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MICRO-ORGANISMS AND DISEASE.

WITH THE PUBLISHERS1 ^/ .

COMPLIMENTS.

MICRO-ORGANISMS AND DISEASE

AN

INTRODUCTION INTO THE STUDY OF
SPECIFIC MICRO-ORGANISMS

BY

E. KLEIN M.D. F.R.S.

Lecturer on General Anatomy and Physiology in the
Medical School of St. Bartholomew's Hospital London

THIRD EDITION REVISED
WITH ONE HUNDRED AND TWENTY-ONE ENGRA VINGS

MACMILLAN AND CO.
1886

The Right of Translation is Reserved

RICHARD CLAY AND SONS,

BREAD STREET HILL, LONDON",

Bungay, Suffolk.

TO

JOHN SIMON, C.B., D.C.L., LL.D., F.R.S.,

THIS BOOK

BY

THE AUTHOR.

PREFACE TO THE THIRD EDITION.

A LITTLE over a year has passed since this book
appeared, and a third edition has become necessary. In
this I have made a number of alterations and additions.
Some of the methods of preparing nutritive-media have
been simplified, while others have been described more
in detail. The methods of staining have been revised,
and a number of facts bearing on septic and specific
organisms, gained during the past year or two, have been
added. I trust that this revision will add to the value
of the work.

E. KLEIN.

Nwember, 1885.

PREFACE.

THE following book, with few additions, is a reprint of
a series of articles that have appeared in the Practitioner
(1884). Not pretending to be in any sense an exhaustive
treatise it will, I hope, nevertheless serve to illustrate most
of the important points bearing on investigations into the
life history of micro-organisms connected with infectious
diseases.

I must not omit to mention that most of the investi-
gations recorded here were carried out for the Medical
Department of the Local Government Board during the
past ten years.

E. KLEIN.

CONTENTS,

PAGE

INTRODUCTION . i

CHAPTER I.

MICROSCOPIC EXAMINATION

CHAPTER II.

PREPARATION OF CULTURE MATERIAL 15

CHAPTER III.

VESSELS AND INSTRUMENTS USED IN CULTIVATIONS . 26

CHAPTER IV.

PREPARATION OF CULTURE-MEDIA FOR INOCULATION ... 32

39701

xii CONTENTS.

CHAPTER V.

PAGE

METHODS OF INOCULATION 38

CHAPTER VI.

MORPHOLOGY OF BACTERIA • 54

CHAPTER VII.

MICROCOCCUS 57

CHAPTER VIII.

BACTERIUM . . . 88

CHAPTER IX.

BACILLUS 96

CHAPTER X.

BACILLUS: NON-PATHOGENIC FORMS .... 108

CHAPTER XL

BACILLUS: PATHOGENIC FORMS . Il8

CONTENTS.

CHAPTER XII.

PACK

VIBRIO . l82

CHAPTER XIII.

SPIROBACTERIUM 184

CHAPTER XIV.

YEAST FUNGI : TORULACE^, SACCHAROMYCES 189

CHAPTER XV.

MOULD FUNGI : HYPHOMYCETES OR MYCELIAL FUNGI .... 194

CHAPTER XVI.

ACTINOMYCES 204

CHAPTER XVII.

ON RELATIONS OF SEPTIC TO PATHOGENIC ORGANISMS . . . 207

CHAPTER XVIII.

VITAL PHENOMENA OF NON-PATHOGENIC ORGANISMS .... 232

xiv CONTENTS.

CHAPTER XIX.

PAGE
VITAL PHENOMENA OF PATHOGENIC ORGANISMS 244

CHAPTER XX.

VACCINATION AND IMMUNITY 253

CHAPTER XXL
ANTISEPTICS • 257

INDEX 265

MICRO-ORGANISMS AND DISEASE,

MICRO-ORGANISMS AND DISEASE

INTRODUCTION.

THE relation of micro-organisms to the infectious diseases
is admitted to be very intimate ; and although it may not be
quite so universal as some are inclined to assume, it is never-
theless definitely proved to exist as regards some of the
infectious maladies affecting man and brutes. In order to
pass in review all the ascertained facts and observations in
this vast and constantly-growing field of pathology, and to
appreciate and to assign their true value to the many
observations bearing on this relation of micro-organisms to
disease, it is necessary that the reader, and still more the
worker in this field, should be enabled to criticise the ob-
servations and facts brought forward by the numerous
writers on the subject, for otherwise he would probably take
as proved what has really not passed beyond the stage of
possibility. And it is this point which requires the most
careful attention, viz., to be able to see at a glance that,
owing to the imperfect or faulty method of investigation
employed, or that, owing to certain inferences incompatible
with the general laws and general tendency of the well-

B

2 MICRO-ORGANISMS AND DISEASE. [INTRO.

founded and experimentally proved facts, the statements set
forth in a particular observation or series of observations are
not to be accepted.

In all investigations of the relation of micro-organisms to
disease it is necessary to bear in mind that, as Koch 1 has
pointed out, no observation can be said to be complete, or,
one should rather say, in no instance can it be said to have
been satisfactorily proved, that a particular infectious disease
is due to a particular micro-organism if any one of the
following conditions remains unfulfilled : — (i) It is absolutely
necessary that the micro-organism in question is present
either in the blood or the diseased tissues of man or of an
animal suffering or dead from the disease. In this respect
great differences exist, for in some infectious diseases the
micro-organisms, although present in the diseased tissues, are
not present in the blood ; while in others they are present
in large numbers in the blood only or in the lymphatics only.
These points will be considered hereafter in the special
cases. (2) It is necessary to take these micro-organisms
from their nidus, from the blood or the tissues as the case
may be, to cultivate them artificially in suitable media, i.e.
outside the animal body, but by such methods as to exclude
the accidental introduction into these media of other micro-
organisms ; to go on cultivating them from one cultivation
to another for several successive generations, in order to
obtain them free of every kind of matter derived from the
animal body from which they have been taken in the first
instance. (3) After having thus cultivated the micro-
organisms for several successive generations it is necessary
to re-introduce them into the body of a healthy animal
susceptible to the disease, and in this way to show that this
animal becomes affected with the same disease as the one
1 Die Mihbrand-impfung, Cassel and Berlin, 1883.

INTRO.] MICRO-ORGANISMS AND DISEASE. 3

from which the organisms were originally derived. (4) And,
finally, it is necessary that in this so affected new animal the
same micro-organisms should again be found. A particular
micro-organism may probably be the cause of a particular
disease, but that really and unmistakably it is so can only be
inferred with certainty when every one of these desiderata
has been satisfied.

It will be my aim in the following pages, first to describe
the methods that may be employed with success in investiga-
tions bearing on the relation of micro-organisms to disease ;
secondly, to describe in conformity with reliable observations
the morphology and physiology of the micro-organisms that
bear any relation to disease ; and thirdly, to enumerate the
observations that have been made in recent years to prove the
existence of such an intimate relation. Last, but not least,
we shall consider the precise relation of the particular micro-
organisms to the causation of disease.

B 2

CHAPTER I.

MICROSCOPIC EXAMINATION.

FOR the examination of micro-organisms good high powers
are essential, at the least a power magnifying 300 to 400
linear diameters. Zeiss' D or E and Zeiss' or Powell and
Lealand's oil immersion i-i2th or i-i6th inch will be found
sufficient for all purposes. In the case of tissues stained with
aniline dyes a good substage-condenser such as Abbe's or
Powell and Lealand's, is invaluable. I use Zeiss' stand
with Abba's condenser, open diaphragm, and plane mirror.
As Koch1 pointed out, and what is now universally acted
upon, stained specimens mounted in Canada-balsam
solution or Dammar varnish, when examined on an Abbe's
condenser, show the micro-organisms with extreme clearness
and sharpness.

The examination of the morphological characters of an
organism is carried out on fresh unstained, as well as on fresh
stained, microscopic specimens. Although the latter method
is, for reasons hereafter to be mentioned, by far the most
perfect and reliable one, it is nevertheless important to

1 Die Aetiologie d. Wundinfectionskrankheiten, p. 34, Leipzig, 1879.
Translated as Traumatic Infective Diseases (New Syd. Soc.), London,
1880.

CH.i.] MICROSCOPIC EXAMINATION. 5

ascertain as far as possible the appearances, chemical
reactions, and general morphology of perfectly fresh speci-
mens. Blood, juices, tissues, and fluids in which the micro-
organisms have been growing, are subjected directly, without
any previous preparation, to microscopic examination. With
artificial nourishing media in which micro-organisms have
been growing, the examination of fresh specimens is of
great importance, for the reason that the organisms can be
easily identified and their size and general morphological
characters be more correctly ascertained than after drying,
hardening, and staining. Besides, the chemical reactions
can be satisfactorily studied in fresh specimens only. All
one has to do is to draw up with a capillary pipette or to take
up with the point of a needle a drop or particle of the
material, to place it on an object-glass, and to cover it up
with a thin cover-glass. Where one has to deal with liquids,
such as artificial nourishing fluids, blood serum, tissue-
juices, secretions, transudations and exudations, no addition
is required. In the case of more solid material, such as
solid artificial nourishing material, bits of tissue, &c., the
addition of a drop of neutral previously well-boiled saline
solution (of 0*6 to 075 per cent.) is advantageous .although
not absolutely necessary, since by pressing down the cover-
glass a layer of the material sufficiently thin for examination
can be obtained. In some instances a bit of tissue .can be
teased out into fine particles by means .of two clean needles.
Where it. is a question of micro-organisms sufficiently con-
spicuous by their shape, size, and general appearance, their
identification in the fresh condition is not difficult ; this is
the case with bacilli, actinomyces, and mycelia, but in the
case of micrococci, especially when isolated ox in couples,
and lying in blood, juices, or tissues, their recognition is
often extremely difficult. When in laige clumps, such as

6 MICRO-ORGANISMS AND DISEASE. [CHAP.

larger or smaller masses of zoogloea, or when in the shape
of chains, the identification is not difficult ; but in the more
isolated state they are not easily recognised, owing, as a rule,
to the presence of granules or particles of various kinds,
from which morphologically their distinction is well-nigh
impossible. In such cases there are certain rules of thumb,
if I may say so, which assist, although they do not abso-
lutely insure, the diagnosis. These are the micro-chemical
reactions. The addition of liquor potassae leaves micro-
organisms quite unaltered, whereas fatty and most albuminous
granules alter or altogether disappear by it. Acetic acid
from 5 to 10 percent, strong does not affect micro-organisms,
but albuminous and other granules become in most instances
altered. These two re-agents, I think, are as reliable as
any others ; if they fail, then others like alcohol, chloroform,
sulphuric ether, &c., are not of any greater help, but the
latter re-agents may be used, for instance, when it is a
question between fat-granules and micrococci, or crystals
and bacilli.

Micro-organisms- have a great affinity for certain dyes,
especially aniline dyes, and therefore these are used with
great success to demonstrate their presence, and to
differentiate in many instances morphological details which
in the unstained condition are not discernible. The
staining is effected on fresh unaltered organisms, or after
they have been dried. In the first instance the process is
carried out thus : — A microscopic specimen is made, and to
it is added afterwards drop after drop of the dye, passing
it through the specimen in the usual way of applying fluids
to a microscopic specimen, i.e. by adding with a capillary
pipette the dye at one margin of the cover-glass and sucking
it up with a strip of filter-paper applied to the opposite
margin of the cover-glass. When the staining has taken

i] MICROSCOPIC EXAMINATION. 7

place the excess of the dye is washed away with salt
solution, water or alcohol, or both, as the case may be (see
below). Unless the organisms are embedded in continuous
masses of solids, this method gives good results. In the
latter case, say if they are embedded in a microscopic lump
of tissue, or in a particular spot of a fine section of a fresh
tissue, it is necessary, after having placed the lump or
section on an object-glass, to drop the dye on to this previous
to putting on the cover-glass. After some minutes the dye
is allowed to run off by inclining the object-glass, and then
the washing is proceeded with till all the excess of the dye
is removed ; the mounting is then done by placing a drop
of water or salt solution on the specimen and covering
it with a cover-glass. In the case of sections through
fresh and hardened tissues containing micro-organisms,
the method of staining and of permanently mounting
them as a whole is more complicated, and will be detailed
presently.

When one has to deal with coherent masses of micro-
organisms, present either in natural media (i.e. animal tissue)
or artificial cultivations, such as zoogloea and pellicles of
micrococcus or bacterium, these can be bodily transferred to
a watch-glass, stained, washed, and mounted without much
difficulty, either for immediate or permanent use. The
permanent specimens are made in this way: — Place the
section or pellicle in a watch-glass containing the dye, leave
it there till deeply tinted, take out with a needle or the like,
wash in water, then in alcohol, leave here for five minutes
or more till most of the excess of the colouring- matter is
removed, then lift it on to an object-glass, spread well out,
place on it a drop of clove-oil, and after a minute or two
drain off the clove-oil, add a drop of Canada-balsam solution
(in chloroform or benzol), and cover with a cover-glass. In

8 MICRO-ORGANISMS AND DISEASE. [CHAP.

some special instances, such as the bacilli of leprosy and
tuberculosis, double staining is required. With other
organisms, such as the bacilli of glanders or tuberculosis,
the washing is carried out, not with water but with acid
(acetic acid and nitric acid respectively). All the details
will be stated when dealing with these special organisms.

The method extensively and successfully used for the
demonstration and preservation of microscopic specimens
of micro-organisms in fluids, as blood, pus, and juices, is
that of Weigert and Koch, which consists in spreading out
on a glass slide or cover-glass a very thin film — the thinner
the better — of the fluid (artificial or natural culture medium),
blood, pus, or juice, and drying it rapidly by holding it for
ten to twenty seconds over the flame of a spirit-lamp or gas-
burner. The most successful preparations are obtained
when the heating is carried on for such a time that the film,
having become opaque at first, rapidly turns transparent.
Several drops of the aniline dye to be used are then poured
over the specimen, and after remaining on it from five to
thirty minutes or more are poured off.

The cover -glass specimen is then well rinsed with distilled
water, dried over the flame, and mounted in Canada-balsam
solution or Dammar varnish — of course always bearing in
mind on which surface of the cover-glass the film has been
spread. If the film has been well dried in the first instance
washing in water is quite sufficient, but if the drying has been
insufficient a good deal of diffuse staining of the ground
substance has taken place, and then of course the cover-glass
specimen must be also washed in alcohol sufficiently long to
remove this undesirable staining, then washed in water, dried
and mounted. In some instances washing with alcohol
removes also the dye from the bacteria, but as a rule it is
better to first over-stain the cover-glass specimen, then wash

I.] MICROSCOPIC EXAMINATION. 9

well in alcohol so as to remove the dye from all except
the bacteria, but do not wash with alcohol too long, then
rinse in distilled water, dry and mount.

The most useful dyes in the examination of animal tissues
for bacteria are those aniline dyes that are soluble in water ;
these are preferable to those soluble in alcohol only. They
have all great affinity for cell nuclei (Hermann) and belong
to the group of neutral or basic aniline colours. Methyl-
blue, methyl-violet, vesuvin, Bismarck -brown, magenta,
fuchsin, gentian-violet, Spiller's purple, rosaniline, Hum-
boldt's red (purple), are the dyes most commendable.

For staining of cover-glass specimens, as well as for sections
made of fresh tissues, the above dyes can be advantageously
used in the following manner : 2 to 5 grammes of the solid
dye are rubbed up in a mortar with 10 ccm. of absolute
alcohol ; add then gradually, while mixing, warm distilled
water, to bring up the total to 100 ccm. ; filter and keep in
stoppered bottle. For use, filter a little of the dye into a
watch-glass. For staining sections of tissues that have been
hardened, the above dyes prepared with Weigert's aniline
oil are preferable; they are prepared thus: (a) Make a
saturated watery solution of pure aniline (aniline oil) by
mixing in a bottle one part of aniline oil with three parts
of distilled water ; shake well every half hour for four to
six hours, decant the water as the oil settles to the bottom.
The decanted fluid is the saturated watery solution of aniline.
Of this take 100 ccm. Add to this (b) a saturated alcoholic
solution of fuchsin, magenta, gentian-violet, Humboldt's
red, methyl-blue or methyl-violet, n ccm. ; mix well, filter
into stoppered bottle. The sections are left in this dye for
from a few minutes to several hours (Humboldt's red
requires only a few seconds). Different bacteria require
different periods to stain. As a rule warming the dye

io MICRO-ORGANISMS AND DISEASE. [CHAP.

facilitates the staining of the bacteria; occasionally, also,
the addition of a few drops of liquor potassse. All sections,
after having been sufficiently stained, are transferred to and
washed in water, then methylated spirit, then in absolute
alcohol, then clarified in clove-oil, and finally mounted in
Canada-balsam (dissolved in chloroform, or better still in
benzol) or in Dammar varnish.

In order to bring out by the dye more conspicuously the
bacteria present in fluids or tissues various methods are used,
all of which are based on the principle that the bacteria have
an affinity to the dye which is greater than that of the tissue-
elements. Hence after staining, the tissue-elements may
be decolourised without abstracting the colour from the
bacteria. Cover-glass specimens or sections, after having
been well stained with a dye, are subjected to various
decolourising re-agents, whereby the tissue-elements become
deprived of the dye, but the bacteria retain it. Although in
some instances this is not easy of achievement, since by
such decolourising processes also the bacteria are liable to
lose the stain, it nevertheless is possible in the majority
of instances. In many cases prolonged washing in alcohol
absolutus and in clove-oil is sufficient to abstract the dye
from the tissue-elements, but in some special cases, owing to
peculiar chemical properties possessed by certain bacteria,
the decolourising process requires special methods. Of these
the following are the most useful : —

i. In some instances the specimens (cover-glass specimens
and particularly sections) are stained in one dye, then
washed in alcohol till quite pale, then transferred to a
contrast dye. As contrast dyes are to be regarded blue
and red, or red and brown, or blue and brown, or violet
and brown. In some cases only the bacteria retain the first
dye, the tissue-elements become stained by the second dye.

I.] MICROSCOPIC EXAMINATION. n

A similar result is often obtained by mixing the two dyes,
and then using them like a single dye ; hereby occasionally
the bacteria are found to take one colour, while the tissue-
elements take the contrast dye.

2. One of the most useful methods for staining bacteria
in sections of hardened tissues is Gram's method. Sections
are kept for ten minutes in absolute alcohol, are then placed
in any of the above mixtures of aniline oil and dye (fuchsin,
magenta, Humboldt's red or gentian-violet, methyl-blue or
methyl-violet), and kept here for from two to five minutes or
more ; they are then washed in alcohol for from one to three
minutes, and are then transferred into the following solution :
one part of iodine, two parts of iodide of potash, 300 parts of
distilled water ; they are kept here till their colour completely
changes (as a rule into dark purple), they are then transferred
into alcohol till all colour has apparently gone. If success-
ful, such sections when examined under the microscope,
show only the bacteria stained, while the tissue-elements are
quite colourless. To bring out these latter more strikingly
the sections are stained in a contrast dye, vesuvin or
Bismarck-brown, if red, violet, or blue has been used as the
first dye.

3. Ehrlich's method, used specially for demonstrating
tubercle-bacilli and leprosy-bacilli. — The specimens, after
having been well stained (from a few minutes to several
hours) with fuchsin or magenta aniline oil dye (see above),
are transferred into a 30 per cent, mixture of nitric acid ;
according to Friedlander a mixture of three parts of nitric acid
in 100 parts of alcohol is equally good. A 10 per cent,
watery solution of nitric acid is quite strong enough. All
bacteria except the tubercle-bacilli and leprosy-bacilli lose
the dye by this treatment. The preparations are then
stained for contrast in vesuvin or Bismarck -brown.

12 MICRO-ORGANISMS AND DISEASE. [CHAP.

For other methods to stain tubercle-bacilli see the chapter
on the latter.

4. Koch's method. — According to this the sections, after
having been stained, are transferred to a saturated solution
of carbonate of potash to which previously an equal volume
of water has been added. The preparations remain here
for from five to ten minutes, are then washed in water,
alcohol, clove-oil, and finally mounted in Canada-balsam
solution or Dammar varnish.

5. Lustgarten's1 method, used for the demonstration
of the syphilis-bacilli. — The sections are stained for from
twelve to twenty-four hours at ordinary temperature, and
then for an additional two hours at 40° C. in aniline oil gentian-
violet ; they are then washed for a few minutes in absolute
alcohol, and then transferred to a 1*5 per cent, solution of
permanganate of potash for ten seconds, then for the same
period into a watery solution of pure sulphurous acid; wash
in distilled water, repeat the above process of placing the
sections first into the permanganate of potash solution, then
into the sulphurous acid water till they become apparently
quite colourless. Only the syphilis-bacilli, tubercle-bacilli
and leprosy-bacilli, are able to retain the dye ; other
bacteria lose it by being subjected to the permanganate.

De Giacomi 2 has improved this method of decolourising
by oxidation. Cover-glass specimens made of syphilis material
are stained with warm fuchsin for a few minutes, are then
washed in water to which a few drops of solution of iron
perchloride have been added, then placed into concentrated
solution of iron perchloride till the preparations have lost all
colour; they are then stained for contrast in vesuvin or
Bism arck-bro wn.

1 Lustgarten, Med. Jahrbiicher der K.K. Ges. d. Aerzte, Vienna, 1885.

2 De Giacomi, Schweizer Correspondezblatt, xv. 12.

i.] MICROSCOPIC EXAMINATION. 13

A. Gottstein1 places sections of syphilis material for
twenty-four hours in fuchsin or aniline oil gentian-violet ;
wash with distilled water, then place them for a few seconds
into a pure or dilute solution of liquor ferri, then wash in
alcohol, clarify in clove-oil, mount in Canada-balsam.

It may not be unnecessary to point out, that if sections are kept for
many hours in the staining fluid, there may be found in them micro-
organisms (particularly bacilli) which have been accidentally introduced
into them by the solutions of aniline dye. Many of these, particularly
when used alkaline, contain organisms, and if the sections are kept
in them for many hours, notably in warm weather, bacteria will be
found to have not only invaded the tissue but to have multiplied
therein.

In examining fresh or hardened tissues for micro-organisms
it is necessary to make thin sections, which can be easily
done with the aid of any of the microtomes in common use,
amongst which Williams's microtome, and especially Dr.
Roy's ether-spray freezing microtome, are no doubt the best
and easiest to manipulate. As regards hardened material, it
is necessary to remember that the hardening must be carried
out properly, small bits — about a half to one cubic inch — of
tissue being placed in alcohol, or better, in Miiller's fluid, and
kept there ; in the first instance, for two to five days ; in the
second, for from one to three weeks or more. Then small
bits are cut out, of which it is desired to make sections.
Those hardened in spirit must be soaked well in water to
enable the material to freeze, then superficially dried with
blotting-paper, and then used for cutting sections with Roy's
microtome. Those hardened in Muller's fluid are at once
superficially dried with blotting-paper and cut. When
making sections with Williams's freezing microtome it is
necessary to soak the material first in gum mucilage and then

1 A. Gottstein, Fortschrittc d. Medizin, Berlin, 1885, No. 16, p. 545.

14 MICRO-ORGANISMS AND DISEASE. [CH. r.

to freeze and to cut. Fresh tissues are at once cut with the
freezing microtome, the sections placed in a 0-6 per cent,
saline solution, floated out and well spread out, and then
stained by transferring them in this condition, i.e. well
spread out, into a watch-glass containing the dye. The
sections of hardened tissues are floated out in water, well
spread out, and then transferred to the dye or dyes as the
case may be.

It is necessary to prevent too much shrinking of the
sections, especially those of fresh tissues ; for this reason it
is advisable to float the sections in the saline solution or water,
as the case may be, on a broad lifter or spatula, to spread
them well out upon it, and to transfer them carefully into
the dye, then into the dish with water used for washing off
the excess of the dye, to transfer them, well spread out on the
lifter, to alcohol, then after several minutes to oil of cloves,
and finally on to a glass slide, on which they are mounted in
the usual manner with Canada-balsam solution, the excess of
clove-oil being previously drained off.

It is advisable, although not absolutely essential, to keep
the sections in a well-spread-out condition for a few seconds
in alcohol before placing them into the dye.

Microphotography, by which microscopic specimens of
bacteria are photographed, have hitherto yielded results so
unsatisfactory, that even Koch, who first introduced it, has
abandoned it in lieu of accurate drawings made in the usual
manner.

CHAPTER II.

PREPARATION OF CULTURE MATERIAL.

ARTIFICIAL cultivations of micro-organisms in suitable
nourishing media in the incubator (Fig. i) at temperatures

FIG. i — INCUBATOR, WITH PAGE'S REGULATOR.

A. Page's Regulator. — This consists of a tube filled with mercury, and immersed
in the water surrounding the chamber of the incubator. In the upper part of the
tube, above the mercurial column/ is a fine open glass tube, having near the lower

16 MICRO-ORGANISMS AND DISEASE. [CHAP.

end a fine hole. When the temperature of the water rises, the mercurial column
rises, and at a certain temperature rises above th» lower open end 01 the small inner
glass tube just mentioned. If this point is reached, then the burner at C receives
only the amount of gas that passes through the fine lateral hole of that inner glass
tube. If the temperature of the water falls, the mercury falls, and the lower end of
the inner glass tube becomes again free, and now the burner at C receives a much
greater supply of gas. If so, the temperature of the water again rises, the mercury
rises, obstructs the lower end of the inner glass tube, the supply of gas is reduced to
what can pass through the fine lateral hole, and consequently the temperature again
falls, and so on. To adjust the regulator it is necessary when the thermometer in-
dicates the required degree of temperature to push the outer large glass tube, and
with it the inner tube, of the regulator so far down that the top of the mercurial
column just obstructs the free end of the inner glass. The temperature then regulates
itself for the reasons stated previously. These regulators are sufficient for all
practical purposes when it is not a question of small differences in temperature, since
they are tolerably constant within one or two centigrades. The trouble one ex-
periences in the working of these and other similar regulators arises from the incon-
stancy of the main gas supply, this, as is well known, varying within wide limits.
The stopcock, E, obviates this to a limited extent ; when this is put at an angle of
45° only a limited amount of gas passes from the main supply tube to the regulator,
and therefore the variations in pressure of the gas are not felt to their full extent.
A Sugg's regulator interposed between E and the main supply tap is very useful.

B. Thermometer to indicate the temperature in the cha:nber.

C. Gas burner.

D. Chamber of incubator.

E. Stopcock to regulate, when required, the supply of gas.

F. Main supply. — The upper, lower, right and left walls of the incubator are
made of a double layer of tin ; between the two is water. The front and back of
the chamber are closed by a movable glass plate.

An excellent incubator for constant temperature is made by the Cambridge
Scientific Instrument Company. It has a double gas supply : one small permanent
flame, and a second one subject to the regulator.

varying between 20° and 38° C., are necessary in order to
study more accurately the life-history of the septic as well
as the pathogenic organisms. Moreover, large numbers of
them become available in a short time, and their relation to
disease can be tested more conveniently. For if it should
be found that, having carried on outside the animal body
successive cultivations of a particular organism, the re-
introduction of this cultivated organism into the animal body
is again productive of the same disorder as before, then the
conclusion becomes inevitable that this organism is intimately
related to the causation of the disease. It must be conceded
that after several successive cultivations in fluids any hypo-
thetical substance supposed to be the materies morbi, and
introduced at first from the blood or tissues, being in a very
diluted condition in the first cultivation, would after several

II.] PREPARATION OF CULTURE MATERIAL. 17

cultivations be practically, lost. But if this last cultivation
should be found to act in the same manner pathogenically,
i.e. if every droplet of it, charged with the new brood of the
organism, nevertheless possesses full pathogenic power, then
it is logical to say that this pathogenic property rests with
the organism. For this and other reasons it is of essential
importance to be able to carry on successive cultivations
of one and the same organism without any accidental con-
tamination or admixture, /.<?. it is necessary to carry on pure
cultivations.

ARTIFICIAL CULTIVATION MEDIA.

A. — FLUIDS.

As fluid nourishing material the following are used with
preference : —

i. Broth made from Meat — pork, beef, rabbit, chicken. — The
connective tissue and fat are first cut out from the fresh meat
— in the case of rabbit or chicken the whole animal without
head or viscera is used — and then placed in water and boiled.
Generally for each pound half an hour's good boiling is
allowed. With regard to the quantity of water, each pound
of meat ought to yield ultimately at least one pint of broth.
When boiled, the broth is allowed to stand, the fat is skimmed
off, and the broth well neutralised, or even made faintly
alkaline by adding liquor potassae, or, better still, carbonate
of sodium.

The fresher the meat the less acid (sarcolactic acid) is in
the broth before neutralisation. The broth is then filtered
through a filter, previously overheated (see below), into flasks
previously sterilised (see below). As a rule beef broth is

c

iS

MICRO-ORGANISMS AND DISEASE. [CHAP.

clear, but if not it is filtered again. If not clear then, it is
allowed to stand for several hours. A fine sediment is found
at the bottom of the vessel, and from this the clear super-
natant fluid is decanted into a sterilised vessel. The broth,
if not clear after the first filtering, can be cleared by boiling
it with the broken shell and white of egg. The now clear
fluid is filtered again. The flasks which receive the broth,
are well plugged with sterilised cotton-wool (see below). In

. 2. — A BUNSEN BUK^R WITH
ROSE FOR BOILING FLUIDS IN
TEST-TUBES.

FIG. 3. — A FLA-<K. CONTAINING STERILE
STOCK FLUID.

this state the flask is placed over a Bunsen burner (Fig. 2)
on a wire netting and boiled for half an hour or more ;
during the boiling the cotton-wool plug is lifted out for half
its length. The flask ought not to contain more broth than
about one-half or two-thirds of its volume, to prevent the
broth from rising too much and wetting the plug. When
turning off the flame the plug is pushed down so as fully
to plug the neck and mouth of the flask ; a beaker with
sterile cotton-wool cap is placed over the mouth of the flask

IL] PREPARATION OF CULTURE MATERIAL. 19

(Fig. 3), and this is allowed to stand for one night. Next
day the boiling is repeated for half an hour or more in the
same manner as before. If the meat has been fresh and the
vessels and cotton-wool have been sterile, twice boiling is
found sufficient to destroy every impurity. But to make
sure, the broth is placed in the incubator and kept there for
twenty-four hours at a temperature of 32° to 38° C., and then
boiled on the next day for half an hour in the usual way.
The supposition is made, that if by any chance after twice
boiling the broth it should contain unchanged spores of
bacilli — the only organisms that will resist boiling, although
they do not resist boiling for more than half an hour — the
spores would germinate into bacilli when kept for twenty-
four hours in the incubator at 32° to 38°, and these would
then be killed by the third boiling. As a matter of fact I
have not as a rule found any contaminating germs survive
the second boiling. It is of course to be borne in mind
that during the first as well as second and subsequent boiling
the cotton-wool plug is not removed from the mouth of the
flask, but is only raised out half its length from the neck.
The cotton-wool and the cotton-wool cap and beaker are
replaced immediately or simultaneously with the turning off
of the burner. This broth so prepared is placed in the
incubator at 32° to 38° C. and kept there from one to three
weeks. If, as is generally the case, it remains limpid, it is
considered completely sterile.

2. Peptone and Sugar Solution. — Beef peptone (Savory
and Moore's) is dissolved in distilled water, over a burner,
to the amount of about 2 per cent. ; to the solution is added
cane-sugar to the amount of about i per cent. ; so that every
100 ccm. of the fluid contains two grammes of peptone and
one gramme of sugar. When dissolved it is well neutralised
and then filtered (the vessels being of course also in this, as

C 2

20 MICRO-ORGANISMS AND DISEASE. [CHAP.

in all other cases, sterilised by overheating) into flasks, and
treated in the same manner as the broth.

An excellent fluid is beef broth with i per cent, of peptone,
the mixture is then made faintly alkaline, boiled and filtered
into sterile flasks, then well boiled to serve as stock, or to
be at once decanted into test-tubes.

3. Buchner's Fluid. — 10 parts of Liebig's extract, and 8
parts of peptone, in 1,000 parts of water.

4. Hydrocele Fluid (Koch). — A new or well sterilised (by
over-heating) trocar and cannula are used for the tapping ;
to the cannula is fixed an india-rubber tube that has been
soaking in strong carbolic acid solution for forty-eight hours.
The distal end of the tube is introduced carefully and rapidly
into the neck of a sterilised flask plugged with sterile cotton-
wool, and the fluid thus allowed to flow into the flask to about
two-thirds of its volume. This fluid is then decanted into
sterile test-tubes (plugged with sterile cotton-wool), each tube
receiving about 5 to 8 ccm. The tubes are then exposed in
the incubator to a temperature of from 55° to 60° C. for
three to five hours on five or six consecutive days.

5. Blood Serum (Koch). — A glass cannula and india-rubber
tubing are soaked for forty-eight hours in strong carbolic acid ;
the cannula is tied into the carotid artery of a healthy horse,
and the arterial blood, after opening the clip at the proximal
end of the artery, is allowed to flow into sterile flasks, or
cylinders with stoppers. After letting the blood stand for
12 to 24 hours in a refrigerator or in an ice box, the serum
is taken off by means of large sterile glass pipettes and
introduced into sterile test-tubes, each receiving about 5 to
8 ccm. The test-tubes, plugged with sterile cotton-wool, are
then exposed in the incubator to a temperature of 58° to 62*
C. in the same manner and for the same time as the
hydrocele fluid was.

II. J PREPARATION OF CULTURE MATERIAL. 21

6. Urine is neutralised and sterilised by boiling for 20 to
30 minutes like broth.

7. Milk (ordinary) is sterilised by gentle and careful
boiling for 20 to 30 minutes.

Of less common use are :

8. Pasteur's Fluid. — In 100 parts of distilled water are
dissolved 10 parts of pure cane-sugar, i part of ammonium
tartrate, and the ash of i part of yeast.

9. Cohris Fluid. — 100 ccm. of distilled water, i gramme
of ammonium tartrate, no sugar, and instead of the ash of
yeast are substituted (A. Mayer) 0-5 gramme of potassium
phosphate, 0*5 gramme of crystallised magnesium sulphate,
0*05 gramme ot (tribasic) calcium phosphate. These two
fluids are sterilised in the same manner as the broth and
peptone solutions. Pathogenic organisms do not thrive in
either of these two fluids.

B. — SOLIDS.

The solid media have the great advantage over the fluids
that in the former artificial cultures can be carried out more
easily ; as, owing to the resistance the solid basis offers to
the growth of the organisms, they remain more limited
to the spot or spots on which they are sown, and therefore
can be watched more easily ; besides, an accidental contami-
nation, i.e. a growth appearing at a spot at which no sowing
was made, can be recognised at once. These advantages
are perhaps of the greatest use when it is intended to
grow the organisms on a surface exposed to the influence
of air — of course protected from contamination with other
organisms.

These advantages of solid media have been very minutely

22 MICRO-ORGANISMS AND DISEASE. [CHAP.

pointed out by Koch in his researches on pathogenic
bacteria.1

As solid media are used :

1. Slices of Boiled Potato or Boiled White of Egg or Paste
(Fokker, Schroter, Cohn, Wernich). — A boiled potato or a
boiled unshelled egg is cut in half with sterile scalpel, and
the cut surface is inoculated. Immediately after, it is placed
on a clean glass plate and covered with a bell-glass, the
edges of the latter being fixed on the former by vaseline or
grease, the chamber is kept moist by a piece of wet blotting
paper being placed inside the bell-glass. The progress of
the growth of a particular organism or of different organisms
sown out at a particular spot or line on the surface of these
substances can be easily watched with the unaided eye.

2. Gelatine (Brefeld, Grawitz, Koch). — This is used ad-
vantageously as a mixture with broth, peptone, beef-extract,
blood serum, or hydrocele fluid. Koch, who introduced
this mixture, used it for the cultivation of bacteria on solids,
to be exposed to the air ; the proportion of gelatine in the
mixture was 2 to 3 per cent. But this mixture, although
solid at ordinary temperature, does not keep solid in the
incubator, not even at 20° C. I have found that at least
7-5 per cent, of gelatine must be contained in the mixture
to keep it solid at 20° to 25° C. Above this temperature
not even 1 1 per cent, gelatine will keep solid.

Nutrient Gelatine, most useful for the growth of all kinds
of bacteria, is prepared in this way :

One pound of lean beef is cut up, to it is added one pint
of water, and is kept boiling in the digester or any other
vessel for from half to three-quarters of an hour. After
having been strained through fine calico it is filtered through
paper into a beaker ; bring up by adding water to 600 ccm. ;
1 Mittheilungen d. k. Gtsundheitsamtes, i. 1881.

II.] PREPARATION OF CULTURE MATERIAL. 23

add to this 60 grams of the finest gold label gelatine cut up
in small pieces, 6 grams of peptone and 6 grams of common
salt. Dissolve on waterbath, but do not let the water boil ;
neutralise with carbonate of soda or, better, liquor potassae
till faintly alkaline ; boil for half an hour, filter by hot filter,
(see Fig. 6) into a sterile flask plugged with sterile cotton-wool,
and bring it up to boiling point, at which it is kept for a few
minutes. This can be kept as stock gelatine, or can be
decanted at once into sterile test-tubes plugged with sterile
cotton-wool. Keeps solid up to about 25° C.

Prepared in this manner the nutrient gelatine passes easily
and comparatively rapidly through filter paper on hot-filter.

The same 10 per cent, nutrient gelatine can be of course
obtained if broth is already made, e.g. broth in a stock
flask, by adding the above-named quantities of gelatine
peptone and salt to 600 ccm. of the broth ; further process
is as above.

3. If it is necessary to expose the cultivation to higher
temperatures than 25° C., the nutrient gelatine cannot be used
as a solid medium. Solid blood serum or solid hydrocele
fluid (Makins) or solid Agar-Agar mixture (Koch) must then
be employed.

The first, i.e. the serum of blood, and the second, i.e. the
hydrocele fluid, can be made solid by heating the above sterile
serum or hydrocele fluid in tubes (see page 20) gradually
up to 68° or 70° C. When this temperature is reached
the material soon turns solid, losing slightly its limpidity,
but is sufficiently transparent for all practical purposes. By
heating it rapidly, or heating it above 7 2°, it becomes solid,
granular, and opaque. Of course, once thus made solid it
cannot be liquefied again, and therefore must be already
contained in the vessels (test-tubes and small flasks) in which
the growth of organisms is to be carried on. Or blood serum

24 MICRO-ORGANISMS AND DISEASE. [CHAP.

and hydrocele fluid can be rendered solid by exposing the
sterilised material (see above), in sterile plugged test-tubes, to
a moderate heat — e.g. in the incubator at 32° to 38° C. — for
several weeks. Through evaporation the material is rendered
solid. Thus treated it retains its limpidity in a perfect
manner.

AGAR-AGAR, or Japan isinglass, can be obtained1 in the
shape of thin transparent lamellae, or more usually as masses
of transparent narrow bands. For cultivation purposes the
following mixture is prepared :

1. Place in a beaker Agar-Agar 10 grams, common salt 10
grams, distilled water 900 ccm. Dissolve over the flame and
boil for twenty minutes, strain, and filter through hot-filter
into a sterile flask plugged with sterile cotton-wool.

It ought to be mentioned, that in all cases where any material (broth,
peptone sugar, nutrient gelatine, or Agar-Agar solution) is filtered into
a sterile flask the cotton-wool plug is taken off, and the glass tube of
the filter having been inserted into the neck of the flask, the cotton-
wool is so replaced that it well guards the mouth of the flask.

2. Place into a clean beaker 100 ccm. of distilled water, add
10 grams of .peptone, and two small tins of Brand's meat
extract ; dissolve over flame, add liquor potassae till faintly
alkaline, boil, filter, and add the filtrate to the above Agar-
Agar solution. For the two tins of Brand's, 10 grams of
Liebig's meat extract can be substituted. Thus a mixture is
obtained which contains, besides meat extract, i per cent, of
Agar-Agar, i plr cent, of peptone and i per cent, of salt.
This is then boiled for half an hour and can be kept as stock
material or can be at once decanted into sterile test-tubes.

1 Messrs. Christy and Co., of 155 Fenchurch Street, have succeeded
in obtaining for me large quantities of this material from Paris. I
understand from my friend Dr. R. Maddox that this substance is in
reality what the French call Gelose.

II.] PREPARATION OF CULTURE MATERIAL. 25

It keeps solid up to a temperature of 50° C., i.e. a tempera-
ture higher than is ever necessary for the growth of bacteria.
It becomes liquid at higher temperatures, and in case of
necessity can be again subjected to boiling. Before con-
sidering it as perfectly sterile it ought to be kept like all
other materials for from several days to several weeks in the
incubator at 32° to 38° C. If quite limpid after this time it
may safely be considered as sterile.

Amongst all the solid media, I have found this mixture of
Agar-Agar and peptone meat extract to be excellent in many
respects. It is beautifully limpid and solid, and an excellent
nourishing material. Agar-Agar alone without the admixture
of peptone is not satisfactory as a culture medium.

The materials which I now prefer for the cultivation of
pathogenic and septic bacteria are : i. Broth (beef) and
peptone; 2. Solid nutritive gelatine; 3. Solid Agar-Agar
peptone meat extract ; 4. Fluid and solid serum of blood ;
all faintly alkaline.

CHAPTER III.

VESSELS AND INSTRUMENTS USED IN CULTIVATIONS.

ALL vessels (flasks, test-tubes, beakers, filters, calico) to be
used are first thoroughly sterilised by overheating. In the
case of flasks and test-tubes, this can be done by exposing
them thoroughly in all parts to the open flame of a large
Fletcher's burner ; while thoroughly heated the mouth is
plugged with a good long plug (i to 2 inches) of sterile
cotton-wool, this being pushed in by means of overheated
forceps. The plug in all cases must not be loose, but also
not too firm — an error in the latter direction being of course
preferable to one in the former. The cotton-wool plug may,
if long enough, be single ; or, if short ones are used, double.
Or the flasks and test-tubes are placed in an air-chamber
(see Fig. 4) heated by a large Fletcher's burner for several
hours, up to between 130° and 150° C. In the case of small
flasks and test-tubes this process is of course much more
convenient, since a large number can be heated simul-
taneously. Beakers and glass filters to be used merely for a
temporary operation are placed over a wire net on a tripod
and heated by the flame of a Bunsen's burner. In the case
of test-tubes which are to receive cultivation-fluids, I generally
expose them, after having been cleaned and dried, in the air-

CH. in.] VESSELS, ETC., USED IN CULTIVATIONS. 27

chamber for several hours (three to six) to a temperature of
from 130° to 150° C. : while hot they are taken out seriatim,
plugged with the sterile cotton-wool, and replaced in the air-
chamber, and heated again for several hours. All this, and
other operations to be described below, may appear to some
rather tedious and unnecessarily complicated, but it cannot
be too strongly insisted on that in these matters one cannot

FIG. 4. — HOT-AIR CHAMBER FOR STERILISING TEST-TUBES AND COTTON-WOOL.

An iron chamber with double wall, the inner chamber having separate folding
doors. In the inner chamber are placed the test-tubes, glasses, &c., and the
cotton-wool, the latter in a loose condition. Both sets of doors are closed, and
the apparatus heated by a large Fletcher's burner. A thermometer passing from
the inner chamber through the upper wall indicates the temperature of the chamber.

be too scrupulous. A slight relaxation may, and occasionally
is, followed by disastrous consequences in the shape of
accidental contamination, and consequent loss of material
prepared at the cost of much labour and time. Long
experience in these matters has taught me that, although in
some instances less scrupulous care has not been followed

28 MICRO-ORGANISMS AND DISEASE. [CHAP.

by bad results, still I have had also many unpleasant failures
owing to slight laxity in these matters.

Several weeks' work may be annihilated by a single omis-
sion. Sometimes one is perhaps in a slight hurry, and does
not think the want of an additional heating of the test-tube
or cotton-wool or an additional boiling of the fluid will be
followed by any bad consequences. But, alas, nature does
not take into account our convenience, and failure is our
reward. If in any kind of experiments " overdoing ". is an
error in the right direction, it is in these very experiments in
the cultivation of micro-organisms.

The cotton-wool used for plugging flasks and test-tubes is
prepared by pulling up loosely a quantity of good cotton-
wool and exposing it in a loose state in the air-chamber to a
temperature of 130° to 150° C. for several hours for several
successive days. The cotton-wool ought to be just brown,
i.e. just singed. Too much charring makes it brittle, and
it is then difficult to make of it a satisfactory plug. The
plug used should not be too firm and not too loose : in the
former case it is not easy to lift it up quickly, and in the
latter it does not close sufficiently well. Cotton-wool that
has been kept, say only for a day or two in the air-chamber
for three or four hours is not absolutely sterile ; nor is
cotton-wool that has been kept in a compressed state in the
air-chamber for any number of days. The central portions
remain under these conditions quite white and are not
sterile. No cotton-wool that is not just brown, i.e. just
singed, is safe from risk of impurity. No cotton-wool
steeped in absolute alcohol, strong carbolic acid, or any
other disinfecting fluid, for ever so many days or weeks,
can be absolutely relied on.

As stated above, a plug of sterile cotton-wool tolerably
firm, of about one to two inches, or two plugs of about one

Hi.] VESSELS, ETC., USED IN CULTIVATIONS. 29

inch each, are used for the plugging of the flasks and test-
tubes. An assertion such as that made by Dr. Williams at
the British Association (Biological Section, September 1883),
that cotton-wool plugs are not reliable, because
they do not protect the fluids in the vessels
plugged with them from accidental air-contami-
nation, is to be accepted only as applying to
very loose plugs and to cotton-wool not properly
sterilised. To good firm plugs of sterile cotton-
wool it evidently cannot apply, since all the
results of all workers in this field (Pasteur,
Sanderson, Cohn, Koch, Klebs, Buchner, and
many others) are against it.

Instruments^ such as the points of needles, and
forceps, used in the processes of cultivation, lift-
ing up cotton-wool plugs, making cotton-wool
plugs, inoculations, &c., must be heated in the
open flame of a Bunsen burner, if they are to
be absolutely relied on for cleanliness. Scissors
and knives used for cutting tissues which are
intended for inoculation, ought to be likewise
scrupulously clean. One ought to keep a
special set of instruments, the blades of which
are capable of being heated in the open flame
without being spoilt.

Syringes used for cutaneous, subcutaneous, or
other inoculations, ought to be capable of being
overheated. The ordinary Pravaz syringe of
vulcanite not being capable of undergoing this
process, Koch has devised a glass syringe
similar to the Pravaz syringe. I do not use any syringe for
inoculation, but prefer using each time a fresh capillary
glass pipette made just before the inoculation. Into this

j

i-

Q S

30 MICRO-ORGANISMS AND DISEASE. [CHAP.

pipette I draw the droplet to be used for inoculation, and
having made a very small incision — about J of an inch —
through the skin, the pointed end of the pipette is pushed
forward into the subcutaneous tissue for about half an inch
or one inch and then the fluid is blown out into the tissue:

FIG. 6.— HOT-WATER FILTER FOR FILTI-RING NUTRITIVE GELATINE OR
AGAR-AGAR MIXTURE.

In this way I am always absolutely safe from any contami-
nation with a previously used virus, which might possibly
adhere to one or other part of a syringe.

The fine point of capillary pipettes (Fig. 5), used for in-
oculation of animals, or for drawing out a drop of fluid of a

HI.] VESSELS, ETC., USED IN CULTIVATIONS. 31

cultivation in a flask or test-tube, or for inoculating material
contained in a test-tube or flask, are thus made : while one
hand holds the bulb of the pipette, the other holds one end,
and putting at some distance from this end the tube into an

FIG. 7.— STEAMER FOR STERILISING CULTURE MATERIAL CONTAINED IN

TEST-TUBES.

i. Wire-net to hold the test-tubes ; 2. Tin vessel ; 3. Wire diaphragm to hold i ;
underneath it is water ; 4. Lid; 5. Gas-burner.

ordinary flame and quickly drawing it out, a point of extreme
fineness can be made. The same is done with the other end
Such a pipette can be considered as practically closed at both
ends.

CHAPTER IV.

PREPARATION OF CULTURE-MEDIA FOR INOCULATION.

WE have on a former page described the methods to obtain
sterile stock of nourishing media suitable for artificial cul-
tivations. The solids, as serum gelatine, serum and hydro-
cele fluid, must, before solidification, be placed in test-tubes
and small flasks, and then sterilised in the manner above
described, to be made ready for establishing cultures, i.e. for
inoculation. The Agar-Agar mixture however can, like
broth, peptone mixture, beef extract solution, and gelatine
mixtures, be kept as stock in large flasks. When thus sterile
these latter can be decanted into a number of test-tubes or
small flasks, in which the cultivation is to be carried out.
Gelatine mixtures (gelatine and broth, gelatine and peptone,
gelatine and beef-extract) and the Agar-Agar mixture, must
of course be liquefied over a flame before being ready for
decanting. The test-tubes most suitable are about six inches
long, and should not be less than about one inch broad ; the
flasks are about of the capacity of one to two ounces, and
ought to have a neck of comparatively good width. The
test-tubes receive the fluids for about one and a-half to two
and a-half inches in depth, the flasks for about one-fourth to
one-third of their bulk. All these test-tubes and flasks with

CH. iv.] PREPARATION OF CULTURE-MEDIA. 33

their cotton-wool plugs, before receiving the material, should
be thoroughly sterilised by overheating. As I mentioned in
the previous chapter, this ought to be well borne in mind, for
starting with a sterile nourishing fluid — i.e. one that has been
kept in the stock flask for several days to several weeks in
the incubator at a temperature of from 32° to 38° C. and that
has remained perfectly clear and limpid — and working with
thoroughly sterilised test-tubes and cotton-wool plugs — very
little care is required to obtain sterile material ready for
inoculation. To start with a stock of nourishing material,
however well sterilised, and to decant it into test-tubes with
cotton-wool plugs not absolutely sterile must lead to failure.
I have seen this happen over and over again, and all the
material decanted became consequently contaminated and
thereby useless for inoculations. The test tubes and flasks
must be well cleaned, then dried, placed in the air-chamber,
and kept there exposed for several hours to a temperature of
from 130° to 150° C. on several successive days, or they
may be thoroughly heated in all parts over the open flame
of a gas-burner. The same applies to the cotton-wool, as
mentioned in a former chapter. The test-tubes and flasks
are plugged by means of clean forceps with the cotton-wool
which is just brown, and then replaced in the air-chamber
and again heated for several hours on two or three occasions
up to a temperature of 130° to 150° C., or they may be well
heated over the open flame of the burner. To decant sterile
stock fluid into these test-tubes and flasks I proceed thus :
A clean beaker with spout, covered with a clean glass plate,
is placed on a wire net on a tripod over the flame of a
Bunsen burner, and thoroughly heated for half an hour or
so ; then it is allowed to cool, and when cool, the plug of the
stock flask is lifted with forceps, and some of the sterile
fluid quickly poured from the flask into the beaker. The

D

34 MICRO-ORGANISMS AND DISEASE. [CHAP.

plug is replaced in the neck of the stock flask and the
beaker covered with the glass plate. Of course the quantity
poured into the beaker should be large enough to supply the
required number of test-tubes or small flasks. The stock
flask containing still some fluid, having been opened for
however short a time, has of course been exposed to air-
contamination, and therefore must be treated accordingly, if
the fluid left in it is to serve as sterile nourishing material on
a future occasion. Consequently it is subjected to boiling
for from fifteen to thirty minutes, then placed in the incubator,
boiled again the next day and put back in the incubator,
where it is left at a temperature of from 32° to 38° C. for
several days. If after a week or so the fluid remains limpid,
it is of course to be considered sterile.

Next, the fluid that has been poured into the beaker
(covered with the glass plate) is poured as quickly as possible
into the test-tubes, one after the other, by lifting with clean
forceps the plug and pouring in the fluid to a depth of
one and a half to two and a half inches, and the plug
replaced.

During this procedure contamination with air-organisms, if
there be any about, becomes inevitable. To lessen this
chance as much as possible, it is necessary to lift the plug
with clean forceps, to pour the fluid as rapidly as is practicable
into the test-tube or flask, and to replace immediately the
cotton-wool plug. Further, it is necessary to bear in mind,
that the atmosphere is not at all times and everywhere
equally contaminated (see Prof. Tyndall's observations). I
generally avoid undertaking this process on windy days, and
when I do it, I generally close windows and doors and keep
the air in the room as still as possible. I do not do it in a
room in which recently (say an hour or two previously) the
floor, walls, or tables have been swept.

iv.] PREPARATION OF CULTURE-MEDIA.

35

I have opened under these conditions the plugs of test-
tubes containing sterile material, and kept them so for a
time varying from one to ten seconds, and in some in-
stances I have not seen more than from 10 to 20 per cent,
contaminated, often less.

Now, having filled the required number of test-tubes and
flasks with the desired quantity of fluid, I subject these
seriatim to boiling. By means of an ordinary test-tube

FIG. 8. — A BEAKER CONTAINING A NUMBER OF CULTURE-TUBES PLUGGED
WITH COTTON-WOOL.

holder I hold them above a very small flame until the fluid
boils, and keep it so boiling for from two to five minutes.
During this process of boiling the cotton-wool is only slightly
pulled up, and immediately before ceasing to boil the plug is
again replaced, aud pushed down with a clean glass rod.
Then the test-tube is placed (of course upright) in a beaker
at the bottom of which a layer of cotton-wool — a sort of

D 2

36 MICRO-ORGANISMS AND DISEASE. [CHAP.

cushion — has been placed. When finished, the test-tubes in
the beaker are all transferred to the incubator and kept
there for from twelve to twenty-four hours at a temperature
of 32° to 38" C. Then the boiling is repeated once more.
After this they are kept in the incubator for several days to
several weeks. I generally keep them there from two to three
weeks, and all those in which the fluid has remained limpid
and clear are considered sterile and ready for use. As a rule,
starting with sterile stock fluid, and using thoroughly sterile
test-tubes and cotton-wool plugs, after once or twice boiling
after decanting there ought to be no loss of tubes through
accidental contamination with air-organisms (during de-
canting). Sometimes, however, I have had loss to the
amount of 5 per cent, or more, but then there was always a
hitch of some kind traceable. To decant under carbolic acid
spray is not practicable and possesses many unpleasant draw-
backs, besides, in some instances when I used it there was
really a greater percentage of contaminated tubes than with-
out it. I therefore do not use the spray.

A simple method is to subject the whole number of test-
tubes into which the nutritive material had been decanted
(broth, peptone broth, nutritive gelatine, Agar-Agar mixture,)
to a steamer (see Fig. 7). The test-tubes are placed into the
wire net (see figure), the top of the group of test-tubes is
covered with tinfoil, so as to protect the plugs from becoming
wet, and then the wire net is placed into the steamer — the
water at the bottom of which has been previously heated to
boiling, — the lid is put on and the steaming is kept up for
from fifteen to twenty minutes. This is repeated on one or
two successive days. I have not seen any tubes go bad,
after they have thus been steamed on three successive days
each time for twenty minutes. Placed in the incubator and
kept at a temperature of 35° to 38° C., from some days to one

iv.] PREPARATION OF CULTURE-MEDIA. 37

or two weeks, they remain free of any growth and are to
be considered sterile.

Test-tubes containing solid nourishing material are
generally kept sufficiently inclined during solidification of the
material (see a former chapter) to allow the material to spread
into a layer of large area ; although useful, it is not essential.

When test-tubes with sterile fluid blood-serum are to be
subjected to the process of solidification (see page 23), it is
advisable to keep the tubes in a slanting position, so as to
allow the serum to spread out into a layer which is suffi-
ciently transparent even after solidification. For this
purpose I place these test-tubes into a double-walled tin
trough, about five inches broad, twenty inches long ; between
the two walls and also inside the trough is water of the tem-
perature of 62° to 65° C. The test-tubes are placed side by
side, so that they rest with the bottom part on the floor,
with the top part on the opposite ledge of the trough. The
difference of level is such, that it allows the serum to spread
out in the test-tubes into a large area. By means of burners
the temperature of the water is then gradually raised to
68° to 70° C.

In order to protect the solid materials, contained in test-
tubes, from drying up, it is advantageous to keep them in
large wide-mouthed bottles with well-fitting (by vaseline,
grease, or otherwise) glass stoppers. This is particularly
necessary in the case of test-tubes, containing Agar-Agar
mixture or blood-serum, and which are to be kept in the
incubator for many days or weeks at a temperature of 35° to
3 C.

CHAPTER V.

METHODS OF INOCULATION.

HAVING now in test-tubes and small flasks sterile material
ready for inoculation, it is necessary to describe the mode
of inoculating the same. .

i. Inoculations from Artificial Cultures. — The first and
simplest is the case where it is required to inoculate a new
tube or flask with a definite organism that has been growing
previously in a culture-tube ; that is to say, where it is
required to establish from an artificial cultivation a new
and further artificial cultivation. Take a freshly drawn-out
capillary pipette, with a fine point, as described in a former
chapter ; draw up with clean forceps slightly the top part of
the cotton-wool plug of the old tube or flask, push carefully
and gently one of the pointed ends of the capillary pipette —
the other can be broken off blunt — through the remaining
part of the cotton-wool plug, and push it downwards till it
emerges into the culture-fluid, or, if this be solid material,
till it reaches the spot or place where the organism is growing ;
allow a small droplet to ascend into the capillary pipette,
which it readily does by capillarity ; or if a larger quantity is
required draw it up by gently sucking at the outer end of
the capillary pipette. Then draw the capillary pipette

CH. v.] METHODS OF INOCULATION. 39

altogether out of the tube and cotton-wool plug, and push
this latter down with the forceps into its former position.
Immediately after this proceed to inoculate the new culture-
tube by doing exactly the same as before, viz., draw up
slightly with the forceps the top part of its cotton-wool plug,
push through the remainder of this plug the pointed end of
the capillary pipette, i.e. the one containing the droplet of
the material to be sown, and push it into the material at the
bottom of the test-tube or flask. A trace of the sowing
material flows out by itself, or, if a large quantity is required,
it is carefully blown from the pipette, but, of course, not
so that the tube is emptied by the blowing. If the sowing
is to be carried out on the surface of solid material, the
seed is deposited on the surface ; if in the depth, the end of
the pipette is pushed down into the depth of the material
and the seed there deposited. The pipette is then altogether
withdrawn and the plug replaced as before. The new
tube is then placed in a beaker on a cushion of cotton-wool,
and exposed to the required temperature in the incubator.

If we have, however, a culture-fluid or any fluid that con-
tains, as the microscopical examination proves, various species
of organisms, which we wish to isolate, then the method of
Klebs of " fractional cultivation," or the method of Lister
and v. Nageli of " dilution," or better still, Koch's method
of " plate-cultivation," is resorted to.

The " fractional cultivation " consists in the attempt to
isolate by successive cultivations the different organisms that
have been growing previously in the same culture. If we
take up by means of a capillary pipette a trace of the culture-
fluid, and inoculate with traces of it in the manner above
described a series of new culture-tubes containing various
nourishing material, and expose these tubes in the incubator
to a definite temperature, say 35° C., then the chances are

40 MICRO-ORGANISMS AND DISEASE. [CHAP.

that in the first twenty-four or forty-eight hours not all the
different species of organisms sown out will have increased
equally in numbers in all tubes ; most probably only one
species in each tube, i.e. the one that grows best in this
particular medium and at this particular temperature, will be
found to have increased to an enormous extent, while the
others have made little or no progress as yet. The nourishing
fluid appears turbid, and filled chiefly with the one kind of
organism. Now draw out with a fresh capillary pipette a
minute droplet of this new culture and inoculate with a trace
of it a new culture-tube. The chances are that you inoculate
only one kind, that is, the one which is most abundant or
perhaps is solely present. After twenty-four hours' incubation
this new tube contains now probably only one kind of
organism. To make it quite certain, inoculate from this a
new culture-tube in the same manner, and now you probably
have sown only a single species. In this manner by
continued transferrence it is possible to obtain cultures of
only one species of organisms. Many conditions, such as
naked-eye appearances of a particular kind, coloration of the
culture-medium, formation of a pellicle, the quantity of
growth in a given time, soon indicate whether we have the
desired single species ; in some instances it is, however,
extremely difficult to isolate after this method.

The method of " dilution " means diluting the culture-fluid
containing the various species to a very large extent with
some sterile indifferent fluid, such as well-boiled saline
solution of 0-6 per cent., and then inoculating new tubes
with a droplet of this greatly diluted material. For this
purpose draw into a rather large pipette a tiny droplet of the
old culture-fluid, then pass the pointed end of this pipette
into a test-tube or flask (plugged) containing well-boiled saline
solution, and draw up a quantity of this solution so as to

v.] METHODS OF INOCULATION. 41

greatly dilute (looo-fold or more) the droplet of culture-
fluid, and with this inoculate then a series of new culture-
tubes containing different nourishing material, using always
only a trace for inoculation. In this way it is probable that,
owing to the great dilution, the trace of a droplet of this
mixture used for the new inoculation contains only one
species. Using a series of new culture-tubes and inoculating
them thus, after twenty-four hours of incubation it will be
found that some tubes have not received any seed, others
only one species. If it be required to dilute the original fluid
greatly, say if it teems with different organisms, then a droplet
of this is placed into a large flask containing the well-boiled
saline solution, so that a dilution of i in 1,000,000 or more
can be effected.

The two methods, i.e. that of fractional culture and of
dilution, may be successfully combined in this way : from
the first or second new culture, established after the method
of fractional cultivation, in which after twenty-four or thirty-
six hours one species greatly predominates, draw out with a
large capillary pipette a droplet, and dilute this to a great
extent with the saline solution, as described above, and now
inoculate with a trace of this mixture a new culture-tube.
Or, if after twenty-four hours' incubation the microscope
reveals in this further culture more than one species, continue
the process of dilution and inoculation for a further generation.
Thus it is possible to obtain cultures of only one species,
although the original fluid contained several species of
organisms.

One of the best methods for isolation is that of the plate-
cultivation introduced by Koch in connection with the
isolation of the choleraic comma bacilli. A test-tube
containing sterile nutritive gelatine as above prepared is
liquefied by gentle heat, best by being kept in water of about

42 MICRO-ORGANISMS AND DISEASE. [CHAP.

40° C., then the plug is lifted and the gelatine inoculated
with a mere trace of the bacterial mixture, either by means
of the point of the capillary pipette or of the overheated and
cooled point of a platinum wire ; the plug is replaced and the
gelatine shaken so as to distribute uniformly the bacteria
that had been introduced. A shallow glass dish with flat
bottom and ground edge, and covered with a similar but
slightly larger dish, has previously been sterilised in the oven
and then allowed to cool ; the liquefied nutrient gelatine
inoculated with the trace of the bacterial material is then
poured out into the lower dish so as to form a thin layer
at its bottom ; the lifting off of the dish-cover, the pouring
in of the gelatine, and the replacing of the cover, ought to
occupy only a moment.

This apparatus is then placed on a glass plate, to which,
by means of greased edge, a bell-glass can be fixed, on the
interior of which is a piece of wet blotting-paper. In this
way a closed moist chamber is established. The whole is
then put into an incubator, the temperature of which does
not reach above 21° or 22° C. (or the temperature of the
room in the warm months), in order to insure the gelatine
setting and remaining so. If a trace of material containing
various species of bacteria is thus distributed into several cc.
of gelatine, each species will start a separate colony after a
few days' growth, and the individual colonies, if different,
will be apparent by different characters, according to shape,
colour, size of the colonies, and according to whether they
liquefy the gelatine or not during their growth. In order to
insure success, it is necessary to infect the original gelatine in
the test-tubes with only a trace of the bacterial mixture ; if
too many bacteria are introduced, their colonies sprouting up
are too numerous and soon become confluent. But if the
experiment is successful, from the individual and separate

METHODS OF INOCULATION.

43

colonies, it is then easy by re-inoculation of gelatine tubes,
or other nutritive material, to start pure subcultures of the
different species. It must be borne in mind that not all
bacteria can be isolated by this method, for some species
of pathogenic organisms require for this growth a higher
temperature than the one at which the nutrient gelatine

FIG. 9.— PLATE-CULTIVATION.

1. Glass plate.

2. Bell-glass.

3. Wet filter paper.

4. Glass dish containing the plate-cultivation on a thin layer of nutritive gelatine.
This glass dish is covered by a second glass dish.

remains solid, while others refuse altogether to grow in
gelatine, or grow only two slow. In the latter case no success
can be looked for, since those bacteria which grow much
faster crowd out the others that require a long time to
come up.

In such cases, particularly when one has to deal with

44 MICRO-ORGANISMS AND DISEASE. [CHAP.

bacteria that do not grow in gelatine at the temperature at
which this latter remains solid, the same method of plate-
cultivation can be used, but substituting the gelatine by the
Agar-Agar mixture above mentioned, care must be taken
not to proceed with the inoculation of the Agar-Agar mixture
before the temperature has fallen to about 50° C. All other
manipulations remain the same.

2. Inoculations with Blood, Juices, and Tissues. — To
establish a cultivation from blood of a dead animal, cut open
the thorax by removing the sternum with clean scissors, cut
open the pericardial sac, pierce with the pointed end of a
fresh capillary pipette the wall of the right ventricle or right
auricle, and allow a drop or two of blood to ascend into the
pipette, or if a larger quantity is required suck it up. With-
draw the pipette and inoculate new culture-tubes as above.
Or, if blood of a large vein is required, separate the vessel
with clean instruments, and make a small incision with clean
scissors and push the pointed end of the capillary pipette well
forward. If juice of a lymphatic gland, or spleen, or other
parenchymatous organ be required, pierce the organ after
having washed its surface with strong solution of perchloride of
mercury (Koch), with the pointed end of a capillary pipette,
then push it into the part required for a little distance, and
squeezing the organ press a drop or two of the juice into it.
The same procedure is adopted when the pus of an abscess
is required, the wall of which can be pierced with the pointed
end of the capillary pipette. If not, a slight incision is made
and the pipette introduced through this into the abscess.
If blood of a living animal is required, expose a vessel with
clean instruments, make a small incision with clean scissors,
push through this incision the pointed end of the capillary
pipette well forward, and allow the blood to rise into the
capillary tube. If blood of a living human being is required,

v.] METHODS OF INOCULATION. 45

clean well with soap and water and then with strong carbolic
acid or perchloride of mercury solution the tip of a finger,
make a venous congestion in the last phalanx by compressing
it with a corner of a handkerchief, prick the volar skin
of the phalanx with a clean (overheated and cooled) needle,
and plunging the pointed end of the pipette into the drop
of blood, allow a droplet to ascend into the capillary tube of
the pipette.

If solid tissues or parts of tissues are required, e.g. the
base of an ulcer, a tubercle of the liver, spleen, or lung, it is
possible to squeeze into the capillary tube of a pipette, after
pushing its pointed end into the part, a small droplet of
juice of the part required ; but if this be not practicable, i.e.
if a solid particle be required, then follow Koch's method.
This is as follows : Cut with clean scissors or scalpel into the
part, dig out rapidly with the point of a needle or platinum
wire previously overheated in the flame of a burner a small
particle, and quickly introduce this into the culture-tube to
the place required, e.g. surface or depth of a solid or fluid
nourishing material. Of course in this case the cotton-wool
must be altogether lifted, and therefore contamination with
organisms is possible. But inoculating several tubes at once
and performing the operation quickly, one always succeeds
in getting some of the tubes without any air-contamination.
I have made numerous inoculations with solid particles
(tubercles) in this manner, and like Koch have seen only a
small percentage of tubes going bad through contamination
with air-organisms.

The same plan, i.e. of using the clean point of a needle or
platinum wire for taking up the material to be used for
inoculation, is resorted to if one has to deal with the culture
in solid nourishing material, on or in which the organisms
are growing that we want to transplant either for inoculation

46 MICRO-ORGANISMS AND DISEASE. [CHAP.

of a new tube or of an animal. A useful method, which does
not require the lifting out of the plug at all, and which can
easily be employed in the last case, is this ; deposit from the
pointed end of a capillary pipette a droplet of some sterile
fluid (broth or thoroughly-boiled saline solution) on the
spot of the solid medium on which the organisms are growing,
then scratch this spot with the end of the capillary pipette in
order to get the organisms off from the solid basis and
mixed with the drop of fluid deposited there, then let this
drop again ascend into the end of the capillary pipette,
and withdraw this altogether. All this can be done with-
out lifting out the cotton-wool plug of the test-tube or flask
in which the growth is proceeding.

If one has to use a particle of tissue the surrounding
portions of which are probably contaminated by putrefactive
organisms, e.g. a tubercle in the lung or a tubercle in the
spleen, it is well to follow Koch, and to disinfect the
surrounding parts by just washing them with a dilute solution
of corrosive sublimate, and then to remove these parts with
clean scissors so as to obtain the central particle which one
wishes to use for inoculation : of course one must not steep
the whole organ in sublimate solution, since this would
naturally destroy all organisms.

All these methods can be easily modified according to the
requirements of the special cases, and it is not necessary
here to give more than what has already been described in
the preceding.1

In order to observe in a microscopic specimen the
gradual changes in the growth of a micro-organism, there
are several methods employed. In all of them it is of

1 Compare also Koch, Untersuchungen iiber patJiogene Bacterien, in
Berichte aus dem k. Gesundheitsamle, Berlin, 1881 ; and Die Aetiologie
d. Tuberculose, Berlin, klin. Wochenschrift, No. 15, 1882.

v.] METHODS OF INOCULATION. 47

course necessary to keep the specimen heated up to the
desired temperature.

The simplest method consists in sowing the organisms on
a suitable nourishing material in a small glass cell, fit to be
placed on the stage of a microscope and to be there observed
even with high powers, similar to those cells which Koch has
used in his studies on bacillus anthracis. Such a glass cell
consists of a glass slide, in its centre a concave pit, not too
large, and capable of being quite closed up by an ordinary
cover-glass, the edges of which fasten by means of clean
paraffin or olive oil. Place with a clean needle a speck of
spleen pulp of an animal dead of anthrax into a drop of
nourishing material, fluid or solid, on the centre of a clean
cover-glass, the edges of which have been prepared as just
mentioned, and fasten this on the above slide so that the
specimen faces the concave pit: expose this so prepared
specimen to a constant temperature, either by placing it in
the incubator and examining it with the microscope from
hour to hour, or on the warm stage (Strieker, Ranvier) used
in histological work for directly observing the influence
of temperature on the various cells and tissues ; or, place
it simply on the stage of the microscope and expose the
whole (i.e. microscope and all) in a suitable warm chamber
(after Klebs), but so that the chamber allows light to pass
by means of a small window to the mirror of the
microscope, while the eyepiece is so arranged as to project
through a hole in the upper wall of the chamber. The plan
which I generally follow is with slight modifications that
of Koch.

A glass cell (Fig. 10) is made by cementing a glass ring,
f-J inch in diameter and about \-^, inch high, on to an
ordinary glass slip. The chamber of this cell is well cleaned
with absolute alcohol. A thin cover-glass, square or round,

48 MICRO-ORGANISMS AND DISEASE. [CHAP.

about one inch in breadth, is well heated by holding it for
a few seconds over the flame of a gas-burner or spirit-lamp.
On the upper edge of the above glass ring is placed with a
camels' hair brush a thin layer of clean olive oil ; a droplet
of water is deposited on the bottom of the cell in order to
keep this afterwards well supplied with moisture ; a drop of
the sterile nourishing material (broth, aqueous humour,
hydrocele fluid, blood- serum, liquefied gelatine mixture,
liquefied Agar-Agar mixture, &c.,) is then deposited by means

FIG. 10. — A GLASS CELL, FOR OBSERVING UNDER THE MICROSCOPE THE
PROGRESS OF GROWTH OF MICRO-ORGANISMS.

The upper figure shows the cell in perspective ; the lower figure in profile or cross
section.

A. Glass slide.

B. Cover-glass.

C. Glass ring forming the wall of the chamber.

P. Drop of nourishing material in which the micro-organisms grow.

of a capillary pipette on to the centre of the cover-glass ;
then the point of a capillary pipette or needle containing the
material it is desired to sow is rapidly plunged into the drop
of the nourishing material (or if this is solidified is deposited
in lines or points on the drop of nourishing material), the
cover-glass inverted and placed on to the glass ring : the
layer of olive oil keeps the edges of the cover-glass air-tight
on the glass ring. This cell is then placed into the incubator

v.] METHODS OF INOCULATION. 49

and exposed there to the desired temperature. Microscopic
examination is carried out from time to time to watch the
progress made. This can be done with high powers, since
the growth is taking place on the lower surface of the
cover-glass.

Although contamination with air-organisms is not excluded,
still it is possible by making several specimens at the same
time and operating rapidly, to obtain pure cultures. This
glass cell can be also watched on a warm stage, or in a Klebs'
warm chamber.

M. Nachet of Paris has designed a glass cell, in which the
drop of nourishing material is deposited on to the bottom of
the cell, the glass slip being here replaced by a very thin
glass ; but then there is a peculiar arrangement in the micro-
scope, by which the lower surface of the glass cell, i.e. the one
nearest to the growth, is directly subjected to microscopic
observation.

After what has been said above about inoculation of solid
and fluid nourishing media with solid matter, it is not
necessary to dwell specially on the method of inoculation
with earth or similar substances.

3. Examination of Water for Micro-organisms. — Most
water contains bacteria of some kind, as has been shown by
direct experiment by Burdon-Sanderson.1 If any sample of
water is to be examined for micro-organisms, particularly
bacterial forms, it is allowed to stand for a few hours, till
most of the particulate matter is settled, and then with
a capillary pipette a little of the fluid and sediment is drawn
out and used for (a) microscopic specimens to be examined
fresh ; (b) microscopic specimens prepared after the Weigert-
Koch method, i.e. by spreading out on a cover-glass a thin
layer, drying it. staining it with suitable aniline dyes, e.g.
1 Reports of the Medical Officer of the Privy Council, 1870.

50 MICRO-ORGANISMS AND DISEASE. [CHAP.

Spiller's purple, gentian violet, methyl blue, or magenta,
washing with water, then spirit, then distilled water, then
drying, and finally mounting it in Canada-balsam solution.
(c) Test-tubes containing sterile nourishing material (broth,
Agar-Agar mixture, gelatine mixture, Cohn's or Pasteur's
fluid) are inoculated in the manner described previously, i.e.
by piercing the cotton-wool plug with the pointed end of the
capillary pipette. These test-tubes are then exposed in the
incubator, and after one or two days or more, a sample is
withdrawn with a capillary pipette, and used for microscopic
examination. As a rule, after a day or two of incubation we
can already distinguish with the unaided eye whether there
are any organisms present, the nourishing fluid either being
uniformly turbid — this is generally the case — or there being
a growth at the bottom of the fluid. But of course the
microscopic examination only shows what kind of organisms
are present. New cultivations are made from this one, if any
are required, (d) A good plan of recognising easily that
there are present various kinds of organisms in such cultures
is one similar to that recommended by Professor Angus
Smith.1 Sterile gelatine broth or gelatine only, contained in
sterile test-tubes plugged with sterile cotton-wool, is liquefied,
but of course not heated to more than about 35° to 40° C.
then inoculated with the water (to be tested), by means of
the capillary pipette ; after inoculation the gelatine is mixed
by shaking the test-tube slightly. In this way the organisms
present in the water are distributed in the gelatine. Then
the gelatine is allowed to set and is kept in this solid state.
The organisms being distributed in the gelatine, after some
days' growth are noticeable as clusters which gradually
increase in extent and are distributed in various parts of the

1 Sanitary Record, p. 344, 1883.

v.] METHODS OF INOCULATION. 51

medium. The various species, owing to difference of growth,
form clusters differing in aspect, size, and arrangement.

A good plan of ascertaining the relative number of certain
bacteria present in given samples of water is that by means
of plate-cultivations. A definite small quantity of the
water is added to a definite quantity of liquefied sterile
nutrient gelatine contained in a sterile test-tube ; shake well
and pour out on a glass plate or glass dish to be kept under
bell-glass in a moist chamber, i.e. after the manner of an
ordinary plate cultivation. After being kept at a temperature
of about 21° to 22° C. notice the number of colonies that
have sprung up, the number of different colonies (size, shape,
colour, and whether liquefying the gelatine or not), and from
these an index is obtained of the approximate number of
such bacteria that have been originally present in a given
quantity of the water. But it must be borne in mind that
the number of colonies is no absolute index of the number
of bacteria originally present in the water, for the following
reasons : (a) not every colony that makes its appearance on
the plate cultivation — even granted it is due to the growth of
a single species, but which is not always the case — owes its
origin to one single individual, since for instance micrococci
bacteria and bacilli may occur in the original water as zoogloea
and chains, and these cannot by any amount of shaking be
broken up into single elements ; (b) not all bacteria introduced
into the gelatine come up as colonies, since not all of them
are capable of growing in the gelatine, and not all can thrive
at the temperature at which the gelatine remains solid • (c)
the liquefaction of the gelatine by some of the colonies and
not by others does not indicate different species, since this
depends sometimes on the nature of the nutrient gelatine,
and to the fact whether the growth takes place in the depth
or on the surface; (d) accidental contamination with organisms

£ 2

52 MICRO-ORGANISMS AND DISEASE. [CHAP.

of the air during the preparation of the plate cultivation
cannot be prevented, and if the air happens to contain a
good many organisms, the total number of colonies appear-
ing in the plate cultivation exceeds the number of bacteria
present in the water tested.

Professor Warden of Calcutta (Chemical News, Nos. 1340 to
1344) and Dr. Percy Frankland (Proceedings of the Royal
Society, No. 238) have made valuable experiments on the
examination of bacteria present in water by means of
plate cultivations.

4. Examination of Air. — The simplest plan to test for the
presence of organisms in the air is to draw out the cotton-
wool plug of several test-tubes or flasks containing the sterile
nourishing material, or, if this be boiled, potatoe, paste, or
gelatine (see p. 20), to expose their surface, and to leave it
thus for variable periods, from a few seconds to several
minutes. Then replace everything and expose the material
to incubation, or keep it only at the ordinary temperature of
the room. Another method is to collect the particles present
in the air on glasses moistened with pure glycerine (Maddox),
and then to make microscopic specimens or inoculate tubes
with this glycerine.

A method which is very useful is the one recommended by
Cohn and Miflet.1 The principle of it is, that by means of
an aspirator, an air-pump of any kind — e.g. a Sprengel pump,
or simply the fall of water — air of a particular locality is drawn
into one, two, or more Wolff's bottles (each with the ordinary
two bent glass tubes), connected with one another by short
pieces of indiarubber tubing, and containing the sterile
material in which the organisms are required to grow. All
bottles and tubes are of course sterile ; the plugging of the
tubes after the air has passed is done with sterile cotton- wool.
1 Zeitsthr-f. Biol. d. Pft. iii. i, p. 119.

v.] METHODS OF INOCULATION. 53

Any given quantity of air for any length of time can be
passed through a series of such bottles, the one that receives
the air first being of course most contaminated.

The bottles are after the experiment placed in the incubator,
if required, the outer end of their tubes being plugged with
cotton-wool.

Miquel l has carefully described many ingenious methods
for the study of air-organisms.

1 Les Organismes vivants de T Atmosphere, Paris, 1883.

CHAPTER VI.

MORPHOLOGY OF BACTERIA.

BACTERIA are minute organisms not containing chlorophyll,
and multiplying by fission — hence the term schizomycetes (v.
Nageli). They are composed of a kind of protoplasm, the
mycoprotein of Nencki, and are invested with a membrane,
which is composed chiefly of cellulose and a certain amount
of mycoprotein (Nencki).

Their contents are transparent and clear, but sometimes
contain minute bright granules of sulphur (Beggiatoa).
Owing to the cellulose membrane they resist the action of
acids and alkalies. Many species of bacteria — micrococcus,
bacterium, spirillum — are able by rapid multiplication to form
colonies ; the individuals are then embedded in a hyaline
gelatinous matrix produced by them, this is also mycoprotein,
Some species are possessed of one or two straight or slightly
spiral cilia or flagella, and thereby they are capable of loco-
motion, darting through, or spinning round, in the fluid in
which they are suspended. Such is the case with some
kinds of bacteria, bacilli, and spirilla.

Bacteria grow best when left undisturbed ; movement of
the vessel in which they grow is not advantageous. Light
and electricity do not appear to have a decided influence
on some bacteria, since they grow well in the light. Accord-
ing to Cohn and Mendelssohn,1 strong electric currents have
a noxious influence on the growth of micrococci.
1 Cohn's Beitr. z. Biol d. Pfl. Bd. iii. i.

CH. vi.] MORPHOLOGY OF BACTERIA. 55

Engelmann1 describes a bacterium photometricum, the
motility of which directly depends on light ; it ceases in
the dark, Duclaux found that exposure to direct sunlight
injures the life and growth of some bacteria, both septic and
pathogenic.

Some bacteria require free access of oxygen, and are
called aerobic (Pasteur) ; others grow without free oxygen,
and are anaerobic (Pasteur). All require for their growth
certain nourishing material containing carbon and nitrogen.
Water is an essential element for them, and a certain temper-
ature is in many instances a stimulant of their growth. Most
pathogenic bacteria require for their propagation a temperature
varying in the different cases between 18° and 40° C. The
bacteria obtain their nitrogen from organic compounds •
some are capable of obtaining it from compounds as simple
as ammonium tartrate ; others, especially pathogenic organ-
isms, require much more complex combinations, such as
occur in the animal body. Carbon they obtain likewise
from organic compounds, such as carbohydrates, amongst
which sugar is the chief, and vegetable acids combined as
salts are also to be mentioned. It is essential for all that
certain inorganic salts, phosphates, potassium and sodium
salts, should be present, since their own substance contains a
large percentage of it — 4 to 6 per cent.

While all are capable of disintegrating organic combina-
tions containing nitrogen, they in their turn help to produce
certain chemical products, which in some cases are definite
for a definite species (see below). Such is the case with the
various bacteria connected with the fermentations producing
lactic acid, butyric acid, and acids belonging to the aromatic
series. On many bacteria connected with putrefaction, and
also on some pathogenic organisms, these chemical products
1 Unters. aus. d. physiol. Labor, Utrecht, 1882.

56 MICRO-ORGANISMS AND DISEASE. [CH. vi.

have a deleterious effect. Small quantities impede their
growth, and sufficiently large quantities kill them altogether.

Most bacteria are killed by heat below the temperature of
boiling water, many of them when exposed for several hours
to a temperature above 50° to 60° C. Exceptions are the
spores of bacilli, which in some instances (spores of hay
bacillus, Cohn) require exposure to the heat of boiling point
for as much as half an hour. By raising the boiling point
above 100°, it does not require more than a few minutes to
kill them (Sanderson).

Drying destroys most bacteria, except the spores of bacilli.
Freezing destroys likewise most bacteria, except the spores of
bacilli, which survive exposure to as low a temperature as
- 15° C., even when exposed for an hour or more. No spores
survive exposure to a temperature of 120° C.

Amongst those substances which inhibit the growth of, or
altogether destroy the bacteria, are carbolic acid, salicylic
acid, thymol, &c. ; corrosive sublimate is one of the most
powerful, since even solutions as weak as i : 15,000 inhibit
the growth of some bacteria after exposure for thirty minutes.

The best classification of bacteria is that given by Cohn,1
and this we shall adopt : (i) spherobacteria or micrococci ;
(2) bacteria or microbacteria ; (3) bacilli or desmobacteria ;
(4) spirilla, (5) spirochsetse. There are also various kinds
which approach one or the other of these, e.g. ascococcus,
sarcina, leptothrix (Beggiotoa), cladothrix, streptothrix, &c.
(see below).

I shall not attempt to give an exhaustive description of the
morphological characters of all micro-organisms, but shall
limit myself to those forms which are related in some way or
other to diseases.

1 Beitr. z. Biol. d. PJl. Bd. i.

CHAPTER VII.
MICROCOCCUS (Hallier, Cohn).

BY the specific term micrococcus is understood a minute
spherical or slightly oval organism (sphere bacterium, Cohn),
that like other bacteria divides by fission (schizomycetes),
and that does not possess any special organ, cilium or
flagellum, by using which it would be capable of moving
freely about. Micrococci, like other granules when suspended
in a fluid medium, show (Brownian) molecular movement-
Micrococci propagate always by simple division, never by
any other means, e.g. gemmation and spores. All assertions
to the contrary are based on incorrect observations. All
micrococci possess a delicate membrane of cellulose, and
owing to this resist the action of alkalies and acids. The
contents are homogeneous and highly refractive while active,
pale when inactive. They consist like those of other bacteria
of mycoprotein (Nencki). The size of micrococci varies
within considerable limits, say 0^0005 to 0*002 millimetres,
or even a little more. Micrococci vary greatly as regards
both size and mode of growth. All multiply by slightly
elongating and then dividing by a transverse constriction
into two : a dumb-bell ; each of these again divides into two,
either transversely or in the same direction as before. The

58 MICRO-ORGANISMS AND DISEASE. [CHAP.

new elements of successive divisions may remain connected,
and thus form a chain (or mycothrix, Itzigsohn and Hallier ;
torulaform string, Cohn), or they separate into single
organisms or dumb-bells. In some species there is a
pre-eminent tendency to form chiefly dumb-bells, in
others to form shorter or longer chains generally more
or less curved, streptococcus (Billroth).

Such exquisite chains one meets with sometimes in serum
of blood exposed to the air for some days, and in pleural and

.•

A ••'

. x.n

..... •••**:

FIG. ii. — MICROCOCCI OF PUTRID

HUMAN SPUTUM.

1. Single micrococci and dumb-bells.

2. Short chains.

3. A long chain.

4. A zooglcea.

This and all subsequent figures are
drawn under a magnifying power of
about 700 diameters except stated other-
wise.

is

V

FIG. 12.— FROM THE SAME PUTRID
SPUTUM AS IN PREVIOUS FIGURE.
THE MICROCOCCI ARE LARGER.

1. Dumb-bells.

2. Sarcinae.

3. A small zoogloea, in reality consisting

of four sarcina-groups.

peritoneal exudations of animals dead for a few days. 1
have seen in an artificial culture made by my friend Mr. A.
Lingard from a blister in a rabbit's ear the most exquisite
convolutions of threads of micrococci. (See Fig. 13.)

A dumb-bell is also called a diplococcus (Billroth).
Between the individuals of a dumb-bell there is always
noticeable a short pale intervening bridge.

VIL] MICROCOCCUS. 59

Some species are specially characterised by dividing into
a dumb-bell ; if each of the elements divides again trans-
versely into a dumb-bell ; a group of four (tetrade or sarcina-
form) is thereby produced. Some species are occasionally

FIG. 13.— PART OF A CONVOLUTION OF CHAINS OF MICROCOCCI ; FROM AN
ARTIFICIAL CULTIVATION STARTED WITH THE SERUM OF A BLISTER OF A
RABBIT'S EAR.

met with, particularly in products of air-contamination, in
which the four individuals are closely pressed against one
another, and then each assumes more or less the shape of a
cube, a true sarcina (see below). But each of these cubes

2 2 »f * ' f 2 5

st §l S2 SI «» «»

~ a* ft •• •• go e$

«8 *f» gl 85 §»

FIG. 14. — GIANT MICROCOCCI, FROM FIG. 15. — SARCINA-MICROCOCCUS, FROM

SAME PUTRID SPUTUM AS IN PRE- AN ARTIFICIAL CULTIVATION.

vious FIGURES. rp, , r ,

i. Ine elements of each sarcma-group

1. Dumb-bells. of four appears single.

2. Division of dumb-bells into sarcina. 2. The elements incompletely divided

3. Incomplete division into sarcina. into secondary groups.

3. Each element of the previous groups
has divided into four small micro-
cocci.

divides into four small micrococci arranged as a small sarcina,
so that a sarcina-within-sarcina-form results.

In many instances the individual members resulting from
division remain closely adherent without any definite arrange-

60 MICRO-ORGANISMS AND DISEASE. [CHAP.

merit, and thus form smaller or larger continuous masses,
zooglcea or colonies, in which the individuals appear embedded
in a hyaline gelatinous matrix ; the amount of this varies in
the different species ; in some there is little of the matrix
actually visible, the micrococci being in close juxtaposition,
in others it is easily recognised, the interstices between the
individuals being measurable.

In some of the pigmented species (see below) the interstitial
matrix contains the pigment. Zoogloea masses always present
themselves as uniformly granular, the granules or micrococci
being of the same size.

True micrococci never elongate to form rods, although in
certain rod-like bacteria the individual elements sometimes
assume the shape of spherical elements (see below).

Some species of micrococci form after some days a pellicle
on the surface of fluid nourishing material, although there is
also an abundance of these micrococci in the depth of the
nourishing material. This pellicle is composed of zooglcea,
and after some time bits of it, or the whole, sink to the
bottom of the fluid medium. Micrococci that thus form
pellicles are pre-eminently aerobic (Pasteur), i.e. require
a great deal of free oxygen, which they receive from the air
to which they are exposed on the surface of the nourishing
material. Other species do not require free oxygen
(anaerobic, Pasteur), and therefore grow well in the depth
and do not form a superficial pellicle. There is a marked
distinction in this respect between different species. The
micrococci occurring in connection with disease are
anaerobic.

When cultivated in suitable fluids, they produce after a day
or two general turbidity \ growing in solid nutritive gelatine
some produce liquefaction of the gelatine, others do not.

Micrococci may be divided, according to their chemical

VIL] MICROCOCCUS. 61

and physiological function, into : (a) septic, (b) zymogenic,1
(c] chromogenic, and (d) pathogenic micrococci.

(a) The septic micrococci are micrococci that occur with
other septic bacteria, wherever there is decomposition of
organic matter in solids or in fluids. There exists a large
number of species of such micrococci, differing from one
another in size and mode of growth. They are widely
distributed in the air, and contamination by air is often
followed by the appearance of micrococci. They also occur
in the body of man and animals wherever there is dead
tissue, in which they grow well and copiously. Of this kind are
the micrococci found in ordinary pus (Ogston), in the normal
oral cavity (on the filiform papillae of the tongue and on the
mucous membrane), in the bronchial secretion in ordinary
catarrhal exudations (nasal cavity, bronchi, &c.), on the free
surface of intestinal and other ulcerations, in the cavity of the
small and large intestine, and in the epidermis of the normal
skin (Bizzozero).

(b) Zymogenic micrococci are micrococci associated with
definite chemical processes, (a) Micrococcus ureas, causing
the ammoniacal fermentation of urine (aerobic, Pasteur),
occurs singly, as dumb-bells or chains, and as zoogloea. ((3)
The micrococcus of the mucoid wine fermentation produces
(Pasteur) a peculiar mucoid change in wine and beer, and
occurs chiefly in chains, (y) The micrococcus causing phos-
phorescence in putrid meat and fish (Pfliiger) forms chiefly
zooglcea (aerobic).

(c) Chromogenic micrococci (Schroter, Cohn). — These
micrococci are characterised by their power of forming

1 I adopt this term from Flitgge : Ferments und Mikroparasiten,
Leipzig, 1883.

62 MICRO-ORGANISMS AND DISEASE. [CHAP.

pigment of various colours. They grow well at ordinary
temperatures, and occur chiefly as zoogloea ; they differ from
one another by forming different pigments. The thicker the
layer the more marked is the pigment. This is either
soluble in water or it is insoluble, in the latter case it re-
mains limited to the cells and their interstitial substance.
The cells are spherical (Micrococcus prodigiosus, chlorinus,
fulvus) or slightly elliptical (M. luteus, auriantiacus, cyaneus,
violaceus). They are all aerobic and produce this pigment
only when there is free access of air. They grow best on
boiled potato, bread, paste, and boiled-egg albumen. They
can be transplanted, and always produce the same pigment.
When growing and kept in the depth of a solid nourishing
material, i.e. removed from the free surface, they grow as

FIG. 16. — OVAL MICROCOCCI WHICH POSSESS A BLUE COLOUR, MICROCOCCUS
CYANEUS, SINGLY AND IN DUMB-BELLS.

colourless micrococci. They abound in the air — in some
localities and at certain seasons more than at others, (a)
Micrococcus prodigiosus is blood-red, the colour is lodged
not in the micrococci but in the interstitial substance, and
is insoluble in water, soluble in alcohol ; it occurs chiefly as
zoogloea, in the shape of smaller or larger droplets. The
cells are the smallest of all pigment-micrococci. (ft) Micro-
coccus luteus is yellowish, and the pigment is insoluble in
water. It occurs also in fluid nourishing material, forming
a pellicle. I have met with it in the air, and have sown it
in fluid pork-broth, where it -grew very abundantly at a
temperature of 32° to 38° C. It was found as single cells
or dumb-bells, and formed a thick pellicle on the surface,

VIL] MICROCOCCUS. 63

which after some time sank down into the fluid, the pellicle
retaining a pale yellow colour, (y) Micrococcus auriantiacus
grows on boiled-egg albumen, chiefly as zoogloea. The
pigment is soluble in water. (8) Micrococcus cyaneus,
violaceus, chlorinus, and fulvus, produce blue, violet, green,
and brown pigment respectively. The first two grow
well as zooglcea of elliptical cells on boiled potatoes, the
third on boiled-egg albumen, and the last is met with on
horses' dung.

Clathrocystis roseo-persicina (Cohn), peach-coloured bac-
terium, Bact. rubescens (Lankester), is an organism of about
0*0025 mm- m diameter, spherical or oval and of a bright
red colour. The cells differ from micrococcus prodigiosus,
not only in their greater size and their intrinsic colour, but
also in this — that having formed zooglcea-masses there are
gradually developed cavities or cysts therein, which are filled
with water, while the coloured cells occupy the periphery.
The cysts ultimately break up. Together with this organism
occur other pink-coloured organisms described by Cohn as
monades.

Monas vinosa, spherical cells about 0*002 — 0*003 mm- in
diameter.

Monas Okenii, cylindrical cells, 0*008 — 0*005 mm. long,
0*005 mm. broad, flagellate.

Rhabdomonas rosea, spindle-shaped, 0*004 mm. broad,
0*02 — 0*03 mm. long, flagellate.

Monas Warmingii, spindle-shaped, 0*008 mm. broad,
0*015 — 0*020 mm. long, flagellate.

Ascococcus. — Billroth first described certain peculiar spheri-
cal, oval, or knobbed masses of minute micrococci, which
he found in putrid meat infusion. Each of the masses is
enveloped in a resistant firm hyaline capsule of about 0*0 10

64

MICRO-ORGANISMS AND DISEASE. [CHAP.

to 0-015 mm- thickness. The masses are of various sizes,
from o'02 to o'oy mm. in diameter, and are composed of
small spherical micrococci. Cohn found them also in his
(Cohn's) nourishing fluid (see Chapter II. A. 7), where they
produce the peculiar smell of cheese. They are capable of
changing acid nourishing material into alkaline. Cohn called
the organism ascococcus Billrothi.

FIG. 17. — Ascococcus BILLROTHI (AFTER COHN).

Sarcince Ventricidi. — Goodsir was the first to describe in
the vomit of some patients, peculiar groups of four cubical
cells, with rounded edges, and closely placed against one
another. These sarcincz ventriculi are of a greenish or reddish
colour. The diameter of the individual cells is about 0*004
mm. They are found in the contents of the stomach of man
and brutes in health and disease, where the groups of four
cells form smaller and larger aggregations. Occasionally
small sarcinae occur on boiled potatoes, egg albumen, and
gelatine exposed to the air. These sarcinae are considerably

VIL] MICROCOCCUS. 65

smaller than the sarcina ventriculi, and when in large
quantities have a yellowish tinge. Like the sarcina ventriculi
they are in groups of four, and these again occur in larger
or smaller aggregations and zoogloea. I have cultivated
them successfully through many generations in pork broth,
beef broth, mixture of gelatine and broth, at ordinary
temperatures and in the incubator ; more easily however
at ordinary temperatures.

(d) Pathogenic micrococci. — Many of these are associated
with disease. In the pus of open wounds,1 and in that of
closed abscesses, occur micrococci, singly, in dumb-bells,
and in colonies or short chains,2 but there are certain acute
inflammations, e.g. that produced by subcutaneous injection
of turpentine, the pus of which does not contain micrococci
or any other organism.3

The secretion of open ulcers, such as occur in ordinary
acute inflammations of the skin and mucous membranes, in
ulcerations of the throat due to scarlatina, in every ulceration
of the intestinal mucous membrane, in the lymph of the
vesicles of the skin and mucous membranes of the mouth
occurring in various kinds of inflammations, there are almost
always present micrococci in dumb-bells, often also in
beautiful chains. In the ulcers and abscesses they often form
continuous masses, i.e. zooglcea, encroaching upon the tissue
of the base of the ulcer. To this category belong the
minute micrococci (about 0*0005 mm. in diameter) which
Klebs described as microsporon septicum, found in and
around wounds. The spread of purulent inflammation in

W. Cheyne, Path. Trans, vol. xxx.

2 Ogston, " Micrococcus in Acute Abscesses," Brit. Med. Journ.
March 12, 1881.

3 Uzkoff, Virchow's Archiv, vol. 86, i. p. 150.

65 MICRO-ORGANISMS AND DISEASE. [CHAP.

connective tissues and in parenchymatous organs is often, if
micrococci are present in the original focus, associated with
a corresponding spreading of the micrococci ; these easily
grow into all the spaces and crevices of the tissues, but
whether this spreading of the micrococci is merely of
secondary importance, i.e. concomitant with or subsequent
to the spreading of the inflammation, or whether it is the
primary cause as some assume, is not clear, and requires
definite experimental proof.

FIG. 18. — FROM THE BASE OF AN ULCER OF THE Mucous MEMBRANE OF THE
LARNYX IN A CHILD THAT DIED OF ACUTE SCARLATINA.

1. Nuclei and fibres of the tissue.

2. Zoogkea of mlcrococci-

In all cases of diarrhoea the secretions of the bowels swarm
with micrococci. In typhoid fever, clumps of micrococci
may be found very extensively on the ulcerations of the
bowels, and in the mucous membrane surrounding the
ulcerations, and may be even traced into the mesenteric
glands and the spleen.1

In dead tissues within the living body, such as occur after
embolism, and in the case of various infectious maladies,
micrococci may be found in colonies, i,e. as zoogloea,

1 Klein, Reports of the Medical Officer, 1876. Letzerich, Sokoloff,
Fischel, &c.

VIL] MICROCOCCUS. 67

in the blood-vessels and in the parts around. The same
holds good for the disseminated abscesses and necroses
occurring in connexion with surgical pyaemia. In this
malady masses of micrococci have been found in many of the
affected organs.1

Wassilieff 2 has shown that these micrococci only occur
after the death of the tissue or tissues, that in these they may
multiply so as to form extensive colonies, and that therefore
the presence of these micrococci is only a secondary
phenomenon.

FIG. 19. — CAPILLARY BLOOD-VESSELS OF NECROTIC MASSES FROM THE LIVER
OF A MOUSE. THE CAPILLARIES ARE DISTENDED BY, AND FILLED WITH

ZOOGLCEA OF MlCROCOCCI.

In pneumonia accompanying certain infectious maladies,
e.g. typhoid fever, tuberculosis, and even in severe catarrhal
pneumonia, large masses of micrococci may occur in the air-
cells. In those cases where lobules and whole lobes become
transformed into solid structures — grey hepatisation — masses
of micrococci may be found in the air-cells, and even growing
into the blood-vessels in which stasis had set in. Such is
the case in pleuro-pneumonia of cattle and in the pneumonia
of swine fever. Pasteur has cultivated micrococci which

1 "Report of the Committee of the Pathological Society," Path.
Trans, vol. xxx.

2 Centralb. f. d. med. Wiss. No. 52, 1881.

F 2

68 . MICRO-ORGANISMS AND DISEASE. [CHAP.

were said to occur in the blood of pigs affected with the
disease known in France as rouget, and which Pasteur con-
sidered identical with the disease known in this country as
swine fever or swine plague. Pasteur asserts also that with
these micrococci artificially cultivated he has reproduced the
disease in swine. It appears, however, from an investigation
by Schiitz1 that rouget is identical with the disease known
in Germany as erysipelas of swine, and that this disease is
caused not by a micrococcus at all, but by a small bacillus.
This disease differs altogether from swine fever in its symp-
toms and course. Compare also Chapter XL " Bacillus
of Swine Fever."

0

FIG. 20. — FROM A PREPARATION OF THE BLOOD OF A CHILD ILL WITH
INFANTILE DIARRHCEA.

1. Blood-discs.

2. Dumb-bells of micrococci.

Micrococci occur always normally in large quantities in
the fluids (saliva and mucus, &c.) of the nasal and oral
cavities, pharynx, larynx, and trachea ; they are derived no
doubt from the atmosphere. On the papillae filiformes of
the tongue they form in some cases large masses.2 Pasteur 3
has inoculated rabbits with the saliva of a child that suffered
from hydrophobia, and having cultivated artificially the

1 Arbeiten aus d. k. Gesundheilsamte, Berlin, 1885.

2 Butlin, "Fur of the Tongue, " Proc. Roy. Soc. 1880.

3 Comptes Rendus, xlii.

vii.] MICROCOCCUS. 69

micrococci present in this saliva, thought to have discovered
that a micrococcus (microbe speciale) 1 is the cause of
hydrophobia. That saliva of the healthy dog and of man
inoculated subcutaneously into rabbits sometimes produces
death in these animals (Senator) had entirely escaped his
notice, and Sternberg 2 has proved this in an extensive series
of experiments. His own saliva proved sometimes fatal to
rabbits. They die of what is called septicaemia, and Stern-
berg thinks it due to the micrococci ; but this is not to be
considered as satisfactorily proved.

All these micrococci stand therefore in no definite causal
relation to the respective maladies, but are probably only of
secondary importance.

The following micrococci are considered to stand in an
intimate relation to specific diseases : —

i. Micrococcus variola et vaccinia. — Chauveau3was the
first to prove experimentally that in vaccinia and in variola
the active principle is a particulate non-diffusible substance.
Burdon Sanderson confirmed and extended this.4 Cohn5
proved that the lymph of vaccinia and variola contains
numerous micrococci. Weigert 6 showed for human small-
pox, Klein 7 for sheep-pox, that the lymphatics of the skin
in the region of the pock are filled with micrococci, and

1 It is not quite clear whether this murobe speciale is a dumb-bell
micrococcus or a bacterium termo ; it is quite possible that it is the
latter, viz. a rod constricted in the middle. If so, it would appear
identical with the bacterium that produces Davaine's septicaemia in
rabbits (see Chapter VIII.).

2 Bulletin of the National Board of Health, U.S.A. April 30, 1881.

3 Comptes Rendus, 1868.

4 Reports on the Intimate Pathology of Contagion.

5 Virchow's Archiv, 1872. Keber, Hallier, Ziirn.
J Med. Centralb. 1871.

Phil. Trans. 1874.

70 MICRO-ORGANISMS AND DISEASE. [CHAP.

Pohl-Pincus 1 traced their passage through the epidermis at
the point of vaccination in the calf. The micrococci are very
minute, according to Cohn's estimate 0*0005 mm. and less
in diameter, single or in dumb-bells, or in shorter or longer
chains, or in small groups. When cultivated on the warm

FIG. 21. — MICROCOCCUS IN THE FRESH LYMPH OF HUMAN SMALL-POX.

1. Singly.

2. In dumb-bells.

3. In short chains.

stage, they form very long chains and colonies. In connexion
with this it must be mentioned, as stated on a former page,
that similar micrococci occur also in the fluid contents of
vesicles in the skin produced by various non-infective
inflammations. To make it sure that the micrococci are the

FIG. 22. — LYMPHATIC VESSEL FROM THE SKIN OF A POCK IN SHEEP-POX.
The vessel is filled with micrococci.

active principle, i.e. the causa morbi, it would be necessary
to artificially cultivate them through several generations,
and then, by re-inoculating them, to reproduce the
disease. This essential link in the evidence is, however,
still wanting.2

1 Vaccination, Berlin, 1882.

2 See the prize announcement of the Grocers' Company, London, 1883.

VIL] MICROCOCCUS. 71

2. Micrococcus erysipelatosus. — The micrococcus is very
minute, smaller than that of vaccinia. Lukomsky * showed
that, at the margin of an erysipelatous zone, that is the part
where the disease is progressing and marked by the character-
istic redness and swelling, the lymphatics of the skin are
filled with zoogloea of micrococci, and the injection of these
vessels keeps pace with the progress of the erysipelatous
process. Orth 2 cultivated these micrococci artificially, and
with such cultures produced by inoculation erysipelas in
rabbits. Fehleisen 3 placed this beyond any doubt, inasmuch
as he produced successive cultures of these micrococci
(derived from the lymphatics of erysipelatous human skin),
and then by re-inoculation produced the disease not only in
rabbits but also in man. Fehleisen found the micrococci
only in the lymphatics of the affected parts, and these he
cultivated artificially for fourteen generations — which it took
two months to do — on peptonised meat-extract gelatine, and
solid serum. The micrococci form a whitish film on the top
of the nourishing material, and when inoculated into the skin
(ear) of rabbits, a characteristic erysipelatous rash makes its
appearance after from thirty-six to forty-eight hours, and
spreads to the root of the ear, and further on to the head
and neck. The animals do not, however, die from it. In
the human subject he produced typical erysipelas after
inoculation with the pure cultivated micrococcus in fifteen
to sixty hours. These inoculations were justifiable because
they were undertaken with a view to cure certain tumours.
Thus one case of lupus, one case of cancer, one case of
sarcoma, were considerably affected, and to the good of the
patient.

1 Virckow's Archiv, vol. 60.

2 Archivj. exp. Path. Bd. i. 1874.

3 Die Adiologie d. Erysipels, Berlin, 1883.

72 MICRO-ORGANISMS AND DISEASE. [CHAP.

Fehleisen also in several instances carried out successfully a
second inoculation within a few months. The same observer
also found that a 3 per cent, solution of carbolic acid and
a i per cent, solution of corrosive sublimate destroys the
vitality of this micrococcus.

3. Micrococcus diphtheriticus. — Buhl, Hiiter, and Oertel
had shown that in diphtheria the membranes include
micrococci. Oertel l found this micrococcus in large
numbers, not only in the diphtheritic membranes of the
organs of the throat and in their neighbourhood as well as
in the surrounding lymphatics, but also in the blood of the
general circulation, in the kidney, and in the muscles. The

FIG. 23.— PORTION OF A DIPHTHERITIC MEMBRANE.
Numerous micrococci present.

micrococci are about 0*00035 too'ooi mm. in diameter, are
slightly oval, occur singly or in dumb-bells or in short chains ;
they form also continuous masses of zooglcea in the shape
of spherical or cylindrical clumps, and as such they
penetrate and destroy the surrounding connective and
muscular tissues. In severe cases they are found blocking
up the capillaries of the glomeruli and the uriniferous tubules
of the kidney.

•Besides micrococci there occur in the diphtheritic mem-
branes also other (rod-shaped) bacteria, but these are evidently

1 "Experim. Unters. uber Diphtheric," Deutsches Archiv f. klin.
Med. Bd. viii. 1871.

VIL] MICROCOCCUS. 73

only accessory.1 Cultivations and inoculations with pure
cultivations of this micrococcus are still wanting.

4. Micrococcus pneumonia. — In acute croupous pneumonia
there occur in the affected lungs large numbers of micrococci ;
Klebs, Eberth, Koch, Leyden, and others have seen them,
but Friedlander 2 first pointed out their constant occurrence.
According to this observer they are oval, each possessed of a
capsule, about o'ooi mm. long, and occur in the sputum
singly, but especially as dumb-bells or diplococci, as chains,
and as zooglcea. Ziehl 3 found them in very large crowds in
the sputum, giving to this in the early stages the peculiar
characteristic brownish " prune-juice " tint. But this state-
ment cannot be correct, since this tint may be very pro-
nounced although the sputum contains only a limited number
of the micrococci. According to this observer, they are very
numerous only in the beginning of the illness ; after the
critical stage they decrease in numbers.

Grifrlni and Cambria saw the micrococci also in the blood.
Salvioli found that their number increases after the third day ;
on the ninth or tenth day they quite disappear.

G. Giles 4 found them in many cases of pneumonia (in
India), both in the sputum and in the blood. Cultivations on
boiled potato yielded good crops. These cultivated micro-
cocci injected into the subcutaneous tissue of rabbits produced
pneumonia.

Salvioli and Zaslein 5 cultivated the micrococci (derived

1 Compare also Klebs, Archiv f. exp. Path. iv. ; Letzerich, Vtrcfoixfs
Archiv, vol. 68; Nassiloff, ibid. vol. 50; Eberth, Zur Kenntniss -d.
bact. Mycosen, 1872 ; Wood and Formad, Report of National Board of
Health, U.S.A. 1882. 2 Virchovfs Archiv, vol. 87.

3 Centralb. f. med. Wiss. No. 25, 1883.

4 Brit. Med.Journ. July 7, 1883.

5 Centralb. f. med. Wiss. No. 41, 1883.

74 MICRO-ORGANISMS AND DISEASE. [CHAP.

from the blood) in meat broth, meat extract solution, &c., at
37° to 39° C., and obtained good crops of them, with which
they produced by inoculation in seven rabbits and six white
rats, typical pneumonia yielding the characteristic micro-
cocci. These micrococci stain best in a mixture of Bismarck
brown and methyl violet, but they srain also very well in
gentian violet.

Quite recently Friedlander and Frobenius * cultivated the
micrococci in nutritive gelatine, and obtained good crops.
The shape of such growths is nail-like, consisting of a vertical
streak, the nail-pin corresponding to the channel of inocu-
lation, and in connexion with this is a disc-shaped patch —
the nail-head — extending on the surface of the gelatine.
Inoculations of these capsulated pneumococd into the lung
tissue of rabbits produced no result, into that of mice produced
pneumonia and pleurisy after twenty-four to forty-eight hours.
In guinea-pigs the results were not so decisive. About half of
the animals escaped, the others died with dyspnoea, the
blood, lungs, and pleural exudations containing the same
pneumococci. From my own observations I cannot accept
these statements without qualification, for I find : That even
in typical cases of croupous pneumonia of man, the micrococci
may be absent or may be only very scarce even between the
third and ninth day; that typical sputum of croupous
pneumonia does not in many instances produce disease
in animals on inoculation ; and that the disease produced
in rabbits and mice is of the nature of septicaemia,
due to a specific septicaemic micrococcus not necessarily
always present in the sputum and lungs of human croupous
pneumonia.

It seems therefore clear, that when sputum produces
on inoculation disease and death of rodents, this is due to
1 Berichte d. physiclog. Gesellschaft in Berlin, Nov. 9, 1883.

VII.]

MICROCOCCUS.

75

the accidental admixture of a capsulated micrococcus, which,
according to Dr. Sternberg of Baltimore, is probably identical
with the one occasionally present in the fluid of the mouth
even of healthy persons. Capsulated micrococci, identical
in morphological respects and mode of growth in artificial

^-y "/Al'ifs^/^rlni

/ A v \i '(^)^>/|\ /^S|

FIG. 24. — FROM A SECTION THROUGH THE HUMAN LUNG IN ACUTE CROUPOUS
PNEUMONIA.

Three capillary vessels filled with blood are seen surrounding an alveolus which is

filled with fibrin and blood corpuscles, amongst them chains of micrococci.

Magnifying power 700. (Stained with gentian violet.)

cultivations, occur in other conditions not connected with
croupous pneumonia.

Bruylants and Verriers l assert that they have successfully
cultivated the micrococci of pleuro-pneumonia of cattle.

1 Bull, de PAcad. Belg. 1880.

76 MICRO-ORGANISMS AND DISEASE [CHAP.

More recently l T. Poels and Dr. W. Nolen, in Rotterdam,
assert that they have ascertained that in pleuro-pneumonia of

FIG. 25.— FROM A PREPARATION OF BLOOD OF RABBIT DEAD AFTER INOCULATION

WITH SPUTUM OF ACUTE CROUPOUS PNEUMONIA.

Weigert-Koch method, stained with gentian violet. Numbers of blood discs,

between them oval micrococci, surrounded by hyaline capsules.

Magnifying power 700.

I

FIG. 26. — FROM A PREPARATION OF PLEURAL EXUDATION OF A MOUSE DEAD
AFTER INOCULATION WITH BLOOD OF RABBIT MENTIONED IN PRECEDING
FIGURE.

Magnifying power 700.

cattle the pulmonary exudations contain micrococci, which

in their morphology and mode of growth in artificial cultures

1 Central*, f. d. med. Wiss. No. 9, 1884.

VIL] MICROCOCCUS. 77

are identical with the micrococci of human pneumonia. And
they further assert that artificial cultures of the micrococci
derived either from human pneumonia or from pleuro-
pneumonia'of cattle, produce in cattle the typical pleuro-
pneumonia. From my own observations I have reason to
doubt the accuracy of these statements.

5. Micrococcus gonorrhoea. — Micrococci have been found
in the pus of gonorrhoea. Neisser,1 and later Bokai and
Finkelstein,2 described them as spherical organisms of about

FIG. 27.— Two LARGE SCALY EPITHELIAL CELLS OF GONORRHCEAL Pus.

The epithelial cells are covered with micrococci, chiefly in dumb-bells, some in
sarcina form.

ofoo8 mm. diameter, generally forming dumb-bells, or sarcina-
like colonies of four. Several such groups form a zooglcea.
They adhere to the pus-corpuscles and epithelial cells. They
stain easily and well in methyl violet and gentian violet.
Bockhart 3 has succeeded in artificially cultivating these
micrococci, and in producing with them the disease by

1 Centralb. f. d. med. Wiss. No. 28, 1879.

2 Prager med. chir. Presse, May, 1880.

3 Sitzimgsberichte der phys.-med. Gesellsch. in Wttrzburg, Sept. 1882.

78 MICRO-ORGANISMS AND DISEASE. [CHAP.

inoculation. The pathogenic character of these micro-
cocci is denied by Sternberg.1

Aufrecht 2 reports the case of an infant twelve days old who
died with suppuration of the umbilical vein and liver. The
liver cells and the interlobular tissue were crowded with
micrococci (shown in sections by means of a 2 per cent,
watery solution of Bismarck brown). These micrococci
corresponded in size to the micrococcus gonorrhoea, and he
thinks it probable that they were derived from the vagina of
the mother ; during birth they might have got into the
umbilical vein, there caused inflammation, and thence passed
into the liver.

6. Micrococcus endocarditicus. — Micrococci in the form of
zoogloea have been seen in endocarditis ulcerosa. They some-
times form plugs in the blood-vessels of the muscular tissue
of the heart (Heiberg,3 Maier,4 Eberth,5 Koster,6 Klebs7).
Heiberg saw the micrococci forming chains in the muscle
of the heart, in the detritus of the ulcerations of the
endocardium, in the plugs in the vessels of the spleen and
kidney.

7. Micrococcus scarlatina. — In scarlatina Coze and Feltz 8
described micrococci as occurring in the blood ; as I have
mentioned above, I have seen them in the ulcerations of
the throat,9 and quite recently Pohl-Pincus 10 described very
minute micrococci adhering to the scales of the desquamating
epidermis in scarlatina. They form small colonies, and stain

1 Medical News, No. 16, 1884.

2 Centralb.f. d. mcd. Wiss. No. 1 6, 1883.

3 Vvrckmtfs Archiv, vol. 56. 4 Ibid. vol. 62.
5 Ibid. vol. 57. 6 Ibid. vol. 72.

7 Archivf. exp. Path. Bd. 9. 8 Malad. refect. 1872.

9 Report of the Medical Officer of the Privy Council for 1876.
10 Centralb.f. d. med. Wiss. No. 36, 1883.

vii.] MICROCOCCUS. 79

violet with a saturated solution of methyl violet. Their
diameter is very small, only about 0-0005 mm. The same
micrococci were noticed by Pohl-Pincus in the throat-dis-
charge.1 See Bizzozero's statements mentioned on p. 61.

8. In cattle plague, also called rinderpest, micrococci
have been found in the lymphatic glands by Klebs (1872)
and by Semmer in the blood and lymphatic glands (1874 and
1881). In conjunction with Archangelski,2 Semmer
cultivated the micrococci obtained from the lymphatic glands
of a sheep dead of inoculated rinderpest, in beef broth, in
meat-extract solution, and in mixture of broth, peptone, and
gelatine at 37° to 39° C. The micrococci grew very copiously
as zooglcea and in chains. With these micrococci (of a first
transfer or cultivation) a calf was inoculated, and died after
seven days from rinderpest. The cultures when transferred
lose gradually their virulence from one generation to the
next, but animals (sheep) inoculated with these are protected
against further virulent disease. Further, cultures exposed
for an hour to a temperature of 46° or 47° C. become greatly
attenuated in their action, and sheep inoculated with virus
thus attenuated are protected against virulent material.
Temperatures of - 10° to - 20° C. annihilate the activity of
rinderpest organisms. The specific nature of these micro-
cocci of rinderpest cannot, however, be considered at all
established as in the case of those mentioned above, e.g.
the micrococci of erysipelas.

9. In puerperal fever micrococci have been found in the
form of zooglcea by Heiberg,3 in all affected organs — endo-
cardium, lung, spleen, cornea, in a case of panophthalmitis

1 Seen already by McKendrick, Brit. Med. Journ. 1872.

2 Centralb.f. d. med. Wiss. No. 18, 1883. 3 Leipzig, 1873.

8o MICRO-ORGANISMS AND DISEASE. [CHAP.

puerperalis, and in the kidney, forming casts in the urini-
ferous tubules and emboli in the blood-vessels. Laffler1
found zooglcea and chains of micrococci in two cases of
puerperal fever associated with brain-softening. In both
cases emboli, due to micrococci, were found in the
surroundings of the softened part of the brain. Emboli
of micrococci were also here found in the vessels of the
kidney.

10. In pernicious anczmia Frankenhauser 2 described the
occurrence of micrococci (?) in the blood of pregnant women
suffering from this anaemia, not uncommon in Zurich. These
micrococci were very large, about one-tenth of the broad
diameter of a red blood-corpuscle, and some were provided
with a flagellum (?). Some were divided in two. In the
blood of the liver they occurred in large numbers. Franken-
hauser's description makes it very difficult exactly to under-
stand what he saw. He also states that these micrococci
were probably derived from decayed teeth, from which all
his patients suffered.

Eppinger3 described micrococci as occurring in acute
yellow atrophy of the liver.

11. In the syphilitic mucous patches of several patients
Aufrecht found a micrococcus, forming generally dumb-bells
and staining very deeply in fuchsin.4 Birch-Hirschfeld 5
confirmed this.

12. Microcoaus of acute infectious osteomyelitis. — Dr. Becker
has made, in the laboratory of the Berlin Imperial Sanitary

1 Breslauer drztl Zeitschrift, 1880.

2 Centralb.f. d. med. Wiss. No. 4, 1883.

3 Prater Viertelj. 1875.

4 Centralb.f. d. med. Wiss. No. 13, 1881.

5 Ibid. No. 44, 1882.

vii.] MICROCOCCUS. 81

Office, a series of important experiments on the micro-
organisms discovered by Schiiller and Rosenbach. He
collected pus from five cases of acute osteomyelitis in which
the abscesses had not been opened, and cultivated the micro-
cocci contained in it on sterilised potatoes, coagulated serum,
and gelatine-peptone. In the latter case, the pus was intro-
duced by means of needles into the mass, which was then
kept at the temperature of the room during three or five
days. After that time, the punctures made by the needles
assumed the appearance of white streaks, around which the
gelatine gradually liquefied and took an orange colour.
After a few days more, the mass gave out a smell like sour
paste, and the microscope revealed the presence of large
numbers of micrococci, having the same appearance as those
found in the pus. A small quantity of the mass was mixed
with sterilised water and injected into the peritoneal cavity
of some animals ; they died in a very short time of acute
peritonitis. The same fluid injected into the jugular vein
caused acute septicaemia and death ; but nothing abnormal
was found in the bones in either case. Dr. Becker then in-
jected a small quantity of the same fluid into the jugular
veins of fifteen rabbits, after having, some days before,
fractured or bruised the bone of one of the hind legs. At
the end of the first week a swelling was formed at the seat
of the bruise or fracture, the animals lost flesh, and died
after a few days. On dissection, large abscesses were found
around and in the bones, and in several cases metastatic
abscesses had formed in the lungs and kidneys. Numerous
colonies of micrococci were discovered in the blood and pus
of the animals upon which the experiments were made.
This micrococcus is identical with the micrococcus pyogenes
aureus of Rosenbach. When growing on Agar-Agar mixture
it forms flat irregular patches of an orange colour.

G

82 MICRO-ORGANISMS AND DISEASE. [CHAP.

13. Koch1 described various kinds of micrococci inti-
mately connected with certain destructive (pyaemic) processes
in mice and rabbits, (a) Micrococcus of progressive necrosis
in mice. Injecting into the ear of mice — white mice, or
better, field mice— putrid fluids, he observed a necrosis of
the tissues of the ear (skin, cartilage) starting from the point
of inoculation and gradually spreading on to the surrounding
parts and killing the animal in about three days. As far as
the necrosis reaches, the tissue is crowded with micrococci,
chiefly in the form of chains and zooglcea. The individual
cells are spherical, of about 0-0005 mm. in diameter. I
may mention that I have found a somewhat different micro-
coccus virulently active on mice. I have inoculated a
number of white mice subcutaneously in the tail with a
small micrococcus cultivated through several generations,
and apparently derived from an artificial cultivation in pork
broth, but due to accidental contamination. These micro-
cocci, after having been cultivated in pork broth through
several generations, were used in infinitesimal doses for the
inoculation of the above mice. In two instances I have
seen that the inoculation was followed after two or three
days by purulent inflammation at the seat of inoculation,
but apparently not spreading beyond it. But as time went
on inflammation and abscess in the lungs set in and the
animals died after about a week. On making longitudinal
sections through the tail, it was found that in most of the
lymph-spaces and lymph-vessels of all parts of the cutis and
subcutaneous tissue, far away from the seat of inflammation,
there were densely crowded masses of the same minute
micrococci as were used for inoculation. And these crowds
of micrococci could be traced to the seat of inflammation,

1 Untersuchungen iiber die Aetiologie d, WundinJections-Krankheiten,
Leipzig, 1878.

VIL] MICROCOCCUS. 83

where they extended amongst the inflammatory products in
great masses. The abscesses in the lungs were filled with
the same micrococci. Inoculated into the skin of fresh
mice, it again produced death by pyaemia. This micro-
coccus may therefore be called the micrococcus pyamice of
mice, (ft) Micrococcus causing abscesses in rabbits. Putrid
blood injected into the subcutaneous tissue of the rabbit
often produces suppurative abscess which, spreading, kills
the animal in about twelve days. In the wall of the abscess

FIG. 28. — FROM A SECTION THROUGH THE TAIL OF A MOUSE INOCULATED INTO
THE SUBCUTANEOUS TISSUE OF THE TAIL WITH ARTIFICIALLY CULTIVATED
MICROCOCCUS.

The part here illustrated is a good distance from the ulceratlon.

1. A capillary blood-vessel filled with blood-corpuscles.

2. Fat cells.

3. Groups of micrococci filling the lymph-spaces of the connective tissue.

are found continuous masses of zoogloea of micrococci.
The pus is infectious. The micrococci are spherical, and of
a very minute size, measuring only about 0*00015 mm. in
diameter. ' (c) Micrococcus causing pyamia in rabbits.
Skin of a mouse was macerated in distilled water for two
days, and of this fluid a hypodermic syringeful was injected
under the skin of the back of a rabbit. After two days the
animal began to lose flesh and died after 105 hours.
Purulent infiltration spread from the seat of inoculation into

G 2

84

MICRO-ORGANISMS AND DISEASE. [CHAP.

the subcutaneous tissue ; peritonitis ; spleen much enlarged ;
slight pneumonia. A hypodermic syringeful of the blood
of this animal was injected under the skin of a second
rabbit, and this died after forty hours. Post-mortem exa-
mination showed the same lesions as in the first case. In

FIG. 29.— FROM A PYOGENIC MEM-
BRANE COVERING THE SEROUS

COAT OF THE INTESTINE OF A
RABBIT DEAD OF PYAEMIA.

1. A large oval nucleus, probably the

nucleus of a detached endothelial
cell.

2. A pus corpuscle.

The rest of the pyogenic membrane is
beset with small micrococci.

o.

FIG. 30.— PYAEMIA OF RABBIT.

Blood of spleen. Between red blood-
discs three dumb-bells and two
single micrococci are shown.
(Gentian violet staining.)

{The micrococci as here represented
are somewhat too large.]

the blood-vessels of the affected parts were present micro-
cocci, single, as dumb-bells, and in zoogloea ; they were
spherical, about 0-00025 mm- in diameter, (d) Micrococcus
causing septicamia in rabbits. An infusion of meat was

FIG. 31.— OVAL MICROCOCCI FROM THE BLOOD-VESSEL OF THE SPLEEN OF A
RABBIT, DEAD OF KOCH'S SEPTICAEMIA.

prepared ; this was left to putrefy, and of this fluid a quantity
was injected under the skin of the back in two cases. Ex-
tensive gangrene with much oedematous exudation followed,
and death ensued in two days and a-half. The blood, the

VIL]

MICROCOCCUS.

capillaries of the kidney, and the enlarged spleen, contained
numerous oval micrococci. Two drops of the cedematous
exudation-fluid were injected under the skin of the back of
another rabbit. Death followed in twenty-two hours. There

FIG. 32 —CULTIVATION OF FOOT AND MOUTH MICROCOCCUS ON NUTRITIVE
GELATINE, AFTER SEVERAL WEEKS' GROWTH.

was no gangrene here ; but oedema was present, spreading
from the seat of the inoculation. Sub-serous haemorrhages
appeared in the intestines ; and minute haemorrhages were
also present in the cedematous tissue and in the muscles of

86 MICRO-ORGANISMS AND DISEASE. [CHAP.

the thigh and abdomen. The oedematous fluid, the cuta-
neous veins, the capillaries in the kidney, especially those
of the glomeruli, in the lung, and in the spleen, contained
numerous oval micrococci, singly, as dumb-bells, and in
zoogloea. The micrococci measured about 0*0008 to 0*00 1
mm. in their long diameter. These micrococci (taken with
the blood) produced in another rabbit and in a mouse the
same fatal disease.

14. Micrococcus bombycis (Microzyma bombycis, Bechamp).
— Oval micrococci, of about 0*0015 mm< in length, present
in large numbers, singly, and as dumb-bells and chains

FIG. 33. — SAME CULTIVATION AS IN FIG. 32 SEEN UNDER A LENS.

(straight or curved), in the contents of the alimentary canal
and in the gastric fluid of silkworms dead of the " maladie
de mortsblancs, flacherie." — Micrococcus ovatus, Nosema
bombycis. Present in large numbers in the blood and
organs, ova included, of silkworms affected with the disease
called "maladie des corpuscles," "pebrine," or Cornalia's
disease. Cornalia first saw them, afterwards Lebert and
Nageli. Pasteur proved definitely that ingestion as well -as
inoculation of the silkworms with the micrococci produces
the disease. The micrococci are comparatively large, 0^003
to 0*004 mm. long, 0*002 mm. broad; they are very bright
and occur singly, or in dumb-bells, or in small groups.

vii.] MICROCOCCUS. 87

1 5. Micrococcus of foot-and-mouth disease. — In the vesicles
of sheep ill with this disease I find a micrococcus, singly,
in dumb-bells, and in curved chains. It stains well with the
ordinary aniline dyes. It grows well in milk, in alkaline
peptone broth, in nutrient gelatine and in Agar-Agar mixture.
Growing on solid material its growth, besides being extremely
slow, is very characteristic, it forms a film composed of
minute granules or droplets, closely placed side by side, but
not confluent. It is highly sensitive towards antiseptics. It
does not liquefy nutritive gelatine, and in liquids does not
form a pellicle, but nevertheless when grown on solids, its
growth remains limited to the surface. It does not curdle
milk, although it turns the reaction of this latter slightly but
distinctly acid.

CHAPTER VIII.

BACTERIUM (Microbacterium, Cobn).

BY this name Cohn 1 designates a class of minute schizo-
mycetes, which are slightly elongated and oval, or short and
cylindrical, with rounded ends. They divide by fission,
like the micrococci, the individuals elongating and becoming
constricted in the middle. They are capable of sponta-
neous locomotion, being possessed of a flagellum at one or
both ends, with which they perform active spinning and
darting movements (Dallinger). Engelmann has shown
that these movements are only possible in the presence of
oxygen. Bacteria are found also as dumb-bells, i.e. in the
act of dividing, and then appear as rods constricted in the
middle. Occasionally, after rapid division, several remain
connected, thereby forming a short chain. In this state the
terminal elements are flagellate. Bacteria, like micrococci,
are capable of forming zoogloea, the interstitial gelatinous
substance being, as a rule, more copious than in the zoogloea
of micrococci. In this state they form pellicles, in which
the elements are without flagella ; but from the margin of
the pellicles one constantly sees elements separating, be-
coming flagellate, and moving away. In some species

1 Biologic d. Pflanzen, ii. (1872), p. 167.

CH. viii.] BACTERIUM. 89

the zoogloea is dendritically ramified (Zoogl&a ramigera,
Itzigsohn), as seen on the surface of fluids containing
decomposing algae.

i. Septic Bacteria. — With Cohn we distinguish two kinds :
— Bacterium termo and Bacterium lineola.

(a) Bacterium termo. — The elements are short and cylin-
drical, about 0-0015 mm- l°ng> a tnird less *n breadth, and
appear generally as dumb-bells. They are common in
putrefying fluids, indeed they form the essential cause or
ferment of decomposition, being the true saprogenous
ferment (Cohn). They are invested in a thick membrane,

/ *

f

FIG. 34.— BACTERIUM TERMO FROM FIG. 35.— ZOOGLCF.A OF BACTERIUM

AN ARTIFICIAL CULTURE. TERMO.

and are flagellate. With the end of putrefaction they
disappear. They grow well in Cohn's nourishing fluid, and
I have found them as constant inhabitants of unfiltered
distilled water in the laboratory ; so much so that with a
drop of this water I am always able to start a copious growth
of bacterium termo in pork broth, Agar-Agar, &c. When
cultivated in the incubator at 32° to 36° C. in suitable
nourishing material (pork broth, chicken broth,) they
produce a uniform turbidity, and after several days an
attempt at a pellicle, the whole nourishing fluid becoming
thicker. But after from several days to a few weeks the
cultures die, a fact which distinguishes them from all other

90 MICRO-ORGANISMS AND DISEASE. [CHAP.

bacteria. Growing in solid Agar-Agar and peptone mixture,
they produce an imperfect liquefaction, numerous gas bubbles
appearing in the material.

(b} Bacterium lineola ( Vibrio lineola, Ehrenberg, Dujardin),
differs from bacterium termo in being longer and thicker.
The cells are about 0*003 to 0-005 mm. long, about 0-0015
mm. thick. They occur in well-water and stagnant water,
where no distinct putrefaction is going on, and form zoo-
glcea, and pellicles, on the surface of potatoes and various
infusions.

* ~s

FIG. 36. — BACTERIUM LINEOLA. FIG. 37. — BACTERIUM LACTIS.

2. Zymogenic Bacteria. — Two kinds are known : Bacterium
lactis and Bacterium aceti.

(a) Bacterium lactis. — According to Pasteur, these bacteria
are about 0*0015 to o'o°3 mm. long, constricted in the centre;
they form short chains, or even zooglcea, and they are motile.
They produce the lactic acid fermentation, in the course of
which lactic sugar is transformed into lactic acid ; they are
anaerobic. Lister,1 by means of pure cultures, established
experimentally their causal relation to the lactic fermentation
or souring of milk.

(c) Bacterium aceti (Mycoderma aceti} is a little smaller
than bacterium lactis, being about 0*0015 mm' m length,
and often forms chains, and also pellicles, on the surface of
the fluid; it is motile. Pasteur maintains that it is the
ferment of the acetic acid fermentation. Cohn 2 found it in

1 Pathological Soc. Transactions, 1878.

2 Biol. d. Pflanztn, ii. p. 173.

viii.] BACTERIUM.' 91

enormous masses in beer that had become sour; it forms
dumb-bells, seldom chains of four, and sometimes a pellicle
on the surface. Pure cultivations have not been made with
it, and before deciding whether it is the real cause of the
acetic acid fermentation, experiments with such pure
cultures, i.e. inoculations of alcoholic fluids with it, are
required.

3. Pigment Bacteria. — Two kinds have been described :
Bacterium xanthinum and Bacterium aeruginosum.

(a) Bacterium xanthinum l is a bacterium about 0*007 to
o'oi mm. long, motile, single, also in dumb-bells, or short
chains. It produces the yellow colour of yellow milk. Its
pigment is soluble in water, and insoluble in alcohol or ether.
When introduced into boiled milk of neutral reaction, it
multiplies with great rapidity; the milk coagulates after
twenty-four hours ; it is soon teeming with them and turns
yellow. The reaction of the yellow milk is at first acid, but
soon becomes alkaline, and the alkalinity gradually increases.

(b) Bacterium aeruginosum. — In green pus Schrceter dis-
covered a bacterium, Bacterium aeruginosum^ The pigment
is greenish, and not lodged in the cells themselves ; it is
easily diffusible.

4. Pathogenic Bacteria. — Three kinds are described ; the
bacteria of Koch's septicaemia, of Davaine's septicaemia, and
of fowl-cholera.

(a) Bacterium septicaemia (Koch). — By injecting into
rabbits water from the rivulet Pauke, and from putrid mutton,
Koch3 succeeded in producing a rapidly fatal septicaemia,

1 Schroeter, Biol. d. Pflanzen, ii. p. 120; Vibrio synxanthus,
Ehrenberg.

2 Loc. cit. p. 122.

3 Mitth. aus d. k. Gaundh. 1881.

92 MICRO-ORGANISMS AND DISEASE. [CHAP.

which was characterised by the following appearances : — The
blood of all the organs contained very numerous bacteria, the
spleen and lymphatic glands were enlarged, and the lungs con-
gested ; but there were no extravasations and no peritonitis.
The smallest quantity of this blood inoculated into the skin
or cornea of another rabbit produced after an incubation of
ten to twelve hours distinct rise of temperature, and death
after sixteen to twenty hours. The conditions after death
were the same as above. Everywhere the blood contained
the bacteria. They are rods somewhat pointed at both ends,
measuring about 0-0014 mm. in length and 0*0006 mm. in
breadth. When stained, they show at each end a deeply-
tinted granule, the middle part remaining unstained ; for this

FIG. 38. — BLOOD OF PIGEON, DEAD OF SEPTICAEMIA.
Four blood-discs and four bacteria-termo are shown.

reason they are easily mistaken for a diplococcus. Generally
these rods occur singly ; occasionally they form a chain of
two, or more than three.

They have been cultivated successively in beef broth, blood
serum, gelatine, and a mixture of gelatine and broth and
peptone. The cultures have the same virulent properties as
the original blood.

Mice, pigeons, fowls, and sparrows are also very susceptible
to these bacteria ; but guinea-pigs, dogs, and rats resist them
successfully.

The microbe found by Pasteur in human saliva, which he
cultivated, and with which he produced septicaemia in

VIIL] BACTERIUM. 93

rabbits, may perhaps be a bacterium identical with the above,
but this is not definitely settled.

(b) Bacterium of Davaine' s septicamia. — This is a bacterium
which was originally derived by Davaine l from putrid ox-
blood in the warm season. Injected into rabbits it produced
rapidly fatal septicaemia, of the same nature as in the case
just mentioned, the blood teeming with a similar kind of
bacterium as in Koch's septicaemia just described. The
smallest quantity of the blood is again rapidly fatal in its
action. It is distinguished from Koch's septicaemia in the
rabbit by this, that Davaine's septicaemia is easily trans-
missible to guinea-pigs, but not to birds.

'o .

FIG. 39, — BLOOD OF RABBIT, DEAD OF DAVAINE'S SEPTICAEMIA.

Dowdeswell2 has shown that when such blood is
thoroughly sterilised (i.e. when the bacteria are killed), it
has no longer any infective power. Davaine had first shown
that the blood of rabbits dead of this form of septicaemia
bears an enormous amount of dilution without the minutest
quantity of it losing its pathogenic properties. Dowdeswell
has shown that this is easily explained by the enormous
number of bacteria present in every drop of the blood. But
it has been shown by Gaffky and Dowdeswell that there is
no increase in the virulence of the virus when it is passed

1 Bull. d. FA cad. de Med. 1872.

2 Proceedings of the Royal Society, No. 221, 1882.

94 MICRO-ORGANISMS AND DISEASE. [CHAP.

through successive animals, as was maintained by Coze and
Feltz.1

(c) Bacterium of fowl-cholera (microbe du cholera des poules].
— Semmer, Toussaint, and Pasteur2 have shown that this
organism is present in large numbers in the blood and organs
of fowls dead of this malady, which is chiefly characterised
by the following symptoms : — The animals are somnolent,
weak in their legs and wings, and they die under symptoms
of extreme sopor. On post-mortem examination, haemor-
rhage is found in the duodenum. The smallest quantity of
the blood is infective. Pasteur successfully cultivated the
bacteria in neutral chicken broth, at 25° to 35° C, and with
it inoculated the fatal disease. The organism is probably a
bacterium termo, very minute and slightly constricted in the
middle, so that it appears of the shape of an 8. When
cultures of this bacterium 3 are kept for some time (one, two,
three or more months), their virulence becomes diminished
or attenuated (owing, according to Pasteur, to the action of
oxygen), and this diminution of virulence is in direct pro-
portion to the time the culture is kept. The diminution or

1 Strasburg, 1866 ; Paris, 1872.

2 I place this here as a bacterium, but it is not quite decided, and not
quite clear from Pasteur's description, whether the microbe is only a
micrococcus dumb-bell, or a bacterium termo. Compare also Semmer
(Vergleichende Pathologie, 1878), Perroncito (Archiv f. wiss. u. pract.
Thierheilk. 1879. Toussaint (Comptes Rendus, xci. p. 301) considers
the disease identical with Davaine's septicaemia. I am inclined to think
that Pasteur has not used pure cultivations, but had the bacterium of
fowl-cholera and an accidental micrococcus together. The latter would
predominate as time passes on, so that after some days it would far
outnumber the bacterium ; and this is exactly what Pasteur's description
suggests. He says that at first the microbe is rod-shaped, and after a
few days it becomes a dumb-bell of micrococcus. The gradual atten-
uation by time of the virulence of Pasteur's cultures of the microbe
of fowl-cholera may be due to the presence of this contaminating
micrococcus.

3 Trans, of the International Med. Congress in London, 1881, vol. i.
p. 87.

VIIL] BACTERIUM. 95

attenuation shows itself in this — that according to the length
of time the culture is kept, the number of animals killed by
its inoculation gradually diminishes, and it ultimately ceases
to kill at all. Each culture of diminished virulence transmits
its attenuation to the next following culture (?). It is
possible to obtain cultures of such a low degree of virulence
that when inoculated into the skin of a fowl only a local
effect is produced, a peculiar infiltration ; but the animal
survives, and is then protected or " vaccinated " against the
more virulent material. But in order to produce this pro-
tection, it*is necessary that the culture (vaccine) should be of
the proper strength. If it does not produce a local effect it
gives no protection.

In fresh cultures the bacterium is more in the shape of a
rod, constricted in the middle ; in cultures several days old
it looks very much more like a dumb-bell of micrococcus (see
note on previous page).

Babes l has found the bacteria in the tissues and blood-
vessels of animals dead of the disease, both inoculated and
epizootic, in the shape of rods of about 0*0015 to °'o°2 mm.
in length and about 0-00025 mm- ^n thickness; the ends
always staining more deeply than the middle part.

1 Archives de Pkysiologie, July 1883, p. 49.

CHAPTER IX,
BACILLUS (Desmobacteritim, Cohn).

General Characters. — Bacilli are cylindrical or rod shaped
bacteria, which are rounded or square-cut at their extremi-
ties ; they are longer in proportion to their thickness than
bacterium termo, and divide by fission, forming straight,
curved, or zigzag chains of two, four, six, or more elements.
Many species of bacilli in suitable nourishing material grow
by repeated division into longer or shorter chains of bacillus
— filaments or leptothrix. These appear straight or wavy
and twisted, isolated or in bundles ; and although in the fresh
condition they appear of a homogeneous aspect, when suitably-
prepared, as by drying and staining with aniline dyes, they
show themselves composed of shorter or longer cubical,
cylindrical, or rod-shaped protoplasmic elements, contained
in linear series within a general hyaline sheath : between
many of the elements is a fine transverse septum. The
isolated bacilli are likewise composed of a membrane and
protoplasmic contents. These latter appear homogeneous or
finely granular, and when stained with aniline, absorb the dye
very easily and retain it better and longer than the sheath.
According to the stage and the rapidity of their growth, the
bacilli vary much in length ; this is the case not only with

CH. ix.] BACILLUS. 97

the single bacilli and short chains, but also in an eminent
degree with the elements of a bacillus-filament or leptothrix.
In each case, indeed, it is possible to ascertain that all
lengths occur from the cubical or spherical element to the
cylinder or rod. The former elongate into the latter and
then divide. According to whether the division occurs in a
short or long element, the daughter elements are cubical or
spherical in the former, cylindrical or rod-shaped in the

/N,

FIG. 40. — BACILLUS SUBTILIS GROWN IN PORK BROTH.

At i, the elements are thickened. The preparation had been dried and stained with
aniline purple.

latter case. This applies to single bacilli, to short chains,
and to the leptothrix forms.

Tnere are a great many species of bacilli, differing from
one another (a) in the shape of the elements, (b] in motility,
(c) in the power of forming filaments or leptothrix, and
particularly (d) in the thickness and length of the elements.

(a) There are some species of bacilli — e.g. hay-bacillus,
anthrax-bacillus, bacillus of putrid blood, bacillus found

H

98 MICRO-ORGANISMS AND DISEASE. [CHAP.

occasionally in the blood-vessels of dead animals, bacillus of
malignant oedema (Koch), &c. — in which in the single
bacilli and in the chains and filaments, the size of the
elements varies from that of a cubical or spherical mass of
protoplasm to that of a cylinder or rod several times as long
as it is thick. In some species (e.g. tubercle-bacilli), the
elements are almost spherical. There are on the other hand
other species (e.g. bacillus amylobacter) where the elements
are always rods or cylinders. In these cases of short bacilli
it sometimes becomes difficult to say whether one has to
deal with bacilli or bacteria, but the growth of the bacilli
into leptothrix, and particularly their power of forming spores,
is decisive, although neither of these events may happen
owing to peculiar conditions.

(b) Some bacilli (e.g. hay-bacillus, bacillus in common
putrefaction, bacillus growing on surfaces of putrefying
material and tissues, bacillus found in the abdominal organs
after putrefaction has set in, &c.), are possessed of a flagellum
at one end, and are therefore endowed with the power of
locomotion. Other species (e.g. anthrax- bacillus, bacillus of
malignant cedema) are without such power. But even in
the first case the power of locomotion is possessed by the
bacilli only when single or in short chains, not by the longer
chains or leptothrix.

(f) Not all bacilli are capable of forming leptothrix-fila-
ments. This power is possessed in an eminent degree by
certain species, such as the hay-bacillus, the anthrax-bacillus,
the bacillus of malignant oedema, the bacillus found on the
surface of the mucous membrane lining the cavity of the
mouth and tongue (leptothrix buccalis). Other bacilli (e.g.
bacillus amylobacter, leprosy-bacillus, tubercle-bacillus, &c.),
generally do not form leptothrix.

(d) There exists the greatest variety in reference to the

ix.] BACILLUS. 99

thickness of the bacilli; some (e.g. bacillus amylobacter,
and some species occurring in ordinary putrefaction) being
several times as thick as others, like hay-bacillus, anthrax-
bacillus, &c.

Many bacilli and bacillus-filaments (e.g. hay-bacillus, an-
thrax-bacillus) degenerate on growing old, the protoplasmic
elements becoming granular and breaking down altogether
into debris. This may occur to single elements within a
chain or leptothrix ; and then the corresponding part of the
sheath of the chain, owing to the subsequent disappearance
of the debris, becomes empty and devoid of protoplasm.
Longer or shorter portions of a chain or leptothrix may thus
degenerate and become deprived of protoplasm, the sheath
only persisting. These portions become at the same time
thicker, the sheath having swollen up.

Another mode of degeneration consists in the elements
and sheath curling up, swelling up, and ultimately breaking
down into debris. According to Cohn,1 bacilli do not form
zooglcea in the same way as micrococcus and bacterium do.
With all due deference to the authority of Cohn, I must hold
that the bacilli possessed of a flagellum are capable of
forming a true zooglcea. When one inoculates a fluid-
nourishing medium (e.g. broth) with hay-bacillus or other
motile bacillus of common putrefaction, after keeping it for
twenty-four hours in the incubator one notices a uniform
turbidity. After several days one notices that the surface of
the fluid becomes covered with a whitish film ; this, as in-
cubation goes on, thickens into a thick resistant not very
friable pellicle. By shaking the fluid the pellicle becomes
detached from the glass wall and sinks to the bottom of the
fluid ; after another day or two a new pellicle is formed, and
so on until the material is exhausted.

1 Beitr. z. Biologie d. Pflanzen, vol. i?.

H 2

ioo MICRO-ORGANISMS AND DISEASE. [CHAP.

Any part of this pellicle examined under the microscope
shows itself to be a zooglosa in the true sense of the word,
vast numbers of shorter or longer bacilli crossing and inter-
lacing and lying embedded in a gelatinous hyaline matrix.
As with bacterium termo, one occasionally notices at the
margin of the mass one or other bacillus wriggling itself free
and darting away. And in the case of non-motile bacilli,
putrefactive and others, I have also seen distinct formations
of zooglcea, having the shape of spherical or oval lumps of
various sizes composed of a hyaline jelly-like matrix, in which
are embedded the bacilli in active multiplication.

In those species in which the bacilli are capable of forming
leptothrix (leptothrix buccalis, hay-bacillus, anthrax-bacillus)
the filaments may form dense convolutions. When in these
convoluted filaments spores are formed (see below) and the
sheaths of the filaments swell up and become agglutinated
into a hyaline jelly-like substance, the spores appear to form
a sort of zooglcea.

Bacilli are killed by drying, but it is necessary to bear in
mind that they must be exposed to the drying process in thin
layers (Koch). At the temperature of boiling water they are
invariably killed, but not their spores. Even heating them
from half an hour to several hours at a temperature above
55° or 60° C. kills them. Freezing also kills them, but not
their spores. Carbolic acid, corrosive sublimate, thymol,
&c., kill them.

One of the most striking phenomena in the growth of
bacilli is their power of forming spores. These are generally
oval when fully developed, spherical when immature ; they
are of a glistening appearance, and take the ordinary dyes
either with difficulty or not at all ; they are generally a little
thicker than the bacilli within which they have developed.
Their formation always takes place in this way : in one or

ix.] BACILLUS. 101

other elementary cubical, spherical, or rod -like mass of pro
toplasm there appears a bright dot; this enlarges at the
expense of the protoplasm until in its fully developed state
it has an oval shape. The whole of the protoplasm of an

FIG. 41. — THE SAME BACILLUS AS IN PRECEDING FIGURE.
At i, spores have made their appearance.

element is not consumed in this process, a small trace always
remaining unused at one or both ends. The sheath enlarges
and the bacillus looks much thickened ; then the sheath
breaks, and the spore with the remnant of protoplasm

'

FIG. 42.— THE SAME BACILLUS AS IN FIG. 43.— BACILLUS SUBTILIS OF HAY

PRECEDING FIGURE. INFUSION.

Some of the spores are germinating At i, spores are germinating into

into bacilli. bacilli.

becomes free. Soon this remnant disappears, if it had not
disappeared while the spore was still contained within the
sheath, and now the spore is free. Under the most
favourable conditions a spore may be formed in each

102 MICRO-ORGANISMS AND DISEASE. [CHAP.

elementary mass of protoplasm, or it may be only in a small
number. In the first case : a consecutive series of spores is
present in the bacilli, two spores if the bacillus is composed
of two elementary cells, four in a chain of four elementary
cells, or a vast number in a leptothrix. In the second case :
a bacillus composed of two or four elementary cells may
contain only one spore at one end or in the middle, or one
at each end, or two together in the middle ; in the leptothrix
spores are seen only at comparatively long intervals. The
position of the spore in the bacillus is generally so that the
long axis of the spore is parallel to that of the bacillus ; but
exceptionally it may be placed obliquely or even transversely.
The bacilli in which spore-formation has set in are always
much thicker, twice or more, than those in which no spore-
formation has occurred ; and as has been stated above, the
sheath swells up and remains for some time as a hyaline
gelatinous capsule around the spore, but sooner or later this
is also lost and the spore becomes quite free. When spore-
formation has taken place in a convolution or in a mass of
leptothrix, and after the sheaths of the bacilli have become
swollen up into a gelatinous matrix, it looks as if we had a
zooglcea, in which the bright oval spores form the particular
elements embedded in a more or less hyaline gelatinous
matrix. But even in these cases on careful analysis it is
noticed that the spores have a linear or serial arrangement,
being originally developed in filaments.

This spore-formation occurs in many species of bacilli, and
it closes the cycle of the life-history of the bacilli. But it
does not take place under all circumstances. In the case of
many bacilli, e.g. hay-bacillus, anthrax-bacillus, bacillus of
putrefaction, spore-formation occurs only when there is an
ample supply of oxygen, e.g. when the bacilli grow on the
surface of the nourishing material (Cohn, Koch). It has

ix.] BACILLUS. 103

nothing whatever to do with the exhaustion of the nourishing
material, as Buchner seems to think ; for if the conditions ot
spore-formation are given, amongst these particularly the
exposure to the air, bacilli will commence to form spores
long before the nourishing material is exhausted. I will
here mention a particular instance to show this.

Take a test-tube with Agar-Agar peptone, such as has been
mentioned in a former chapter as fit for inoculation ; inoculate
the surface of the Agar-Agar with hay-bacillus or anthrax-
bacillus, place it in the incubator, and keep it there at a
temperature of 30° to 35° C. After 36 to 48 hours you will
find the surface covered with a good crop of bacilli and lepto-
thrix, and in some of them spore -formation is already going
on with great vigour. For several days after, the amount of
leptothrix increases, and in the filaments large numbers of
spores are formed. This goes on for several weeks, long
before the nourishing material becomes exhausted. But
during all this time the spore-formation is limited only to the
surface ; the filaments growing into the deeper strata remain
without spore-formation. The same observation can be made
with gelatine mixtures of peptone, broth, &c., in the test-tube
or in the glass cell described and figured in Chapter V. In
the case of the gelatine mixture it is particularly instructive
to watch this process, since it clearly proves that the free access
of air is essential for the formation of spores. For if the
anthrax-bacillus be grown on the (solid) mixture of gelatine
and broth described in a former chapter and kept at the
ordinary temperature of the room or in the incubator at not
more than 22° to 25° C., the spore-formation on the surface
occurs only as long as the material remains solid. Anthrax-
bacillus as it grows liquefies the gelatine mixture ; in conse-
quence of this after some days the superficial layers of the
material become fluid, and the bacillar growth, sinking to

104 MICRO-ORGANISMS AND DISEASE. [CHAP.

the bottom of the fluid layer, is thus removed from the surface.
The spores which were freely formed while the growth went
on on the surface, germinate again into bacilli, but because
these have now sunk into the depth, although rapidly
multiplying and growing into filaments, they cease to form
any spores.1

Bacilli which are not possessed of the power of locomotion
(i.e. are without a flagellum) when sown into the depth of a
fluid or solid material, if they have no chance, accidental or
otherwise, of reaching the surface, do not as a rule form
spores; but there are some such bacilli which, although
not growing on a free surface, nevertheless form spores,
e.g. the bacillus butyricus or amylobacter (Prazmowski).
Some putrefactive bacilli occurring after death in the
abdominal organs (intestine, kidney, spleen, liver), and
in fluid exudations within the peritoneal and pleural
cavities, show also spore-formation ; probably they get
their oxygen from the tissues. Anthrax-bacillus, however,
never forms spores except it is growing well exposed to the
outer air.

The bacilli which are possessed of a flagellum (e.g. hay-
bacillus, bacillus of putrefaction) generally form a pellicle
on the surface, and in this pellicle copious spore-formation
goes on.

The spores first formed, when shaken down into the
fluid, again germinate into bacilli, and there multiply.
The last pellicle formed in a culture, before exhaustion,
represents the last crop of spores ; and these, owing to the
exhaustion of the nourishing fluid, remain as spores, only
capable of germinating into bacilli when new nourishing
material is added, or when they are transplanted to new
nourishing material.

1 Report of the Medical Officer of the Local Government Board, 1881.

ix.] BACILLUS. 105

It is a rule that wherever the spores are formed they ger-
minate into bacilli if they have access to nourishing material ;
but if not, or if the nourishing material is exhausted, they
remain as spores. Spore-formation does not take place at
low temperatures. Koch found in the case of anthrax-bacillus
that a temperature below 12° C. prevents the formation of
spores. Pasteur states that in the case of anthrax-bacillus
spore-formation does not take place above 40° C. ; never for
instance at 42° or 43° C. Koch gives 43° as the upper limit ;
but I have found that both in the case of hay-bacillus and
anthrax-bacillus the bacilli lorm spores copiously even at a
temperature of 44° C. Moisture is an essential element
in the formation of spores.

The spores represent the seeds capable of retaining life
and of germinating into bacilli even after what would appear
the most damaging influences (that is, damaging to all other
kinds of organisms and to the bacilli themselves), such as
long lapse of time, drying, heat, cold, chemical re-agents, &c.
Spores retain the power to germinate into bacilli after the
lapse of long periods, and there is no reason to assume that
these periods have any limit ; it makes no difference whether
they are kept dry or in the mother-liquid.

The temperature of boiling water, while it kills micrococci,
bacteria, and bacilli themselves, does not affect the vitality of
the spores. Cohn (loc. tit.*) found spores of hay-bacillus still
capable of germination even after boiling ; boiling for half an
hour or more killed them. Prazmowski found that the spores
of bacillus butyricus (amylobacter) are killed by five minutes'
boiling. In the case of bacillus subtilis, e.g. hay-bacillus, I
found that boiling for half an hour does invariably kill them,
but ten minutes is not to be relied on. Exposing the spores
of anthrax-bacillus to a temperature of oc to — 15° C. for one
hour did not kill them. Antiseptics, such as carbolic acid

106 MICRO-ORGANISMS AND DISEASE. [CHAP.

(5 to 10 per cent), strong solutions of phenyl-propionic acid
and phenyl-acetic acid, corrosive sublimate (i : 10,000),
although the spores were kept in these fluids for twenty-four
hours and more, did not kill them.

The spores of bacillu-santhracis are killed by one to two
minutes' boiling in water or salt-solution.

This great resistance of spores to low and high tempera-
tures, to acids and other substances, is due to this, that the
substance of each spore is enveloped in a double sheath : an
internal sheath probably of a fatty nature, and an external
one probably of cellulose ; both are very bad conductors
of heat.

Owing to the fact that spores resist the action of boiling
water, if not prolonged for ten minutes, and that the other
bacteria (such as micrococcus, bacterium, and bacillus itself)
are killed by the temperature of boiling water if kept at this
temperature for a few seconds, it is possible to separate the
spores of bacilli from the other organisms. All one has to do
is to subject the fluid containing these various organisms to
the temperature of boiling water for a few seconds. All
except the spores of bacilli will be thereby killed, and
thus the fluid becomes free of all other organisms except
the spores.

When spores are sown in a nourishing material, fluid or
solid, and when this is exposed to a temperature of about 32°
to 38° C., the spores after the lapse of a few hours, in some
cases six (spores of anthrax-bacillus), in others two to four
hours (spores of hay-bacillus), in others more than six hours,
are seen to germinate, each spore growing into a bacillus. In
the case of solid nourishing material the presence of moisture
is essential.

In this germination what one sees is this : the spore
increases in thickness, it then loses its dark contour at one

ix.] BACILLUS. 107

pole or at one of the long sides, and at this point a pale
projection appears. This projection increases in length
and gradually becomes as long as a bacillus, the investment
of the spore gradually fading away. This new bacillus soon
divides into two, and so on.

The spores are capable of germinating independently of
the free access of air.

It has been shown by Engelmann that the presence and
renewal of oxygen as well as a certain concentration of the
nutritive material is essential for the motility of those bacteria
that are possessed of cilia, i.e. that are possessed ot
locomotion.

As long as the bacteria are living, their protoplasm does
not combine (stain) with nitrate of silver solution, only after
death does this become possible. Hereby an index is
furnished for ascertaining whether, and which, bacteria in a
given sample are living, and which are dead. There is no
difference in this respect, i.e. in respect of the different
reaction of nitrate of silver on living and dead protoplasm,
between the protoplasm of bacteria and that of other vegetable
or animal tissues.

All bacteria, pathogenic and non-pathogenic, require for
their growth and multiplication oxygen, which they either
obtain from the medium in which they grow, and which
oxygen is dissolved in those media, or if this is consumed
or absent it is obtained by the bacteria in the process of the
chemical decomposition of the carbohydrates and proteids
present. Dr. Dupre1 has shown that the presence or
disappearance of oxygen (air) dissolved in water is a precise
gauge, in the first case of the absence, in the second of the
growth, of microphytes.

1 Report of the Medical Officer of the Local Government Board, 1884.

CHAPTER X.

BACILLUS : NON-PATHOGENIC FORMS.

SEPTIC bacilli.

(a) Bacillus subtilis (hay-bacillus). — The elementary rods
are of various lengths from 0*002 to 0*006 mm., and are

FIG. 44.— FROM A CULTURE OF BACILLUS SUBTILIS (HAY-BACILLUS).

Various forms between single bacilli and leptothrix.

Magnifying power about 700.

about 0-002 mm. in thickness. According to Cohn 1

at a temperature of 21° C. division into two requires

1 Loc. dt.

CH. x.] BACILLUS. 109

about one hour and a quarter, at 35° C. only about twenty
minutes.

The bacilli are capable of forming leptothrix filaments.
The bacilli when single are possessed of one flagellum, or
sometimes of two, one at each end. After division the
individual bacilli remain connected, each possessing a
flagellum at the free end. Each of them divides again
into four, so that a chain of four is formed. But they may
separate again or may go on dividing, remaining united,
and thus forming a longer or shorter filament. Not all

FIG. 45. — FROM A CULTURE OF BACILLUS SUBTILIS (HAY-BACILLUS), WITH
COPIOUS FORMATION OF SPORES.

1. Mass of spores embedded in hyaline matrix.

2. Bacilli.

3. Single bacilli containing each a spore : the sheath of the bacilli is well seen.

Magnifying power about 700.

bacilli possess the flagellum, many of them being for a time
in a resting state.

The bacilli form a dense resistant pellicle on the
surface of the nourishing medium, and in this copious
spore-formation takes place. If shaken when growing in a
fluid, the pellicle falls to the bottom, and soon a new pellicle
is formed.

Spore-formation is independent of any deficiency of nourish-
ing material. The spores are oval, bright, of about o'ooi to
0*002 mm. in length, and about 0*0006 to 0*001 mm. in

no MICRO-ORGANISMS AND DISEASE. [CHAP.

thickness. They do not stain in ordinary dyes, and hence
form a great contrast to the bacilli.

This bacillus is very common and widely distributed ; it
occurs in almost every organic substance rich in nitrogenous
compounds which is left exposed to the air to decompose.
The best material is hay-infusion. An infusion, cold or
hot, of hay is made in a beaker or flask ; the fluid is filtered,
covered with a glass plate, and left to stand in a warm
place. After a day or two it swarms with bacillus
subtilis, which is also called hay-bacillus, since ordinary
hay contains multitudes of its spores. For this reason
even boiling of the fresh infusion for a few minutes does
not sterilise it.

sn'i oj

FIG. 46. — GERMINATION OF SPORES INTO BACILLI.

a. Spores of a small kind.

b. Spores of a larger kind of bacillus subtilis.

Magnifying power about 700.

The bacillus grows well in every fluid that contains the
necessary salts and nitrogenous compounds ; thus all kinds
of broth, all kinds of animal fluids (hydrocele, blood-serum,
&c.), gelatine, peptone solution, &c., are suitable nourishing
media.

The spores of the hay-bacillus are widely distributed
in the air, and most contaminations by air are due to its
spores.

(b) Bacillus ulna. — By this name Cohn * designates certain
species of bacilli, stiffer and thicker than those of bacillus
subtilis. The individual elements are about o'oi mm. long,
1 Loc. cit. p. 177.

X.]

BACILLUS.

in

and 0*002 mm. thick. They are motile, just like bacillus
subtilis. Although they form chains they do not form proper
leptothrix. They occur in putrid fluid. They are very
common in the ichor produced by injecting ammonia or other
substances producing sloughing and necrosis of the subcu-
taneous tissue in the guinea-pig.

FIG. 47. — BACILLUS ULNA, IN THE CAPILLARIES OF THE HUMAN LIVER.
POST-MORTEM CHANGE.

1. Liver cells, somewhat swollen.

2. Bacilli.

Magnifying power 300.

(c) bacillus septicus occurs in earth, in putrid blood, and
in many putrid albuminous fluids. It is non-motile, and is
capable of forming leptothrix. The thickness varies from
o'oo4 to o-oi mm., and its length depends on the number
of elements contained in a row. The shortest are about 0*004
mm. There are various species, differing from one another
in the thickness of the elements. They are all anaerobic.
The elements, whether in the short rods or in the leptothrix
filaments, are cubical or rounded. The rods and filaments
are markedly rounded on the ends. It forms spores

112

MICRO-ORGANISMS AND DISEASE. [CHAP.

independently of free access of air. The spores are oval, and
differ in thickness according to the thickness of the bacilli
they are formed in. The bacillus is found occasionally in
the blood-vessels of man and animals after death. In a
nourishing fluid, in which micrococcus, bacterium termo, or
bacillus subtilis grows, they have no chance of growing,
and even when numerous at first they soon disappear.

Fie;. 48.— STREPTOTHRIX FOERSTERI
(AFTER COHN).

FIG. 49.— CLADOTHRIX DICHOTOMY
(AFTER COHN).

(d) Streptothrix and Cladothrix. — Cohn * found in a
concretion of the human lacrimal canals long, pale, smooth,
apparently branched threads, either straight or twisted ; they
were finer than the threads of leptothrix buccalis ; he called
them Streptothrix Foersteri. They are probably identical
morphologically with Cladothrix dichotoma. This latter
1 Beitr. z. Biol. d. Pflanzen, vol. i. p. 186.

x.] BACILLUS. 113

occurs in pond-water containing decomposing organic matter.
It consists of long whitish threads fixed on chlorophyll -con-
taining algae. The threads when fresh appear smooth, pale,
occasionally granular, and on staining they are seen to be
composed of shorter or longer bacilli just like the leptothrix
form of bacillus subtilis ; but they are thicker than the
bacillus subtilis. Occasionally the ends of the threads are
seen not as linear series of bacillar rods, but like bacillus
anthracis and the bacillus of blue milk (see below) as
chains of torula-like spherical elements. From the threads
single motile bacilli are seen to come off. The threads are
only apparently branched, since the branches are threads
merely stuck on to other threads sideways at an acute angle.
A bacillus may be seen to stick to a thread and then to grow
out by continuous divisions into a long chain of bacilli, thus
forming, as it were, a side-branch. Some of the threads are
wavy and curved; most of them are, however, straight.
Zopf1 claims to have observed that the threads of the
cladothrix gave rise to micrococcus, bacterium, bacillus, and
spirillum ; and states that each of these is again capable of
growing into the threads of the cladothrix. But these
observations were not made after exact methods.

(e) Beggiatoa. — In stagnant water, particularly in sulphur-
containing water, peculiar oscillating colourless threads are
met with of the thickness of o'oooi to o'oi6 mm. ; they
contain highly refractive granules, which Cohn (Beitrdge zur
Biol. d. Pfl. i. 3) has shown to be composed of sulphur.
After dissolving these granules it is seen that each thread is
septate, being composed of a sheath and transverse septa at
regular intervals, by which the threads appear made up of a

1 Zur Morphologic der Spaltpflanzen, Leipzig, 1882 ; see also
Cienkowski.

I

ii4 MICRO-ORGANISMS AND DISEASE. [CHAP.

series of short cylindrical elements. There are a number
of species varying from one another in the thickness of the
threads.

FIG. 50. — THREADS OF CLADOTHRIX DICHOTOMA HIGHLY MAGNIFIED AND
STAINED WITH SPILLER*S PURPLE.

1. Threads of bacilli.

2. Tprula-forms.

The sheath is everywhere well seen.

Zymogenic bacilli.

Amongst these there is one species definitely known,
namely the Bacillus butyricus (Bacillus amylobacter?- Clos-
tridium bitty ricum, ferment butyrique, Pasteur). This bacillus

1 Prazmowski, Leipzig, 1880 ; van Tieghem, Bulletin de la Socitte
Boianique, vol. xxiv. 1877.

x.] BACILLUS. 115

has the same morphological characters as regards length
and thickness of the rods, as regards power to form lepto-
thrix, and as regards motility, as the bacillus subtilis. It
is capable of forming zoogloea, and is anaerobic, since it
grows well and forms spores copiously even when not ex-
posed to the air. After the rods have gone on dividing and
forming chains and filaments for some time, they swell up,
become granular and oval with more or less pointed ends,
and the formation of oval spores sets in. In this state the
oval rods are about 0-002 to 0-003 mm- thick, and the spores
are about 0-002 to 0*003 mm- l°ng an(* o'ooi mm. thick.
In solutions of starch, dextrin, or sugar the bacillus forms

FIG. 51. — CLOSTRIDIUM BUTYRICUM, OR BACILLUS BUTYRICUS.
Some of the spindle-shaped forms include an oval spore.

butyric acid. The fermentation of butyric acid in old milk
and ripening cheese is due to this bacillus. Cellulose is de-
composed by it, and hence its great importance in the diges-
tive process of herbivorous animals, in whose stomach and
intestine it is very common. It is very common also in
substances containing starch.

Iodine produces a characteristic blue staining in the pro-
toplasm of the bacillus. In young rods the colour produced
by iodine is blue, in older rods it is violet.

E. Kern described (Biolog. Centralbl. ii. p. 135) a bacillus
under the name of dispora caucasica, which he found in the

I 2

u6 MICRO-ORGANISMS AND DISEASE. [CHAP.

Caucascus, and which is used as ferment to produce from
cow's milk a peculiar drink called "kephir" or "hyppO."
The bacillus is similar to the bacillus subtilis, but is dis-
tinguished from it and all other bacilli by this, that every
bacillus forms two spores, one at each end, hence the name
dispora. But after recent investigations it appears that this
bacillus is accidental, the fermentation being produced by
saccharomyces mycoderma (see Chapter XIV.)

FIG. 52. — BACILLUS SYNCYANUS (AFTER NEELSEN).

1. Typical bacilli, motile.

2. Non-motile rods invested in a gelatinous capsule.

3 and 4. Bacilli in which spore-formation is going on.
5. Torula-form of the bacillus.

Pigment bacilli.

(a) Bacillus ruber.1 — This appears as minute rods, iso-
lated or in twos and fours, and motile. It was found on
boiled rice by Frank. Its colour is red, and contained in
the bacilli themselves.

(b) Bacillus erythrosporus. — Motile isolated rods and
leptothrix. It was found in meat-extract solutions and on

1 Cohn, Frank, Beitr. z. BioL d. Pflanzen, vol. iii. p. 181.

x.] BACILLUS. 117

decomposing albumen, and forms pellicles. In the rods are
found oval spores.1

(c) Bacillus syncyanus (Neelsen) causes the blue colour of
milk after the milk has become acid ; it grows well in am-
monium lactate. It consists of motile rods, single or in
short chains. Neelsen 2 saw the bacilli assuming a torula-form,
the individual cells being hourglass-shaped or oval, or even
spherical (compare Bacillus anthracis, Chapter XL). The
rods appear also as a non-motile variety, and are then found
to be invested with a thick hyaline gelatinous envelope.
The rods form bright oval spores, being at the same time
swollen up and ovoid. In Cohn's nourishing fluid they are
capable of forming leptothrix, in which occur here and there
huge spherical or oval swellings, which, according to Neelsen,
probably represent gonidia.

1 Cohn and Miflet, Beitr. z. Biol. d. Pflanzsn. vol. iii. p. 128.

2 Beitr. z. Biol. d. Pflanzen, vol. iii. p. 187.

CHAPTER XL

BACILLUS: PATHOGENIC FORMS.

PATHOGENIC bacilli.1

(a) Bacillus of septicaemia of mice (Koch). — By inoculating
ordinary house mice with minute quantities of putrid fluids

FIG. 53. — FROM A SECTION THROUGH THE LUNG OF A MOUSE DEAD OF KOCH'S

SEPTICAEMIA.

1. Small vessel filled with blood; the white blood-corpuscles are filled with very

minute bacilli.

2. Interalveolar tissue : in it a white corpuscle filled with the bacilli. Magnifying

power 700.

3. A white blood-corpuscle more highly magnified, 1000.

(Stained with magenta.)

Koch found that occasionally one or another of these animals
showed signs of conjunctivitis and sopor, and finally death
followed in about forty to sixty hours. In these cases slight

1 What has been said of the micrococci associated with open wounds
and abscesses applies also to bacilli ; i.e. there are often bacilli present
in the secretions of open wounds, and in the tissue of the base of
ulcers ; and as the inflammation spreads, so also do the bacilli gradually

CH. XL] BACILLUS: PATHOGENIC FORMS. 119

oedema is found at the seat of inoculation ; the spleen is
large; in the cedematous' tissue and in the blood-vessels,
large and small, numbers of minute bacilli are found, chiefly
contained in the white blood-corpuscles, but also free. They
are very minute, about 0*008 to o'ooi mm. long, to 0*0001
to 0*0002 mm. thick, isolated or in couples, or in chains of
four or more. The smallest quantity of this blood invariably
kills, with the same symptoms, house mice and sparrows, but

FIG. 54. — FROM A SECTION THROUGH THE SMALL INTESTINE OF A MOUSE DEAD
OF SEPTICAEMIA.

The figure represents a section through a small vein in the submucous tissue, filled
with blood. At i, there is a homogeneous substance and in it numerous bacilli,
but these bacilli are much larger than the bacilli of Koch's septicaemia in the
mouse.
Magnifying power about 700. (Stained with methylene blue and vesuvin.)

not field mice. Rabbits inoculated with these bacilli in the
skin of the ear or the cornea show only a local inflammation,
and the affected tissues contain numerous bacilli of the
same kind. Such animals after the local effect has passed
off, are protected against any further attack by the same

invade the surrounding tissues. To mention one series of cases only, in
ulcerations and in inflammations of the mucous membrane of the
stomach and intestine, large numbers of bacilli are occasionally found
on the surface of the inflamed parts, and gradually invading the inflamed
tissue. Von Recklinghausen ( Virc how's Archiv, vol. xxx,), von Wahl
(ibidem , vol. xli.), saw minute pustular nodules in the inflamed gastric
mucous membrane which were full of bacilli. Whether the presence
and growth of these bacilli was the primary cause or only a concomitant
symptom (due, for example, to the loss of active vitality of the tissue)
remains to be proved.

120 MICRO-ORGANISMS AND DISEASE. [CHAP.

bacilli. Koch cultivated these bacilli artificially on mixtures
of aqueous humour and gelatine, of gelatine and peptone (i
per cent.), salt (o'6 per cent. NaCl), and sodium phosphate
in sufficient quantity to just produce an alkaline reaction.
The bacilli grow well on this mixture, and by repeated and
rapid division form peculiar branched series.

YIG. 55. — FROM A SECTION THROUGH A LYMPHATIC GLAND OF MAN DEAD OF
SEPTICAEMIA.

1. A blood-vessel which at one place is distended. by and filled with minute bacilli.

2. Lymph-corpuscles.

3. Degenerated lymph-corpuscles.

Magnifying power 700. (Stained with gentian violet.)

(b) Bacillus of septic&mia of man. — In several cases of
human septicaemia I have found in the blood-vessels of the
swollen lymphatic glands large numbers of minute bacilli,

XL] BACILLUS : PATHOGENIC FORMS. 121

slightly thicker than those just mentioned. They form con-
tinuous masses, both in the capillaries and in the minute
veins, amounting in some cases to veritable emboli. They
occur isolated or in short chains, their length about 0*00 1 to
0*0025 nim., their thickness about 0*0003 to 0*0005 mm-
Arloing and Chauveau (mentioned in the British Medical
Journal, Jan. 12, 1884) found in gangrenous septicaemia
around wounds short bacilli, some containing one or two
spores, which they consider as the true cause of the gangrene.
They are destroyed when fresh by a temperature varying
between 90° and 100° C. ; after drying, a temperature of
i20°C. is required.

FIG. 56. — FROM A SECTION THROUGH THE MESENTERIC GLAND OF A PERSON
WHO DIED OF TYPHOID FEVER.

1. Capillary blood-vessel filled with blood-corpuscles.

2. Large lymph-cell.
3- Nuclei.

4. Bacilli.

Magnifying power 700.

(c) Bacillus of typhoid fever of man. — Klebs 1 described in
the inflamed Peyer's glands, in the mesenteric glands, larynx,
and lungs of patients dead of typhoid fever, certain bacilli,
which are about 0*0002 mm. thick and of various lengths,
forming filaments up to 0*05 mm. long. These bacilli form
1 Archivf. exp. Path. vol. xii.

122 MICRO-ORGANISMS AND DISEASE. [CHAP.

spores. Eberth l found in about 50 per cent, of cases of
patients dead of typhoid fever, in the mesenteric glands and
spleen, peculiar short bacilli, rounded at their ends, and
occasionally slightly constricted in the middle ; some of them
contained spores. The bacilli stain very freely with methyl-
violet. It is, however, doubtful whether these bacilli can
be considered as necessarily and intimately connected with
typhoid fever, seeing that they are not constant, and only
occur in the mesenteric glands and spleen, i.e. in localities
into which an immigration of putrefactive bacilli from the
bowels may easily take place ; especially when we remember
that in fatal cases of typhoid fever there constantly occur
severe sloughing and necrosis of the Peyer's glands. (See
end of Chapter XVIII.) The bowels in typhoid fever always
contain innumerable masses of micrococci in colonies ; and
these micrococci are found not only in the tissue of the
intestinal mucous membrane but also in the mesenteric
glands and spleen.3

(rf) Bacillus of choleraic diarrhoea from meat -poisoning.—
In July, i88o,3 there occurred in Welbeck, Notts, an exten-
sive outbreak of diarrhoea among over seventy-two persons
who had partaken of beef and ham sandwiches sold at
Welbeck on the occasion of a sale of timber and machinery
on the estate of the Duke of Portland. The infection
showed itself after an incubation-period varying from twelve
hours or less to forty-eight hours or more. The first
symptoms were a sudden feeling of languor, nausea, griping
in the abdomen, in some cases giddiness and fainting, and

1 Virchoirfs Archiv, vols. Ixxxiii., Ixxxvii. See also Koch, Mittheil.
a. d. k. Gesundheitsamte, i. 1881 ; and Gaffky, ibid. 1882.

2 Klein, Reports of the Medical Officer of the Privy Council, 1875.

3 Report by Dr. Ballard in the Reports of the Medical Officer of the
Local Government Board, 1880.

XL]

BACILLUS: PATHOGENIC FORMS.

123

pain in the trunk. Then followed pain in the abdomen,
diarrhoea, and vomiting, the diarrhoea being most constant.
Four cases ended fatally. On. post-mortem examination

FIG. 57. -FROM A SECTION THROUGH THE KIDNEY OF A CASE THAT DIED
AFTER MEAT-POISONING AT WELBECK.

The figure represents part of a glomerulus of a Malpighian corpuscle, in which
some of the capillary blood-vessels are filled with the bacilli. Magnifying
power 700.

1. Capsule of Malpighian corpuscle.

2. Capillaries filled with bacilli.

3. Capillaries empty.

4. Bacilli contained between capillaries.

enteritis and pneumonia were most prominent. Part of the
kidney was examined in microscopic sections, and it was
found that many of the tubuli uriniferi contained hyaline

124 MICRO-ORGANISMS AND DISEASE. [CHAP.

casts ; that the capillaries of the glomeruli of the Malpighian
corpuscles, and the afferent arterioles, contained numbers of
bacilli, some of the capillaries being distended by and
plugged with masses of bacilli densely aggregated. In
February, 1881, a similar but less extensive outbreak
occurred at Nottingham, among fifteen persons that had
partaken of certain baked pork. The symptoms were
similar to those in the Welbeck outbreak. One case ended
fatally. Post-mortem: bloody exudation in pericardium,
intense pneumonia, mesenteric glands enlarged, enteritis,
Peyer's glands enlarged. Bacilli similar to those of the
above case were found in the blood, in the pericardial
exudation, in the juice and in the bloody fluid rilling the

FIG. 58. — ISOLATED BACILLI IN A SMALL ARTERY OF THE SAME KIDNEY AS IN
PRECEDING FIGURE.

Some bacilli contain spores.

alveolar cavities of the inflamed lung, in the vessels of the
kidney, in the submucosa of the inflamed Peyer's glands of
the small intestine, in the blood-vessels of the spleen and
around them.

The bacilli vary in length between 0*003 and 0*009 mm- >
their thickness is about 0-0013 mm. They are rounded at
their extremities, single or in chains of two, and some con-
tain a bright oval spore, situated in the centre or at one end,
and about o'ooi mm. thick. This was the case with the
bacilli in the glomeruli of the kidney of the Welbeck case.
The bacilli containing spores were thicker than those without
them,

XL] BACILLUS: PATHOGENIC FORMS. 12$

Experiments by feeding and inoculation made on dogs and
cats, rabbits, guinea-pigs, and mice, with the ham that had
done the mischief in the Welbeck case produced positive
results. In all cases we found pneumonia and haemorrhage
in the liver, peritonitis in some, spleen enlarged in most.
The bacilli found in this ham were cultivated in the incubator
in white of egg, and after two days' cultivation four white
rats, and several guinea-pigs and white mice were inoculated,
and they became ill after twenty-four hours ; they were quiet,
did not feed well, and were more or less soporous. When
killed the spleen was found enlarged, and in the lungs were
found haemorrhage and hyperaemia, and in some cases
extensive pneumonia.

Blood, pericardial exudation, and lung juice from the fatal
Nottingham case inoculated into ten animals (guinea-pigs
and white mice) produced fatal results in six, the other four
were killed : but in all there was severe pneumonia, in eight
out of the ten there was peritonitis, in four also pleuritis, and
in two in addition enlargement of the liver and spleen. Bacilli
were found in the blood and exudations of these animals.
On cultivating blood and lung juice from the above case a
crop of bacilli was produced, which on inoculation proved
very poisonous in the same way as in the previous cases.1

(e) Bacillus malaria. — Klebs and Tommasi-Crudeli 2 de-
scribed a bacillus occurring in the soil of the Roman
Campagna, which they cultivated on gelatine. The rods
are about 0-002 to 0*007 mm- l°ng > tne7 grow in cultures
into long leptothrix filaments composed of short joints. The
rods form spores either in the centre or at their ends. They
grow well also in other media, e.g. albumen, urine, and glue.

1 Compare also Huber, Archivf. klin. Med. xxv.

2 Archiv f. exp. Pa'.h. vol. xi.

126 MICRO-ORGANISMS AND DISEASE. [CHAP,

Quite recently Marchiafava and Celli x found that the red
blood discs of patients affected with recent malaria contain
peculiar homogeneous bodies, possessed of amoeboid move-
ment, in size a fraction of that of the blood discs. They call
them hamoplasmodium malaria. Sometimes these plasmodia
include pigment granules assimilated from the pigment of
the blood discs. Such pigmented plasmodia had been
already noticed by Laveran. Marchiafava and Celli found
that malaria blood containing the plasmodia is capable of
producing intermittent fever in man after intravenous in-
jection. The blood corpuscles of a person so infected again
contain the haemoplasmodia.

(/) Bacillus of ulcerative stomatitis in the calf. — In the
Lancet of May, 1883, A. Lingard and E. Batt described
peculiar bacilli in ulcerations occurring on the tongue and
buccal mucous membrane of the calf. " The typical ulcer
in advanced cases consists of a sore with free overhanging
edges. On section through the sore the tongue is found
necrosed to a considerable depth." " Whenever the sore
touches any other part of the mouth or cheek, the disease is
communicated and rapidly spreads. In some cases similar
necrotic changes had taken place in the lung. The line of
junction of the necrotic with the healthy tissues was found
to be occupied by a dense mass of bacilli having the appear-
ance of a dense phalanx advancing upon the healthy tissues.
The disease has been proved capable of transmission (to the
rabbit and mouse) by injection of the bacilli in question,
which are equally numerous and virulent after passing
through several generations by inoculation."

The disease often ends fatally in calves.

The best method of staining the bacilli was found to be
1 Fortschritte d. J/#/..Nos. xi., xviii., xxiv., 1885.

XL]

BACILLUS : PATHOGENIC FORMS.

127

this : The sections, both those prepared from the ulcerations
of the calf's tongue and from the inoculated tissues of the
rabbit, are immersed in a mixture of magenta and methyl
blue, then washed in spirit, and after clarifying in clove-oil
are mounted in Canada-balsam solution. The bacilli are

I't/Jf V

!&/«£/

FIG. 59 — FROM A SECTION THROUGH NECROSED AND ADJOINING INFLAMED PARTS
OF THE EAR OF A RABBIT, INOCULATED WITH MATTER TAKEN FROM
ULCERATIVE STOMATITIS OF THE CALF.

1. Necrosed part.

2. Inflamed tissue.

3. Bundles of bacilli.
Magnifying power 700. (Stained with magenta.)

stained deep pink, the inflamed tissue blue. The bacilli
appear as thin rods in rows, thus forming a leptothrix-like
growth. In some of the long filaments the individual bacilli
are not well shown. The filaments are either straight or
more or less curved. The length of the single bacilli varies

128

MICRO-ORGANISMS AND DISEASE. [CHAP.

from 0*004 mm- °r IGSS to o'ooSmm. or more; the thickness
is about o'ooi mm. Many of them contain spores. In the
ear of the rabbit they invade the connective tissue as well as
the cartilage over the whole extent of the ulceration and its

FIG. 60. — FROM A SECTION THROUGH TONGUE OF CALF, ULCERATIVE STOMATITIS.

1. Muscular fibres.

2. Inflamed tissue.

3 Bundles of the bacilli.
Magnifying power 700. (Stained with magenta.)

neighbourhood. Mr. Lingard found the same bacilli,
having the same arrangement, in a case of noma in the
human subject.

(g) Bacillus of glanders. — In 1882 Schiitz and Loffler 1
ascertained the occurrence of peculiar bacilli in the nodules
1 Dtu'sche med. Wochenschrift, 52, 1882.

XL]

BACILLUS : PATHOGENIC FORMS.

129

of the nasal mucous membrane and internal organs, such as
the lung, spleen, and liver, of horses dead or dying from
glanders. These bacilli are very minute, being of about the
size of tubercle-bacilli (see below), and are brought out by
staining the tissues with a concentrated watery solution of
methylene-blue, and after this washing with very dilute acetic

FIG- 61. — FROM A SECTION THROUGH THE CARTILAGE OF RABBIT'S EAR IN WHICH
ULCERATION HAD BEEN PRODUCED BY INOCULATION WITH NECROSED MATTER
OF CALF'S TONGUE.

1. Cartilage capsules.

2. Bundles of good bacilli.

3. Bundles of degenerating bacilli.
Magnifying power 700. (Stained with magenta.)

acid. They succeeded in artificially cultivating the bacilli
in the incubator at 38° C, on solid sterilised serum of horses'
and sheep's blood, using for the purpose particles (see below
under " Tubercle-bacilli ") of nodules of the lung and spleen
of a horse dead of glanders. After two days (i.e. on the

130 MICRO-ORGANISMS AND DISEASE. [CHAP.

third day) there appeared on the surface of the inoculated
material the first traces of the growth in the form of minute
transparent droplets which consisted entirely of the
characteristic bacilli. Cultivating these through several
generations or transferences, and then inoculating with them
a horse, rabbits, guinea-pigs, and mice, positive results were
obtained, especially in the guinea-pigs, which appear very
susceptible to the disease. On the site of the subcutaneous
inoculation appears an ulcer with indurated base, speedily
enlarging and spreading; other ulcers follow in the

FIG. 62. — Pus OF A PULMONARY ABSCESS IN A HORSE DEAD OF GLANDERS.

1. The nuclei of pus cells.

2. The glanders-bacilli.

Magnifying power 700. (The preparation had been stained with methylene-blue.)

neighbourhood, the neighbouring lymphatic glands become
swollen, and the general infection follows, in the form of
nodules and ulcers on the nasal septum, and nodules in the
internal organs. In the guinea-pig a characteristic tumour
of the testis, ovary, and vulva is often observed. In all
these cases the diseased tissues and organs contained the
characteristic bacilli.1

1 Other writers on the bacilli of glanders are Drs. Bouchard, Caoitan,
Charvin, in the Revue medicate Jran<;aise, Dec. 30, 1882. N. P.
\VassilieflT, in Deiitsche med. Woch. II, 1883, observed the bacilli in
human glanders.

XL] BACILLUS : PATHOGENIC FORMS. 131

(//) Bacillus of swine plague. — In a report to the medical
officer of the Local Government Board for 1877-1878, 1 have
shown that in this acute infectious disease the lungs, intes-
tines, and serous membranes are commonly found affected.
The lungs show lobular and lobar pneumonia, the large
intestine contains haemorrhages and ulcers, while the serous
membranes and lymph-glands are inflamed. Occasionally,
but not often, also the skin shows irregular red patches. I
have shown that in this disease the diseased organs contain
a form of bacillus, which only by its small size differs from

FIG. 63.— FROM A SECTION THROUGH THE INFLAMED INGUINAL LYMPH-GLAND

OF A PlG DEAD OF SwiNE PLAGUE.

1. A capillary blood-vessel filled with bacilli.

2. Reticulum of adenoid tissue.

3. A lymph-cell.

Magnifying power 700. (Stained with Spiller's purple.)

bacillus subtilis. I have also shown that particles of the
diseased organs or artificial cultures of this bacillus when
inoculated into pigs, mice and rabbits, produce the disease
with multiplication of the bacillus, while pigeons, fowls, and
guinea-pigs are unaffected by cultivations of the bacilli or by
the original virus.

Pasteur maintains that in an infectious disease of the pig
known in France as rouget and chiefly characterised by red
patches of the skin there is present in the blood a dumb-bell

K 2

132 MICRO-ORGANISMS AND DISEASE. [CHAP.

micrococcus, which, when artificially cultivated yields
material which, inoculated into pigs, is said to produce the
disease. Mice, rabbits, and pigeons are susceptible to the
disease, but in rabbits after several transferences the virus
becomes attenuated, and with this a mild form of the
disease can then be produced, protecting the animal thus
operated upon from a subsequent severe attack.

From the recent investigations on this subject by Schiitz l
it is quite clear, that the disease that in France is called
rouget and in Germany erysipelas of swine, is an altogether

FIG. 64. — FROM A PREPARATION OF BRONCHIAL Mucus OF A PIG DEAD OF
SWINE PLAGUE.

1. Detached epithelial cells of alveoli.

2. Bacilli.

3. Micrococci.

Magnifying power 700. . (Stained with Spiller's purple.)

different disease from what in this country and America is
called swine-fever, or swine-plague, or pig-typhoid. The
symptoms and course of the disease are totally different in the
two maladies. Schiitz has shown that the disease rouget or
erysipelas is due to a minute bacillus which in size, mode of
cultivation, and effect on inoculation, is almost identical
with the bacillus of mouse-septicaemia (Koch). Schiitz ex-
amined some of the attenuated lymph sent by M. Pasteur,

1 Mitth. aus d. k. Gesundheitsamte, Berlin, i. 1885.

XL] BACILLUS: PATHOGENIC FORMS. 133

and found that it contained the dumb-bell micrococcus
described by M. Pasteur ; but this was accidental contamina-
tion ; there was present the minute bacillus, and the action
of such lymph was due not to the micrococcus but to this
bacillus.

Artificial cultivations made in broth and hydrocele fluid
from diseased organs of the pig,, mouse, and rabbit, after an
incubation of twenty-four hours at temperatures ranging
between 30° and 42° C. contain the above rods, which crowd

FIG. 65 —FROM A SECTION THROUGH THE KIDNEY OF RABBIT DEAD OF SWINE
PLAGUE, SHOWING A MALPIGHIAN CORPUSCLE, THE CAPILLARIES OF THE
GLOMERULUS BEING TRANSFORMED INTO HYALINE IMPERMEABLE CYLINDERS.

i. Bacilli.
Magnifying power 500. (Stained with Spiller's purple.)

the nourishing fluids, all being rather short, about croc 2 to
0*003 mm. long, and all possessed of the power of active
locomotion, such as is known to be possessed by the septic
bacterium termo and bacillus subtilis. During the following
days of incubation, while the rods multiply, many of them
lose their motility, grow longer, up to 0*005 mm- an<^ more,
and in some of the longer samples bright spores make their
appearance, one spore at one or both ends or sometimes in
the centre.

134 MICRO-ORGANISMS AND DISEASE. [CHAP.

From these cultivations new cultivations may be made and
carried on through successive generations, all cultures behav-
ing in the same manner ; and in all of them the rods only
are present, and show exactly the same changes as in the
parent culture.

The smallest droplet of any of these cultivations produces
the disease in pigs, mice, and rabbits. The mice and rabbits
die with exactly the same appearances and with the same

FIG. 65. — BLOOD OF FRESH SPLEEN OF A MOUSE THAT DIED OF SWINE PLAGUE.

1. Blood discs.

2. A large nucleus.

3. Groups of minute bacilli.

4. Long bacilli.

5. Dumb-bells of bacilli.

Magnifying power 700. (Stained with gentian violet.)

anatomical lesions as when they are inoculated with material
directly taken from the diseased organs of a pig dead of swine
plague. Those animals generally die on the fifth, sixth, or
seventh day, and on post-mortem examination show a charac-
teristic swelling of the spleen, a characteristic disease of the
liver (chiefly coagulative necrosis of tracts of the liver tissue),
and inflammation of the lungs.

Inoculations of suitable sterilised nourishing fluids made

XL]

BACILLUS: PATHOGENIC FORMS.

J35

from the spleen, liver, and lung of such animals always result
in producing a copious crop of the characteristic bacilli, as

FIG. 67. — FROM A SECTION THROUGH A NECROTIC PATCH OF THE LIVER OF A

MOUSE DEAD OF SwiNE PLAGUE.

1. Tracts of liver cells shrunk.

2. Capillary blood-vessels filled with very small micrococci,

amongst which are seen the bacilli.

3. Bacilli only.

Magnifying power 700. (Stained with'Spiller's purple and magenta.)

do those made with the lung and bronchial glands of pigs
dead of swine plague ; but from the blood of the pig the
cultivations do not as a rule succeed, nor as a rule from the

r//»

FIG. 68. — BACILLI OF SWINE PLAGUE,

FROM AN ARTIFICIAL CULTURE,

AFTER FORTY-EIGHT HOURS' IN-
CUBATION.

Magnifying power 700. (Dried and
stained with Spiller's purple.)

FIG. 69. — BACILLI OF SWINE PLAGUE,
FROM AN ARTIFICIAL CULTURE,
DURING SIXTH DAY OF IN-
CUBATION.

i and 2. Bacilli.

3. Bacilli in which spores have been

formed. Magnifying power 700.

(Fresh specimen.)

blood of mice ; occasionally, however, those from the blood
of rabbits dead of the disease do succeed.

136 MICRO-CRGANISMS AND DISEASE. [CHAP.

Quite recently I have ascertained that pigs inoculated
with artificial cultures of these rods (started from the pig,
mouse, or rabbit dead of the plague) or with the diseased
organs of a mouse or rabbit, suffer from a mild form of the
disease, which after one or two weeks passes off completely.
I have had pigs that had been twice inoculated, the first
time with artificial cultures, the second time with diseased
organs of mouse and rabbit, and each time the pigs suffered
from a mild form of the disease. They were then inocu-
lated a third time with the juice of the diseased (fresh) lung
of a pig dead of the plague ; this time also they showed
distinct symptoms of the disease, but after a few days to a
week they completely recovered. If normal (or not pre-
viously inoculated) pigs are inoculated with matter from the
diseased fresh lung of a pig dead of the plague, they as a
rule die from a virulent form of the disease. But in the
above case they were protected by previous inoculations,
not altogether against a new attack but against a fatal
attack.

(i) Bacillus Leprce. — Armauer Hansen1 first ascertained
the existence of large numbers of minute bacilli in the
peculiar large leprosy-cells of Virchow, which occur in the
nodules of leprous patients. Neisser confirmed this, and
considerably extended our knowledge of the bacilli, showing
that they can be readily stained pink with fuchsin or with
Ehrlich's acid solution of eosin-hsematoxylin. The bacilli
are fine rods about 0*004 to 0*006 mm. long and less than
0*001 mm. thick. They are pointed at their ends, and
always occur in masses within the large leprosy-cells of the
leprous tubercles of the skin and internal organs. But they

1 Vin how's Anhiv, vol. Ixxix. ; and Quart. Journ. of Micro. Sd.
1880.

XL]

BACILLUS: PATHOGENIC FORMS.

137

are also present in the interstitial tissue of the nervous
branches in the anaesthetic variety of the disease.1 Some
bacilli are motile, others not ; some possess bright oval
spores, and others are more or less beaded, owing to local
collections of the protoplasm within their sheath. Neisser
and Armauer Hansen have cultivated them artificially in

FIG. 70. — FROM A SECTION THROUGH THE LARYNX OF A PATIENT DEAD OF
LEPROSY.

Huge cells in fibrous connective tissue ; the ce3s axe filled with the leprosy

bacilli.
Magnifying power 600. (Stained with magenta and vesuvin.)

blood:serum and in solutions of meat-extract. Neisser has
also shown that the characteristic leprosy-cells are only
wandering cells modified by the growth and multipli-
cation in them of the bacilli. In the blood the bacilli do

1 Compare also Cornil, Union Medicate, iSSi, Nos. 178, 179, and
Babes, Archives d. Physiologic, July, 1883.

138 MICRO-ORGANISMS AND DISEASE. [CHAP.

not occur, and they spread probably only by way of the
lymphatics.

Inoculation experiments on domestic animals and monkeys
have hitherto failed.1 Damsch2 maintains, however, that

FIG. 71. — BACILLI OF THE SAME PREPARATION AS IN PRECEDING FIGURK.
More highly magnified, 1000.

he was able, by inoculation with leprous tissue into the
peritoneal cavity and into the skin, to produce in cats a

IT/

FIG. 72. — CELLS OF THE LEPROSY NODULES OF MAN, FILLED WITH THE
LEPROSY BACILLI (AFTER NEISSER).

distinct increase and sprouting of the bacilli. Preparations
of leprous nodules of the larynx and skin made by my

FIG. 73.— FROM AN ARTIFICIAL CULTURE OF BACILLUS OF LEPROSY
(AFTER NEISSER).

friend, Mr. A. Lingard, and stained with Weigert's solution
of magenta and vesuvin, showed the leprosy-bacilli com-
pletely filling all the cells, small and large, spherical and

1 Kbbner, Virchouts Archiv, vol. Ixxxviii. ; Hansen, ibidem, vol. xc.

2 Virchow's Archiv, vol. xcii.

XL]

BACILLUS : PATHOGENIC FORMS.

139

spindle-shaped, contained between the connective-tissue
bundles.

In a section through the liver of a bird (Rhed) that died
in the Zoological Gardens in London, prepared by Dr.
Gibbes after his method of staining for tubercle-bacilli, there
were seen innumerable aggregations of larger and smaller
pink masses (visible to the unaided eye as dots of the size
of a pin's point to that of a pin's head or millet-seed, and

FIG. 74.— FROM A SECTION THROUGH A NODULE OF THE LIVER OF RHEA.
r. Cells of various sizes filled with minute bacilli ; owing to the smallness of the
bacilli and to their being crowded in the cells and owing to the comparatively
low magnifying power (300) the bacilli appear like dots.

(Stained with fuchsin and methyl-blue.)

larger). Under the microscope these pink masses were seen
to be composed of cells of various sizes, each filled with an
enormous number of what appeared under a high power
very short bacilli, much shorter than tubercle-bacilli. But
they gave the same reaction as tubercle-bacilli. Here and
there isolated cells of various sizes could be seen filled with
the bacilli. In the large cells the cell-outline was becoming

140 MICRO-ORGANISMS AND DISEASE. [CHAP.

indistinct, and in some the cell-substance was seen to break
down, whereby the bacilli became free. In these respects,
in the size, distribution, and character of the bacilli, there
exists a remarkable similarity between the nodules in leprosy
and the nodules just mentioned.

FIG. 75. — Two CELLS OF THE LEPROSY (?) NODULES IN THE LIVER OF A
BIRD (RHEA).

The cell-substance is crowded with minute bacilli, similar to leprosy-bacilli
Magnifying power 700. (Stained with magenta.)

(/) Bacillus of malignant oedema (Koch), vibrion septique
(Pasteur). By inoculating mice, rabbits, and especially
guinea-pigs subcutaneously with a comparatively large
quantity of earth, or of putrid fluid, one occasionally
produces death in twenty-four to forty-eight hours. This
form of septicaemia is also called " Pasteur's septicaemia,"
and is of course distinct and different from Davaine's
septicaemia.1 At the seat of the inoculation and spreading
from it into the subcutaneous tissue of adjoining parts there
is much discoloration and occasionally haemorrhage ; a
turbid offensively- smelling ichor fills the spaces of the
subcutaneous tissue, and in it are found large numbers of
bacilli, some motile, others not. The lungs are hypersemic

1 Rosenberger maintains (Centralblatt f. </. med. Wiss. 4, 1883) thnt
the blood and exudation-fldds of rabbits dead of Davaine's or Pasteur's
septicaemia can be effectually sterilised by heat \vithout losing their
f-pecific action, reproducing on injection into fresh animals the disease
with the recurrence of the organisms characteristic of the disease.
Dowdeswell, however,, states (Proceedings of the Royal Sociey, 221,
1882) that this is not the case, for on really effectual sterilisation by
heat the organisms are killed, and the fluids become innocuous.

XI.]

BACILLUS: PATHOGEN 1C FORMS.

141

and have small haemorrhagic spots. The spleen .is invariably
enlarged and haemorrhagic spots are often noticed on the
peritoneum of the abdominal organs, and there is some
peritoneal .exudation. The blood of the spleen, <of the
liver, lung, and intestine, the serous coating of the abdominal
organs, and the peritoneal exudation, contain .the same
bacilli as the subcutaneous exudation. Many of them

FIG. 76.— BLOOD OF A GUINEA-PIG DEAD OF KOCH'S MALIGNANT

1. Red blood discs.

2. White corpuscles.

3. Single bacilli.

4. Chain of long bacilli.

5. Leptothrix.

Magnifying power 700. (Stained with gentian violet.)

include spores. By injecting the bacillus into the peritoneal
cavity of guinea-pigs death is produced rapidly, especially
after passing it through two generations, so rapidly indeed
that the animals often die within sixteen hours (Burdon
Sanderson and Klein). In all these instances a viscid
transparent slightly but spontaneously coagulable exudation,
poor in white and red corpuscles, is found in the peritoneal

142 MICRO-ORGANISMS AND DISEASE. [CHAP.

cavity, and the peritoneum of all parts is highly inflamed.
Bacilli are present in it in enormous numbers, many of them
containing spores. The blood of the heart does not contain
bacilli immediately after death, but has them some hours
after.

The bacilli in question are about 0*003 to °'°°5 mm. long
and a little over o'ooi mm. thick ; they are rounded at their
ends ; they form chains of two and more, and these chains
are straight or broken. They also form leptothrix, straight,
or more commonly curved. The bacilli have been artificially
cultivated by Pasteur 1 in blood-serum and in neutral solution
of Liebig's meat-extract. Gafifky 2 grew them on potatoes at
38° C. The artificial culture is capable of producing the
malignant osdema, but it is always necessary to inject more
than minimal quantities. The bacilli grown in fluids outside
and inside the body form spores without free supply of air,
and are therefore anaerobic (Pasteur).

In human faecal matter there are always present innumerable masses
of bacteria — micrococci, single and dumb-bells, and in clumps of
zoogloea, bacterium termo, and various species of bacilli, varying in
thickness, length, and in motility, some being motile, others not. It
has been shown by Bienstock (Centralbl. f. med. Wiss. 1883, p. 949)
that a bacillus can be cultivated from normal human faeces which in
many respects resembles the bacillus of malignant cedema ; it produces
death in mice, but without the symptoms of malignant cedema.

Professor Rossbach has maintained ( Centralblatt f. d. med. Wiss. 5,
1882) that when a solution of papayotin (the juice of Carica papaya] is
injected into the veins of a rabbit, the animal dies, and shortly after
death — even so short a time as fifty minutes after the injection — there
are found in the blood large numbers of bacteria. Dowdeswell, how-
ever, states (Practitioner, May, 1883) that solutions of papayotin con-
tain as a rule the spores of a motile bacillus which in all respects
resembles bacillus subtilis ; in artificial cultures in 10 per cent, solutions
of papayotin, in blood-serum, and in broth, these spores develop into

1 Bull, de FAcad. 1877. 2 Mittheil. a. d. k. Gesundh. 1880.

XL] BACILLUS : PATHOGENIC FORMS. 143

bacilli which form leptothrix filaments, and in them spores soon make
their appearance. Filtered papayotin solutions, when injected into the
blood of rabbits, kill like unfiltered ones, but neither during life nor
after the death of the animals could any organisms be detected in the
blood. It appears to follow from these experiments, that papayotin
solutions contain spores, and that these spores are those of a bacillus
subtilis which does not possess any specific pathogenic properties.

(k) Bacillus of symptomatic anthrax (Ger. Rauschbrand,
Fr. charbon symptomatique, Arloing, Cornevin, and Thomas,
Bull, de lAcad. 1881 ; Eng. black leg, quarter- evil]. This

FIG. 77.— BLOOD OF A GUINEA-PIG DEAD OF SYMPTOMATIC ANTHRAX.

Blood-corpuscles and between them several bacilli.
Magnifying power 700. (Stained with Spiller's purple.)

disease, which is not uncommon in cattle, generally ends
fatally and is very infectious. It is characterised by haemorr-
hagic effusion (or " tumour ") in the subcutaneous and inter-
muscular tissues of one or other, or both, anterior or posterior
extremities, in consequence of which the movements of the
animal so affected become greatly impeded. The animals
generally die in the course of the second or third day after
infection. The subcutaneous tumour contains numerous
bacilli, as do the abdominal and thoracic viscera.

The bacilli are about the size of those of malignant anthrax

144 MICRO-ORGANISMS AND DISEASE. [CHAP.

or a little thicker ; they are rounded at their ends and often
include at one end a bright oval spore ; spores are present
also in the bacilli of the internal organs ; (as will be shown
below, this is not the case with the bacillus of malignant
anthrax.) 'The bacilli are either single or form short chains.
Some of the bacilli are motile.

Inoculations with them into the subcutaneous tissue of
guinea-pigs, rabbits, sheep, and calves always prove fatal, the
same subcutaneous haemorrhagic effusions being produced.

Injections of small quantities of bacillus-containing
material into the veins produce only a slight febrile
disorder; large doses produce death. Animals in which
by intravenous injection of small doses slight illness has
been produced are afterwards protected against the fatal
dose. But minimal doses injected subcutaneously also
produce only a slight transitory swelling, and the animal so
treated is afterwards protected against the fatal dose (Arloing,
Cornevin, and Thomas). The spores of the bacilli when
heated up to 85° C. for six hours lose their virulence (Arloing,
Cornevin, and Thomas).

(/) Bacillus anthrads. — Pollender,1 Brauell,2 Davaine,3 and
then Bollinger 4 recognised in the blood of animals dead of
malignant anthrax the presence of stiff short and long rods,
which Davaine called bacteridie du charbon. They were
identified by Cohn5 as bacilli in morphological respects
similar to bacillus subtilis, except that the bacilli anthracis
are non-motile.

Koch 6 showed the ubiquitous distribution of these bacilli

1 Viertdj. f. gericht. Med. 1855.

2 Virchoufs Archiv, vol. xiv. 1858.

3 Comptes Rendus, Ivii. 1863.

4 Med. Cenlralblatt, June, 1872.

5 Beitr. z. B'.ol. d. Pflanzen, vol. ii. « Ibid. vol. iii.

XL] BACILLUS: PATHOGENIC FORMS. 145

in the blood of the organs, and especially of the spleen. He
succeeded in cultivating the bacilli artificially, by placing a
bit of such a spleen in a drop of aqueous humour, and
watching the growth of the bacilli under the microscope.
In this manner he ascertained that the rods multiply by
division, and that they grow into long, homogeneous-looking,
straight or twisted filaments in which after some time, and
with free access of air, bright oval spores make their appear-
ance, while the filaments become homogeneous and swollen.

/ (
\

FIG. 78. — HEART'S BLOOD OF A MOUSE DEAD OF ANTHRAX.

1. Blood-discs.

2. White blood-corpuscle.

3. Bacilli anihrac.s.
Magnifying power 700. (Fresh specimen.)

These spores become free, and when artificially cultivated or
injected into a rodent animal, germinate into the character-
istic bacilli ; these elongate and divide, and in artificial
cultures again grow into the long leptothrix filaments, which
again form spores. Koch ! saw in preparations of aqueous
humour kept at 35° C. in the incubator the spores germin-
ating after three to four hours. The single bacilli as they
present themselves in the blood measure between 0*005 and

1 Beitr. z. Biol. d. fflanzen, vol. ii. part ii. p. 288.

L

145 MICRO-ORGANISMS AND DISEASE. [CHAP.

0*02 mm. in length, and o.ooi to 0-0012 in thickness ; they
are truncated.1 The spores produced by growing the bacilli

1 IG. 79.— PART OF A BLOOD CLOT FROM THE HEART OF A MOUSE DEAD OF
ANTHRAX.

Magnifying power 500. (Stained with Spiller's purple.)

with free access of air are about o'ooi mm. thick, and about
o'oo2 to 0.003 mm. long. They are not stained by the
ordinary dyes and differ herein from the bacilli.

In the human subject malignant anthrax occurs as " woolsorter's
disease " ; for the aetiology and pathology of this malady see Spears
(Reports of the Medical Officer of the Local Government Board, 1881 and
1882) and Greenfield (ibid. 1881). It occurs also in sorters of hides.

All rodents and herbivorous animals are susceptible to anthrax ; rats
are, however, infected with difficulty, pigs are very insusceptible, and
so are dogs and cats. Infection of animals can be produced by in-
oculation into the skin and subcutaneous tissue intravascular injections,
and by inhalation of spores (Buchner, Untersuchungen iiber niedere
Pilze, by Prof. v. Nageli, 1882, p. 178). In wools^rter's disease the
usual mode of infection is by inhalation of spores adhering to the wool
of the fleeces of animals (sheep, goats) dead of anthrax. As in rodents
infected with anthrax, so also in man, the blood-vessels of all organs
contain the bacilli, and extravasations of the infected blood are frequent

1 It is generally assumed that the bacilli are the same ii all animals
affected with splenic fever, but this is most undoubtedly not the case,
as has been already pointed out by Huber (Deutsche med. Woch. 1881);
the bacilli of the guinea-pig are thicker than those of the mouse or
sheep, and these again are thicker than those of the rabbit.

XL] BACILLUS : PATHOGENIC FORMS. 147

in many parts of the body. The presence of bacilli in the extravasa-
tions into the mucous membrane of the trachea and bronchi does not
necessarily mean that these parts represent the points of entrance of
the bacilli into the system, as Greenfield seems to regard as self-evident
(Reports oj the Medical Officer of the Local Government Board, 1881).
As a matter of fact I find in every lung of mouse, rabbit, and guinea-
pig, dead after subcutaneous inoculation with anthrax, bacilli anthracis
in the alveolar cavities and in the smaller and larger bronchi. Ingestion
of bacillar material is sometimes foil >wed by anthrax, but in these cases
abrasions in the mucous membrane of the mouth, pharynx, or gut, may
have been the real place of entrance. Mice fed with anthrax material
do not become infected (Klein, ibid. 1881). But the reported cases
of intestinal mycosis (see for the Ikerature of this subject, Koch,
"^Etiologie d. Milzbrandes," Mittheil. a. d. k. Gesundheitsamte, 1881,)
seem nevertheless to indicate that such a mode of infection, namely,
by the alimentary canal, is not excluded. Compare also Falk, Virchow's
Archiv, vol. xciii. From recent observations by Koch and others, it
has become clear, that infection by the alimentary canal can be readily
produced with spores.

Rodents inoculated with the bacillus of the blood or
spleen of an animal dead of anthrax, or with the bacillus or
spores of an artificial culture, die generally within forty-eight
hours ; in some instances in twenty-four to thirty hours, in
other exceptional instances after forty-eight to sixty hours.
The blood in all instances contains the bacilli, the spleen is
large and full of bacilli, and so are the blood vessels of most
other organs, the exudations, and the urine. In the placenta
of a pregnant guinea-pig dead in consequence of inoculated
anthrax, I have seen that the bacilli kept strictly as a rule
within the maternal blood-vessels, and are wholly absent in
the blood of the vessels of the foetus. Subcutaneous in-
oculation or injection into the cutis of the minutest quantity
of bacillus-containing material (blood or artificial culture)
invariably produces death. Subcutaneous injection of
bacillus-containing material in the guinea-pig almost always
produces a characteristic cedema, spreading sometimes over

L 2

148 MICRO-ORGANISMS AND DISEASE. [CHAP.

a large area. The cedematous fluid is clear and contains
only a few bacilli.

Archangelski ( Centralblatt /. d. med. Wits. 1883, p. 257) claims to
have ascertained that if an animal be inoculated with anthrax, many
hours before the bacilli appear in the blood, there are present numbers
of spores. Just before death they all become changed into the bacilli.
He further maintains that those spores taken from the blood can be
shown to multiply by division, and without changing into bacilli, by
cultivating them artificially with exclusion of oxygen. I have shown,
however (Reports of the Medical Officer of the Local Government Board
for 1883), that none of these assertions are borne out by actual observa-
tion, and that they are erroneous.

FIG. 80. — FROM A PREPARATION OF HEART'S BLOOD OF A GUINEA-PIG DEAD
OF ANTHRAX.

1. Red blood-disc--.

2. White corpuscle.

3. Bacilli anthracis.

Magnifyirg power 700. (Stained with Spiller's purple.)

Any fluid containing proteid material is a suitable nutrient
medium for the bacilli ; they grow abundantly at all tem-
peratures between 15° and 43° C, best between 25° and
40° C. They elongate and divide rapidly, and the bacilli
grow out into long curved and peculiarly twisted filaments
which often form bundles, the individual filaments being
twisted round one another like the strands of a cable.

The bacillus anthracis grows best in neutral fluids, and to

XL]

BACILLUS : PATHOGENIC FORMS.

149

a lesser extent also in alkaline fluids which contain proteid
material. When growing in neutral nourishing fluids, it
forms on the bottom of the fluid characteristic fluffy whitish
masses, which are convolutions of the characteristic filaments.
These appear homogeneous in the fresh state, their ends
being slightly thicker and rounded. Examined in pre-
parations made after the Weigert-Koch method (i.e. drying
of a thin layer and staining it with aniline dyes, washing in

FIG. 81. — FROM AN ARTIFICIAL CULTURE OF BACILLUS ANTHRACIS.

Convolutions of thread*, eacK c imposed of bacilli.
Magnifying power 300. (Stained with Spiller's purple.)

water, then in spirit, then again in distilled water, and then
drying and mounting in Canada-balsam solution), all the
bacilli and their filaments are seen to be composed of a thin
hyaline sheath, and in this is a row of cubical or rod- shaped
masses of protoplasm taking the dye very readily. Accord-
ing to the length of the bacilli the number of these elemen-
tary masses of protoplasm varies. Some of the rod-shaped

150 MICRO-ORGANISMS AND DISEASE. [CHAP.

elements appear constricted in the middle, preparatory to
division. Between each two elements is a fine septum.

Bacilli anthracis when growing at ordinary temperatures
on a solid medium (e.g. a mixture of gelatine and broth, or
Agar-Agar and peptone) show a very peculiar modification,
inasmuch as some of the elements assume a spherical or oval

FIG. 82. — FROM AN ARTIFICIAL CULTURE OF BACILLUS ANTHRACIS, CARRIED ON
AT ORDINARY THMPERATURE AND ON SOLID (GELATINE) MATERIAL. TORULA
FORM.

Magnifying power 450. (Stained with Spiller's purple.)

shape, a torula-form, and as such they multiply by gemmation
and division, and form clusters or arrange themselves in
chains. By and by each of these spherical elements
elongates into a rod, and when all elements have undergone
this change we have the typical smooth filament of the

XL] BACILLUS: PATHOGENIC FORMS. 151

leptothrix form. Some of the elements in such a filament
remain for a long time of a spherical shape, and are much
larger, looking like the sporangium of a nostoc-alga. The
most interesting forms are those where an ordinary smooth
filament of anthrax- bacillus at its growing ends shows itself
to be composed of a chain of torula-elements. Such torula
forms occur also in ordinary cultivations in fluid media at
temperatures of 20° to 30° C., but not by any means so often
as at ordinary temperatures and in a solid medium. These
torula-cells are about 0-0013 to 0*0026 mm. in diameter. The
torula-forms are very virulent, but in an animal always
assume the ordinary shape of the typical bacillus.1

As has been mentioned in treating of pigment bacilli, such
a torula-form has been also observed by Neelsen in the
bacillus that causes the colour of "blue milk;" and Zopf-
has observed it also in cladothrix dichotoma.

I have also observed this torula-modification in the
filaments of septic bacilli, in a bacillus that I found growing
accidentally in pork-broth. The bacillus had the same
morphological characters as the bacillus subtilis of hay-
infusion, and also formed a pellicle composed of filaments.
In some of the filaments the large torula-like cells could be
seen here and there interposed between cubical and
cylindrical cells.

On inoculating fluid media (e.g. broth of any kind or pep-
tone fluid) with the bacilli anthracis, either those of the
blood or of the spleen of an animal dead of anthrax, and
shaking the fluid so as to distribute the bacilli uniformly
through the fluid and exposing this to a temperature of from
25° to 40° C., it will be noticed that after twenty-four to forty-

1 Klein, Quart. Journ. of Micro sc. Science, April, 1883.

2 Zur Morphologic d. Sfalfpjianzen, ii. and Die Spaltpihe^ Breslau,
1883.

152 MICRO-ORGANISMS AND DISEASE. [CHAP.

eight hours incubation, the fluid is uniformly turbid, owing
to the rapid multiplication of the bacilli. These are shorter
or longer typical anthrax-bacilli. But as incubation proceeds
all the bacilli grow into filaments, and these being heavier
sink to the bottom of the fluid and form the characteristic
whitish fluffy convolutions. But on inoculating dilute broth,
care being taken that the inoculating material, whether

FIG. 83. — FROM A PREPARATION OF THE BLOOD OF SPLEEN OF A GUINEA-PIG
DEAD OF ANTHRAX.

1. White bhod-corpu<=cle.

2. Red blood-discs, shrunken.

3. Chains of hacillus anthracis.

4. Degenerating bacilli, the sheath only being preserved.
Magnifying power 700. (The preparation has been stained with gentian-violet.)

consisting of blood-bacilli, bacilli of a culture, or spores of
culture-bacilli, is deposited at once on the bottom of the
fluid, and this is not shaken up, it will be noticed on incu-
bation that the fluid remains limpid. All the growth, in
the shape of the fluffy whitish masses, takes place at the
bottom.

XL] BACILLUS: PATHOGENIC FORMS. 153

After a few days' incubation, no matter what the tempera-
ture is, many of the bacilli and their leptothrix-filaments
show signs of degeneration, consisting in the granular disin-
tegration and absorption of the protoplasmic contents of the
bacilli and their filaments, at first only here and there, but
by and by over longer pieces. Such bacilli and leptothrix-
filaments appear in such places as if empty. This is also
noticed in the bacilli of the blood and spleen of an animal
inoculated with anthrax even at the point of death or soon
after death, if the number of bacilli is great.

FIG. 84. — FROM AN ARTIFICIAL CULTURE OF BACILLUS ANTHRACIS IN BROTH

AFTER MANY PAYS INCUBATION.

The threads are swollen and curled up, and in many places the protoplasm has

disappeared, leaving the sheath and septa distinct
Magnifying power 700. (Stained with Spiller's purple.)

Another form of degeneration consists in the filaments of
bacilli becoming much curled and swollen, and finally
disintegrated into an amorphous de'bris.

As long as the bacilli grow in the depth of a fluid they
never form spores, but when grown on the surface with free
access of air, or on solid media (e.g. serum gelatine, gelatine
broth, Agar-Agar, potato, &c.), the bacilli, having developed
into filaments, proceed to form spores. But they may form
spores even in fluid media if by some accident, either by

154 MICRO-CRGANISMS AND DISEASE. [CHAP.

sticking to the glass vessel containing the fluid or by means
of a cotton-wool fibre, some of the bacilli remain on the
surface of the fluid. This formation of spores is not due to
exhaustion of the nourishing medium, as is maintained by
Buchner — it has, in fact, nothing to do with it — but repre-
sents the last stage in the life-history of the bacilli, provided
they have an ample supply of oxygen. If this latter con-
dition is not fulfilled, as when they are grown at the bottom
of a fluid, the bacilli gradually degenerate as mentioned above.
Spore-formation occurs, cceteris paribus, at all tempera-
tures between 18° and 45° C. Koch found 15° C. the lower
limit. Pasteur states that in a nutrient medium exposed to
a temperature of 42° to 43° C. the bacilli are not capable of
forming spores ; but this is not correct, for when the bacilli
are growing on the surface of the nutrient medium, they form
spores even at a temperature of 44° to 44° 5 C., as I have con-
clusively shown by growing them on Agar-Agar and peptone
mixture. The spore-formation consists in the appearance
of a bright glistening spherical body in the protoplasm of an
elementary mass or cell ; this body gradually enlarges till it
reaches its full size, becoming at the same time oval. The
bacilli at these points are thicker than where no spore-
formation has set in. Under the most favourable conditions,
each cubical or rod-shaped mass of protoplasm includes one
spore, in which case the bacillar filament contains an almost
unbroken row of spores ; but in other cases only an elemen-
tary mass here and there contains a spore, the rest breaking
down and becoming absorbed. In the first case also, the
protoplasm of the elements almost entirely disappears, the
sheath swelling up and becoming hyaline, and only the
bright spores remaining. Their linear arrangement, however,
still indicates that they were formerly contained in one
filament.

XL]

BACILLUS : PATHOGENIC FORMS.

155

If bacilli grow in the depth of a fluid medium, they do
not form spores, as has been stated above ; and as we have
also seen, as new bacilli appear, or the old filaments increase
in length, degeneration sets in. This degeneration gradually
affects greater and greater numbers, and when the fluid is

FIG. PS. — FROM AN ARTIFICIAL CULTURE IN NEUTRAL PORK-BROTH OF
BACILLUS ANTHRACIS, wifn COPIOUS FORMATION OF SPORES.

Magnifying power 700. (Stained with Spiller's purple.)

exhausted for the formation of new bacilli, it necessarily
follows that the whole growth gradually becomes involved in
the process of degeneration, the whole mass becoming
smaller, and finally only de'bris is left. Such cultures,
namely, those in which the degeneration involves the whole

156 MICRO-ORGANISMS AND DISEASE. [CHAP.

mass of the bacilli, are quite innocuous when inoculated into
animals, or into fresh nourishing media. But as long as
there are any good protoplasmic elements of the bacilli left,
the culture is virulent to rodents, with the exception of mice,
as will be stated presently ; and it is capable, when trans-
ferred to new suitable nourishing media, of starting new
cultures that prove virulent to all rodents and sheep.

The same holds good of the bacilli in the blood and organs
of an animal dead of anthrax, provided the animal be not
opened, and its organs, exudations, or urine be not exposed
to the free air ; for the bacilli not exposed to the air gradually
degenerate, and the blood and organs of such an animal
although at first deadly poison to other susceptible animals,
become at length quite innocuous. Systematic observation
has shown me that small animals, such as mice and guinea-
pigs, when kept unopened or buried in earth, become quite
innocuous after five to eight days, the anthrax-bacilli having
by this time, by degeneration, altogether disappeared from the
blood, spleen, and other organs. Pasteur's statement that
in animals dead of anthrax and buried, the bacilli form
spores, that these spores are taken up by earthworms and
carried to the surface of the soil, where they are deposited
with their castings and thus are capable of infecting animals
grazing or sojourning on this soil, is not borne out by the
above observations. And further, Koch has proved l by
direct experiment that spores of anthrax-bacilli when mixed
with earth in which worms are present, are not taken up by
these creatures.

Drying bacilli of the blood or of a culture in a thin layer
invariably kills them, but the spores remain unaffected.

The bacilli of the blood of a rodent dead of anthrax are

. J Miltheil. a. d. k. Gcsundhdtsamte, 1881.

XL] BACILLUS: PATHOGENIC FORMS. 157

always thinner than the bacilli cultivated in a neutral fluid
medium like pork-broth.

Cultivation of the blood-bacilli at temperatures varying
between 20° and 40° C. in any suitable nourishing material,
solid or fluid, however many transferences (new cultivations
or so-called new generations) be made, always yields a crop
of virulent bacilli. It is absolutely incorrect to say, as
Buclmer1 and Greenfield2 maintain, that continued transfer-
ence weakens the action of the bacilli ; as long as the cultures
remain pure, not contaminated and finally suppressed by
accidental innocuous bacilli, the anthrax-bacilli retain their
full virulence.

Cultures of the blood-bacilli at 20° to 38° C. in fluid media,
e.g. neutral pork-broth, during the first or second week, are
virulent to mice, guinea-pigs, and rabbits ; but after that
they lose their power on mice, provided the growth takes
place only in the depth and no spores are formed ; but they
retain it, as regards guinea-pigs and rabbits, as long as they
contain good bacilli at all. 3 But fresh cultures made of
such bacilli invariably produce a growth which is fatal to all
rodents during the first or second week.

Pasteur has stated that blood-bacilli which have become
attenuated in virulence by exposure to 42° or 43° C. for
twenty days are capable of starting new cultures of attenu-
ated virus. This I question, for I find that such a culture
starts new cultures of virulent bacilli ; in the same way the
bacilli of a culture that is only " vaccine " for sheep, when it
is inoculated into a guinea-pig kills it with anthrax, and then
yields bacilli that are fatal to sheep.

1 Ueber d. Erzeug. dcs Milzbrandes, Munich, 1880.

2 Proceedings of the Royal Society, June 17, 1880.

3 Klein, Reports of the Medical Officer of the Local Government Board,
1881.

i58

MICRO-ORGANISMS AND DISEASE. [CHAP.

Blood-bacilli exposed to a temperature of 55° C. or to a
solution of ^ to i per cent, of carbolic acid, lose their viru-
lence (Toussaint). Chauveau found that exposure to a tem-
perature of 52° C. for fifteen minutes, or of 50° C. for twenty

FIG. 86. — NETWORK OF CAPILLARIES FILLED WITH BACILLUS ANTHRACIS ; FROM
THE OMENTUM OF A RABBIT DEAD OF ANTHRAX.

1. Extravasation of the ba:illi.

2. Capillaries filled with the bacilli.

Magnifying power 350.

minutes, destroys the virulence of the blood-bacilli. Pasteur 3
ascertained that by cultivating blood -bacilli in chicken-broth
at 42° to 43° C. they lose their virulence after twenty days'

1 Cumptes Rendus, 1881 ; Transactions of the International Medical
Congrtss in London, 1881, vol. i.

xi] BACILLUS: PATHOGENIC FORMS. 159

cultivation, not as Pasteur thinks owing to the action of
oxygen, but owing to the high temperature ; and when such
bacilli are injected into sheep and cattle they do not kill
though they induce sometimes a slight illness. After this ill-
ness has passed off, the animals are protected against viru-
lent anthrax. But with reference to this "vaccination," it
must be borne in mind that twenty days' cultivation of blood-
bacilli at 42° to 43° C. does not always yield attenuated virus, J
and also that sheep and cattle not killed by inoculation of
attenuated virus produced by Pasteur's method 2 or by other
means (see below), although they are protected against viru-
lent anthrax, remain so only for a limited time, probably
about nine months.

In all these experiments with the anthrax-bacilli it is
necessary to bear in mind that by passing the bacilli through
different species of animals they become endowed with
different qualities, and that bacilli which are fatal to some
are not fatal to all animals. While, for instance, the blood-
bacillus of sheep or cattle dead of anthrax invariably pro-
duces death when inoculated into sheep or cattle, after
passing through white mice 3 it loses this virulence for sheep
and cattle. The blood of white mice dead of anthrax does
not kill sheep ; it produces only a transitory illness and the
animals are, for a time at least, protected against virulent
anthrax. The blood of guinea-pigs dead of anthrax produces
illness, sometimes de|ith, in cattle, but as a rule does not kill
(Sanderson and Duguid), and the blood of the biscachia of

1 Klein, Reports of the Medical Officer of the Local Government Board,
1882.

2 Pasteur thinks that such cultures remain free of spores because of
the temperature of 42° — 43° C. ; but this is not so, as has been pointed
out above ; the state nent only holds good so long as the bacilli are
prevented from growing on the surface.

'4 Klein, Reports of the Medical Officer of the Local Government Board,
1882.

i6o

MICRO-ORGANISMS AND DISEASE. [CHAP.

South America does not kill cattle, while it gives them a
transitory illness, and after this immunity for a time.1 Again,

FIG. 87. — FROM A SECTION THROUGH THE KIDNEY OF A RABBIT DEAD OF
ANTHRAX.

The capillaries of the cortex are naturally injected with the Bacillus anthracis.

1. A glomerulus.

2. Capillaries surrounding the convoluted uriniferous tubules not shown here.

Magnifying power 450. (Spiller's purple. )

Pasteur's " vaccine," which as a rule (but not without
exception) does not kill sheep or cattle, is fatal to ro-

1 Roy, Nature, December, 1883.

XL] BACILLUS: PATHOGENIC FORMS. 161

dents.1 From all this it follows that as regards virulence the
bacilli anthracis differ in the different species of animals, and
in them acquire different qualities. A culture that does not
kill mice, such as an artificial culture of blood-bacillus after
one or two weeks' incubation at 20° to 35° C., or a culture
that for other reasons, as when attenuated by heat or anti-
septics, does not produce fatal anthrax in guinea-pigs, fails
to give to these animals any immunity whatever. Rodents,
so far as my experience goes, either die of inoculation with
anthrax-bacilli or they do not die ; but they are not
provided with immunity by the attenuated virus.

Koch 2 maintains that in neutral chicken-broth the bacilli
growing at 42° C. lose their virulence in thirty days, and at
43° C. in six days, first for rabbits, then for guinea-pigs, and
lastly for mice. I am quite sure from my own observations,
that these results are not uniformly obtained, since I have
seen anthrax-bacilli very virulent both for rabbits and
guinea-pigs even after growing for thirty-six days at 42° '5 C.

Bacillus anthracis is capable, as we have seen, of growing
well outside the body, and, when well supplied with oxygen
from the air, of forming spores which represent the per-
manent seeds. Thus if animals, such as sheep and cattle,
die of anthrax in a field, the bacilli in the effusions of
such animals (e.g. urine, blood, effluvia from the mouth and
nostrils) always contain numbers of the bacilli, and these will
be able to grow indefinitely on the surface of the soil, there
being always present a large amount of suitable nourishing

1 Klein, Reports of the Medical Officer of the Local Government Board,
1882. Similar results have been obtained by Gaft'ky (Mittheil. a. d. k.
Gesundheitsamte, 1882).

2 Ueber d. Milzbrandimpfung, 1882.

Spores of bacillus anthracis stand heating to 100° C. in the dry state
for over an hour without being killed ; in the moist state, e.g. exposed
to steam at 100° C., they are killed after fifteen minutes' exposure
(Koch).

M

1 62 MICRO-ORGANISMS AND DISEASE. [CHAP.

material, as vegetable and animal decaying matter, and since
free access of air is always insured they will eventually form
spores. Such soils, owing to the presence of these spores,
will remain a permanent source of infection to sheep and
cattle sojourning on them (Koch).

(m) Bacillus tuberculosis (Koch). — In all cases of tubercu-
losis in man, cattle (Perlsucht) and monkeys, of tuberculosis

FIG. 88. — FROM A PREPARATION OF HUMAN TUBERCULOUS SPUTUM, STAINED

AFTER THE EHRLICH-WEIGERT METHOD.
The nuclei are stained blue, the tubercle-bacilli pink. Magnifying power 700.

artificially produced (by inoculation with human or bovine
tuberculous matter) in cats, guinea-pigs, rabbits, and rats, and
in spontaneous tuberculosis in birds (hens), Koch * found in
the fresh state, and particularly after staining with methylene-
blue and vesuvin, peculiar fine bacilli, some with bright oval
spores, some without, some smooth and homogeneous -
looking, others more of a beaded appearance. One cubic
1 Berliner klin. Wochenschrift, xv. 1882.

XL] BACILLUS: PATHOGENIC FORMS. 163

centimetre of a concentrated alcoholic solution of methylene-
blue is mixed with 200 ccm. of distilled water; to this are
added two ccm. of a 10 per cent, solution of caustic potash.
In this solution the fresh or hardened sections or particles of
tubercles are kept for half an hour if heated up to 40° C., or for
twenty-four hours if not heated. After this the preparation
is stained for two minutes in a filtered concentrated watery
solution of vesuvin, then washed in distilled water. On ex-
amination with a -jL oil immersion lens and Abbe's condenser
it will be found that all the elements are stained brown with
vesuvin except the bacilli, which are blue. A still more suc-
cessful and more delicate reaction is shown by the bacilli if
the preparation is stained after Ehrlich's method. About
5 ccm. of pure anilin (anilin oil) are well mixed with 100 ccm.
of distilled water and filtered ; to this is added a saturated
alcoholic solution of fuchsin, and with this the preparation is
stained for a quarter to half an hour. It is then washed for
a few seconds in a mixture of one part of nitric acid and two
parts of water, and then is well washed in distilled water.
The preparation when now examined shows no trace of
colour except in the tubercle-bacilli, which retain the red
colour of the fuchsin. The tissue may now be stained either
with vesuvin or methylene-blue, which makes the ground-
work brown or blue, but the bacilli remain red. This reaction
after washing with nitric acid is exceedingly delicate, and is
perfectly characteristic and trustworthy, as all putrefactive
organisms become discoloured by the washing with nitric
acid, the tubercle-bacilli only retaining the colour. There
are other methods which are very good ; those of Weigert and
of Gibbes l are very quick and trustworthy in their action.

Weigert has devised a staining fluid which gives very beau-
tiful results and is very useful for staining sections, fresh or
1 Lancet, August 5, 1883.

M 2

1 64 MICRO-ORGANISMS AND DISEASE. [CHAP.

hardened ; it is as follows : — Take of a 2 per cent, watery
solution of gentian-violet 12 ccm., and of a saturated watery
solution of anilin oil 100 ccm. Mix. This is used like an
ordinary staining-fluid for the first stain. For the second or
contrast stain the following solution is used : —

Bismarck brown i gramme.

Spiritus vini rectificati (sp. gr. '830) . 10 ccm.

Distilled water . . . . 100 ccm.

The sections remain in a few drops of this solution for
fifteen minutes. This method yields the finest specimens of

FIG. 89. — FROM A PREPARATION OF CASEOUS MATTER FROM PULMONARY
DEPOSITS IN BOVINE TUBERCULOSIS, STAINED AS IN PRECEDING FIGURE.

Magnifying power 700.

tubercle-bacilli in sections through tuberculous growths that
I have seen ; unfortunately the colour of the bacilli is very

liable to fade.

In the case of tuberculous sputum, or similar matter, a

small droplet or particle is spread out in a thin layer on the
cover-glass, well dried by passing it over the gas-flame of a
Bunsen burner, and then stained in the way described in
Chapter I. Sections of tubercles, fresh or hardened, are
stained without first drying.

XL]

BACILLUS: PATHOGENIC FORMS.

165

In all cases of human tuberculosis, particularly in the
sputum, in caseating scrofulous glands, in bovine tubercles, in
artificially-induced tubercles and caseating glands of rodents,
the tubercle-bacilli have been shown to exist. They are
most numerously found in the caseous masses in the lung
found in bovine tuberculosis. Here Koch found them not
only scattered through the caseous masses, but also in the
well-known giant-cells ; in some cases they form a more or
less regular zone in the peripheral portion of the cell. But

FIG. 90. — FROM A SECTION THROUGH TUBERCULOUS DEPOSITS IN THE LUNG OF

A Cow.

Two giant-cells and two small cells containing tubercle-bacilli.
Magnifying power 700.

according to Koch the bacilli by and by disappear again from
the giant-cells.

The bacilli do not show any motility and often include
granules considered to be spores ; they thus would be capable
of forming spores within the body. Owing to these, human
phthisical sputum retains its virulence even after drying.
Koch cultivated the bacilli artificially, i.e. outside the body,
and by carrying on the cultivation for several successive trans-
missions succeeded in isolating and clearing them from the

166 MICRO-ORGANISMS AND DISEASE. [CHAP.

tuberculous tissue. These pure bacilli, no matter how many
times they have been transferred, no matter how far removed
from their original breeding-ground, always produced the
characteristic disease when inoculated into suitable animals.
The cultivation succeeded equally with material derived from
human tubercles, from bovine tubercles, and from the artifici-
ally-induced tuberculosis of guinea-pigs. The bacilli grow
well at a temperature varying between 37° and 39° C. in
solid serum, Agar-Agar peptone mixture, and solidified
hydrocele fluid.1 (See Chapter II.) An incision is made
into a tubercle with clean (overheated) scissors and a particle
of a tubercle is taken up with the point of a clean (overheated)
needle and deposited on the top of one of these sterile solid
media kept in a test-tube plugged with sterile cotton-wool.
After keeping it for ten days to a fortnight in the incubator at
37° to 39° C. the first traces of growth make their appearance
in the shape of small dry whitish scales, which gradually
increase in size until they coalesce. These scales are made
up of the typical tubercle-bacilli lying closely side by side ;
some of the bacilli are longer, others shorter, and many of
them have spores. New cultures may be established
from these bacilli. Inoculation with them or with further
cultivations into the subcutaneous tissue, peritoneal or pleural
cavity of guinea-pigs and rabbits, produces after three, four,
or more weeks, the typical lesions characteristic of artificial
tuberculosis ; namely, swollen lymphatic glands near the
seat of inoculation, with subsequent caseation and ulceration ;
enlargement of the spleen due to numerous whitish tubercles,
the larger ones caseous ; enlargement of the liver, which is
mottled by the presence of uniformly distributed whitish

1 Solidified hydrocele fluid has been successfully used for the cultiva-
tion of the tubercle-bacilli, not by Koch, but by my friend Mr. Makins
of St. Thomas's Hospital.

XL] BACILLUS : PATHOGENIC FORMS. 167

points and streaks, which by and by become confluent and
caseous ; tuberculosis of the peritoneum ; isolated tubercles
in the lungs, at first grey and transparent, then caseating in
the centre ; enlargement and subsequent caseation of the
bronchial glands.

Owing to the fact that the tubercle-bacilli require for their growth
high temperatures (37° to 39° C.), it is evident that, unlike some other
pathogenic organisms, they do not thrive in the outside world in
temperate climates.

Inoculation with the pure bacilli into the anterior chamber
of the eye of rabbits and guinea-pigs produces the character-
istic tuberculosis described by Cohnheim and Salomonsen.
After an incubation oifrom two to three weeks there appears
on the iris a crop of minute grey tubercles enlarging and
undergoing caseous degeneration. Later on general tubercu-
losis of the eyeball and other organs follows. So that Cohn-
heim's assertion, that only tuberculous matter implanted into
the anterior chamber of the eye can produce this outbreak of
a crop of tubercles on the iris, is, by Koch's observations,
strengthened in the highest degree; the tubercle-bacilli
present in, and characteristic of, true tubercles are thus
manifestly connected with the real cause of the morbid growth.
A large number of pathologists have, since the publication
of Koch's paper, devoted themselves to various parts of
this question of the relationship of the tubercle-bacilli
to the tuberculous process, and have, with few exceptions,
verified Koch's observations. The chief opposition,
leaving out of account those who, either from imperfect
technical skill in the manipulation and staining of the
bacilli, or by reason of the inadequate number of their
observations, have denied Koch's statements, comes mainly
from observers who, like Toussaint, Klebs, and Schiiller,
maintain that tuberculosis is due to a micro-organism which

i68 MICRO-ORGANISMS AND DISEASE. [CHAP.

is a micrococcus and not a bacillus, or who, like Schot-
telius and others, do not admit that human and bovine
tuberculosis are the same, and are, therefore, not inter-
changeable, which they ought to be if in both the same
bacillus occurs, and if this bacillus is the vera causa morbi.
But there can be no doubt that a vast number of competent
observers have fully verified Koch's dictum, that the
tubercle-bacilli are specific and different from other bacilli,
except those of leprosy, as regards their chemical nature
(compare their behaviour to nitric acid) ; and that wherever
they are present in the sputum we have to deal with real
tuberculosis, wherever after repeated examinations they are
found to be absent there is no tuberculosis. This has by
this time, although not much more than a couple of years
has elapsed since Koch's first publication, become in
the hands of all competent workers a matter of daily
practical application, especially as regards the examina-
tion for bacilli of the sputum of patients suspected of
tuberculosis.

The other equally important part of Koch's discovery,
namely, the artificial cultivation of the tubercle-bacilli and
the production with them of tuberculosis, has also been
verified by Weichselbaum.1 Weichselbaum also ascertained 2
that in acute miliary tuberculosis of man the blood contains
the bacilli.

An important series of observations was published by
Mr. Watson Cheyne in the Practitioner for April 1883,
in which he proved, (i) That the organs of rabbits
and guinea-pigs suffering from the tuberculosis induced
by Toussaint's cultivations from the blood of tuberculous
animals, which cultivations Toussaint considered to be
those of micrococci, turned out, on careful microscopic
1 Wiener med. Blatttr, 1883. 2 Ibid. 10, 1884.

XL] BACILLUS: PATHOGENIC FORMS. 169

examination and suitable staining, to contain the typical
tubercle-bacilli ; (2) That inoculations with cultures of
Toussaint's pure micrococci not containing any tubercle-
bacilli did not produce tuberculosis in animals ; (3) That
Koch's assertions as regards the constant occurrence of
the tubercle-bacilli in the tubercles of animals artificially
tuberculised are quite correct ; (4) That material other
than tuberculous does not produce tuberculosis, that is

FIG. 91. — FROM A SECTION THROUGH A TUBERCLE OF THE LUNG FROM A CASE

OF ACUTE MILIARY TUBERCULOSIS IN A CHILD.

Several alveoli are seen filled with debris ; in the centre of this are numerous nuclei,
and amongst them the tubercle-bacilli. Magnifying power about 350.

to say, that the cases of artificial tuberculosis in guinea-
pigs observed by Wilson Fox and Burdon Sanderson,
and in the older experiments of Cohnheim and Fraenkel,
viz. those in which chronic inflammation and caseation
(i.e. artificial tuberculosis) was thought to have been
induced by other than tuberculous matter (e.g. by non-
tuberculous caseous matter, setons, indifferent substances

1 70 MICRO-ORGANISMS AND DISEASE. [CHAP.

like bits of gutta-percha inserted into the peritoneal cavity,
&c.), were really due to accidental contamination with
tuberculous material.

According to my own experience extending over a very
large number of cases of human miliary tuberculosis and
tuberculosis of cattle, I cannot for a moment accept the
statement that the bacilli found in the two affections are
identical ; for I find that in the two diseases their morpho-
logical characters and distribution are very different. The
bacilli of human tuberculosis are conspicuously larger than
those of the tuberculosis of cattle, and in many instances
more regularly granular. As is seen in Figs. 88-90, those
of human sputum are nearly half, or at least one-third,
as large again as those of the caseous masses from the lungs
of cattle.

The bacilli in the tuberculous deposits of cattle are always
contained in the cells ; the larger the cell the more numerous
the bacilli. This fact comes out very strikingly in thin and
well-stained sections. Around many of the smaller and larger
clumps of bacilli the cell-outline is still recognisable, and when
the cell disintegrates, as it does sooner or later, the bacilli
become free in groups ; in this respect there exists a
remarkable similarity between leprosy and bovine tuber-
culosis. But in the human tubercles the bacilli are always
scattered between the cells.

I cannot agree with Koch, Watson Cheyne, and others,
who maintain that each tubercle owes its origin to the
immigration of the bacilli, for there is no difficulty in
ascertaining that in human tuberculosis, in tuberculosis
of cattle, and in artificially-induced tuberculosis of guinea-
pigs and rabbits, there are met with tubercles in various
stages — young and adult — in which no trace of a bacillus
is to be found ; whereas in the same section caseous

XL] BACILLUS: PATHOGENIC FORMS. 171

tubercles may be present containing numbers of tubercle-
bacilli.

Schuchardt and Krause1 have found tubercle-bacilli, though
sparingly, in fungoid and scrofulous inflammations ; Demme 2
and Doutrelepont 3 found the tubercle-bacilli also in the
tissue of lupus. But the bacilli occurring in the lupus- tissue,
as far as I am able to see, are morphologically different

a

FIG. 92.— FROM A SECTION THROUGH THE KIDNEY OF RABBIT DEAD OF
ARTIFICIAL TUBERCULOSIS.

a. Blood-vessel filled with caseous matter, and in it numerous tubercle-bacilli.

b. Nuclei of cells of the tuberculous new growth.

c. Capillary vessel in cross section.

Magnifying power 700.

from the tubercle-bacilli. In a preparation made of the
juice of lupus-tissue, large transparent cells with several
nuclei are found, in the cell-substance of which are noticed

1 Fortschritte d. Med. 9, 1883.

2 Berliner klin. Woch. 15, 1883.

3 Monatshefte f. practische Dermatologie, 6, 1883.

1/2 MICRO-ORGANISMS AND DISEASE. [CHAP.

FIG. 93.— FROM THE SAME KIDNEY AS IN PRECEDING FIGURE.

a. Large artery filled with caseous matter, and in it numerous tubercle-bacilli.

b. Coat of artery.

c. Nuclei of the tuberculous new growth. d. A Malpighian corpuscle.

Magnifying power about 500.

FIG. 94.— FROM THE JUICE OF LUPUS-TISSUE PREPARED AFTER THE KOCH-

WEIGERT METHOD OF DRYING A THIN LAYER ON A COVER-GLASS.

Magnifying power about 700.

XL]

BACILLUS : PATHOGENIC FORMS.

173

groups of thickish, short bacilli, thicker and shorter than
tubercle-bacilli. These bacilli are either placed singly or in
chains of two.

Subcutaneous inoculation of or feeding with human and bovine
tubercular (caseous) matter of guinea-pigs and rabbits produces general
tuberculosis, but there are certain differences both as regards the
anatomical lesions as well as duration whiclj show that the two diseases

FIG. 95. — PLATE CULTIVATIONS IN NUTRITIVE GELATINE, AFTER THREE DAYS'
GROWTH AT 20° C , SEEN WITH THE UNAIDED EYE.

1. A colony of microccus.

2, 3, 4. Colonies of cholera comma-bacilli.
The clear part is due to liquefaction of the gelatine.

are not quite identical. While rabbits are extremely susceptible to
bovine tuberculosis, in consequence of inoculation or feeding, and while
they develop rapidly a general tuberculosis, they are less susceptible to
human tuberculosis, the disease taking a slower course and involving
fewer organs, and these to a lesser degree.

It is well established that bovine tuberculosis is transmissible to pigs,
cattle, sheep, monkeys, rodents, &c., by ingestion (Johne, Deutsche

174 MICRO-ORGANISMS AND DISEASE. [CHAP.

Zeitschrift f. Thierheil, &c., 1883), but it is not experimentally proved
that human beings can contract human tuberculosis by feeding on milk
and flesh derived from tuberculous animals.

(n) Bacillus of Syphilis. — Lustgarten described (Med. Jahrb. derk. k.
Gfsellsch. d. Aerzte. Vienna, 1885) peculiar bacilli as occurring in
syphilitic products. They resemble in size and aspect very much the
tubercle-bacilli ; their ends are slightly thickened, and they often show
nodosities ; these bacilli are never found free between the tissue elements,
but always inclosed in cells, generally singly or in couples, or rarely in
groups, but their total number in a given section is always small. The
peculiarities they show in their mode of staining, and which they share
with tubercle-bacilli and leprosy- bacilli, have been mentioned in
Chapter I. De Giacomi (Schweizer Correspondenzblatt, xv. 12) has
fully confirmed the statements of Lustgarten. His method of staining
the syphilis-bacilli has been mentioned in Chapter I.

Doutrelepont and Schiitz (Deutsche Med. Woch. 1885, No. 19) have
also demonstrated the occurrence of these same bacilli by simply stain-
ing sections made of syphilitic tissues in a watery I per cent, solution
of gentian- violet with subsequent contrast staining by safranin.

On the other hand Cornil, and particularly MM. Alvarez and Tavel,
state that a bacillus identical in mode of staining, size, and aspect with
the one described by Lustgarten as the specific syphilis-bacillus, has
been found by them in some normal secretions (Brit. Med. Journ.
Oct. 17, 1885).

(0) Bacillus of Foulbrood. — Messrs. F. Cheshire and W. Cheyne
described (Microsc. Journ. August, 1885) a peculiar bacillus which
occurs in the tissues and juices of bees, and especially their larvae, which
sometimes in beehives become affected with, and die of, the disease
known as ' ' foulbrood. " This bacillus shows certain peculiarities in its
mode of growth in nutritive gelatine and Agar-Agar, and is capable of
forming spores. With such cultivations the disease was reproduced in
healthy bees.

(£) The Comma-Bacillus of Asiatic Cholera. — Koch has
discovered in the evacuations of persons affected with acute
Asiatic cholera a peculiar vibrio, which he called comma-
bacillus, and of which he states that it is intimately connected
with the causation of the disease. Koch isolated these
comma-bacilli from the mucus-flakes of the fluid contents

XL] BACILLUS : PATHOGENIC FORMS. 175

of the ileum by the method of plate-cultivation in nutritive
gelatine described in Chapter V. The appearances presented
by these comma-bacilli in such plate-cultivations are charac-
teristic ; they are shown in Fig. 95, but it is also shown
there that a similar character is exhibited by some micro-
cocci. These comma-bacilli multiply readily in the mucus-
flakes taken from the lower part of the ileum of a person dead
of acute cholera if these flakes are placed and kept on moist
linen in a moist chamber.

The comma-bacilli grow and multiply well in neutral
broth, neutral nutritive gelatine, neutral Agar-Agar mixture.

FIG. 96. — FROM A PREPARATION OF Mucus FLAKES OF THE FLUID IN THE
ILEUM OF A CASE OF TYPICAL CHOLERA.

Numbers of comma-bacilli of different lengths are shown amongst numbers of small
straight bacilli. Magnifying power about 700.

An alkaline condition is not essential, as maintained by Koch,
although, like other bacteria, notably putrefactive bacteria,
they grow more luxuriantly if the medium is faintly alkaline.
They grow well in milk, on potato (although the reaction
of this latter is acid), and I have seen them grow even in
faintly acid peptone broth.

The comma-bacillus of Koch is a curved rod of almost
uniform thickness, sometimes slightly pointed at the
extremities, its length about half that of a tubercle-bacillus,
its thickness about the same as that of the latter. But
the comma-bacilli vary in curvature and length within

176 MICRO-ORGANISMS AND DISEASE. [CHAP.

considerable limits, some being just curved while others are
almost semicircular, some being twice and three times as
long as others. They are motile, and divide transversely.
In neutral Agar-Agar mixture, kept at ordinary temperature,
length-divisions may be observed (see Fig. 98). After trans-
verse division they may remain joined end to end, and then
forming an S-shaped corpuscle. But sometimes, particularly
in artificial cultivations in broth, they remain joined end to
end even after repeated division, and then form either a

FIG. 97.

From an Artificial Cultivation of chole-
raic Comma-bacilli in Gelatine
Peptone. Magnifying power 700.
Most of these are single curved
bacteria, a few are joined end to
end in twos, thus forming S-shaped
organisms ; and a few are in chains
of several placed end to end.

O

A*

: *i

FIG. 98.

From an Artificial Cultivation of chole-
raic Comma-bacilli in Agar-Agar
Peptone at the ordinary tempera-
ture of the room after several
weeks. The Comma-bacilli change
by vacuolation into planoconvex,
then biconvex, and finally circular
organisms ; these by division give
origin to two semicircular comma-
bacilli. Magnifying power about
700.

wavy or spiral-like organism. But the type is represented
by a single curved rod. For this reason it is not correct
to speak of them as comma-bacill, since they correspond to
what is generally considered a vibrio or spirillum.

The comma-bacilli are present, amongst crowds of other
putrefactive bacteria, in very varying numbers in the choleraic
evacuations, sometimes very scarce, sometimes numerous ; in
the mucus-flakes taken from the cavity of the lower part of
the ileum of typical rapidly fatal cases of cholera very soon

XL]

BACILLUS: PATHOGENIC FORMS.

177

after death they are present in small numbers, in the upper
part of the ileum and in the jejunum they are either very
scarce or altogether absent. The longer the examination is
delayed, of course within certain limits, the more likely are
the comma-bacilli to be found numerously in the flakes, but

FIG. 99.

Part of a test-tube containing gelatine-peptone ; in it pure cultivation of choleraic
comma-bacilli. The funnel-shaped opening of the channel in which the growth
of the comma-bacilli is going on contains a long air bubble.

not to the exclusion of other bacteria. They are generally
absent from the mucous membrane itself, inclusive of the
epithelium of the surface loosened but not detached. No
organisms of any kind are found in the tissue of the intestine,
in the blood, and other tissues; putrefactive bacteria,

N

iy8 MICRO-ORGANISMS AND DISEASE. [CHAP.

including comma-bacilli, are capable of growing after death,
and even before, into the clefts and spaces of the intestinal
wall from the internal cavity.

The mucus-flakes of the small intestine, taken from a
typical rapidly fatal case immediately after death, contain
besides detached epithelial-cells, numbers of lymph cor-
puscles, some perfect, others swollen up and disintegrating.

FlGS. 100 AND 101.

Pure cultivation of choleraic comma-bacilli in gelatine-peptone-broth. The two tubes
had been inoculated at the same time with the same comma-bacilli, and were
kept under precisely the same conditions. In both the surface of growth is
marked by a depression. At the bottom of the growth is a whitish precipitate of
masses of comma-bacilli. The rest of the channel is filled with almost clear
liquefied gelatine.

Soon after death all disintegrate. These corpuscles contain,
in varying numbers within their protoplasm, straight minute
bacilli much smaller than the comma-bacilli, being only
half or a fourth their length, and more or less pointed.

But these bacilli are constantly found also free ; singly, in
chains, and in smaller or larger groups.

XL]

BACILLUS : PATHOGENIC FORMS.

179

These small, straight bacilli are non-motile, and when
grown artificially they form spores. Neither the comma-
bacilli nor these small bacilli show in their mode of growth,
in artificial cultivations in various media, greater peculiarities
than other kinds of bacteria. The peculiar mode of growth
of the comma-bacilli in gelatine mixtures is shown in the
accompanying woodcuts.

Pure cultivation of choleraic comma-bacilli in gelatine-peptpne-broth. The inocula-
tion had been made, not in a channel, as in the previous figures, but on the
surface. There is also here a depression of the growth on the surface, and in the
extent of the growth the gelatine is liquefied, with a whitish precipitate at the
bottom.

Both the comma-bacilli and small straight bacilli grow
well in alkaline and neutral media, and are not killed by
weak acids although they do not show growth in them, or
only to a very limited degree. Neither the comma-bacilli
nor the straight small bacilli can be considered as connected
with the cause of cholera.

The result of the experiments performed by Nicati and

N 2

i8o

MICRO-ORGANISMS AND DISEASE. [CHAP.

Rietsch, by Koch and others, viz., death following in some
of the animals after injection of choleraic mucus-flakes or of
cultivations of comma-bacilli into the cavity of the small
intestine, were not due to cholera, but were probably due
either to the operation or to septic poisoning. Rodents,
carnivorous animals, and monkeys must be considered
insusceptible to cholera.1 There is direct evidence that the
water contaminated with choleraic evacuations only, and of
course with comma-bacilli, when drunk by a large number
of persons did not produce cholera ; there is no definite

FIG. 103.

From a preparation of mucus flakes of the ileum of a case of typical cholera.
Comma-bacilli and minute straight bacilli, singly and in masses . Three lymph-
corpuscles containing in their interior numerous small straight bacilli.

evidence that a cholera patient elaborates ready virus and
passes it out with the evacuations.

Comma-bacilli of various species have been discovered in
other diseases of the alimentary canal ; in the fluid of the

1 In connection with all the experiments said to have yielded positive
results on guinea-pigs, it must be mentioned that a variety of bacteria
are known, which although derived from normal but putrid secretions,
act very poisonously on rodents, e.g. the bacillus isolated by Bienstock
from normal human faecal matter ; the dumb-bell micrococcus isolated
by Pasteur and Sternberg from normal saliva, by Escherich from the
stool of milk-fed infants, by myself from sputum of pneumonia.

XL] BACILLUS : PATHOGENIC FORMS. 181

mouth of normal persons (Lewis) ; in old cheese (Denike).
The comma-bacilli found by Finkler and Prior in old stools
of cholera nostras differ in size and mode of growth from
Koch's comma-bacilli. So do those found in diarrhoea due to
other causes. But some species in the fluids of the mouth are
identical with Koch's comma-bacilli in their mode of growth.
The small straight bacilli above described are probably
identical with those mentioned by Emmerich, and which
are regarded by this observer as the true cause of cholera.
On guinea-pigs they act very poisonously.

Koch has shown that if guinea-pigs be kept for twenty-four hours
without food, then 5 cc. of a 5 per. cent, solution of sodic carbonate, and
after twenty minutes, 10 cc. of a broth cultivation of choleraic comma-
bacilli be injected into the stomach, and immediately after this I cc.
of (German) tincture of opium for each 200 grammes weight of the
animal is injected into the peritoneal cavity, the majority of these
animals die after a day and a half to three days. The small and large
intestine are found distended by, and filled with a clear fluid containing
numerously the choleraic comma-bacilli. Experiments of this nature
have no bearing whatever on the question of the causal relation of the
comma-bacilli to cholera in man, for the following reasons :

(1) It is quite gratuitous to consider the disease from which those
animals die as cholera, for during life they do not show any of the
special symptoms characterising cholera.

(2) Koch himself has proved by numerous experiments that guinea-
pigs treated in the above manner, but omitting the injection of opium
tincture into the peritoneum, do not become ill at all, although even
twenty hours after the introduction of the comma-bacilli, the stomach and
small and large intestine contain them numerously in a living state, as
is proved by the successful plate cultivations made by Koch himself
from the contents of these organs.

(3) Death of the animals has been produced after precisely the same
method by Finkler's comma-bacilli, and even by Denike's cheese
comma-bacilli.

(4) Death does not ensue unless tincture — not watery solution — of
opium be injected into the peritoneal cavity.

From all this it follows that the choleraic comma-bacilli are powerless
to produce disease in guinea-pigs, even if present in the small and large
intestine in a living state ; and that a previous pathological state of the
intestine, such for instance as is produced by the injection into the
peritoneal cavity of considerable quantities of tincture of opium,
enables the comma-bacilli to undergo multiplication.

CHAPTER XII.

VIBRIO.

VIBRIONES are characterised by being rod-shaped, but
not straight; they are more or less wavy; and they are
motile.

FIG. 104. — VIBRIO RUGULA
(AFTER COHN).

FIG. 105.— VIBRIO SERPEXS,
ISOLATED (AFTER COHN).

(a) Vibrio rugula consist of rods of about 0*008 to o-oi6
mm. in length, and curved either like a C or like an S.
They are single, or form chains of two. Their protoplasm
is always slightly granular. They are found in putrefying
organic substances, and often form continuous masses, the
individuals interlacing in all directions.

CH. xii.] VIBRIO. 183

(b) Vibrio serpens. — This is also a septic organism, much
thinner and longer than the previous one, more wavy, as
a rule, curved into a single or double wave. The length

FIG. 106 — VIBRIO SERPENS IN SWARMS (AFTER COHN).

varies between 0*011 and 0*025 mm- I* *s motile ; and also
forms continuous masses, the individuals interlacing in all
directions.

NOTE. — Many of the longer pathogenic bacilli, as bacillus of anthrax,
of symptomatic charbon, of tubercle, of ulcerative stomatitis, &c.,
occasionally present themselves in forms closely resembling vibriones.

CHAPTER XIII.

SPIROBACTERIUM (Spirillum}.

SPIRILLA are filaments of a spiral shape, motile, and
owing to their shape follow a spiral course when moving.
They are probably capable of forming minute bright spores.

FIG. 107. — SPIRILLUM TENUE, (i) SINGLY AND (2) IN SWARMS (AFTER COHN).

i. Septic spirilla. — These are found in all kinds of
putrefying organic substances, and are of three kinds.

(a) Spirillum tenue. — This is much finer and more wavy
than vibrio serpens, the turns being closer together and
spiral. Its length varies between 0-002 and 0*005 mm> j ^
often forms continuous felted masses ; it is motile.

CH. XIII.]

SPIROBACTERIUM.

185

Occasionally the spirilla grow to a great length — two, three,
and more of them forming a chain ; the individual spirilla are
not arranged in a linear series, but folded into a zigzag. This
form, which in reality is not a special kind of spirillum, is
called by Cohn 1 spirochceta plicatilis. The spirillum found
in the tartar of the teeth is of this form, spirochata denticola,
But there exist all intermediate forms between a single

FIG. ioS.— SPIRILLUM UNDULA
(AFTER COHN).

FIG. IOQ — SPIRILLUM VOLUTANS
(AFTER COHN).

spirillum tenue and a spirochaeta. In stained specimens the
construction of the spirochaeta from several spirilla tenua is
very distinct.

(£) Spirillum undula is much thicker and shorter than
the former ; there are all forms between such as are only
1 Bdtrage zur Biologie d. Pflanzen, vol. ii.

1 86

MICRO-ORGANISMS AND DISEASE. [CHAP.

half a turn to such as are of a whole turn of a spiral. It is
motile and forms chains of two or more elements, occurring
also in continuous masses, occasionally held together by a
hyaline interstitial substance.

/'

r£

FIG. no. — BLOOD OF RELAPSING FEVER (HUMAN).

Blood-corpuscles and spirilla Obermeyeri.

Magnifying power 700. (After Koch.)

(c) Spirillum volutans. — These organisms are giant
spirilla; long and thick, with granular protoplasm; 0^025
to 0*03 mm. long; motile, and with a flagellum at
each end.

2. Pigment spirilla.

(a) I have seen on paste a spirillum, morphologically
identical with spirillum undula; it is of a pale pink

XIIL] SPIROBACTERIUM. 187

or rosy colour.1 It is motile, and forms a kind of
zooglcea, the individuals being closely placed and there-
fore producing a rosy colour of a more decided tint. Where
they form continuous masses, the naked eye can detect the
rosy tint.

r \

FIG. in. — BLOOD OF APE INOCULATED WITH BLOOD SHOWN IN PRECEDING
FIGURE.

Blood-corpuscles and spirilla.
Magnifying power 700. (After Koch.)

(b) Spirillum sanguineum (Ophidomonas sanguined Ehren-
berg). — This was observed by Cohn and Warming 2 in
pondwater. Morphologically it is identical with spirillum
volutans. It is motile, with a flagellum either at one

1 "On a Rose-coloured Spirillum," Quar. Journ. of Micr. Sci., vol.
xv. New Series.

'2 Bcitr. z. Biol. d. PJIanzen, vol. i.

188 MICRO-ORGANISMS AND DISEASE. [CH. xin.

or both ends. Warming occasionally saw two and three
flagella at one end. It is about 0*003 mm. thick ; all
forms occur between such as have half and such as
have two and a half turns of a spiral. Lankester also
saw the same kind of organism amongst his peach-coloured
bacteria.1

3. Pathogenic spirilla.

Spirillum Obermeyeri (of relapsing fever) is morphologically
identical with spirillum tenue (or spirochaeta plicatilis of
Cohn). It was discovered in great numbers by Obermeyer 2
in the blood of the general circulation in patients suffering
from relapsing fever. The spirilla disappear from the blood
during the non-febrile stages, gradually decreasing in
numbers. They are motile ; they come out well in
specimens of blood made after the Weigert-Koch method
of drying the blood in a very thin layer, and then staining
with methyl-violet or Bismarck-brown.3 H. V. Carter 4
succeeded in producing relapsing fever in monkeys by
inoculation with human blood containing the spirillum
Obermeyeri. The blood in the monkey contained the same
spirilla in great numbers. Koch has cultivated artificially
the spirilla Obermeyeri, and saw them growing into long
spiral threads.5

1 Quarterly Journ. of Micr. Science, vol. xiii. New Series.

2 Centralb. f. med. Wiss. IO, 1873.

3 Weigert, Deutsche med. Woch. 1876; Heydenreich, Berlin, 1877.

4 Lancet, 1879, vol. i. p. 84 ; and 1880, vol. i. p. 662.

5 Deutsche med. Woch. 19, 1879.

CHAPTER XIV,

YEAST FUNGI : TORULACE^, SACCHAROMYCES.

YEAST, torula (Pasteur), or saccharomyces^ is not a bac-
terium, but belongs to an altogether different order of fungi
— the Blastomycetes. It consists of spherical or oval cells,
very much larger than the largest micrococci, and as in the
case of these each cell consists of a membrane and contents.
The contents are either homogeneous or finely granular pro-
toplasm ; in the latter case there are generally present one,
two, or more small vacuoles.

There are a great many species of Torula, varying from
one another morphologically chiefly in their size, and
physiologically by their action on various fluids (see below).

The cells multiply in suitable media by gemmation, a
minute knob-like projection appearing at one side of the cell,
and enlarging till it reaches nearly the size of the original or
mother-cell. It finally becomes altogether constricted
off from this latter, or having reached its full size remains
fixed to the mother-cell, and each cell again producing
by gemmation a new cell. In this way aggregations
of four, six, eight, or more cells are formed, which may
be arranged either as a chain when the production proceeds

igo MICRO-ORGANISMS AND DISEASE [CHAP.

in a linear manner, or as a group if the gemmation takes
place laterally.

Under varying conditions of growth, e.g. on transplanting
ordinary yeast growing in sugar-containing fluids on to potato,
but sometimes also in the same nutritive fluid, it is observed
that some of the yeast cells enlarge twice, thrice, and more
times ; they then form in their interior two, three, or more
small cells by endogenous formation ; these new cells are

FIG. 112. — TORULA, OR SACCHAROMYCES.

In the lower part of the figure an ascospore and four isolated spores (after Rees)

are shown.
Magnifying power about 700.

regarded as spores1 — the mother-cell being an ascospore
— and become free by finally bursting the membrane
of the mother-cell. On sowing these new cells into
sugar-containing fluids they multiply by the process of
gemmation.

Classifying them according to physiological function there
are various species of torula or saccharomyces. They all

1 T. de Seynes, Comptes Rendus, 1866; Rees, Sot. Zeitschr. 1869;
Hansen, Carisberg Labor at. 1883.

xiv.] YEAST FUNGI. 191

have the power to split up sugar into alcohol and carbonic
acid, but this power is not possessed by all to the same
degree.

(a) Saccharomyces cerevisice (torula cerevisice). This is the
ordinary yeast used in the production of beer. The in-
dividual full-grown cells vary in diameter from 0*008 to
o'oi mm. ; they form beautiful long chains. They produce
ascospores.

(b) Saccharomyces vim is very common in the air, and pro-
duces alcoholic fermentation of grape-juice ; it is therefore
the proper yeast of wine-production. Its cells are elliptical,
slightly smaller than the former ; it forms ascospores.

(c) Saccharomyces pastorianusis of various kinds (Hansen):
in some the cells are about o-oo2 to 0-005 mm- m diameter,
in others larger. Some form ascospores, others do not.
Most of them can be found in wine-fermentation and in
cider- fermentation, but only after the first alcoholic fermen-
tation is completed. They are very common in the air. I
have sown a Saccharomyces, which was contained in ordinary
water, on solid nourishing media (gelatine, and gelatine
and broth). It grew up copiously and formed groups of a
distinct pink colour. When growing in the depth of the
nourishing medium it grew as a colourless torula, no
ascospores were formed, multiplication taking place by
gemmation only.1

(ct) Saccharomyces mycoderma (mycoderma mm). This
yeast is found forming the scum or pellicle on the surface of
wine, beer, and fermented cabbage (Sauerkraut) ; its cells
are oval, about 0*006 mm. long and 0^002 broad. It forms
chains ; the ascospores are two or three times larger. It has
nothing to do with the alcoholic fermentation, and is not to

1 Quart. Journ. of Micr. Science, 1883.

192

MICRO-ORGANISMS AND DISEASE. [CHAP.

be confounded with mycoderma aceti^ which is a bacterium
and the efficient cause of acid fermentation in wine and
beer.

The saccharomyces mycoderma does not grow well in the ,
depth of liquids, but when sown into a liquid of acid re-
action and containing but little sugar, Cienkowsky saw the

FIG. 113. — SACCHAROMYCES MYCODERMA, OR OIDIUM ALBICANS.

(After Grawitz.)
From an artificial cultivation in dilute nourishing material.

d. Branched mycelium.
fa. Torula stage.
//3. Mycelial stage.

cells elongating into cylindrical elements ; each of which by
gemmation produced a new cell which also elongated, and
so on till a linear series of cylindrical cells was formed,
separated from one another only by a thin septum ; a mass
of filaments very much resembling a mycelium was thus

1 Nageli, see chapter viii. 2.

xiv.] YEAST FUNGI. 193

formed. The cylindrical cells give origin by gemmation to
spherical and elliptical torula-cells.

Such a growth, in which the torula-cells are capable of
forming a sort of mycelium, was formerly called oidium, and
as o'idium albicans is recognised as the cause of " thrush ; "
the well-known white patches which occur on the mucous
membrane of the mouth and pharynx in suckling infants
and debilitated patients.

Grawitz 1 has proved by observations on artificial cultures
that this fungus is identical with the oidium variety of Sac-
charomyces mycoderma ; the cells are spherical or cylindrical,
the former about 0*003 to °'005 mm- in diameter, the latter
up to o'O3 or 0-05 mm. long. As above described it forms
mycelium-like filaments from which, by lateral and terminal
gemmation, spring spherical or oval torula-cells. It also
forms ascospores containing four to eight spores.

1 Virchvufs Archiv, vol. Ixx.

CHAPTER XV.

MOULD-FUNGI I HYPHOMYCETES OR MYCELIAL FUNGI.

OF this class of fungi only those are of special interest to
the pathologist which in some way or other are connected
with disease. The fungi consist of branched and septate
threads or hyphae ; each filament or hypha is composed of a
row of cylindrical cells, consisting of a membrane and clear
protoplasm, the individual cells being separated from one
another by a thin transverse septum ; they increase in
number by fission, and in this way the filaments increase in
length. The growing ends of the hyphae are filled, not with
transparent, but with highly-refractive protoplasm. Some
cells, by budding out laterally, produce cylindrical threads,
which subdivide into a series of cylindrical cells, these by
division and lengthening forming a new branch-hypha. The
filaments form by their branches an interlacing feltwork,
called thallus or mycelium. The mycelial fungi which in-
terest us, belong to the order known to botanists as the
Ascomycetes. They are characterised by the fact that one or
other branch of the mycelial-hyphae produces at its end a
series of spherical or oval cells — the conidia-spores or con-
idia. In addition to this some of the hyphae form peculiar
large mother-cells, or sporangia, in the interior of which

CH. XV.]

MOULD-FUNGI.

195

spores are formed by endogenous formation. When these
sporangia are cylindrical or club-shaped, they include eight
spores, and are called asci ; the spores being ascospores. All
conidia or spores by germination grow into the mycelial
threads which become septate or subdivided into a row of
cylindrical cells; these by division cause the lengthening of
the mycelial threads.

FIG. 114.— OIDIUM LACTIS.
Mycelium and spores.

(a) Oidium lactis. — Here the mycelium is composed of
septate branched filaments of various thicknesses. Some
branches of the mycelium at their ends or laterally at a sep-
tum produce by division a series of spherical or oval conidia-
spores, about 0*007 to o'oi mm. long. These ultimately
become isolated, and then germinate into a short cylindrical

O 2

[96

MICRO-ORGANISMS AND DISEASE. [CHAP.

filament, which subdivides by transverse septa into a series
of cylindrical cells ; these by continued growth and division
give origin to the ordinary septate branch-hyphae. The
formation of conidia proceeds at the ends of these in the
same manner as before. The oi'dium lactis forms a whitish
mould on milk, bread, paste, potato, &c.

FIG. 115. — FUNGI FROM A FAVUS-PATCH (NEUMANN

Favus, Herpes tonsurans, and Pityriasis versicolor of man
and animals, are, according to the researches of Grawitz,1 due
to a fungus in morphological respects identical with oi'dium
lactis. In favus it is known as Achorion Schoenleini, in
Herpes tonsurans as Trichophyton tonsurans, in Pityriasis
versicolor as Microsporon furfur. Grawitz has shown by
artificial cultures on gelatine, that the spherical or oval

1 Virchow's Archiv, vol. Ixx. p. 560.

xv.] MOULD-FUNGI. 197

conidia germinate into shorter or longer cylindrical filaments,
which by subdivision form septate mycelial hyphae. These
and their branches give origin in their turn to spherical or
oval spores or conidia. They, as well as the hyphae, differ
in size in the various species.

Malcolm Morris and G. C. Henderson,1 on the other hand,
maintain, that by artificial cultivation of the spores of
Trichophyton in the substance of gelatine-peptone, at tem-
peratures varying from 15° to 25° C., these grow into
branched septate mycelial filaments, which by their mode of
fructification are seen to be identical with the mycelium of
Penicillium. Compare also with Babes.2

(b} Aspergillus. — Some of the branches of the mycelium
of this fungus assume an upright position, are thicker and
not at all, or only slightly, septate, and at their end form
flask-shaped enlargements, from which grow out radially
short cylindrical cells — basidia ; and these again at their
distal or free ends produce chains of spherical spores or
conidia. This is a very common mould, and according to
differences in the coloration of the mycelium and spores is
subdivided into different species ; A. glaucus^ candidus,
flavescens, fumigatus, &*c.

Besides this mode of spore-formation (asexual), there is
another (sexual), which according to De Bary consists in
this : some terminal branch of the mycelium becomes
twisted like a spiral, this is considered the female organ of
fructification or carpogonium ; from the same thread branches
grow towards the carpogonium ; one of these branches
becomes fused with the terminal portion of the carpogonium
called the ascogonium, while the others — the pollinodia —
branch and surround the carpogonium like a capsule : the

1 Journal of the Royal Microscopical Society ', April

2 Archives de Physiologic, 8, 1883, p. 466.

II, 1883.

198

MICRO-ORGANISMS AND DISEASE. [CHAI\

FIG. 116.— ASPERGILLUS GLAUCUS (AFTER DC. BAKY).
A, Hypha, the end of which, c, bears
st. The basidia.
as. Ascogonium.

FIG. 117.— E. PERITHECIUM, HIGHLY MAGNIFIED
as. Ascogonium.
•w. Cells of the pollinodia.

xv.] MOULD-FUNGI. 199

whole organ is now called a perithedum. Finally the asco-
gonium by rapid division gives origin to a number of oval
septate tubes, inside of which by endogenous formation
numerous spores make their appearance.

Grohe l was the first to show that the introduction of the
spores of some species of aspergillus into the vascular
system of rabbits sometimes produces death, with symptoms
of metastasis into the various organs due to localised foci,
where these spores grow into mycelial filaments. Lichtheim 2
showed that such mycoses in rabbits cannot be produced by
the spores of Aspergillus glaucus^ but by those of Aspergillus
flavescens and fumigatus. Grawitz 3 studied this process
more minutely, and found that, no matter whether the spores
are injected into the vascular system or into the peritoneal
cavity, there are established in the kidneys, liver, intestines,
lungs, muscles, and occasionally in the spleen, marrowbones,
lymphatic glands, nervous system, and skin, minute meta-
static foci, due to the growth of the spores into mycelial
filaments with imperfect organs of fructification, but no
spore-formation. Grawitz thought that the spores of ordi-
nary moulds (penicillium and aspergillus) are capable of
assuming these pathogenic properties if cultivated at higher
temperatures (39° to 40° C.), and in alkaline media. These
fungi, as is well known, grow well at ordinary temperatures
and in acid media, and are then innocuous when introduced
into the animal body ; but by gradual acclimatisation they
can also be made to grow at higher temperatures and in
alkaline media, when they assume pathogenic properties,
becoming capable of resisting the action of living tissues
and of growing in them. This view has been proved to be

1 BerL klin. Woch. 1871.

2 Ibid. 9 and 10, 1882.

3 Virchow's Archiv, vol. Ixxxi. p. 355.

200 MICRO-ORGANISMS AND DISEASE. [CHAP.

FIG. 118.— FROM A SECTION THROUGH THE KIDNEY OF A RABBIT DEAD THIRTY-
SIX HOURS AFTER THE INJECTION OF SPORES INTO THE JUGULAR VEIN.

F. Fat droplets. T. Tyrosin crystals.

In the upper part of the figure is a metastatic focus composed of Aspergillus spores
and mycelium. In the lower half of the figure the urinary tubules and two
Malpighian corpuscles are seen. (After Grawitz.)

incorrect by Gaffky,1 Koch,2 and Leber.3 Those spores
that do exert such pathogenic properties are not at all

1 Mittheil. a. d. kais. Gesundhdtsamie, 1880.

2 Berl. klin. Woch. 1881. 3 Ibid. 1882.

xv.] MOULD-FUNGI. 201

dependent on such acclimatisation, and are not ordinary
moulds, but a distinct species of aspergillus (Lichtheim), which
grows well at higher temperatures (38° to 48° C.), and the
spores of which under all conditions of growth are capable
of producing in rabbits the mycosis in question.

(c) Penidllium. — In this fungus hyphae, which are not
septate, grow out from the mycelium ; from the end of each
of these arise like the fingers of the hand a number of short
branched cylindrical cells, which give origin to chains of
spherical spores.

The following two fungi belong to the order of fungi
called Phy corny cetes.

(d) Mucor is characterised by this, that from the mycelium
hyphae grow out which are not septate, and at the end of
these a large spherical cell originates, sporangium, in which
by endogenous formation a large number of spherical spores
are developed ; the wall of the sporangium giving way, the
spores become free.

(e) Saprolegnia ; colourless tubular threads, forming gela-
tinous masses on living and dead animal and vegetable
matter in fresh water. The cylindrical or flask-shaped ends
of the threads — xoosporangia — form in their interior numbers
of spherical or oval spores — zoospores, possessed of locomo-
tion (one flagellum at each pole) and which finally escape
from the threads. These zoospores after some time become
resting, surround themselves with a membrane, and finally
germinate into a cylindrical mass which becomes transformed
into the mycelium. Besides this asexual there is, however,
a second or sexual mode of fructification, consisting in this :
At the end of a mycelial thread a cell grows up into a
spherical large ball, the oogonium. From the same thread,
thin threads — antheridia — grow towards the oogonium, with
the protoplasm of which they merge. This latter then

202

MICRO-ORGANISMS AND DISEASE. [CHAP.

differentiates into a number of spherical masses, the oospores,
which become invested with a membrane. These become
free and then germinate and grow into a mycelium.

FIG. 119. — SAPROLEGNIA OF SALMON DISEASE.

A sporangium filled with zoospores ; in connection with them several young
mycelial threads.

Saprolegnia grows on the skin of living fish, and causes
here severe illness often terminating in death. Thus the

xv.] MOULD-FUNGI. 203

salmon disease, as Professor Huxley has shown,1 is caused
by this parasite. The zoospores of this salmon saprolegnia
are, however, as Huxley has shown, as a rule non-motile.
The hyphae of the fungus traverse the epidermis in the
diseased patches of the salmon, and they bore through the
superficial layer of the derma, a stem-part being situated in
the epidermis, and a root-part in the derma ; each of these
elongates and branches out. " The free ends of the stem-
hyphae rise above the surface of the epidermis and become
converted into zoosporangia, more or fewer of the spores of
which attach themselves to the surrounding epidermis and
repeat the process of penetration." In saprolegnia associated
with the salmon-disease Professor Huxley observed only the
asexual mode of fructification.

An important case of general " mycosis mucorina " in
man, ending in death, has been recently described by Dr.
Paltauf (Virchow's Archiv, vol. 102, 3, p. 543). From the
alimentary canal of the patient an invasion of the internal
organs by the mycelium and spores of a kind of mucor
occurred, leading to the formation of metastatic inflam-
matory foci in the Peyer's glands, lungs, pharynx, larynx,
cerebrum, and cerebellum. In all these organs were found
smaller and larger foci of inflammation caused by the
presence of non-septate, branched mycelial threads and of
sporangia, showing that the fungus belonged to the group of
mucor.

1 Proceedings of the Royal Society, No. 219, 1882.

CHAPTER XVI.

ACTINOMYCES.

IN cattle there occurs a fatal disease, which is characterised
by the formation of firm nodules of various sizes, due to a
growth of small cells. In the centre of the nodules lie
dense groups of peculiar club-shaped corpuscles — actinomyces
— radiating from a firm homogeneous centre, and joined to
this by longer or shorter, single or branched, filamentous
stalks. Each of these actinomyces-corpuscles appears homo-
geneous, and of a bright slightly greenish lustre. These
masses consist of what is called Actinomyces (Bellinger), and
the disease is termed actinomycosis. In cattle the disease
manifests itself by firm tumours in the jaw, in the alveoli of
the teeth, and particularly by a great enlargement and
induration of the tongue — "wooden tongue." On making
sections through this latter organ there are found present in
all parts microscopic tumours of small-cell growth. In the
centre of each tumour is a clump of actinomyces. This
clump is surrounded by a zone of largish cells, with one to
four nuclei, The periphery of the tumour is made up of a
fibrous capsule, with spindle-shaped cells. Occasionally the
tumours are to be seen also in the skin and in the lung ; in
the latter organ they appear as whitish nodules, easily

CH. XVI.]

ACTINOMYCES.

205

FIG. 120. — FROM A SECTION THROUGH THE TONGUE OF A Cow DEAD OF

ACTINOMYCOSIS.

A nodule is shown composed of round cells, in the centre is the clump of actinomyces
surrounded by large transparent cells. Magnifying power 350.

FIG. i2i.— A CLUMP OF ACTINOMYCES MORE HIGHLY MAGNIFIED, 700.

mistaken for tubercles.1 Bellinger first described the
disease in cattle.2 Israel3 was the first to point out a

1 Pflug, Centralbl. f. med. Wiss. 14, 1882 ; Hink, ibid. 46, 1882.

2 Ibid. 27, 1877.

3 Virchow's Archiv, vols. Ixxiv. and Ixxviii.

206 MICRO-ORGANISMS AND DISEASE. [CH. xvi.

disease in man characterised by metastatic abscesses
(spreading it seems from a primary abscess of the jaw) in
various internal organs, due to the presence of a fungus,
which afterwards was identified as actinomyces, and Ponfick l
has clearly established that in man it is not a rare disease.

According to careful observations, Johne 2 succeeded in
transmitting the disease from cattle to cattle by inoculation,
but not by feeding. He also found 3 in twenty out of twenty-
one healthy pigs examined, the actinomyces present in the
crypts of the tonsils.

Israel 4 succeeded in transmitting the disease to a rabbit
by inserting into the peritoneal cavity a piece of a human
antinomyces-tumour.

R. Virchow quite recently,5 in conjunction with O. Israel
and Duncker, ascertained that pork occasionally contained
whitish chalky nodules, larger than those due to trichinae,
and containing in their interior the actinomyces.

O. Israel6 claims to have succeeded in artificially cul-
tivating the actinomyces on solid ox-serum ; in fluid media
the growth does not succeed, owing to the swelling up and
death of the actinomyces-corpuscles.

1 Die Actinomykose des Menschen, Berlin, 1881.

2 Deutsche Zeitschr. f. Thiermedicin, vii. 1881.

3 Centralbl.f. med. Wiss. 15, 1881.

4 Ibid, xxvii. 1883.

5 Virchvu?s Archiv, vol. xcv. p. 544.

6 Ibid. vol. xcv. p. 142.

CHAPTER XVII.

ON RELATIONS OF SEPTIC TO PATHOGENIC ORGANISMS.1

THERE is hardly any question which to the pathologist and
sanitary officer can be of greater importance than the relation
of septic to pathogenic organisms. To the pathologist the
life history of a micro-organism, outside and within the animal
body, must ever remain an important field of inquiry ; to the
sanitary officer all conditions affecting the life and death of
those organisms which produce, or at least are intimately
bound up with, infectious diseases, such as the distribution
and growth of these micro-organisms outside the animal
body, the agencies which affect it in a favourable and
unfavourable sense, are the points which he has particularly
to consider in dealing with the spread and prevention of
infectious maladies. Now, it is known of many micro-
organisms, both those that are associated with putrefactive
processes as well as those that are bound up with infectious
disease, that temperature, the medium in which they grow,
presence and absence of certain chemical compounds are
capable of materially affecting them. I need not for this

1 The greater part of this chapter is copied from an interim report
by myself to the Medical Officer of the Local Government Board,
1884.

2o8 MICRO-ORGANISMS AND DISEASE. [CHAP.

purpose enumerate all that is known already by direct experi-
ment, but will only limit myself to reference to the researches
of Schroter, Colin, and Wernich on that group of micro-
organisms known as pigment bacteria, i.e. bacteria which
only under certain conditions, notably temperature and soil,
produce definite pigments (Cohn's Beitrage zur Biologic d.
Pflanzen} ; to those of Hansen (Carlsberg Laboratory) on
yeast ; to those of Neelsen on the bacilli producing the blue
colour of milk, the bacillus syncyanus (Beitr. zur Biol. d.
Pflanzen, iii. 2, p. 187) ; to the works of Toussaint, Pasteur,
Chauveau, Koch, and others on the bacillus anthracis;
Arloing, Thomas, and Cornevin on the bacillus of symptomatic
charbon ; of Koch on the bacillus of tuberculosis ; of Israel
on actinomyces, and many others ; and particularly would I
refer to the many valuable suggestions and considerations
expressed by v. Nageli in these respects in his book, Die
niederen Pilze, Miinchen, 1877 and 1882.

While from these observations it would appear that both
septic and pathogenic micro-organisms are capable of suffering
some modifications in their morphological and physiological
behaviour, it is nevertheless still an open question whether
an organism which under ordinary conditions is only
associated with putrefactive changes in dead organic
material, and which cannot under these ordinary conditions
grow and multiply within the living body, can, under certain
extraordinary circumstances, become endowed with the
power of growing and multiplying within the body of a living
animal, creating there a pathological condition, inducing
there an infectious disease.

Three distinct septic micro-organisms have, after numerous
experiments and careful observations, been mentioned, as
being capable when growing under certain extraordinary
conditions of assuming pathogenic properties. These three

xvii.] SEPTIC AND PATHOGENIC ORGANISMS. 209

organisms are : (A) the common bacillus of hay infusion
is said by Buchner to be capable of transformation into
bacillus anthracis ; (B) a bacillus subtilis, present in the air,
which, although quite harmless in itself, assumes distinct
pathogenic properties when growing in an infusion of the
seeds of Abrus precatorius, becoming hereby endowed with
the power of causing severe ophthalmia (Sattler) ; (C) a
common mould, aspergillus, which harmless in itself, when
grown on neutral and alkaline material at about body-
temperature (38° C.) assumes, according to Grawitz, very
poisonous properties, producing in rabbits inoculated with it
death, with metastasis of aspergillus and its spores in the
various internal organs.

There are in the literature of micro-organisms other cases
mentioned, in which such a transformation has been supposed,
but without any experimental proof, and we need not there-
fore trouble ourselves more about them.

Let us now review seriatim the above three cases :

(A) Dr. Hans Buchner in a paper, which for many reasons
may be considered an important one, " Ueber d. experim.
Erzeugung des Milzbrandcontagiums, &c.," published in the
Sitzungsberichte d. math, physik. Classe d. k. Bair. Akademie d.
Wiss. 1880, Heft iii. p. 369, states that he succeeded in
transforming the common bacillus of hay infusion, the hay
bacillus, into the bacillus anthracis.

The hay bacillus and the bacillus anthracis rank together
morphologically under that form which Cohn has named
bacillus subtilis.

The two are, however, not quite identical in morphological
respects. The hay bacillus is a minute rod or cylindrical-
shaped bacillus, which by elongation and division produces
chains and further threads just like the bacillus anthracis, but
in the latter (i.e. bacillus anthracis) the bacilli and their

p

210 MICRO-ORGANISMS AND DISEASE. [CHAP.

threads are composed of cubical elements, as is shown in
stained specimens, and as has been mentioned in a former
chapter, whereas in those of the hay bacillus the elements
are rods or cylinders. I have seen, however, many of the
short hay bacilli which being constricted, i.e. in the act of
division, appear as two short more or less cubical elements
placed end to end. It is generally assumed that in hay
bacillus the bacilli are always rounded at their ends, whereas
the bacilli anthracis are as if straight cut at their ends ; but
this is not universally the case, since I have seen in cultures
the bacilli anthracis with distinctly rounded ends. But,
speaking generally, the hay bacillus is a rod more distinctly
rounded at its ends, the bacillus anthracis of the blood is
not so.

The bacillus anthracis is slightly thicker than the hay
bacillus. In artificial cultivations carried on in neutral
broth the bacillus anthracis is about twice as thick as the
hay bacillus growing in the same fluid, and when both are
growing in neutralised hay infusion the two are very
conspicuously different from one another, and can at a
glance be distinguished from one another ; the hay bacillus
being about half the thickness of the bacillus anthracis. In
stained specimens, too, the latter is beautifully made up
of a row of cubical cells, whereas the former consists of
cylinders only.

True, the bacillus anthracis is not always of the same
thickness, for, as I have shown, when growing in neutral
pork broth it is decidedly thicker than in the blood of an
animal dead of anthrax. And also in the blood of different
animals the bacillus anthracis slightly varies in thickness, for
in the guinea-pig's blood it is slightly thicker than in that of
the rabbit or sheep.

The hay bacillus is motile, possessed of a flagellum, and

XVIL] SEPTIC AND PATHOGENIC ORGANISMS. 211

therefore capable of locomotion ; the bacillus anthracis is
not motile. I am quite aware that Cossar Evvart (Quarterly
Journal of Microscopical Science, April 1878) states that he
has seen in a specimen kept artificially heated under micro-
scopic observation that the bacillus anthracis, at first non-
motile, is capable of becoming motile. At one or both ends
a flagellum grows out from its body. But this observation
is unreliable, since Ewart did not guard himself in any way
from the accidental introduction of septic bacilli, many of
which are motile. Besides, he says of the bacilli, which he
figures as anthrax bacilli, that they are connected with one
another by two fine threads, and that they probably separate
from one another and each retains one filament, which is its
flagellum. But his observations, so far as they have appli-
cation to anthrax bacilli, are capable of quite a different
interpretation. In every specimen of blood and in every
artificial culture bacilli can be seen, in which at one place
or more the protoplasm is wanting, owing, as I have shown,
to degeneration ; in such places only the empty sheath is
present and of course in fresh specimens this gives the
appearance as if the two protoplasmic portions of the
bacillus were connected with one another by two fine
threads, i.e. the sheath being transparent is seen here
edgeways.

In no instance has the bacillus anthracis been observed
to be motile. I have examined thousands of specimens of
fresh bacillus anthracis in the blood and in artificial cultures,
and I have never seen anything that in the least would lead
me to differ from this proposition.

As regards the spores they are of the same aspect and
size in both the hay bacillus and bacillus anthracis. The
threads in good cultures form in both cases the same bundles
more or less twisted and forming convolutions, but in certain

p 2

212 MICRO-ORGANISMS AND DISEASE. [CHAP.

cultures of the bacillus anthracis, e.g. in broth in which the
growth is limited to the bottom of the fluid, the convolutions
and the twisted condition of the threads are more pronounced,
more cable-like.

Hay bacillus being motile, every culture of it is uniformly
turbid, the bacilli being capable of moving about, and after a
day or two of incubation at 35" C. they form a distinct pellicle
on the surface of the fluid, which in further stages becomes
complete and thick. These pellicles are composed of a
dense feltwork of threads of the bacilli, and in them spore-
formation is going on.

By shaking the fluid the pellicle sinks to the bottom, and if
the fluid is not exhausted yet, a new pellicle is formed of the
same nature.

In no culture of hay bacillus are there ever observed those
cloudy, fluffy, whitish and transparent convolutions that are
seen in cultivations of bacillus anthracis carried on at the
bottom of fluid broth, and which have been so accurately
described by Pasteur.

Both the hay bacillus and the anthrax bacillus when
growing on gelatine mixtures liquefy the gelatine ; both
when growing in meat broth turn the at first colourless fluid
in the course of incubation to an amber, and further to a
brown, tint.

The hay bacillus is capable of thriving well in acid
solutions, it grows copiously in hay infusion, which is of a
distinct acid reaction ; the bacillus anthracis, although capable
of making a slight progress in acid hay infusion, does
not get far, for degeneration soon sets in ; it thrives best in
neutral solutions. Hay bacillus thrives also very well in
neutral solutions.

Buchner states, that by successive cultivation of bacillus
anthracis under constant variation of the nutritive material,

XVIL] SEPTIC AND PATHOGENIC ORGANISMS. 213

he saw it assume gradually the properties of hay bacillus.
Thus he saw that its mode of growth gradually changed,
inasmuch as instead of forming, as the typical bacillus
anthracis does, fluffy convolutions at the bottom of the fluid-
nourishing medium, it gradually showed a tendency to stick
to the glass and to the surface of the fluid, and to form a
sort of pellicle just like the hay bacillus does. This I consider
to be an erroneous interpretation of an easily explained and
simple fact. It does not want any of the many successive
generations of bacillus anthracis, in which Buchner says he
has achieved this transformation ; it simply requires two
nourishing fluids in both of which the bacillus anthracis will
thrive well, but which fluids differ in specific gravity. Let
Buchner do as I have done, let him take two test-tubes, both
containing sterile broth, but in one the broth concentrated,
in the other dilute. Let him inoculate the two test-tubes
with bacillus taken from the same blood, say of a guinea-pig
dead of anthrax, let him place them in the incubator at a
temperature of 35° to 38° C. After two or three days, and
more decidedly later, he will notice this very difference in
the aspect of the cultures that he lays so much stress on
as indicating a change in the physiological character of the
bacillus. One test-tube, containing the dilute broth, shows
the typical fluffy convolutions at the bottom of the fluid ; while
the other, containing concentrated broth, shows a distinct
attempt at the formation of a pellicle. Let him now take
out a droplet from this second test-tube and inoculate with
it two test-tubes of the same nature as above, i.e. one
containing concentrated broth, the other dilute broth. After
two or three or more days of incubation he will find exactly
the same differences as above.

Buchner states that the bacillus anthracis when carried
through a large number- of successive cultures, at a tempera-

214 MICRO-ORGANISMS AND DISEASE. [CHAP.

ture of 35° to 37° C, gradually loses its pathogenic properties.
Of this assertion I have said already a great deal in my
Report for 1881 — 1882, and I mention it here merely in
connexion with Buchner's other assertions. I have shown,
that even assuming that Buchner has had in all his cultures
the true bacillus anthracis, but for which there is no definite
proof, as Koch has so ably pointed out in his critical review
of Buchner's work (Mittheilungen aus dem k. Gesundheitsamte,
Berlin, 1881, End. I.), Buchner having tested his cultures
on white mice only, has fallen into a serious error, for, as I
have shown (Reports for 1881 — 1882), a culture of bacillus
anthracis may have become quite harmless to white mice, but
be still very virulent to other animals. In fact, therefore,
Buchner's result does not require for its achievement more
than one culture, provided this has been kept for several days
or weeks without spore-formation, as was the case in
Buchner's experiments.

As regards Buchner's statement that by successive cul-
tivation of bacillus anthracis at 35° to 37° C., this assumes
the morphological and physiological characters of hay bacillus,
I agree with Koch in regarding this as a complete error. If
the cultures are quite safe from contamination nothing of
the sort ever happens. I have now for several years carried
on such cultures, and have not seen anything of the sort.
It is of course clear that if by any accidental contamination,
say at the time of inoculating a fresh tube, a motile septic
non-pathogenic bacillus, with which, or with the spores of
which, the air sometimes abounds, is introduced, every new
culture established from this one will abound in this bacillus
and as it grows quicker and more easily than the
bacillus anthracis, the next cultivations become barren of
all the bacillus anthracis, and only the non-pathogenic motile
bacillus will be found present. This criticism has been

XVIL] SEPTIC AND PATHOGENIC ORGANISMS. 215

applied by Koch to Buchner's experiments, and I must fully
endorse it.

But there is a much more serious statement of Buchner's
— serious, because if true in nature, it is dreadful to contem-
plate to what amount of anthrax man and brute may become
subject — viz., that he maintains to have succeeded in trans-
forming the hay bacillus into bacillus anthracis, by carrying
the former through many generations under ever varying
change of soil. It is needless to detail here all these experi-
ments of Buchner, since I do not attach any great value to
them, and I should not have troubled myself much about
them, were it not that one meets in mycological literature,
particularly on the part of botanists, an acceptance of
Buchner's statement that hay bacillus can change into the
pathogenic bacillus anthracis (see Zopf, Die Spaltpilze,
Breslau. 1883).

I have repeated Buchner's experiments on rabbits, guinea-
pigs, and white mice. I have grown the hay bacillus in
various kinds of broth, in gelatine broth mixtures, in hydro-
cele fluid, in peptone fluid, in Agar-Agar and peptone, at
temperatures varying between 30° and 38° C, and I have,
to put it shortly, never seen that it shows the least tendency
to change its morphological characters, or that it ever
assumes any morphological or physiological character like
the bacillus anthracis. I consider this a perfectly hopeless
task, and I feel sure any one might as soon attempt to
transform the bulb of the common onion into the bulb of
the poisonous colchicum.

But Buchner states that with his cultures of hay bacillus
carried through many generations under varying conditions
of soil, he inoculated white mice, which died under symptoms
of anthrax, and whose blood contained the typical bacillus
anthracis. I do not for a moment doubt that he really had

216 MICRO-ORGANISMS AND DISEASE. [CHAP.

mice dying from anthrax after inoculation with cultures of
hay bacillus, but I question the admissibility of his interpre-
tation. I believe that some accidental contamination of the
culture of hay bacillus with anthrax spores or otherwise may
have occurred and have got overlooked. How liable one
kind of infective material is to be invaded by foreign infective
matter may be understood from the following examples of
its actual occurrence.

It is now admitted on all hands that the results of
Villemin in producing what is called artificial tuberculosis in
guinea-pigs, by inoculating the animals subcutaneously with
cheesy matter derived from human and bovine tuberculosis
or from a guinea-pig suffering from artificial tuberculosis,
cannot be produced by any other means ; it cannot be pro-
duced by ordinary, i.e. non-tubercular cheesy or other pus,1
nor by setons (as once thought by Wilson Fox and Sanderson)
setting up chronic caseous inflammations in the skin of
guinea-pigs, nor by chronic mechanical irritation, e.g. inser-
tion into the peritoneal cavity of bits of gutta-percha or
other substances producing chronic peritonitis (as was
thought by Cohnheim and Fraenkel), but, as Cohnheim
now tersely puts it, tuberculosis can be produced only by
matter derived from a tuberculous source, and anything
that produces this tuberculosis is derived from a tuberculous
source. Dr. Wilson Fox, after the very important experi-
ments performed by Dr. Dawson Williams, according to
which chronic inflammation in the skin of guinea-pigs
produced by setons, is in no case followed by tuberculosis,
has conceded that in his earlier experiments there must have
entered some error in the use of the materials. Cohnheim
has conceded the same. It is clear that these observers,
while working at the same period with both tuberculous and
1 Compare Watson Cheyne, Practitioner, April 1883.

xvii.J SEPTIC AND PATHOGENIC ORGANISMS. 217

non- tuberculous matter, must have had, in the course of
experiments with the latter substance, accidental contamina-
tion with the former, and hence had the guinea-pigs inoculated
by them with non-tuberculous matter nevertheless affected
with tuberculosis. Dr. Williams, who had no contamination
to fear, working with non-tuberculous matter only, had
consequently no accidental contamination. This shows us
how dangerous, as regards reliability of results, it is to work
in one laboratory with different infective materials at the
same period.

I have myself experienced some very curious results
bearing on this very point. During the last year I have
seen the following cases of accidental contamination occur.
I work in the laboratory of the Brown Institution, which
comprises a suite of rooms. Although working extensively
on anthrax, I generally limit myself to one room only. A
friend of mine, who one day injected into a vein of a
guinea-pig blood taken from a blood-vessel of a dog
suffering from distemper, found, to his great disappointment,
the guinea-pig dead after two days under the typical
symptoms of anthrax, the blood of this animal teeming
with the characteristic bacilli. The hypodermic syringe
used in this experiment for injection had not been previously
used by me in my anthrax experiments, since I never use a
syringe in my inoculations, but only glass pipettes freshly
made and drawn out into a fine tube. The experiment was
performed in the room adjoining the one in which my
anthrax investigations were being carried on, but I was in
the habit of making every day a good many specimens of
anthrax cultivations and spores, so that there must have been
a good many of these spores distributed on the table and
floor, and probably found their way into the wound of the
guinea-pig at the time the above experiment was made.

218 MICRO-ORGANISMS AND DISEASE. [CHAP.

Another gentleman working in the laboratory of the Brown
Institution intended to inoculate several guinea-pigs with
human tubercles. For this end he mashed up in saline solu-
tion, in a clean mortar, a bit of human lung studded with
tubercles. He did this in my room on the same table on
which I was working with anthrax. One of these guinea-
pigs, inoculated with human tubercle, died before the second
day was over of typical anthrax. Its blood was teeming with
the bacillus anthracis. Such an accidental anthrax of a
guinea-pig inoculated with tuberculous matter occurred
several times. In all cases freshly drawn-out glass capillary
pipettes had been used for performing the inoculation, and
also the other instruments had been carefully cleaned before
the inoculation.

I myself had the following accidental contaminations : —
A guinea-pig had been inoculated -with a culture of
bacillus anthracis, which I did not expect would produce
anthrax, the culture not being capable of starting new cul-
tures, the bacillar threads being all in a state of degeneration.
The animal, of course, remained unaffected. Some weeks
afterwards inspecting the guinea-pig, to my surprise, I found
the inguinal lymphatic glands at the side of the former
inoculation greatly swollen, filled with cheesy pus. The
animal was killed, and was found to be affected with general
tuberculosis, the cheesy matter of the tubercular deposits
containing the tubercle bacilli. Comparing my notes on
this animal with those of my friend Lingard, we found that
on the very day on which I inoculated the animal with my
anthrax culture we had inoculated several other guinea-pigs
with tuberculous matter. This tuberculous matter was
prepared in the same room in which I prepared the fluid
for my anthrax inoculation, but the instruments in the two
sets of experiments had not been the same.

xvii.] SEPTIC AND PATHOGENIC ORGANISMS. 219

A rabbit was inoculated with a culture of bacillus an-
thracis which I did not expect would produce anthrax. The
animal remained unaffected with anthrax, but died after
four weeks with the symptoms of extremely well-marked
tuberculosis — in fact, the best marked case that I have seen
— of both lungs, spleen, liver, and kidney. The tubercular
deposits contained the tubercle bacilli.

Also in this instance inoculations with tuberculous matter
had been going on at the same time, when I meant to have
inoculated nothing else but a culture of anthrax bacilli.

I think all these facts taken together prove unmistakably
that working with two contagia in the same laboratory and at
the same period, accidental contamination is of no rare
occurrence. And this applies with equal force to Buchner's
experiments. Buchner worked extensively with anthrax
cultures in the same laboratory, and at the same time he had
those successful cases of anthrax in mice which he thought
to have inoculated with cultures of hay bacillus, and acci-
dental contamination probably was the result. Buchner
himself has experimentally shown that anthrax virus in the
shape of spores can by inhalation produce anthrax, and, there-
fore, this is another argument against his above cases of
positive results. I am. assuming that his cultures of hay
bacillus were really free of spores of bacillus anthracis : but,
seeing that his anthrax cultures were probably contaminated
with hay bacillus, I do not see why, by some chance, one of
his tubes which he thought he inoculated with hay bacillus
should not have been accidentally contaminated with the
spores of bacillus anthracis, of which there must have been
many about in the air of the laboratory.

If Buchner could show us that in a laboratory, in which
for some considerable time anthrax cultures, anthrax
animals, and examinations of anthrax bacilli had not been

220 MICRO-ORGANISMS AND DISEASE. [CHAP.

carried on, cultivation of hay bacillus ultimately yields a fluid
which produces typical anthrax, then I should be perhaps
prepared to concede his proposition of a transmutation of
hay bacillus into bacillus anthracis. Such a proposition is
of the widest importance, and therefore its proof ought to be
beyond cavil, there ought to be no chance of a possibility
of error. Such proof Buchner has not given, and I cannot
therefore accept his interpretation.

(£) The second instance in which the transformation of a
common septic into a specific or pathogenic organism has
been experimentally achieved, or I should rather say has been
stated to have been achieved, is the jequirity bacillus. In
1882 the well-known ophthalmologist M. L. de Wecker in
Paris drew attention to the therapeutic value of the seeds
or beans of Abrus precatorius, a leguminosa common in India
and South America. The people of Brazil use it under the
name jequirity as a means to cure trachoma, or granular
lids. De Wecker after many experiments found that a few
drops of an infusion made of these seeds causes severe
conjunctivitis, in the course of which, no doubt, trachoma
is brought to disappearance and cure, and it is accordingly
on the Continent and in this country now used for this
therapeutic object. [I am informed by my friend Dr. T.
Lewis, formerly of India, now pathologist at the Netley Army
Medical School, that the people in some parts of India know
the poisonous properties of these seeds, and use it for
inoculating with them subcutaneously, cattle ; in consequence
a severe inflammation is set up, and the animals die of some
sort of septicaemia. This is done for the sake of simply
obtaining the hides of the beasts.]

Sattler, in a very important and extensive research ( Wiener
medic. Wochenschrift, N. 17-21, 1883, and Klin. Monatsbl.f.

XVIL] SEPTIC AND PATHOGENIC ORGANISMS. 221

Augenheilk. June 1883) ascertained that when an infusion of
the jequirity seeds is made of the strength of about half per
cent., this infusion after some hours to a few days contains
numerous bacilli, motile, capable of forming spores, and in
all respects identical with a bacillus subtilis. The bacilli
are about 0*00058 mm. thick, and from 0*002 to 0^0045 mm.
long. They form a pellicle on the surface of the infusion,
and in the bacilli of this pellicle active spore formation is
going on. The bacilli grow and multiply well at a tempera-
ture of about 35° C., but also, only slower, at ordinary
temperature. Sattler cultivated artificially the bacilli on
blood-serum gelatine and meat extract peptone gelatine, both
solid media, and continued their growth through several
successive cultivations. Both the infusions of the jequirity
and the bacilli taken from these artificial cultures inoculated
into the conjunctiva of healthy rabbits produce severe
ophthalmia, leading to the production of great cedematous
swelling of the conjunctiva and eyelids, and temporary
closure of the latter, and to the secretion of purulent
exudation. Both the exudation and the swollen lids are
said to contain infective bacilli and their spores. Sattler
ascertained by many experiments, that none of the bacilli and
the spores distributed in the atmosphere had those specific
properties, viz., to excite ophthalmia, as long as they grow
in other than jequirity fluid, but having had access, i.e. having
entered the jequirity infusion, assume here this specific
power. There is no doubt that Sattler worked the whole
problem with great care, worked out all points connected
with it in great detail, and for this reason his work was
considered to have for the first time unmistakably established
that a harmless bacillus, owing to the particular soil in which
it grew, assumes definite specific or pathogenic properties.
To me this jequirity bacillus had a great interest, since I was

222 MICRO-ORGANISMS AND DISEASE. [CHAP.

particularly anxious to get hold of such an organism, in
order to see whether and how far it can again be made
harmless. For if ever there was a good case, a case in
which a previously harmless micro-organism had by some
peculiar conditions become specific, this was a case ; and
therefore it must be here possible by altering its conditions
of life again to transform it into a harmless being. The
theoretical and practical importance of such a case must be
evident to every one who has at all devoted any thought to
the relation of micro-organisms with disease. The whole
doctrine of the infectious diseases, I might almost say, is
involved in such a case, for if in one case it can be-
unmistakably proved that a harmless bacterium can be trans-
formed into a pathogenic organism, i.e. into a specific virus
of an infectious malady, and if this again can under altered
conditions resume its harmless property, then we should at
once be relieved of searching for the initial cause in the
outbreak of an epidemic. But in that case we should be
forced to contemplate, as floating in the air, in the water, in
the soil, everywhere, millions of bacteria which, owing to
some peculiar unknown condition, are capable at once to
start any kind of infectious disorder, say anthrax (Buchner),
infectious ophthalmia (Sattler), and probably a host of
other infectious diseases, and thus to form the starting-
point of epidemics. And the only redeeming feature,
if redeeming it can be called, in this calamity would be
the thought that the particular bacterium would by and
by, owing to some accidental new conditions, again become
harmless.

These were the reasons, and good reasons I think they
were, which prompted me to inquire into the jequirity
bacillus and jequirity ophthalmia, and after a very careful
and extensive series of experiments, to be described presently,

xvii.] SEPTIC AND PATHOGENIC ORGANISMS. 223

I have proved beyond any doubt that the jequirity bacillus,
per se, has no more power to create an infectious ophthalmia
than Buchner's hay bacillus had of creating anthrax.
The following experiments prove this conclusively : —
The seeds of jequirity ( Abrus precatorius) are crushed
and powdered, the perisperm is removed, and of the rest an
infusion is made of about the strength of half per cent-
with distilled water, previously boiled and contained in a
flask previously sterilised (by heat) and plugged with sterile
cotton-wool. The infusion is made while the water is still
tepid. After half an hour the infusion is filtered into a
fresh sterile flask, plugged with sterile cotton-wool, the access
of air being limited as much as possible. This is effected
by keeping the cotton-wool in the mouth of the flask around
the end of the glass filter. The filtered fluid is of a slightly
yellowish-green colour, and is almost neutral and limpid.
A small quantity is withdrawn with a capillary glass pipette
freshly drawn out, and from this several test-tubes containing
sterile nourishing material (peptone solution, broth, Agar-
Agar and peptone) are inoculated ; and from the same
pipette, and at the same time, several eyeballs of healthy
rabbits are inoculated, by placing a drop or two of the
infusion under the conjunctiva bulbi. The test-tubes are
placed in the incubator and kept there at 35° C. After
twenty-four hours all eyeballs are intensely inflamed, the
eyelids closed and swollen, and a large amount of purulent
secretion is present in the conjunctival sac, but all the test-
tubes remain perfectly limpid ; no growth has made its
appearance, and they remain so.

In a second series the infusion prepared in the above
manner is used fifteen minutes after it is made and used as
above, for inoculation of test-tubes and eyeballs. The fluid
in the test-tubes after incubation remains limpid, the eyeballs

224 MICRO-ORGANISMS AND DISEASE. [CHAP.

all become inflamed. In both series the amount of fluid
inoculated into the test-tubes is more than twice as great
as that injected into the eyeballs. From this it is quite
clear that the fluid used for inoculation of the test-tubes was
barren of any micro-organisms, and nevertheless it possessed
a powerful poisonous principle. I do not mean to say that
the infusion as a whole contained in the flask contains no
organisms, but that the small quantity of the fresh infusion
that was used for the inoculation of the test-tubes and eyeballs
contained none is absolutely certain. When such a flask
is placed in the incubator, after twenty-four to forty-eight
hours or later there are found in it large quantities of bacilli,
the spores of which must have entered from the air during
the process of preparing the infusion. The bacilli are such
as described by Sattler ; they soon form spores in the usual
way. Such an infusion is very poisonous, just like the fresh
one. Sattler has shown, and this is easily confirmed, that
the spores of these bacilli stand boiling for a few minutes
without losing their power to germinate. Consequently, if
such a poisonous infusion full of bacilli and spores be boiled
for half a minute the spores are not killed ; proof for this :
that if with a minute dose of this spore containing boiled
infusion any suitable sterile nourishing material in test-tubes
be inoculated, and then these test-tubes be placed in the
incubator at 35° C., after twenty-four to forty-eight hours
the nourishing fluids are found teeming with the jequirity
bacilli; but no amount of this material produces the least
symptom of ophthalmia. Every infusion of jequirity loses its
poisonous activity by boiling it a short time, \ to i minute,
and hence the above result.

In this respect the poisonous principle of jequirity infusion
comports itself similar to the pepsin ferment, which, as is
well known, is destroyed by short boiling.

xvn ] SEPTIC AND PATHOGENIC ORGANISMS. 225

If an infusion is made as above, and after fifteen minutes
it is filtered and then subjected to boiling for J to i minute,
it will be found to have become absolutely non-poisonous,
but not sterile : placing it in the incubator after twenty-four to
forty-eight hours, vast numbers of the jequirity bacillus are
found in it. But no amount of this fluid is capable of
producing the slightest symptom of ophthalmia.

A large per-centage of the rabbits, whose conjunctiva has
been inoculated with the fresh unboiled poisonous infusion,
die after several, three to eight, days. The eyeballs and
eyelids are intensely inflamed, as stated above, the skin and
subcutaneous tissue of the face, neck, chest, and even
abdomen, is found enormously cedematous, the pericar-
dium, pleura, lungs, . and peritoneum very much inflamed,
their cavities filled with a large quantity of exudation.
The exudations of the conjunctiva, pericardium, peri-
toneum, the cedematous skin and subcutaneous tissues contain
no infective property, and as a rule no bacilli or spores of
any kind, if examined in the living animal or immediately
after death.

There is one point which requires careful consideration ;
it is this : Sattler states that he has cultivated the bacillus,
taken from a poisonous jequirity infusion, through several
successive generations on solid material, and with the new
cultures he was able to produce the jequirity ophthalmia.
I have no doubt whatever that this is really the case, but it
bears an interpretation different from the one Sattler gave
it. Sattler, and most pathologists, would, of course, say this :
if any micro-organism taken from a soil that possesses
infective properties be carried through many successive
artificial cultivations, all accidentally adhering matter would
hereby become so diluted that it may be considered as
practically lost ; that is to say, the organisms of the further

Q

226 MICRO-ORGANISMS AND DISEASE. [CHAP.

generations have become altogether free of that matter. If
the organisms of these further generations still possess the
same poisonous property as the original material, then we
must conclude that this poisonous principle is identical with
the micro-organism. I do not agree with this whole chain
of propositions, although I agree with some parts. If a
micro-organism be carried through several successive cultiv-
ations in a fluid medium, always using for inoculation of a
new culture an infinitesimal dose, and as nourishing medium
a comparatively large quantity of fluid, then, no doubt,
carrying on the cultivations through four, five, or six successive
cultures, any accidentally adhering original matter becomes
practically lost, and if then the organism still possesses the
same poisonous action to the same degree as the original
material, then no doubt the conclusion that organism and
poison are in this case identical becomes inevitable. But
this is not the case with the jequirity bacillus. Taking from
a poisonous jequirity infusion full of the bacilli one to two
drops, and inoculating with it a test-tube containing about
four to five cc. of nourishing material, and using this at once
without previous incubation, we find that even a few drops of
this so diluted fluid still possess poisonous action. Precisely
the same result is obtained when taking from a perfectly fresh
jequirity infusion, i.e. before any organisms have made their
appearance, one to two drops, and diluting them with four
to five cc. of distilled water, and using of this diluted fluid
one to two drops for inoculating the conjunctiva of healthy
rabbits, severe ophthalmia will be the result. Carrying
on the cultivation of these bacilli started from a poisonous
infusion, for a second generation in fluid medium, no trace
of any poisonous action can be now detected, any quantity
of such a cultivation is incapable of producing ophthalmia.
Sattler used in his cultivations solid nourishing material,

XVIL] SEPTIC AND PATHOGENIC ORGANISMS. 227

on the surface of which he deposited his drop of poisonous
jequirity infusion containing the bacilli ; after some days'
incubation, the bacilli having become greatly multiplied,
he took out from this second culture a drop, and transferred
it to a new culture-tube of solid material, and so he went on :
every one of these cultures possessed poisonous action.
Clearly it would, since he always used part of the original
fluid deposited on the surface of the solid nourishing material.
Part of this (being gelatine) became by the growth liquefied,
but considering that Sattler started with infusions of
considerable concentration — he left the seeds for many
hours and days in the infusion — it is not to be wondered
at that this would bear a considerable amount of dilution,
and still retain its poisonous properties. From all this we
see, then, that the jequirity bacillus per se has nothing to do
with the poisonous principle ofj the jequirity seeds, but that
this principle is a chemical ferment in some respects (in its
inability to withstand boiling) similar to the pepsin ferment.1

(C) The third case, in which an experimental attempt has
been made to transform a common septic into a specific or
pathogenic micro-organism, is exemplified by the common
mould, aspergillus, a mycelial fungus. But since this point
has been discussed already in Chapter XV. I need not here
enter into it again ; suffice it to say that certain species of
aspergillus possess the power of making in the rabbit a

1 Since this has been in print, I became aware that Messrs. Wardei
and Waddell published in Calcutta during the present year a most
valuable memoir, detailing a large number of observations on the
jequirity poison, which are in complete harmony with my own observa-
tions. They have definitely proved, that the active principle is a
proteid — abrin — closely allied to native albumen ; that its action is
similar to that of a soluble ferment, that it can be isolated, and that it
is contained, not only in the seeds bnt also in the root and stem of
Abrus precatorius.

Q 2

228 MICRO-ORGANISMS AND DISEASE. [CHAP.

general mycosis, and this power they possess ab initio ; other
kinds of aspergillus do not possess this character and cannot
acquire it under any conditions.

Thus also this third case of a transformation of a common
into a specific organism due to altered conditions of growth
falls to the ground.

It might be now asked, how about those cases in which by
injection of very small quantities of putrid organic substances,
pyaemia or septicaemia has been produced in rodents ? Take
the case of Davaine's septicaemia in rabbits. This disease
has been produced in rabbits by Davaine, Coze and Feltz,
and by many other observers, by injecting into the subcu-
taneous tissue of healthy rabbits small quantities of putrid
ox's blood. The rabbits die in the course of a day or two,
and their blood is found teeming with minute organisms,
which prove to be bacterium termo ; every drop of this blood
possesses infective properties ; when inoculated into a rabbit
it produces septicaemia with precisely the same appearances
as before. Pasteur and Koch have succeeded in producing
septicaemia in mice and rabbits, and especially in guinea-pigs,
by inoculating them subcutaneously with garden earth or with
putrid fluid. This is Pasteur's septicaemia, or Koch's malig-
nant oedema ; it is characterised by oedema at the seat of
inoculation, and spreading hence in the subcutaneous tissue
of the surrounding parts. The animals die generally in
twenty-four to seventy-two hours.

Koch has produced by injection of small quantities of
putrid fluids into the subcutaneous tissue of mice a peculiar
septicaemia ; the animals sometimes die in forty to sixty hours,
and the white corpuscles of the blood are found crowded
with exceedingly minute bacilli. Koch succeeded also in
producing a pyaemia in rabbits by injection of putrid fluids,
and this pyaemia is characterised by zoogloea of minute

XVIL] SEPTIC AND PATHOGENIC ORGANISMS. 229

micrococci being present in the blood-vessels. Further, a
progressive necrosis in mice by inoculating them with putrid
fluids, the necrosis being due to the growth of micrococci
and spreading from the seat of inoculation, and destroying
as they spread all the elements of the tissue. All these
cases have been minutely described by Koch in his
classical work, Die Aetiologie der Wundinfectionskrank-
heiten, Leipzig, 1879. I have in addition mentioned in
Chapter VII. § 13 a micrococcus causing abscess and
pyaemia in mice.

Now do these cases prove that septic micro-organisms,
living and thriving in putrid organic fluids, can, when intro-
duced into the body of animals, owing to some peculiar
unknown condition, so change as to produce a fatal infections
disease? I must say, with Koch, who has very ably
discussed all these points, ' No.' Those organisms which
are connected with the above morbid processes possess this
pathogenic power ab initio, not due to any peculiar condition
of growth.

Amongst the legion of different species of micrococci and
bacilli occurring in putrid substances, the great majority are
quite harmless ; when introduced into the body of an animal
they are unable to grow and to multiply, and therefore are
unable to produce any disturbance. But some few species
there are which, although ordinarily growing and thriving in
putrid substances, possess this power, that when introduced
into the body of a suitable animal they set up here a specific
disease.

One of the best studied cases is that of the bacillus an-
thracis. This organism is capable of growing well and
copiously outside the body of an animal, it thrives well
wherever it finds the necessary conditions of temperature,
moisture, and nitrogenous material ; when it finds access to

230 MICRO-ORGANISMS AND DISEASE. [CHAP.

the body of a suitable animal it produces the highly infectious
fatal malady known as anthrax. The micrococcus of ery-
sipelas is now well known through the admirable researches
of Fehleisen to be capable of existence and multiplication
outside the animal body ; it grows well in artificial cultures,
so does the tubercle bacillus of Koch, so does the bacillus
which I described of swine-plague, mentioned in a former
chapter, and so do other micro-organisms. Davaine's
septicaemia in rabbits, Koch's septicsemia in mice, &c., &c.,
cannot be produced by every putrid blood or putrid organic
fluid, only by some, only now and then, i.e., when the
particular micro-organism capable of inducing the disease
is present in those substances, and then only when it finds
access into a suitable animal. Davaine's septicaemia of
rabbits cannot be induced in guinea-pigs, Koch's septicaemia
of mice cannot be induced in guinea-pigs, anthrax bacilli
cannot induce the disease in dogs, and so with the other
micro-organisms.

We conclude then from this that some definite micro-
organisms, although as a rule existing and growing in various
substances of the outside world, have the power when finding
access into the body of a suitable animal to grow and thrive
here also, and to induce a definite pathological condition.
But this power they have ab initio. Those that do not
possess this power cannot acquire it by any means whatever.
Just as there are species of plants .which act as poisons to
the animal body, and other species of plants which, although
belonging to the same group and family, and although very
much alike to the others, have no such power and cannot
acquire such a power by any means, so there are micro-
organisms which are pathogenic while others are quite
harmless. The latter remain so no matter under what
conditions and for how long they grow.

xvii.] SEPTIC AND PATHOGENIC ORGANISMS. 231

I have made a series of experiments with the view to
obtain pure cultivations of definite septic micro-organisms :
various species of micrococci, bacterium termo, and
bacillus subtilis, of which the morphological characters
could with precision be ascertained and which at starting
were tested to be barren of any power of inducing disease.
I have cultivated these in pure cultivations for many
generations, and under varying conditions, and then I have
inoculated with them a large number of animals (mice,
rabbits, and guinea-pigs) ; and to put it briefly, I have not
found that hereby any of them acquired the least pathogenic
power.

CHAPTER XVIII.

VITAL PHENOMENA OF NON-PATHOGENIC ORGANISMS.

As has been stated in a former chapter, all putrefaction of
organic matter is associated with micro-organisms. It is now
generally admitted, because based on a large number of
exact experiments (by Schwann, Mitscherlich, Helmholtz,
Pasteur, Cohn, Burdon Sanderson, Lister, W. Roberts,
Tyndall, and many others), that organic matter kept safe
from becoming contaminated with micro-organisms of the
air, water, and filth, remains free of them, and consequently
of the form of decomposition which is generally considered
as putrefactive ; namely, the changing of proteids into soluble
peptones ; then the splitting up of these into leucin and
tyrosin ; further the decomposition of these and other
crystallisable nitrogenous bodies into comparatively low
compounds. These in their turn by oxidation ultimately
yield ammonia, and its salts and nitrates of inorganic ele-
ments, with the simultaneous development of certain gases,
e.g. ammonia, sulphurreted hydrogen, and other products,
belonging to the aromatic series. The view now generally
entertained is that the organisms cause disintegration of
nitrogenous compounds by withdrawing from them certain
molecules of nitrogen, building up with these their own pro-

CH. xvm.] NON-PATHOGENIC ORGANISMS. 233

toplasm. Similarly carbohydrates and inorganic salts, as
phosphates, potassium, and sodium salts, are dissociated by
them, inasmuch as they require a certain amount of carbon,
phosphorus, potassium, and sodium, for building up their
own bodies. In this process of decomposition certain
alkaloids are produced, the composition of which is not
accurately known, and which are called by the collective
name of ptomaines (Selmi and others). These alkaloids are
known to have a poisonous (toxic) effect when introduced in
sufficient quantities into the system of a living animal. Very
possibly the poisonous property of some articles of food, that
have undergone putrefaction or some unknown kind of
fermentation, is caused by some ferment, the product of
micro-organisms ; (sausage-poisoning, poisoning by bad fish
and other articles). Brieger (Die Ptomaine, Berlin, 1885)
has isolated from putrid proteid materials several alkaloids
and has studied more accurately their toxic properties.

Gaspard, Panum, Bergmann, Billroth, Burdon Sanderson,
and many others had already shown, that by putrefaction of
animal substances, a substance can be obtained — the septic
poison or sepsin — by various chemical processes which in
themselves are destructive of every living micro-organism ;
this substance injected into the vascular system of animals,
especially dogs, in sufficient quantities, produces a marked
febrile rise of temperature, and is capable of causing death
with the symptoms of acute poisoning, e.g. shivering, vomiting
and purging, spasms, torpor, collapse and death. After
death is found severe congestion and haemorrhage of the
lungs and intestine, particularly the duodenum and rectum ;
haemorrhage in the pleura, pericardium, peritoneum, and
endocardium. This putrid infection, identical with poisoning
by ptomaines, leads to death in twelve to twenty-four hours,
or even less. On injecting smaller quantities only a febrile

234 MICRO-ORGANISMS AND DISEASE. [CHAP.

disturbance is noticed, severe symptoms and death only
following after injection of considerable quantities, such as
several cubic centimetres of putrid fluid. There is a priori
no reason why something like putrid intoxication should not
occur as a pyaemic infection in the human subject ; if, for
instance, at an extensive wound, e.g. after amputation of a
limb, a large surface of suppurating tissue is established, on
which, as is well known, numbers of putrefactive organisms
are capable of growing, it is possible and quite probable, that
here these organisms produce the septic poison, which when
absorbed into the system in sufficient quantities produces
septic intoxication. From this affection septicaemia proper,
due to absorption of a specific organism by a small open
wound or a vein, which increases within the body, and
therefore is a living, growing, and self-multiplying, en-
tity producing septicaemia, must be carefully distin-
guished.

These putrefactive processes must be distinguished from
certain fermentative processes, in the course of which by
introducinga definite micro-organism — zymogenic organism —
into a definite substance, definite chemical products are pro-
duced. Thus the torula cervisiae or saccharomyces intro-
duced into a solution containing sugar, produces alcoholic
fermentation, i.e. oxidation and splitting up of sugar into
alcohol and carbonic acid.

The bacterium lactis introduced into substances containing
lactic sugar, moist, or grape sugar, produces by oxidation a
conversion of the sugar into lactic acid and carbonic acid (?).
A micrococcus (see a former chapter) produces, according to
Pasteur, the conversion of dextrose into a sort of gum, called
by Bechamp viscose, and recognised by Pasteur as the cause
of the viscous change of wine and beer. The urea in the
urine is converted by the micrococcus ureae (Pasteur) into

xviii.] NON-PATHCGENIC ORGANISMS. 235

carbonate of ammonium. Solutions containing starch,
dextrin, or sugar, infected with the bacillus amylobacter
yield, as mentioned in a former chapter, butyric acid.
The same bacillus converts glycerine (Fitz) into butyric
acid, ethyl-alcohol, &c. Alcohol is oxidised by the
presence of a definite micrococcus (Pasteur) into acetic
acid.

A species of minute bacillus subtilis produces from fats
butyric acid (Pasteur, Cohn), and many kinds of micro-
organisms from pigmentary bodies, e.g. those producing the
blue colour of milk.

What the chemical influence of pathogenic organisms on
animal tissues may be is not yet known ; and even when they
grow outside the body, i.e. in artificial cultures, it is not yet
known what their chemical effect on the nourishing material
is, except that, as is the case with all other organisms, putre-
factive and pathogenic, they continue to grow and multiply
as long as there are present the necessary substances, i.e.
until the medium is " exhausted."

From the enormous number of micro-organisms present
in the outer world, it is clear that the role they play in the
disintegration of higher organic bodies into lower compounds,
as well as in the building up of new compounds, is a very
important one ; to mention but one series, to wit, the enor-
mous importance they have for the vegetable kingdom in
reducing nitrogenous compounds to soluble nitrates of in-
organic salts, so essential to the existence and growth of our
common field crops (Laws).

One of the most interesting facts observed in the growth of
septic micro-organisms is this, that the products of the
decomposition started and maintained by them have a most
detrimental influence on themselves, inhibiting their power
of multiplication, in fact, after a certain amount of these

236 MICRO-ORGANISMS AND DISEASE. [CHAP.

products has accumulated, the organisms become arrested in
their growth, and finally maybe altogether killed. Thus the
substances belonging to the aromatic series, indol, skatol,
phenol, and others, which are produced in the course of
putrefaction of proteids, have a most detrimental influence
on the life of many micro-organisms, as has been shown by
Wernich and others (see Antiseptics).

It is not well known whether in all or in some of these
instances the organisms produce the chemical effects by
creating a special zymogen or ferment, and by this create the
chemical disturbance, or whether they merely dissociate the
compounds by abstracting for their own use certain mole-
cules ; but this much is known, that in consequence of this
chemical disturbance definite chemical substances are pro-
duced. It is quite possible that the pathogenic, like the
zymogenic, organisms have this special character, that if the
soil (animal body) contains a certain chemical substance, they
are capable of growing and thus capable of producing a
definite zymogen or ferment.

In many cases of putrefactive and zymogenic organisms
a definite soil may be capable of furnishing suitable material
for various organisms at the same time ; as a matter of fact
this is what one constantly meets with in ordinary putrefac-
tion of vegetable and animal matter, which teem with various
species of micro-organisms. But as a rule it will be observed
that one species is more apt to find a suitable soil in this
substance than others ; and then it will be found that this one
organism, above all others, grows more numerously ; and when
it has done growing, that is, when it has exhausted its own
peculiar pabulum, another organism, not dependent on this,
but on some other substance present, makes a good start
and multiplies accordingly. Thus one finds constantly, that
a fluid, supposing it contains a variety of proteids, carbo-

xvili.] NON-PATHOGENIC ORGANISMS. 237

hydrates, and salts, having become infected with micrococcus,
and various species of bacilli, in the first days or weeks
chiefly micrococci are present ; afterwards when the micro-
cocci have done multiplying and sink to the bottom of the
fluid, this latter gradually becomes filled with a variety of
bacilli. Or if micrococcus and bacterium termo be sown at
the same time in a suitable fluid, we find that at first only
the bacterium termo increases rapidly ; afterwards, when this
has ceased multiplying, the micrococcus takes the field. In
still other cases, as in putrid blood, exudation fluid, par-
ticularly in putrefying solid parenchymatous or other
substances, various kinds of micro-organisms grow on
simultaneously in different parts of the material.

The same holds good for the zymogenic organisms. A
solution containing sugar is a very fit soil for saccharomyces ;
when this has exhausted the material, i.e. when all the sugar
has been split up into alcohol and carbonic acid, the latter
escaping as gas, then the material is ready for the organisms
capable of converting the alcohol into acetic acid. The two
kinds of organisms may be, however, growing at the same
time, the saccharomyces leading.

Septic or putrefactive organisms then, like zymogenic and
pathogenic organisms, are c&teris paribus dependent for
their growth on the presence of the suitable nourishing
material. And, as we have seen, they differ materially from
one another in this respect; while putrefactive organisms
find in almost all animal and vegetable fluids the substances
necessary for nutrition, the zymogenic and pathogenic
organisms are much more limited in these respects. Where
there is no alcohol present the organisms producing the
acetic acid fermentation cannot grow; where there is no
sugar or a similar substance present the saccharomyces
cannot grow, and so also a particular pathogenic organism —

238 MICRO-ORGANISMS AND DISEASE. [CHAP.

the bacillus anthracis — cannot grow in the living tissues of
the living pig, dog, or cat, but grows well in those of rodents,
ruminants, and man; the bacillus of swine-plague grows
well in the pig, rabbit, and mouse, but not in the guinea-pig
or man.

Septic organisms differ also from pathogenic organisms in
this, that the former are capable of growing in fluids contain-
ing only simple nitrogenous compounds, e.g. tartrate of am-
monia, whereas pathogenic organisms require more complex
combinations, proteids, or allied nitrogenous substances.
Thus, for instance, in Cohn's and Pasteur's fluids septic
micrococcus, bacterium, and bacillus grow well and copiously,
but pathogenic organisms absolutely refuse to grow in them ;
even bacillus anthracis, which appears the least selective,
cannot make a start in it. Some organisms, e.g. tubercle-
bacilli, require the most complex nitrogenous compounds ;
they refuse to grow, for instance, in broth in which anthrax-
bacilli, bacilli of swine-plague, micrococcus diphtheriticus
and erysipelatous can grow well.

All septic and zymogenic organisms properly so-called,
and described in former chapters, differ in this essential
respect from pathogenic organisms, that the former two
absolutely refuse to grow in the living tissues of a living
animal.

As was stated in former chapters, it is not at all uncommon
to find masses of micrococci in tissues which during the life
of the subject have become dead or necrotic, or so severely
changed by inflammation or otherwise that they may be con-
sidered as practically dead. In the diseased, necrotic
intestine, the liver, the spleen, in abscesses, in the subcu-
taneous, submucous, or parenchymatous tissues, masses of
micrococci have been noticed which in no way bear any
intimate relation to disease, merely finding in the dead or

xviii.] NON-PATHOGENIC ORGANISMS. 239

severely diseased tissues a suitable nidus for their growth
and multiplication. But they may be present even in
organs which show no severe disorganisation ; thus, for
instance, in fatal cases of small-pox, typhoid fever, pyaemia,
even infantile diarrhoea, masses of micrococci may be found
in and around the blood-vessels in the liver and spleen. In
all these cases the micrococci are capable of growing,
because owing to the severe general disorder these tissues
have before the actual death of the patient lost their
vitality, and, consequently, are unable to resist the immigra-
tion and settlement of the micrococci. Of the same
character are the masses of bacilli one meets with some-
times in the intestinal wall, liver, and mesenteric glands
after death from severe disorder of the bowels, e.g. typhoid
fever and dysentery. I cannot for a moment accept the
view of Klebs and Koch, that the presence of the bacilli
mentioned in a former chapter necessarily stands in any
causal relation to typhoid fever, seeing that they are not
constant, and particularly that they are found in organs
directly connected with the intestines, which we know are
in this disease in an intense state of disorganisation.

The question arises : Where do the micrococci and
bacilli come from which are thus capable of settling in a
disorganised • tissue even during the life of the subject ?
There can be no doubt that, as regards the intestinal wall,
the mesenteric glands, the liver, and the spleen, the organisms
could readily, in cases of severe disorganisation of the
intestine, immigrate from the cavity of the bowel, where
they are normally present, into the wall of the intestine, and
moreover be absorbed together with the products of dis-
organised tissue into the mesenteric lymphatic glands, the
liver, and the spleen. Further, it is not difficult to explain
that if a focus of inflammation or necrosis be set up at

240 MICRO-ORGANISMS AND DISEASE. [CHAP.

various internal places in consequence of emboli carried
from an inflammatory focus to which micrococci or bacilli
from the outer world have access, e.g. the skin, alimentary
canal, respiratory organs, these internal places or metastases
would harbour the same organisms, and as soon as disintegra-
tion— abscess, caseation, or necrosis — takes place in these
metastases, also the imported organisms would multiply to a
great extent, the tissue being shut out from the circulation
and practically dead.

All this I say is not difficult of explanation if we bear in
mind that the products of an inflammatory focus to which
organisms have ready access from the outer world become
themselves a ready nidus for these organisms ; and when
some of these products are absorbed and taken into the
general circulation to act as emboli and thus to set up
inflammation in distant regions, the organisms, embedded
in and shielded by those products from any destructive
action the living blood in the circulation may be capable of
exerting, are thus transported to the new foci of inflamma-
tion and disintegration, resulting from the emboli. All this
is self-evident, and does not require more proof than what
is already known, and it follows from such considerations
that the presence of micrococci and bacilli in the tissues of
internal organs, in severe cases of disease, when some of the
organs become disorganised before the actual death of the
body, or in secondary foci of severe inflammation and
necrosis, may have no connection whatever with the original
cause of the disease or necrosis, but may be, and probably is,
simply due to a transportation and immigration of non-
pathogenic putrefactive organisms.

A most striking case of this kind I met with in mice dead
of swine-plague ; the bowels were severely inflamed, and in
the liver there were present necrotic patches, an almost

xviii.] NON-PATHOGENIC ORGANISMS. 241

constant symptom of the disorder ; in such necrotic patches
the capillary blood-vessels are sometimes, not always, found
distended and plugged with the zoogloea of putrefactive
micrococci, which have nothing to do, specifically, with the
real disease (see a former chapter).

The cavity of the alimentary canal, small and large
intestine, especially the latter, contains under normal con-
ditions innumerable masses of putrefactive micro-organisms.
These being much smaller than chyle-globules, must of
necessity become as easily absorbed as the latter by the
lacteals, and by these are carried into the general circulation ;
but being putrefactive they are unable to resist the action of
the normal blood, and therefore in the healthy condition
perish. But if there be in any part a focus of disorganisation
they can settle there and propagate, provided they get there
through the blood in a living condition. Many experiments
prove that they cannot pass unscathed through the normal
healthy blood, and therefore it is not probable that they
would reach such a focus in a living state ; but let them be
well inclosed in a solid particle, say of disorganised tissue,
and then carried through the vascular system, and we can
quite understand that in this state, i.e. in and with that
particle, they may reach the distant focus in a living state,
and if in this focus the conditions are favourable for their
growth, e.g. if there is inflammation and necrosis, we may
expect them to multiply accordingly.

It is now admitted by most competent observers that in
the healthy and normal state the blood and tissues contain
no micro-organisms whatever, and that the assertions to the
contrary are due to errors in the experiment, i.e. to accidental
contamination. I will on this point merely refer, amongst
many others, to the observations of Watson Cheyne 1 and
1 Pathological Transactions, vol. xxx.

R

242 MICRO-ORGANISMS AND DISEASE. [CHAP.

F. W. Zahn.1 Consequently it cannot be maintained that if
in any focus of disintegration micro-organisms make their
appearance they are derived from those normally present ;
we must, on the contrary, assume that putrefactive organisms
can be imported from parts connected with the outer world
into distant localities in which disorganisation of tissues has
taken place.

It is clear from the foregoing that after death micro-
organisms will readily immigrate into the various tissues, and
in this respect those organs situated near places where under
all conditions micro-organisms exist will be the first to be
invaded by them; e.g. the lungs, from micro-organisms
present in the bronchi and air-cells derived from the outer
air, the walls of the alimentary canal, the mesenteric glands,
the peritoneal cavity, the liver and the spleen. The bacilli
possessed of locomotion are particularly to be mentioned in
this respect, but other non-motile bacilli and micrococci also
find their way into these organs ; thus Koch 2 saw only a
few hours after death bacilli (non-motile) present in the
blood of the arteries of a healthy person who had died by
strangulation.

Quite recently Bizzozero has shown (Centralbl. f. d. med. Wiss. No.
45, 1885) that in perfectly normal and living rabbits the lymphatic
tissue of the Peyer's glands of the intestine contains bacteria, either free
between the lymph-cells, or aggregated in groups within the protoplasm
of certain large lymph-cells. I have myself had opportunity to verify
this statement. In a perfectly healthy rabbit immediately after killing
it, the peritoneum and outer muscular coat is stripped off, then with
a clean and sterilized scalpel a scraping is made from the lymphatic
tissue of the deepest part of a Peyer's gland, of course care being
taken that the mucous membrane lining the intestinal cavity remains
unopened. With the scraping cover-glass specimens are then made,

1 Virchow's Archiv, vol. xcv.

2 Pathogene Micro -organismen.

xviii.J NON-PATHOGENIC ORGANISMS. 243

dried, stained with gentian-violet, and mounted. In such specimens
large cells are found, the protoplasm of which is filled with beautiful
small bacilli. ' When, therefore, speaking of the occurrence of bacilli
or other bacteria in the tissue of the lymphatic glands or other parts of
the wall of the bowels of a person dead of some disease or other, it is
necessary to bear in mind the above facts, viz., that already under
perfectly healthy conditions, even during life, bacteria can make their
way from the internal cavity into the tissue of the intestinal wall.

Metschnikoff has pointed out (Virchow's Archiv, vol. 97, 3, p. 502)
that amoeboid cells in the blood and connective and lymphatic tissues
are capable of embodying bacteria introduced into the tissues, and he
called those cells phagocytes. "While there is no doubt that bacteria
like other granules and particles can be swallowed by amoeboid cells, it
is manifestly going too far to say, as Metschnikoff is inclined to do,
that the phagocytes play an important part in neutralizing the action of
pathogenic bacteria introduced into the blood and tissues by quickly
swallowing and destroying those bacteria. Where in a tissue pathogenic
bacteria find the suitable conditions for growth and multiplication, they
can do successful battle against the amoeboid cells, but where those
conditions do not obtain, the bacteria linger and die, and like other
particles can be swallowed up by the amoeboid cells. That the presence
of bacteria in the protoplasm of amoeboid cells does not indicate that
the former are being removed or destroyed and their action neutralized
by the latter, but exactly the contrary, is proved in Koch's septicaemia
of mice, in bovine tuberculosis, in leprosy, and in other diseases
detailed in former pages (see Chapter XI.).

The assumption that where the leucocytes (phagocytes) are capable
and sufficiently numerous to take up and destroy the pathogenic bacteria,
no disease follows and immunity is the result, is contrary to some
elementary facts, e.g. however small the number of anthrax bacilli or
tubercle bacilli introduced into the blood of a susceptible animal,
infection sets in with certainty, while no infection follows in an animal
unsusceptible to the disease however large the dose of these bacilli.
No one would be bold enough to say that the white blood-cells differ
in the two animals in numbers and characters. Or to put it more
strongly : it would be absurd to say that in a sheep which has passed
through a mild form of anthrax, and as is well known has hereby become
unsusceptible to a second attack, the leucocytes have altered in number
and character, so that before the first attack they have been unable to
swallow up and destroy the anthrax bacilli, but by the first attack had
become endowed with this new power.

R 2

CHAPTER XIX.

VITAL PHENOMENA OF PATHOGENIC ORGANISMS.

As has been stated in the preceding chapter, the specific
micro-organisms have the great differential character that
they are capable of existing and propagating themselves in
healthy living tissues. In those species in which the com-
plete series of proofs has been furnished to establish the fact
that the micro-organisms are intimately associated with the
cause of the malady (e.g. malignant anthrax, tuberculosis,
swine-plague, erysipelas), in which it has been shown beyond
doubt : (a) that an animal suffering from the malady contains
in definite distribution the particular micro-organism, (b)
that the micro-organisms, cleared by successive artificial
cultures from any adhering hypothetical chemical virus, when
introduced into a suitable animal produce the malady, (c)
that every such affected animal contains the micro-organism,
in the same distribution and relation to the diseased organs
as the original animal dead after disease — in those instances,
I say, the only way of understanding the effect of the micro-
organisms is to assume, what is actually the case, that the
micro-organisms introduced into the living tissue go on
multiplying, and directly or indirectly, i.e. themselves or
by their products, as will be stated below, produce certain

CH. xix.] PATHOGENIC ORGANISMS. 245

definite disorders in the different parts. In the most
favourable cases (anthrax, tuberculosis), a single organism
introduced into a suitable locality in the animal body will be
capable of starting readily a new brood. But in other cases
it is necessary that an appreciable number of the organisms
be introduced in order to start a brood. This was the case
in some of the septicaemic processes in rodents studied by
Koch.1 The period between the time of introduction of
the organism into the body (blood, skin, or mucous mem-
branes, subcutaneous tissue, lungs, alimentary canal) and
the production of the new brood large enough to produce a
definite effect locally or generally, corresponds to the
incubation-period of the disease, and, as is well known,
there is in this respect a great difference in the different
diseases. Thus in anthrax the introduction of the bacilli
into the subcutaneous tissue of a suitable animal is followed
after from sixteen to twenty-four hours or more by a local
effect (oedematous swelling), and a few hours after by general
constitutional illness, when bacilli can as a rule be found in
the blood. On the other hand, in tuberculosis after the
introduction of the bacilli tuberculosis into the subcutaneous
tissue, the nearest lymph-glands show the first signs of
swelling and inflammation after one week or even later, and
the general disease of the internal viscera does not follow
until one, two, or more weeks have elapsed. This is also
borne out by observations of the behaviour of these bacilli
in artificial cultures ; whereas a suitable material inoculated
with the bacillus anthracis and kept at the temperature of
the animal body (38° to 39° C.), shows already after twenty-
four hours a good crop of the bacilli ; in the case of the
tubercle-bacilli the first signs of a new brood are not noticed,
as Koch has pointed out, and as I have had in several

1 Infectionskrankheiten, loc. cit.

246 MICRO-ORGANISMS AND DISEASE. [CHAP.

instances occasion to verify, before ten to fourteen days have
passed.

One of the most important points, and the most difficult
of comprehension, is this power of the pathogenic organisms
to resist the influence of the healthy tissues of the living
animals, a power which we said above is not possessed by
the non-pathogenic organisms. A careful analysis shows at
the outset that this power of the pathogenic organisms is
not possessed by them indiscriminately, for while a particular
species is in some animals capable of overcoming the
influence of the living tissue, i.e. to multiply and to produce
the particular disease, in other animals it is not capable of
doing this, and hence the animal remains unaffected — it is
said to be not susceptible to the disease. Thus, for instance,
the bacillus anthracis when introduced into a human being
or a herbivorous animal, is capable of multiplying and of
producing anthrax, whereas in carnivorous animals and even
in the omnivorous pig it is not capable of doing so. Or
again, the bacillus of swine-plague while capable of produc-
ing the disease in swine, rabbits, and mice, is not capable of
doing so in man, bird, the guinea-pig, or carnivorous animals.
Now, where are we to look for this difference in behaviour ?
The tissues and juices of a pig when obtained as infusion or
otherwise are just as good a nourishing material for the
bacillus anthracis as the tissues and juices obtained from a
herbivorous animal ; artificial cultures of the former and of
the latter behave in exactly the same manner, both as regards
copiousness of growth and virulence of the bacilli. Again,
artificial cultures of the bacillus of swine-plague made in
juice of the tissues of the guinea-pig or fowl are exactly the
same as those made of the juice of the tissues of a rabbit
or pig. The tissues, therefore, per se cannot be said to possess
any inimical action on the organisms. The living condition

xix.] PATHOGENIC ORGANISMS. 247

per se can also not have this power, since we see that the
power to overcome the influence of the living tissue is
precisely the great distinguishing character of pathogenic
organisms. There remains, therefore, only one thing, that
is that there is something or other present in a particular
tissue to which this latter owes its immunity, and this some-
thing must of necessity be connected with the tissue while
alive, as we said before. Now, the life of the tissue in the
pig cannot be different from that of the mouse, if by life is
understood the function of the tissue, the connexion with
the vascular and nervous system, and all the rest of it. The
subcutaneous connective tissue has no different function, no
different relation to the vascular and nervous system in the
pig from what it has in the mouse, and nevertheless we find
that it behaves so differently in the two cases towards the
bacillus anthracis. This something then, which inhibits the
growth and multiplication of the bacillus anthracis in the
tissue of the pig but not in the mouse, must be something
which, although dependent on the life of the tissue, is not
identical with any of the characters constituting the life of the
tissue, but must be some product of that life. To assume
then, as is done by some observers, that the living state of
the cells per se is the inhibitory power does not cover the
facts, as we have just shown. The most feasible theory
seems to me to be this, that this inhibitory power is due to
the presence of a chemical substance produced by the living
tissues. It does not require any great effort to conceive,
and it does not seem at all improbable, that the blood and
tissues of the pig contain certain chemical substances which
are not present in the mouse, substances which like so many
others chemistry is not yet capable of demonstrating. But
that there exist vast and gross differences in the chemical
constitution of the blood and tissues of different species

248 MICRO-ORGANISMS AND DISEASE. [CHAP.

of animals there can be no reasonable doubt ; it is a fact
with which physiological chemistry is quite familiar.

We arrive then, after all this, at the conclusion that owing
to the presence in the blood and tissues of particular chemical
substances, present only during life, and a result of the life of
the tissue, the organisms in a particular case cannot thrive and
produce the disease. And further, that for each particular
species of organism there is a particular chemical substance
required to exert this inhibitory power, for, as we have seen,
while the anthrax-bacillus is not capable of thriving in the
pig, it does well in the guinea-pig, while the bacillus of swine-
plague thrives well in the pig, it does not in the guinea-pig.
The incapability of non-pathogenic organisms to thrive in
healthy living tissues would on this theory be explained
by the assumption that these chemical substances present in
every healthy living tissue are inimical to all putrefactive
organisms.

What we have said hitherto refers only to the healthy
living tissues. It is quite possible to imagine that owing to
the presence of a particular chemical substance in the healthy
living tissue, a pathogenic organism is not able to thrive in
a particular animal ; but under certain abnormal conditions,
when for instance owing to a diseased or altered state of the
tissue that substance is absent, the organism might be enabled
to exist and multiply and to produce the disease. Supposing
it to be true that a person so long as he is healthy and well
is able to successfully withstand the growth of a particular
pathogenic organism, he is then unsusceptible to the disease ;
but we can understand that if the alimentary canal or the
lungs be diseased, then the organism passing into the bowels
by ingestion or into the lungs by inspiration would find there
a tissue in which the necessary inhibitory substance, present
in the healthy state, might be absent, and the organism

xix.] PATHOGENIC ORGANISMS. 249

would be capable of thriving and of producing the
disease.

The next point to consider is the relation of the specific
micro-organism to the essence of the disease, or in other
words the question whether the organism itself is the virus or
whether this latter is the product of the former, something
in the same way as the septic ferment ir, the product of the
putrefactive organisms 1

That the virus in the infectious diseases is intimately con-
nected with the organisms is proved by the fact that the
virus introduced into the body multiplies ad infinitum ; for
instance, in anthrax or tuberculosis after the introduction of
an infinitesimal dose, we find the disease (malignant anthrax
or general tuberculosis respectively) sets in in its virulent
form ; in the first case every drop of blood teems with the
bacillus anthracis ; in the second (tuberculosis) every tuber-
culous caseous particle in the lymph -glands, lungs, spleen
and liver contains the bacilli ; in both instances crops of the
bacilli are produced in the afflicted body, and every particle
of the tissue containing the bacilli is capable of starting the
disease when introduced into a fresh subject. Moreover the
artificial cultures of the pure bacilli are possessed of the
same pathogenic power. The same holds good for leprosy,
for erisypelas, for swine-plague, &c. So that the proposition
that the organisms are intimately connected with the virus
must be considered as well established.

But even after this it remains an open question whether
the organism is identical with the virus, or whether the
organism is concerned in elaborating the virus — a sort of
ferment ; and further, whether the virus being the latter's
product, is obtainable apart from the organism.

Let us start with the proposition that the virus is a product

250 MICRO-ORGANISMS AND DISEASE. [CHAP.

of the organism, a sort of non-organised ferment, but not
the organism itself, although this latter is essential for the
creation of the latter.

Inoculating a few bacilli anthracis into the subcutaneous
tissue of a suitable animal, e.g. a guinea-pig, we find after
twelve to twenty-four hours the first indications of illness,
consisting in a local swelling and a general rise of the body-
temperature. At this time there are present in the local
swelling bacilli, but only in small numbers ; in the blood the
bacilli are very scarce indeed, so scarce that it is difficult to
meet with one bacillus in an appreciable quantity of blood.
By this time then the bacilli could not have produced the
change by their " numbers " alone. Immediately before
death, sometimes some hours, we find in most instances the
blood teeming with the bacilli, but this is by no means in all
cases ; I have seen a considerable number of deaths from
typical anthrax in the mouse, guinea-pig, rabbit, and sheep,
occurring from forty-eight to sixty hours after inoculation, in
which the number of bacilli of the blood and tissues was
extremely small; they were present, but only here and
there was there one to be found. That the bacilli are present
some hours before death in the shape of spores, as has been
maintained by Archangelski, I have disproved in a former
chapter. In those cases in which the bacilli are scarce even
in articulo mortis and immediately after death, the scarcity is
not due to the bacilli having already degenerated, since the
degenerating bacilli are not noticeable in any way in these
instances. It remains then to assume that death occurs in
these cases not owing to the presence of the bacilli in num-
bers ; that is to say, that it is neither owing to their appro-
priating from the blood-corpuscles the available oxygen
necessary for their multiplication (Bellinger) and thus pro-
ducing death by asphyxia, nor to their mechanical effect in

xix.] PATHOGENIC ORGANISMS. 251

plugging up the capillaries of vital organs, a theory upheld
by some observers from the fact that in most cases the
capillaries appear filled with the bacilli, and in some cases
in extensive regions, lung, kidney, and spleen, the capillaries
are almost occluded by the bacilli. We must assume, then,
that although as a rule immediately before death all the con-
ditions are present to enable the bacilli to multiply readily
and to produce a large crop, this is not necessarily connected
with the cause of death, being in fact in consequence of the
animal being in articulo mortis ; but that the immediate
cause of death is the chemical alteration produced by the
bacilli in the blood and tissues. For producing this effect it
is not necessary to have more than a certain number of the
bacilli. As soon as this number is reached death follows.
The same may be said of other pathogenic organisms.
Thus for instance in the case of tubercle-bacilli, after the in-
troduction of these into the subcutaneous tissue of a guinea-
pig, multiplication takes place, and after they reach a certain
number,, the nearest lymph-glands become swollen and
inflamed and then caseous : but this stands in no relation to
the number of the bacilli, for in some instances the micro-
scopic examination reveals only very few bacilli, they are
scattered in very small numbers over very wide areas. And
the same is observed in the tuberculous deposits of the
internal organs. In some of them the bacilli are exceedingly
scarce, while in others neither more nor less advanced they
are numerous. Here also we must assume that as soon as
a certain, perhaps even small, number of the bacilli have
been produced, the chemical effect produced is sufficient to
be the cause of a certain pathological change. In glanders
the nodules in the skin and lung reveal sometimes, even
under the most careful examination after approved methods,
the presence of but very few bacilli. In swine-plague, in

252 MICRO-ORGANISMS AND DISEASE. [CH. xix.

the lungs, which in severe cases are enormously affected,
sometimes only very few of the pathogenic organisms can
be discovered. It follows then that the pathological con-
dition brought about by the organisms is not due to the
direct action of their numbers ; but is an indirect sequence,
brought about by definite chemical alterations in the blood
or tissues, as the case may be.

In this we may assume two theories as possible ; (a) It is
possible that these chemical effects are produced by the
presence and growth of the organisms, as truly as in the
alcoholic fermentation of sugar the alcohol produced is a
result of the presence of the yeast ; this change is only in
so far a product of the organism as this, in its multiplication,
assimilates some molecules of carbon and hydrogen, which
it abstracts from the sugar, and in consequence of which the
sugar yields alcohol ; but it is not, as it were, a secretion of
the organism, a special ferment, (b) But it is likewise
possible that the organism elaborates a special ferment,
which, after a certain amount has been produced, sets up the
particular pathological changes. From these considerations
it follows that the virus cannot be considered independent
of the organism ; we cannot assume that the two can have
a separate existence ; for, as we have just now shown,
the most feasible assumption, and the one borne out by
observation, is that owing to the multiplication of the
organisms, certain chemical changes are produced in the
blood and tissues, or that a special ferment is created, which
sets up the anatomical changes characteristic of the particular
disease.

CHAPTER XX.

VACCINATION AND IMMUNITY.

WE have in the foregoing chapter tried to show that owing
to the presence in the normal blood and tissues of a living
animal of some chemical substances varying in the different
species and inimical to particular pathogenic organisms, the
latter when introduced into the tissues of the particular
species, cannot thrive, and that it is for this reason that the
animal is not susceptible to the corresponding disease. Now,
how do we explain the fact that a human being or an animal
having been once the subject of a particular infectious disease,
becomes thereby in some cases unsusceptible to a second
attack ? The oldest and perhaps the most favoured theory to
explain this immunity is that which assumes that during the
first attack the organisms growing in the body consume, or
are instrumental in eliminating or destroying, some chemical
compound necessary for the existence and multiplication of
the organism. As soon as this substance has become con-
sumed or destroyed the organisms cannot further multiply,
and therefore the disease ceases ; and further, that owing to
the subsequent absence of this same chemical compound, a
new infection by the same organisms is not possible, i.e. the
individual is protected. Thus this theory puts the case on a

254 MICRO-ORGANISMS AND DISEASE. [CHAP.

level with, say, the relation of the saccharomyces to the
alcoholic fermentation ; as long as a solution contains sugar,
the saccharomyces is capable of multiplying, but as soon as
all the sugar has disappeared as such, i.e. has become split
up into alcohol and carbonic acid, the fermentation ceases,
the solution being now exhausted as regards the sac-
charomyces ; a new charge of saccharomyces put into the
solution is not capable of multiplication. This theory, then,
to explain the immunity, is generally spoken of as the
Exhaustion Theory.

On careful analysis, it will be found that it is not capable
of explaining all the facts of the case. As we mentioned in
a former chapter, cattle inoculated with blood of a guinea-
pig dead of anthrax become affected with anthrax, which,
although not fatal, is nevertheless sometimes very severe.
The animal recovers, and is now, for a time at least, protected
against a second attack. But there is absolutely no ground
for the assumption that if any infusion of the tissues of this
animal were made, the bacillus anthracis sown in it would
not thrive luxuriantly, seeing that bacillus anthracis grows
on almost anything that contains a trace of proteids. Simi-
larly when of the tissues of a guinea-pig, or mouse or rabbit,
dead of anthrax, an infusion is made, and this is used as
nourishing material for bacillus anthracis in artificial cultures,
it is found that these latter thrive splendidly. The same
fact I have observed in the case of swine-plague. There is
then no reason whatever for assuming that, if after one
attack of illness the blood and tissues become an unfavour-
able soil for a second invasion of the same organism, this
should be due to the exhaustion 01 some necessary chemical
compound.

There is another theory, commonly spoken of as the
Antidote Theory (Klebs). According to this, the organisms

XX.] VACCINATION AND IMMUNITY. 255

growing and multiplying in the body during the first attack
produce, directly or indirectly, some substance which acts as
a sort of poison against a second immigration of the same
organism. I am inclined to think that this theory is in har-
mony with the facts. There is nothing known, from the
observations before us, which would negative the possibility
of the correctness of this theory ; nay, I would almost say
all our knowledge of the life of the micro-organisms points
to the conclusion that the different species are associated
with different kinds of chemical processes, and that as a
result of the activity we find different chemical substances
produced.

The different fermentations connected with the different
species of fungi afford striking illustrations of this view.
According to this theory, we can well understand that — just
as in the case of an animal, say a pig, unsusceptible to
anthrax — the unsusceptibility being due to the presence in
the blood and tissues of a particular chemical substance
inimical to the growth of the bacillus anthracis — so also in
the case of a sheep or ox that has once passed through
anthrax — there is now present in the blood and tissues a
chemical substance inimical to the growth and multiplica-
tion of the bacillus anthracis whereby these animals become
possessed of immunity against a second attack of anthrax.

Whether this chemical substance has been elaborated
directly by the bacilli, or whether it is a result of the chemical
processes induced in the body by the bacilli during the first
illness, matters not at all ; it is only necessary to assume
that the blood and tissues of the living animal contain this
chemical substance.

Some observers (Grawitz, &c.) are not satisfied with this
theory, but assume that owing to the first attack the cells of
the tissues so change their nature that they become capable

256 MICRO-ORGANISMS AND DISEASE. [CH. xx.

of resisting the immigration of a new generation of the same
organism. There is absolutely nothing that I know of in
favour of such a theory ; it is impossible to imagine that the
cells of the connective tissues, of the blood and of other
organs, owing to a past attack of scarlatina, become possessed
of new functions or of some new power, as, for instance, a
greater power of oxidising or the like. Connective tissue-
cells, blood-corpuscles, liver-cells, and other tissues are, so
far as we know, possessed of precisely the same characters
and functions after an attack of scarlatina as before.

On the whole then, we may, it seems, take it as probable,
that owing to the presence in the normal blood and tissues
in a living animal of a chemical substance inimical to the
growth of a particular micro-organism, this animal is un-
susceptible to the disease dependent on the growth and
multiplication of this micro-organism ; and further, that in
those infectious maladies in which one attack gives im-
munity against a second attack of the same kind, one attack
produces a chemical substance in the blood and tissues
which acts inimically to a new immigration of the same
organism ; hence the animal becomes unsusceptible to a
new attack, or is " protected." This is not the case with all
infectious maladies, for, as is well known, in a good many
instances a single attack does not protect against a second ;
and, as is also well known, a first attack may protect but
only for a limited period, or for a period greatly differing in
different individuals. All this would be explained by our
theory in the same way as it is explained by the other
theories; viz., when one attack does not protect, no inhibi-
tory chemical substance has been produced ; while in those
diseases in which one attack does protect only for a limited
period, the necessary inhibitory substance has only lasted
for a limited period, and so on.

CHAPTER XXI.

ANTISEPTICS.

IN former chapters we. have on several occasions men-
tioned that a variety of substances and conditions are
capable of exerting a detrimental influence on the life and
growth of micro-organisms. Amongst these are — The
presence of certain substances in the nutrient soil, the
temperature, and some chemical products, such as those
belonging to the aromatic series, phenol, indol, skatol, &c.
The presence of certain substances in the nourishing
material is, as we have seen, an essential condition, cceteris
paribus, for the growth and multiplication of micro-organisms.
Thus pathogenic organisms do not thrive in an acid medium,
they cannot thrive if proteids or allied compounds and cer-
tain inorganic salts are absent : putrefactive and zymogenic
organisms, on the other hand, or, at any rate, some of them,
are capable of thriving well in acid media (e.g. the bacillus
subtilis in acid hay-infusion, the micrococcus ureae in acid
urine). Further, many (not all) pathogenic organisms can-
not thrive unless they are exposed to a certain degree of
warmth ; they thrive best at blood-heat, while putrefactive
and many zymogenic organisms thrive well at ordinary
temperatures, though of course their growth is more rapid

s

258 MICRO-ORGANISMS AND DISEASE. [CHAP.

at higher temperatures, such as 30° to 38° C. Heat above
50° or 60° C. arrests the growth of and even kills many
organisms, except the spores of bacilli, which, as we find on
a former page, survive even when exposed to the tempera-
ture of boiling water for several minutes. The presence of
carbolic acid, phenol, thymol, salicylic acid, perchloride of
mercury, &c., restrain even when in great dilution the growth
of micro-organisms.

In any inquiry into the influence of one substance or
another on micro-organisms it is necessary to bear in mind
that the influence of certain conditions on the micro-
organisms may be a twofold one : (i) the condition may be
unfavourable to the growth of the organism in question, and
(2) the condition may be fatal to the life and existence of it.
The second condition involves, a fortiori, the first ; but the
reverse is not the case. Owing to the failure to distinguish
between these two propositions a great deal of confusion has
arisen on the subject. One hears constantly this or that
substance is an " antiseptic," meaning by this a substance un-
favourable to the growth of micro-organisms, or a substance
is a " germicide," implying by this that this substance kills the
organisms ; but when one comes to analyse the observations
that are said to establish this reputation for a particular sub-
stance, one finds that the substances in question have really
only a restraining effect on the growth of the organisms.

jBy sowing any micro-organism into a nourishing medium,
to which has been added a certain substance (e.g. carbolic
acid to the amount of i per cent.), and exposing this
medium to the conditions of temperature, moisture, &c.,
otherwise favourable to the growth of the organism, if we
find that after the lapse of a due period the growth is
retarded or altogether inhibited, the conclusion is drawn
that this substance (viz., the carbolic acid of i per cent.) is

XXL] ANTISEPTICS. 259

a germicide. There is nothing more fallacious than this
method of reasoning ; a great many micro-organisms can be
exposed to a i per cent, solution of carbolic acid for hours
without in the least being affected, for on being then
transferred to a suitable nourishing medium they grow and
thrive well. Similarly by placing the spores of bacillus
anthracis in a proteid medium containing perchloride of
mercury of the strength of i in 300,000, it is found (as
Koch has shown) that the spores are absolutely incapable of
germinating. But if from this the conclusion is drawn, that
perchloride of mercury of the strength of i in 300,000 is
a germicide, I should most strongly dissent, for perchloride
of mercury of o'i per cent, is not a germicide for all
spores any more than vinegar ; for on placing the spores
of bacillus anthracis in a proteid medium, to which so much
vinegar or any other acid has been added as makes it
decidedly acid, ' it will be found that the spores do not
germinate.

In order to pronounce a certain substance a germicide in
the strict sense of the word, it is necessary to place the
organisms in this substance for a definite time, then to
remove them thence, and to place them in a suitable
nourishing medium ; if they then refuse to grow the conclu-
sion is justified that the exposure has injured or destroyed
the life of the organisms. In the case of pathogenic
organisms a substance to be pronounced a germicide must
be shown to have this power, that when the organism is ex-
posed to the substance and then introduced into a suitable
artificial medium it refuses to grow; and it must also be
shown that when introduced into a suitable animal it is in-
capable of producing the disease which the same organism,
unexposed to the substance in question, does produce,

I have made a good many observations on the influence

s 2

260 MICRO-ORGANISMS AND DISEASE. [CHAP.

of antiseptics on micro-organisms, both putrefactive and
pathogenic, and I have found that many assertions hitherto
made on this subject, treated in the above light, are abso-
lutely untrustworthy and erroneous.

Various species of putrefactive micrococci, bacterium
termo, bacillus subtilis, various pathogenic micro-organisms,
as bacillus anthracis, bacillus of swine-fever, absolutely
refuse to grow in media to which is added phenyl-propionic
acid, or phenyl-acetic acid, to an amount so small as i in
i, 600 ; but if the same organisms are exposed to these
substances in much stronger solutions, i in 800, i in 400,
or even i in 200, and then transferred to a suitable
nourishing material, it is found that they have completely
retained their vitality, they multiply as if nothing had been
done to them. I have exposed the spores of bacillus
anthracis to the above acids of the strength of i in 200 for
forty-eight hours and longer, and then inoculated guinea-pigs
with them, and I found that the animals died of typical an-
thrax in exactly the same way as if they had been inoculated
with pure spores of the bacillus anthracis.

Koch has published a large series of systematic and mo?t
valuable observations l made in testing the influence on
spores of bacillus anthracis of a large number of antiseptics
(thymol, arsenate of potassium, turpentine, clove-oil, iodine,
hydrochloric acid, permanganate of potassium, eucalyptol,
camphor, quinine, salicylic acid, benzoic acid, and many
others), and amongst them he found perchloride of mercury
to be the most powerful, since even a solution of i in
600,000 is capable of impeding, one of i in 300,000 of com-
pletely checking, the germinating power of the spores. To
regard these substances, from these observations, in any way
as germicides for the spores of bacillus anthracis would be
1 MitthdL aus. d. k. Gesundheitsamte, Berlin, 1881.

xxi.] ANTISEPTICS. 261

no more justifiable than to consider weak vinegar as such.
Perchloride of mercury in a solution of i in 300,000 is no
more capable of interfering with the life and functions of the
spores of bacillus anthracis than water or salt solution, for
the spores may be steeped in that solution for any length of
time, and yet on being transferred to a suitable medium they
grow and multiply splendidly, and when inoculated into
rodents they produce fatal anthrax with absolute certainty.
With my friend Dr. Blyth, Medical Officer of Health for
the Marylebone district in London, I have tried the action
of a number of substances in common use as antiseptics (e.g.
Cal vert's fluid, pure terebene, phenol 10 per cent., per-
chloride of mercury o'i per cent.) on the spores of bacillus
anthracis, exposing these in comparatively large quantities of
the above fluids (the two being well mixed) for several hours,
and then inoculating guinea-pigs with them (spores and anti-
septic). The animals died with symptoms of typical anthrax,
the blood teeming with the bacillus anthracis.

These substances cannot therefore be considered germi-
cides for the spores of bacillus anthracis, any more than
water.

In all these inquiries, particularly in those upon patho-
genic organisms capable of forming spores, the influence of
the substances must be judged not merely by their action on
the organisms, but also on the spores ; for, in this very case
of the bacillus anthracis, the bacilli taken from the blood of
an animal dead of anthrax are killed after an exposure of
say ten minutes to a solution of phenyl-propionic acid of the
strength of i in 400, or even i in 800, whereas the spores
of the bacilli (produced in artificial cultures) withstand com-
pletely exposure to this acid of any strength and for any
length of time.

It is not my object to pass here in review all that has

262 MICRO-ORGANISMS AND DISEASE. [CHAI\

been done in this interesting field of research, important
because of its obviously great practical consequence. The
work hitherto done has been enormous, but, I fear, of less
utility than at first sight appears, for in most of it the point
most prominent in the mind of the worker was to ascertain
whether the particular antiseptic, mixed with the nourishing
medium in a solution of definite strength, has or has not the
power of inhibiting the growth of the micro-organisms.
This point no doubt is of some interest, and perhaps of
great interest, but whether a particular substance is an anti-
septic in the proper sense of the word, i.e. whether on ex-
posing the organisms to this substance in a solution of
definite strength and for a definite period, the organisms
become afterwards incapacitated from growing or multiply-
ing ; or still more, whether or not the substance is a
germicide, i.e. is capable of altogether annihilating the life of
the organisms ; these are questions which require special
attention, and represent a wide and rich field of inquiry ;
but, as far as I can see, have received only in very few
instances due attention.

The disinfecting power of dry heat and steam on various pathogenic
and non-pathogenic organisms has been studied by Koch and his
coadjutors {Mittheil. atts. d. k. Gesundheifsamte, Berlin, 1881). Dr.
Parsons has described in the supplement to the fourteenth annual Report
of the Medical Officer of the Local Government Board, 1884, a series of
observations made conjointly with myself on the influence of dry heat
and steam on bacillus and spores of anthrax, bacillus of swine-fever and
bacillus of tuberculosis. Pieces of flannel received the fluids containing
the above organisms (separately), and were then exposed to heat. After
this the organisms were squeezed out of the flannel previously steeped in
salt solution, and this fluid was then injected into suitable animals and
the effect watched, and compared with the result of the inoculation wiih
the same materials not exposed to heat. The results of these experiments
were (/.^.p. 225) : " The spores of bacillus anthracis lost their pathogenic
power after exposure for four hours to a temperature a little over the

xxi.] ANTISEPTICS. 263

boiling-point of water (212° to 216° F.), orfor one hour to a temperature
of 245° F. Non-spore bearing bacilli of anthrax (blood or culture) and of
swine-fever, were rendered inert by exposure for an hour to atemperature of
212° to 218° F.,and even five minutes exposure to this temperature sufficed
to destroy the vitality of the former, and impair that of the latter." The
tubercle bacilli were killed by exposure to dry heat for five minutes.
Exposure to steam at 212° F. is destructive, even after five minutes, to*
all the pathogenic organisms tested, including the spores of bacillus
anthracis.

The action of four solutions, phenylpropionic acid and phenylacetic
acid, of phenylpropionate of soda and of sulpho-carbolate of soda, on
bacillus anthracis, of blood and cultures, sporeless and spore-bearing, of
phenylpropionic acid in conjunction with peptone, of acid reaction
(sulphuric acid) on the life and growth of bacillus anthracis, the action
of phenylpropionic and phenylacetic acid and sulpho-carbolate of soda on
human and bovine tuberculous matter, of the action of chlorine and
sulphurous acid gas on swine-fever virus is fully described in the Report
of the Medical Officer of the Local Government Board, 1884, in a series
of papers by myself, Mr. Laws, and Mr. A. Lingard.

The action of perchloride of mercury, although powerful, is not such as
maintained by Koch (Mittheil. aus. d. k. Gesundheitscimt', Berlin, 1881).
While Koch asserts that this substance in solutions of I in 100,000 has the
power to kill sporeless bacillus anthracis, I find that exposure of the
bacillus anthracis taken from the blood of an animal dead of anthrax to
a solution of perchloride of mercury in water in the proportion of
i in 20, coo or I in 25,000 for one hour does not impair the action of the
bacilli. Solutions of I in 10,000 for thirty minutes' exposure kill the
bacilli. But there exists a difference in resisting power according to the
virulence of the bacilli themselves.

The bacilli of blood of an anthrax animal are capable of growing and
of producing infective bacilli in broth peptone, and in nutrient gelatine to
which perchloride of mercury has been previously added in the propor-
tion of I in 25,000 or I in 30,000. The spores of bacillus anthracis are
capable of germinating and producing good and active crops of bacilli
in broth peptone, and in nutritive gelatine to which perchloride of
mercury has been added in the proportion of I in 25,000 or I in 30,000.
Koch mentions I in 300,000 as inhibiting the power of the germination
of the spores of bacillus anthracis.

The spores of non- pathogenic bacilli and of some pathogenic bacilli
(bacillus anthracis) resist the action of perchloride of mercury in solutions
of i in 10,000 even after exposure for four days.

264 MICRO-ORGANISMS AND DISEASE. [CH. xxi.

Non-pathogenic organisms have a markedly greater resisting power to
phenylpropionic and phenylacetic acid and to perchloride of mercury
than pathogenic.

Koch's comma bacilli of Asiatic cholera and Tinkler's comma
bacilli behave in this respect like non-pathcgenic organisms. Sporeless
non-pathogenic bacilli have a lesser resisting power than non-pathogenic
nnicrococci.

INDEX.

ABRIN, 227

Abrus precatoritTS, 220
Abscess in rabbit, 83
Achorion Schoenleini, 196
Actinomyces, 204
Acute inflammations, 65
Aerobic, 55
Agar-Agar, 24
Air examination, 52
Amylobacter, 114
Anaemia perniciosa, 80
Anaerobic, 55
Anilin dyes, 9
AniLn oil, 9
Antheridia, 201
Anthrax bacillus, 144
Antiseptics, 257
Aromatic products, 236
Artificial cultures, 38
Artificial tuberculosis, 169
Afd, 195

Ascococcus Billrothi, 64
Ascogcnium, 198
Asco.nycetes, 194
Ascosppres, 195
Aspergillus, 197
Attenuation, 158

BACILLUS, general characters of, 96

septic, 108

zvmcgenic, 1 14

chromogenic, 116

pathogenic, 118
Bacterium, general characters of, 88

termo, 89

lactis, 90

lineola, 90

Bacteria, pathogenic, 91
Bacteridia, 144
Basidia, 197
Beggiatoa, 113
Broth, preparation of, 17
Buchner's fluid, 20

CAPILLARY pipette, 29
Caibolic acid, 258
Carpogc nium, 197
Cattle plague, 79
Charbon symptomatic, 1 43
Cheyne's observations, 168
Che lera bacillus, 174
Choleraic diarrhoea, 122
Cladothrix dichotoma, 112
Clathrocystis, 63
Clostndium butyricum, 115
Comma bacillus, 174
Cohn's fluid, 21
Comdia, 194
Cotton-wool, 28

DAVAINE'S septicaemia, 93
Desm bacteria, 96
Diphtheria micrococcus, 72
Diplococcus, 58
Di^pora caucasica, 115
Dumb-bell micrococcus, 57

EHRJ.ICH'S method of staining, 163
Endocarditis ulcerosa, 78
Erysipelas micrococcus, 71

FAVUS fungus, 196
Flasks, sterilisation of, 26
Fcot-and-mouth disease, £7
Foul-brood, 174
Fowl cholera, 94

GELATINE, 22

Gelose, 24

Germicide, 258

Glanders baollus, 128

Glass cell, 47

Gonorrhoea micrococcus, 77

266

INDEX.

HAY ba-illus, 138
Hay infusion, no
Herpes tonsurans, 196
Hot-air chamber, 27
Hydr cele fliud, 20
Hyphae, 194

Nourishing media, 17

fluids, 17

s >lids, 21

preparation of, 17

sterilisation of, 19
Nosema bombycis, 86

IMMUNITY, 251
Incubator, 15
Indol, 236

Infectious diseases, 2
Inoculation, method of, 38

JEQUIRITY bacillus, 221
Jequirity infusion, 223
Jequirity ophthalm.a, 221
Jequirity seeds, 220

KLEBS'S method of cultivation, 39
Koch's malignant nedemu. 140
Koch's meth d of cultivation, 41

inoculation, 45

staining, 163

progressive necrosis, 82

septicaemia of mouse, 118

of rabbit, 84
Koch-Weigert method, 8

LEPROSY bacillus, 136

Lept ithrix, 96

Leptr thrix buccalis, 98

Lister's method of cultivation, 40

CEDEMA, malignant, 140
Oidium albicans, 193
Oi'dium lactis, 195
Oogonium, 201
Oospores, 202
Ostto-nyelitis, 80

PAPAYOTIN, 142
Pasteur's fluid, 21
Pasteur's septicaemia, 140
Pasteur's vaccin charbonner.x, 100
Pathogenic organisms, 2

viial phenomena of, 244
Penicillium, 201
Peptone, 19
Perithecium, 199
Phenol, 236

Pityriasis versicolor, 196
Plasmodium malarias, 126
Pleuro pneumonia, 76
Pneumococcus, 74
Polli lodia, 197

Progressive necrosis in mous; 82
Ptomaines, 233
Puerperal fever, 79
Pyaemia of mouse, 83

rabbit, 83

MALARIA bacillus, 125
Malaria plasmodium, 126
Malignant oedema, 140
Meat extract, 20
Meat poisoning, 122
Microbe of fowl cholera, 94
Micrococcus, 57

general character, 57

chromogenic, 61

pathogenic, 65

septic, 61

zymogenic, 61
Microsporon furfur, 196
Microzyma bombycis, 85
Monas, 63
Mucor, 201
Mycelium, 194
Mycoderma aceti, 90
Mycoderma saccharomyces, 191
Mycosis intest.nalis, 147
Mycothrix, 58

NAGELI'S method of culture, 40
Noma, 128

SACCHAROMYCES, 191
Salmon disease, 203
Saprolegnia, 201
Sarcina, 59, 64
Scarlatina micrococcus, 78
Schiz <mycetes, 54
Septic ferment, 233
Septicaemia of man, 120

mouse, 118

rabbit, 84
Serum of blood, 20
S.Ik-worm diseases, 86
Spherobacterium, 57
Spirillum, 184

septic, 184

pigment, 186

pathogenic, 188
Spirochasta, 185
Sporangium, 194
Spores, general characters, zoc

formation of, 101

germinat.on, 106

of anthrax bacilli, 154
Stomatitis ulcerativa, 126
Sterilisation of instruments, 26

INDEX.

267

Streptococcus, 58
Streptothrix, 112
Swine plague, 131
Syphilis bacillus, 174

TEST-TUBES, 32

Thalltis, 194

Thrush, 193

Torula, 187

Torula form of bacillus, 150

Trichophyton tinsurans, 196

1 ubercul. sis bacillus, 162

Typhoid fever bacillus, 121

micrococcus, 61

VACCIN charbonneux, 160
Vaccination, 253

Vaccinia and Variola micrococcus, 69

Vibrio, 182

Virus, general character of, 249

WATER examination, 49
XANTHINUM bacterium, 91
YEA.ST fungus, 189

ZXXJLCEA, 60

Zooglcea ramigera, 89

Zo^spores, 201

Zymjgen, 236

Z>'inogenic bactwia, gc
bacilli, 114
micrococci, 61

THE END.

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