. — M.

XI. — Alkaline Aniline Water Solutions.
Sodium hydrat. (1 per cent.) . . 1
Aniline oil water .... 100. — M.
And add —

Fuchsin, or methyl-violet powd. . 4
Cork well. Filter before using.

O

34 ESSENTIALS OF BACTERIOLOGY.

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, i. 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. •*

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
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.

Removing Excess of Stain. The surplus stain is washed oil*
by dipping the glass in water, distilled water always best,
though ordinary running water is admissible.

The water is removed by drying between filter paper or

METHOD OF STAINING SPECIMENS. 35

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
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.

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.

3t> ESSENTIALS OF BACTERIOLOGY.

Cutting. The microtome should be able to cut sections
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.

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-side.

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-

Dith 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
section is then taken, avoiding the errors, if any ; and having
reached this stage proceeded Avith as follows : —

Dish IV, alcohol, 2 to 3 seconds to remove the water in the
tissue.

V. A few drops of oil of clores, 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.

STAINING AND MODIFICATIONS. 37

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. Ilinse off alcohol with water.

V. Contrast color with either eosin, picro-carmine, orbismark-
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 rabbit septica3mia.
" of malignant O3dema.
" of pneumonia (Friedlander).
" of glanders.
Diplococcus of gonorrhoea.
Spirillum of relapsing fever.
Gram's Method for Tissues (modified by GUnther).

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.

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.

38 ESSENTIALS OF BACTERIOLOGY.

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-t'uchsin 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 methyliu
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. Carlol.-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.

Flagella Stain, with Loffkr^s Mordant.— I. A few drops of the
mordant (No. viii.) 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. Aniline water fuchsin (neutral) held in flame about H
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.

Spoi'ogenic bodies stain quite readily, and in order to distin-
guish them from spores Ernst uses alkaline nuthylin blue, slightly
warmed.

Then rinse in water.

Contrast with cold bismark -brown.

The spores are colored bright blue, the spore granules a dirty
blue, being mixed with the brown, which colors also the bacteria.

Kiihntfs Method. — In sections, the alcohol used sometimes de-
colorizes too much. To obviate this K-tihm mixes the alcohol
with the stain, so that while the section is beting anhydrated it
is constantly supplied with fresh dye.

Weiyert uses aniline oil to dehydrate instead of alcohol, and
here, too, it can be used mixed with the dye.

METHODS OF CULTURE. 39

General Double Staining for Sections.
I. Stain (watery dyes) . . . 10 to 15 minutes.
II. Acetic acid and water (1 to 4) . £ minute.

III. Alcohol 2 to 3 minutes.

IV. Contrast stain, usually picro-car-

mine or eosiu . . . . 2 to 3 minutes.

Y. Alcohol i minute.

VI. Clove oil. Canada balsam.

Instead of coloring with the contrast last, it can be used first,
then alcohol one-half minute, followed by the bacteria stain, acid
water, alcohol, clove oil, and Canada balsam in succession.

The stains for special bacteria will be given when treating of
the same.

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.

40

ESSENTIALS OF BACTERIOLOGY.

Second. To maintain this degree of disinfection 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
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
not too moist with the sublimate.

Heat. Heat is the best agent we possess for general use.
Dry heat and moist heat are the two forms employed.

FIG. 11.

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,

METHODS OF CULTURE.

41

and | foot deep. In them, glass-ware, cotton, and paper can be
sterilized. When the cotton is turned slightly brown it usually
denotes sufficient sterilization. All instruments, where prac-
ticable, should be drawn through flame of alcohol lamp or Bun-
sen burner.

Moist Heat. — Steam of 100° C. in circulation has been shown
to be a very effective application of heat.

Koch's Steam-chest, The best
way of obtaining circulating
steam is by aid of Koch's appa-
ratus. This consists of a cylin-
drical tin chest about 2£ feet
high and about | foot in diame-
ter ; divided in its interior by a
perforated diaphragm, a, an up-
per chamber for the, c, steam and
a lower one for water, 6. Two
or more gas-burners placed under-
neath the chest, which stands on
a 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 10 to
15 minutes, or longer, as required.

The autoclave of Chamberland
allows a temperature of 120° C,
to be obtained, and is much used
in Pasteur's laboratory.

Instead of sterilizing for a long
time at once, successive steriliza-
tion is practised with nutrient
media, so that the albumen will
not be too strongly coagulated,
three days in succession.

FIG. 12.

Koch's Steam-chest.

Fifteen minutes each day for

42 ESSENTIALS OF BACTERIOLOGY.

Fin. 13.

Chamberland's Autoclave with pressure.

Fractional Sterilization of Tyndall. Granted that so many
spores originally exist in the object to be sterilized, it is sub-
jected to 60° C. 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
GO0 heat will destroy them, and so at the end of a week, using

METHODS OF CULTURE.

43

four hours' application each day, all the spores originally present
will have germinated aud the bacteria destroyed.

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.

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
brush, placed obliquely to drain, and when dry corked with

FIG. 14.

FIG. 15.

Wire-Cage.

Cotton plugged Test-Tubes.

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.

44 ESSENTIALS OF B A CTERIOLOG Y .

CHAPTER VIII.

NUTRIENT MEDIA.

OF the many different media recommended and used since

bacteriology becaiwe 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 Loffler). A cooked infusion of chopped
beef made slightly alkaline with carbonate of soda. Prepared
as follows : 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 or straining cloth, the white
meat remaining in the cloth 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
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 liltered 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.

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 con-
tents in the steam-chest. A tin pail with perforated bottom

NUTRIENT MEDIA. <J5

makes a good container in which they can be lowered in the
Koch's oven. 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 of 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.

Next placed in 1 to 500 solution of bichloride of mercury for
| hour, Then in the steam-chest for f hour.

In the meantime, a receptable is prepared for them. This is
called the moist chamber.

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.

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.

FIG. 16.

Moist chamber for potatoes.

The operator now prepares his own hands, rolling up his coat
sleeves and carefully washing his hands, then taking a potato
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

46 ESSENTIALS OF BACTERIOLOGY.

the moist chamber, when they of their own weight fall aside,
the cut surface uppermost. They are then ready for inoculation.

Fio. 17.

Method of slicing potato. (After Woodhead and Hare.)

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.

Manner of Inoculation. With a platinum rod or a spatula
(sterilized) the material is spread out 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. 18.)

From the original or first slice, a small portion including some
of the meat of the potato is spread out 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 individuality.

This is the principle carried on in all the cultivations. It is
a physical analysis.

SOLID TRANSPARENT MEDIA.
FIG. 18.

47

Method of inoculation. (Woodhead and Hare.)

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.

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 materials. %

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.

Koeh-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.

48

ESSENTIALS OP BACTERIOLOGY.

FIG, 19.

This is placed in a flask and gently heated until the gelatine

is dissolved.

Neutralization with the soda and then cooking in water-bath

for 1 hour or more until the 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, nnd 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.
19.)
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 Icng 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.

Hot-water filter.

SOLID TRANSPARENT MEDIA. 49

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
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 the gelatine can be mixed with milk, blood, serum,
urine, and agar-agar.

The nutrient gelatine bouillon can also receive additions iu
the shape of glycerine (4 per cent, to 6 per cent, being added),
or reducing agents to take up the oxygen present.

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.

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. The reaction is taken, very little of the alkali being
sufficient to neutralize it.

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.
4

50

ESSENTIALS OF BACTERIOLOGY.

Cotton can be used here instead of filter-paper, or filtering
entirely dispensed with by making use simply of decantation.

As Agar, at its best, is seldom clear, a little more or less opaque-
ness, when a great deal of trouble and time can be saved
thereby, will not harm. The test-tubes are filled as with the
gelatine, and sterilized in the same manner. AVhile 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
bottom, or a little white sediment remain encysted in the centre ;
this cannot easily be avoided, nor does it form any serious obstacle.
Glycerine Agar. The addition of 4 per cent, to 6 per cent, 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 majority of bacteria develop,
and some with preference.

Preparation of Nutrient Blood Serum.
If the slaughter of the animal can be super-
vised, it were best to have the site of the
wound and the knife sterilized carefully,
and then sterile flasks placed to receive the
blood directly as it flows.

It is placed on ice forty-eight hours, and
then the serum is drawn out with sterile
pipettes into test-tubes ; these are placed
obliquely in an oven where the temperature
can be controlled and maintained at a cer-
tain degree.

Incubators or Brood-ovens. Incubators
or brood-ovens, as such ovens are called,
consist essentially of a double-walled zinc
or copper chest, the space between the walls filled with water.
The oven is covered with some impermeable material to pre-

FIG. 20.

Flask to receive blood

SOLID TRANSPARENT MEDIA.

51

vent the action of surrounding atmosphere. (Fig. 22. ) It is sup-
plied with a thermometer arid with a regulator. The regulator

FIG. 21.

Incubator for blood serum.

is connected with the Bunsen burner, and keeps the thermometer
at a certain height.

There are several forms of regulators in use, and new ones
invented continually.

The size of the flame is regulated by the expansion ot mer-
cury, which, as it rises, lessens the opening of the gas supply.
The mercury contracting on cooling allows more gas to enter
again. (Fig. 23.)

Koch has invented a safety burner, by which the gas supply is
shut off should the flame accidentally have gone out.

Coagulation of Blood Serum, The tubes of blood serum
having been placed in the brood-oven, are kept at a tempera-

52

ESSENTIALS OF BACTERIOLOGY.

ture of 65° to 68° C., until coagulation occurs ; then removed
and sterilized.

Fio. 22.

Babe's incubator.

Thermo-regulators.

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.

J31ood serum, formerly much more used than now, was especi-

INOCULATION OF GELATINE AND AGAR. 53

ally applicable to the culture of tubercle bacilli. The glycerine
agar has now superseded it.

Human blood serum derived from placenta, serum from ascitic
fluid, and ovarian cysts are prepared in a similar manner to the
above.

Other Nutrient Media. Milk, urine, decoctions of various
fruits and plants, and lately for cultivating anaerobic bacteria,
eggs.

Fresh Egg Cultures, after Hiippe. 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
opening is covered with a piece of sterilized paper, and collodion
over this.

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.

Test Tube Cultures. The gelatine, agar, or blood serum having
solidified in an oblique position, is smeared on the surface with
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
fingers 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 c^re 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 : —

64 ESSENTIALS OF BACTERIOLOGY.

The 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).

FIG. 24.

Manner of holding tubes for inoculation : a, tube with material ; b, tube to be
inoculated ; c, cotton plugs.

From the first dilution three drops are taken into a third tube,
which becomes the second dilution.

The plugs of cotton must be replaced after each inoculation,
and during the same must be carefully protected from contami-
nation.

INOCULATION OF GELATINE AGUE.

55

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. 25.

FIG. 26.

Manner of holding plugs.

plug opposite its tube. They are now read}' for spreading on
glass plates.

Glass Plates. The larger the surface over which the nutrient
medium is spread the more isolated will
the colonies be ; window glass cut jn rec-
tangular plates 0x4 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. 20.) 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.

A

Iron box for glass plates.

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. 27.) The glass plate taken out of the iron box is placed
under the bell-glass. The tube containing the gelatine is held

56 ESSENTIALS OF B ACTERrOLOGY.

in the (lame a second to singe the cotton plug, and the plug
removed, the edges of the tube again flamed, the bell-glas.s
lifted, and the inoculated gelatine carefully poured on the plate,
leaving about one-third inch margin from the borders ; the lips

FIG. 27.

Nivellicr leveling and cooling apparatus.

of the tube being sterile can be used to spread the media 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. 28.

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 CJmmbcr Prepared Out of Two Glass Dishes, as for
tJie Potato- Culture*. Then 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

INOCULATION OF GELATINE AND AGAR. 57

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- FIG* 29.

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 Petri saucerg
the lower dish, covered by the larger one,

and placed in some cool place, different saucers being used for
each dilution.

This method is ver}' useful for transportation ; the saucers can
be viewed under microscope similar to the glass plates.

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.

NOTE.— The peptone nutrient gelatine, blood serum and agar
can now be purchased already prepared, thus saving a great
deal of time and making unnecessary the purchase of consider-
able apparatus.

58

ESSENTIALS OF BACTERIOLOGY.

CHAPTER XI.

THE GROWTH AND APPEARANCES OF COLONIES.

FIG. 30.

Naked eye 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 ap-
pearances, from that of a small dot,
like a fly speck, to that resembling
a small leaf. Some are elevated,
some depressed, and some, like cholera, cup-shaped, umbilicated.
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. 31.)

GROWTH AND APPEARANCES OF COLONIES. 59

Impression or "Klatseh" 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

FIG. 31.

FIG. 32.

Microscopic appearances
of colonies.

Klatsch preparations.

stained or examined so. The Germans give the name of
"Klatsch" to such preparations. Many beautiful pictures can
be so obtained.

Fishing. To obtain and examine the individual members of
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.

60 ESSENTIALS OP BACTERIOLOGY.

It is necessary to transfer the bacteria to fresh gelatine about
every six weeks, lest the products of growth and decay given
oft* 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 0x3* gen.
FIG 33 Several measures have been adopted of which the
easiest and most serviceable are the following : —

Liborius's High Cultures. The tube is filled about
f 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 ana?robic germs will
grow7 from the bottom upward, and any rcrobins
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
£ gelatine, the material being obtained by breaking
test-tube with the culture.

Hesse's Method. A stab culture having been made
with ana?robic germs, gelatine in a semi-solid condi-
Liborius's tjon {s p(nire(i jnto the tube until it is full, thus dis-

IIU'tlllMl. - . - . T,. _ . .

placing the air. (r ig. .54. )

Esmarch's Method. Having inoculated a tube with the
microbe the gelatine is rolled out on the walls of the tube, a
"roll culture," and the rest of the interior filled up with gela-
tine, the tube being held in ice water in the meanwhile. The
colonies develop upon the sides of the tube and can be easily
examined microscopically.

CULTIVATION OF ANAEROBIC BACTERIA.

61

Gases like Hydrogen to replace the Oxygen. Several arra nge-
ments for passing a stream of hydrogen through the culture : —

Frankel puts in the test tube, a rubber cork containing tw°
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. 35.) Hiippe uses eggs as described
in Chapter IX.

FIG. 34.

FIG. 35.

FIG. 3G.

Hesse's method.

Frankel's method.

Buchner's method.

Use of JSrobic 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

62 ESSENTIALS OF BACTERIOLOGY.

subtilis or some other aerobic germ. The subtilis does not allow
the oxygen to pass by, appropriating it to itself.

Buchner's Method. The test tube containing the culture
is placed within a larger tube, the lower part of which con-
tains an alkaline solution of pyrogallic acid or some other agent
which absorbs oxygen. The tube is then closed with a rubber
stopper. (Fig. 37.)

CHAPTER XIII.

THE WAY IN WHICH BACTERIA AFFECT THE ANIMAL ORGANISM.

BACTERIA affect the animal organism by depriving the cells
of the body of oxygen and nitrogen which they appropriate to
themselves for their maintenance.

They do more than this, however, for in their secretions and
excretions the main potency lies.

Ptomaines, or Cadaveric alkaloids, was the name first applied
to those bodies formed during putrefaction, but now used for
all alkaloids or bodies of a basic nature formed by bacteria.
Many of these ptomaines when introduced into the body give
rise to the same set of symptoms as the bacteria themselves do,
so that we may say, bacteria affect the animal body chiefly through
certain toxic principles which they produce and which can be isolated.

Toxines and Toxalbumens. Late researches claim two classes
of products for bacteria— the one toxic and destroyed by heat ;
the other anti-toxic, having a direct action upon the tissue and
preventing further infection. Then proteids or toxalbumens,
products extracted from pure cultures, which, like ptomaines,
produce symptoms similar to those of the bacteria itself. They
are amorphous and have no basic action, giving, however, all
the reactions of a proteid or albumen.

Filtration of Cultures. These products are isolated from the
culture after the bacteria themselves have been separated.

A filter consisting of a cylinder of porcelain, asbestos or
kaolin, through which the culture fluid passes, the bacteria
remainin^ behind, is called the Pasteur-Chamberland filter.

THE ANIMAL ORGANISM.
FIG. 37.

63

Pasteur-Chamberland filter with pressure.
A. Container. H. Filter. K. Porcelain. P. Air-pump.

64 ESSENTIALS OF BACTERIOLOGY.

(Fig. 37.) The culture can be forced through or allowed to filter
slowly.

The gerraless liquor is then treated with various agents,
alcohol and acetic acid being that used for the loxalbumen of
diphtheria, and a white amorphous powder is at length obtained.
These agents have different effects in different doses, and are
used also to establish an immunity.

Toxic Bacteria. Those bacteria which produce toxic agents
outside of the body, and will not develop in the body, are
called toxic bacteria. They are pathogenic only in the sense
that their products, when accidentally introduced into the body,
cause mischief.

Infectious Microbes. Those bacteria which can develop and
do develop in the animal body, and there generate products
injurious to the same, are called infectious bacteria, or pathogenic
bacteria.

The Variations of Pathogenesis, The same animals under
different circumstances can be differently affected by the same
germ.

The ordinary white mouse is not acted upon by the bacillus
of glanders.

If, however, glycosuria be produced in the mouse in any way,
it speedily becomes attacked by the bacillus.

Different animals are differently affected by the same germs.
As we said before, ordinarily the white mouse is not acted upon
by the bacillus of glanders, but the house mouse is at all times.

The bacterium may first become active when mixed with
certain chemicals, it having been harmless before.

Attenuation or Weakened Virulence. Bacteria can be les-
sened in action either temporarily or permanently, or made in-
active entirely without destroying them. There are the natural
decay and loss of strength ; and successive cultivation in artificial
media for a long time of the same germ also destroys its potency.
But artificial means can be used, such as the use of chemical
agents added to the nutrient soil, or by passing the germ through
animals who are in some sense immune, and are less affected than
the animals for whom it is strictly pathogenic.

Thus the bacillus of swine-erysipelas, which is quite virulent

THE ANIMAL ORGANISM. 65

for pigs, when passed through rabbits loses much of its power,
and again introduced into pigs will sicken them but slightly.
Sunlight or any other agent that is destructive to germs will
also weaken them when used cautiously.

Heat is the surest agent to lessen the action.

The longer it takes to produce the attenuation, the more lasting it is.

The grade of virulence will oftentimes remain through suc-
cessive generations.

Some of the attenuations have been named according to the
animal that they will affect ; thus, Mice-anthrax is a culture of
anthrax which has been exposed to a temperature of 42.0 C. for
twenty days, and which will destroy nothing larger than mice.
A culture exposed for ten days will kill nothing larger than
rabbits, etc.

The only explanation that can be given of attenuation is that
the microbes, though similar in appearance, differ, in that the
weaker ones give rise to less toxic products ; they have been
exhausted.

Nageli makes use of the simili of the sweet and bitter almond,
the one poisonous, because it contains amygdalin, but both pos-
sible to be borne on the same tree, and looking alike in every
particular.

The Resistance of the Animal Organism to Bacteria. The
body is in some sense resistant to bacteria ; to some more, to
others less ; and this resistance has been variously explained.

Chemical Theory. The greater or less alkalinityof the blood di-
minishing or increasing the virulence is the explanation of some.

The Theory of the Action of the Serum of the Blood, It has
been lately shown that the serum of the blood has a direct in-
hibiting action on all bacteria ; and this is directly dependent
upon the quantity and quality of albumen in it. It was for-
merly thought the salts of the blood were the main factors, but
these only serve to keep the albumen in good condition.

The serum will hinder the growth of germs, and when bac-
teria are injected directly into the blood, they soon disappear.
They cannot osmose ; they collect in the liver, spleen, and
marrow of bones, and the corpuscles aid growth. The serum
of different animals acts differently upon the same microbe, .
5

66 ESSENTIALS OP BACTERIOLOGY.

Cellular Theory of Metschinkoff, The phagocytes, as he
terms them, are auti-microbie. They are the soldiers which
endeavor to destroy the enemy. If the cells are strong, they
become the victors ; if the bacteria are stronger, the bacteria
conquer and eat up the cells.

But this theory, though having many supporters, is opposed
on sufficient grounds, the one reason being that whenever cells
become the residence of live bacteria they suffer ; and if Mets-
chinkoff and others have seen bacteria directly enter cells and
disappear, it is that they were destroyed before, and that the

leucocytes only acted as scav-
FlG' 38' engers. (Fig. 38.)

And the late researches
with the serum of the blood
freed of its cellular elements,
and being directly anti-bac-
teric, would seem to place the
phagocytes in the background.
The matter is, however, by
no means settled.

Phagocytes of Metschinkoff: a. Bacillus m , , ,

entering ; 6. Bacillus inclosed. T° Sum UP. We have P"th°-

genic microbes such as give

rise to products injurious to the animal organism. The infec-
tious ones can overcome the natural resistance of the animal
body and develop therein. The bacteria can have this activity
lessened or destroyed by agents which are injurious to their
products, so as to render them inactive.

CHAPTER XIV.

IMMUNITY.

THE natural or acquired power of resistance to bacterial
influences is called immunity.

Natural. As we have mentioned before, certain animals are
naturally not acted upon by bacteria that affect other animals.
We say the animal or person is immune by nature.

IMMUNITY. 67

Acquired. But immunity can be acquired by various means.
We know that one case of smallpox usually protects against
other attacks and so with morbilla and scarlatina. This is
through disease.

Acclimatization Immunity. Various diseases, which strangers
to a climate become affected with, do not trouble the natives.

Artificial Immunity. By the attenuated virus, as with anthrax.

Inoculations with sterilized cultures, the germs being destroyed.
Even certain chemical substances when injected give immunity
from certain germs, and when albumen prepared in certain ways
was injected, immunity also obtained.

The various theories which have been made to explain the
phenomena of immunity are all unsatisfactory. Some say that
a first attack destroys the agents which are necessary for the
disease to arise. Others say that certain bacterial products
remain in the body, and prevent a return of the diseases — act as
guards. Some recently claim that the soil is rendered unfit for
the further development of the bacteria, after injection of some
of the active principles of the bacterium. Some place it in
the blood of the animals, it exerting a direct germicidal action.

It has not yet been satisfactorily explained, perhaps, in the
late activity of bacteriological workers, some solution of this
important question will be arrived at.

Cure of Infectious Diseases with Bacteria and their Products.
Antagonism. It has been known, and is easily demonstrated,
that the growth of one bacterium near another results often in
the destruction of one, a direct antagonism existing.

Rabbits suffering with anthrax were injected with large quan-
tities of streptococcus of erysipelas (pyogenes), and a cure
effected, those not so treated dying.

Several other diseases have been so treated in animals with
interesting results.

Toxalbumen Injections. When diphtheria is produced in
animals, an injection of the toxalbumen of diphtheria will cure
the same, and if injected first, diphtheria will not arise.
Tetanus has been cured and prevented in a similar manner.

Tuberculin. Dixon, in 1889, found, by injecting products of
tuberculosis cultures in glycerine, that Guinea-pigs so treated,

68 ESSENTIALS OF BACTERIOLOGY.

suffering from tuberculosis, were cured ; control animals dying.
Koch, in 1890, applied this method to man, and the final results
are still sub juclice.

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.

CHAPTER XV.

EXPERIMENTS UPON ANIMALS.

THE smaller rodents and birds are the ones usually employed
for inoculation, as rabbits, Guinea-pigs, rats and 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 the}r multiply rapidly. Therefore it is best to have them in
some large stall or inclosure. They can be fed upon all sorts of
vegetables and grasses, and require but little attention.

EXPERIMENTS UPON ANIMALS. 69

Methods of Inoculation, Z Inhalation.— Imitating the natural
infection, either by loading an atmosphere with the germs in
question or by administering them with a spray.

//. Tlirougli 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 " tubercnline," 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.
(Pig. 40.)

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.

70

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

FIG. 39.

Manner of making intravenous injections in the rabbit.

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
aponeurosis cut through where the skull is the thinnest. Then
the bone carefully trephined, and the dura exposed. In Rabies
inoculation, the syringe containing the hydrophobia 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-
tines after they have been exposed, by carefully opening the

EXPERIMENTS UPON ANIMALS. 71

abdomen. This is done in order to avoid the action of the
gastric juice.

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 away 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.

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-
patkoycnic being the main divisions. It is usual to describe the
non-pathogenic first.

Non-Pathogenic Bacteria. We can give but a few of the more
important varieties.

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.

-F(/rrn.— 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.

(72)

NON-PATHOGENIC BACTERIA. 73

The 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.

Gases. — A trimethylamin odor arises from all culture.

Stain. — Takes all aniline 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. Auto-
matic movements present ; facultative ancerobin.

Growth. Gelatine. — Liquefy rapidly.

Colonies. — Bound, or oval, granular margins ; brilliant red
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. FlG- 40'

Stain. — Takes all dyes.

Bacillus Mesentericus Vulgatus.

The common potato bacillus of
Flugge.

Habitat. — Surface of the soil, on
potatoes, and in milk.

Form. — Small thick rods with
rounded ends, often in pairs.

Properties. — Very motile ; pro-
duce abundant spores; liquefy Colony of Bacillus Mesentericus
gelatine. 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.

74 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 Bury).

Origin.— Found on cooked cabbage.

Form. — Large rods, four times as long as they are broad,
2.5 p. Thick rounded ends. Chains with ten or more mem-
bers often formed.

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. 41.

Bacillus Megaterium, with spores.

Stab Culture.— Funnel-shaped from above downwards.

Potato. — Thick growths, 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. 75

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.

Stab Culture.— Soon a 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 Zopfi. (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; aerobic; 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.

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. — Very motile ; dissolves gelatine.

Growth. — Rapidly ; strongly aerobic.

Plate.— Round, gray colonies, with depressed white centre.

76 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.

Bacillus Spinosus. (Luderitz.)

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.

Grotcth. — The growth occurs at ordinary temperatures only
when the oxygen is excluded. Very strongly anaBrobic. 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 anterobic, must be cultured with the usual care taken
with anserobins.

Some Bacteria found in Milk. Bacillus Acidi Lactici.
(Iliippe.)

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.— Slowly ; is facultative anaerobic. Grows first at 10° C.

NON-PATHOGENIC BACTERIA. 77

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 with 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.

Growth.— Quickly, at 35° to 40° 0., with oxygen.

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.)

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.

78 ESSENTIALS OF BACTERIOLOGY.

FIG. 42. Properties. — Very 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
I S .- h) iodine will color blue some portions of the
1 >H bacillus ; therefore it has been called amy-

(P 1 li « fofracter.

\ « Growth. — It is strongly anicrebic, and

^ B| j has not yet been satisfactorily cultivated.

f i Bacillus Lactis Cyanogenus. Bacterium

Bacillus Amyiobacter. 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 Red 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. 79

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.

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.

Stab Culture. — Cone-like liquefaction containing air, and the
pigment, in separated granules, lying towards the bottom.

Stroke Culture on Agar.— A violet, ink-like covering which
remains for years.

Bacillus Coeruleus. (Smith.)

Origin. — Schuylkill water.

Form. — Very thin rods ; 2.5 /*. long, 0.5 /t*. wide ; often in
threads ; spores were not found.

Properties. — Liquefies gelatine; produces a very deep-blue
pigment.

Growth. — Slowly, with oxygen, at ordinary temperature.

Plate. — Round colonies on the surface of bluish color.

80 ESSENTIALS OF BACTERIOLOGY.

Stab Cultures. — A cup-shaped liquefaction along the needle
thrust, with a sparse growth, the liquefied portion appearing
blue.

Fluorescent Bacteria. Several kinds present in water.

Bacillus Erythrosporus. (Eidarn.)

Origin.— Drinking water and putrefying albuminous solutions.

Form. — Slender rods often in short threads, with spores of
oval shape, and appearing as if stained with fuchsin.

Properties. — Motile ; does not dissolve gelatine ; produces a
greenish-fluorescent pigment which appears yellow in reflected
light, but green on transmitted light.

Growth. — Somewhat quickly ; facultative anaerobic ; growth
only at ordinary temperatures.

Plates. — White colonies, with greenish-yellow fluorescence
around each colony. Under microscope the periphery appears
radiated.

Stab Cultures.— Good growth along the needle thrust; the
whole gelatine gives out the fluorescence.

Bacillus Fluorescens Liquefaciens.

Ch'iyin. — Water, and from conjunctival sac.

Form. — Very fine little rods ; no spores.

Properties. — Motile ; forms a greenish-yellow fluorescent pig-
ment ; liquefies gelatine.

Growth.— Rapid only at ordinary temperatures, and strongly
aerobic.

Plates. — Round colonies, cup-shaped depressions, the solid
gelatine that remains becoming colored with greenish-yellow
fluorescence.

Stab Culture.— On the surface, air-bubble depressions ; the
white colonies in the bottom of these depressions, and the solid
gelatine around the inoculation shining with the fluorescence.

Phosphorescent Bacteria. Six varieties of phosphorescent
bacteria have been described ; they are found usually in sea-
water, or upon objects living in the sea.

Bacillus Phosphorescens Indicus. (Fischer.)

Origin. — Tropical waters.

Form. — Thick rods, with rounded ends, sometimes forming
long threads.

NON-PATHOGENIC BACTERIA. 81

Properties. — Very motile ; liquefying gelatine at a tempera-
ture of 25° to 30° C., with oxygen and a little moisture, and in
the dark, a peculiar electric-blue light develops a phosphores-
cence.

Growth. — Slowly ; must have oxygen ; does not grow under
10° C. or over 50° C.

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 and blood-serum.

Bacillus Phosphorescens Indigenus, (Fischer.)

Origin. — Waters in the northern part of Germany. It differs
from the Indian bacillus, in that it grows at a temperature of
5° C., and does not develop upon potatoes or blood-serum.

Bacillus Phosphorescens Gelidus. (Fb'rster.)

Origin. — Surfaces of salt-water fish.

Form. — Short, thick rods, looking oval sometimes ; zoogkea
are 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 0° and 20° 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
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 twenty-four hours.

The other three varieties require glucose in the culture before
they give out any glow. They are Bacterium Pflugeri, Bact.
Fischeri, and Bact. Balticum. 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,
dadothrix, and beggiatoa.
6

82 ESSENTIALS OF BACTERIOLOGY.

Crenothrix Kiihniana. (Rabenhorst.) Long filaments joined
at one end ; little rod-like bodies form in the filaments ; and
these break up into spores.

ZoogUea 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.

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.

Several large spirilla and vibrio live in bog and rain-water,
but our space does not suffice to describe them.

Bacterium Ureee.

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 Ureae. (Pasteur and Van Tieghain.)

Orirjin. — Decomposed urine and in the air.

Form. — Cocci, diplococci, and steptococci.

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
is reached.

Colonies on Plate. — On the surface like a drop of wax.

NON-PATHOGENIC BACTERIA. 83

Stab Cultures. — Looks like a very delicate thread along the
needle thrust.

Spirillum. Spirillum Rubrum. (Esmarch.)

Origin. — Body of a mouse dead with septicaemia.

.Form.— Spirals of variable length, long joints, tlagella 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 Roll 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.

Sarcina. 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. (Schroter.)

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

84 ESSENTIALS OF BACTERIOLOGY.

all on the surface, a few separated colonies following the needle
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.)

Orifjin. — Stomach of man and animals.

Form. — Colorless, oval cocci, in groups of eight and packets
of eight.

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.

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 the}' 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.)— Bayer and Da-
vaine, in 1850, first described this bacillus ; but Pasteur, and
later -Kbc/t, first gave it the importance it now has.

Synonyms. — Bacteridie du charbon (Fr. ), Milzbrand bacillus
(germ) ; bacillus of splenic fever, or malignant pustule.

Ori'jin.— In blood of anthrax-suffering animals.

PATHOGENIC BACTETUA. 85

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.

FIG. 43.

Anthrax bacilli in human blood (fuchsin staining), Zeiss 1-12 oil immersion.
No. 4 ocular taken from Vierordt.

Properties. — Liquefies gelatine ; immotile ; the spores are very
resisting, living twenty years.

Growth. — Grows rapidly, between 12° C. and 45° C., and re-
quires plenty of oxygen, not growing without it ; 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
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 fiaky masses
at the bottom.

Potato.— A. dry creamy layer, and when placed in brood-oven,
rich in spores.

86

ESSENTIALS OF BACTERIOLOGY

Varieties. Asporogenic. — By cultivation in gelatine, contain-
ing 1 to 1000 ac-carbolic, a variety develop that cannot produce
spores. Also involution farms, differing from the usual type.

FIG. 44.

FIG. 45.

Stab Cultures of Anthrax in Gelatine.

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 Gram's method very nicely.

Pathoyeneste.— When mice are inoculated with anthrax mate-
rial through a wound in the skin, they die in twenty-four hours

PATHOGENIC BACTERIA. 87

from an active septicaemia, the point of inoculation remaining
unchanged. The following appearances then present them-
selves : —

Peritoneum.— Covered with a gelatinous exudate.

Spleen. — Very much swollen, dark red, and friable.

Liver. — Parenchymatous degeneration.

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 inhaled, and then a
pneumonia is caused, the pulmonary cells containing the bacilli ;
when the spores are inhaled, a general infection occurs.

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 can also occur 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.

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.

88 ESSENTIALS OF BACTERIOLOGY.

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 tree in nature.

Bacillus Tuberculosis. (Koch.)

This very important bacillus was first described, demonstrated,
and cultivated by Koch, who made his investigations public on

FIG. 4G.

V

O

( >

s

s

^

X

Tubercle bacilli in sputum, carbol-fuchsin, and methylin blue. Zeiss 1:12 oil
immersion.

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

PATHOGENIC BACTERIA. 89

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.

Growt h.— 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 throe
weeks. It is facultative anaerobic. On gelatine it does not
form a growth.

Colonies on Mood 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 Ayar. — J$y adding four to six per cent, glycerine to
ordinary agar-peptone medium, Nocard and Roux 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.
The cotton plugs of the tubes are FIG. 47.

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

the ruga? of the stomach, and some- ^0

times looks like crumbs of bread moist-
ened. The impression or "Klatsch" preparation shows under

90

ESSENTIALS OF BACTERIOLOGY.

the microscope a thick curled-up centre around which threads
are wound in all directions. And these fine lines show the
bacilli in profusion.

FIG. 48.

S tf\ r~

,$\v^

$\

Klatsch preparation.

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
very good nurture ground.

Varieties. — Dixon, of Philadelphia, has obtained branched
forms of bacilli which he believes to be degenerated weakened
ones.

In Sputum.— Little granules arranged like streptococci, which

PATHOGENIC BACTERIA. 91

take the characteristic stain, and look as if the protoplasma had
been destroyed that enclosed them.

FIG. 49.

Growth on Agar.

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.

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-
np 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 ;
otherwise we let it stay, in cold solution until the next morning
the advantages of which will be later on described.

TJie Rapid Method.— (B. Frankel's method modified by Gab-
bet.) The principle is to combine with the contrast stain the

9 ESSENTIALS OF BACTERIOLOGY.

decolorizing agent ; but the preparations are not permanent ;
the method, however, is very useful.

Two solutions are required : one of Ziehli's carbol-fuchsin ;
the other Gabbet's acid methylin blue. (See No. X., on
page 33.)

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 live minutes (cold),
or two minutes in the hot, immediately then transferred to the
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.

Aiwtlwr Raitid Method. — This method possesses the advan-
tage of giving permanency to the preparations ; but the bacilli
are distorted and ugly crusts form.

Three dishes are required : —

The first contains nitric acid and water 1-4.

The second alcohol.

The third distilled water.

They are arranged one after the other in the above order.

Two staining solutions must be at hand, carboi-fuchsin and
watery methylin blue. The cover-glass containing the dried
sputum is passed three times through the flame, then covered
with a, few drops of carbol-fuchsin, and held in the forceps over
the flam?, so that the stain will boil upon the glass. With a
pipette the dye is constantly added and kept boiling for about
one minute. It is then decolorized by holding it in the first
dish until it appears brownish-black, then directly into the
second dish, when the alcohol peals off the red color in little
clouds, and it becomes nearly colorless ; about five seconds
suffice. Then it is placed in the third dish, the water washing
oft' the alcohol. If the color of the preparation is now deeper
than a slight pink, it is again dipped into the acid, alcohol, and
back into the water, careful not to hold it too long in the above
solutions. The contrast stain is now applied, a few drops of the
methylin blue solution left on cold for two minutes being suffi-
cient. The glass is now dried and mounted on the slide in

PATHOGENIC BACTERIA. 03

Canada balsam. Examined with oil-immersion. The tubercle
bacilli red, all else blue.

Slaw Method. — When perfect permanent preparations are
desired and the bacilli to be seen unaltered, the slow method is
to be preferred, and it is to be recommended whenever the time
allows. It consists simply in allowing the carbol-fuchsin to work
upon the preparation a number of hours. We usually place the
cover glass with the dried sputum and which has been drawn
through the flame three times, in a little dish containing enough
dye to allow the glass to be immersed. We do this about 5 or 6
o'clock P.M., and the next morning the preparation is ready
for decolorizing, the process being the same as described above,
viz., 25 % nitric acid, alcohol and water, and the contrast stain
methylin blue. We thus avoid the formation of ugly crusts,
the bacilli are not distorted, the specimen is permanent and
very clear.

Biederfs 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
(he 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.

Staining Bacillus Tuberculosis in Tissue (sections}. — The general
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 offuchsin or aniline

94 ESSENTIALS OF BACTERIOLOGY.

water fuchsin can be used, but the sections must remain in the
stain over night.

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.

Patliogenesis. — 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 shmcs 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 ihe ab-
sence of a tubercular process. It is their presence only that
warrants a positive declaration.

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.

M«n is infected as follows : —

Throutjh wounds.— Local tuberculosis.

Throwjk nutrition. — Milk of tuberculosis cows.

Phthisical patients swallowing their own sputum and causing
an intestinal tuberculosis.

Inhalation. — This is the most usual way, probably constitu-
ting the cause in ,9(t of the cases.

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 clanger can be avoided.

PATHOGENIC BACTERIA. 95

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 also
by nutrition.

Immunity. — No one can be said to be immune, though per-
sons who have been greatly weakened would offer less resistance
than health}'' individuals.

Products Of Tubercle Bacilli. The last year has developed
some wonderful facts in relation to this important deadly bacillus.
In 1889, Dr. Dixon, then Professor of Hygiene at the Univer-
sity of Pennsylvania, spoke of a method of curing tuberculosis
in guinea-pigs and with products obtained from the bacillus ;
not much was thought of this statement at the time.

In August, 1890, Koch, before the Medical Congress claimed
that he also had been able to cure tuberculosis in guinea-pigs,
and would be able to give some interesting facts later on. In
November he claimed that he had obtained reactions in man
similar to those in the guinea-pig, and believed that a cure was
at hand.

In the excitement which followed this statement, the greatest
hopes were raised and the impossible expected. In January,
1891, Koch made public the manner of preparing the lymph or
u Tuberculin" or u Kochin," as it was variously called : old cul-
tures of tubercle bacilli mixed with 60 per cent, glycerine and
filtered through a Chamberlain-Pasteur filter, the filtrate thus
obtained being a dark-brown liquid, sp. gr. somewhat higher than
water, an odor like "beef extract," a sweetish taste, not soluble
in alcohol; according to Jollas, containing 50 per cent, water,
and showing a strong Biuret reaction ; he thinks it therefore a
toxalhumen. 1 milligramme of the lymph is supposed to contain
but ysnffo milligramme of the active principle. Dixon's lymph
is obtained in a very similar manner, and no doubt contains the
same principle.

Dixon recommends instead of using the pure culture for
obtaining the lymph, the tuberculosis lung of calf, a portion of
which is treated with water and glycerine, and then filtered
through Chamberlain-Pasteur filter without pressure.

Manner of Usiwj Koch's Lymph.— One milligramme of the

96 ESSENTIALS OF BACTERIOLOGY.

Koch's lymph is injected under the skin of one suffering with
a tubercular process, and in a few hours to a few days, a rise of
temperature, tightness about the chest, and exaggerated cough-
ing spells take place, the symptoms varying in intensity;
usually a secondary rise occurs on the following day. The
dose has been gradually increased until the reactions subsided,
and GOO milligrammes have then been borne without any reaction.

On Lupus the process could be watched and was very char-
acteristic ; a peculiar redness after the first injection, and after
a few more injections scabs formed, and an apparent cure seemed
to be obtained, but relapses were common and but very few
authentic cures if any can now be had.

Koch believed that the tuberculosis tissue was rendered
necrotic by this toxic principle, making the soil unfit for the
bacilli which then perished or were expectorated.

Virchow dampened the excitement and ardor by showing a
great diffusion of fresh miliary tubercles in the bodies of persons
who had died and who had been treated with the lymph. Cool,
careful, and untiring study and time taken together will, we
trust, bring a happy solution and a genuine remedy.

Lepra Bacillus. ( H a n sen . )

Origin. — In 1880 Armauer Hansen declared, as the result of
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-Uffreduzzi have obtained growths upon
blood serum to which peptone and glycerine had been added.
The growth is very slow, requiring about eight days at a tem-
perature of 37° C.

Colonies. — Small grayish round spots, under microscope ap-
pearing like a close-netted spider web around a firm centre.

Stab Cultures. — Show a waxy-like growth along the needle track.
/.— They resist the decolorizing action of acids as the

PATHOGENIC BACTERIA. 97

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.

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
nodules contain the bacilli in large numbers.

Method of Infection. — Not yet determined ; the air, soil, water,
and food of leprous districts have been carefully examined with-
out result.

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.

In the same year Alvarey and Tavel and Matterstock found
a similar bacillus reacting in the same way to Lustgarten's color
method in normal secretions, especially in the smegrna of the
prepuce.

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.

Origin. — In the cells of syphilitic tissue, in the secretion of
syphilitic ulcers, and in the smegma of the prepuce and vulva.

Form. — Small slender rods similar in appearance to tubercle
bacilli, sometimes swelled at the ends and curved S-shaped.

Colorless oval spaces also present, which Lustgarten calls
spores.

Growth. — As before mentioned, they have not yet been culti-
vated.

Staining. — Lustgarten's method : —

1. Aniline water gentian violet, 12 to 24 hours, and then 2
hours longer in brood oven.

98 ESSENTIALS OF B ACTEK IOLOG Y.

2. Rinsed in alcohol. 2 to 3 minutes.

3. Aqueous solution potass, permang. (1£ per cent.). 10
seconds.

4. Aqueous solution of sulphuric acid. 2 seconds.

5. Aq. destil. to wash.

Numbers three, four, and five repeated, until the section is
colorless. Then alcohol, oil of cloves, and Canada balsam as
usual.

De GiacornVs method:—

1. Aniline water fuchsia. 24 hours.

2. Rinse in dilute tr. ferri clilor. sol.

3. Decolorize in concentrated tr. ferri chlor.

4. Wash in alcohol, oil of cloves, Canada balsam, etc.

For cover-glass preparations, wash in water instead of alcohol.

Tubercle and lepru bacilli are colored by this method also,
but syphilis bacilli become decolorized if washed with acids.

Puthogenests. — No pathological actions have yet been definitely
proven. They are found in greater quantities the younger the
infection is.

Bacillus of Glanders. (Bacillus Mallei, Loffler-Shiitz.)

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 present.

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-agur
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 glycerine-agar and blood serum small
transparent drops of whitish or grayish color, which soon
coalesce to form a broad band.

PATHOGENIC BACTERIA. 99

Potato. — An amber-colored honey-like growth which gradually
turns red.

Staining. — Since the bacillus is very easily decolorized, some
special methods have been recommended.

Loffler^s.— (For cover-glass preparations.)

1. Alkaline methylin blue (Loffler's). a 5 minutes.

2. Acetic acid with a few drops of tropseoliii. 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. gij. — M.

The sections are kept in this 5 seconds.
Kuhne's method. Coverglaxs.

1. Warm carbol-blue 2 min.

2. Decolorized in weak sol. of muriatic acid (10 parts to 500).

3. Washed in water.
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 gtts. of turpentine. 5 min.

6. Turpentine, xylol, Canada balsam.

If contrast stain, add 5 gtts. of safranin (Bisniark-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.

Manner of Infection.— Glanders being a highly contagious dis-
ease, it requires but a slight wound to allow it to gain entrance.

100 ESSENTIALS OF BACTERIOLOGY.

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.

Bacillus of Diphtheria. (Klebs-Lbffler.)

Origin. — In diphtheritic membrane, by Loftier, 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.

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, between temperature
of 20° and 40° C. They are facultative anaerobic ; they grow
quite rapidly and profusely.

Colonies on Gelatine Plates.— At 24° C. little round colonies,
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 Loffler). — Blood serum 3 parts, and bouil-
lon 1 part ; the bouillon contains peptone, 1 per cent. ; chloride
of sodium, £ per cent. ; and dextrin, 1 per cent.

On this medium a very thick yellowish-white layer occurs on
the surface, and isolated colonies in the upper strata.

Staining. — Is not colored by Gram's method. Stained best
with Loffler's alkaline methylin-blue.

Pathogenesis. — 'By inoculation, animals, which naturally are
not subject to diphtheria, have had diphtheritic processes de-
velop at the site of infection ; hemorrhagic oedema then follows,
and death.

In rabbits 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-

PATHOGENIC BACTERIA. 101

ner, the diphtheria bacillus, then gains entrance and the disease
develops.

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.

Eoux 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.

Brieger and Frankel, through frequent precipitation of the
culture bouillon with acetic acid and alcohol, obtained a white,
amorphous body, which gave all the reactions of an albumen,
and being highly toxic, they gave it the name of tox-albumen.
It is soluble in water and decomposed by higher temperatures.

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.

Behring found several ways to make animals immune. One
method was to infect them with diphtheria and then inject tri-
chloriodine into them, which prevented them from dying, and
they were then immune.

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.

Streptococcus in Diphtheria. Streptococci have been found
quite constant in diphtheria, but they resemble the strepto-
coccus pyogenes, and have no specific action.

Bacillus of Typhoid or Enteric Fever. (E berth-Gaff ky.)

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 long 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

1C2 ESSENTIALS OF BACTERIOLOGY.

less deeply than some similar bacilli. Spores have not yet been
found ; small oval spaces appear in some of the degenerated
bacilli just at one end, but these bacilli are less resistant than
those without this so-called spore ; they do not liquefy gelatine.

FIG. 50. FIG. 51.

"

, i/ /•'

, Typhoid fever bacillus in pure cul- Bacillus of typhoid fever.

ture. 650 diameters.

Growth.— They are facultative anaerobic ; grow best at 37°
C., but can also develop at ordinary room temperature. All
nutrient media can be used as culture ground. They develop
chiefly on the surface, and very slowly.

Colonies on Gelatine Plates.— Two forms ; the ones near the
surface spread out like a leaf, transparent with bluish fluor-
escence. The deeper ones appear as whetstone crystals of
uric acid, 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.
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.

PATHOGENIC BACTERIA. 103

Milk. — They grow very well in milk without producing any
visible changes in its composition.

Carbolized-Gelatine.— Gelatine which has added to it j^ per
cent, carbolic acid will allow the typhoid bacillus to develop,
other similar bacilli being destroyed.

Staining. — Colored with the ordinary aniline dyes, when they
are warmed ; since they are easily decolorized, acids should be
avoided.

Grain's method is not applicable. Tissue sections stained as
follows : —

Alkaline 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
faces 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, }7ellow,
pasty layer is formed thereon.

In Water. Bacilli have been found which also resemble
typhoid bacilli, and one must be very careful 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 gtts. 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

104 ESSENTIALS OF BACTERIOLOGY.

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.

Patfiogenesis. — Lower animals have not 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.

Products. — Brieger found a ptomaine in the cultures which he
named typhotoxin with the formula C9H17NO2. 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.

Bacillus Neapolitanus. (Emmerich.)

Origin. — During the cholera epidemic in Naples, in 1884,
Emmerich found this bacillus in the blood and intestinal dis-
charges of cholera-suffering patients. He supposed it to be the
real cause of cholera ; but since then it has been shown to he
nothing more than the Faeces bacillus which Brieger described,
and which is found in faeces of healthy persons, in the air and
various putrefactive processes.

Form.— Very much like the typhoid bacillus, short rods with
rounded ends with oval spaces in them as the typhoid.

Properties. — Immobile, differing thus markedly from typhoid.
Do not liquefy gelatine.

Growth. — They are facultative anaerobic ; they grow more
rapidly than the typhoid, and endure cold and heat better than
they do.

PATHOGENIC BACTERIA.

105

Colonies. — They are exactly the same as typhoid — the same
whetstone-shaped deep ones and the leaf-shaped surface ones.

Potato. — A thick yellow-brown pasty layer is formed instead
of the transparent almost invisible growth of the typhoid
bacil'us.

Staining. — Do not take Gram. Fuchsin stains them easily.

Pathogenesis. — When large quantities injected into guinea-
pigs, they die at times, sometimes with intestinal symptoms,
sometimes without.

CHAPTER III.

FIG. 52.

PATHOGENIC BACTERIA — CONTINUED.

Spirillum Cholerae. (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
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
is broad, and possesses at each end a
flagella.

Properties. — They are very motile ;

liquefy gelatine. They are easily affected by heat and dryness.
Spores have not been found, though some (Hiippe) claim arthro-
spores.

Comma bacillus, pure cul-
ture. 600 diameters.

106

ESSENTIALS OF B A CTERIOLOG! Y.

FIG. 53.

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.

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-
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 well
and grow in sterilized milk
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.

Staining. — They are colored well with watery aniline solu-

Cholera dejections upon a damp sheet, a.-
Formed bacilli. 600 diameters. (Koch.)

PATHOGENIC BACTERIA. 107

tions. The flagella can be well seen by staining according to the
flagella stain.

Pathoyenesis. — Experiment animals are not subject to cholera
Asiatica, but by overcoming two obstacles Koch has produced
choleraic symptoms in guinea-pigs. Nicati and Rietsch pre-
vented peristalsis and avoided the acidity of the stomach juices
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.

Comma bacillus in mucus, from a case of Asiatic cholera.

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-

108 ESSENTIALS OF BACTERIOLOGY.

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.

Products. — " Cholera red." When chemically pure nitric or
sulphuric acid is added to nutrient peptone cultures of the
cholera bacillus a rose-red color is produced. This will riot 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.

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 spi-
FlG- 55' ^ rillum of cholera ; it differs from

£& it, however, in many ways, and
^ >vS'"/5* nas been found in healthy per-

£ ' *

Jfife Form.— Somewhat thicker than

^';;'oV the cholera vibrio, otherwise

VMr"' about the same form ; it forms

^' the long spirilla less often. Has

Flagella.

Spirillum Finkleri. 700 diameters. _. . ...

(FiQgge.) Properties.— It is very motile.

Liquefies gelatine in a short time.

Groicth. — It grows quickly at ordinary room temperature. It
is facultative aerobic.

Colonies o?i Gelatine PJates.— Round, finely granular colonies,
which in twenty-four hours are ten times as large as the cholera
colonies, and in forty-eight hours the whole plate is liquefied,

PATHOGENIC BACTERIA.

109

FIG. 56.

it being then impossible to distinguish any separate colonies.
The microscopic appearances in no way resemble the cholera
colony.

Stab Cultures. — The gelatine is liquefied
from above downwards, like a stocking in
appearance, and in three days 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.

Patlicgenesis. — Foic man it has no spe-
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. (Den eke.)

Origin. — In 1885 Deneke found in old
cheese a spirillum very similar in appear-
ance to the cholera spirillum.

Form. — The same as the cholera vibrio.

Properties. — Very motile, liquefy gela-
tine.

Growth. — They grow quicker than the
cholera, and slower than the Finklcr ; they are also facultative
aerobic.

Colonies.— They at first resemble cholera colonies ; they have,
however, a yellow-green iridescence, and are somewhat more
irregular ; also grow more rapidly.

Slab Cultures.— A thick line along the needle-track and the
yellow colonies forming at the bottom, on the surface a bubble
of air similar to the cholera. The gelatine is all liquid in two
weeks.

Potato. — At brood-heat a thin yellow membrane, but not
always constant.

Staining, as cholera bacillus.

Stab Culture. (Finklcr-
Prior.)

110 ESSENTIALS OF BACTERIOLOGY.

PathcHjenesis.— 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
septicccmia." 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
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.

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.

PATHOGENIC BACTERIA. Ill

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 now called it " Diplococcus Pneumonia," and
believed it to be the real cause of pneumonia. It has been found
in many other serous inflammations, and also in the mouth of
healthy persons.

Streptococcus pyogenes and staphylococcus pyogenes aureus have
been found in some cases.

FIG. 57.

Pneumo-bacillus of Friedlander, with capsule.

Pneumo-bacillus (Pneumococeua). (Friedlander.)

Origin. — In the lung of a croupous-pncumonia person, by
Friedlander, in 1882.

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-

112

ESSENTIALS OF BACTERIOLOGY.

Fro. 58.

ing then as little buttons, with a porcelain-like shimmer, the
edges smooth.

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. 58. 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-
nia. Stab Culture.
(Nail Culture.)

cone. ale. gent, violet, 50.0

1. Stain in warm ^ aqua, 100.0

acetic acid, 10.

for 24 hours. M.

2. Rinse in one per cent, acetic acid.

3. Alcohol to dehydrate. Mount in balsam.

The capsule will be found stained a light bine, the bacillus a
deep blue.

Pathogentsis.— Animals are not affected unless the culture is
injected intrapleura.

Pneumobacilhis of Frankel. (A. Frankel and Weichselbaum.)
Synonyms. — Pneumococcus ; Diplococcus of Pneumonia ; Mi-

PATHOGENIC BACTERIA.

113

crococcus of sputum septicaemia ; Micrococcus Pasteuri ; Diplo-
coccus lanceolatus.

Origin. — A, Fraukel found it in the sputum of pneumonic
patients, thinking it at first to be the micrococcus of sputum
septicaemia ; 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. 59.

Bacillus of Pneumonia in Saliva. (After Biondl.)

Properties. —They are without self-movement ; do not liquefy
gelatine.

Growth.— Grow only at high temperature, 35° C. ; are facul-
tative anserobic. 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.
8

114 ESSENTIALS OF BACTERIOLOGY.

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.

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
Friedlande-r bacillus.

Pathogenesis. — Rabbits and guinea-pigs, if subcutaneously in-
jected, die in the course of a couple of days with septicaemia.
(0.1 c.cm. 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 pel-
cent, of croupous pneumonia they are found and usually only
during the existence of the u prime juice" sputum, i. e., the first
stage.

Fia. 60.

/ ,x

*
t

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 it, it

PATHOGENIC BACTERIA.

115

Fio. 61.

requiring some slight injury to allow this ever-present germ to
develop the disease.

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 Friedlauder.

Micrococcus Tetrageims. (Koch. Gaffky).

Origin. — Koch found this microbe in the cavity of a tubercu-
lous lung. Gaft'ky, in 1883, studied its patho-
genic actions and gave it the name it now
bears.

.Form. — Cocci which are gathered in the tis-
sues in groups of four, forming a square, a
tetrad. See Fig. 62. In artificial culture,
sometimes found in pairs. A capsule of light
gelatinous 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 porcelain-like ap-
pearance ; under low power they are seen very
finely granulated.

Stab Culture.— Small round separated colo-
nies along the needle-track, and on the sur-
face a button-like elevation, a form of "nail
culture." See Fig. 58.

Potato. — A thick slimy layer which can be
loosened in long shreds.

Staining. — Colored with the ordinary aniline
stains. Gram's method also applicable.

Pathogenesis. — White mice and guinea-pigs stab Culture,
die in a few days of septicaemia when injected Micro^8 tetra~

116 ESSENTIALS OF BACTERIOLOGY.

with the tetragenus cultures, and the mierococcus 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. Pfeitfer.

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 gela-
tine cultures.

Growth. — At ordinary temperatures, quite rapidly ; facultative
anserobin.

Gelatine Plates. — Oval points, and like a porcelain button on
the surface.

Stub 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 ; then care-
fully decolorizing with acetic acid, the capsules are seen rad or
light violet around the deeply-tinged bacillus. Gram's method
not applicable.

Patlwgenesis. — Subcutaneously injected in mice, they die in
48 hours. Rabbits die whon a large quantity is injected into
the circulation. The blood and juices have a peculiar stringy,
fibrinous consistence.

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 procer-ses, and
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 foreign bodies and chemicals,
and a discharge occurs in them even when every aseptic and

PATHOGENIC BACTERIA.

117

antiseptic precaution has been taken ; but such a discharge is
free from bacteria, and no more like pus than a benign growth
is like a malignant one.

Streptococcus Pyogenes. (Bosenbach.) 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, Kosenbach

FIG. 62.

Streptococcus pyogenes in section of skin.

described an identical coccus which has been found in nearly
all suppurative conditions.

Form. — Small cocci singly and in chain-like groups. Spores
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.

Stab 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.

118 ESSENTIALS OF BACTERIOLOGY.

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 eft'ect
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.

In erysipelas the cocci reside in the lymphatic glands and
ducts. They have not been found in the blood. In air, soil, and
putrefying matters they have been often discovered.

Staphylococcus Pyogenes Aurens. (Boscnbach.)

Origin. — Found very commonly in pus (80 per cent, of all sup-
purations), in air, water, and earth ; also in sputum of healthy
persons.

FIG. 63.

1. (48 hours.) Colonies of micrococcus pyogenes aureus. 2. (5 days.)

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.

PATHOGENIC BACTERIA.

119

FIG. Gl.

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 anuerobin.

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 yellow granular
mass, the gelatine being all liquid.

Stroke Culture on Agar. — The pigment dif-
fused over the surface where the growth is, in
moist masses.

Potato. — A thin white layer which gradu-
ally becomes yellow and gives out a doughy
smell.

Staining. — Very readily colored with ordi-
nary 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 become
plugged with masses of cocci, infarct occur in kidnej^ and liver,
and metastatic abscesses form in viscera and joints. Garre, by
rubbing the culture on his forearm, caused carbuncles to appear.

Fracturing a long bone in an animal and then injecting the
staphylococcus into a large vein, as the jugular, will produce
osteomyelitis. Becker isolated this microbe from several cases
of osteomyelitis, and thought it a specific germ, giving it the
name of 4' micrococcus of osteomyelitis. "

Suppuration is nearly always produced by this microbe, and
it is found in the majority of suppurative processes.

Stab culture. Micro-
coccus pyogenes
aureus.

120 ESSENTIALS OF BACTERIOLOGY.

Micrococcus Pyogenes Albus. (Kosenbach. ) Similar in every
respect to the pyogenes-aureus, except that it does not form a
pigment.

Micrococcus Pyogenes Citrens. (Passet.) This staphylo-
coccus liquefies gelatine 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. It was found in pus, but
gave no action in animals.

Micrococcus Cereus Flavus. (Passet.) A lemon-yellow
colored growth after a sbort time, otherwise not differing from
cereus albus.

Micrococcus Pyogenes Tenuis. (Rosenbach.)

Origin.— Found in the pus of large inclosed abscesses.

Form.— Cocci, without any especial arrangement.

Properties.— Not much studied.

Growth. — It was cultivated on agar, on which it formed in
clear thin colonies ; along the needle-track an opaque streak,
looking as if varnished over.

Bacillus Pyocyaneus. (Gessard.)

Synonyms. — Bacterium a3ruginosum, bacillus fluorescens.
(Schroter.) The bacillus of bluish-green pus.

Origin. — Found in 1882 in the green pus in pyocysemia.

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.

Growth. — Develops readily at ordinary temperature, growing
quickly and mostly on the surface ; it is an-obic. 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
shimmer forming on the surface, and the gelatine having a deep
fluorescence.

PATHOGENIC BACTERIA. 121

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
then 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 Gonorrhoea. Gonococms. (Neisser.) In 1879
Neisser demonstrated the presence of this
germ in the secretion of specific urethritis. 'IG' '

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 Seinmel (roll)-form.
Four to twelve such pairs are often found
together.

Properties. — No movement of their owrn.
Culture. — On gelatine-agar or potato they Gonococci in gon-
do not grow, and only upon human-blood orrhoeai pus. Ani-
serum have they given any semblance of a
growth. The temperature must be between
33° and 37° C., and the growth occurs very slowly and sparsely.

122 ESSENTIALS OF BACTERIOLOGY.

In three days a very thin, almost invisible, moist yellowish
growth, seeming to be composed of little drops.

Under low power small processes are seen shooting out from
the smooth border.

FIG. 66.

(Jonococci in pus.

It requires to be then transferred to fresh media, as it quickly
perishes.

Staining.— Colored easily with all ordinary aniline stains.

Gram's method is not ai>plicable, this being one of its main
diagnostic features.

The following method for coloring cover-glasses is recom-
mended 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 gonorocci nre 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 ; the}' are

PATHOGENIC BACTERIA. 123

distinguished from the gonococcus, in that they are colored with
Gram's method, whereas the micrococcus of gonorrhoea is not.
Therefore it is always necessary, after having first found these
peculiar-shaped microbes, to apply Gram's stain, and if they are
then not found one can safely say it is the gonococcus.

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 gonorrhoeal rheumatism
is said to be caused by the lodgment of the cocci in the joints.

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 extending its influence, having
gained entrance through the vagina. It is found chiefly in the
superficial layers of the mucous membrane.

Similar Microbes found in the Urethra and Vagina.

Micrococcus Citreus Conglomerates. (Bumm.) Very similar
to the gonococci in form, they are, however, easily cultivated,
and form yellow colonies which dissolve the gelatine and grow
quite rapidly \ the surface of the gelatine is at first moist and
shiny, but later on wrinkled. They are colored with Gram's
method, and have no special pathological action. They are 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.— Im motile ; do not liquefy gelatine.

Growth. — Very slow at ordinary temperature, but more rapid

124 ESSENTIALS OF 13 ACTEKIOLOG Y .

at brood-heat. The colonies are as 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.—Noue 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 with 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.

Secondly.— Not stained with Gram's method.

Thirdly. — lief using to grow readily upon gelatine.

All the similar bacteria being easily cultivated.

These characteristics, taken in tolo, 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. AVlu n
the germ so tested is found, the process can be called vj,< -iji
without a doubt.

Bacillus of Tetanus. (Nicolaicr-Kitasato.)

Origin.— Nicolaier found this bacillus in the pus of a wounH
in one who had died of tetanus, describing it in 1884.

Kitasato has since then been able to isolate and cultivate this
germ. (1889.)

PATHOGENIC BACTERIA.

125

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.

FIG. 67.

Bacillus of Tetanus with spores.

Properties. — Not 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°
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 flourishes.

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.

High 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

126

ESSENTIALS CF li ACTERIOLOGY.

the gelatine liquefies, and then the growth settles at the bottom
of the tube.

FIG. 68. FIG. 69.

Appearance of culture of bacillus
of tetanus after agitating the lique-
fied gelatine. (Frftnkel and Pfeif-
fer.)

Six days' culture of bacillus
of tetanus in gelatine (deep
stab). (Frftnkel and Pfeiffer.

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.

PATHOGENIC BACTERIA. 127

Bouillon. — Adding glucose to the bouillon gives a medium in
which an abundant growth occurs.

Staining.— All the ordinary stains, Grain'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.

Four ptomaines among them : tetanin, tetanotoxin, and spas-
motoxin ; also a toxalbumen.

Immunity. — Kitasato, by inoculation of sterilized cultures,
has been able to cause immunity from the effects of virulent
bacilli.

Habitat. — The bacillus is present in garden earth, in manure ;
and even from mortar it has been isolated.

The earth of special districts seems to contain the bacilli in
greater quantities than in others.

Bacillus (Edematis Maligni. (Koch.)

Vibrion Septique. — ( Pasteur. )

Origin. — In garden earth, found lately also in man, in severe
wounds when gangrene with osdema had developed. Identical
with the bacillus found in Pasteur's septicaemia.

Form. — Bods somewhat smaller than the anthrax bacilli, the
ends rounded very sharply. Long threads are formed. Very
large spores which cause the rods. to become spindle- or drum-
stick-shaped.

Properties. — Very motile ; liquefy gelatine ; do not produce
any foul gaseous products in the body.

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
glancing colonies with fluid contents, under low power, a mass

128

ESSENTIALS OF B A CTERIOLOd Y .

of motile threads in the centre, and at the edges a wreath-like
border.

FIG. 70.

FIG. 71.

Cultures in agar of malignant
Oedema, after 24 hours, at 37° C.
(Frftnkel and Pfeiffer.)

Culture in gelatine of malig-
nant (Edema. (Frftnkel and
Pfeiffer.)

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.

PATHOGENIC BACTERIA. 129

Agar. — The gases develop more strongly in this medium, and
the odor is more prominent.

Guinea-Pig Bouillon. — In an atmosphere of hydrogen cloud-
ing of the entire culture medium without any flocculent pre-
cipitate until third day.

Staining. — Arc 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 covered and saturated with a clear red
serous exudate free from smell. This contains 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. 6., in the serous coverings of the different organs ;
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
bod}'. 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.— IK produced by injection of the sterilized cul-
tures, and also the filtered bloody serum of animals dead with
the disease.

Spirillum of Relapsing Fever. (Obermaier.)

Syn. Spirocheete Obermaieri.

Origin. — Found in the blood of recurrent fever patients,
described in 1873.

Form.— Long, wavy threads (16 to 40 ^ 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-

loO ESSENTIALS OF BACTERIOLOGY.

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.

Bacillus Malariae. (Klebs and Tommaci-Crudcli.)

Origin. — These two observers have found a germ present in
malarial persons in the blood, which produced an intermittent
fever in animals which had been inoculated with such blood.
They were also found in the soil of the Roman Campagna.
Very little importance is at present attached to this germ, but
at the time of its discovery, 1879, it was thought to be the cause
of malaria.

Hsematozoa of Malaria. Certain micro-organisms are found
in the blood of persons suffering from malaria, and have lately
been very carefully studied. They do not belong to bacteria,
being really of animal origin, among the protozoa; but because
they are described in the larger works on bacteria, it is neces-
sary that they be considered here.

Synonyms. IJamutomonas Malarice (Osier). Plasmodium Ma-
larice (Laveran).

Form. — Various shapes have been described, and whether
they are all of one micro-organism or several distinct organisms
is not yet definitely settled. In the cell they have been found
intra-corpuscular, and outside of the cell extra-corpuscular. Three
varieties of the intra-corpuscular have been noted.

1st. A kind without pigment, and having amoeboid movements,
occupying about one-third of the red corpuscle. It is probably
the first stage of the organism.

2d. Pigmented amvcboid variety. The pigment probably ob-
tained from the blood-corpuscle, which is faded in color ; more
than one may be present in the same corpuscle.

3d. The pigment set free in grains, or surrounded by large
homogeneous bodies which constantly change their outline.

The extra-corpuscular bodies present several peculiar forms,
and are supposed to be derived from the intra-corpuscular ones.

1st. Tlie scmilunar bodies rf Laveran ; spherical and crescent-

PATHOGENIC BACTERIA.

131

shaped and motionless, containing within them little particles of
pigment, arranged in the centre and having movement. These
groups of pigment are at times found free in the serum.

FIG. 72.

.0

0

0

Extra-corpuscular.

Intra-corpuscular.
VARIOUS FORMS OF PLASMODIA.

2d. Finely granular masses of protoplasm, which assume vari-
ous flower-like shapes, usually in the form of a rosette pigment
in the centre ; at the end of a paroxysm it falls to pieces, the
pigment being then set free.

3d. Oml bodies nearly the size of a red corpuscle, with long
motile flagella (Carter).

Cultivation of these organisms has not yet been attained.

Staining and Examination of Blood. —Take the blood of a per
son subject to malarial fevers, just before a paroxysm. Having
first carefully cleansed the finger, a ligature is applied, arid the

132 ESSENTIALS OF BACTERIOLOGY.

drop of blood drawn with a needle, brought on a well-cleaned
cover-glass, and immediately covered with a second cover-
glass. This is now examined with a strong objective (dry sys-
tem) by day-light.

If, now, a stained preparation is wanted, the cover-glasses are
slid apart, passed three times through the flame, and a concen-
trated solution of methylin-blue left on for a few minutes.

Still better is it to allow a drop of methyliu-blue solution in a
little ascitic fluid to flow slowly on the cover-glass before the blood
has become dry. The finer structure will then be more plainly
brought out. Laveran recommends the strong objective of the
dry system for examining.

Pathogenesis. — These organisms have been found only in
malarial diseases, and they have been constantly found. Malarial
paroxysms have been produced in a healthy person by inocu-
lation of blood containing such organisms. They disappear
under the use of quinine.

Golgi finds certain types constant in tertian, and others again
peculiar to quartan.

Some, however, hold all these various forms as nothing more
than changed blood-corpuscles. The impossibility of obtaining
a pure culture leaves the question still in doubt.

Grassi and Fektti claim to have produced in sparrows and
man, by injection of the blood of malarial persons, malarial
fever, and found the specific parasites for the different forms.
The amoeboid or intra-corpuscular give rise to the typical inter-
mittent fevers. In the crescent-shaped extra-corpuscular, pro-
ducing the dumb ague or irregular fever, three different amoebae
were found.

Hsemoeba praecox produces the quotidians.
" vivax u u tertians,

malaria? " " quartans.

They place them with the Rhizopoda.

BACTERIA PATHOGENIC FOR ANIMALS. 133

CHAPTER IV.

BACTERIA PATHOGENIC FOR ANIMALS BUT NOT FOR MAN.

Bacillus of Symptomatic Anthrax. (Bellinger 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.

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 growth first 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

134 ESSENTIALS OF BACTERIOLOGY.

of infection. The bacilli are found in great numbers in the
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. " Rauschbrand" is the German name; " Charbon synip-
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 add 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 avitidus. Bacillus of fowl septicaemia.

Origin. — In 1879 Perroncito observed this
FIG. 73. cocci-like bacillus in diseases of chickens, and

*> *° Pasteur, in 18SO, 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
Chicken cholera auout twice as long as it is broad, the ends
bacillus. slightly rounded. The centre is very slightly

influenced by the aniline colors, the poles
easily, so that in stained specimens the bacillus looks like a
dumb-bell or a figure-of-eight. (Microbe en huit.)

Properties. — They do not possess self-movement ; do not
liquefy gelatine.

BACTERIA PATHOGENIC FOR ANIMALS. 135

Growth. — Occurs at ordinary temperature, requiring oxygen
for development. It grows very slowly.

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. — Methyliu 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 fteces becoming mixed with
the food.

136 ESSENTIALS OF BACTERIOLOGY.

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
from it and each other but very little. They have been described
by various observers and found in different diseases.

1st. Bacillus of American Swine Plague. Syn. Hog cholera;
infectious pneumonia. (Billings, Detmers, and Salmon.)

2d. Bacillus of Swedish-Danish Swine Plague. Swine </////<-
theria. (Selander. )

3d. Bacillus of Swine Plague. (Loffler, Scliutz.)

4th. Bacillus of Texas Fever. Cattle plague. (Billings.)

5th. Bacillus of the Steer Plague. (Oreste and Armanni.)

6th. Bacillus of the French Swine Plague. (Coruil and
Chantemesse.)

7th. Bacillus Cuniculicida. Rabbit septicaemia. (Gaffky.)

8th. Bacillus of Duck Cholera. (Cornil and Toupet;)

Differences. The various bacilli found in swine plague differ
from each other very slightly.

They are but little active in/ourfs, very fatal in pigs and mice.

In pigs, hemorrhages occur in all the viscera, the mucous mem-
brane of the intestines being ulcerated and necrosed.

The cultures of the Swedish swine plague— No. 2 of the above
list— resemble the typhoid bacillus. The form and cultures of
the others are hardly to be distinguished from those of the
chicken cholera.

The bacillus of duck cholera (No. 8) is harmless for chickens,
but fatal to ducks ; otherwise identical with chicken cholera.

Bacillus of Rabbit Septicaemia. Similar in action to the
chicken cholera.

The other properties are identical with those of the microbe
of chicken cholera.

Bacillus of Erysipelas of Swine. (Loffler, Schiitz. ) Sch m ;,„ -
rollaufbacillus (German). Rouget du pore (French).

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 /*, looking at first like little needle-like
crystals. Spores have not been found.

BACTERIA PATHOGENIC FOR ANIMALS.

137

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 diarrhoaa 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.

FIG. 74.

Bacillus Murisepticus.

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.

138 ESSENTIALS OF 13 ACf ERIOLOG Y .

Bacilli are found in the blood and all the viscera.

One attack, if withstood, protects against succeeding ones.

Immunity. — Has also been attained by injecting vaccines of
t\vo 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.

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
"mat depis."

Form. — Very small cocci seldom in chains.

Properties, immotile ; liquefying gelatine.

Growth.— Growth occurs best between 20° and 37° C., is very
rapid, and irrespective of ox}'gen.

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 Cwture. — 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.

PatJwgenesis. — If a pure culture is injected into the mammary
gland of sheep, a " mat 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.

BACTERIA PATHOGENIC FOR ANIMALS. 139

In rabbits abscesses at the point of infection, but no general
affection.

Bacillus Alvei. (Cheshire and Cheyne.) Bacillus melittoph-
tharus. (Colm.)

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.

Growth. — Grows best between 20° C. and 37° C., very slowly ;
cerobic.

Gelatine Plates. — Small grooves are slowly formed, which unite
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-bubbles 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 larvse, 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 Amylivorus. (Bun-ill.)

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

140 ESSENTIALS OF BACTERIOLOGY.

microbes which he grouped under the common name of Protons,
which have the putrefying properties ascribed to B. Termo.

Proteus Vulgaris.

Origin. — In putrid animal matter, in meconium 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
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.

Fungi exist without chloropliyl.

Sacchuromycetes or Yeasts increase through budding ; the
spores 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 Cerevisiae. (Torula Cerevisice.) This is the
ordinary beer yeast.

Form. — Kound 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

(141)

142 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 "mould"
on wine, preserves, and "sour krout."

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.

Ayar 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 occurs.

Growth.— Not liquefying ; snow-white colonies on gelatine
plates.

Stab CuHure. — 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.—ln man the parasitic thrush, or " white mouth,"
is caused by this fungus. In the white patches the spores and
filaments of this microbe can be found. Rabbits receiving an

YEASTS AND MOULDS. 143

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 : Pendllium, Mucor, and
Aspergillus.

Pencillium Glaucum.

Origin. — The most widely distributed of all moulds, found
wherever moulds can exist.

Form. — From the mycelium, hyphens spring which divide into
basidia (branches), from which tiny filaments arise (sterygmata),
arranged like a brush or tuft. On each sterygma 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 pencillium 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-
lar head, a spore-bulb, or Sporangium.. The spore-bulb is par-
titioned onMnto 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
" ccilumella" behind.

Growth. — Takes place at higher temperatures on acid media.

It is not Pathogenic.

Achorion Schonleinii.

Tricophyton 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 Schb'nleinii, was
discovered by Schonlein in 1839, in Favus, and is now known as
the direct cause of this skin disease.

144 APPENDIX.

Origin. — Found in the scaly crusts.

Form. — Similar to oidiuiu lactis..

Growth. — Is very sparse. On gelatine round white masses
inclosed by a zone of liquefied gelatine.

In milk it is destroyed.

Pathoyenesis. — Causes favus in man.

Tricophyton Tonsurans. Found, in 1854, by Bazin, in Tinea.

Form. — Similar to the achorion or i'avus 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.

Patlwyenesis. — Herpes tonsurans and the various tinea3 are
produced by this fungus.

Microsporon Furfur. Found in tinea versicolor, almost iden-
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 hyphen has formed upon its further end a bulb,
from which pear-shaped sterygmata arise and bear upon their
ends the conidia or spores.

Growth. — Best upon fruit juices. Non-pathoyeni\ The mould
is green. AsperyUhtifiavua 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 sealing-wax around the edges. Yeasts take methy-
line-blue stain very well.

Ray Fungus. A division containing the actinomyces. (Bol-
lenger and Israel.)

Oriyin.— In actinomycosis of man and cattle, in the growth.

RAY FUNGUS. 145

Form. — In the pus or scrapings, little yellow grains about
the size of a pin's head are seen by the naked eye. When one of
these points is flattened out between the cover-glass and slide
and placed under microscope (200 x), aster-shaped figures will

Kir,. 75.

Actinomyces.

be seen, the centre thick, radiating from it, little hyphens, which
become thicker and rounder at their peripheral end. These
bottle-shaped hyphens are supposed to be the spore-bearing
organs. Some of these may have separated from the main body
and lie loose or attached to it by a very thin filament.

Growth.— Develops only at brood heat and by exclusion of
oxygen.

In Agar. — After several weeks a yellowish growth was ob-
tained, but this consisted mainly of mycelia, the club-shaped or
conical rays not forming.

In eggs a growth developed when the method of Hueppe was
carried out.

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-
10

146 APPENDIX.

en ing, 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 : —

Ziebel's carbol-fuchsin, ten minutes. Rinse in water.

Cone, alcohol sol. of picric acid, li ve minutes. Rinse in water.

Alcohol, 50 per cent. , fifteen minutes. Alcohol absolute, clove
oil, balsam.

The rays stained red, the tissue yellow.

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.

147

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. 76.

cylinder. The eeroscqpe, 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

148

APPENDIX.

FIG. 77.

outer end. The aspirator consists of two ordinary wash-bottlrs
connected with each other by a rubber tube, C. 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
through a sieve with meshes 0.25 millimetres 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. 149

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, t. e., the one nearest the aspirator.

Varieties Found in Air. The only pathogenic ones found with
any constancy are the staphylo-coccus 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. Sarcina,
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.

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 2 germs, if one of these two be a typhoid bacil-
lus. 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

150 APPENDIX.

germ can be proven to be present in tbat particular water, tbe
epidemic may be pa>t.

Purity of Waters. Tbe purest water we have is tbe natural
spring water— water that has slowly filtered its way through
various layers of gravel and sand and cornes finally clear and
sparkling from tbe 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
tbat will insure this, since tbe 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
using chiefly tbe 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 materinls 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 ; tbe number
of germs in a cubic centimetre was 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. 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.

AIR, SOIL, AND WATER. 151

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, 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 pas-
sage in the form of an aspirator or air-pump. (See Fig. 37.)

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.

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 should, or rather
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, which, as before stated,
should not be later than an hour after collection. The sample
is placed in a sterilized 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 in it 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 thoroughly 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 to three days
examined. If the germs are equally divided, there should be

152 APPENDIX.

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.

To count the colonies which develop upon the plates, a spe-
cial apparatus has been designed, for, unaided, the eye cannot
see them all.

Wolfhugel'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.

Varieties Found. The usual kinds found are non-pathogenic,
but, as is well known, typhoid and cholera are principally spread
through drinking water, and many other germs may and do
find their way into the water. Many of the common varieties
give rise to fluorescence, or produce pigment.

Eisenberg gives 100 different varieties as ordinarily found.
As mentioned before, 2 bacteria to a cubic centimeter, one of
them typhoid, give more danger to a water than thousands of
non-pathogenic one?. When, however, more than 200 bacteria
to the c.c. are found, such a water ought not to he considered
potable. Distilled water forms often a good medium for some
bacteria.

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.

AIR, SOIL, AND WATER. 153

Frankel's Borer. Eriinkel has devised a small apparatus in
the form of a borer, which contains near its lower end a small
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 by 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.

CONCLUSION. In tracing thus briefly the characteristics of
the more important bacteria, and the various methods used in
studying them, we are conscious of the very superficial manner
in which this has been done. We excuse ourselves, however,
on the ground that this work is but a wedge with which to enter
upon the study, or, for those who do not care to proceed further,
an eminence from which a fair view of the ground can be ob-
tained. In this, its humble mission, we trust it may meet with
success.

INDEX.

ABBE'S condenser, 26
Achorion Schouleinii, 143
Actinomyces, 144
Actinouiycosis, 145
M robins, 24

facultative, 24

obligative, 24
Agar-agar, 49

bouillon, 49

glycerine, 50
Air, examination of, 146
Anaerobins, 24

facultative, 24

obligative, 24
Aniline dyes, 30

oil, 38

oil water, 31

Animals for experiment, 68
Anthrax, 84
Arthrospores, 18
Asbestos filter, 150
Aspergillus fumigatus, 144

glaucus, 144
Asporogenic bacteria, 22
Attenuation, 64
Autoclave of Chamberland, 41

BACILLUS acidi lactici, 76
alvei, 139
amylobacter, 77
anthracis, 84
avicipis, 134
butyricus, 77
capsule, 116
coeruleus, 79
comma, 105
cuniculicida, 136
erythrosporus, 80
faeces, 103
fluorescens, 120

liquefaciens, 80
hay, 75

Bacillus, indicus, 73
Klebs-Loffler, 100
lactis cyanogenus, 78

erythogenes, 78
lepra, 96
malariae, 130
mallei, 98
megaterium, 74
melittoptharus, 139
mesentericus vulgatus, 73
Milzbrand, 84
murisepticus, 138
mycoides, 74
Neapolitanus, 103, 104
cedernatus maligni, 127
of American swine plague, 136
of anthrax, 84
of bluish-green pus, 120
of chicken cholera, 134
of diphtheria, 100
of duck cholera. 136
of fowl septicaemia, 134
of French swine plague, 136
of glanders, 98
of mouse septicaemia, 138
of rhinoscleroma, 115
of steer plague, 136
of Swedish-Danish plague, 136
of swine erysipelas, 136
of swine plague, 136
of symptomatic anthrax, 133
of syphilis, 97
of tetanus, 124
of typhoid fever, 101
phosphorescens gelidus, 81

indicus, 80

indigenus, 81
pneumo-, of Frankel, 112

of Friedlander, 111
potato, 73
prodigiosus, 72
pyocvaneus, 120

0,121

(155)

156

INDEX.

Bacillus, ramosus, 74 '

root, 74

smegma, 97

spinous, 76

subtilis, 75

tuberculosis, 88

violaceus, 79
Bacteria, 17

antagonism of, 67

asporogenic, 22

as remedial agents, 67

desmo-, 17

effect on body, 62

fluorescent, 80

in air, 146

in milk, 76

in water, 79, 149

infectious, 64

influence upon — of age, 25

of electricity, 24
of light, 24
of oxygen, 24
of temperature, 23

life of, 23

micro-, 17

non-pathogenic, 25, 64

of hemorrhagic septicaemia, 136

of pneumonia, 110

origin of, 23

pathogenic, 25, 64

phosphorescent, 80

similar to cholera bacillus, 108

sphere-, 17

spiro-, 17

staining of, 30

structure of, 18

toxic, 64

unstained, 27

vital actions of, 24
Bacteridie du charbon, 84
Bacterium acidi lactic! , 77

aeruginosam, 120

Balticum, 81

Fischeri, 81

Pflugeri, 81

syncanum, 78

termo, 139

ureae, 82

zopfi, 75

Beggiatoa alba, 82
Benches for glass plates, 56
Biedert's method of collecting bac-
teria, 93
Black-leg, 133
Blight, 139

Blood serum as media, 50

theory, 65

Bouillon, agar, 49
gelatine, 47
preparation of, 44
sterilization of, 44

Bread mash, 47

Brood-oven, 50

Brownian movements, 19

CATTLE plague, 136
Cell contents, 18
Cell wall, 18
Cellular theory, 18, 66
Charbon symptomatique, 134
Charcoal filter, 150
Chemical theory, 65
Cholera, 105

red, 108

Cladothrix dichotoma, 82
Classification, 17
Clostridium, 21

butyricum, 77
Cohn's system, 17
Cotton plugs, 43
Cover-glass specimens, 34
Crenothrix, 81

KUhuiana, 82
Cultivation, 39

artificial, 39

methods of, 39

of anaerobins, 60
Cultures, appearances of, 58

egg, 52

filtration of, 62

glass plate, 52

glass-slide, 52

potato, 45

rolled, 57

test-tube, 52

DE BARY'S system, 18
Decolorants, 31
Diphtheria, 101

Diplococcus albicans amplus, 123
tardissimus, 123
lanceolatus, 113
of pneumonia, 111, 113
Disease, cure of bacteria, 67
Disinfectants, 40

heat as, 40
Drying specimens, 34

INDEX.

157

EUDOSPORUS, 18
Enteric fever, 101
Esmarch's method, 60

tubes, 57
Experiments on animals, 68

FERMENTATION, 25
I Fevers, 143
Filter, asbestos, 150

Chamberland, 63

charcoal, 150

cotton, 50

hot water, 63

Pasteur, 63

sand, 148

sponge, 150
Filtration of cultures, 62

of water, 150
Fishing, 59
Fission-fungi, 17
Flagella, 19

staining for, 38
Fluorescence, 25
Foul-brood, 139
Frankel's borer, 153

method for anrerobins, 61

stain for tubercle, 91
Fuchsin, carbol, 31,33
Fungi, 141
Fungus, ray, 144

thrush, 142

flABBETT'S stain, 33
IJ Gas formation, 25
Gelatine, 47

bouillon, 47

clouding of, 48

paste, 35

sterilization of, 49
Gelatinous membrane, 18
Germination, 21
Gonococcus, 121
Gonorrhoea, 121
Gram's stain, 33
Guinea-pigs, 68

H^EMATOMONAS malarise, 130
Haematozoa of malaria, 130
Hanging drop, 29
Heat, 40
dry, 40

leat, moist, 41
iemorrhagic septicaemia, 136
rlerpes tonsurans, 144
ilesse's method for air, 146

for anaerobins, 60
log cholera, 136
Homogeneous lens, 26
Hot-air oven, 40
Hot-water filter, 63
Huppe's method, 61

IMMERSION lens, 26
i. Immunity, 66

acquired, 67

artificial, 67

natural, 66
Incubators, 50
Inoculation, 69

cutaneous, 69

in eye, 70

intra-duodenal, 70

intra-peritoneal, 69

intra-tracheal, 70

intra-venous, 69

of cerebral membranes, 70

subcutaneous, 69
Iodine, 31
Iris blender, 27
Iron box for plates, 55

KLATSCH preparations, 59
Kochin, 95
Koch's lymph, 95
rules, 68
stain, 32
steam-chest, 41
Kiihne's stain, 33
method, 38

LACTIC acid, 134
Laveran, semilunar bodies of,

130

Leprosy, 96
Liborius's method, 60
Liquefaction of media, 25
Locomotion, 18
Loffler's alkaline stain, 32
blood serum, 100
mordant, 33
Lupus, 96
Lustgarten's method, 97

158

INDEX.

MALARIA, 130
Material from animals, 71
Media, fluid, 44

nutrient, 44

solid, 45

transparent, 47
Metschnikoff's theory, 66
Microbe en huit, 134
Micrococci similar to gonococcus,

123
Micrococcus amylivorus, 139

cerus albus, 120
flavus, 120

cholera gallinarum, 124

citreus conglomeratus, 123

Indicus, 73

of gonorrhoea, 121

of mal de pis, 138

of osteomyelitis, 119

of sputum septicaemia, 111

Pasteuri, 113

pyogenes aureus, 118
citreus, 120

subflavus, 124

tenuis, 120

tetragenus, 115

ureae, 82
Micro-organisms of suppuration,

116

Microscope, 26
Microsporon furfur, 143
Moist chamber, 45, 56
Mordants, 31
Moulds, 141, 143

examination of, 144
Mouse septicaemia, 138
Movements, vibratory, 19
Mucor mucedo, 143
Mycoprotein, 18

NAIL culture, 112
Nivellier apparatus, 55
Nutrient media, 44

OIDIUM, 142
albicans, 14?
lactis, 142

Odors In cultures, 25
(Edema malignans, 127
Oil immersion, 26
Oxidation, 24

PARASITES, 23
L facultative, 'J3
Pasteur filter, 63
Pencillium glaucum, 143
Petri's sand filter, 148

saucers, 57
Phosphorescence, 25
Pigmentation, 25
Plasmodium malaria?, 130
Platinum needles, 28
Pneumo-bacillus, 111
Potato cubes, 46

cultures, 45

inoculation of, 46

in test-tubes, 46

mash, 47

Products of tubercular bacilli, 95
Proteus, 140

mirabilis, 140

vulgaris, 140

zenkeri, 140
Ptomaines, 24, 62
Putrefaction, 25

RABBIT septicaemia, 136
Rausch brandy, 134
Reduction, 24
Relapsing fever, 129
Reproduction, 19
Rouge t du pore, 136

QACCHAROMYCES albicans, 141
ij cerevisia?, 141

mycoderma, 143

niger, 141

rosaceus, 141
Saprophytes, 23
Sarcina alba, 84

aurantica, 84

flava, 84

lutea, 83

rosea, 84

ventriculi, 84
Schizomycetes, 17
Schweinerotlauf, 136
Slides, concave, 29
Soil, examination of, 152
Solutions, composite, 31

formulae of, 32

stock, 30, 32

strong, 32

weak, 31

INDEX.

159

Soor, 142
Spasmotoxin, 127
Specimens, cover-glass, 34

cutting of, 36

drying of, 34

Klatsch, 59

permanent, 35
Spirillum, 17

choleras, 105

concentricum, 83

Finkleri, 108

of relapsing fever, 129

rubrurn, 83

tyrogenum, 109
Spirochaete obermaieri, 129
Spores, arthro-, 18, 22

contents of, 21

endo-, 18, 20

formation of, 18, 20

requisites for, 22

resistance of, 22

staining of, 37, 38 '
Stain, alkaline, 31, 32

alkaline aniline water, 33

Gabbet's, 33

Gram's, 33

Koch's, 32

Kuhne's, 33

Loffler's, 32

picro-carmine, 33

Ziehl-Nielsen, 33
Staining, Ernst's method of, 38

De Giacomi's, 98

general method of, 34

Gram's method of, 37

Kuhne's method of, 39

Loffler's method of, 99

Lustgarten's method of, 97

of flagella, 38

of spores, 37, 38

of sporogenic bodies, 38

of tissue sections, 36

rapid method for tubercle, 92

slow method of, for tubercle, 93

solutions, 30

special methods of, 36

Weigert's method of, 38
Staphylococcus, 18

pyogenes aureus, 118

Sterilization, 39

fractional, 42
Streptococci, 18

in diphtheria, 101
Streptococcus pyogenes, 117
Suppuration, 116
Swine diphtheria, 136

erysipelas, 136

plague, 136
Syphilis, 97
System of Cohn, 17

of De Bary, 18

rpEST-TUBES, 43
1 Tetanin, 127
Tetanis, 124
Tetanotoxin, 127
Thermo-regulator, 51
Thrush, 142
Tinea, 144

Toxalbumens, 62, 67
Toxines, 62

Tricophyton tonsurans, 143
Tuberculin, 67, 95
Tuberculosis, 88
Typhoid fever, 101
Typhotoxine, 104

VIBRIO, Finkler-Prior, 108
Metschnikoff, 110
Vibrion septique, 127

WATER, bacteria in, 77, 152
examination of, 149
Weigert's method of staining, 38
Wire cages, 43
Wolfhugel's apparatus, 152

YEASTS, 141
JL examination of, 144

F/IEHL'S solution, 33
Zj Zooloea, 18

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have been omitted. The sections devoted to optical principles and
the normal and abnormal refraction of the eye in large portion have
betn written by Dr. James Wallace, Chief of the Eye Dispensary of
the University Hospital. The chapter devoted to the application
of the shadow-test has been prepared by Dr. Edward Jackson. The
book will be suitably illustrated by a number of wood-cuts, many of
them from cases in the practice of the author, in addition to which
there will be several chromo-lithographs.

IN PREPARATION.

DISEASES OF WOMEN.

BY HENRY J. GARRIGUES, A.M., M.D.,

Professor of Obstetrics in the New York Post-Graduate Medical School and
Hospital ; Gynaecologist to St. Mark's Hospital in New York City ; Gynae-
cologist to the German Dispensary in the City of New York; Con-
sulting Obstetrician to the New York Infant Asylum; Obstetric
Surgeon to the New York Maternity Hospital ; Fellow of
the American Gynaecological Society ; Fellow of the
New York Academy of Medicine ; President of the
German Medical Society of the City of New
York, etc. etc.

It is the intention of the writer to provide a practical manual on
Gynaecology, for the use of students and practitioners, in as concise a
manner as is compatible with clearness.

Syllabus of Obstetrical Lectures

In the Medical Department, University of Pennsylvania,

BY RICHARD C. NORRIS, A.M., M.D.,

DEMONSTRATOR ON OBSTETRICS IN THE UNIVERSITY OF PENNSYLVANIA.

Price, Cloth, Interleaved for Notes . , , $2.00 Net,

The New York Medical Record of April 19, 1890, referring to this
book, says : " This modest little work is so far superior to others on
the same subject that we take pleasure in calling attention briefly to
its excellent features. Small as it is, it covers the subject thoroughly,
and will prove invaluable to both the student and the practitioner as
a means of fixing in a clear and concise form the knowledge derived
from a perusal of the larger text-books. The author deserves great
credit for the manner in which he has performed his work. He has
introduced a number of valuable hints which would only occur to one
who was himself an experienced teacher of obstetrics. The subject-
matter is clear, forcible, and modern. We are especially pleased with
the portion devoted to the practical duties of the accoucheur, care of
the child, etc. The paragraphs on antiseptics are admirable ; there
is no doubtful tone in the directions given. No details are regarded
as unimportant ; no minor matters omitted. We venture to say that
even the old practitioner will find useful hints in this direction which
he cannot afford to depise."

9

READY SHORTLY.

SAUNDERS'

Pocket Medical Formulary.

BY

WILLIAM M. POWELL, M.D.,

Attending Physician to the Mercer House for Invalid Women, at Atlantic

City, N. J. ; Late Physician to the Clinic for the Diseases of Children

in the Hospital of the University of Pennsylvania and St. Clement's

Hospital ; Instructor in Physical Diagnosis in the Medical

Department of the University of Pennsylvania, and

Chief of the Medical Clinic of the Philadelphia

Polyclinic.

Containing about 2000 Formulae, selected from several
hundreds of the best-known authorities.

A concise, clear, and correct record of the many
hundreds of famous formulae which are found scattered
through th°! works of the

Most imminent Physicians and Surgeons

of the world ; particularly helpful to the student and
young practitioner, as it gives him a taste for writing his
prescriptions in an elegant and correct manner, thus avoid-
ing incompatible and dangerous prescriptions. The use
of this work is to be recommended even to the older prac-
tioner, as through it he becomes acquainted with numerous
formulae which are not found in the text-books, but have
been collected from among the

Rising Generation of the Profession, College Professors, and

Hospital Physicians and Surgeons.

10

NOW READY.

NEW AND REVISED EDITIONS OF

SAUNDERS'

QUESTION COMPENDS,

Arranged in Question and Answer Form.

The Latest, Cheapest, and Best
ILLUSTRATED SERIES OF COMPENDS EVER ISSUED.

THE ADVANTAGES OF QUESTIONS AND
ANSWERS. — The usefulness of arranging the subjects in
the form of Questions and Answers will be apparent,
since the student, in reading the standard works, often is at
a loss to discover the important points to be remembered,
and is equally puzzled when he attempts to formulate ideas
as to the manner in which the Questions could be put
in the Examination-Room.

These small works, which can be conveniently carried in the pocket,
contain in a condensed form the teachings of the most popular
text books.

The authors are nearly all connected with the various colleges as
Demonstrators or Lecturers, and are therefore thoroughly conver-
sant, not only with the wants of the average student, but also with
the points that are absolutely necessary to be remembered in
the Examination-Room. These books are constantly in the hands
of their authors for revision, and are kept well up to the times, their
fast sale allowing them to be almost entirely rewritten whenever
necessary, instead of having to wait for the edition to be sold, as is
the case with an ordinary text-book.

11

No. 1.

ESSEITIALS OF PHYSIOLOGY,

H. A. HARE, M.D.,

Professor of Therapeutics and Materica Medica in the Jefferson Medical Col.

lege of Philadelphia; Physician to St. Agnes' Hospital and to the

Medical Dispensary of the Children's Hospital ; Laureate of

the Royal Academy of Medicine in Belgium, of the

Medical Society of London, etc. ; Secretary

of the Convention for the Revision of

the Pharmacopoeia, 1S90.

NUMEROUS ILLUSTRATIONS.

Third Edition, Revised and Enlarged.

Price, Cloth . . $1.00; Interleaved for Notes . . $1.25.

Unii-ersity Medical Magazine,
October, 1888.— " Dr. Hare has
admirably succeeded in gather-
ing together a series of Ques-
tions which are clearly put and
tersely answered."

Pacific Medical Jmirnal, Octo-
ber, 1889.—" Hare's Physiology
contains the essences of its sub-
ject. No better book lias ever
been produced, and every stu-
dent would do well to possess a
copy."

Times and Register, Philadel-
phia, October ft, 1889.—" In the
second edition of Hare's Physi-
ology all the moredifficult points
of the study of the nervous sys-
tem have been elucidated. As
the work now appears it cannot
fail to merit the appreciation of

Specimen of Illustrations. the overworked student."

Journal of the American Association, November 23, 1889. — " Hare's
Physiology — an excellent work ; admirably illustrated ; well calcu-
lated to lighten the task of the over-burdened undergraduate."

12

No. 2.

ESSENTIALS OF SURGERY.

CONTAINING, ALSO,

Venereal Diseases, Surgical Landmarks, Minor and Operative Su~-
gery, and a Complete Description, together with full Illustra-
tions, of the Handkerchief and Roller Bandage.

BY EDWARD MARTIN, A.M., M.D.,

Clinical Professor of Genito-Uriiiary Diseases, Instructor in Operative Sur-
gery, and Lecturer on Minor Surgery, University of Pennsylvania;
Surgeon to the Howard Hospital ; Assistant Surgeon to the
University Hospital, etc. etc.

PROFUSELY ILLUSTRATED.

FOURTH EDITION,

Considerably enlarged by an Appendix containing Ml directions
and prescriptions for the preparation of the various mate-
rials used in ANTISEPTIC SURGERY ; also sev-
eral hundred recipes covering the medical

treatment of surgical affections.
Price, Cloth, $1.00. Interleaved for Notes, $1.25.

Me.lical and Surgical Rejjorter,
February, 1889. — " Martin's Sur-
gery contains all necessary essen-
tials of modern surgery in a com-
paratively small space. Its style
is interesting and its illustrations
admirable."

University Medical Magazine,
January, 1889.— "Dr. Martin has
admirably succeeded in selecting
and retaining just what is neces-
sary for purposes of examination,
and putting it in most excellent
shape for reference and memor-
izing."

Kansas City Medical Record. —
"Martin's Surgery. — This admir-
able compend is well up in the
most advanced ideas of modern

surgery." Specimen of Illustrations.

13

No. 3.

ESSENTIALS OF ANATOMY,

Including the Anatomy of the Viscera.

BY CHARLES B. NANCREDE, M.D.,

Professor of Surgery and Clinical Surgery in the University of Michigan,

Ann Arbor ; Corresponding Member of the Royal Academy of

Medicine, Rome, Italy ; Late Surgeon Jefferson

Medical College, etc. etc.

ONE HUNDRED AND FORTY FINE WOODCUTS

THIRD EDITION.
Enlarged by an Appendix containing over Sixty Illustrations of

the Osteology of the Human Body.
The whole based upon the last (eleventh) edition of

GRAY'S ANATOMY.
Price, Cloth, $1.00. Interleaved for Notes, $1.25.

American Practitioner and
News, February 16, 18b9.

" Nancrede's Anatomy. —
For self-quizzing and keep-
ing fresh in mind the
knowledge of Anatomy
gains at school, it would
not be easy to speak of it
in terms too favorable."

Southern Californian Practi-
tioner, January 18, 1880.
" Nancrede's Anatomy. —
Very accurate and trust-
worthy."

American Practitioner and
Neu:s, Louisville, Kentucky.
" Nancrede's Anatomy. —
Truly such a book as no
student can afford to be
without."

Specimen of Illustrations.

14

No. 4.

Essentials of Medical Chemistry

ORGANIC AND INORGANIC.

CONTAINING, ALSO,

Questions on Medical Physics, Chemical Physiology,
Analytical Processes, Urinalysis, and Toxicology.

BY

LAWRENCE WOLFF, M.D.,

Demonstrator of Chemistry, Jefferson Medical College ; Visiting Physician

to German Hospital of Philadelphia ; Member of Philadelphia

College of Pharmacy, etc. etc.

SIXTH THOUSAND.

Price, Cloth, $1.00. Interleaved for Notes, $1.25.

Cincinnati Medical News, January, 1889. — " Wolff's Chemistry.- -A little
work that can be carried in the pocket, for ready reference in solving difficult
problems."

St.. Joseph's Medical Herald, March, 1889.— "Dr. Wolff explains most
simply the knotty and difficult points in chemistry, and the book is therefore
well suited for use in medical schools."

Medical and Surgical Reporter, November, 1889. — "We could wish that
more books like this would be written, in order that medical students might
thus early become more interested in what is often a difficult and uninterest-
ing branch of medical study."

Registered Pharmacist, Chicago, December, 1890.— " Wolff 's Chemistry."
— " The author is thoroughly familiar with his subjects. A useful addition to
the medical and pharmaceutical library."

15

No. 5.

ESSENTIALS OE OBSTETRICS,

BY W. EASTERLY ASHTON, M.D.,

Obstetrician to the Philadelphia Hospital

NUMEROUS ILLUSTRATIONS. SIXTH THOUSAND.

Price, Cloth, $1.00. Interleaved for Notes, $1.25.

A

Specimen of Illustrations.

Southern Practitioner, January, 1890. — Ashton's Obstetrics. — An excellent
little volume containing correct and practical knowledge. An admirable com-
pend, and the best condensation we have seen."

Chicago Medical Times. — " Ashton's Obstetrics. — Of extreme value to stu-
dents, and an excellent little book to freshen up the memory of the practi-
tioner."

Medical and Surgical Reporter, January 26, 1889. — " Ashton's Obstetrics.
— A work thoroughly calculated to be of service to students in preparing for
examination.7*

.\nr York M&liral Abstract, April, 1890.— «« Ashton's Obstetrics should be
consulted by the medical student until he can answer every question at sight.
The practitioner would also do well tb glance at the book now and then, to
prevent his knowledge from getting rusty."

16

No. 6.

ESSENTIALS

OP

Pathology and Morbid Anatomy.

C. E. A It JI A XI) SEMl'LE, B.A, M.B., Cantab., L.S.A., M.R.C.P., Innil.,

Physician to the Northeastern Hospital for Children, Harkney ; Pro-
fessor of Vocal and Aural Physiology and Examiner in Acous-
tics at Trinity College, London, etc. etc.

ILLUSTRATED. FOURTH THOUSAND,

Price, Cloth, $1.OO. Interleaved for Notes, $1.25.

From the College and (Jlimcul Record,
September, 1889. — " A small work upon
Pathology and Morbid Anatomy, that re-
duces such complex subjects to the ready
comprehension of the student and practi-
tioner, is a very acceptable addition to
medical literature. All the more modern
topics, such as Bacteria and Bacilli, and
the most recent views as to Urinary Path-
ology, find a place here, and in the hands
of a writer and teacher skilled in the art
of simplifying abstruse and difficult sub-
jects for easy comprehension are rendered
thoroughly intelligible. Few physicians
do more than refer to the more elaborate
works for passing information at the time
it is absolutely needed, but a book like this
of Dr. Semple's can be taken up and perused continuously to the profit and
instruction of the reader."

Indiana, Medical Journal, December. 1889. — "Semple's Pathology and
Morbid Anatomy. — An excellent compend of the subject from the points of
view of Green and Payne."

Cincinnati Medical News, November, 1889. — Semple's Pathology and Mor-
bid Anatomy. — A valuable little volume — truly a muUnm. in paivo."

17

Specimen of Illustrations.

No. 7.

ESSENTIALS

OF

Materia Medica, Therapeutics,

AND

PRESCRIPTION WRITING.

HENRY MORRIS, M.D.,

Late Demonstrator, Jefferson Medical College ; Fellow College of Physicians,

Philadelphia; Co-editor Biddle's Materia Medica ; Visiting

Physician to St. Joseph's Hospital, etc. etc.

SECOND EDITION. FOURTH THOUSAND.

Price, Cloth, $1.00. Interleaved for Notes, $1.25.

MEDICAL AND SURGICAL REPORTER, October, 1889.

"Morris* Materia Medica and Therapeutics. —One of the best compends in
this series. Concise, pithy, and clear, well-suited to the purpose for which it
is prepared."

GAILLARD'S MEDICAL JOURNAL, November, 1889.

" Morris* Materia Medica.— The very essence of Materia Medica and Thera-
peutics boiled down and presented in a clear and readable style."

SANITARIUM, New York, January, 1890.

"Morris* Materia Medica.— A well-arranged quiz-book, comprising the
most important recent remedies."

BUFFALO MEDICAL AND SURGICAL JOURNAL, January, 1890.
"Morns' Materia Medica. — The subjects are treated in such a unique and
attractive manner that they cannot fail to impress the mind and instruct in
a lasting manner."

18

Nos. 8 and 9.

Essentials of Practice of Medicine,

BY HENRY MORRIS, M.D.,

Author of " Essentials of Materia Medica," etc.

With an Appendix on the Clinical and Microscopical
Examination of Urine.

BY LAWRENCE WOLFF, M.D.,

Author of "Essentials of Medical Chemistry," etc.

COLORED (VOGEL) URINE SCALE AND NUMEROUS
FINE ILLUSTRATIONS.

8ECOND EDITION,

Enlarged by some THREE HUNDRED Essential

Formulae, selected from the writings of the

most eminent authorities of the

Medical Profession.

COLLECTED AND ARRANGED BY

WILLIAM M. POWELL, M.D.,

Author of "Essentials of Diseases of Children.''

Price, Cloth, &2.OO. Medical Sheep, $*2.5O.

SOUTHERN PRACTITIONER, Nashville, Tenn., January, 1891.
"Morris* Practice of Medicine. — Of material aid to the advanced student
in preparing for his degree, and to the young practitioner in diagnosing affec-
tions or selecting the proper remedy."

AMERICAN PRACTITIONER AND NEWS, Louisville, Ky.f January, 1891.
"Morris' Practice of Medicine. — The teaching is sound, the presentation
graphic, matter as full as might be desired, and the style attractive."

SOUTHERN MEDICAL RECORD, January, 1891.

"Morris* Practice of Medicine is presented to the reader in the form of
Questions and Answers, thereby calling attention to the most important lead-
ing facts, which is not only desirable, but indispensable to an acquaintance
with the essentials of medicine. The book is all it pretends to be, and we
cheerfully recommend it to medical students."

19

No. 10.

ESSENTIALS OF GYNAECOLOGY,

EDWIN B. CRAIGIN, M.D.,

Attending Gynaecologist, Roosevelt Hospital, Out- Patients' Department
Assistant Surgeon, New York Cancer Hospital, etc. etc.

58 FINE ILLUSTRATIONS.
SIXTH THOUSAND.

Price, Cloth, $1.OO. Interleaved for Notes, $1.25.

m

•m*

Specimen of Illustrations.

Medical and Surgical Re-
porter, April, 1890.— "Craig-
gin's Essentials of Gynaecol-
ogy.— This is a most excel-
lent addition to this series
of question compends, and
properly used will be of
great assistance to the stu-
dent in preparing for ex-
amination. Dr. Craigin is
to be congratulated upon
having produced in com-
pact form the Essentials of
Gynaecology. The style is
concise, and at the same
time the sentences are well
rounded. This renders the
book far more easy to read
than most compends, and
adds distinctly to its value."

College and Clinical Record,
April, 1890. — " Craigin's
Gynaecology. — Students and
practitioners, general or spe-
cial, even derive information
and benefit from the perusal
and study of a carefully
written work like this."

No. 11.

Essentials of Diseases of the Skin,

BY HENRY W. STELWAGON, M.D.,

Clinical Lecturer on Dermatology in the Jefferson Medical College, Philadel-
phia; Physician to Philadelphia Dispensary for Skin Diseases; Chief
of the Skin Dispensary in the Hospital of University of Penn-
sylvania; Physician to Skin Department of the Howard
Hospital ; Lecturer on Dermatology in the Women's
Medical College, Philadelphia, etc. etc.

74 ILLUSTRATIONS, many of which are original.

FOURTH THOUSAND.

Price, Cloth, $1.00. Interleaved for Notes, $1.25.

Specimen of Illustrations.

New York Medical Journal, May, 1890.— "Stelwagon's Diseases of the
Skin.— We are indebted to Philadelphia for another excellent book on Derma-
tology. The little book now before us is well entitled "Essentials of Derma-
tology," and admirably answers the purpose for which it is written." The
experience of the reviewer has taught him that just such a book is needed.
We are pleased with the handsome appearance of the book, with its clear
type, good paper, and fine wood-cuts."

No. 12.

ESSENTIALS

Minor Surgery, Bandaging, and
Venereal Diseases,

BY EDWARD MARTIN, A.M., M.D.,

Author of "Essentials of Surgery," etc.

82 ILLUSTRATIONS, mostly specially prepared for this wcrk.

Price, Cloth, $1.00. Interleaved for Notes, $1.25.

Medical News, Phila-
delphia, January 10,1891.
"Martin's Minor Surgery,
Bandaging, and Venereal
Diseases. — The best con-
densation of the subjects
of which it treatsyetplaced
before the profession. The
chapteronGenito-Urinary
Diseases, though short, is
sufficiently complete to
' ~Y"\ make them thoroughly
I acquainted with the most
_ / / advanced views on the
subject."

Nashville Journal of
Medicineaiid Surgery, No-
vember, 1 890. — ' 'Martin 's
Minor Surgery, etc., should
be in the hands of every
student, and we shall per-
sonally recommend it toour
students as the best text-
book upon the subject."
Pharmaceutical Era, Detroit, Michigan, December 1, 1890. — "Martin's
Minor Surgery, etc. — Especially acceptable to the general practitioner, who
is often at a loss in cases of emergency as to the proper method of applying a
bandage to an injured member."

22

Specimen of Illustrations.

No. 13.

ESSENTIALS

OJT

Legal Medicine, Toxicology,

HYGIENE.

BY

C. E. ARMAND SEMPLE, M.D.,

Author of " Essentials of Pathology and Morbid Anatomy."

130 ILLUSTRATIONS.

Price, Cloth $1.OO.

Interleaved for Notes .... 1.25.

SOUTHERN PRACTITIONER, Nashville, May, 1890.
"Seraple's Legal Medicine, etc. — At the present time, when the
field of medical science, by reason of rapid progress, becomes so vast,
a book which contains the essentials of any branch or department of
it, in concise, yet readable form, must of necessity be of value. This
little brochure, as its title indicates, covers a portion of medical science
that is to a great extent too much neglected by the student, by reason
of the vastness of the entire field and the voluminous amount of matter
pertaining to what he deems more important departments. The lead-
ing points, the essentials, are here summed up systematically and
clearly."

MEDICAL BRIEF, St. Louis, May, 1890.

" Semple's Legal Medicine, Toxicology, and Hygiene. — A fair sample
of Saunders' valuable compends for the student and practitioner. It
is handsomely printed and illustrated, and concise and clear in its

teachings."

23

No. 14.

ESSENTIALS OF

Refraction and Diseases of the Eye,

BY EDWARD JACKSON, A.M., M.D.,

Professor of Diseases of the Eye in the Philadelphia Polyclinic and College for
Graduates in Medicine ; Member of the American Ophthalmological So-
ciety; Fellow of the College of Physicians of Philadelphia; Fel-
low of the American Academy of Medicine, etc. etc.

AND

Essentials of Diseases of the Nose and Throat,

BY E. BALDWIN GLEASON, M.D.,

Assistant in the Nose and Throat Dispensary of the Hospital of the University

of Pennsylvania ; Assistant in the Nose and Throat Department of the

Union Dispensary; Member of the German Medical Society,

Philadelphia ; Polyclinic Medical Society, etc. etc.

TWO VOLUMES IN ONE. PROFUSELY ILLUSTRATED.

Price, Cloth, 91.OO. Interleaved for Notes, $1.25.

University Medical Mag-
azine, Philadelphia, Octo-
ber, 1890. — "Jackson and
Gleason's Essentials of Dis-
eases of the Eye, Nose, and
Throat. — The subjects
have been handled with
skill, and the student who
acquires all that here lays
before him will have much
more than a foundation for
future work."

New York Medical Rec-
ord, November 15, 1890.
— "Jackson and Gleason
on Diseases of the Eye,
Nose, and Throat. — A

valuable book to the be-
Specimen of Eye Illustrations.

ginner in these branches,

to the student, to the busy practitioner, and as an adjunct to more thorough
reading. The authors are capable men, and as successful teachers know
what a student most needs"

114

No. 15.

ESSENTIALS

DISEASES OF CHILDREN,

BY

WILLIAM M. POWELL, M.D.,

Attending Physician to the Mercer House for Invalid Women, at Atlantic
City, N. J. ; Late Physician to the Clinic for the Diseases of Chil-
dren in the Hospital of the University of Pennsylvania and
St. Clement's Hospital ; Instructor in Physical Diag-
nosis in the Medical Department of the Uni-
versity of Pennsylvania, and Chief of
the Medical Clinic of the Phil-
adelphia Polyclinic.

Price, Cloth . . . . . . $1.OO.

Interleaved for Notes .... 1.25.

AMERICAN PRACTITIONER AND NEWS, Louisville, Ky., December 20, 1890.
" Powell's Diseases of Children. — This work is gotten up in the
clear and attractive style that characterizes the Saunders' Series. It
contains in appropriate form the gist of all the best works in the de-
partment to which it relates."

SOUTHERN PRACTITIONER, Nashville, Tennessee, November, 1890.
"Dr. Powell's little book is a marvel of condensation. Handsome
binding, good paper, and clear type add to its attractiveness."

ANNALS OF GYN^COLOGY, Philadelphia, December, 1890.
14 Powell's Diseases of Children. — The book contains a series of im-
portant questions and answers, which the student will find of great
utility in the examination of children."

No. 16.

ESSENTIALS

EXAMINATION OF TJEIIE.

BY

LAWRENCE WOLFF, M.D.,

Author of "Essentials of Medical Chemistry," etc.

COLORED (VOGEL) URINE SCALE AND NUMEROUS
ILLUSTRATIONS.

Price, Cloth

75 Cents.

Specimen of Illustrations.

UNIVERSITY MEDICAL MAGAZINE,

June, 1890.

** Wolff 's Examination of the
Urine. — A little work of decided
value."

MEDICAL RECORD, New York,

August 23, 1890.
"Wolff's Examination of
Urine. — A good manual for
students, well written, and
answers, categorically, many-
questions beginners are sure
to ask."

MEMPHIS MEDICAL MONTHLY, Memphis, Tennessee, June, 1890.
"Wolff's Examination of Urine. — The book is practical in char-
acUr, comprehensive as is desirable, and a useful aid to the student

in his studies."

20

No. 18.

ESSENTIALS

PRACTICE OF PHARMACY,

BY

LUCIUS E. SAYRE,

Professor of Pharmacy and Materia Medica in the University of Kansas.

Price, Oloth, $1,00. Interleaved for Notes, $1.25,

ALBANY MEDICAL ANNALS, Albany, N. Y., November, 1890.
" Sayre's Essentials of Pharmacy covers a great deal of ground in
small compass. The matter is well digested and arranged. The
research questions are a valuable feature of the book."

AMERICAN DOCTOR, Richmond, Va., January, 1891.
" Sayre's Essentials of Pharmacy. — This very valuable little manual
covers the ground in a most admirable manner. It contains practical
pharmacy in a nutshell."

NATIONAL DRUG REGISTER. St. Louis, Mo., December 1, 1890.
" Sayre's Essentials of Pharmacy.— The best quiz on pharmacy we
have yet examined."

WESTERN DRUG RECORD, November 10, 1890.

"Sayre's Essentials of Pharmacy. — A book of only 180 pages, but
pharmacy in a nut-shell. It is not a quiz-compend compiled to en-
able a grocery clerk to ' down' a board of pharmacy ; it is a finger-
post guiding a student to a completer knowledge."

27

SAUNUERS' QUESTION-COMPENDS.

In Preparation. Ready about September 1, 1891.

No. 17.

Essentials of Diagnosis,

No. 19

Essentials of Hygiene.

ILLUSTRATED.

BY ROBERT P. ROBINS, M.D.

No. 20.

Essentials of Bacteriology.

ILLUSTRATED.

BY M. V. BALL, M.D.

No. 21.

Essentials of Nervous Diseases and Insanity,

ILLUSTRATED.
BY JOHN C. SHAW, M.D.

No. 22.

Essentials of Medical Physics.

ILLUSTRATED.
BY FRED. J BROCKWAY, M.D.

No. 23.

Essentials of Medical Electricity.

ILLUSTRATED.

BY DAVID D. STEWART, M.D., and EDWARD S. LAWRENCE, M.D.

OTHERS PREPARING.

28

The Fiske Fund Prize Essay for 189O.

THE

SURGICAL TREATMENT

OF

WOUNDS AND OBSTRUCTION

OP THE

INTESTINES.

BY

EDWARD MARTIN, A.M., M.D.,

Clinical Professor of Genito-Urinary Diseases, Instructor in Operative Sur-
gery, and Lecturer on Minor Surgery, University of Pennsylvania;
Surgeon to the Howard Hospital ; Assistant Surgeon to the
University Hospital, etc. etc.
AND

HOBART A. HARE, M.D.,

Professor of Therapeutics, Jefferson Medical College ; Attending Physician
to St. Agnes' Hospital.

ILLUSTRATED.

Price, Cloth $2.00, Net.

" In presenting this Essay upon the Surgical Treatment of Wounds
and Obstruction of the Intestines to the Trustees of the Fiske Fund,
it is proper to outline the scope of our work, and to state "briefly the
facts and lines of original research upon which our conclusions are
based. For over two years we have made experiments in the labo-
ratory upon these subjects, and have carried out in every detail all
the methods and modifications of operations that have been published
or which have occurred to us in the course of our own studies. . . .
In addition to the original work involved in studying so important
a branch of surgery as the one before us (and which will be found
represented, graphically, in part at least by a number of tracings),
we have collected and placed before the reader what we believe to be
the fullest statistics yet collected upon gunshot wounds of the abdo-
men."— EXTRACT FROM PREFACE.

29

INDEX.

PAGE

ANNOUNCEMENT . . . i

AMERICAN TEXT-BOOK OF SURGERY . . . . 2, 3
VlERORDT AND STUART'S MEDICAL DIAGNOSIS . . 4
KEATING'S NEW UNABRIDGED DICTIONARY or MEDICINE 5
SAUNDERS' POCKET MEDICAL LEXICON .... 6
NANCREDE'S ANATOMY AND MANUAL OF DISSECTION . 7
DESCHWEINITZ'S DISEASES OF THE EYE .... 8

GARRIGUE'S DISEASES OF WOMEN 9

NORRIS' SYLLABUS OF OBSTETRICAL LECTURES . . 9
SAUNDERS' POCKET MEDICAL FORMULARY . . .10
SAUNDERS* SERIES OF QUESTION COMPENDS . . .11

HARE'S PHYSIOLOGY 12

MARTIN'S SURGERY ....:... 13

NANCREDE'S ANATOMY 14

WOLFF'S CHEMISTRY 15

ASHTON'S OBSTETRICS 16

SEMPLE'S PATHOLOGY, ETC 17

MORRIS' MATERIA MEDICA 18

MORRIS' PRACTICE OF MEDICINE 19

CRAGIN'S GYNAECOLOGY 20

STELWAGEN'S DISEASES OF THE SKIN . . . .21

MARTIN'S MINOR SURGERY, ETC 22

SEMPLE'S LEGAL MEDICINE. ETC 23

JACKSON AND GLEASON'S DISEASES OF EYE, XOEI:, AND

THROAT - 24

POWELL'S DISEASES OF CHILDREN ... .25

WOLFF'S EXAMINATION OF URINE ... .26

SAYRE'S PRACTICE OF PHARMACY . . 27

WORKS IN PREPARATION AND IN PIM>- . . .28
MARTIN AND HARE'S WOUNDS AND OBSTRUCTION OF

THE INTESTINES - 29

30

THE

CLIMATOLOGIST.

A MONTHLY JOURNAL OF MEDICINE

DEVOTED TO THE

Relation of Climate, Mineral Springs, Diet, Pre-
ventive Medicine, Race, Occupation, Life
Insurance and Sanitary Science
to Disease.

EDITED BY

JOHN M. KEATING, M. D.
FREDERICK A. PACKARD, M. D. CHAS. F. GARDINER, M. D.

ASSOCIATE EDITORS:

NORMAN BRIDGE, M.D.,

Los Angeles, Cal.
VINCENT Y. BOWD1TCH, M.I).

Boston, Mass.
SAML. R. BURROUGHS, M.D.,

Raymond, Tex.
J. WELLINGTON BYEKS, M.D.,

Charlotte, N. C.
J. M. DACOSTA, M.D.,

Philadelphia, Pa.
CHARLES DENISON, M.D.,

Denver, Colo.
GEORGE DOCK, M.[>.,

Galveston, Texas.
WM. A. EDWARDS, M.D.,

San Diego, Cal.
J. T. ESKRIDGE, M.D.,

Denver, Colo.
S4MUEL A. FISK, M.D..

Denver, Colo.
W. H. GEDDINGS, M.D.,

Aiken,S. C.
JOHN B. HAMILTON, M.D.,

Chicago, 111.
T. S. HOPKINS, M.D.,

Thomasviile, Ga.
FREDERICK I. KNIGHT., M.D.,

Boston, Mass.
R. L. MACDONNELL, M.D.,

Montreal, Canada.

FRANCIS MINOT, M.D., Moaton.Muss.
ALFRED L. LOOM1S, M.I) , ,

New York City.
HENRY M. LYMAN, M.D.,

Chicago, Ills.
WILLIAM OSLER, M.D.,

Baltimore, Md.
WILLIAM PEPPER, M.D.,

Philadelphia, Pa.
BOARDMAN REED, M.D.,

Atlantic City. N. J.
J. REED, JR., M.D.,

Colorado Springs, Colo.
GEORGE II. ROHE, M.D.,

Baltimore. Md.

KARL VON RUCK, M.D.,

Asheville, N. C.
FREDK. C. SHATTUCK, M.I).,

Boston, Mass.
S. E. SOLLY, M.D.,

Colorado Springs, Colo.
G. B. THORNTON, M.D.,

Memphis, Tenn.
E. L. TRUDEAU, M.D.,

Saranac Luke, N. Y.
J, B. WALKER, M. D.,

Philadelphia. Pa.

J. P. WALL, M.D., Tampa, Florida.
, M.D.,

JAMES C. WILSON,

Philadelphia, Pa.

Yearly Subscription $2.OO. Single Numbers 2O Cts.

W. B. SAUNDERS, Publisher,

913 Walnut Street, Philadelphia, Pa.

EXTRACT FROM THE INTRODUCTION IN THE OPENING

NUMBER OF

"THE CLIMATOLOGIST."

AUGUST, 1891.

" THE object of this JOURNAL is to promote original investi-
gation, to publish papers containing the observations and ex-
perience of physicians in this country and Europe on all matters
relating to CLIMATOLOGY, MINERAL SPRINGS, DIET, PREVENTIVE
MEDICINE, RACE, OCCUPATION, LIFE INSURANCE, AND SANITARY
SCIENCE — and in that way to supply the means by which the
general practitioner and the public at large will become better
acquainted with the diseases of this country and Europe, and
better armed to meet the requirements of their prevention or
cure. The study of these subjects in this country is exciting
great and increasing interest, and all admit that, from the little
knowledge already possessed of its resources, possibly every
known combination of atmospheric condition, soil, altitude, cli-
mate, or mineral springs, is to be found on this continent. It is
confidently expected that such a. journal \v\\\ receive encourage-
ment and be an authority upon all questions which are included
in its title.

" Original papers upon diseases of localities — those incident
to occupation, race, or climate, the study of epidemics, the
questions of proper food, of the water supply, its potability
and distribution, matters relating to drainage and diseases de-
pendent on it — as well as experimental studies, or laboratory
investigations on bacteriology, will form a prominent portion
of the material presented during the year, and it is to be hoped
that physicians of all sections of the country will send papers
upon these or any other subjects which will be of general in-
terest.

" Special attention will also be paid to the subject of health
resorts, descriptions of Sanitariums with special reference to
their suitability to certain cases, and the proper selection ot
patients likely to be benefitted by them. The utmost care will
be taken that this JOURNAL shall assume and maintain the
highest scientific character. It will be absolutely independent
in its principles— -fair towards all. It will depend for its main-
tenance upon the support given to it by the prcfession, as it is
not published in the interest of any special section or clique."

POCKET MEDICAL LEXICON;

OR,

Dictionary of Terms and Words used in Medicine and Surgery,

BY JOHN M. KEATING, M.V.,

Editor of "Cyclopedia of Diseases of Children," etc.; Author of the
"New Pronouncing Dictionary of Medicine,"

HENRY HAMILTON,

Author of "A New Translation of Virgil's yEneid into English Verse;'
Co-author of a "New Pronouncing Dictionary of Medicine."

Price, 75 Cents, Cloth. $1.00, Leather Tucks.

This new and comprehensive
work of reference is the outcome
of a demand for a more modern
handbook of its class than those
at present on the market, which,
dating as they do from 1855 to
1884, are of but trifling use to
the student by their not con-
taining the hundreds of new
words now used in current lit-
erature, especially those relat-
ing to Electricity and Bacteri-
ology.

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(From Appendix to Medical Lexicon.)

Annals of (rt/nwcoloyy, Phila-
delpliia, December, 189O.

Saunders' Pocket Medical Lexi-
con—a very complete little work,
invaluable to every student of
medicine. It not only contains a
very large number of words, but
also tables of etymological factors
common in medical terminology ;
abbreviations used in medicine,
poisons and antidotes, etc.

RETURN TO the circulation desk of any
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NOW READY.

MEDICAL DIAGNOSIS.

DR. OSWALD VIERORDT.

Professor of Medicine at the University of Heidelberg ^formerly Privat
at University of Leipzig ; Professor of Medicine and Director of the
Medical Polyclinic.at the University of Jena.

Translated, with Additions, from the Second Enlarged Germar
Edition, with the Author's Permission.

BY :

FRANCIS H. STUART, A.M., M.D.,

Member of the Medical Society of the County of Kings, N. Y. ; Fellow of the

New York Academy of Medicine ; Member of the British

Medical Association, etc.

NUMEROUS COLORED AND WOOD ENGRAVINGS.
Price, Cloth, $4.00; Sheep, $5.OO.

In this work, as in no other hitherto published, are given fall and
accurate explanations of the phenomena observed at the beds!
is distinctly a Clinical work by a master teacher, characterized by thor-
oughness, fulness, and accuracy.

It is a mine of information upon the points that are so
often passed over without explanation.

The student who is familiar with its contents will have a sound foun-
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The author gives a complete though brief presentation of the Micro-
organisms, whose recognition and discrimination are made pos^
cultivation, and inoculation, and which, through the labors of those
eminent bacteriologists, PASTEUR, KOCH, and others, h,
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the cure of disease.