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HEINEMANN'S

Scientific Ifoanbboofcs

A KNOWLEDGE of the practical Sciences has now
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The fifth volume, 'A Manual of Bacteriology/ by
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MANUAL OF BACTERIOLOGY. ByA.B. GRIFFITHS,

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LONDON: WM. HEINEMANN, 21 BEDFORD STREET, W.C.

^einemann'0 Scientific

A MANUAL OF BACTERIOLOGY

GRIFFITHS

T71TI7ERSIT7

Ibeinemann's Scientific 1ban£>booh0.

A MANUAL OF

BACTERIOLOGY

A. B. GRIFFITHS, Ph.D., F.R.S.E., F.C.S.
IV

TJIIVBRSITT

LONDON
WILLIAM HEINEMANN

1893

BIOLOGY
UBRAKY

46709

TO
ERNEST A. GRIFFITHS, ESQ.

OF HER MAJESTY'S CONSULAR SERVICE IN JAPAN,
MEMBER OF THE ASIATIC SOCIETY OF JAPAN,

ETC. ETC. ETC.

THIS WORK
IS AFFECTIONATELY DEDICATED BY

HIS BROTHER

UHI7BRSIT7

PREFACE

IN preparing this volume I have endeavoured to
meet the requirements of those who are desirous of
obtaining a knowledge of the nature and doings,
for good or for evil, of those minute beings which
are termed microbes or bacteria.

The mystic words ' microbes ' and ' bacteria ' have
been hurled at the popular head with so much
emphasis and so little explanation that it would
not be surprising to find many people living
under the misapprehension that they are minute
' fiery serpents/ which are always on the look-out
for victims, and crawl about them day and night.
Not a few people feel comforted by the knowledge
that microbes, harmless or harmful, belong to the
vegetal rather than to the animal kingdom. Such
knowledge takes away the element of repulsiveness
arising from the notion of microbes being internal
animal parasites or eutozoa.

Although microbes are minute plants, they are
capable of giving rise to some of the most deadly

xii PREFACE

diseases to which human flesh is heir. Conse-
quently, a knowledge of the science of microbes, or
bacteriology, is now incumbent on all medical men,
sanitary engineers, chemists, physiologists, and
biologists ; and even intelligent householders would
be all the better if they had a general knowledge of
the subject detailed in the following pages.

My sincere thanks are due, and are here most
gratefully tendered, to Dr. E. Klein, F.RS. ; Prof.
P. F. Frankland, F.E.S. ; Prof. A. Gautier (of Paris) ;
Prof. L. Brieger (of the University of Berlin);
Prof. C. Tommasi-Crudeli (of the University of
Eome) ; Prof. I. Giglioli (of Portici, near Naples) ;
Dr. Eoux (of the Pasteur Institute) ; Dr. P. Miquel
(of Paris); Dr. T. Lauder Brunton, F.RS.; Mr. W.
Watson Cheyne, F.E.C.S. ; Dr. G. Sims Woodhead,
F.E.S.K ; Dr. C. Zeiss (of Jena) ; and Messrs. F. E.
Becker & Co. (of London) for valuable aid in various
parts of the book.

In conclusion, it is hoped that this volume may
contribute something towards a proper understand-
ing and an intelligent appreciation of the important
and far-reaching subject of bacteriology.

A. B. GEIFFITHS.

EDGBASTON,

January 1893.

CONTENTS

CHAPTER I

INTRODUCTION : Koch's Canons — Vivisection— General
Properties of Microbes — Products of Microbian Activ-
ities— Sizes, Weights, and Reproductive Powers of
Microbes, etc., ......

CHAPTER II

BACTERIOLOGICAL LABORATORY AND ITS FITTINGS :
The Edinburgh Laboratory — The Pasteur Institute—
The Microscope — Microphotographic Apparatus —
Dissecting Instruments — Microtomes — Sterilisers —
Incubators — Cultivation Tubes, etc. , .

CHAPTER III

METHODS OF CULTIVATING, STAINING, AND MOUNTING
MICROBES, ETC. : Cultivation Media — Cultivation
Methods — Staining Preparation sand Tissues — Harden-
ing, Imbedding, Cutting, and Mounting Preparations
— Methods of introducing Microbes into Living
Animals— The Unit of Microscopical Measurements,
etc., ...... 49

CHAPTER IV

THE ORIGIN, CLASSIFICATION, AND IDENTIFICATION OF
MICROBES : Pleomorphism— Modes of Reproduction—
The Classifications of Cohn, Zopf, Baumgarten, Hueppe,
and De Bary, etc., ..... 98

CONTENTS

CHAPTER V

THE BIOLOGY OF MICROBES, ETC. : Micrococci — Bacteria
— Bacilli— Spirilla — Spirochaetae— Yeast-Fungi, etc. , .

CHAPTEE VI

INFECTIOUS DISEASES AND MICROBES : Yellow Fever —
Hydrophobia— Erysipelas — Puerperal Fever — Influ-
enza — Pneumonia — Scarlatina — Leprosy — Syphilis —
Tetanus — Malaria — Typhoid Fever — Cholera —
Glanders — Diphtheria — Tuberculosis — Anthrax —
Actinomycosis— Thrush, etc., . . . .

CHAPTER VII

MICROBES OF THE AIR : Examination of Air — Number
of Dust Particles in Air — Air of Lincoln, Paris,
London, etc. — Air of Country Places, etc.,

CHAPTER VIII

MICROBES OF THE SOIL : Examination of Soils— Soils
of Lincoln, Manchester, London, Paris, Dieppe, New
Zealand, New York — Microbes and Leguminous
Plants— Nitrification, etc., . . 276

CHAPTER IX

MICROBES OF WATER : Examination of Waters — Water
from Rivers Witham, Irwell, Thames, Lea, Seine,
Marne, Isar, Spree— Self-purification of Rivers— Sand
Filtration— Sterilisation of Water by Electricity,
Heat, and Filtration through Porous Porcelain —
Classification of Waters, etc., . . 286

CONTENTS xv

CHAPTER X

PAOE

PTOMAINES AND SOLUBLE FERMENTS : Properties of the
Ptomaines— Extraction of the Ptomaines — The Non-
oxygenous Ptomaines — The Oxygenous Ptomaines —
The Leucomaines — Albumoses, etc., . . . 305

CHAPTER XI

GERMICIDES AND ANTISEPTICS : Metallic Salts— Halogen
Elements — Aromatic Compounds — Oxidising Com-
pounds— Miscellaneous Germicides — Concluding Re-
marks, ....... 325

APPENDIX, ...... 332

INDEX, . 343

UNIVERSITY

CHAPTEK I

INTRODUCTION

DURING the past ten years or so there is hardly a
subject which has received so much attention as the
Science of Bacteriology — the Study of Microbes.
No one need wonder that the scientific world has
been so busy in such a fruitful field of research,
for it has not only been demonstrated that microbes
play important parts in the processes of fermenta-
tion, putrefaction, nitrification, etc., but that many
of these lowly beings are intimately connected with
infectious diseases.

Phthisis, diphtheria, cholera, malaria, glanders,
scarlatina, etc., have been proved to be the result?
of the action of certain microbes on the blood an*
tissues.

Infectious diseases being due to the action of cer-
tain microbes, it is necessary to isolate the microbes
and to study them apart from the body. Hence
the necessity of obtaining a pure culture of any
particular microbe (i.e. its freedom from other
microbes, etc.) before we can accurately study its

2 A MANUAL OF BACTERIOLOGY

mode of growth, multiplication, and the products it
may give rise to. In fact, Dr. E. Koch l has laid
down the following canons _to ascertain whether a
microbe is, directly or indirectly, the causa causans
of a particular disease: —

(1.) The microbe in question must have been
found either in the blood, lymph, or tissues of the
man or animal which is suffering from, or who has
died of, the disease.

(2.) The microbe taken from this medium (blood,
tissues, etc.), and artifically cultivated in certain
media, 'must be transferred from culture to culture
for several successive generations, taking the pre-
cautions necessary to prevent the introduction of
any other microbe into these cultures, so as to
obtain the specific microbe, pure from every kind of
matter proceeding from the body of the animal
whence it originally came.

(3.) The microbe, thus purified by successive cul-
tures, and reintroduced into the body of a healthy
animal capable of taking the disease, ought to re-
produce the disease, in the animal, with its char-
acteristic symptoms and lesions.

(4.) Finally, it must be ascertained that the
microbe in question has multiplied in the system
of the animal thus inoculated, and that it exists in
greater number than in the inoculating medium.

Microbes are everywhere present — in the air, in
the earth, and in waters ; in and on food, clothes,
etc. ; consequently they gain admittance into the
bodies of man and animals. These microbes do not

1 Die Milzbrand-impfung, 1883.

INTRODUCTION 3

necessarily give rise to disease, for many are harm-
less, although they may be present in the blood and
tissues. Not even in the case of an infectious disease,
where a certain microbe is present, can one say that
it is the cause of that disease. Not until Koch's
canons are fulfilled, is the experimenter justified in
saying that any particular microbe is pathogenic or
disease-producing.

From what has been said, it will be seen that
bacteriology, as applied to disease, is dependent
upon observation of, and experiments upon, living
matter. Among phenomena of so complex a char-
acter as infectious diseases, simple observation goes
but a very little way, and our knowledge of all the
most important truths of bacteriology, as applied to
these diseases, has been obtained by experimentation
upon living animals.

Vivisection is necessary for a proper interpreta-
tion of the phenomena. But ' every now and again
a loud outcry is raised against this method, partly
from ignorance and partly from prejudice. Many —
probably most — of the opponents of experiments on
animals are good, honest, kind-hearted people, who
mean well, but either forget that man has rights
against animals as well as animals against man, or
are misled by the false statements of the other class.
These are persons who, blinded by prejudice, regard
human life and human suffering as of small import-
ance compared with those of animals, who deny that
a man is better than many sparrows, and who, to the
question that was put of old, " How much, then, is
a man better than a sheep?" would return the reply,

4 A MANUAL OF BACTERIOLOGY

" He is no better at all." Such people bring un-
founded charges of cruelty against those who are
striving, to the best of their ability, to lessen the
pains of disease both in man and also in animals, for
they, like us, are liable to disease, and, like us, they
suffer from it.' 1

Without vivisection, the important researches of
Pasteur, Koch, Klein, and others could not have
been conducted; in fact, vivisection is absolutely
necessary to ascertain the pathogenic nature of any
microbe.

We now proceed to detail the general properties,
etc., of microbes. All microbes contain two principal
parts — a cell-wall or limiting membrane and a
semi-fluid contents — the protoplasm. The cell-wall
is composed of cellulose — a carbohydrate having the
empirical formula C6 H10 05. The protoplasm ap-
pears to vary somewhat in its chemical composition;
for, in some microbes, this complex substance is
devoid of sulphur and phosphorus, whereas in
others, both of these elements are present. The
protoplasm which is devoid of sulphur and phos-
phorus, has been termed mycoprotein by Nencki,
and has entirely different reactions from the proto-
plasm containing sulphur and phosphorus.

Microbes are capable of giving rise to various
products, such as acids, alkaloids, colours, enzymes,
albumoses, etc. This property depends upon the
present potentialities of the protoplasm (in each
case), and the inter-relation of its various functions,
and these again result from, or are modified by, the

1 Dr. Lauder Brunton in Nature, vol. xliv. p. 331.

INTRODUCTION 5

adjustment which takes place between an organism
and its environment. For instance, the cholera
bacillus grown on albumin produces toxines or
alkaloids, and is pathogenic; on potatoes, it gives
rise to a brown pigment and is chromogeuic ; while
on sugar it produces butyric acid, and is con-
sequently zymogenic or fermentive (Hueppe). The
action of gases, heat, light, electricity, and various
antiseptics have the power of altering the common
properties of a microbe ; but in every case the usual
products, etc., are formed when the microbe is once
more transferred to its natural mode of life. ' Every
organism has more potentialities or modes of action
than those which are actually in operation at any
given time, and when the environment is changed
one or other of these potentialities may come into
action, replacing, more or less completely, a former
one.' The extent of the powers of adaptation of
an organism depend on its potentialities and their
capacity of extension, and these vary, in each case,
enormously, a view in perfect consonance with the
results which experiments have already yielded.

As microbes differ in their actions, they likewise
differ in their dimensions ; and, as a general rule,
they vary from about 0*0005 mm. to 0'05 mm. in
length or diameter, as the case may be. Dr. F.
Cohn calculated that one bacterium (Bacterium
termo) weighs 0*000,000,001,571 milligramme, or
that six hundred and thirty-six millards of bacteria
would weigh one gramme, or six hundred and
thirty-six thousand milliards a kilogramme; and
the late Professor J. Clerk Maxwell stated that the

6 A MANUAL OF BACTERIOLOGY

smallest organised particle visible under the micro-
scope contains about two million molecules of
organic matter.

The reproductive power of microbes is most
prolific; and Cohn has calculated that a single
microbe at the end of three days would have in-
creased to nearly forty-eight billions, a mass which
would weigh no less than 7500 tons. But this
astounding rate of reproduction is kept in check by
the limited supply of food, as well as by various
circumstances which make the environment unsuit-
able for such a rapid rate of increase. ' As a con-
sequence of their enormous fecundity, it will be
readily understood that they are ubiquitous. Every
surface teems with them; all natural waters are
infested by them ; even the skin of the most washed
of mankind ; even the moisture of the sweetest
mouth harbours them by the million ! One thing,
however, they cannot stand, and that is boiling.
Boil them or the stuff in which they are nourishing,
and they cease to live — or, in other words, the liquid
or solid substance so treated is sterilised. By means
of sterilised nutriment we can test any object for
the presence of microbes or bacteria, as they are
sometimes called. We prepare a broth suitable for
their nourishment, and sterilise it. If kept her-
metically sealed (as are preserved vegetables and
tinned meats), no microbes will appear in the broth.
Touch the broth with any stick or stone, or add to
it a drop of purest spring water, and it will, after a
few hours, swarm with microbes and putrefy. This
was the discovery of Theodore Schwann, also cele-

INTRODUCTION 7

brated for his cell-theory. He showed fifty years
ago that what we call 'putrefaction' is not the
result of death, but of life. The unpleasant smell
and the disintegration of dead bodies, whether of
plants or animals, is entirely due to microbes — it is
the accompaniment of their digestion. If you
destroy all the microbes present by means of boiling
heat, and then prevent the access of new microbes
(which are blown about in the dust of the air), dead
bodies never putrefy. Supposing that by the fiat of
an omnipotent Being all microbes could be annihi-
lated, the earth's surface would soon be covered
with dead bodies remaining unchanged year after
year, century after century. The seas and lakes
would be choked with them, and we should have to
use them for paving our roadways and building our
houses. But worse than that, all the carbon and
nitrogen which living things use in turn in their
successive occupation of the earth's surface from
generation to generation, would soon be tied up.
There would be no food for the green plants : herbi-
vorous creatures would cease to exist. The con-
templation of these imaginable horrors gives us
some notion of the part played by microbes in the
order of nature.'

CHAPTEK II

THE BACTERIOLOGICAL LABORATORY AND ITS FITTINGS

BEFORE describing the necessary apparatus, etc., for
the proper investigation of bacteriological problems,
we give a general account of the laboratories of the
Koyal College of Physicians, Edinburgh, and those
of the Pasteur Institute, Paris : these being chosen
as typical examples of bacteriological laboratories.

The Edinburgh Laboratory. — The ground floor of
this laboratory, which is situated in Lauriston Lane,
contains a workshop, stores, and a room set apart
for experimental physiology. The latter is 32
feet long, 18 feet wide, and 14 feet high: it is
fitted with tables for microscopical work ; a respira-
tion apparatus driven by water-power ; recording
apparatus ; galvanometer and other electrical ap-
pliances ; and sink and draining apparatus. ' On
each microscope table, which is painted black and
hard varnished, a white band about four inches
broad is painted, four inches from the edge of the
tables. Some of the tables, instead of being
varnished, are covered with plate glass, painted as
above on the under surface, and imbedded in felt.
On these glass-covered tables the microscope stands

THE BA CTERIOLOOICA L LA BORA TOR Y 9

on a felt circle, to diminish the risk of breakage
when the bell jar is lowered over the microscope/

On the second floor there are five rooms, three of
which are occupied by the laboratory assistant. Of
the other two rooms, one is used as a library and
museum ; while the other is the director's private
room. The former room is fitted with an Oertling's
balance, a barometer graduated in inches and
millimetres, a thermometer with Fahrenheit and
centigrade scales, and a large spectroscope. This
room is also used for the meetings of the committee.

The third floor (counting from the basement)
contains six rooms. ' The first of these, a small one,
is used as a still-room ; the still is connected with
the water-pipe and is self -feeding, so that to obtain
a supply of distilled water all that is necessary is to
turn on the tap and light the Bunsen burner.' A
second room — the chemical room — is fitted with a
good supply of gas and water, working benches,
evaporating chamber, sandbaths, and the necessary
apparatus and reagents for the analysis of water,
air, food, and for physiological chemical work. The
next room is fitted with a table for histological
work, but is chiefly used for blow-pipe work, metal
injections (Cathcart's method), imbedding in paraffin
and celloidin, and section-cutting by means of the
microtome, etc. The same room is also used as a
store for some of the glass apparatus. The next
room is used as a store for chemical reagents. This
is followed by another small histological room ; and
finally, a room is set apart for the estimation of
urea, albumin, and glucose in urines.

A MANUAL OF BACTERIOLOGY

The fourth and top story contains three well-
lighted rooms (Fig. 1). The south room is the true
bacteriological laboratory (Figs. 1 and 2), and is fitted

with tables, micro-
scopes, sterilisers,
incubators, and the
apparatus neces-
sary for research in
the various branch-
es of bacteriology.
The other two
rooms on the top
story are fitted for
histological work.

The original cost
for the whole equip-
ment of the ' Edin-
burgh Laboratory '

CUPBOARD was only fSSO.1

BACTERIOLDG/CAL ffi ^ Of course this la-
boratory, being a
public one, has
been fitted for
many workers ; but
a good private
laboratory, suit-
able for one's own

ROOK'

/H CUB AT OR
STOVl

*'- — ,*-»—• 27 fT . — -

. i. PLAN OF BACTERIOLOGICAL
LABORATORY, ETC.

'-?

fitted at less than a fourth of the above-mentioned
amount.

1 See Dr. G-. Sims Woodhead's paper in Proc. Roy. Physical
Soc., Edinburgh, vol. ix. p. 521.

12 A MANUAL OF BACTERIOLOGY

The Pasteur Institute. — This celebrated institu-
tion (see frontispiece) is not simply an hospital for
the treatment of persons suffering from hydrophobia
or rabies, but is a building set apart for the study
of micro-biology in all its branches. The Pasteur
Institute is situated in the Eue Dutot — not far from
the Cimetiere Montparnasse — on the south side of
Paris. It is the most perfect building of its kind
in the world ; the cost of erection, fitting, and
endowment being £100,000. The anti-rabic de-
partment forms a relatively small portion, there
being in addition an important department, in
which are prepared vaccines for the prevention of
several of the infectious diseases of cattle — rouget
de pore (swine fever), anthrax, etc, — as well as
laboratories, lecture-rooms, and a large library. In
the same building is the residence of M, Pasteur,
who naturally takes the greatest interest in the
work of the institute.

The Pasteur Institute covers an area of 11,000
square metres, and consists of two blocks, running
parallel, one behind the other. These blocks are
united by a long corridor. On the first floor of the
front block is a room used as a library and council
chamber ; and the second floor of the same block is
entirely occupied by the attendants and servants of
the establishment. On the right of this block is M.
Pasteur's residence. The block in the background is
divided into two wings, each about 25 metres long,
and 15 metres from back to front. In the right
wing, on the ground floor, are the rooms set apart
for the anti-rabic treatment, and a laboratory in

THE BACTERIOLOGICAL LABORATORY 13

which the preparation of the virus is carried on.
This laboratory is always maintained at a tempera-
ture of 23° C. In the left wing, on the ground
Hoor, is a lecture-theatre for biological chemistry, a
laboratory, and a room set apart for photographing
microbes, etc. At the end of the block (i.e. at the
back) are two rooms (one on each side of a central
corridor) used as aquaria. The remaining portion
of this block is occupied as a store-room and
a general laboratory; the latter being used for
the preparation of the various cultivating media,
glass-blowing, etc. The second story is, likewise,
divided into two halves ; on the left is the micro-
biological department, and on the right that of
practical biology. On the same floor there is a
large laboratory fitted with sterilisers, incubators,
evaporating chambers, etc. : in fact, this room is
used for the growth of all kinds of microbes.
Joining this room is a smaller laboratory, out of
which one steps into the museum. In addition,
there is a chemico- biological laboratory and a
lavatory. The third story of the rear block com-
prises two series of rooms, which are all used for
research ; the left wing is occupied by the depart-
ment of applied or practical bacteriology, while the
right wing is devoted to the study of comparative
micro-biology or bacteriology. Each of these de-
partments is fitted with incubators, sterilisers, and
other bacteriological apparatus.

In each of the departments of the institute there
is a director's private room and laboratory.

Besides the two main blocks there are separate

14 A MANUAL OF BACTERIOLOGY

buildings, etc., in the grounds of the Institute.
Among these are the cages for the accommodation
of animals; a special house for the reception of
dogs ; stables, etc., for large animals ; a rabbit-
house ; a run, etc., for hens ; and an aviary. All
these places are kept in a state of perfect cleanliness.
This important establishment would not be com-
plete without a crematory ; this consists of two large
furnaces, situated in one corner of the grounds, whicli
are used for destroying all useless animal matter.

The Pasteur Institute accommodates fifty workers,
and is open to foreign as well as French scientific
and medical men. Besides being an institute for
research, it is also used for the instruction of pupils
in both general and special methods of bacterio-
logical investigation.

The above is only a general account of the
Pasteur Institute ; but the reader desirous of ob-
taining fuller information is referred to the Annales
de I'lnstitvt Pasteur, 1889.

Having given a description of the Edinburgh
and the Paris laboratories, we now proceed to
describe the various apparatus and appliances used
in the study of bacteriology.

The Microscope. — In our experience the best
microscopes suitable for the study of 'les infini-
-ment petits ' are those made by Carl Zeiss, of Jena
(Fig. 3). These instruments are monocular micro-
scopes, and consist of the usual parts — the stand,
eye-pieces, and objectives. Very few workers in
bacteriology use the binocular microscope, because
there is a great -loss of light, and the definition of

THE BA C TERIOLOGICA L LA BORA TOR Y 15

high-power objectives is impaired when this instru-
ment is used. It is believed by some that when
the monocular microscope is used continuously the
eyes are apt to become fatigued. The student
should learn to
keep both eyes
open when work-
ing with the
monocular micro-
scope. There are
several ways by
which this may
be effected. One
is by having a
black sheet of
paper near to the
second eye; an-
other plan is to
put the hand be-
fore the eye.
Perseverance is
all that is needed.
One evening is
quite enough to
make any one
skilful, if he is
determined to '
succeed. The
student need no
more fear seeing things on the table with the
second eye than seeing the crown of his head, unless
he is^training for drawing objects -on the table by

FIG. 3. ZEISS' MICROSCOPE.

16 A MANUAL OF BACTERIOLOGY

means of a camera lucida from the instrument,
whilst with one eye he looks at the object, and
with the other draws the figure. A very little
reflection will convince any one how desirable it
is to keep the nerves of the eye as nearly in their
right position as possible ; for an undue strain is
caused if they are strained, and the sight is injured
if much work is done.

The object-glass or objective (Fig. 3 A) is the
most important part of the microscope ; conse-
quently it is necessary to have good lenses to do
satisfactory work. The objectives are known as low
and high powers — but for bacteriological work, the
microscope should be provided with the following
objectives (Zeiss') : —

D and E (dry lenses), J (water immersion), ^
(oil immersion). Zeiss' lenses give perfect defini-
tions, and everything there is to be seen can be
made out with the highest powers. Oil-immersion
lenses are taking the place of water high powers, as
they need no correction for the thickness of the
cover-glass, and are therefore much easier to use ;
1 the only drawback is that the essential oil (e.g. cedar
oil) used will dissolve Canada balsam, Dammar
varnish, and many of the sealing fluids, and it is
necessary to cover them with Hollis' glue, which is
not acted on by cedar oil.' Of Zeiss' high powers
the ^ oil-immersion lens is the best, and may be
thoroughly recommended for bacteriological research.
Oil-immersion lenses possess far greater brilliancy
and definition than the water and dry lenses [such
as Zeiss' K and L (water), and F (dry)]. In using

THE BA GTE RIO L OGICA L LA BORA TOR Y 1 7

oil-immersion lenses, a drop of cedar oil is placed
on the front glass, the lens in use is then lowered
on to the slide until contact is made. The lens is
then focussed by the fine adjustment (Fig. SF)
until the object is seen sharply defined.1

It is desirable that the bacteriologist's microscope
should be fitted with a revolving triple nose-piece
(Fig. 3 A); by this means three objectives (of
different magnifying power) can be brought succes-
sively into position, without unscrewing.

The eye-piece or ocular is also an essential part
of the microscope (see Fig. 3c); and for bacterio-
logical research, the whole (five in number) of Zeiss'
huyghenian eye-pieces are recommended. The fol-
lowing table shows the magnifications of Zeiss'
objectives and eye-pieces with a tube of 155 milli-
metres in length — i.e. the Continental microscope
with a short tube : —

1 Zeiss no longer makes the ^ oil-immersion objective ; this
lens has now been superseded by the introduction of a new
series of objectives — the apochromatic lenses — made of the new
glass. These lenses are said to excel the ordinary objectives,
by giving almost perfect achromatism and sharpness of image
over the whole visual field (see Abbe's paper in Sitzungxberichte
d. med.-naturw. Gesettschaft zu Jena, 1886).

A MANUAL OF BACTERIOLOGY

EYE-PIECES.

II.

III.

IV.

Oil-immersion Water-immersion -P. , . ,.
objectives. objectives. Drv obJectlves-

«i

a 2'

««

a
aa
A,AA
B, BB
C,CC
D, DD
E
F
G
H
J
K
L

A
A

4-12
30

7-17
41

10-24

71
130

97
175

130
235

120

145

195

270

360

175

230

320

435

580

270

355

490

670

890
1350

405

540

745

1010

260

340

470

640

855

320

430

590

805

1075

430
570

570
760

785

1070

1430
1900
2570

1045

1425
1930

770

1030

1415

260

340

470

640

855

380

505
810

695

950

1265

605

1110

1515

2020

Zeiss' J objective is equal to an English TV inch,
while his B, 0, D, DD, E, and ^ oil-immersion are
equal to an English 1, J, J, J, J, and ¥V inch respec-

THE BACTERIOLOGICAL LABORATORY 19

lively. The medium objectives are issued in two
different forms, with a greater or less aperture
according to the purpose for which they are required.
Those with a large aperture (distinguished by double
lettering), possess with equally perfect definition a
considerably higher resolving power, and permit
of greater magnification being obtained by the use
of the stronger eye-pieces. Nevertheless, the work-
ing distance in BB, CO, DD, although relatively
large, is perceptibly less than in the corresponding
series of smaller aperture, and the former are more
sensitive to differences in thickness of the cover-glass
and object than the latter. Therefore, B, C, and D
are recommended as the more suitable for working
glasses in histological and anatomical research,
particularly when the next stronger dry lens is
available for higher magnification.

The magnifications of the English objectives and
eye-pieces with a ten-inch tube (i.e. the English
microscope with a long tube), are given in the
following table : —

A MANUAL OF BACTERIOLOGY

EYE-PIECES.

OBJECTIVES.

4 inch.

120

150

100

150

190

100

133

200

250

170

227

350

440

254

333

500

625

270

360

540

675

450

600

900

1125

500

666

1000

1225

700

940

1350

1640

The above will serve as approximately correct
tables for ordinary work, but if the exact magnifying
power of any objective is required it must be
specially tested.

The proper illumination of microscopic objects is
of the highest importance, and that suitable for one
class may be altogether unfit for another. Daylight
is the best light to use for bacteriological work ;
but if one is working at night or in the winter, a
paraffin lamp is required. It is essential that the
flame should be steady and of moderate size.
Parallel rays may be advantageously thrown on the
mirror (Fig. SD) of the microscope by means of a
bull's-eye condenser, placed so that the flame is
nearly in the focus. For comparatively low powers,
a fla.t or concave mirror may be used to reflect the
light, but for higher powers it is essential that the
light should be concentrated by means of an Abbe's

THE BACTERIOLOGICAL LABORATORY

•21

substage condenser (Fig. 3fi). This condenser, first
described by Professor Abbe (of the University of
Jena),1 is of very short focus, and collects the light
reflected by the flat mirror into a cone of rays of
very large aperture, and projects it on the object.
Abbe's condenser is focussed on the object by coarse
and fine adjustments. ' When the whole field is to
be examined, the lamp is used with the whole
breadth of the flame, but when a small portion is to be

FlG. 4. MlCROPHOTOGRAPHIC APPARATUS.

(After Carl Zeiss.)

specially examined with a high power, it is necessary
to turn the lamp so that the edge of the flame is pre-
sented, by which the light is very much intensified.
The correct distance at which to place the lamp can
only be found out by practice. A piece of blue glass
should be interposed between the lamp and the con-
denser : this can be done by having it fitted into the
condenser or by having a separate stand ; different
shades of blue will be found useful for various objects.

1 Archivfur Mikr. Anat. vol. ix. p. 496.

22 A MANUAL OF BACTERIOLOGY

The blue colour is a great help to the eyes, and
also throws up the stained specimen with more dis-
tinctness.'

Microplwtograpliic Apparatus. — The application of
photography as a means of illustrating microscopic
preparations has been, on the whole, successful.
Koch, Crookshank, Van Ermengern, and others, have
produced beautiful photographs of microbes and
sections of diseased tissues. For this purpose many
different kinds of apparatus have been devised ; but
one of the best is represented in Fig. 4. It consists
of two tables (A and S) for the microscope and
camera respectively ; two diaphragm carriers (E and
F) for use with sunlight ; an electric lamp (C) ; a
holder for taking absorption cells (H); a water
chamber for absorbing heat rays (T); a camera (K);
a collective lens-system for projecting the image of
the carbon points on the focussing screen (L) — this
is required when the electric lamp is used ; a micro-
scope (M) ; and focussing apparatus (a, b, b', h).
This apparatus can be used with sunlight,1 lime-
light, electric-light, and lamp-light. For micro-
photographic purposes, microscopic preparations are
best when stained yellow, black, or brown, and
mounted in either Canada balsam (dissolved in
xylol) or a saturated solution of potassium acetate.
Several authors have recommended the use of the
isochromatic dry-plates, and first-class photographs
have been obtained by them.2

1 When sunlight is used, a heliostat is also necessary.

2 See Crookshank's Photography of Bacteria; and Van Enn-
engem in Bulletin de la Soc. Beige de Microscopie, No. 10, 1884.

THE BACTERIOLOGICAL LABORATORY 23

Another method for obtaining illustrations of
microscopic preparations is by means of the camera
lucida. Among the best of these instruments,
suitable for bacteriological purposes, are those of
Zeiss and Nachet. ' Combined with the use of a
micromillimetre objective, the camera lucida affords
also a simple method for the measurement of
bacteria.'

The third and last method for obtaining illustra-
tions of microscopic preparations is drawing by
hand. If a white piece of card-board or smooth
drawing-paper is fixed at the same level as the stage
of the microscope ; by keeping both eyes open — one
for looking at the object through the microscope,
and the other for looking at the piece of card-board
— an image of the object is seen on the card, which
can be readily traced with a pencil. For drawing
bacteria, etc., no pencil is so well adapted as
Windsor and Newton's HHHH; the blacklead
being brought to its final point by gentle rubbing
on the surface of the finest ground glass, or,
better still, a very fine hone. For inking the pencil
drawings, the finest etching pens should be used —
perhaps the best are those made by Joseph Gillott ;
and the same maker's No. 303 is also a very fine-
pointed pen. In addition to the pencils and pens
—Indian-ink, water-colours, and brushes are neces-
sary. With practice and patience, very accurate
drawings of microscopic preparations can be made
by hand.

Dissecting Instruments. — For the dissection of
diseased organs, tissues, etc., certain instruments are

A MANUAL OF BACTERIOLOGY

necessary. Figs. 5 and 6 represent various kinds of
scalpels, microscopic needles, knives, scissors, and

FIG. 5. DISSECTING KNIVES AND NEEDLES.
A to F are used in microscopic dissections.
G to J are used in ordinary dissections.

forceps ; and Fig. 7 illustrates a very useful form of
dissecting microscope.

THE BA CTERIOLOGICAL LA BORA TOR Y 25

The mode of carrying out a dissection for bac-
teriological purposes is as follows : Animals either
artificially inoculated with pathogenic microbes or
those naturally suffering from infectious diseases

FIG. 6. DISSECTING SCISSORS AND FORCEPS.

FIG. 7. DISSECTING MICROSCOPE.

should be dissected as soon after death as possible.
In dissecting, every precaution must be adopted to
exclude putrefactive or other microbes. The dis-
section should be performed in a perfectly still

26 A MANUAL OF BACTERIOLOGY

room with closed doors ; and the instruments used
in the dissection must be previously sterilised in
the hot-air steriliser or the Bunsen flame. The
animal under examination (e.g. a mouse, rabbit,
guinea-pig, etc.) is pinned out on a slab of gutta-
percha previously washed in a solution of mercuric
chloride (corrosive sublimate). It is now bathed in
a stream of the same germicidal agent ; and after
having cut away the hair with sterilised scissors, the
seat of inoculation, etc., should be examined first,
and any pathological characteristics should be noted.
If there is any exudation, it should be used for
inoculating purposes and microscopical examination.
To examine the internal organs, place the animal
on its back and make an incision extending (if
necessary) from the abdominal to the thoracic
region. The organ under examination should be re-
moved from the body-cavity, with sterilised scissors
and forceps ; and after removal it should be washed
with mercuric chloride. The organ is now incised,
and the fluid, or a portion of the organ itself (i.e.
from the cut) should be used for inoculating various
cultivation media. If the blood of the animal is
required, it is best obtained from a vein by making
an incision with sterilised scissors, and then insert-
ing a sterilised capillary pipette or a platinum
needle. The blood so obtained should be examined
microscopically, and various cultivation media
inoculated with it. If the cultivations are con-
taminated by the presence of other microbes, frac-
tional plate-cultivation must be resorted to, in order
to isolate the pathogenic microbe.

THE BACTERIOLOGICAL LABORATORY 27

After dissection, the organs, etc., may be pre-
served in absolute alcohol, i.e. if they are required
for future examination ; and all useless matter
should be destroyed, and, finally, the hands, instru-
ments, and table disinfected.

FIG. 8. SCHANZE'S MICROTOME.

Such is the mode of carrying out a dissection on
a dead animal; but to obtain microbes from the
living animal or from man, these may be isolated
from pus and other discharges, or from the blood.
These fluids must be obtained with all the necessary

28 A MANUAL OF BACTERIOLOGY

precautions to prevent external microbes gaining
an entrance.

Microtomes. — These instruments are used for cut-
ting sections of organs, tissues, etc. ; and there are
many forms in use. Fig. 8 represents Schanze's
microtome, and it is a most useful instrument for
cutting sections imbedded in celloidin.

The Cambridge rocking microtome 1 is an instru-
ment for producing ribbons of sections imbedded
in paraffin. The razor is supported and clamped in
front of a brass tube containing the imbedded
object. This tube fits tightly on to the end of a
cast-iron lever ; and is made to slide backwards or
forwards so as to bring the imbedded object near
to the razor. By an arrangement of pivots, milled
screws, and a milled wheel, the lever is moved for-
wards, and the object to be cut is therefore brought
across the edge of the razor : when the lever is made
to move backwards the section is cut. The values
of the teeth on the milled wheel are as follows :

1 tooth of the milled wheel = ^57 of an inch = '000625 mm.

2 teeth ,, ,, = ^J^ ,, ='001250 mm.
4 j, ,, ,, = 10000 55 — '0025 mm.

16 ,, ,, 15= urfto 55 = '01 mm.

On working this microtome the sections should
adhere together so as to form a ribbon. The work-
ing of this instrument requires very little skill on
the part of the operator ; consequently it is to be
recommended to those who require very thin sec-

1 Made by the Cambridge Scientific Instrument Company,
St. Tibb's Row, Cambridge.

THE BACTERIOLOGICAL LABORATORY 29

tions of diseased organs, etc. Dr. Sims Woodhead
has somewhat modified the Cambridge rocking
microtome by adding a solid end to the brass tube
into which ' dies ' of various sizes, with roughened
surfaces, can be screwed. ' This does away with the
inconvenience of having to " melt in " the imbedded
tissue into the tube. A dozen of the dies may
be used, and to each of these a piece of tissue
may be fused, and kept ready for cutting at any
time. '

Besides the microtomes just mentioned there are
those of Korting, Eeichert, and Jung, which are
principally used in France and Germany. When
tissues are to be examined in the fresh state,
either Koy's or Williams' freezing microtome should
be used for section-cutting. In the former instru-
ment the tissue is frozen by means of an ether
spray ; while in the latter the frozen tissue is pro-
duced by a mixture of ice and salt. There is no
doubt that Eoy's microtome is the better instrument
of the two, as the freezing of the tissue only
occupies from thirty to forty seconds ; and this
microtome may also be used for cutting objects
imbedded in paraffin and not requiring freezing —
in other words, the instrument can be used both
as a freezing and a non-freezing microtome.

Tissues imbedded in paraffin or a mixture of
white wax and olive oil may be cut by hand with
a hollow-ground razor. The razor is dipped in
dilute alcohol and then drawn diagonally across the
mass (containing the specimen) with a steady
sweep. Before cutting each section the razor

A MANUAL OF BACTERIOLOGY

should be dipped in dilute alcohol. 'Great care
is required in cutting sections by hand, to hold the
razor firmly yet lightly, so as to cut them thin and
at the same time even, and this cannot be done
without a great deal of practice/ The author has,
in his possession, sections of the human brain vary-

FIG. 9. KOCH'S 'STEAM STERILISER.

ing from the y oV o to the T^¥ of an incn in thick-
ness which were hand-cut by Dr. E. Palmer, who
was formerly the resident physician to the Lincoln
County Asylum, Lincoln. To obtain such sections
requires skill and practice, therefore it is better to
use the microtome.

THE BACTERIOLOGICAL LABORA TORY 31

Sterilisers. — In a study like bacteriology, all
vessels, instruments, etc,, used in the cultivation
of microbes, must, before use, be rendered perfectly
sterile. It cannot be too firmly impressed upon
the mind that the only way to obtain pure cultiva-
tions of microbes, is the complete sterilisation of
all vessels arid instruments used by the experi-
menter. For the accomplishment of this object
steam, hot-air, Btmsen or spirit flames, and germi-
cides are used as sterilising agents.

Fig. 9 represents Koch's steam steriliser ; and is
used for sterilising test-tubes, flasks, and for cook-
ing potatoes. It is a cylindrical vessel of stout
tin plate, with a copper bottom, provided with a
conical lid, brass tubulure for the insertion of a
thermometer, a grating, water gauge, tap, and a
receiver with perforated bottom for cooking potatoes
(b). The cylinder (which is 20 in. high and 10
in. diameter) is divided into two compartments
(a and c). The lower one contains boiling water,
while the steam therefrom passes into the upper or
sterilising compartment. The cylinder is heated
from below by a Bunsen's or Fletcher's burner.
Steaming is usually kept up for from fifteen to
twenty minutes ; and this operation is repeated on
three successive days each time for twenty minutes.
By such steaming the various cultivation media,
etc., are rendered sterile, i.e. free from microbes. A
later form of this steriliser contains three compart-
ments instead of two. Two of these are used as
sterilising compartments, while the lowest one con-
tains the boiling water, which is always kept at a

32 A MANUAL OF BACTERIOLOGY

constant level. Both forms of Koch's ' steriliser are
covered externally with felt.

The so-called steam digesters or ' autoclaves '
are chiefly used in France. They are made of
stout copper, and are used for sterilising sealed
flasks (containing bouillon) under pressure. The

temperature in
these digesters
often rises as high
as 120°C.

Besides the
above - mentioned
steam sterilisers,
there are those of
Herrmann and
Hirschberg, Ost-
walt, Muencke,1
and Woodhead.2
All these are use-
ful instruments,
and are to be re-
commended for
the bacteriological

FIG. 10. HOT-AIR STERILISER.

laboratory.

Two forms of hot-air sterilisers are represented
in Figs. 10 and 11 respectively, and are used for the
sterilisation of flasks, test-tubes, cotton- wool, etc.
The former consists of a double wall of sheet-iron,
and the inner dimensions are 12 in. x 10 in. x 10

1 Dingler's Polytechnisches Journal, 1885, Bd. 257, p. 283.
'2 Proceedings of Royal Physical Society of Edinburgh, vol.
ix. p. 524.

THE BACTERIOLOGICAL LABORATORY 33

It is heated by a gas-burner, and is made to
hang against the wall of the laboratory.

The steriliser represented in Fig. 1 1 l is either
heated by paraffin-oil or by gas. It consists of a

I I /O Q '&<^L> 4j f

H/g? 5> 0 & Q O

-^FFTT'^W

Fio. 11. HOT-AIR STERILISER HEATED BY PARAFFIN OIL OR GAS.

(Devised by Mrs. A. B. Griffiths.)

A, Thermometers. B, Copper Shelf with Holes of different sizes. C, Mica

Window. D, Iron support for Oven over Flame. E, Paraffin Oil

Lamp. F, Screw to raise Wick. G, Wire Gauze.

sheet-iron chamber, provided with shelf containing

1 First described by the author in the Proceedings of the
Poyal Society of Edinburgh, vol. xiv. p. 105.

34 A MANUAL OF BACTERIOLOGY

a series of holes of different sizes; by this means
the tubes or flasks are placed in a vertical posi-
tion in the steriliser. It may be stated that all
good hot-air sterilisers should allow the tubes to
be placed in a vertical rather than a horizontal
position. By this means the heated air rises in the
inverted tubes, flasks, etc., and the current so
formed (in each tube, etc.), destroys all the microbes
and spores present therein.

The hot-air sterilisers of Koch, Muencke, Pasteur,
and Klein are all good sterilisers. Dr. Klein's con-
sists of an iron chamber with double wall and
double folding-doors. In the inner chamber are
placed the test-tubes in a horizontal position, and
the cotton-wool above them. After closing the doors
the steriliser is heated by a Fletcher's gas-burner.

' Test-tubes (to be sterilised) should be exposed
to the full heat of the chamber for several hours.
After this they should be taken out of the steriliser
while hot, plugged with sterilised cotton-wool, and
then reheated for a few hours longer. Beakers and
glass funnels may also be sterilised in the hot-air
steriliser, or by being heated over a Bun sen flame.
To prepare sterilised cotton-wool, place the wool in
a loose condition, and heat it in the hot-air steri-
liser to a temperature of about 150°C. for several
hours on several successive days.'1 Over-heating
the cotton-wool in the hot-air steriliser to the above
temperature until singed has proved invariably and
absolutely safe for all cultivations.

1 See Dr. A. B. Griffiths' book, Researches on Micro-Organisms,
p. 14(Bailliere&Co.).

THE BACTERIOLOGICAL LABORATORY 35

To use cotton-wool, flasks, and tubes disinfected
by prolonged steeping in alcohol, carbolic acid solu-
tion, and other chemicals, is not absolutely reliable ;
and many failures have been the results of such
methods of sterilisation. Therefore, 'it cannot be
too thoroughly insisted on that the flasks and test-
tubes, and especially the cotton- wool used as plugs
for the vessels,
should be thor-
oughly sterilised
by over-heating,
for cultivations
are as often con-
taminated by
this not being
properly carried
out as by the
non-sterility of
the nourishing
fluids or the acci-
dental entrance
of organisms
from the air.'

For the sterilis-
ation of scalpels,
forceps,platiuum

needles, etc., the Bunsen flame is the best way of
cleansing them ; but, unfortunately, the naked flame
is most destructive to the blades of scalpels; to
obviate this, Israel's case was devised. It is a sheet-
iron box (with lid), in which the scalpels, etc., are
exposed to a temperature of 150°C. in the hot-air

FIG. 12. SERUM STERILISER.

36 A MANUAL OF BACTERIOLOGY

steriliser for an hour or so. By this device the
blades are not injured.

For the preparation of solidified sterile blood
serum two pieces of apparatus are necessary ; these
are represented in Figs. 12 and 13. Fig. 12 is the
serum steriliser, and consists of a double-walled
cylinder, 13 inches in height and 11 inches in dia-
meter, made of stout tin, with a copper bottom.

FIQ. 13. SERUM INSPISSATOK.

This cylinder is provided with a double-walled lid,
having a tubular prolongation of stout copper, tap,
and gauge : the whole being surrounded with thick
felt. The apparatus is divided internally into four
compartments ; and into these are placed the test-
tubes, or glass capsules, containing the blood serum.
Between the two walls of the cylinder is a layer of
water, which is heated from below ; while the water

THE BACTERIOLOGICAL LABORATORY 37

in the lid (i.e. between the two walls) is heated by
means of the prolongation (see Fig. 12). It will be
seen that the whole apparatus is essentially a hot-
water jacket. The temperature of the steriliser
should be maintained for an hour at 60° C. on five
or six successive days. By this means the fluid
serum is completely sterilised, but it is not solidi-
fied. To solidify the serum the piece of apparatus
represented in Fig. 13 is required. It is a double-
walled case, also made of stout tin (13J in. longx
13 J in. widex4j in. deep). It is provided with a
copper bottom, glass cover, water-gauge, and ther-
mometers ; there is also an arrangement by which
this inspissator, as it is called, can be fixed at the
angle required; this being necessary to give the
serum a sloping surface. The tubes, etc., containing
the sterile but fluid serum, are placed in the inspis-
sator ; and this apparatus (like the serum steriliser)
containing water (between the two walls) is heated
from below. To coagulate the serum, and to solidify
nutrient agar-agar, a temperature between 65° and
68° C. should be maintained ; but as soon as solidi-
fication takes place the tubes should be removed
from the inspissator. Solidified blood serum is used
for the cultivation of Bacilhis tuberculosis, Bacillus
mallei, and a few other microbes ; but we shall refer,
in detail, to the various cultivation media and the
methods of cultivation, in the next chapter.

Incubators. — Many microbes are capable of being
cultivated at the ordinary temperature of the
laboratory; but certain microbes require a higher
temperature for their proper development and mul-

A MANUAL OF BACTERIOLOGY

tiplication. For the latter purpose various ovens or
incubators have been devised. One of the most

FIG. 14. BABES' INCUBATOK.

convenient forms is the incubator of Dr. Babes
(Fig. 1 4). It consists of a rectangular, double-walled

THE BACTERIOLOGICAL LABORATORY 39

chamber, covered on five sides with felt, but in
front the felt forms a loose flap, which can be raised.
The interspace between the two walls is filled with
water, which is heated from below. The incubator
has two glass doors, a moveable shelf, a water-gauge,
and a gas-regulator.

Among other good incubators are those of Pas-
teur,1 Eohrbeck,1 Klein,2 Gautier, Abel, D'Arsonval.
and Hueppe. Whatever form of in-
cubator is preferred, it is essential
that it should be provided with a
gas or heat regulator. The acting
agent in most regulators of this de-
scription is either a membrane,
mercury, or electricity. Tieftrunk's,
Giroud's, and Elster's are membrane
gas -regulators ; Eeichert's, Page's,
Schiitz's, Fraenkel's, and Meyer's
are mercury heat - regulators ; and
Schlosing's is a membrane heat-regu-
lator. Fig. 15 represents Keichert's
mercury heat - regulator. It is a Flo> 15>
tube with two lateral arms (a and REICHERT-S

,x ,, .. f , \ , . REGULATOR.

0); the upper portion of which is
extended into a funnel-like arrangement, bearing
the arm b. Into this funnel-like opening fits a
hollow T piece. ' One arm of the T piece is open,
and connected with the gas supply; the vertical

1 For figures of these incubators see Dr. Griffiths' Researches
on Micro-Organisms, pp. 19 and 20.

2 See Dr. Klein's Micro- Organisms and Disease (3d ed.),
p. 15.

A MANUAL OF BACTERIOLOGY

portion terminates in a small orifice, and is also
provided with a minute lateral opening.' The tube
and arm a contain mercury. Reichert's regulator is
fixed into the roof of the incubator, so that its

FIG. 16. KARL ABEL'S INCUBATOR.
(With thermo-electric regulator.)

lower portion projects either into the water chamber
or into the interior of the incubator. 'When the
incubator reaches the required temperature, the

THE BA CTERIOLOGICA L LABOR A TOR Y 41

mercury is forced up by means of the screw in the
lateral arm, until it closes the orifice, at the extre-
mity of the vertical portion of the T piece. The
gas which passes through the lateral orifice is suffi-
cient to maintain the apparatus at the required
temperature. If the temperature of the incubator
falls the mercury contracts, and the gas passing
through the terminal orifice of the T piece, increases
the flame of the burner, and the temperature is
restored.' Page's regulator resembles somewhat the
regulator just described : both are simple and useful
forms for the bacteriological laboratory. By such
devices the various incubators, may be maintained
at a temperature which is almost constant; the
slight differences (say of one or two degrees Centi-
grade) are due to the variations in the pressure of
the gas supply ; but this inconstancy is remedied by
first passing the gas through a pressure - regulator
(such as Moitessier's).

In addition to the above-mentioned regulators,
there are two forms which are worked by the agency
of the electric current. Babes'1 and Abel's2 are
thermo-electric regulators ; the latter being repre-
sented in Fig. 16. These are useful regulators ; but
for general work those of Keichert and Page are
specially recommended.

Cultivation Tubes, etc. — Fig. 17 represents an im-
portant series of glass tubes, flasks, etc., used in
the cultivation of microbes in liquid media. These

1 Centralblatt fur Bakteriologie und Parasitenkunde, 1888.

2 Ibid,, 1889, p. 707.

A MANUAL OF BACTERIOLOGY

are first carefully washed with soap and water,
then with a boiling solution of potassium per-
manganate, to which a few crystals of oxalic acid
are added. They are then rinsed with distilled
water, and are allowed to drain on a rack for some
time, after which they are carefully plugged with

FIG. 17. CULTIVATION TUBES, FLASKS, ETC.

A, Gayou's Tube. B, Gayon and Dupetit's Tube. C, Chamber-land's Tube.
D, Sternberg's Bulb. E, Aitken's Tubes. F, Pasteur's Flask with Cap.
G, Pasteur's Bulb Pipette. H, Pasteur's Test-tube. I, Miquel's Double
Tube. J, Lipez's Tube. K, Miquel's Bulb Tube. L, Pasteur's Pipette
Flask. MO, Pasteur's Flasks. N, Lister's Flask. P. Chamber-land's Pipette.
R, Duclaux's Tube. S, Miquel's Filter Flask. T, Pasteur's Double Tube.

cotton-wool, care being taken that the wadding
inside the neck is perfectly smooth and firm, the
tuft outside being large enough to overlap well
the lip of the test-tube (Woodhead). They are

THE BACTERIOLOGICAL LABORATORY 43

then ready for the nutrient fluid and subsequent
sterilisation.

Fig. 17 F and H represent Pasteur's flask and
tube, both of which are provided with caps. The
narrow portion of each cap contains a plug of
cotton-wool. De Freudenreich's flask is somewhat
similar to that of Pasteur. These are used for the
cultivation of microbes in bouillon. In Pasteur's
pipette flask (Fig. 17 L), the tube above the bulb is
contracted twice, and on either sides of these con-
tractions there are plugs of cotton-wool. The portion
below the bulb is bent twice and is drawn out to a
capillary point. The flask is charged with bouillon
and inoculated by aspiration ; and then the capillary
point is sealed in the Bunsen flame. Miquel's1
bulb tubes (Fig. 17 K and i) are similar devices.

The tubes and flasks T, M, K, P (Fig 17) are pro-
vided with lateral arms drawn out to fine points,
and with necks plugged with cotton-wool. They
are filled by aspiration and are convenient for storing
sterilised bouillon. ' The sealed end of an arm is
nipped off with sterilised forceps, the sterile bouillon
aspirated into each limb, and the arm again sealed
in the flame; a series of such tubes and flasks can
be arranged upon a rack on the working table.'

Sternberg's bulbs (Fig. 17 D) are generally kept
in stock in the bacteriological laboratory. They
are readily prepared by blowing a bulb on a piece
of glass-tubing, and then drawing the tube out to
a fine point which is hermetically sealed. To fill
a bulb, it is first slightly heated, then the sealed

1 Lea Organismes Vivants de I' Atmosphere, 1883.

44 A MANUAL OF BACTERIOLOGY

point nipped off, and the open end dipped beneath
the surface of the culture fluid. As the bulb cools
the fluid is drawn into it. The neck of the bulb
is again sealed, and the fluid contained therein is
sterilised by repeatedly boiling the bulb in a water-
bath. It is then placed in an incubator for three
or four days. If the contents remain transparent
and clear, there is no doubt that the fluid has
been properly sterilised. Many of these bulbs,
containing sterilised bouillon, should be kept in
stock.

It may be mentioned that Chamberland's tube
(Fig. 17 c) is filled and sterilised in the same
way as Sternberg's bulb.

Sir Joseph Lister's flask (Fig. 17 N) is used for
the storage of culture fluids. The fluid is introduced
into the flask, the neck plugged with cotton-wool,
and the fluid sterilised by repeated boiling. When
a portion of the sterile fluid is required, all that
is necessary is to pour it through the lateral arm of
the flask : this is done by simply tilting the flask.
When the flask regains its erect position a drop of
the fluid remains behind in the fine opening of the
arm ; and thereby prevents the regurgitation of
unfiltered air. After the removal of a portion of
the fluid, a cap of cotton- wool is tied over the lateral
opening, and the residue in the flask is kept for
future use.

Aitken's tube (Fig. 17 E) is a modification of the
ordinary test-tube. It has a lateral arm whose ex-
tremity is hermetically sealed. The nutrient fluid
is introduced through the open end of the tube,

THE BACTERIOLOGICAL LABORATORY 45

which is then plugged with cotton-wool. The
fluid is sterilised by heating in the usual way ;
and is inoculated by nipping off the sealed end
of the lateral arm, and introducing the inoculating
needle through the orifice. The needle deposits
the material on the opposite side of the tube : it is
then withdrawn and the lateral orifice again sealed.
The fluid is then tilted so as to wash down the
inoculating matter. The inoculated tube is then
placed in an incubator.

The remaining tubes, flasks,
and pipettes (see Fig. 17) are
ati used in the cultivation of
microbes. Some are used for
storage purposes ; while others
are used as culture tubes,
flasks, etc. A very good stor-
age flask has been recently
described by Dr. Sims Wood-
head.1 This flask (Fig. 18) was
devised in order to do away FIG. is.

.., ., , , ,11 WOODHEAD'S STORAGE FLASK.

with the troublesome method
of filling test-tubes, etc., with a pipette. A large
flask (containing bouillon) is fitted with an india-
rubber stopper with two holes. ' Through these
pass two tubes, one with a thistle-head tube run-
ning to near the surface of the fluid, i.e. about
two-thirds of the distance down into the flask,
the other passing just through the stopper. To the
shorter tube is fitted a piece of india-rubber tubing

1 Proceedings of Royal Physical Society of Edinburgh, vol. ix.
p. 537.

46 A MANUAL OF BACTERIOLOGY

on which is a Mohr's clip, and to the other end
of this tubing is fitted a piece of glass tubing
with a constricted orifice. A plug of carefully
sterilised cotton wadding is pushed into the thistle-
head, the india-rubber stopper is pushed into the
neck of the flask, and then a sheet of cotton
wadding is placed over the whole of the tubes
and the mouth of the flask, and is held in position
by an india-rubber band. The flask is placed in a
steam steriliser, where it may be left for a sufficient
length of time to allow of it becoming perfectly
sterilised. It is filled nearly a third full with
bouillon or gelatine, after carefully removing the
sheet of wadding and the stopper ; these are then
replaced, and the whole is again sterilised as usual.
When the gelatine or bouillon is to be drawn off
into test-tubes, the flask is inverted and held in a
retort stand, the sheet of wadding is carefully re-
moved and folded, the glass nozzle is inserted into
the mouth of the test-tube, the clip is opened and
the gelatine or bouillon escapes ; all the air passing
into the flask, being filtered through the wadding
in the thistle-head tube, is thoroughly sterilised.
If the whole of the gelatine or bouillon is not with-
drawn, all that is necessary is to replace the sheet
of wadding (care having been taken to preserve
the inner surface, by folding it inwards). There is
no necessity to sterilise after this has been once
done, all that is necessary subsequently is to heat
sufficiently to render the peptonised gelatine fluid ;
but this is not required if the stock flask con-
tains bouillon. This apparatus is specially useful

if ' ^^

ITJiriVERSITYf

THE BACTERIOLOGICAL LABORATORY 47

for milk, as the cream always rises to the surface
and is so left to the last/

In all the vessels previously mentioned the
nutrient fluid is sterilised by heat ; but in certain
cases it is necessary to sterilise the fluid without the
application of heat : this is performed by means of
the apparatus devised by M. Chamberland, of the
Pasteur Institute. The fluid is forced by a hand-
pump through porous porcelain ; and by this means
it is sterilised.

In addition to the apparatus, etc., already men-
tioned in this chapter, a well - fitted laboratory
should contain: gas-burners with mica chimneys,
water-baths, hot-water filters, platinum needles,
wire cages for test-tubes, test-tube stands, glass
damp chambers, graduated cylinders, glass dishes
and capsules, thermometers, syringes, meat press,
' glass benches,' desiccators, anatomical jars, iron
box for glass plates, mouse cages, beakers, glass rods,
glass and india-rubber tubing, chemical balance and
weights, as well as the various nutrient materials,
stains, hardening, imbedding, and mounting mate-
rials, and chemical reagents. The last are useful
for the extraction and analysis of ptomaines and
similar bodies.

Although we have detailed the most important
pieces of apparatus for the bacteriological labora-
tory, there are others of importance, but as these
are only used for special purposes they will be
described later in the volume, i.e. in their proper
places.

48 A MANUAL OF BACTERIOLOGY

It only remains for us to say on this subject that
all the apparatus, etc., used by the English, French,
and German schools of bacteriologists may be ob-
tained from Messrs. F. E. Becker & Co., 33 Hatton
Wall, Hatton Garden, London.

CHAPTER III

THE METHODS OF CULTIVATING, STAINING, AND
MOUNTING MICROBES, ETC.

Cultivation Media. — There are two forms of media
used in the cultivation of microbes — one fluid ajid
the other solid. The fluid media were first used by
the French school, while the latter (i.e. the solid
media) were originated by Dr. E. Koch and his fol-
lowers. Both fluid and solid media have their own
special advantages, and both are now used in every
bacteriological laboratory.

Of the fluid media, the first to be described is
bouillon (beef, pork, or chicken broth). This
medium is prepared in the following manner : — one
pound of lean beef (pork or chicken) is minced by
passing it through an ordinary mincing or sausage
machine. The minced beef is thoroughly mixed
with lOOOcc. of distilled water, and the mixture
allowed to stand for twenty-four hours. It is again
thoroughly mixed, and then boiled for about an
hour. As the fluid is always more or less acid, it is ^
necessary to render it neutral or slightly alkaline,
this being done by the addition of a solution of pure
sodium carbooate. The point at which the fluid

A MANUAL OF BACTERIOLOGY

becomes neutral or slightly alkaline is easily ascer-
tained by the ordinary test-papers (litmus and tur-
meric). It is essential to neutralise any acids,
because they are well known to interfere with the
growth of many microbes. The extract so obtained
is strained through fine linen, and finally filtered
through Swedish filter paper. If the filtrate is still

acid, add a little more
sodium carbonate solu-
tion ; remove the fat by
skimming; add distilled
water to make up to the
original bulk; and again
filter (by means of a hot-
water filter, Fig. 19) into
a large storage flask or
into sterilised test-tubes
provided with sterilised
plugs of cotton -wool.
These vessels and their
contents are then heated
in the steam steriliser
for half-an-hour on each
of three successive days.
Sometimes the beef ex-

ria. iy. HOT-WATER *ILTEB.

tract or bouillon is modi-
fied by the addition of other materials. Dr. P. Miquel
adds common salt in such proportions as to make
a 0'5 per cent, solution. MM. Eoux and Nocard
add glycerine to the bouillon before it is finally
sterilised. This glycerine-bouillon is an excellent
medium for the growth of Bacillus tuberculosis.

THE METHODS OF CULTIVATING MICROBES 51

Liebig's extract (5 to 1000) and Cibil's extract of
beef (20 to 1000) may also be used for the same
purposes as bouillon; but these extracts require
very careful sterilisation by Professor Tyndall's
method of discontinuous heating.

Liquid blood serum is used in drop-cultivations,
etc. It is obtained by collecting the blood of a
healthy sheep, calf, or horse, in sterilised flasks or
glass cylinders with stoppers. The vessel or vessels
containing the blood are placed in an ice-box or in
ice-cold water for about twenty-four hours, when
the separation of the clot will be completed. The
fluid serum is then transferred, by sterilised pipettes
(see Fig. 1 7 G), into sterilised test-tubes provided
with cotton-wool plugs. The test-tubes and their
contents are then heated in a serum steriliser for an
hour or two at 60° C. on six successive days. Up
to this point the serum forms a fluid medium ; but
in the majority of cases blood serum is used as a
solid medium. To solidify it, the serum (contained
in test-tubes, watch-glasses, or capsules) is placed
in an inspissator, kept at a temperature between 65°
and 68° C., until solidification takes place.

Milk is also used as a fluid medium. It is best
sterilised at 120°C. in a steam digester or an auto-
clave. By this means it is readily sterilised in
about fifteen minutes. Milk can also be sterilised
in the steam steriliser at 100°C., but it is necessary
to heat it for an hour on the first day, and for thirty
minutes on each of the following two days, that is
(unless an autoclave is used), milk must be sterilised
by discontinuous heating.

52 A MANUAL OF BACTERIOLOGY

Various infusions of hay, wheat, cucumber, and
turnip, and decoctions of malt, prunes, raisins, and
horse-dung are used as cultivation media. They
are sterilised by being heated in the steam steriliser
for thirty minutes on three or four successive days.
The mucors grow well in decoctions of malt and
horse-dung ; various Aspergilli in a decoction of
malt and prune-juice ; and an infusion of hay is a
useful medium for the growth of Bacillus suUilis.

Urine and other fluids of the body are used as
cultivation media: these are sterilised after the
manner described for bouillon.

Besides the above-mentioned fluid media, there
are two others which are useful for the growth of
certain microbes and moulds. One of these is Pas-
teur's fluid, which contains 10 parts of pure cane-
sugar, 1 part of ammonium tartrate, the ash of 1
part of yeast, and 100 parts of distilled water. The
other is known as the Cohn-Mayer fluid, which con-
tains in 100 cc. of distilled water half a gramme each
of magnesium sulphate and potassium phosphate,
one gramme of ammonium tartrate, and 0*5 gramme
of tricalcium phosphate. Pasteur's and Cohn-
Mayer's fluids are sterilised by the method of dis-
continuous heating • or if they are placed in sealed
flasks and sterilised in an autoclave, the sterilisation
is complete in about fifteen minutes. Both of these
fluids are useful media for the cultivations of the
various species of Torulce or yeasts. Test-tubes,
flasks, etc., are filled with fluid media by means of
sterilised pipettes ; or, better still, the fluid media
can be run directly into the cultivation vessels by

THE METHODS OF CULTIVATING MICROBES 53

using Woodhead's storage flasks, which have already
been described. For the inoculation of various
media, pieces of platinum wire, either mounted in
sealed glass-tubing, or unmounted, are used. They
are sterilised by being heated in the Bunsen flame.
To inoculate any medium, the sterilised needle, or a
capillary pipette (Klein), is first dipped into the

A. :B.

-JT

FIG. 20. INJECTION SYRINGES.
(A, Dr. Petri's. B, Dr. Klein's. C, Dr. Koch's.)

inoculating substance, and then transferred to the
medium. Where Aitken's tubes and similar devices
are used the medium contained therein is inoculated
by using unmounted sterilised needles. These are
dipped into the inoculating substance and then
dropped into the fluid medium. To inoculate

54 A MANUAL OF BACTERIOLOGY

animals, platinum needles or injection syringes (Fig.
20) are used; but in every case these instruments
must be thoroughly sterilised before use. As Petri's
and Koch's syringes cannot be heated without de-
struction, they are sterilised by being immersed in
a solution of mercuric chloride or mercuric iodide ;
and after this these syringes should be washed with
sterilised hot water.

In addition to the above, ' glass needles are espe-
cially useful when anaerobic microbes are being
dealt with, as the smooth surface of the glass does
not allow of oxygen (air) being carried down with
it along the track, which closes up as soon as the
needle is withdrawn.'

We now proceed to describe the solid media
beginning with nutrient gelatine. This is made
according to the process already described for the
preparation of bouillon, except that after the filtra-
tion of the neutral or slightly alkaline fluid, 100
grammes of the best gelatine,1 10 grammes of pep-
tone (albumin), and 5 grammes of common salt are
added. The gelatine is allowed to soften and
dissolve gradually by gently heating the mixture in
a water bath. The nutrient gelatine is then steril-
ised as usual, and filtered into tubes or flasks where
it solidifies. The tubes and flasks being filled with
the nutrient gelatine must be sterilised in a steam
steriliser for a quarter of an hour on three successive
days. If these tubes show no signs of turbidity
after about a week's incubation, they may be con-
sidered sterile.

1 Coignet's gold label gelatine is the best for this purpose.

THE METHODS OF CULTIVATING MICROBES 55

Solid egg albumin is sometimes used as a cultiva-
tion medium. The white of an egg is poured on to
a slab of glass (sterilised), where it is coagulated and
sterilised by being heated in the steam steriliser,
or in the hot-air steriliser if tho temperature be
properly regulated. Solid egg albumin (Fig. 21) is

FIG. 21. MICROCOCCUS CHLORINUS.

Growing on sterilised white of egg, after a fourth attenuation.
(The white of egg coagulated and sterilised upon a slab of blackened glass.)

readily inoculated by means of a platinum needle
containing the inoculating material. Micrococcus
chlorinus grows very well on this medium.

Dr. F. Hueppe's method of cultivating on egg
albumin is different from the above. It is as
follows: — The shell is first disinfected with a solu-

A MANUAL OF BACTERIOLOGY

tion of mercuric chloride ; a hole is chipped at one
end of the egg, and the membrane cut through with
a pair of sterilised scissors. The exposed egg
albumin is inoculated by means of a platinum or
glass needle. The opening is covered with a piece
of sterilised paper or cotton wool, which is then
painted over and sealed with surgical collodion.
The egg is then placed in an incubator.

Cooked potatoes
are also used for cul-
tivation purposes.
The potatoes
(smooth -skinned)
are scrubbed and
the so-called eyes
removed by a sharp
knife. They are now
soaked for twenty
minutes in a solu-
tion of mercuric
chloride (1 in 1000);
washed in water,
and then cooked in
a steam steriliser for
thirty minutes. After cooling, the potatoes are cut
by a knife previously sterilised in the naked flame,
or in Israel's box placed in a hot-air steriliser. The
potatoes are cut 1 through the middle, and the two
halves of each potato are then placed in previously
sterilised damp chambers (Fig. 22).

1 The hands during this operation should have been pre-
viously dipped into a solution of mercuric chloride.

FIG. 22. DAMP CHAMBER.
(For plate-cultivation, etc.)

THE METHODS OF CULTIVATING MICROBES 57

The potatoes are inoculated by means of a plati-
num needle or scalpel containing the inoculating
material which is streaked over the surfaces of the
potatoes. Second, third, and fourth attenuations
may be made from potato-cultivations. Sometimes
these cultivations require placing in an incubator,
while at other times the growth readily forms at
the ordinary temperature of the laboratory. Potatoes
form a good medium for the cultivation of numerous
microbes, especially the putrefactive and chromo-
genic forms.

Another solid medium for the cultivation of
microbes is agar-agar.1 This substance is an excel-
lent substitute for nutrient gelatine ; for the latter
melts at about 26° C., consequently it cannot be
used for the cultivation of certain microbes requir-
ing a much higher temperature for their proper
growth and development. Agar-agar remains solid
up to 50° C. Sterilised nutrient agar-agar is pre-
pared by a similar method to the one already
described for preparing nutrient gelatine, with the
exception that 20 grammes of agar-agar are used
instead of the 100 grammes of gelatine. Although
nutrient agar-agar remains solid up to 50° C., it is
surpassed in this property by blood serum. Blood
serum solidifies at 70° C., and always remains solid.
The method for preparing solid blood serum has
already been described. Both nutrient agar-agar
and solid blood serum are suitable media for the
growth of certain microbes requiring a higher tem-
perature than usual.

1 Consists of the dried fragments of certain Algae.

58 A MANUAL OF BACTERIOLOGY

A good medium for the growth of chromogenic
microbes is made of ground rice. The late Dr.
Isidor Soyka's formula for the preparation of this
medium is as follows : — 1 0 grammes of ground rice,
15 cc. of milk, and 5 cc. of neutral beef bouillon.
These ingredients are made into a paste, which is
transferred to covered glass dishes or small flasks.
The dishes or flasks are then sterilised (in the steam
steriliser) for half an hour on three successive days.

Bread-paste is also used as a medium for the
cultivation of microbes. It is prepared in the fol-
lowing way : — The crumb of a loaf is broken into
small pieces, dried in an oven, and rubbed through
a fine sieve. The finely-divided bread is then placed
in a sterilised flask, to the depth of half an inch,
sterilised water being added until the bread is
thoroughly moistened. After replacing the cotton-
wool plug the flask (or flasks) is sterilised in the
steam steriliser for the same length of time as rice-
paste. The flask containing either bread- or rice-
paste can be reversed, and is readily inoculated by
means of a platinum needle.

To inoculate solid culture media ' the test-tube or
flask is held inverted in the left hand, and the plug
of cotton wool is twisted once or twice in the mouth
of the test-tube to break down any adhesions
between it and the neck of the vessel. If the plug
is at all dusty, it is well to singe the surface by
passing it rapidly through a flame before removing
it from its position. The plug is removed and held
between two of the unoccupied fingers of the left
hand, great care being taken that no part of the

THE METHODS OF CULTIVATING MICROBES 59

plug that passes into the test-tube shall come in
contact with any source of infection other than the
air itself. At the same time this portion of the plug
is directed downwards, in order to avoid any falling
germs that may be present in the atmosphere. The
platinum or glass needle, with its charge of seed
material, is plunged straight into the gelatine mass,
then carefully withdrawn and the plug replaced.
Where the seed material is also in solid gelatine, the
two tubes may be held inverted in the left hand,
one between the thumb and finger, the other between
the first and second, the plugs being held between
the second and third and third and fourth fingers '
(Woodhead).

The macroscopical appearances of the test-tube
cultivations should always be noted, for many
microbes give rise to characteristic growths. Some
microbes wholly or partially liquefy the nutrient
medium, while others have not this property ; but
may give rise to pigments, etc., in the medium or
media in which they are growing.

Cultivation Methods. — If the original fluid under
examination contains different microbes, and it is
desired to separate them, so as to obtain pure culti-
vations of one or all of the microbes present in the
original fluid, one of three methods may be used for
this purpose. The three methods are known as —
plate-cultivations, fractional cultivations, and the
dilution method.

In order to utilise the method of plate-cultivation,
about three tubes containing sterilised nutrient
gelatine or agar-agar are placed in a water-bath

60 A MANUAL OF BACTERIOLOGY

heated to 40° C. or 55° C. respectively, so as to melt
the medium in each tube. The tubes are then care-
fully inoculated with a mere trace of the original
fluid. The cotton-wool plugs are replaced, and the
tubes rolled about so as to distribute the microbes
throughout the media. The contents of the tubes
are quickly poured into the lower portion of the
same number of Dr. Petri's double dishes (Fig. 23 B)
or glass plates (Fig. 23 A). The dishes or plates
(which should have been previously sterilised) are
then placed in a damp chamber (see Fig. 22). The
damp chamber, with its contents, are removed to an

FIG. 23. APPARATUS FOR PLATE-CULTIVATION.
A, Glass Bench with'Plates. B, Petri's Double Dish.

incubator, and remain there for several days at
about 23° C., or higher if agar-agar is used (i.e.
according to the temperature required for the
growth of the microbes).

In a few days or so each species will have
started a separate growth or colony in different
parts of the solidified plate of nutrient gelatine, or
agar-agar. The individual colonies are recognisable
according to certain macroscopical appearances, such
as colour, shape, liquefaction or non-liquefaction of
the medium, and the size of the colonies. By plate-
cultivation the different species of microbes (i.e. in

THE METHODS OF CULTIVATING MICROBES 61

a microbian mixture) separate themselves from each
other ; and from these colonies pure cultivations of
each microbe may be obtained by carefully re-
inoculating a number of tubes containing sterilised
nutrient gelatine or agar-agar. Plates of gelatine or
agar-agar (Fig. 24) may also be reinoculated in a

FIG. 24. A PLATE-CULTIVATION OF SPIRILLUM TYBOOENUM.

A, Colonies growing on nutrient gelatine (sterilised).

B, The spirillum x 1265.

similar manner. First, second, and third attenua-
tions may be obtained by this mode of cultivation.
Both the macroscopical and microscopical appear-
ances should be noted. To examine the growth
under low power one of the plates should be placed
upon the stage of the microscope, and the appear-

62 A MANUAL OF BACTERIOLOGY

ances carefully observed under Zeiss' B, C, and D
objectives, or any similar low powers. After this a
cover-glass preparation should be made by rubbing
a needle, previously dipped into the growth on the
plate, on a clean cover-glass. A drop of sterilised
water is now added; the cover-glass is allowed to
dry ; then passed three times through the Bunsen
flame ; and finally stained with a drop of fuchsine
or some other aniline colour. The cover-glass pre-
paration should be temporarily or permanently
mounted, according to the methods described later
in this chapter. After mounting, the preparation
should be examined under high powers, such as
Zeiss' J and T^. It should be borne in mind that
the eye has to be trained in order to see objects
distinctly with such high powers ; but, it may be
remarked, that ' in all extremely delicate work with
high-power lenses, the first difficulty is the greatest.
If once an object has been seen, however difficult, it
is immensely easier to see it again. On the other
hand, there is as great a diversity in different
individuals in the sensitiveness of the retina, as there
is in the sensitiveness of the olfactory, or auditory
nerves. It is impossible to enable some persons to
see objects beyond a certain limit of minuteness ; as
it is to enable others to detect certain scents, or hear
notes pitched higher or lower than a given point.'

The fractional cultivation method consists in the
attempt to isolate, by successive cultivations, the
different organisms that have been growing pre-
viously in the same culture. A number of tubes
containing various cultivation media (sterilised) are

THE METHODS OF CULTIVATING MICROBES 63

inoculated with a mere trace of the original microbian
mixture, and are then placed in an incubator for a
couple of days or so. It will then be noticed that
the different species of microbes (sown in each tube)
will not have increased equally in numbers in all
the tubes (due, of course, to the nature of the
medium, the temperature, and the period of incuba-
tion). It is possible that only one species will have
developed, so far, in each tube. With these tubes
a similar number of tubes are re-inoculated, and so
on. By this fractional method of cultivation pure
growths are ultimately obtained. For further
information concerning this method the reader is
referred to Dr. Kleb's paper in the Archiv fur
Exper. Pathologic, 1873.

The dilution method consists in greatly diluting a
drop of the original microbian mixture with some
sterile saline solution (0*5 °/0). A series of tubes,
containing different cultivation media (sterilised),
are each inoculated, by means of a platinum needle
or glass pipette, with a mere trace of the diluted
mixture. After about thirty hours' incubation,
growths (most likely of one species only), make
their appearance in some of the tubes. The original
microbian mixture or fluid maybe diluted a thousand-
or even a million-fold, if the original fluid teems
with different microbes. The dilution method has
been largely used by Dr. P. Miquel in his examina-
tions of the different waters in and around Paris.

By the fractional, dilution, and plate methods,
cultures containing many different species of
microbes are capable of being separated one from

64 A MANUAL OF BACTERIOLOGY

another. Sometimes a combination of the fractional
and dilution methods is used for the same purpose.
The methods of cultivating anaerobic microbes
are somewhat different from the above ; as the air
I must be excluded from the cultivation apparatus.
In the cultivation of Bacillus cholerce Asiaticce, Koch
made use of plate cultivations on which very thin
sheets of glass or mica were placed before the
gelatine was perfectly set. By this means the
colonies of microbes grow out of contact with the

air.

A second method for excluding air (i.e. oxygen)
is to allow the microbes to grow under the receiver
of an air-pump which has been exhausted of air.

A third method is to allow the microbes to grow
in an atmosphere of carbonic anhydride or hydrogen
gas. Another method consists in inoculating a
cultivation tube with the anaerobic microbe, and
then covering the surface of the medium with a
layer of sterilised oil.

Dr. Eoux has also devised two methods for this
object. One of these methods is to fill a sterilised
pipette with sterilised nutrient gelatine. Both ends
of the pipette are hermetically sealed. To inoculate
the gelatine, one end of the pipette is nipped* off,
the inoculating material introduced, by a fine glass
needle, into the gelatine, and finally the open end
of the pipette is again sealed. By this device the
microbes grow anaerobically. The second method
of Dr. Eoux is to boil a quantity of agar-agar in a
test-tube ; and after quickly cooling, the medium is
inoculated with the anaerobic microbe. A layer of

THE METHODS OF CULTIVATING MICROBES 65

melted nutrient gelatine is now poured on the
surface of the agar-agar, and when it is cooled a
drop of a bouillon cultivation of Bacillus siibtilis is
run on to the surface from a capillary pipette. The
tube is then sealed, or the cotton-wool plug is
rendered impervious by being luted with warm
paraffin- wax. The object of growing Bacillus subtilis
is that it uses up the oxygen at the surface ; con-
sequently the microbe below receives none, or, in
other words, it is able to grow anaerobically. To
obtain inoculating material from Roux's tube, it is
broken at the bottom and a sterilised needle inserted
into the lower growth.

FIG. 25. DROP-CULTURE CELL.
(With arrangement for admitting Gases into the Cell)

In place of the Bacillus subtilis, the layer of
nutrient gelatine is covered with a solution contain-
ing one part of pyrogallic acid to ten parts of a
solution of potassium hydroxide (10 per cent.). The
potash solution of pyrogallic acid may be replaced,
with advantage, by a 3 per cent, solution of ferrous
sulphate, or a 2 per cent, solution of cuprous
chloride ; both of these compounds (the author has
found) prevent the entrance of air.

A drop culture forms a useful method for study-
ing the growth and multiplication of microbes under
low or high power objectives. For this purpose a
glass cell is required. This is made by cementing

66 A MANUAL OF BACTERIOLOGY

a sterilised glass ring ( j in. diam. x | in. high) to a
microscopic slide, which has been thoroughly cleaned
and sterilised (Fig. 25). The upper edge of the ring
is moistened with olive oil or vaseline ; and the cell is
covered over by means of a thin cover-glass, previously
sterilised by passing it through a Bunsen flame.
The surface of the sterilised cover-glass (A) contains
a drop of bouillon or other medium, along with the
microbes for examination. A drop or two of
sterilised water should be deposited at the bottom
of the cell ; i.e. upon the upper surface of the glass
slide. This arrangement forms a miniature damp
chamber, in which the growth of microbes may be
watched even under the highest powers. After the
examination of the cell and its contents, it may be
placed in an incubator until it is required again for
microscopical examination.

To study the action of heat on drop cultures, the
warm stages of Schafer, Eanvier, Israel, Schultze,
Strieker, etc., are often used upon the fixed stage of
the microscope.1 The action of various gases on
drop-cultures may be watched by a modification of
the glass cell as represented in Fig. 25. The gases
enter through B. The author has used this device
during his researches on the action of certain gases
on Bacillus tuberculosis. The action of the voltaic
current or discharges of faradaic electricity may be
observed by simple modifications of the drop- culture
cell.

1 An excellent piece of glass apparatus is used by the Rev.
W. H. Dallinger, F.R.S., for ascertaining the thermal death
point of microbes. (See Proc. Roy. Soc., 1878.)

THE METHODS OF CULTIVATING MICROBES 67

Drop-cultures form ready means for studying the
complete life-history of any microbe.

The methods for examining fluids and fresh tissues
are as follows: (1) blood, urine, saliva, pus, tears,
culture-fluids, and other liquids containing microbes,
are easily examined microscopically by placing a
drop of the liquid on a glass slide and covering it
with a thin cover-glass; (2) when microbes for
examination are growing on plates of nutrient
gelatine, a small portion of the culture should be
taken up on a sterilised platinum or glass needle
and placed in a drop of sterilised water on a glass
slide. After thinning, the preparation is covered
with a cover-glass and examined under low and
high powers ; (3) for the microscopical examination
of fresh tissues, they should be teased out with
sterilised needles in dilute glycerine or salt solution
(sterilised), then temporarily mounted, in either
liquid, on a glass slide and covered with a thin
cover-glass. If there is an excess of glycerine or salt
solution round the edges of the cover-glass, it must be
removed by placing small pieces of filter or blotting-
paper in contact, which will soon absorb the super-
fluous fluid, but the paper must not be left too long
or it will drain the fluid from under the cover-glass.
In the examination for micrococci and other small
microbes, the tissues should be first treated with
acetic acid, and then with a solution of potassium
hydroxide (potash), the object being to dissolve and
disintegrate fatty and albuminous globules which
might be mistaken for microbes. Alcohol and ether are
also useful agents for dissolving small globules of fat.

68 A MANUAL OF BACTERIOLOGY

It has been recorded that a certain foreign medi-
cal professor mistook minute globules of fat for so
many micrococci ; and certainly the illustrations in
his paper indicated that such was really the case.
Therefore, let all bacteriologists, young and old, be
very sceptical at times as to what they think they
see with the highest powers of the microscope.
Without wishing to detract an iota from the honesty
of purpose and truth of our fellow-workers, we are
sure that a good deal unintentionally has been said
to have been seen with the microscope which has
never been seen at all. We set to work longing to
discover something newer than the last new thing.
We hope to find it, we begin to think we have
found it, and we may go so far as to make ourselves
believe we really did see it once. The event must
be recorded ; we proclaim it, and in so doing pro-
pagate error. Therefore, let it be borne in mind
that to use the highest powers with accuracy re-
quires continual practice ; even when the retina of
the eye is sensitive enough to appreciate light-waves
proceeding from such organisms as the smallest
micrococci.

Staining Cover-glass Preparations and Tissues. — To
prepare a cover-glass preparation for staining, a
sterilised cover-glass is smeared with the microbian
matter (solid or liquid), or with blood, pus, etc., by
means of a sterilised needle or capillary pipette.
The excess of material is squeezed out by means of
an additional cover-glass placed over the original
one. The two glasses are then separated, each bear-
ing a small portion of the microbian matter. After

THE METHODS OF STAINING MICROBES 69

drying for a few minutes, they are passed rapidly
(three or four times) through a Bunsen flame. To
stain the preparations they are allowed to float
(with the prepared side downwards) on the surface
of an aqueous solution of methyl violet, gentian
violet, or magenta, for a short time. After this the
cover-glasses are washed with water, then spirit,
and finally with sterilised distilled water. They
are then drained, dried, and mounted in Canada
balsam or any suitable medium. The preparation
must now be set aside to dry, and when thoroughly
dry it is ' ringed ' or sealed with Hollis' glue.

Before continuing the description of the various
methods of staining, we describe the preparation of
several staining fluids : — (1) Gentian violet stain is
prepared by rubbing 2 grammes of gentian violet in
a glass mortar with 10 cc. of alcohol (sp. gr., 0*83),
in which has been dissolved 2 cc. of aniline oil. To
this is added 90 cc. of distilled water ; (2) Koch's
methyl violet stain contains the following ingre-
dients:— Aniline water, 100 cc. ; an alcoholic solu-
tion of methyl violet, 11 cc. ; and absolute alcohol,
10 cc. ; (3) the stain known as Bismarck brown is
prepared by dissolving 2 grammes of Bismarck
brown in 15 cc. of alcohol, and then adding 85 cc.
of distilled water ; (4) haematoxylin solution con-
tains 2 grammes of haematoxylin, 2 grammes of
alum, and 100 cc. each of alcohol, glycerine, and
distilled water; (5) the methylene blue stain is
prepared by dissolving 2 grammes of methylene
blue in the same quantities of alcohol and water as
are required to prepare the Bismarck brown stain ;

70 A MANUAL OF BACTERIOLOGY

(6) Eanvier's picro-carmine stain contains 1 gramme
of carmine, 3 cc. of ammonia, 10 cc. of distilled
water, and 200 cc. of a cold, saturated solution of
picric acid ; (7) vesuvin stains are prepared by dis-
solving 3, 4, or 5 grammes of vesuvin in 100 cc. of
distilled water ; (8) Dr. Gibbes' solution for double
staining contains 2 grammes of magenta and 1
gramme of methyl violet, which are triturated in a
glass mortar with 15 cc. of alcohol (in which has
been dissolved 3 cc. of aniline oil). To this mixture
is added 15 cc. of distilled water; (9) Gram's iodine
solution is prepared by dissolving 1 gramme of
iodine and 2 grammes of potassium iodide in 300
grammes of distilled water; (10) LofHer's stain con-
tains 30 cc. of a concentrated alcoholic solution of
methylene blue, and 100 cc. of an aqueous solution
of potassium hydroxide (1 in 10,000) ; (11) an eosin
solution is prepared by dissolving 5 grammes of
eosin in 100 cc. of distilled water.

We now continue the description for staining
microbes and tissues. To stain tissues containing
microbes, place them in either an aqueous solution
of methyl violet (2 -2 5 grammes in 100 cc. of water),
or one of gentian violet (containing the same
strength of solution), and allow them to remain in
the solution for some hours. When deeply stained,
wash in water to remove the excess of the stain,
and then lay them out flat in methylated spirit, and
let them remain until no more colour comes away.
Transfer them to absolute alcohol, and then oil of
cloves, and mount in Canada balsam (Gibbes).

To double stain bacilli which produce spores, the

THE METHODS OF STAINING MICROBES 71

cover-glass preparation should be floated for half
an hour on the surface of a small quantity of hot
magenta and aniline stain.1 The magenta is dis-
charged from the bacilli by washing in water, in
alcohol, or weak nitric acid, according to the species.
The preparations are then treated (for three or four
minutes) in a solution of methylene blue, and
finally washed with water, drained, dried, and
mounted in Canada balsam or other mounting
media. By this method the spores are stained red,
while the bacilli are blue.

Koch's method for staining tubercle bacilli is as
follows: — Cover-glass preparations of the sputum,
etc., are placed in a solution containing 1 part of a
concentrated solution of methylene blue, 2 parts of
a potash solution (10 per cent.), and 200 parts of dis-
tilled water. The preparations remain in the solution
(heated to 40° C.) for twenty-four minutes. They
are then washed in water, and placed in an aqueous
solution of vesuvin for two or three minutes ; again
washed, and subsequently treated with alcohol, oil
of cloves, and finally mounted in Canada balsam.
Koch's method stains the bacilli blue, and the
nuclei, etc., brown. ' All the other forms of bacteria
which Koch has as yet examined in this way are
stained brown, with the exception of the bacilli
found in leprosy, which also retain the methylene
blue in preference to the vesuvin. These bacilli

1 This stain is prepared by mixing together 5 cc. of aniline
oil and 100 cc. of distilled water. The mixture is filtered, and
to the filtrate is added a concentrated alcoholic solution of
fuchsine or magenta, until a precipitate begins to ba formed.

72 .A MANUAL OF BACTERIOLOGY

may also be stained by other aniline dyes if the
solution be made alkaline by the addition of caustic
potash or soda.'

To stain the flagella of certain microbes, Koch
recommends that the cover-glass preparations should
be floated on a concentrated aqueous solution of
hsematoxylin. They are then transferred to Muller's
fluid,1 or to a five per cent, solution of chromic acid.
By using either of these reagents the flagella are
stained a brownish-black colour.

On the other hand, Dr. Dallinger 2 does not think
that Koch's method of staining brings out the
flagella well. Dallinger uses high powers and the
microbes alive.

Dr. Crookshank has, however, succeeded in photo-
graphing the flagella by staining with a concentrated
alcoholic solution of gentian violet. The prepara-
tion is then rinsed in water, dried, and mounted in
Canada balsam.

Gram's method for staining microbes in tissues is
as follows : — The sections containing the microbes are
soaked in absolute alcohol for twelve minutes, and
then placed in a gentian-violet-and-aniline solution 3
for about three minutes. The sections are then
placed in a solution of iodine (in potassium iodide)
for several minutes, or until they are of a brown
colour. After this they are transferred to absolute

1 This fluid contains 2 grammes of potassium bichromate,
1 gramme of sodium sulphate, and 100 cc. of distilled water.

2 Journal of Royal Microscopical Society, 1878, p. 172.

3 This is similar to Koch's methyl-violet-and-aniline stain,
except the methyl violet is replaced by gentian violet.

THE METHODS OF STAINING MICROBES 73

alcohol until decolourised ; they are then placed in
oil of cloves, and finally mounted in Canada balsam.
As Gram's method only gives a faint colour to
the tissues, they may be stained a deeper colour
by immersing the sections (after decolourising with
alcohol) in an aqueous solution of vesuvin, eosin, or
Kanvier's picro-carminate of ammonia. They are
finally washed in alcohol, and mounted as already
described.

One of the best methods for staining cover-glass
preparations is the one devised by Ehrlich. The
cover-glass preparations are made to float (with the
prepared face downwards) in a solution of fuchsine
made in the following manner : 5 cc. of aniline oil
and 100 cc. of distilled water are mixed together and
filtered. To the filtrate is added a concentrated
alcoholic solution of fuchsine. The preparations re-
main in this solution for fifteen minutes ; they are
then washed in nitric acid (one part of nitric acid to
two parts distilled water) and rinsed in distilled
water. An after-stain of methylene blue or vesuvin
gives the nuclei, etc., a blue or brown colour, while
the tubercle-bacilli or other pathogenic microbes are
stained red. The elegance of this method is that
the tubercle-bacilli impregnated with fuchsine resist
the action of nitric acid, whilst the saprophytic
microbes (present in sputum and saliva), nuclei,
etc., are immediately decolourised by the acid.
Both Ehrlich's and Koch's methods are also applic-
able for staining tubercular and other tissues.

The Ehrlich- Weigert is another method for stain-
ing microbes in situ. The tissues are placed in a

74 A MANUAL OF BACTERIOLOGY

warm solution of aniline-methyl-violet,1 and then
decolourised with nitric acid (one in two). The
tissues may be stained brown by immersing them in
an aqueous solution of Bismarck brown or vesuvin.
In this case the microbes are blue and the tissues
brown. Other aniline colours may be used, but the
decolouriser is nitric acid. The stained sections are
washed, cleared in oil of cloves, and then mounted
in Canada balsam.

In the Baumgarten method cover-glass prepara-
tions of sputum are placed in a very dilute solution
of potassium hydroxide (potash), and after being
slightly pressed on the microscopic slides they are
ready for examination. By this method the bacilli
(tubercle) are seen in the unstained condition. This
is a quick method of examining phthisical sputum,
as it does not take more than ten minutes.

Gibbes' rapid double-staining method is applicable
for staining sections as well as cover-glass prepara-
tions. No decolourising agent is used, while the
double-staining process is performed in one opera-
tion. The preparations are allowed to remain in a
warm aniline-magenta-methyl- violet solution for five
minutes, or in the case of sections for several hours.
They are washed in methylated spirit until no more
colour comes away. The preparations are now
dehydrated in absolute alcohol, dried and mounted
in Canada balsam dissolved in xylol. By this

1 The above solution is prepared by mixing together 100 cc.
of a saturated aqueous solution of aniline and 11 cc. of a satu-
rated alcoholic solution of methyl violet. The filtered mixture
is the Ehrlich-Weigert stain.

THE METHODS OF STAINING MICROBES 75

method the tubercle-bacilli and certain other patho-
genic microbes are stained red, while the putrefac-
tive bacteria and micrococci are blue. This method
is a rapid one, and is, consequently, recommended
for the busy medical man.

The Ziehl-Neelsen method of staining the tubercle-
bacilli is a modification of the Ehrlich-Weigert
method already described. The cover-glass prepara-
tions or sections are stained in the following dye :
1 gramme of fuchsiue is dissolved in 10 cc. of
absolute alcohol, and to this is added 100 cc. of an
aqueous solution of carbolic acid (5 per cent.). The
mixture is then heated. In the hot dye sections are
stained in six or seven minutes ; and cover-glass
preparations are stained in about three minutes.
The preparations or sections are now placed for a
second or so in 90 per cent, alcohol, then in dilute
sulphuric acid (25 per cent.), when the pink colour is
replaced by a yellowish brown. The preparations,
etc., are then transferred to a solution of lithium
carbonate. They are afterwards stained in an
aqueous solution of methylene blue, cleared in oil
of cloves, and mounted in Canada balsam. This
method (also known as the carbol-fuchsine method)
gives excellent results.

To ascertain the presence of tubercle-bacilli in
tuberculous milk, the best plan is to pass the milk
through one of the ordinary centrifugal machines
used in the dairy ; and then to take the sediment
(after the separation of the cream and skim milk)
for examination. In lieu of a centrifugal machine,
the milk should be allowed to stand for about

A MANUAL OF BACTERIOLOGY

twenty-four hours in a chemical separator (Fig. 26)
surrounded with ice. The sediment (containing the
bacilli) is drawn off from the separator by means of
a tap (see Fig. 26) ; and a few drops of the sediment
are dried on a cover-glass, and examined in the
ordinary way.

Dr. W. Ktihne's methylene blue method is one of
the best means of staining for general purposes. It
^ is prepared by dissolving 1'5
grammes of methylene blue in 10
cc. of absolute alcohol; and 100
cc. of an aqueous solution of
carbolic acid (5 per cent.) are
added. Preparations are stained
in this dye from five minutes to
two hours ; and sections remain
in it for twenty-four hours. They
are washed in water, followed by
acidulated water,1 and are then
transferred to a solution of
lithium carbonate (5 per cent.).
They are again washed in water, dehydrated in abso-
lute alcohol, placed in aniline oil, and transferred
to terebene for two or three minutes. After
this treatment the preparations are washed in xylol,
and finally mounted in Canada balsam. This stain
is useful for the bacilli of leprosy, glanders, tuber-
culosis, arid almost any microbe.

Cover-glass preparations of anthracic blood, etc.,
are floated on a hot alcoholic solution of fuchsine

FIG. 26.
CHEMICAL SEPARATORS.

1 Two or three drops of hydrochloric acid to 100 cc. of
distilled water.

THE METHODS OF STAINING MICROBES 77

for thirty minutes. They are then decolourised in
weak hydrochloric acid, and after - stained with
methylene blue. By this means the spores are
stained red and the bacilli blue.

Anthrax- bacilli and spores may also be stained
with an aqueous solution of gentian violet, fuchsine,
or any of the aniline dyes ; if the cover-glass pre-
paration is first passed ten or eleven times through
the Bunsen flame.

Sections of anthracic tissues are well stained by
Gram's method, and after - stained with picro-
carminate of ammonia, or eosin.

The bacillus of glanders is stained by the method
of Schlitz. The sections are placed in an alcoholic
potash solution of methylene blue 1 for twenty-four
hours. They are then washed in acidulated water,2
transferred for five minutes to 50 per cent, alcohol,
ten minutes to absolute alcohol, clarified in oil of
cloves, and finally mounted in Canada balsam. As
already stated, the bacillus of glanders (Bacillus
mallei) may be stained by Klihne's methylene blue
method.

There are three principal methods for staining the
bacillus of syphilis. (1) Lustgarten's method con-
sists in placing the sections of syphilitic tissues, etc.,
for about twenty-four hours in a solution containing
100 cc. of aniline-water (5 per cent.) and 11 cc. of a
saturated alcoholic solution of gentian violet. They
are now heated for two hours at 60° C. After this

1 This stain contains equal parts of a concentrated alcoholic
methylene blue solution and a solution of potash (1 in 10,000).
- Water containing 5 per cent, of acetic acid.

78 A MANUAL OF BACTERIOLOGY

treatment, the sections are placed for three or four
minutes in absolute alcohol, transferred to a solution
of potassium permanganate (1*5 per cent.) for ten
minutes, and decolourised by immersion in con-
centrated sulphurous acid. The sections are then
dehydrated in absolute alcohol, clarified in oil of
cloves and mounted in Canada balsam. (2) The
next method is that of Doutrelepont and Schiitz.
The sections of syphilitic tissues containing the
bacilli are immersed in an aqueous solution of
gentian violet (1 per cent.), and are after-stained
with an aqueous solution of safranin (1 per cent.).
(3) The last method is that of De Giacomi, in which
the preparations are immersed in a hot solution of
fuchsine containing a drop or two of ferric chloride.
They are then decolourised in a concentrated solu-
tion of ferric chloride, and after - stained with
Bismarck brown or vesuvin. In both the Doutrele-
pont-Schiitz and De Giacomi methods, the prepara-
tions (after staining) are dehydrated, clarified, and
mounted in the usual way.

Sections of tissues containing the Bacillus leprce
are stained by immersion in a solution of fuchsine
in aniline-water. They are then decolourised in
hydrochloric acid (33 per cent.), and after-stained
with methylene blue. Another method is, first to
tie a piece of thread around the base of one of the
leprosy nodules, so as to cut off the blood supply ;
then with a fine-pointed scalpel (see Fig. 5) a small
puncture is made, when a clear fluid exudes. From
this fluid, cover-glass preparations are made. Cover-
glass preparations and sections of leprosy tissues

THE METHODS OF STAINING MICROBES . 79

may be stained by the methods of Ehrlich, Ziehl-
Neelsen, and Gram.

The method (devised by Dr. Loftier) for staining
the Bacillus diphtherice consists in placing the
sections in Loffler's alkaline methylene blue (already
described) for about five minutes. The excess of
stain is removed by very dilute acetic acid (0'5 per
cent). They are then dehydrated in alcohol, clari-
fied in cedar oil, and mounted in Canada balsam.
Sections may also be stained by Gram's method;
and Dr. Klein has produced beautiful stained sec-
tions of diphtheritic membranes * by staining them
with rubin 2 and methyl blue. By this method the
bacilli are stained blue, while the nuclei and necrotic
substances of the membranes are stained red.

To stain the Bacillus typhosus (the microbe of
typhoid fever), there are several methods in use.
For tissue-staining, the method of Gram may be
used. Some bacteriologists recommend steeping
the sections for twenty-four hours in methylene
blue; but this stain possesses the disadvantage of
quickly fading. The colour, however, may be fixed
by placing the sections either in a solution of picro-
carminate of ammonia, or of iodine dissolved in
potassium iodide, or in ammonium picrate. Dr.
Kiihne's method consists in allowing the sections to
remain for some time in a concentrated aqueous
solution of oxalic acid, washing them in water, and
afterwards staining with methyl blue dissolved in a

1 See Report of Medical Officer of the Local Government Board,
1889-90, p. 143.

2 Rubin is rosaniline nitrate.

80 A MANUAL OF BACTERIOLOGY

solution of ammonium carbonate (1 per cent.). To
demonstrate the spores of this bacillus, cover-glass
preparations and tissue-sections must be placed
in a hot solution of fuchsine. They are then
decolourised with nitric acid (see Ehrlich's method),
after-stained with methylene blue, and mounted as
usual, after dehydration and clarification in the
media already described.

The most important methods for staining the
Micrococcus pneumonice are as follows: — (1) By the
method of Gram. (2) Cover-glass preparations of
pneumonic sputum and exudations are treated
with acetic acid, stained with gentian violet, and
temporarily mounted in distilled water, or water
and glycerine, i.e. for immediate examination; or
they may be dried and permanently mounted in
Canada balsam.

Cover-glass preparations of gonorrhceal pus,
blood, or of artificial cultivations of the Micrococcus
gonorrhcece are readily stained with an aqueous
solution of fuchsine. This method may be also
used for demonstrating the presence of the same
micrococcus in the tears of new-born infants suffer-
ing from purulent ophthalmia of gonorrhoeal origin.

The cholera bacillus (Bacillus cholerce Asiaticce) is
stained by the following methods : (1.) The dis-
charges, etc.. containing the microbe are spread and
dried on a cover-glass. They are then stained with
an aqueous solution of fuchsine, washed with water,
dried, and mounted in Canada balsam. (2.) The
hardened sections x of the intestines are placed for

1 Hardened in absolute alcohol.

THE METHODS OF STAINING MICROBES 81

twenty-four hours in a strong aqueous solution of
inethylene blue ; and finally treated in the usual
way. (3.) 'The best method yet described of de-
monstrating the cholera bacillus in the discharges
of the intestines is that recommended by Cornil
and Babes, who spread out one of the small white
mucous fragments on a microscopic slide, and then
allow it to dry partially ; a small quantity of an
exceedingly weak solution of methyl violet in dis-
tilled water is then flowed over it, and it is flat-
tened out by pressing down on it a cover-glass,
over which is placed a fragment of filter paper,
which absorbs any excess of fluid at the margin
of the cover-glass. Cholera bacilli so prepared
and examined with an oil-immersion lens
(Zeiss' ^o homog., Oc. 3 or 4) may then be seen ;
their characters are the more readily made out
because of the slight stain they take up, and
because they still retain their power of vigorous
movement, which would be entirely lost if the speci-
men were dried, stained, and mounted in the
ordinary fashion/

For staining cover-glass preparations of the blood
of patients suffering from relapsing fever (i.e. con-
taining the Spirillum Obermeieri), fuchsine, gentian
violet, and Bismarck brown have been used with
considerable success. Sections of the brain, liver,
lungs, kidneys, etc., of monkeys or human beings
dead of the disease, are best stained with Bismarck
brown, vesuvin, or chrysoidine.

Cover-glass preparations of the blood, exuda-
tions of the throat, etc., from cases of scarlatina

82 A MANUAL OF BACTERIOLOGY

(i.e. containing the Micrococcus scarlatince) are stained
with a saturated solution of methyl violet. The
micrococci, adhering to the scales of the desqua-
mating epidermis in such cases, are also stained
with the same dye. As the scarlatina micrococcus
has been found in diseased cow's milk,1 such milk
should be treated by the method described for the
examination of tuberculous milk (see p. 75).

Bacillus lutyricus is best stained with a solution
of iodine in potassium iodide.

Actinomyces is usually stained by Plant's method.
Sections of nodules, tumours, etc. (from cases of
Actinomycosis) are immersed for ten or twelve
minutes in a stain containing two grammes of
magenta, 3 cc. of aniline oil, 20 cc. of alcohol
(sp. gr. 0*83), and 20 cc. of distilled water. The
stain (with the sections) is warmed to 45° C. The
sections are rinsed in water, and after- stained in a
strong alcoholic solution of picric acid for about
eight minutes. They are then immersed in water
for five minutes, in alcohol (50 per cent.) for fifteen
minutes ; and finally passed through absolute alcohol
and oil of cloves, and mounted in Canada balsam.
The tissues containing this fungus may be examined
in the fresh state. A little of the tissue, etc., is
transferred to a microscopic slide, teased out with
needles, and then temporarily mounted in a drop of
glycerine and water.

We have given most of the principal methods
for the examination of microbes. It may be re-

1 See Dr. Klein's Reports to the Local Government Board,
1885-8.

THE METHODS OF MOUNTING MICROBES 83

marked that nearly all microbes can be stained
with the various aniline dyes ; although their
capacity for absorbing these dyes differs consider-
ably. This capacity or affinity for aniline dyes is
of great use to the bacteriologist to ascertain the
presence of microbes, and to differentiate in many
instances morphological details which in the un-
stained condition are not discernible.

Hardening, Imbedding, Cutting, and Mounting
Preparations. — Many medical men and students on
reading the different staining, hardening, imbedding,
cutting, and mounting processes ' which any tissue
has to undergo before it can be examined with
the microscope, will be inclined to think it very
tedious work. It is, however, a mere matter of
routine, and when once this routine is established,
the whole thing is comparatively simple. It takes
very little time to change the hardening fluid, and if
the student gets into the habit of looking over the
bottles on the shelf every morning where he keeps
tissues in the process of hardening, a glance at the
labels will show those requiring a change. When
the sections are mounted and examined under the
microscope, he will find himself amply repaid for
all his trouble if he has faithfully carried out the
different processes in every detail. It is always
better to have one or two shelves devoted to those
preparations which require changing; and those
which require fresh fluid often, as for instance
those hardening in chromic acid should be kept
by themselves. Each bottle should be labelled,
and the tissue, date, and hardening fluid clearly

84 A MANUAL OF BACTERIOLOGY

written on the label. Every morning this shelf
should be examined, and the hardening solution
changed in those requiring it, the date being each
time written on the label, so that it may be seen
at a glance how long the tissue has been in the
fluid, and whether the hardening agent ought to be
renewed. Muller's fluid and bichromate of potash
preparations may be placed by themselves, and
need only be looked at occasionally/

The best hardening agents are absolute alcohol,
methylated spirit, Muller's fluid, chromic acid solu-
tion, potassium bichromate solution, and osmic acid.
(1.) Pieces of an organ, etc., should be cut from
J in. to 1 in. cubes, and placed in one of the
hardening solutions. If absolute alcohol or methy-
lated spirit is used the tissues should remain in the
spirit from two to three days. Many delicate
tissues, however, cannot be placed in strong spirit
without shrinking ; to obviate this such tissues are
first placed in dilute spirit (one part of water to
two parts of spirit). In this mixture the tissues
remain about twenty-four hours and are then trans-
ferred to the strong spirit for one or two days.
After this they are ready for imbedding and cutting.
(2.) Muller's fluid is an excellent hardening agent.
To prepare it, dissolve two parts of potassium
bichromate, one part of sodium sulphate, and 100
parts of distilled water. The preparations to be
hardened should remain in the fluid from two to
three weeks. When the fluid becomes cloudy it re-
quires changing ; but it retains its hardening pro-
perties for a long time. The preparations, after

THE METHODS OF MOUNTING MICROBES 85

being hardened in Miiller's fluid, should be washed
in water, and then placed in dilute spirit (one of
water to two of spirit) for about twenty-four
hours. Sometimes the treatment with dilute spirit
is dispensed with, especially if the sections are to
be cut immediately. (3.) Chromic acid solution is
really a mixture of chromic acid and spirit. It
is prepared by dissolving one gramme of chromic
acid in 600 cc. of distilled water. Two parts of this
solution is then mixed with one part of methylated
spirit. The material to be hardened is placed in this
fluid for twenty-four hours ; the fluid is then
changed, and again every third day; the material
being hardened in from eight to twelve days. The
material should not be allowed to become brittle,
which it does if it remains too long in this fluid.
After hardening the material is washed in water,
and the sections cut immediately (i.e. after imbed-
ding), or it is placed in dilute spirit for twenty-four
hours, and then transferred to strong methylated
spirit. In this fluid the material may remain for
an indefinite time ; that is, if it is not required for
immediate use. (4.) A two per cent, solution of
potassium bichromate is sometimes used, especially
where tissues require slow hardening. ' This solu-
tion takes from three to seven weeks to harden,
according to the size of the specimen, and the fre-
quency with which the solution is changed.' (5.)
A 0'5 per cent, solution of osrnic acid is used for
hardening certain preparations — such as the in-
ternal ear. This solution must be protected from
light ; for this purpose the bottle in which it is

86 A MANUAL OF BACTERIOLOGY

kept should be painted externally with black oil
paint.

To decalcify small bones or teeth, they are placed
in Ebrier's or Kleinberg's solution. Ebner's solution
contains five grammes of sodium chloride (salt),
5 cc. of hydrochloric acid, 20 cc. of distilled water
and 100 cc. of alcohol. Kleinberg's solution is
made as follows: 100 cc. of a saturated aqueous
solution of picric acid are added to 2 cc. of strong
sulphuric acid. The mixture is filtered and 300 cc.
of distilled water are added. In either solution the
materials (to be decalcified) remain until sufficiently
softened ; they are then allowed to soak in water, and
finally passed through weak spirit to absolute alcohol.

For cutting sections either by hand or by the
microtome, it is necessary (as a rule) to imbed the
material in one of the imbedding mixtures. If the
material to be cut has been preserved in alcohol, it
is better first soaked in water for about ten hours to
remove the spirit, and then placed in mucilage T for
about five hours. For cutting with the non-freezing
microtomes, the material is imbedded in celloidin
or paraffin, mounted on cork.2 To imbed in celloidin
the hardened material is first placed in a mixture of
alcohol and ether for thirty or forty minutes ; then
transferred to a solution of celloidin (dissolved in
equal parts of alcohol and ether) from two to twenty
hours.3 A cork placed in the clamp of the micro-

1 Mucilage is prepared by making a solution of gum Acacia.

2 If the material is firm enough it is sometimes mounted on
cork without being imbedded.

3 The length of time depends on the nature of the material.
It is longer for spongy structures like the lungs.

THE METHODS OF MOUNTING MICROBES 87

tome is smeared on its upper surface with a solution
of celloidin, which is left to harden. When the
material is ready, it is mounted upon a prepared
cork (i.e. it is placed on the smeared surface) ; and
a little celloidin solution is poured over the material
so as to cover it. The mounted material is now
placed in 70 per cent, alcohol in order to harden
the celloidin (which has a pasty consistence). In a
few hours or so the imbedded material will be
ready for cutting with one of the microtomes already
described. Schanze's microtome is a useful instru-
ment for cutting sections of materials imbedded in
celloidin. In cutting a tissue imbedded in celloidin
or mounted directly on cork, the razor and tissue
should be kept wet with alcohol, and the sections
carefully transferred to alcohol. The sections (if
from celloidin material) are placed in oil of cloves
in order to dissolve out the infiltrated celloidin.
They are then ready for staining, etc.

For fixing pieces vifirm materials directly on corks
either glycerine-gelatine l or gelatine is used. These
substances are liquefied by the application of heat.

Paraffin wax for use as an imbedding material is
first dissolved in chloroform, and then used in a
similar manner to the solution of celloidin already
described. The imbedded material must be cut
perfectly dry, and the sections removed to xylol.

1 Glycerine-gelatine is prepared as follows : to 10 parts of
gelatine add sufficient water to allow the gelatine to swell up ;
pour off the water, and melt the gelatine. To the melted gela-
tine add 10 parts of glycerine, and finally a few drops of some
germicidal agent, preferably carbolic acid. The latter is added
in order to preserve the glycerine-gelatine.

88 A MANUAL OF BACTERIOLOGY

The xylol dissolves out the infiltrated paraffin, and
the sections are then placed in alcohol to extract
the xylol. After this treatment they are ready for
the staining process.

Instead of celloidin and paraffin, wax-and-oil
mixture l and vaseline-and-paraffin mixture are used
for imbedding purposes.

Before alcohol-hardened tissues are cut with the
freezing microtomes they must be soaked in water
for ten minutes, this process to be followed by five
hours' soakage in mucilage. After this they are
frozen and cut with the microtome, whose razor
must be perfectly sharp and free from notches.
Tissues hardened in Muller's fluid (if they have not
been subsequently placed in alcohol) are at once
dried with blotting-paper, then frozen, and finally
cut. Fresh tissues are covered with mucilage,
frozen, and cut. The razor should be moistened
with a solution of gum, and the sections transferred
with a camel-hair brush to warm distilled water for
fifteen minutes — the object being to dissolve out the
mucilage. They are then ready for staining, etc.,
with the exception of sections of fresh tissues, which
should be placed, before staining, in a 0'6 per cent,
saline solution, so as to prevent too much shrinking
of the sections.

In cutting sections with the microtome ' very
little force is required in pushing the razor or knife
through the material, and if it is sharp a very slight
turn of the screw each time will enable one to cut a

1 Equal parts (by weight) of white wax and olive oil are
melted together.

THE METHODS OF MOUNTING MICROBES 89

section, which ought to be so thin as to be almost
invisible.'

After staining, etc., the sections are mounted in
various media on glass slides (3 in. X 1 in.), and
covered with thin cover-glasses.1 ' When high-
power lenses are to be used it facilitates the work
very much to know the exact thickness of the cover-
glass under which the specimen is mounted, and
with very high powers, or those with wide angles of
aperture, the cover-glass must be at least 0'004 in.
to enable the lens to work through it.' All Zeiss'
objectives in fixed mounts are corrected for a cover-
glass of medium thick-
ness (between 0'15
and 0'2 mm., or 0'006
and 0-008 in.). In the
higher series from CC
upwards the thickness
of the cover-glass con-
sistent With the mOSt Flo> .27, zEIS8- COVER-GLASS TESTER.

perfect correction is

indicated on the side of the mount by small figures
(mm.). As a rule, it is sufficient for ordinary work
to use cover-glasses of an estimated medium thick-
ness.

Oil-immersion objectives are within wide limits
independent of the thickness of the cover-glass.
But considerable variations in the thickness of the
cover-glass may be compensated for — by slightly
lengthening the body-tube for thinner cover-glasses ;
and by slightly shortening the body-tube of the

1 The round ones are better than those that are square.

90 A MANUAL OF BACTERIOLOGY

microscope for thicker cover-glasses. Zeiss makes
a good tester (Fig. 27) suitable for the exact
measurement of the thickness of cover-glasses. The
measurement is effected by a clip projecting from a
box ; the reading is given by an indicator moving
over a divided circle on the lid of the box. The
divisions show hundredths of a millimetre, and the
instrument is capable of measuring up to five milli-
metres.

Before use the glass slides and cover-glasses should
be perfectly clean.

Many methods for permanently mounting tissues
and cover-glass preparations have already been
described. For fresh tissues glycerine is often used,
while for hardened tissues the following mounting
media have each their special advantages : —

(a) Canada balsam dissolved in xylol.

(&) Canada balsam dissolved in benzol.

(e) Canada balsam dissolved in chloroform and
turpentine.

(d) Dammar varnish.

After the tissues have been stained, they pass
through the following processes : — Washing off the
excess of stain, dehydrating, clearing or extracting
the infiltrated material used in the imbedding pro-
cess, etc. ; mounting, cementing, or sealing ; and
finally, labelling the slides. The following list gives
the various agents for the above-mentioned pro-
cesses : —

(Water.

(1) Washing agents, . . . J Dilute spirit.

I Absolute alcohol.

(2) Dehydrating agent, . . Absolute alcohol.

THE METHODS OF MOUNTING MICROBES 91

(3) Clearing agents, .

Oil of cloves.
Oil of cedar.
Xylol.

Aniline oil.

Terebene.
( Canada balsam.

(4) Mounting agents, . . . \ Dammar varnish.

I Glycerine.
f Hollis' glue.

«3, Cementing agent,,

iBlack asphalte varnish.

To mount afresh specimen, the section should be
placed with the utmost care in the centre of a glass
slide. The section should not be folded in any part,
therefore it must be carefully spread out with
needles. This must be performed without stretch-
ing the specimen. After this has been done, wipe
off all moisture with a clean cloth. Now take up
' a cover-glass and place a drop of glycerine in the
centre, invert and place it horizontally on the pre-
paration, leaving the weight of the cover-glass to
spread out the glycerine.' If there is an excess of
glycerine round the edges of the cover- glass, it
must be carefully absorbed by filter or blotting-
paper, but on no account should the cover-glass be
removed. To seal, ring, or cement the preparation,
paint round the edge of the cover-glass and a little
way on the slide, a ring of Hollis' glue or Dammar
varnish. Hollis' glue1 is better than Dammar
varnish, for it is not acted upon by the cedar oil
used with oil-immersion lenses. The sealing of

1 Gold size is sometimes used for sealing glycerine prepara-
tions.

92 A MANUAL OF BACTERIOLOGY

microscopic preparations with Hollis' glue or any
other cementing agent is performed with a camel-
hair brush and a turn-table (Fig. 28). The slide is
fixed with the clips of the turn-table, the table re-
volved, and the brush containing the cement is held
in a vertical position, so as to touch the edge of the
cover-glass. By this means a ring of the cement is
deposited, which dries in a day or two. The pre-
paration is permanently sealed, and should now be
labelled and placed in the cabinet.

The various preparations of Canada balsam and
Dammar varnish are prepared as follows : — (a) To
prepare xylol balsam it is necessary to dissolve
Canada balsam in xylol until it has the consistency
of treacle ; (&) benzol balsam is
prepared by first drying the
Canada balsam until it is brittle.
It is then dissolved in benzol
until it has the same consist-
ency as the xylol balsam. If these mounting fluids
get thick on keeping, they are thinned by the addition
of xylol and benzol respectively; (c) chloroform-
turpentine balsam is prepared by dissolving 3 ozs.
of Canada balsam in 1 oz. of chloroform and 1 oz.
of turpentine. If this medium gets thick, it is
thinned by the addition of chloroform ; (d) Dammar
varnish is prepared by first dissolving 1J oz. of
powdered gum Dammar in 1 J oz. of turpentine, and
filtering. At this point \ oz. of gum mastic is dis-
solved in 2 ozs. of chloroform, and the solution
filtered. The two solutions are finally mixed to-
gether, and again filtered.

THE METHODS OF MOUNTING MICROBES 93

These fluids are used for mounting hardened
•tissues, and they should be preserved in stoppered
or well-corked bottles ; while for daily use a small
drop-bottle of each fluid should be placed on a
table set apart for mounting purposes. It may be
mentioned that xylol balsam is the best mounting
fluid for stained microbes ; chloroform-turpentine
balsam acts well with hardened sections ; and benzol
balsam is the most useful solution for general micro-
scopic purposes.

These mounting fluids are all used in the same
manner, therefore a description of mounting in
xylol balsam will also apply to the other fluids.

The sections having been stained and washed,
they are placed for twelve minutes in absolute
alcohol contained in a watch-glass : the alcohol
dehydrates them. They are now drained, and then
placed in oil of cloves to clarify them. While in
this medium they should be carefully straightened
out with needles.1 Having now placed a drop of
xylol balsam in the centre of the slide, it is spread
out with a needle ; then a section is carefully lifted 2
out of the oil of cloves, drained, and placed in the
xylol balsam. A small drop of xylol balsam is
placed on the under surface of a clean cover-glass,
which is lowered on to the section. With practice
and perseverance, slight pressure with the forefinger
is all that is required to produce a slide devoid of

1 Ordinary steel needles mounted in wooden handles.

2 A lifter is made by beating out one end of a copper wire,
and then turning up the broad portion. Lifters made of German
silver may be purchased at Messrs. F. E. Becker & Co., of
Hatton Garden, London.

94 A MANUAL OF BACTERIOLOGY

air-bubbles. To remove these bubbles small air-
pumps have been devised, but they are not to be
recommended ; ' the only thing to be done when an
air-bubble lodges in a cavity of the section, and
refuses to move in any way by gentle pressure, is
to lift the cover-glass, and transfer the section to
oil of cloves, and then remount it.' As the mount-
ing of sections may be performed in the summer,
the xylol balsam is much thinner than usual (due
to the heat), and therefore takes a much longer
time to set. In such cases a mounting clip (Fig. 29)
is useful to keep the cover-glass from moving, i.e.

until the balsam sets.
After this the slide should
be sealed with Hollis'
glue, or some other ce-
menting agent, as already
described.1

FIG. 29. MOUNTING CLIP. . Methods of Introduc-

ing Microbes into Living

Animals. — In such experiments guinea-pigs, rabbits,
mice, fowls, etc., are used. Pure cultivations of
microbes and infectious matter are introduced into
the animal body by the following methods : —

(a) Inhalation.

(b) Swallowing.

(e) Direct inoculation.

(d) Special operations.

(a) An animal is made to inhale the infectious
matter, etc., disseminated by means of a spray ; (b)

1 For further information see Martin's Manual of Microscopic
Mounting.

THE METHODS OF MOUNTING MICROBES 95

the infectious matter is mixed with the animal's
food; (c) the infectious matter is introduced into
the animal body by cutaneous or subcutaneous in-
oculation or injection; (d) by the fourth method
mentioned above (i.e. special operations), the infec-
tious matter may be injected into the duodenum, or
introduced into 'the peritoneal cavity by the per-
formance of abdominal section/ These and other
operations are used as means of introducing micro-
bian matter into the living animal. But it cannot
be too firmly impressed upon the mind that all
operations should be performed with antiseptic pre-
cautions ; and the instruments, as well as the hands
of the operator, should be thoroughly disinfected.

Before closing the present chapter we give a few
remarks on what is known as the unit of micro-
scopical measurement. It has been the general prac-
tice among bacteriologists to give the dimensions of
microbes in terms of a thousandth part of a milli-
metre, which is called a micro-millimetre, and is
known by the symbol fi.1 This unit is of great
importance, for ' it is always easier to conceive the
size of any object, and especially to realise the com-
parative sizes of two objects, when their dimensions
are given in terms of a unit smaller than either;
for instance, it is difficult exactly to comprehend
the length represented by -^ of an inch, and few
people can readily compare such dimensions as TV
and 75^ of an inch. This difficulty vanishes when
the dimensions are expressed as multiples of a small,
properly chosen unit, and not as fractions of a large

1 1 /u=0'001 mm.=Tjr^nr in., or 0*0000393 in.

96 A MANUAL OF BACTERIOLOGY

one. For this purpose a fraction of an inch might
be adopted instead of a fraction of a millimetre
(mm.) ; but, at any rate, in measuring the spores of
fungi, TWOTT °f an incn is too large a unit, and
TWO-OIF of an inch would be inconveniently small.
It happens that, if we take TFOTJ- of a millimetre as
our unit, we can express the size of the spores of all
fungi, etc., in the fewest possible figures. For in-
stance, many micrococci measure about 1 //,, the
spores of Penicillium about 3 //,, the spores of many
Myxomycetes about 1 0 //,, and so on. If we compare
these figures with the following: O'OOl mm., 0'003
mm., O'Ol mm.; or, still more, with these: 0*00004
in., 0*00012 in., 0'0004 in. — we see the great saving
effected in the trouble of writing down the dimen-
sions, quite apart from the greater readiness with
which they can be compared with one another.
But perhaps the difficulty with some is that of
realising and actually applying this unit; we will
therefore give an easy method by which the size of the
micromillimetre may be obtained. Place the micro-
scope in such a position that the image projected
upon a piece of white paper is magnified 254 times :
this can easily be done by a quarter-inch objective,
with the use of the draw-tube, or by placing the
paper at a greater distance than ten inches from the
eye-piece. Let this position be marked, so that the
microscope can be placed in it again at any time.
Now copy on the paper, from a scale, an inch
divided into ten parts, and with a line pen subdivide
each tenth into five equal parts. Then the value of
each of these subdivisions will be 2 /A, and of the

THE METHODS OF MOUNTING MICROBES 97

whole tenth of an inch, 10 /-t. If this scale be care-
fully copied on a piece of thin cardboard or other
suitable substance, the dimensions of any microbe,
etc., drawn by the camera lucida or otherwise on the
paper in that position of the instrument, can be
easily read off in /*s. With the aid of a deeper eye-
piece or higher objective we can magnify the image
508 times, and then each small division of the scale
will represent 1

CHAPTEE IV

THE ORIGIN, CLASSIFICATION, AND IDENTIFICATION
OF MICROBES

SCIENTISTS and non-scientists are agreed that there
was a lifeless period in the history of the earth —
therefore that life had a beginning. But when,
where, and how did life begin? 'As to the time,
there is no evidence whatever. Life is enormously
older than any record of it. Even the higher forms
were developed long before the periods in which we
first find their remains. As to the place, probably
in the polar regions, as Buffon suggested in his
Epoques de la Nature. The earth being a cooling
globe, those regions would be the earliest to reach a
temperature under which life is possible.' During
the past twenty years or so, Buffon's theory has
been supported by Comte de Saporta * and others ;
and it is highly probable that in the earliest zoic
epochs (especially the north polar) regions of the
earth were of a hot and humid nature. Moisture
and heat are essential to life ; therefore life had its
beginnings in water.2 It is probable that lowly
plants (possibly microbes) were the first organised

1 UAncienne, Vtg&ation Polaire.

2 See Professor Moseley in Nature, September 3, 1885.

THE ORIGIN OF MICROBES 99

beings which made their appearance on the earth,
for it is well known that all microbes require mois-
ture, while many live m water or similar media.
From these and other facts it is probable that the
Schizomycetes were the forms of life which originated
in the polar regions of the earth — the other parts of
the earth, at that remote time, being too hot for life
to exist. But if life originated in the particular
part of the earth indicated, this does not explain the
origin of life. How did life begin ? This question
has occupied the thoughts of men in all ages, but if
we regard living and non-living matter as composed
of elements which are common to both kinds of
matter, wherein lies the difference which gives as
one result non-living matter, and as another result
living matter? The difference must lie in the
mixing of these elements. If the first form of living
matter were a microbe it originated either by a
creative act or by spontaneous generation. Both the
theory of creation and that of spontaneous genera-
tion account for the origin of life : in fact, the be-
ginning of life can only be explained theoretically,
for there is no practical or direct proof of how life
originated. On this point Professor Huxley 1 says :
' If it were given me to look beyond the abyss of
geologically recorded time to the still more remote
period when the earth was passing through physical
and chemical conditions, which it can no more see
again than a man can recall his infancy, I should
expect to be a witness of the evolution of living
protoplasm from not living matter. I should expect

i Critiques and Addresses, p. 238.

100 A MANUAL OF BACTERIOLOGY

to see it appear under forms of great simplicity, en-
dowed, like existing fungi, with the power of deter-
mining the formation of new protoplasm from such
matters as ammonium carbonates, oxalates, and
tartrates, alkaline and earthy phosphates, and water
without the aid of light. That is the expectation to
which analogical reasoning leads me ; but I beg you
to recollect that I have no right to call my opinion
anything but an act of philosophical faith.'

Besides the two great theories which account for
the origin of life from mineral matter,1 there are
others, which we now describe. It has already
been stated that putrefaction is the result of life,
not of death — the result of microbian activities 2 —
but formerly many naturalists believed that by
putrefaction the organic elements which had com-
posed the body of the dead animal formed them-
selves by free creative power into independent
beings, which differed entirely from those from
which their material was produced, yet are in every
case animated, and have the power of propagation ;
thus the albumin and fat globules take the form of
microbes, perhaps also of yeasts and moulds, or even
of those little infusorial animals, whose presence
never fails in corruption. This mode of origin has
been called equivocal generation or generatio cequi-
voca.3 Other naturalists dispute the possibility of

1 Those of creation and spontaneous generation.

2 The microbes being introduced from the air, water, etc.

3 The equivocal origin of microbes must be distinguished
from the spontaneous generation, which we have already
alluded to ; for in the latter case there existed no organisms
on the earth.

THE ORIGIN OF MICROBES 101

living beings, however small and simple, ever origi-
nating in any other way than from germinal matter
which sprang from the same form of life ; and they
insist that the belief in the equivocal origin of
microbes is that last remnant of an old superstition,
which the light of science has not entirely banished.
In ancient times it was thought that serpents and
frogs originated from slime, that caterpillars were
generated from decayed leaves, vermin from filth,
and worms from spoiled meat. Now-a-days every
child knows that all these things are fables ; every
housewife knows by experience that no maggots
originate in meat if the blow-fly is prevented by a
wire-screen from entering and depositing its eggs.
They have learned, through careful covering, to keep
away the minute mould- spores, which settle with
other dust from the air, and which colonise on their
preserved fruits ; they know that trichina and tape-
worm only originate from raw or half-cooked pork,
in which these animals were already present in the
embryonic stage. Even the farmer no longer believes
that the grain rust (Puccinia graminis) originates
from chilling, but that it springs from spores which
are scattered by the barberry bushes (Berberis vul-
garis), or other fallen stalks, and that the blight may
be prevented in corn crops, if the seed (before sow-
ing) is steeped in a solution of iron sulphate or copper
sulphate, in order to kill the spores which cling to it.1
Concerning microbes and their related ' fermenta-
tions/ the above-mentioned observations lead without

1 See Dr. Griffiths' book, The Diseases of Crops, pp. 128-132
(Bell & Sons).

102 A MANUAL OF BACTERIOLOGY

doubt to the conclusion that they do not originate
through equivocal generation ; for when nitrogenous
material from the animal or vegetal world is heated
in flasks, even at as low a temperature as 70° C., all
the microbes are killed, and if the entrance of new
germs from outside is in every way prevented, and
it were possible to keep the flasks for ever, no
microbes would ever originate of themselves. On
the contrary, the entrance of a single germ, in each
flask, is sufficient to cause multiplication, and with
it putrefaction. If microbes originate from putrid
matter through equivocal generation, putrefaction
must appear before the microbes ; but experience
shows the contrary, that putrefaction is a conse-
quence of the development and growth of microbes.
"Within the last few years a theory has been advanced
to account for the origin of microbes, which has
caused some sensation, viz., that under certain
conditions the ordinary mould-fungus will give rise
to moving germs of extraordinary minuteness ; such
germs are capable of developing into microbes, into
yeasts, and finally again into the mould-fungus.
When microbes are found in the blood or organs in
certain diseases, the authors of this theory of pleo-
morphism are satisfied that the spores of the com-
mon mould germinate in the human body; that
these spores first swarm as microbes, but under
suitable culture may be nourished into different
species of moulds. However, unprejudiced research
has not given the slightest proof that microbes
stand in any connection with the dev^pment of
yeasts, moulds, or other fungi. They always originate,

THE ORIGIN OF MICROBES 103

as far as we know at present, from spores, etc., of
the same kind (Cohn).

Concerning the doctrine of pleomorphism, it may
be stated that Lankester,1 Van Tieghem, Zopf,
Cienkowski, Billroth, Neelsen, Hauser, and others
have noticed that certain microbes pass through
various phases during their life-histories. And
Sattler, Gravitz, and Blichner, believe that they have
transformed certain non-pathogenic microbes into
pathogenic forms by simply cultivating the former
in different media or under different physical condi-
tions. For instance: Sattler2 states that he has
transformed the non-pathogenic Bacillus subtilis
into a pathogenic form, capable of producing
infectious ophthalmia, by cultivating the microbes
(at 35° C.) in an infusion of jequirity seeds. Gravitz
believed that he had transformed the non-pathogenic
moulds — Aspergillus glaucus, Penicillium glaucum —
into pathogenic forms by cultivating them in
alkaline media at about 40° C. Biichner states
that he has transformed Bacillus subtilis into
Bacillus anthracis and vice versd : ' that by successive
cultivation of Bacillus anthracis under constant
variation of the nutritive material, he saw it assume
the morphological and physiological characters of
Bacillus sultilis!

Klein, Koch, Cohn and others do not accept the
theory of pleomorphism, or the transformation of
microbes ; and Klein 3 has proved most conclusively

1 Quarterly Journal of Microscopical Science, 1873, p. 408.

- Wiener Medic. Wochenschrift, 1883.

3 Micro-Organisms and Diseate, pp. 207-231 (3d ed.).

104 A MANUAL OF BACTERIOLOGY

that no pathogenic microbe is ever transformed into
a non-pathogenic form, or vice versd. In fact, he
says that 'those organisms which are connected
with morbid processes possess this pathogenic
power ab initio ; and are not due to any peculiar
condition of growth.' If a harmless microbe could
be proved capable of transformation into a harmful
form, ' the whole doctrine of the infectious diseases
is involved in such a case ; for if in one case it can
be unmistakably proved that a harmless microbe
can be transformed into a pathogenic organism, i.e.
into a specific virus of an infectious disease, 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
microbes which, owing to some peculiar unknown
condition, are capable at once to start any kind
of infectious disorder, say anthrax (Buchner), in-
fectious ophthalmia (Sattler), and probably a host
of other infectious diseases, and thus to form the
starting-point of epidemics. And the only redeem-
ing feature, if redeeming it can be called, in this
calamity, would be the thought that the par-
ticular microbe would by-and-by, owing to some
accidental new conditions, again become harmless '
(Klein).

The transformation of microbes into different
forms is entirely opposed to the Darwinian law.
To one who has fully comprehended the meaning

THE ORIGIN OF MICROBES 105

and the operation of this law, it will be at once
apparent that there must be error somewhere in the
matter. ' If the law of actual variation/ says Dr.
Dallinger, ' with all that is involved in the survival of
the fittest, could be so readily brought into complete
operation, and yield so pronounced a result, where
would be the stability of the organic world ? Nothing
would be at one stay. There could be no perman-
ence in anything living. The philosophy of modern
biology is that the most complex forms of living
creatures have derived their splendid complexity
and adaptations from the slow and majestically
progressive variation and survival from the simpler
and the simplest forms. If, then, the simplest forms
of the present and the past were not governed by
accurate and unchanging laws of life, how did the
rigid certainties that manifestly and admittedly
govern the more complex and the most complex
come into play ? If our modern philosophy of
biology be, as we know it is, true, then it must be
very strong evidence indeed that would lead us to
conclude that the laws seen to be universal break
down and cease accurately to operate, where the
objects become microscopic, and our knowledge of
them is by no means full, exhaustive, and clear.
Moreover, looked at in the abstract, it is a little
difficult to conceive why there should be more
uncertainty about the life-processes of a group of
lowly living things, than there should be about the
behaviour, in reaction, of a given group of molecules.
The triumph of modern knowledge is a knowledge
— which nothing can shake — that Nature's processes

106 A MANUAL OF BACTERIOLOGY

are immutable. The stability of her processes, the
precision of her action, and the universality of her
laws, are the basis of all science, to which biology
forms no exception. Once establish, by clear and
unmistakable demonstration, the life-history of an
organism, and truly some change must have come
over Nature as a whole, if that life-history be not
the same to-morrow as to-day ; and the same to one
observer, under the same conditions, as to another.

'But the fact that there is no evidence of any
direct relation evolutionary between two such
forms as Bacillus subtilis and Bacillus anthracis, the
fact that there is no ready way either naturally or
artificially of their being changed into each other,
must not blind us to the fact that such an evolutional
relation in the past is eminently probable, nay almost
certain. It may, in all probability must, have
taken an indefinite time in the past to effect ; but
being once effected, the specificity is continued as
in every other form by inheritance.'

There are certain conditions under which a
microbe may appear to have altered its properties.
For instance, Chauveau1 has shown that Bacillus
anthracis loses its virulence when submitted to the
action of compressed oxygen ; but it does not lose
its vaccinal property after this treatment. This new
character is said to be maintained by suitable
cultivation. Although Bacillus anthracis may lose
its virulence under such abnormal conditions as
already alluded to, it does not become a non-patho-
genic microbe, for it still preserves one of the most

1 Comptes Rend/us de P Academic de* Sciences, tome 109.

THE ORIGIN OF MICROBES 107

essential attributes that indicate the infectious
nature of the pathogenic microbe, viz., its vaccinal
property. Besides, Chauveau has further shown
that the non-virulent Bacillus anthracis may be
revivified by degrees when grown in suitable media.
These researches do not point to any transformation
of Bacillus anthracis into a non-pathogenic species,
but simply show that oxygen under pressure is
capable of modifying the microbe's pathogenic
power. In fact, microbes have the power of adapt-
ing themselves to considerable variation of external
conditions; but this does not involve permanent
change in the organism.

Microbes belong to the vegetal kingdom ; in other
words, they are fungi. As they multiply by re-
peated subdivision, and also frequently reproduce
themselves by spores, which are formed endogen-
ously, they are grouped together in a class called
the Schizomycetes, splitting fungi, or Spaltpilze, as
the German naturalists term them.

The forms of microbian cells vary considerably —
they are round, ovate, elliptical, cylindrical, etc.
Microbes live isolated, singly, or in larger or smaller
colonies, or, in many cases, united in pairs, or many
together in threads or groups. Nearly all microbes
possess two different modes of life : one of motion
and another of rest. In certain conditions they are
extremely motile, and when they swarm in a drop
of water or other fluid they move among each other
in all directions; sometimes rotating round their
longitudinal axis, while in other cases the move-
ment is an oscillating one, or the threads alternately

108 A MANUAL OF BACTERIOLOGY

bend and straighten themselves, etc. At other times
the motile microbes become motionless. In this
state many of them aggregate together and excrete
a gelatinous material which entirely envelopes them.
This colony is termed a zoogloea, in fact it is the
resting stage of the particular microbes. In the
zooglcean stage, microbes often produce spores.

Microbes multiply by fission (i.e. division) and
spore-formation. The warmer the air, etc., the
faster proceeds the division, and the stronger the
multiplication ; in a lower temperature it becomes
slower, and ceases entirely at the freezing-point of
water. Their fecundity is enormous, and would, in
a very short time, choke up the earth ; but this
rapid rate of increase is kept in check by the
limited supply of food, climatic conditions, and the
struggle for existence.

As an example of the enormous fecundity of mic-
robes we describe the rate of reproduction of a
common form, viz., Bacillus sultilis. This bacillus
attains a certain length and then divides across into
two. 'Each half grows to the size of the parent,
and then similarly divides, and so on as long as
food and other conditions of their life are present.
Bacillus sultilis has been observed to divide in this
way every half-hour, a rate which gives in twenty-
four hours more than three hundred billion of in-
dividual microbes as the offspring of one parent.
They are extremely minute, varying from 20^0^th
of an inch to the TzyWth of an inch in length.'

As already stated, microbes propagate by fission
and by spore-formation. The following table gives

THE REPRODUCTION OF MICROBES

109

those that are produced only by fission, and those
that multiply by fission and spores : —

Mode of Propagation.

General Remarks.

Micrococci .

Fission

Spherical and oval in
form.

Bacteria .

Fission . ~ .''

Rod- shaped microbes,
generally smaller than
bacilli, and devoid of
spore-formation.

Bacilli . .

Fission and spores

Rod-shaped microbes,
many are provided
with flagella.

Vibriones .

Fission and spores

Curved or more or less
wavy rods provided
with flagella.

Spirilla . .

Fission and spores

Spiral-shaped microbes.

Spirochaetae

Fission and spores

Filamentous and wavy
microbes.

Concerning the reproduction of the micrococci, if
the division takes place in one direction only, the
resulting form (if the two cells remain together) is
a diplococcus, dumb-bell, or colon (:). The diplo-
coccus may again divide, without separation, form-
ing a streptococcus or chain, which may become
curved or even twisted in appearance. Sometimes
the division of these microbes is in two directions,
resulting in four cocci (::), which is termed a meris-
mopedia, or in three directions forming a sarcina-
coccus or sarcina.1

All microbes require for their nutrition and growth

1 A division into a large and an indefinite number of cells is
termed an ascococcus.

110 A MANUAL OF BACTERIOLOGY

oxygen, carbon, nitrogen, certain salts, and water.
Although some microbes are anaerobic, they require
oxygen, which is obtained from the carbohydrates
and albuminoids of the medium in which they live,
or from the free oxygen which may be dissolved in
that medium.

Before considering the various classifications of
microbes, we mention the fact that microbes in
general are sometimes called Bacteria, but as there
is a genus of that name, it is better that the word
should be applied only when one is alluding to
microbes of that genus. The study of microbes
(which includes all forms of Schizomycetes) has been
consequently termed Bacteriology ; but it is an un-
fortunate name, which, at the present time, cannot
well be replaced by another.

Microbes may be simply divided into aerobic1'
and anaerobic 2 forms. Bacillus spinosus and Bacillus
cedematis maligni are examples of the former ; while
Micrococcus candicans and Bacillus sultilis are ex-
amples of the latter kind.

Microbes may be also divided into pathogenic
(disease-producing), septic (putrefactive), zymogenic
(fermentive), and chromogenic (pigment-forming)
forms.

The Schizomycetes, which Sachs includes in his
group the Thallophytes, have been classified by Cohn3
into five genera : —

(1) Spherobacteria or micrococci.

1 Those requiring free access of oxygen (air).

2 Those which do not require free oxygen.

3 Beitrdge zur Biologie der Pjlanzen, 1872 et seq.

THE CLASSIFICATION OF MICROBES 111

(2) Microbacteria or bacteria.

(3) Desmobacteria or bacilli and vibriones.

(4) Spirobacteria or spirilla.

(5) Spirochsetae.

This classification is founded upon the idea that
all the various morphologically or physiologically
distinct forms belong to different species. Koch's
researches with plate-cultivations have given great
support to the classification of Cohn, which, in our
opinion, is the best, that is, from the bacteriologist's
point of view. In such a classification a micro-
coccus produces nothing but a micrococcus, a bacil-
lus nothing but a bacillus, and so on.

Zopf (who is the great apostle of the doctrine of
pleomorphism) divides microbes into four groups :* —

(1) Coccacese.

(2) Bacteriaceae.

(3) Leptothricheae.

(4) Cladothrichese.

The first group contains streptococcus, merismopedia,
sarcina, micrococcus, and ascococcus forms ; in fact
this group only contains cocci. The second group
contains the following genera : — Bacterium, Spiril-
lum, Vibrio, Leuconostoc, Bacillus, and Clostridium.
Most of these forms, according to Zopf, pass through
a coccus stage. The third group contains four
genera : — Crenothrix, Beggiatoa, Phragmidiothrix,
and Leptothrix. This group (like the second) is
believed to possess coccus, rod, and thread forms.
The fourth and last group only contains the genus
1 Die Spallpilze, 1885.

112 A MANUAL OF BACTERIOLOGY

Cladothrix, which shows coccus, rod, thread, and
spirillar forms.

Baumgarten divides microbes into two groups,
each containing three genera : —

Monomorphic Group.

Pleomorphic Group.

Coccus.
Bacillus.
Spirillum.

Spirulina.
Leptothrix.
Cladothrix.

The genus Bacterium is entirely dispensed with in
this classification ; and Fliigge, who modified Cohn's
classification, has submerged the genus Bacterium
into the genus Bacillus, as both these forms were
rod-shaped ; but it should be borne in mind that
the bacteria do not produce spores, whereas in
the bacilli spore-formation is of common occur-
rence.

Hueppe's classification is based on the mode of
reproduction, or, rather, fructification ; and the late
Dr. De Bary divided them into two groups : Mic-
robes which produce endospores, and microbes
which produce arthrospores. But as we know so
little about spore-formation in the Schizomycetes,
Hueppe's and De Bary's classifications are of very
little practical value at the present time.

'The determination of species rests upon the
accumulated evidence afforded by a thorough know-
ledge of their life-history.' The form of the mic-
robe, the physiological, pathological, and other

THE IDENTIFICATION OF MICROBES 113

changes it effects, and the microscopical and macro-
scopical appearances under cultivation, must be
collectively taken into account. This determina-
tion or identification of species will be considered in
the next chapter.

CHAPTEE V

THE BIOLOGY OF MICROBES, ETC.

IN this chapter we describe nearly all the more im-
portant microbes ; but the microbes present in such
diseases as tuberculosis, cholera, diphtheria, scarla-
tina, etc., will be described in Chapter vi.

MICROCOCCI.

Micrococcus prodigiosus. — This microbe, which
measures from 0*5 to 1 //, in diameter, gives rise to a
blood-red pigment when grown on boiled potatoes,
white of egg, starch-paste, bread, agar-agar, and
other media. Fig. 30 represents the macroscopic
and microscopic appearances of -this microbe. It
grows well on agar-agar, which it liquefies. The
pigment, which M. prodigiosus gives rise to, is in-
soluble in water, but soluble in alcohol; and in
many of its reactions it resembles certain aniline
colours.1 This pigment is only produced under cer-
tain conditions, viz., at a temperature of from 20° to
22° C., and after the gelatine or agar-agar has lique-

1 Erdmann in Journal fur Praktische Chemie, 1866 ; and
Schroter in Beitrdge zur Biologie der Pfldnzen, vol. i. p. 109.
114

THE BIOLOGY OF MICROBES, ETC.

115

fied. As the temperature rises to blood-heat M.
prodigiosus loses its power of forming the red pig-
ment ; but forms casein, lactic acid, and probably
other substances. ' When growing and kept in the
depth of a solid nourishing material, i.e. removed

A, Macroscopic
appearance of Mic-
rococcus prodigiosus
on sterilised potato
after four days' cul-
tivation. From a
fifth fractional cul-
tivation.

C, Microscopic
appearance of M.
prodigiosus.
X 1265.?

B, A growth of Micrococcus
prodigiosus in nutrient agar-
agar.

Fio. 30. MICROCOCCUS PRODIGIOSUS.

from the free surface, colonies of this microbe grow
as colourless micrococci.' They are always present
in the atmosphere, and give rise to the phenomena
known as ' bleeding bread,' ' blood-rain/ ' bloody
sweat,' etc.

116 A MANUAL OF BACTERIOLOGY

A desiccation of four months at 32° C. (dry heat) l
does not destroy the vitality of M. prodigiosus;
but when exposed to the action of ozone the microbe
is killed.2 From these facts one can readily under-
stand how it is that M. prodigiosus (as well as other
aerial microbes) is always present in the air of
towns, villages, etc. ; but is never in the air at sea,
for the ozone present in sea-air destroys the microbes.

Micrococcus luteus. — This is another chromogenic
aerial microbe. It is found as single cells, dumb-
bells, or in packets. The cells are 1 '2 p in diameter ;
and they grow rapidly on nutrient gelatine plates
(plate-cultivations) giving rise to a yellow pigment.
The colonies, so produced, are round and slightly
granular in appearance. M. luteus grows in nutrient
agar-agar, bouillon ; on steamed potatoes ; and as
drop -cultures. The pigment produced by this microbe
is insoluble in water, and is unchanged by sulphuric
acid and alkalies. It is also destroyed by the action
of ozone.

Micrococcus chlorinus. — This microbe produces a
yellowish green pigment when grown on sterilised
white of egg (see Fig. 21) and fluid media. The
cells are about 1 ft in diameter. The pigment is
soluble in water, and is decolourised by acids.

Micrococcus aurantiacus. — The cells of this aerial
microbe are 1*5 //, in diameter ; and they occur
singly, in pairs, or in zooglea. On plate-cultivations
they form orange-coloured drops and spots, which

1 Griffiths in Proceedings of Royal Society of Edinburgh, vol.
xvii. p. 262.

2 See Griffiths' Researches on Micro-Organisms, p. 184.

THE BIOLOGY OF MICROBES, ETC. 117

ultimately coalesce into equal-sized patches. On
fluid media they form an orange-coloured pellicle.
M. aurantiacus also grows on steamed potatoes and
white of egg. The pigment is soluble in water.

Micrococcus fulvus. — Cells 1/5 fi in diameter;
they form rusty-red drops and gelatinous masses
on horse-dung.

Micrococcus violaceus. — The cells are 1*4 JJL in
diameter, and occur as bright violet-coloured gela-
tinous drops or patches on the surface of steamed
potatoes exposed to the air.

Micrococcus cyaneus. — The cells are elliptical and
grow on potatoes and fluid media, giving rise to a
blue pigment when in contact with air. The pig-
ment is soluble in water, and the solution is at first
green, but afterwards becomes an intense blue.
Acids convert this pigment into a red colouring
matter, while alkalies turn it green. There are no
characteristic absorption bands shown when a solution
of the blue pigment is examined by the spectroscope.

Micrococcus rosaceus. — The cells are from 1 to 1*5
fjL in diameter, and give rise to a rose-coloured growth
on the surface of nutrient gelatine and agar-agar.

Micrococcus cinnabareus. — This microbe grows very
slowly on the surface of gelatine. At the end of
eight days the colonies appear as small drops of
a red colour ; but ultimately the colour becomes
red-brown. This microbe occurs in twos, threes, and
fours ; and very rarely as an isolated coccus. The
pigment is soluble in water.

Micrococcus hcematodes. — This microbe is some-
times found in human sweat. It grows on steamed-

118 A MANUAL OF BACTERIOLOGY

egg albumin in a damp chamber placed in the
incubator (see Figs. 22 and 14). The pigment pro-
duced is a red colour.

Micrococcus flavus tardigmdus. — Colonies of this
microbe form raised drops of a chrome-yellow colour.
In test-tube cultivations, they form small yellow
beads along the track of the needle. This microbe
does not liquefy the gelatine.

Micrococcus flavus liquefaciens. — The microbe
grows in colonies of a yellow colour, and the cells
form diplococci and zooglea. They liquefy the
gelatine.

Micrococcus versicolor. — Small cocci forming iri-
descent colonies. The colonies are flat, not raised \
and in test-tubes the yellowish colonies have the
appearance of small beads, i.e. along the needle track.
These cocci are found in pairs or in masses.

Micrococcus flavus desidens. — This microbe occurs
in the dust of the atmosphere. The cells are 0*8 p
in diameter, and occur singly, as diplococci, and in
short chains. They form yellow colonies, which
ultimately sink down in the gelatine. The yellow
pigment is only formed at the surface of the gela-
tine, for in the track of the needle the colonies are
white.

Micrococcus citreus conglomerate. — The cells are
1'5 fju in diameter, and occur in the atmosphere and
in blennorrhoeic pus. On gelatine plates they form
citron yellow colonies.

Micrococcus cereus flavus. — The cells are 1'5 //, in
diameter, and occur singly, in lemon-yellow groups,
or in short chains. They are found in pus.

THE BIOLOGY OF MICROBES, ETC. 119

Micrococcus subflavus. — The cells are 0'8 //, in
diameter, and occur singly, in pairs, in tetrads, and
zooglcea groups. On gelatine they form white dots,
which ultimately become yellow and confluent. This
microbe was originally found in vaginal secretions
and lochial discharges.

Micrococcus radiatus. — The cells are 0'8 p in
diameter, and occur singly and in short chains. They
form ' whitish colonies with a yellowish-green sheen.'
The colonies liquefy the gelatine and sink down in
it ; there developing, in the course of a day or two,
a circlet of rays.

Micrococcus pyogenes citreus. — The cells occur
singly, in chains, and masses. They grow on nutrient
agar-agar and gelatine, giving rise to a lemon-yellow
pigment. They are obtained from pus.

Micrococcus pyogenes. — The cells are 1 p in diame-
ter, and occur in chains or diplococci. They form
small colonies which grow slowly ; on plate-cultiva-
tions they are first white, then pale yellow, and
finally become brown. They (the colonies) have no
tendency to run together in either plate, stroke,
or puncture cultivations, except on agar-agar or
blood serum where the mass is thicker in the
centre. They do not grow on potatoes ; and do
not liquefy any medium. They occur in the pus
of acute abscesses.

Micrococcus pyogenes aureus. — This coccus occurs
in osteomyelitis. It grows on boiled potatoes,
nutrient gelatine, agar-agar, and blood serum, giving
rise to orange cultures. This microbe liquefies
gelatine, and the colonies remain limited to the

120 A MANUAL OF BACTERIOLOGY

centre of the liquefying area. M. pyogenes aureus is
0*8 to 0'9 fj, in diameter, and occurs as diplococci,
tetrads, short chains, and in irregular masses. It is
fatal in large doses to guinea-pigs, mice, and rabbits
if injected into the veins or into the peritoneal
cavity. According to Becker, ' when a small quantity
of a cultivation was introduced into the jugular vein
after previous fracture or contusion of the bones of
the leg, the animal died in about ten days, and
abscesses were found in and around the bones,
and in some cases in the lungs and kidneys/ This
microbe peptonises albumin.

Micrococcus urece. — The cells are round or oval,
and measure 1/25 to 2 //, in diameter. They occur
isolated or concatenate or forming a zooglcea on
the surface of the fluid. M. urece secretes a ferment
which causes the ammoniacal fermentation of urea :

CH4N20 + 2H20 = (NH4)2 C03.

The ferment has been isolated (in aqueous solu-
tion), and it is proved that it has the power of
converting urea into ammonium carbonate.1 Besides
this well-known microbe, there are certain bacteria,
and possibly bacilli, which produce a similar re-
action.2

Micrococcus pyogenes albus. — The cells are 0*8 to
0*9 //, in diameter, and occur as diplococci, tetrads,
short chains, or irregular masses. They grow
rapidly on gelatine plates, producing colonies which

1 Dr. Musculus in Comptes Rendus, vol. Ixviii. ; and Dr.
Sheridan Lea in Journal of Physiology, 1883 and 1885.

2 See Dr. MiqueFs paper in the Annuaire de I' Observatoire de
Montsouris, 1889.

THE BIOLOGY OF MICROBES, ETC. 121

are white. In test-tube cultivations, a white mass
is formed along the needle track. About the third
day of growth liquefaction sets in, and ultimately a
white deposit settles at the bottom of the liquefied
gelatine. This microbe is associated with suppura-
tion. It is found in pus, necrotic tissues, etc.

From what has been already stated in this chapter
it will be seen that many micrococci are associated
with wounds, abscesses, etc. Concerning the action
of these microbes, Dr. W. Watson Cheyne l says : —

(1.) There are various kinds of micrococci found
in wounds treated aseptically, differing markedly
from each other in their effects on animals. They
agree in growing best at the temperature of the
body, and in causing acidity and sweaty smell in
the fluids in which they grow. The experiments
(Cheyne's) show that cultivations may be carried on
in fluid media with accuracy.

(2.) The micrococci examined grew best in media
exposed to oxygen gas ; and they grew only with
difficulty in the absence of oxygen. Dr. A. Ogston 2
stated that these micrococci were anaerobic; but
there is no doubt that this statement is erroneous.

(3.) Their effect on animals was not altered by
growth with or without oxygen.

(4.) The effects of these micrococci on rabbits
and man were not similar, some of the most virulent
forms for rabbits causing no deleterious effect in
wounds in man.

(5.) The kidney is apparently an important

1 British Medical Journal, 1884.

2 Ibid., 1881.

122 A MANUAL OF BACTERIOLOGY

excreting organ for microbes (Fig. 31); and microbes
incapable of growing in the blood, may cause serious
effects by growing in the excretory canals. This
may explain some cause of pyelitis.

(6.) Micrococci are always present in acute

FIG. 31. SECTION OF KIDNEY CONTAINING MICROCOCCI (after Watson Cheyne).

To the left is a mass of micrococci ; to the right an inflammatory ring,
and intermediately the necrotic area, infiltrated with micrococci. What
are evidently remains of two kidney-tubules are seen full of micrococci
and leucocytes.

X 375.

abscesses, and are probably the cause of them. In
some cases, the micrococci are the primary cause of
the inflammation and suppuration, as in pyeernic

THE BIOLOGY OF MICROBES, ETC. 123

abscesses; generally, however, they begin to act
after inflammation has been previously induced.
This inflammation may be caused by an injury, by
the absorption of chemically irritating substances
from wounds, by colds, etc.

(7.) There are several different kinds of micrococci
associated with suppuration.

(8.) Micrococci cause suppuration by the produc-
tion of a chemically irritating substance (probably
a ptomaine), which, if applied to the tissues in a
concentrated form, causes necrosis of the tissue,

Fio. 32. MICROBES IN PURPURA.

(Watson Uheyne.)

A, Micrococci. B, Bacilli.

X 2500.

but, if more dilute, causes inflammation and sup-
puration.

Micrococcus in purpura hcemorrhagica. — Watson
Cheyne1 has observed cocci (measuring 1'15 /* in
diameter) in certain cases of purpura hsemorrhagica.
This microbe forms colonies in the blood ; and the
haemorrhages are due to the plugging of the small
vessels by masses of these microbes. The microbes
occur in chains (Fig. 32), and stain well with

1 Transactions of the Pathdogkal Society of London, 1884.

124 A MANUAL OF BACTERIOLOGY

methylene blue. In another case of the same
disease, Watson Cheyne found that certain bacilli
plugged the vessels and gave rise to haemorrhages.
Concerning this disease, he remarks that ' we may
have to do with an infective disease of which the
essence is the entrance of certain specific organisms
into the blood, and their growth in it. It may,
however, be that in these two cases, and in others,
the primary affection is something quite distinct
from microbes, resulting, however, in such an altered
constitution of the fluids of the body, that of the
innumerable organisms present in the mouth and
intestinal tract, certain of them may be able to
penetrate into and live in the blood, form emboli,
and thus lead to the haemorrhages which are so
marked a feature of these diseases.'

Micrococcus variolce et vaccinice. — Micrococci (0'5
p in diameter) have been found in the lymphatics
of the skin (in small-pox,1 cow-pox, and sheep-pox2)
in the vicinity of the pocks. The microbes were
found by Cohn3 in the lymph of vaccina and
variola. No doubt they are the active agent in
small-pox and cow-pox, for if the lymph is filtered
through a Chamberland filter, the filtrate loses its
infectious properties.

The author * has shown that a solution of salicylic
acid acts upon vaccine lymph, and deprives it of
the power of inoculation.

1 Weigert in Med. Centralblatt, 1871.

2 Klein in Philosophical Transactions of Royal Society, 1874.

3 Virchow's Archiv, vol. Iv.

4 Griffiths in Proc. Roy. Soc. Edinburgh, vol. xiv. p. 97.

i°

<>>&

V »-•'•

••H:

:; v.

^. -

'** '

V..- .
8 : /

Fio. 33. VARIOUS MICROBES.

126 A MANUAL OF BACTERIOLOGY

According to Quist,1 artificial cultivations of M.
vaccinice have been used, with success, for vaccina-
tion purposes.

M. vaccinice (Fig. 33, 1) occurs singly, in pairs,
chains, and colonies.

Micrococcus endocarditicus. — This microbe has
been found by numerous observers in masses and
chains in the granulations, blood-vessels, the valves
and muscles of the heart in endocarditis ulcerosa ;
and there is little doubt that the disease is due to
this microbe. M. endocarditicus measures from 0'5
to 1 fjb in diameter, and occurs singly and in chains.
This microbe is capable of assuming the zoogloean
state, and no doubt when in this state it gives rise
to embolism.

The same microbe has been found in the spleen,
kidneys, and urine.

Micrococcus in Measles. — Dr. Keating2 of Phila-
delphia, and subsequently Cornil and Babes,3 have
observed the presence of micrococci (singly and
as diplococci) in the capillary vessels of the skin,
in the catarrhal exudations, and in the blood of
persons suffering from measles. The same microbe
has also been found in the urine during the course
of the disease. This microbe has not yet been
cultivated.

Dr. Salisbury, in 1862, stated that measles was
due to a certain fungus derived from musty straw.
Since that date, the pathogenic nature of Salisbury's

1 St. Peter sburgh Med. Wochenschrift, 1883.

2 Philadelphia Medical Times, 1882.

3 Les Bacteries, 1885.

THE BIOLOGY OF MICROBES, ETC. 127

straw-fungus has been generally discredited, until
the year 1889, when Mr. C. Candler1 argued in
favour of Salisbury's theory — that fungus-dust
from mouldy straw produces a disease resembling
measles ; and that this fungus-dust when introduced
into the human body, develops into microbes (!).
In the great epidemic of measles in Victoria during
the years 1874-75, Candler states that he could not
discover any instance of measles in a dwelling from
which damp straw (in the form of bedding) had
been excluded, but in every house where measles
occurred, the presence of damp straw in the bed-
rooms was easily made out. There is nothing
impossible in the supposition that damp straw
favours the growth of microbes ; and it might con-
ceivably be proved by sufficient evidence that this
is a favouring or even a necessary condition for the
growth of the specific virus of measles. But the
evidence which Candler adduces is quite inadequate
to prove that the cause of measles is a fungus, since
it might just as well be Keating's micrococcus or
any other microbe.

Micrococcus gonorrhcece. — Drs, Neisser,2 Bokai,
and Finkelstein 3 have described micrococci in the
urethral discharge and the pus of gonorrhoea.
These microbes (Fig. 33, 16) measure 0'83 ^ in
diameter, and occur singly, as diplococci, sarcinse,
and in zooglcean groups. They frequently adhere
to the epithelial cells and pus- corpuscles. Dr.

1 The Prevention of Measles, 1889.

2 Centralblatt fiir d. Med. Wissensch., 1879.

3 Prager Med. Chir. Presse, 1880.

128 A MANUAL OF BACTERIOLOGY

Bockhart 1 has artificially cultivated these microbes ;
and has reproduced the disease by inoculation, thus
proving their pathogenic character. A similar micro-
coccus is often found in the purulent ophthalmia
of new-born infants ; and it is possible that such
ophthalmia is, in the majority of cases, of gonorrhceal
origin.

* Aufrecht2 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. These micro-
cocci corresponded in size to Micrococcus gonorrhcece,
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 '
(Klein).

Microccus tetragonus. — This microbe is found in
the sputum of patients suffering from phthisis. It
is only saprophytic in man, but pathogenic in
animals. Mice inoculated with a pure cultivation
of this microbe die in a few days, the microbe after-
wards being found in the various organs of the body.
Micrococcus tetragonus (Fig. 33, 14) measures 1 //, in
diameter, and occurs as tetrads surrounded by a
hyaline membrane. This microbe forms small
white points on nutrient gelatine in about twenty-
four hours, which ultimately run together.

Micrococcus intracellularis meningitidis. — This
microbe has been observed in the pus found at

1 Sitzungsberichte der Pkys. Med. Gesell. Wiirzburg, 1882.

2 CentralUattfur d. Med. Wissensch., 1883.

THE BIOLOGY OF MICROBES, ETC. 129

the base of brain after death in cases of acute
cerebral meningitis. It occurs singly, as diplococci,
chains, and zoogloea ; and it grows on a mixture of
agar-agar and gelatine at the temperature of the
body. This microbe grows better at the surface
than in the deeper layers of the medium, and gives
rise to finely granular and yellowish-brown colonies.
The microbe, when cultivated artificially, only
remains virulent for six days ; and it is said that it
' affects mice, guinea-pigs, rabbits, and dogs.' Like
M. gonorrhcece, this microbe 'is almost invariably
found within the cells contained in the exudation/

Micrococcus lomlycis. — The cells are oval, and
measure 0'5 p in diameter. They occur singly, as
diplococci and chains, and produce the ' flacherie '
or ' schlafsucht ' — one of the silkworm diseases.
Another disease of the same larva is known
as ' pebrine ' ' maladie des corpuscules,' and is caused
by a microbe called Micrococcus ovatus, which
measures about 1*5 //, in diameter. M. ovatus is pre-
sent in large numbers in the blood and organs of
affected silkworms.

Micrococcus of cattle -plague. — Micrococci have
been found in the blood and lymphatic glands of
cattle dead of this disease. They occur singly, as
chains and zoogloea, and grow rapidly in bouillon and
other media at 37° C. Semmer and Archangelski *
have shown that calves inoculated from a pure cul-
tivation of this microbe died in seven days with all
the typical symptoms of cattle-plague or rinderpest.

By successive cultivations, or by exposing culti-
1 Centralblatt fiir d. Mod. Wissensch., 1883.
I

130 A MANUAL OF BACTERIOLOGY

vations for an hour to a temperature of 46° C., the
virulence of this microbe is greatly reduced ; and in
this attenuated or weakened form it has been used
for the protective inoculation of sheep and cattle.

Micrococcus of foot-and-mouth disease. — According
to Dr. Klein, the microbe of this disease occurs
singly, as diplococci, and in curved chains. ' It
grows well in milk, in alkaline peptone broth, in
nutrient gelatine, and in agar-agar mixture. Grow-
ing 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
does not liquefy nutrient 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.' The microbe has been observed in the vesicles
of sheep suffering from the disease.

Micrococcus septicus. — The cells are 0*5 //, in dia-
meter, and occur singly, as diplococci and chains.
Colonies are produced very slowly on nutrient
gelatine; they are seen as minute dots on the
fourth and fifth days in plate and tube cultivations.
They are fatal to mice, rabbits, etc. ; the vessels in
the various organs become plugged with these
microbes, this ultimately forming purulent or
necrotic foci. M. septicus is present in soil.

Micrococcus in gangrene. — Small oval micrococci
have been found in gangrene of the lungs. They
live in colonies, form zoogloaa, and grow on nutrient

THE BIO LOO Y OF MICROBES, ETC. 131

gelatine, giving rise to the characteristic but offen-
sive gangrenous odour. The same microbes have
been observed in various gangrenous tissues, and
also in the blood of patients suffering from ' Clou de
Biskra ' or ' Bouton d'Alep,' which excite gangrene
when injected into rabbits.

Micrococcus perniciosus. — According to Wolff1 this
microbe is the cause of a disease of the grey parrot.
The cells measure 0*8 p in diameter, and occur
singly, in chains and zooglcea. They produce
nodules in the liver, lungs, spleen, and kidneys;
but inflammation around the nodules is entirely
absent. The microbes also occur in the blood. The
disease is said to be fatal to 80 per cent, of the
grey parrots imported into Europe.

Micrococcus insectorum. — This microbe occurs in
the digestive organs of the chinck-bug (Blissus
leucopterus) ; and is probably the cause of an infec-
tious disease of this insect. The cells are obtusely
oval (0'7 to 1 fj, long x 0'55 /JL broad), and occur
singly, in pairs, chains^ or zoogloea. They may be
cultivated in bouillon.

Micrococcus of Tissue Necrosis in Mice. — Dr. E.
Koch observed that a certain micrococcus, isolated
from putrid fluids, when injected into the ear of a
mouse, gave rise to tissue necrosis and death in
about three days. The microbe was not found in
the blood and internal organs. The cells measure
0'5 fi in diameter, and occur in chains and zoogloea.

Micrococcus in whooping-cough. — Whooping-cough
is undoubtedly an infectious disease, and, according

1 Virchow'a Archiv, 1883.

132 A MANUAL OF BACTERIOLOGY

to Dr. Burger,1 oval micrococci are often present
in the pearly phlegm ejected by patients suffering
from this disease. They have not yet been culti-
vated.

Micrococcus in pernicious anaemia. — According to
Frankenhauser2 the blood of pregnant women suffer-
ing from pernicious anaemia contains a large number
of micrococci which appear to be of a pathogenic
character. These micrococci are comparatively of
large size, ' about one-tenth of the broad diameter
of a red blood-corpuscle.' These microbes have not
been cultivated.

Micrococcus of Nitric Fermentation. — Mr. E. War-
ington, F.K.S.,3 has recently isolated from soil a
micrococcus which converts nitrites into nitrates.
But this micrococcus, as well as Frankland's nitrous
bacillus, will be described later, i.e. under the head-
ing of ' the microbes of the soil.'

Micrococci in Pyaemia and Septicaemia. — A con-
siderable number of micrococci (from O'l to I'O //, in
diameter) have been found in various organs, blood,
etc., in pyaemia and septicaemia in the lower animals
and in man.4

Micrococci have also been described in haemo-
philia neonatorium, in ozsena, in acute yellow
atrophy of the liver, in closed abscesses, and in many
other diseased conditions.

1 Berlin Klin. Woch., 1883. See also the Appendix.

2 Centralblatt fur d. Med. Wissensch, 1883.

3 Journal of Chemical Society, 1891, pp. 484-529.

4 See Dr. Watson Cheyne's papers in the British Medical
Journal, Sept. 20, 27, Oct. 4, 1884, and July 31, 1886 ; also Dr.
Crookshank's Manual of Bacteriology (2d ed.)

THE BIOLOGY OF MICROBES, ETC. 133

BACTERIA.

Bacterium termo. — The cells are oblong and
measure 1'5 n in length and about 0'5 //, in breadth
(Fig. 33, 15). Each cell is surrounded with a thick
membrane of cellulose (C6 H10 05) and a flagellum
at each end. Dr. W. H. Dallinger, F.K.S., has
measured the diameter of the flagellum of this
microbe, andjie finds that it is the aoAooth of an
inch, or expressed in decimals 0*00000488526 inch.1
B. termo is one of the commonest forms in water
and putrefying fluids, but it always disappears when
putrefaction terminates, in fact it has been called
the microbe of putrefaction. It has remarkable
powers of vitality; it is most active between 32°
and 36° C. ; at a temperature below 5° C. and
above 46° C. it does not produce putrefaction in
putrescible fluids ; however, above 50° C. it is killed,
but even at so low a temperature as — 1 1 0° C. this
microbe is not destroyed.2 It grows well on
bouillon, agar-agar, etc., and after several days'
incubation a pellicle is formed on the fluid medium.
When grown on solid agar-agar it imperfectly
liquefies the medium, and gaseous products are
generated. On sterilised potatoes B. termo produces
a slimy grey colony. It occurs singly, in pairs, and
zoogloea, and it is readily obtained by placing a
piece of meat in water kept in a warm place for a
few hours. It may be remarked that this microbe
has been considered to be only a phase-form of a
protean species.

1 Journal of Royal Microscopical Society, 1878, p. 174.

2 Giglioli's Fermentive Microbi, p. 50t , „ -^ v.

I w « IVZXwITj

o»-

134

A MANUAL OF BACTERIOLOGY

Bacterium lineola. — This microbe resembles B.
termo in form and movement, only it is much longer
and thicker than that microbe. Each cell measures
from 3 to 5 /A in length and 1*5 //, in breadth, and is
provided with two flagella, one at each end of the
cell. This microbe occurs singly, in pairs, in zoo-

gloea, but never
in chains or ros-
aries. It is found
in well-water and
stagnant water,
where no distinct
putrefaction is go-
ing on ; it forms
pellicles on steril-
ised potatoes and
various infusions.
<^> — " Bacterium allii.
— During the year
1887 the author1
discovered this
microbe in the
greenish slime of
diseased or putre-
fying onions and
allied plants. It measures from 5 to 7 //, in length and
2 /JL in breadth, and occurs singly, in pairs, and forms
zooglcea. It has been named Bacterium allii because
it was originally found on Allium cepa (the onion).
Bacterium allii grows tolerably well on nutrient
agar-agar, and produces a bright green pellicle on

1 Proceedings of Royal Society of Edinburgh, vol. xv. p. 40.

FIG. 34. BACTERIUM ALLII.
A, Growing on agar-agar.

B, The microbe isolated.

THE BIOLOGY OF MICROBES, ETC. 135

the surface of the nourishing medium (Fig. 34). The
green pigment is soluble in alcohol, and an alcoholic
solution gives an absorption spectrum, consisting of
a band extending from the extreme violet to the
blue part (nearly to the Fraunhofer line F) of the
spectrum. There is also an absorption band in the
green, and one in the yellow, part of the spectrum.
The end of the band in the yellow is exactly in the
same position as the D line in the solar spectrum.
Bacterium allii forms an alkaloid or ptomaine from
albuminoid molecules. This ptomaine has the same
chemical composition as hydrocoridine (C10 H17 N).1
Besides the pigment and ptomaine, small quantities
of sulphuretted hydrogen gas are liberated from the
medium on which the microbe lives. The sulphu-
retted hydrogen was proved by the black stain (PbS)
produced upon paper impregnated with a solution of
lead acetate, and also by the yellow stain (CdS)
produced on cadmium paper (CdCl2). B. allii is
best stained with gentian violet. The vitality of
this microbe is remarkable,2 for it still retains its
vitality when exposed, in a dry state, to a tempera-
ture of 32° C. for six months. A pure culture of
this microbe exposed to —15° C. for three days
proved that it was not killed ; but it was killed
after fourteen days' exposure at the same tempera-
ture. An E.M.F. of 3 -3 volts killed B. allii iii ten
minutes.

1 See Dr. Griffiths' papers in Comptes Rendus de I'Acade'mie
des Sciences, tome 110, p. 416 ; and Gentralblatt fur Bakte.riologie
und Parasitenkunde, Bd. vii. (1890), p. 808.

- Proceedings of Royal Society of Edinburgh, vol. xvii. pp.
262-264.

136 A MANUAL OF BACTERIOLOGY

The microbe in question is quite distinct from
the lacillus (giving a green fluorescence) which
Heraeus obtained from soil.1 The bacillus of
Heraeus converts urea into ammonia, while Bac-
terium allii has no such action, for it decomposes
albuminoids (vegetal and animal) with the forma-
tion of a ptomaine among other products.

Bacterium aceti. — This is the microbe which
causes the acetic fermentation according to the well-
known reaction : —

C2H5OH + 02 = H20 + CH3COOH.
It is about 1'5 //, in length, and occurs singly, in
long chains, and forms a pellicle on the surface of
the nutritive fluid. Although Pasteur maintained
that B. aceti was the cause of the acetic fermenta-
tion, and Cohn2 observed the microbe largely in
sour beers, yet not until the commencement of 1886
could any one say with certainty that this microbe
was the real cause of the acetic fermentation. In
that year Mr. A. J. Brown 3 prepared pure cultiva-
tions of the microbe in question, and found that it
does convert alcohol into acetic acid or vinegar.
The author4 entirely indorses the correctness of
Brown's researches. After obtaining pure cultiva-
tions of B. aceti by the fractional and dilution
methods, it was found that these cultivations, when
used to inoculate sterilised ethyl alcohol (6 per cent.)
gave acetic acid in abundance.

1 Zeitschrift fur Hygiene, 1886.

2 Biol. d. Pflanzen, Bd. ii. p. 173.

3 Journal of Chemical Society, 1886, p. 172.

4 Proceedings of Royal Society of Edinburgh, vol. xv. p. 46.

THE BIOLOGY OF MICROBES, ETC. 137

Bacterium lactis. — The cells measure from T5 to
3 IJL in length, and are constricted in the centre like
the figure 8. They occur singly, in long chains,
zoogloea, and like B. aceti, they are motile. Bac-
terium lactis is the cause of the lactic fermentation
or the souring of milk. The sugar of milk or lactose
is converted into lactic acid by the growth of this
microbe (Lister *). Unlike B. aceti, this microbe is
anaerobic. B. lactis, along with other microbes,
plays an important part in the preparation of sauer-
kraut ; and Dr. Baginski has recently shown that it
produces a powerful reducing action in pure cultiva-
tions, where the nutrient fluid was coloured with
methylene blue.

Bacterium decalvaiis. — This microbe was discovered
by Dr. G. Thin 2 in the roots of the hair in cases of
Alopecia areata ; and he supposes that it penetrates
downwards between the root-sheath and the hair,
then passes through the cuticle of the hair, and
ultimately ascends within its substance, causing it
soon to fall off. It measures 1*6 /*, in length, and
occurs usually in pairs.

Bacterium cholercegallinarum. — This is the microbe
of fowl cholera, and it is found in large numbers in
the blood and organs of fowls dead of this disease.
It measures 1*2 to 1-5/4 in length, and the ends are
always stained more deeply than the middle part.
B. cholerce gallinarum is easily cultivated in chicken
broth (neutral) at 25° — 35° C., and when fowls are
inoculated with a drop of this culture they always

i Transactions of the Pathological Society, 1878.
Proceedings of Royal Society, vol. xxxiii. p. 247.

138 A MANUAL OF BACTERIOLOGY

die with the characteristic symptoms of the disease.
If a culture of the microbe is kept for two or three
months its virulence is lessened ; and an attenuated
virus has been successfully used by Pasteur in the
protective inoculation of fowls against this disease.
This microbe is pathogenic in rabbits as well as
fowls, but guinea-pigs have an immunity; it is
aerobic, and is cultivated in contact with sterilised
air or in aerated fluids. In fact, ' its toxic effect has
been supposed to be due to the abstraction of
oxygen from the blood, producing asphyxia.' B.
cholerce gallinarum grows on gelatine as small,
round, white colonies with lemon-yellow centre. It
grows on potatoes at 37°C., producing yellow-grey
drops. M. Duclaux1 has shown that this microbe
produces a ptomaine; but when the ptomaine is
separated, by filtration through porous porcelain
from its microbe it does not produce fowl cholera ;
for it causes a passing sleep, which does not gener-
ally end fatally. From this fact the conclusion
may be drawn that in fowl cholera the microbe is
essentially the active agent in producing the disease.
Bacterium pseudo-pneumonicum. — This microbe
forms greyish-white layers in test-tube cultivations ;
while on gelatine plates the colonies appear as white
dots. It grows on sterilised potatoes at 37° C.,
giving rise to a white, viscid layer; it measures
1*16 fj, in length, and 0*8 p in breadth, and requires
air for its growth. It is only slightly pathogenic.
B. pseudo-pneumonicum occurs in pus taken from
abscesses.

1 Ferments et Maladies.

THE BIOLOGY OF MICROBES, ETC. 139

Bacterium xantMnum. — This bacterium is the
cause of 'yellow milk/ which at first is acid, but
soon becomes alkaline. The pigment produced by
this bacterium is soluble in water, and insoluble in
alcohol and ether. B. xanthinum measures from
07 to I'O ft in length, and forms colonies on pota-
toes.

Bacterium septicus agrigenum. — This microbe
occurs in soils, and measures from 2 to 3 /z, in length.
On plate cultivations it produces colonies of a greyish-
yellow colour, with a yellowish-brown centre. This
microbe is fatal to mice, rabbits, and guinea-pigs.
It multiplies rapidly in the blood, and it adheres to
the red blood corpuscles.

Bacteriumcoli commune. — This bacterium measures
1-7 fi in length and 0'4 p in breadth, and it occurs
in the faeces of infants fed on human milk. It
grows on nutrient gelatine, forming granular colonies
of a yellowish colour. It is fatal to rabbits and
guinea-pigs, causing violent diarrhoea and fever.

Bacterium fcetidum. — This microbe was discovered
by Dr. Thin 1 in the alkaline serous exudation from
the soles of the feet of a person who suffered from
profuse sweating of the feet. It produces a foetid
odour, which is also observable in artificial cultures
of this microbe. B. fcetidum occurs singly, in pairs,
and leptothrix threads. This microbe appears to be
identical with Rosenbach's Bacillus saprogenes ; and
it is readily cultivated in agar-agar and blood-
serum.

Bacterium Neapolitanum. — This microbe occurs as

1 Proceedings of Royal Society, vol. xxx. p. 473.

140 A MANUAL OF BACTERIOLOGY

short-rods with rounded ends, measuring about 1 //,
in length. On nutrient gelatine it forms circular
colonies, which, however, become irregular, granular,
refractive, and of a yellow colour. It was isolated
from certain cases of cholera at Naples ; but it has
nothing to do with the disease, for it is only sapro-
phytic in man.

Bacterium septicum sputigenum. — This microbe is
identical with Sternberg's Micrococcus Pasteurianus
and Frankel's Bacillus septicus sputigenus. It is
present in human saliva, and occurs as short rods,
frequently joined together in chains of five, six, or
seven links. It is usually obtained from the rusty
sputum of pneumonic patients and from severe cases
of empysemia. It gro\^s well in bouillon and on
agar-agar at 34° C., but slowly on gelatine plates.
The colonies are granular and white. This microbe
is pathogenic in rabbits, mice, and guinea-pigs, but
fowls and dogs have an immunity. Dr. Watson
Cheyne l says that it ' apparently loses its virulence
when cultivated outside the body. The blood of
rabbits which have died of this microbe is very
virulent, a small quantity being sufficient to set up
the disease in a fresh animal, but when grown in
meat-infusion, agar-agar, etc., it rapidly (in three or
four days, unless re-inoculated into fresh material)
loses its virulence, and the dose necessary to cause
death increases as the cultivation becomes older.
When it does not cause death it may produce a
slight local effect, and such animals are apparently
protected from a subsequent inoculation with viru-

1 .British Medical Journal, July 31, 1886.

THE BIOLOGY OF MICROBES, ETC. 141

lent material. The animals often do not die for
three or four days after the injection, and generally
exhibit nervous symptoms, sometimes ending in
paraplegia.'

Bacterium indicum. — Is an aerial microbe rod-
shaped with rounded ends. On nutrient agar-agar
it produces a scarlet-coloured growth, but after some
days the growth loses its bright colour, and becomes
purplish, like an old cultivation of Micrococcus pro-
digiosus. On gelatine this microbe liquefies the
medium, and colours it scarlet. It also grows well
on the surface of potatoes.

Bacterium merismopedioides. — Each rod measures
from 1 to 1 '5 //, in thickness. It was first observed
by Dr. Zopf in the river Panke, Berlin, and is said
to divide into long and short rods, and finally into
cocci. This microbe also exists in zooglean form.1

Bacterium Zopfii. — This bacterium, which was dis-
covered by Kurth, measures from 2 to 5 p in length,
and from 0*7 to 1 p in breadth. It is motile, and
occurs in long threads. It grows on gelatine-plates,
developing into thread-like growths in about thirty-
six hours. This microbe was first isolated from the
intestine of fowls.

Bacterium oxytocum perniciosum. — First isolated
from sour milk. Each rod has rounded ends, and
forms yellowish colonies on gelatine plates. Needle
cultures have the characteristic nail appearance.
In milk this microbe produces curdling and an acid
reaction. It measures 1 /JL in length ; and in large
doses it is pathogenic in rabbits.

1 Zopf, Die Spaltpilze (1885) ; and Die Pilze (1890).

142 A MANUAL OF BACTERIOLOGY

Bacterium phosphorescens. — The cells of this mi-
crobe are almost circular, being from 1*3 to 1*9 /JL
long, and lll to 1*7 p broad; each cell is motile,
and surrounded by a gelatinous membrane. It is
readily cultivated on fish broth containing a small
quantity of peptone ; it grows slowly at the ordinary
temperature in peptonised gelatine, or in peptonised
gelatine containing 2 per cent, of glucose, but only
at the surface, and the property of emitting light
depends on the presence of oxygen.1 It also grows
well in 2, 3, and 4 per cent, solutions of sea-salt,
containing 0'25 per cent, of peptones. On shaking,
the phosphorescence becomes intensified, but on
cooling to 0° C. its intensity is somewhat dimin-
ished. The phosphorescence disappears when the
solution is heated to 35° C. for a few minutes, but
re-appears on cooling; it is, however, completely
destroyed by heating at 35° C. for fifteen minutes.

After two or three weeks the culture solutions
become yellowish, and gradually lose their phos-
phorescence; after several weeks phosphorescence
ceases entirely, but the microbes do not die. The
phosphorescence is most probably caused by ferment
action in the presence of oxygen. This microbe
forms colonies on the surface of agar-agar, gelatine,
and potatoes, and also grows in urine and milk.

Bacterium P/£%m'.— This microbe is the most
phosphorescent of all the light-emitting bacteria.
It is distinguished from the preceding form by not
emitting light with peptone and maltose, but it

1 See Hjelt's General Organic Chemistry (English translation),
p. 94.

THE BIOLOGY OF MICROBES, ETC. 143

emits light with peptone and glucose. It measures
from 1'5 to T9 //, in length, and from 1 -3 to 17 //, in
breadth ; these rods have rounded ends, and appear
to divide exceedingly rapidly. The bacterium is
motionless, and occurs singly, and sometimes in
short chains. ' On plates prepared with peptone
gelatine, to which a small quantity of glucose, and
from 2 to 3 per cent, of common salt have been
added, the microbe develops luxuriantly, giving
rise to small, white, mother-of-pearl-like colonies,
about the size of a pin's head, with no surrounding
zone of liquefied gelatine.' This microbe is readily
obtained by placing fresh cod or herring (with moist
surfaces) between a couple of plates, and kept at
about 20° C. for twenty-four or thirty-six hours.
At the end of this time small phosphorescent points
or dots are seen to glow on the surfaces of the fishes.
These dots are colonies of the microbe in question.

Bacterium Fischeri. — Unlike the preceding phos-
phorescent microbes, B. Fischeri liquefies peptonised
gelatine ; and by the addition of a small quantity of
sugar the intensity of the phosphorescence is in-
creased. The microbe is motile, and occurs singly
and in short chains. It grows on agar-agar at a
low temperature (from 0° C. to 15° C.).

Bacterium Balticum. — Like the preceding microbe,
B. Balticum was found in the waters of the Baltic,
and also liquefies peptonised gelatine.

The four forms of phosphorescent bacteria can-
not develop their lighkemitting functions to their
highest point without the presence of some substance
from which carbon may be easily obtained, such as

144 A MANUAL OF BACTERIOLOGY

glycerol, glucose, asparagine, sugar, etc., as well as
peptone. For this reason they have been termed pep-
tone-carbon-bacteria. Beyerinck,1 who has recently
studied these microbes, states that they are best
cultivated in fish broth with sea-water, to which are
added 1 per cent, of glycerol, 8 per cent, of gelatine,
and i per cent, of asparagine.

Photo- Bacterium Indicum. — This microbe occurs
in the West Indian Sea. It liquefies gelatine very
rapidly ; and the greatest intensity of light is given
off when the culture is kept between the tempera-
tures of 30° and 35° C.

Bacterium luminosum. — This microbe, which is
most active at about 15° C., is found in the North
Sea. Both the preceding and the present microbes
give off light in peptonised gelatine without requiring
the presence of sugar or any other carbohydrate, conse-
quently they have been termed peptone-bacteria.

In all these bacteria (phosphorescent) the develop-
ment of luminosity is constantly accompanied by
the transition of peptones into organised, living
matter, under the influence of free oxygen, with or
without the concurrence of another compound con-
taining carbon.

Certain other bacteria, although they do not emit
light, are influenced by it, among these are the two
following microbes : —

Bacterium chlorinum. — The cells are from 2 to 3
fi in length, and are motile. This microbe accumu-
lates in the light, but only when oxygen is absent.

1 Transactions of Royal Academy of Sciences of Amsterdam,
1890.

THE BIOLOGY OF MICROBES, ETC. 145

Bacterium photometricum. — According to Dr.
T. W. Engelmann,1 this microbe is influenced by
light ; in fact, its movements are stated to depend
on light. It produces a red pigment, but the
amount of pigment formed varies with the action of
light. Different coloured lights affect this bacterium
differently, the most powerful being the ultra-red,
the yellow, and part of the green.

Bacterium crassum sputigenum. — This microbe was
originally isolated from sputum ; it also occurs in
the 'fur' scraped from the tongue. It measures 1 /*
in length and 0*8 /A in breadth. Colonies on gelatine
plates appear as grey, viscid drops, and in needle
cultures develop a nail-shaped growth. This mic-
robe is fatal to mice, rabbits, and dogs.

Bacterium pneumonicum agile. — This is the mic-
robe of vagus pneumonia of rabbits. The cells are
short thick rods, which occur singly or in chains of
three or four. This bacterium forms dark granular
colonies on gelatine, which subsequently liquefies.
It also grows on blood serum, bouillon, and potatoes.
The growth on potatoes spreads very rapidly over
the whole surface as a red layer. Pure cultures of
this microbe are fatal to rabbits.

Bacterium violaceum. — This microbe was dis-
covered by Bergonzini,2 and it measures from 2 to 3
p in length, and from 0'6 to 1 p, in breadth. It
occurs on egg-albumin, forming a violet pigment.
This pigment is insoluble in water, and soluble "in

1 PJluger's Archiv, vol. xxx. p. 95; Revue Internal. Science,
tome ix. (1882), p. 469.

2 Ann. Soc. Nat. Moden., vol. xiv.

146 A MANUAL OF BACTERIOLOGY

alcohol and ether. It is said that ether dissolves out
a red-violet pigment, and alcohol a deep blue one.

Bacterium brunneum. — ' Motile rods, producing a
brown colour. They were observed on a rotting
infusion of maize.'

Proteus vulgaris. — This bacterium is found in
abscesses, putrefying organic matter, meconium-
fseces, and in water. The rods are from 1'25 to 3*75
/j, in length, and about 0'6 //, in breadth. The
threads or chains are usually twisted and convo-
luted, and, according to Hauser,1 involution forms
are found — spherical bodies about 1*6 //, in diameter.
This microbe grows rapidly in nutrient gelatine,
causing liquefaction of the gelatine. In test-tube
cultivations, the fluid gelatine, which is at first
turbid, becomes subsequently more or less clear in
the middle, with a' deposit of flocculi at the bottom,
and a slight turbidity at the top. Growing on
gelatine plates, this microbe rapidly forms greyish
masses, which consist of motile and swarming colo-
nies. After forty-eight hours' growth a foul odour
and an alkaline reaction are developed. This
microbe is pathogenic, and produces abscesses or
death according to ,the dose or quantity of the
microbian culture injected into the animal ; with
rabbits and mice inoculation does not cause any
effect, but the injection of quantities varying from
^ to T3o cc. causes death. On this point Watson
Cheyne 2 states ' that TV cc. injected into the mus-
cular tissue was a fatal dose, indeed ¥V cc. almost

1 Ueber Faulniss-Bacterien, 1885.

2 British Medical Journal, July 31, 1886.

THE BIOLOGY OF MICROBES, ETC. 147

invariably killed, though some animals survived it :
^ cc. always caused an extensive abscess, of which
the animals usually died in six to eight weeks.
Doses of less than -g-J-^ cc. did not produce any
effect. We have thus three results, according to the
dose employed. A small dose (below -g-J^ cc.) pro-
duced no effect ; from -^ to -^ cc. caused abscesses,
while above ^V cc- caused death in from twenty-
four to thirty-six hours. Further, the size of the
abscess depended apparently also on the dose, -^
cc. causing only a slight trace of pus, which dis-
appears, while TV causes a large and spreading
abscess, ultimately resulting in the death of the
animal; and the intermediate doses produce inter-
mediate effects. On several occasions I have diluted
the cultivation considerably, and made plate-cultiva-
tions from this diluted material, in order to ascertain
the number of bacteria present by counting the
number of colonies which developed. The result is
that on an average 1 cc. of gelatine cultivations
contained 4,500,000,000 bacteria. Thus, doses up
to 9,000,000 produced no effect; from 9,000,000
up to 112,500,000 caused abscesses, and above
225,000,000 caused death. It is difficult to under-
stand the influence of dose in producing these
effects, but the following seems to be a fair supposi-
tion. Eabbits are not very susceptible to the action
of this bacterium ; in other words, in the struggle
for existence between the bacteria and the cells
which follows the introduction of this bacterium,
the victory will, in most cases, remain with the
cells, and the bacteria will disappear. If, however,

148 A MANUAL OF BACTERIOLOGY

along with the bacteria, a large quantity of their
products (ptomaines) are introduced, these products
interfere with the action of the cells, and enable the
bacteria to get a foothold. If a large number of
bacteria are introduced at one place they grow for a
time till attacked by the cells, and each produces a
small quantity of poisonous material. Where the
number of bacteria is very large this material
destroys the tissues in the neighbourhood, and
enables the bacteria to spread over a large area
before the layer of cells formed around them is able
to form a barrier against their progress. The extent
to which they spread — in other words, the size of
the abscess which results — must, therefore, depend
firstly on the number of bacteria and the quantity
of products introduced in the first instance ; and,
secondly, on the vitality of the animal. It may be
that a very large amount of organisms is introduced
in the first instance, producing such an amount of
poison as to kill the animal in a few hours.' The
investigations of Watson Cheyne, as well as those of
Hauser, undoubtedly prove that a ptomaine of
poisonous properties is formed by these bacteria
(Proteus vulgaris), but there can be no doubt, also,
that these bacteria themselves are truly pathogenic
for rabbits under proper conditions. Watson Cheyne
has also shown that when Proteus vulgaris is grown
in bouillon it acts with less virulence than when it
is grown in nutrient gelatine.

Proteus mirabilis. — This bacterium is something
like the preceding microbe, although somewhat
shorter. It liquefies gelatine much slower than

THE BIO LOOT OF MICROBES, ETC. 149

Proteins vulgaris, and forms granular colonies of a
brownish colour. It also forms zoogloea.

Protem Zenkeri. — This motile bacterium is 1'65 //,
in length and 0*4 /j, in breadth. In plate-cultiva-
tions it gives rise to greyish colonies, but no zooglcea
are formed. There is only a very slight liquefaction
of the gelatine, and no odour is given off from
cultures on gelatine or blood serum ; but there is a
strong smell given off when the microbe is cultivated
in bouillon.

BACILLI.

Bacillus beribericm. — This microbe was discovered
by De Lacerda1 in the blood of patients suffering
from the disease known as beri-beri, kakke, etc.
It occurs singly in long chains and produces spores.
When cultivated in bouillon and then injected into
rabbits this microbe is said to produce all the
symptoms of beri-beri. The disease is characterised
by anaemia, anasarca, degeneration of the muscular
tissues, numbness, pain and paralysis of the extremi-
ties ; and one of its chief habitats is in Japan. It
is prevalent in the Malay Archipelago, the Molucca
Islands, New Guinea, Burmah, Siam, Ceylon, and
India (south and east) ; and it is endemic as well
as epidemic in other parts of the world.

According to Prof. B. H. Chamberlain,2 'kakke
[i.e. beri-beri] is the national scourge of Japan,
and attacks with special frequency and virulence

1 Lancet, February 9, 1884, p. 268.

2 Things Japanese, 1890, p. 188 (Kegan Paul, Trench, & Co.).

150

A MANUAL OF BACTERIOLOGY

young and otherwise healthy men — women much
less often.' De Lacerda believes that the bacillus
is derived from rice which has undergone a peculiar
alteration.

The epidemic spread of this disease is probably
influenced by climate, and seems to coincide with
conditions of high atmospheric moisture and extreme
thermometric variations.1

Bacillus alvei. — This microbe produces the disease

known as ' foul-
brood ' of bees, and
it has been thor-
oughly investigated
by Cheshire and
Cheyne.2 It mea-
sures 4 fjL in length
and 0 '5/4 in breadth,
and the oval spores
which it produces
measure 2'1 //, in
length and 17 JJL in
FIG. 85. BACILLUS ALVEI. breadth. JB. alvei

blood and juices of
the larvae, drones, workers, and queens, and is

1 For further information see Dr. Felkin's paper in Proceed-
ings of Royal Society of Edinburgh, vol. xvi. p. 291 ; Dr. E.
Baelz's paper in Mittheil. Deuts. Gesellschaft fur Natur- und
Volkerkunde Ostasiens, Bd. iii. p. 301 ; Dr. Anderson's paper in
Transactions of Asiatic Society of Japan, vol. vi. ; Dr. Wernich's
Geographisch-medicinische Studien ; Dr. Scheube's Die Japan-
ische Kak-ke; and the Japanese reports by Drs. Takaki and
Miura.

2 Journal of Royal Microscopical Society, 1885, p. 582.

THE BIOLOGY OF MICROBES, ETC.

151

also present in the ova. Numbers of this mic-
robe are seen moving backwards and forwards
in the blood, etc., of larva? attacked with the
disease. Leptothrix forms of the microbe are
common when the dis-
ease is in rapid progress ;
these sometimes measure
250 p in length (Fig.
35). In the juices of
the larval bee during life
these bacilli do not pro-
duce spores, although
after death spores abound.
In test-tube cultivations
the bacilli grow both on
the surf ace of the gelatine1
and along the needle-
track. At the surface
the bacilli form a delicate
ramifying growth, and
along the track whitish
irregular - shaped masses
appear, which slowly in-
crease in size and run
together. In a few days

processes are seen to shoot out from these masses,
which may extend through the gelatine for long
distances from the track, being thickened at various
parts, and clubbed at the ends. If only a very few
bacilli are introduced with the needle, a beautiful
and characteristic growth is obtained, for by this

i The best growth in gelatine is obtained at about 20° C.

FIG. 36. BACILLUS ALVEI.
(Cheshire and Cheyne,)

152 A MANUAL OF BACTERIOLOGY

means groups of bacilli become planted at a con-
siderable distance from each other (Fig. 36). This
appearance is quite characteristic of B. alvei, and is
not seen in the cultivation of any other microbe.
'The bacilli of anthrax and of mouse septicaemia
also spread out from the needle track, but the
appearance of their cultivation is quite different.
In anthrax delicate threads, not clubbed, shoot out
from the track, soon anastomosing with other
threads and forming a delicate network throughout
the gelatine. In mouse septicaemia the appearance
is that of a delicate cloudiness spreading through
the gelatine. These ' foul-brood ' bacilli, growing in
this material, render it liquid after a time, the
liquefaction beginning at the surface and only
spreading slowly downwards, but ultimately the
whole tube becomes liquid. The liquid becomes
yellowish in colour after a time, and gives off an
odour of stale, but not ammoniacal, urine. This
colour and odour are distinctive of the diseased
larvae.'

In plate- cultivations, the bacilli grow out in
series of rods in single file, or in rows of several
side by side. The processes which are formed have
a tendency to form curves and circles. Later on,
the gelatine in the vicinity of the bacilli becoming
liquid, forms a series of channels in which the
bacilli move backwards and forwards.

They grow most rapidly on the surface of nutrient
agar-agar, forming a whitish layer, but the ramify-
ing processes seen on the surface of gelatine do
not occur, or only very imperfectly, in agar-agar.

THE BIOLOGY OF MICROBES, ETC. 153

Here the bacilli arrange themselves apparently side
by side, and producing spores in this position, we
have as a result, after a few days' cultivation, long
rows of spores lying side by side, with here and
there an adult bacillus.

In uiilk they grow well at the body temperature,
and in a few days cause coagulation of the milk ;
and on potatoes they form a dryish yellow layer.
These bacilli also grow in blood serum and in
bouillon.

FIG. 37. BACILLUS ALVEI.
(Cheshire and Cheyne.)

A, Passage of spore into bacillus condition.

B, Passage of bacillus into spore condition.

B. alvei does not grow below 16° C. ; but it grows
most rapidly in cultivating media kept at the
body temperature. Cheshire and Cheyne sprayed
a cultivation of the bacillus in milk over a honey-
comb containing a healthy brood of larval bees, and
succeeded in reproducing the disease known as
' foul-brood.' They also succeeded in infecting adult

154 A MANUAL OF BACTERIOLOGY

bees by feeding them with material containing these
bacilli.

This microbe is best stained with methyl violet ;
but the spores resemble the spores of other microbes
in not taking on the stain. Fig. 37 represents the
passage of a spore into the bacillus condition, and
vice versd.

Bacillus of Grouse Disease. — Dr. Klein 1 has re-
cently proved the microbian nature of grouse disease.
The disease, which is infectious, is caused by a
bacillus measuring T6 x 0'6 //,. It grows well on
agar-agar at 36° to 37° C.2 ; also on nutrient gelatine
and in alkaline bouillon. Klein proved the patho-
genic nature of the microbe by a series of inocula-
tion experiments. The bacillus is readily stained
by Weigert's method.

Bacillus suUilis. — The hay-fever microbe was
originally isolated from an infusion of hay. It
measures 6 x 2 //,, and has slightly rounded ends.
This bacillus occurs singly, in short chains, in lep-
tothrix filaments, and in zooglcea. It forms oval
spores (I'2x0'6 //) ; but spore-formation occurs
only when there is an ample supply of air ; never-
theless it is independent of any deficiency of
nourishing material (Klein). The bacilli when
single possess one or two flagella (Fig. 33, 10).
' The bacilli form a dense resistant pellicle on the
surface of the nourishing medium, and in this
copious spore-formation takes place. If shaken

1 Gentralblatt fur Bakteriologie und Parastienkunde, Bd. vi.
pp. 36 and 593 ; Bd. vii. p. 82 ; and Bd. ix. p. 47.

2 That is, in from two to four days.

THE BIOLOGY OF MICROBES, ETC. 155

when growing in a fluid the pellicle falls to the
bottom, and soon a new pellicle is formed.' This
microbe may be readily obtained by exposing a
previously sterilised infusion of hay to the atmo-
sphere for a short time : the spores being always
present in the air. On plate-cultivations, white
rounded colonies formed, which frequently give rise
to radiating processes. On potatoes and agar-agar
B. subtilis forms a moist, cream-coloured layer,
which ultimately becomes granular and dry. It
grows on blood serum and nutrient gelatine, both of
which it liquefies. B. subtilis is a motile microbe,
and is best cultivated at a temperature of about
30° C. This microbe can withstand a temperature
of — 1 8° C. ; x and its spores have been proved to
have a remarkable power of resisting the influence
of high degrees of heat. For instance, a short ex-
posure to 100° C. does not destroy the vitality of the
spores. However, an E.M.F. of 2'72 volts destroys
both the spores and bacilli.^ The action of ozone 3
on both the spores and bacilli is that they are com-
pletely destroyed ; this fact explains the absence of
this and other microbes in the air at sea — the latter
containing an appreciable amount of ozone.

Bacillus ethaceticus. — This small bacillus (1*5 to
5'1 x 0*8 to 1*0 /A) was discovered by Dr. P. F.
Franklaud, F.E.S.,4 and has the power of decompos-

1 Griffiths in Proceedings of Royal Society of Edinburgh,
vol. xvii. p. 263.
a Griffiths, ibid., vol. xv. p. 45.

3 Griffiths' Researches on Micro -Organisms, p. 184.

4 Proceedings of Royal Society of London, vol. xlvi. p. 345.

156 A MANUAL OF BACTERIOLOGY

ing solutions of mannite, glucose, sucrose, lactose,
starch, glycerol, and calcium glycerate. It has no
fermentive action on dulcite, the isomer of mannite,
which thus furnishes a very striking instance of the
selective power of microbes between the most closely
allied isomeric bodies. The products of the fer-
mentation of the above-mentioned compounds are
essentially alcohol and acetic acid, with a small and
variable proportion of formic acid, together with a
trace of succinic acid. Frankland l represents the
decomposition of glyceric acid (calcium glycerate) by
this microbe as follows :
5C3H604 = C2H5OH + 4CH3COOH + H20 + 3H2

+ 5C02.

The alcohol and acetic acid are produced approxi-
mately in the proportion of one molecule of alcohol
to four molecules of acetic acid.

Bacillus lutyricus. — This is the microbe of the
butyric fermentation ; and it is found in the cells of
laticiferous plants, in milk, and in decaying-plant
infusions, etc. B. butyricus is morphologically like
B. subtilis, but distinguished by the fact that at
certain times it contains starch in its cells. It
measures from 3 to 10 //, in length and 1 p in
breadth : it frequently forms chains, and gives rise
to well-developed spores. When spore-formation is
about to take place the protoplasm of the cell
becomes granular, and at certain points gives rise to
oval spores. This microbe grows on gelatine-plates,
in the deeper layers of the medium, as yellow or
brown colonies of a granular appearance ; and ulti-

i Journal of Chemical Society, 1891, p. 81.

THE BIOLOGY OF MICROBES, ETC. 157

mately the gelatine is liquefied. On agar-agar it
forms a viscid yellow layer ; while in test-tube
cultivations it liquefies the gelatine which becomes
cloudy. B. butyricus grows best between 35° and
40° C. It is the cause of the rancidity of butter
and the ripening of cheese. It decomposes cellulose,
and hence it is of great ' importance in the digestive
process of herbivorous animals, in whose stomachs
and intestines it is very common/

Bacillus ulna. — This species is closely allied to B.
subtilis. It measures 10 X 2 /JL; and occurs singly,
in chains, but it does not form leptothrix. It gives
rise to spores which measure 2 '8 p x 1 p» This
microbe is found in rotting eggs. On the surface
of bouillon it forms thick colonies which ulti-
mately unite, giving rise to a pellicle. It is readily
cultivated on sterilised egg-albumin.

Bacillus of Symptomatic Anthrax. — This microbe
is the cause of the infectious disease known as
quarter-evil, rauschbrand, charbon symptomatique,
etc. The disease affects cattle, giving rise to the
formation of an irregular tumour in the subcutaneous
and intermuscular tissues. There is high fever, and
death generally occurs in about forty-eight hours.
This motile microbe (3 to 5 p x 0'5 to 0*6 //,) is
found in the serous fluids, bile, tumours in this
disease. It has been cultivated in fowl broth to
which small quantities of glycerol and ferrous sul-
phate have been added. It also grows on blood
serum, nutrient gelatine, and vegetable albumin.
As the microbe is anaerobic, it must be cultivated
in an atmosphere devoid of free oxygen. It is best

158 A MANUAL OF BACTERIOLOGY

cultivated at the temperature of the body. Spore-
formation takes place at the ends of the cells.

MM. Arloing, Cornevin, and Thomas l have shown
that the virus is capable of giving immunity to
animals inoculated with it. The following are the
chief facts observed by them : (a.) Injection of a
very small quantity of the virus into the loose con-
nective tissue of any part of the body produces a
temporary illness, and protects the animals, (b.)
Injection of a moderate quantity into the scanty
connective tissue of the tail produces a slight affec-
tion, and confers immunity. Very large doses,
however, may cause death. A moderate quantity
injected into the cellular tissue in other parts of
the body causes death, (c.) Injection into the veins
does not kill, but confers immunity, and the same
result follows injection into the respiratory tract.
(d.) Cultivation does not deprive the microbe of its
virulence, but heating the spores to 85° C. for six
hours destroys their virulence.

On page 117 of their book (loc. cit.), MM. Arloing,
Cornevin, and Thomas state that the following sub-
stances destroy or do not destroy the virulence of
this microbe : —

Do not destroy the virulence.

Destroy the virulence.

Alcohol (90 %).

Glycerol.

Sulphate of quinine (10 %)•

Hydrogen peroxide.

Sodium hyposulphite.

Ammonia.

Tannic acid (20 %).

Salicylic acid (O'l %).
Carbolic acid (2 %).
Boric acid (20 %).
Sodium salicylate (20 %).
Potassium permanganate (5 °/0).
Mercuric chloride (01 %).
Silver nitrate (O'l %).

Du Charbon Bacttrien (1883).

THE BIOLOGY OF MICROBES, ETC. 159

Bacillus ianthinus. — This motile microbe was first
found in water, and differs from B. violaceus (also
found in water) by not liquefying gelatine. It
occurs singly, and in threads. On nutrient gelatine,
agar-agar, and potatoes it produces white spots,
which rapidly become violet. The pigment, which
is soluble in alcohol, is only developed in the pre-
sence of air.

Bacillus violaceus. — This bacillus is also found in
water. It grows as small round colonies on gelatine
plates. These are first white, but rapidly assume a
violet colour. It also grows on agar-agar, blood
serum, and potatoes ; giving rise, on each of these
media, to the same pigment. The microbe is a
motile rod about four times as long as broad, with
rounded ends, and often contains spores.

Bacillus cyanogenus. — This microbe measures 2'5
to 3'5 p x 0'4 IJL ; and occurs in chains and zooglcea.
Spore-formation is also present. In test-tube culti-
vations, it gives rise to a white head, while the
surrounding gelatine becomes blue or dark brown.
In alkaline milk, it gives rise to a slate-coloured
pigment; while in acid or sour milk, a beautiful
blue pigment is developed (in fact, it is called ' the
microbe of blue milk'). On agar-agar, it forms a
brown pigment. It also grows on potatoes, boiled
rice, starch-paste, etc. ; and the colouring matter
which is formed varies with the nourishing
medium. These pigments are freely developed at
from 15° to 18° C., but at 37° C. no colour is formed
at all.

Bacillus erythrospwus. — This bacillus was found

160 A MANUAL OF BACTERIOLOGY

in putrefying albuminous fluids, potable water, etc.
It occurs singly and as leptothrix. On gelatine-
plates, white colonies are formed. The outer zones
of these colonies are of a yellowish-green colour.
On potatoes, this microbe forms brown patches which
do not spread. It produces dirty-red spores.

Bacillus cedematis maligni. — This microbe, ob-
tained from soil, is a pathogenic microbe. Mice,
rats, cats, etc., inoculated with a pure cultivation of
this bacillus, die in a few hours. It measures from
3 to 5 fji x 1 //,, and has rounded ends. It occurs
singly, in chains, and leptothrix (straight or curved) ;
spores are formed ; and the microbe is anaerobic.
It grows well on the surface of a neutral solution of
Liebig's extract of meat at 36° to 38° C., or in
nutrient agar-agar ; but air must be excluded from
the cultivation tubes or flasks.1 For some recent
work concerning the microbe of malignant cedema,
see Dr. Klein's paper in Centralblatt fur Balderiologie
iind ParasitenJcunde, Band x. (1891), p. 186.

Bacillus of rhinoscleroma. — A microbe found in
the tissues of patients suffering from rhinoscleroma
— a disease which gives rises to tumours on the
lips, and nasal and pharyngo-laryngeal regions.
The bacillus measures from 1*5 to 3 //, x 0*5 to 0'8
//, ; it has rounded ends, produces spores, and sur-
rounds itself with an elongated capsule (Fig. 33, 11).
This microbe is readily stained with a solution of
methyl violet.

Bacillus of Indigo Fermentation. — This microbe is
morphologically similar to the bacillus of rhino-

1 See Dr. Griffiths' Researches on Micro- Organisms, p. 235.

THE BIOLOGY OF MICROBES, ETC. 161

scleroma ; and it has been proved by Alvarez 1 to be
the cause of the indigo fermentation and the pro-
duction of indigo-blue. Indigo-blue or indigotin is
the product of several plants belonging to the
Indigofera and other genera. It does not exist
in these plants ready - formed, but is produced
by the decomposition of a glucoside (C26H31N017)
called indican. By the action of Alvarez's bacillus,
indican yields indigo-blue (C8H5NO) and indiglucin
(C6H1006):-

,, + 2 H20 = C8H6NO + 3 C6H1006.

The bacillus of indigo fermentation has been
shown to possess pathogenic properties, and oc-
casions in animals a transient local inflammation,
or death, with visceral congestion and fibrinous
exudations.

Bacillus pyocyaneus. — This microbe is a very
minute, short, thin rod ; and it is said to produce
spores. It occurs in chains of twos or threes, or
collected into irregular masses ; and it has been
isolated from pus of those cases in which the wounds
exhibit a greenish-blue colour. According to Dr.
Gessard,2 B. pyocyaneus produces a greenish pigment
of a definite composition, which has been called
' pyocyanin.' Pyocyanin can be extracted from pus
by means of chloroform. Dr. J. Kunz 3 has grown
this microbe on nutrient gelatine kept for three or
four days at the ordinary temperature, and then for

1 Comptes Rendus de I' Academic des Sciences, tome 105.

2 De la Pyocyanine et de son Microbe, 1882.

3 Monatsheftefur Chemie, Bd. ix. p. 361.

162 A MANUAL <_.'/• Lt

seven days at 35° C. It liquefies the gelatine, which
shows a green fluorescence, and has the specific
smell of blue pus. Kunz extracted from the lique-
fied gelatine pyocyanin and pyoxanthose, but the
liquid still showed a green fluorescence due to a
distinct colouring matter, which is only soluble in
water and alcohol, and is not destroyed by boiling.
Concentrated solutions of this colouring matter
transmit red and green light only, but dilute solu-
tions have no absorptive power. According to
Kunz, pyocyanin contains nitrogen and sulphur.
The green pigment which is formed when this
bacillus is grown on nutrient gelatine is most pro-
bably produced by the oxidizing action of the air
on a chromogen which is formed by the bacillus, as
the pigment is not contained in the bacillary cells.
In gelatine solutions, the green colour disappears
gradually at the ordinary temperature in ten or
fifteen weeks, giving place to a dark reddish-brown
colour, and the reaction becomes strongly alkaline.
B. pyocyaneus grows in milk, and produces a
yellowish-green solution, which becomes intensely
green when ammonia is added.

The chemistry of the microbian pigments is a
subject which has been very little investigated ; but
these pigments are undoubtedly products formed
from the decomposition of albuminoids by the
agency of microbes.

Bacillus septiccemice (rabbit). — This microbe, which
is pointed at both ends, measures T4 x 0*7 p. It
occurs singly and in chains; and it grows in bouillon,
nutrient gelatine, and blood serum. On gelatine-

THE BIOLOGY OF MICROBES, ETC. 163

plates, it produces 'dot-like colonies, and in test-
tubes little spherical masses in the needle track, and
a layer on the free surface/ This bacillus was
isolated by Koch from putrid meat infusion and
river-water. It is innocuous to guinea-pigs and
white rats ; but rabbits, mice, and birds are very
susceptible to the attacks of this microbe.

Bacillus septiccemice (mouse). — This non-motile
bacillus was isolated from garden soil and putrefy-
ing fluids. It measures 1 x O'l /*, and occurs
singly, in pairs, and chains of four or more. It
grows on gelatine-plates, in the deeper layers of the
medium, as delicate white clouds. In test-tube
cultivations, it produces delicate branching growths
along the track of the needle. On agar-agar, lemon-
yellow colonies are formed. This bacillus kills
house-mice in forty to sixty hours; but field-mice
have an immunity.

Bacillus septiccemice (man). — In human septicaemia,
Klein x found in the blood-vessels of the lymphatic
glands certain bacilli which form continuous masses
in the capillaries and small veins. These bacilli
measure 1 to 2*5 //, x 0*3 to 0*5 JJL, and occur singly
or in short chains.

Bacillus diphtlierice vitulorum. — This microbe
measures 2 -5 to 3' 6 x 0*5 /&, and was described by
Loffler as occurring in the diphtheria of calves.
Mice inoculated from a calf died with all the
characteristic symptoms of the disease. The
microbe has not been artificially cultivated.

Bacillus diphtheria columbarum. — This bacillus

1 Micro-Organisms and Disease, p. 120 (3d ed.).

164 A MANUAL OF BACTERIOLOGY

was isolated from the false membrane of the
diphtheria of pigeons. It is a short rod with
rounded ends, and occurs in irregular masses. On
the surface of gelatine, it forms light yellow films,
while in the deeper layers of that medium white
nodules are formed. This bacillus destroys pigeons,
sparrows, mice, and rabbits ; but fowls, guinea-pigs,
rats, and dogs have an immunity.

Bacillus of Diphtheria of Rabbits. — This microbe
measures 3 to 4 yu, X 1 /-t ; it has rounded ends, and
occurs singly, in pairs, or in long chains. On
gelatine-plates it forms grey colonies, which become
brown. It was isolated during growth in 'the
diphtheritic processes of the intestine ; ' and causes
(in rabbits) an inflammatory exudation in the
alimentary canal.

Bacillus cavicida. — This microbe was discovered,
by Dr. Brieger, in faeces and putrefying fluids. The
rods are very small, and they form colonies com-
posed of white concentric rings on gelatine plates. On
potatoes they give rise to dirty yellow masses. They
are fatal to guinea-pigs, but not to mice and rabbits.

Bacillus pyogenes fatidus. — A microbe with
rounded ends, and measuring 1*45 X 0'58 //,, was
isolated from putrid pus. It occurs in pairs or
chains, and is motile and produces spores. On the
surface of gelatine and agar-agar it forms greyish
films, and on potatoes a shining brown growth is
developed. From all these media a strong putrid
odour emanates, but no smell is given off when the
microbe is cultivated in milk. It is fatal to mice
and guinea-pigs.

THE BIOLOGY OF MICROBES, ETC. 165

Bacillus of Swine Erysipelas. — This bacillus has
been obtained from the blood of pigs which have
died of the disease. It measures 1*1 //, x 0*2 p.
In test-tube cultivations it produces a cloudy growth
in the track of the needle. It is fatal to mice,
pigeons, and rabbits, as well as pigs.

Bacillus of Ulcerative Stomatitis in the Calf. —
Drs. A. Lingard and E. Batt1 discovered certain
bacilli in ulcerations on the tongue and mucous
membrane of the mouth of calves (Fig. 33, 13).
They measure 4 to 8 p, x 1 /i, and occur singly and
as leptothrix forms, the filaments of which are
either straight or more or less curved. They con-
tain spores ; and when injected into a mouse or
rabbit they produce a fatal result. These bacilli
are best stained by immersion in a mixture of
methylene blue and magenta.

Bacillus of Swine Plague. — This microbe2 measures
2 to 3 //, in length, and produces spores. It was
observed in the organs of pigs which had died of
swine fever, or swine plague. It is readily culti-
vated in broth and hydrocele fluid at temperatures
ranging between 30° and 42° C. A drop of either
of these cultures inoculated into pigs, rabbits, and
mice produce the disease, with multiplication of
the bacilli ; and ' the animals die with a character-
istic swelling to the spleen, coagulative necrosis of
tracts of the liver tissue, and inflammation of the
lungs.' Pigs inoculated with artificial cultures of
the microbe are protected against a fatal attack.

1 Lancet, 1883.

2 See Klein's Micro -Organisms and Disease, pp. 131-136.

166 A MANUAL OF BACTERIOLOGY

Bacillus putrificus coli. — It was first isolated from
faeces, and measures about 3 //, in length. On gela-
tine it has an opalescent appearance, but finally
becomes a yellow colour. It is motile microbe,
which occurs in long or short threads. Spore-
formation has been observed.

Bacillus epidermidis. — It was discovered in the
fragments of epidermis taken from between the toes-
This microbe measures from 2 '8 to 3 yu, in length,
and 0*3 //, in breadth; it forms spores from 1*2 to
1'5 p in length, and 0'3 to 0'4 p in breadth. It
grows only sparsely on nutrient gelatine and agar-
agar. On potatoes it forms a characteristic super-
ficial skin.

Bacillus of Nitrous Fermentation. — Dr. P. F.
Frankland1 has recently isolated from soil a bacillus
which converts ammonia into nitrites. This mic-
robe will be described under the heading of ' the
microbes of the soil/

Bacillus megaterium. — This microbe was dis-
covered by the late Dr. De Bary on boiled cabbage.
The rods are motile, and measure 10 p, x 2 -5 p.
They occur singly and in chains, and grow on gela-
tine and agar-agar, forming a whitish layer. On
potatoes at 20° C. yellowish-white dots are formed.
B. megaterium is an aerobic microbe, and produces
spores.

Leptothrix luccalis. — This microbe occurs in the
slime of the teeth, on the epithelium of the mouth,
etc. j in other words, it is one of the microbes of the
mouth. It occurs as isolated bacilli or threads,

1 Philosophical Transactions of the Royal Society, 1890, p. 107.

THE BIOLOGY OF MICROBES, ETC. 167

generally arranged in bundles (Fig. 38), which may
be interwoven with one another. Each thread is
divided into short rods, from 1 to 1'2 //, broad, and
from 2 to 10 //, long. This microbe is believed to
be connected with dental caries.

Leptothrix innominata. — This microbe is usually
found on the soft white matter which is deposited
on the teeth. The threads are from 0'5 to 0*8 p in
breadth.

Leptothrix parasitica. — The threads are slender,
not articulated, loosely felted, and for the most part
curled. They measure
from 100 to HO /* in
length, and about 1 ^ in
breadth, and occur both
in still and running water.
This bacillus (Fig. 33, 22)
is best cultivated on in-
fusions of rotting algae and
animal substances. This
microbe is believed by Plo. 38. LEPTOTHBIX BucCAUS.
Zopf and others to give

rise to micrococci, bacteria, etc. ; in other words,
it is a pleornorphic form, but Zopf s observations
were not made after exact methods.

Beggiatoa roseo-persicina. — This is the ' peach-
coloured bacterium ' of Ray Lankester,1 and is really
a sulpho-chromogenic bacillus. It occurs 'on the
surface of marshes, or on water in which algae are
rotting, and sometimes these bacilli are in such

1 Quarterly Journal of Microscopical Science, vol. xiii. p. 408.

168 A MANUAL OF BACTERIOLOGY

quantity that the whole marshes and ponds may be
coloured blood-red by them/ B. roseo-persicina con-
tains dark-coloured sulphur granules, the dark
colour being due to the pigment — bacterio-purpurin
— formed by the microbe. This pigment is in-
soluble in water, alcohol, etc., and when examined
spectroscopically it shows a strong absorption band
in the yellow, a weaker band in the green and blue,
and a darkening in the more refrangible half of the
spectrum.

Beggiatoa alba. — This bacillus occurs as threads
without distinct articulations. The threads are
longer and thicker than leptothrix, and they are
found in marshes and sulphur springs. The cells
(about 3*5 Abroad) of B.alba contain sulphur granules
(Fig. 33, 20), and, according to Cohn and Cramer,1
these granules consist of crystalline sulphur, which
is highly refractive. When these crystalline granules
are disintegrated and examined microscopically,
they are seen to be composed of a number of
rhombic (octahedral) crystals. A variety (B. alba
marina) of this microbe forms a delicate white gela-
tinous membrane on decaying animals and algae in
a marine aquarium.

Beggiatoa nivea. — The threads of this bacillus are
very slender, indistinctly jointed, and form undu-
lated woolly tufts of milky-white colour. B. nivea
occurs in sulphur springs.

Beggiatoa miralilis. — The microbe occurs in sea-
water, forming a white gelatinous scum on decom-
posing algae, etc. The threads are very thick,

1 Beitrage zur Biologie der Pfltinzen, vol. i.

THE BIOLOGY OF MICROBES, ETC. 169

motile, bent and curled in various ways, and they
have rounded ends. They are distinctly articulated
(16 //, broad), and contain sulphur granules.

Besides the four last-mentioned microbes there
are B. leptomitiformisy B. arachnoidea, and B. pel-
lucida, each of which contains sulphur granules.
These microbes play an important part in the elimi-
nation of sulphur and the disengagement of sul-
phuretted hydrogen. The sulphogenic or sulphur-
forming microbes are found in certain waters, and
many of the natural sulphurous waters are due to
the action of these microbes on alkaline sulphates
and organic matter present in such water. The
decomposition of calcium sulphate by sulphogenic
microbes may be represented by the following
equations : —

(a) 3 CaS04 + H20 = S2 + H2S + 3 CaO + 5 02.
(ft) 2 CaS04 = S2 -|- 2 CaO + 3 02.

Sulphogenic microbes are also capable of decom-
posing animal and vegetal albumin with the libera-
tion of sulphur.

Bacillus septicus. — This microbe occurs in soil,
putrid blood, and other fluids. Its breadth varies
from 4 to 10 /i, and its length depends on the
number of elements contained in a row : the shortest
are about 4 /z. It is a non-motile bacillus, capable
of forming leptothrix and spores.

Bacillus of conjunctivitis. — This bacillus was
obtained from the conjunctival sac in cases of con-
junctivitis. It grows on agar-agar plates as pearly
dots, and in bouillon. The latter medium is the

170 A MANUAL OF BACTERIOLOGY

best for the cultivation of this microbe. It measures
from 1 to 2 p in length, and 0*25 //, in breadth.

Bacillus figurans. — This microbe was first de-
scribed by Crookshank,1 and occurs in soil and in
the atmosphere. It has rounded ends, and forms
spores. On the oblique surface of agar-agar it
forms a feather-like growth. On gelatine plates
B. figurans causes ' a cloudy growth, spreading from
various points/ When ' cultivated in nutrient gela-
tine this bacillus forms on the surface visible wind-
ings, from which fine filaments grow down into the
gelatine. They spread out also in almost parallel
lines transversely from the needle track.'

Bacillus Hansenii. — The rods measure 2 '8 to 6 p
x 0'6 to 8 //,, and are best cultivated on steamed
potatoes, where they form a deep yellow layer,
which has the odour of amylic alcohol. Ultimately
the yellow layer dries, and changes to a brown
colour, at the same time forming spores (1*7 JJLX
1-1 /*,). This bacillus occurs on bouillon, wine, and
malt infusions, which have been kept at about
32° C.

VIBRIONES.

These microbes are rod-shaped, but not straight ;
they are more or less wavy, and they are motile.

Vibrio serpens. — This vibrio measures from 1 1 to
25 fju long, and from 0'8 to M //, in breadth. It
occurs in various infusions.

Vibrio rugula. — The rods measure from 6 to 16 yu,
in length, and about 0*5 to 2*5 //, in breadth. They
are curved or S-shaped, and bear a flagellum at each

1 Lancet, 1885.

THE BIOLOGY OF MICROBES, ETC. 171

end. They swarm when causing decomposition in
vegetable infusions. According to Prazmowski,
Vibrio rugula develops a spore at one end of the
cell.

SPIRILLA.

Spirillum tyrogenum. — This spirillum measures
about 0*8 to 1*5 p, in length. On gelatine plates
(see Fig. 24) it forms colonies of a greenish-brown
colour. In test-tubes the gelatine becomes liquid
along the needle-track, while on agar-agar a pale
yellow layer develops. This microbe, which is
non-pathogenic, was isolated, by Deneke, from old
cheese. S. tyrogenum is capable of withstanding a
temperature of — 18° C. for several days.1

Spirillum Finkleri. — The rods are curved, and
they are larger and thicker than the Spirillum
cholera Asiaticce. On gelatine-plates they grow
rapidly, forming small white dots with a brownish
tinge; and the gelatine is liquefied very rapidly.
The fluid (from the liquefaction) becomes completely
turbid, whereas in S. cholerce Asiaticce the upper
part remains clear. In gelatine tube cultivations,
liquefaction occurs in the form of a funnel-shaped
tube, and the fluid becomes turbid. On agar-agar
and potatoes white films or layers are formed. S.
Finkleri was discovered in the dejecta of cases of
cholera nostras, and it was said to be identical with
the Spirillum of Asiatic cholera; but it is quite
distinct. •

1 Griffiths in Proc. Roy. Soc., Edinb., vol. xvii. p. 263 ; and
Researches on Micro-Organisms, p. 176.

172 A MANUAL OF BACTERIOLOGY

Spirillum Obermeieri. — This microbe is the cause
of relapsing fever, and was first discovered by Ober-
meier 1 in the blood of patients suffering from the
disease. Carter 2 reproduced the disease in monkeys,
in whose blood and organs the spirilla were found
in great numbers. This microbe (16 to 40 p long),
which is motile, exhibits spiral forms, and, according
to Albrecht,3 produces spores. 8. Olermeieri (Fig.
33, 7) has been artificially cultivated by Koch.4
The microbe only occurs during the relapses, and is
absent during the non-febrile intervals.

Spirillum tenue. — This spirillum measures from
4 to 15 ^ in length, and about 2'25 p in breadth.
It usually occurs in various infusions, in which it
moves about with great rapidity. It occurs in
swarms or united in a zoogloea.

Spirillum undula. — It measures from 8 to 1 2 yu-
in length, and from 1-1 to 1*4 //, in breadth (Fig. 33,
5). There is a flagellum at each end, and the
microbe is actively motile ; although at times it
forms a zoogloea. It occurs in bog- water and various
infusions.

Spirillum wlutans. — This microbe occurs in marsh
water and various infusions. It measures from 20
to 30 fj, in length, and 1-5 to 2 /*, in breadth (Fig.
33, 6). The protoplasm contains a number of
dark granules, and there is a flagellum at each end.

Spirillum sanguineum. — This was observed by

1 Centralblattfur Med. Wissensch., 1873.

2 Lancet, vol. i. p. 84, and p. 662.

3 St. Petersb. Med. Woch., 1879.

4 Deutsche Med. Woch., vol. xix.

THE BIOLOO Y OF MICROBES^ ETC. 173

Cohu and Warming in pond-water. It is said to be
morphologically identical with Spirillum volutans.
The cells contain numerous red bodies and many
sulphur granules. According to Saville Kent,1 this
microbe is not identical with Ehrenberg's Ophido-
monas sanguinea : the latter being a true monad.

Spirillum concentricum. — This microbe was dis-
covered by Kitasato in putrefactive blood. It grows
rapidly on gelatine-plates, giving rise to greyish-
white round colonies, each of which has concentric
markings. It does not liquefy the gelatine, and is
non-pathogenic.

Besides the above-mentioned spirilla, there are
the following, which occur in brackish and sea
water : S. violaceum, S. Rosenbergii, S. attenuatum,
etc. ; but the reader is referred to the works of
Warming for an account of these microbes.

SPIROCILET.E.

Spirocliceta plicatilis. — This microbe is of extra-
ordinary length— 110 to 225 p (Fig. 33, 19). It
occurs in stagnant water. The threads are arranged
in wavy lines.

Spirochceta gigantea. — The threads are blunt at
both ends. It occurs in sea-water.

YEAST-FUNGI.

These fungi are not microbes (i.e. they are not
Schizomycetes), but belong to an altogether different
order — the Saccharomycetes. They multiply chiefly

Manual of the Infusoria, p. 244.

174 A MANUAL OF BACTERIOLOGY

by gemmation or budding, but they can also produce
spores, especially when they are deprived of nourish-
ment. These organisms occur widely distributed
in air, soil, and water, and they are the cause of the
alcoholic fermentation.

Saccharomyces cerevisice. — This organism is some-
times termed Torula cerevisice, and is the true fer-
ment of beers. The cells are round or oval (8 to 0
fj, long), and are either isolated or united in small
colonies. The spore-forming cells (when isolated)
measure from 11 to 1 4 //, long ; and the spores mea-
sure from 4 to 5 fi in diameter. This organism occurs
in beers brewed by both the high or low systems of
fermentation. Fig. 39, 1 and 2, represent the beer-
ferments. There are two races of this species, high
(1) and low (2) yeasts. The cells of high yeast are
slightly larger and more round than those of low yeast.
Low yeast never rises to the surface of the fermenting
wort, which is kept at a temperature varying from
4 to 5° C. This low fermentation is a slow process,
occupying about fourteen days. The low fermenta-
tion gives rise to ' Lager ' or ' Bavarian ' beer. High
yeast rises to the surface as the fermentation pro-
ceeds, and the wort is kept at a temperature varying
from 16° to 20° C. The fermentation is rapid, and
rarely occupies more than a few hours or so. The
high and low yeasts are not different species. Both
high and low yeasts secrete a soluble enzyme which
converts maltose and sucrose into invert sugars
(dextrose and levulose) : —

CI2H22On + H20 = C6HI206 + C6H1206.
[maltose] [dextrose] [levulose]

THE BIOLOGY OF MICROBES, ETC.

175

Saccharomyces minor. — This organism (Fig. 39, 3)
consists of a spherical cell measuring 6 p in diameter.
It occurs in chains of six or nine cells. The spore-
forming cells each measure from 7 to 8*5 p, in dia-
meter, and contain from 2 to 4 spores, each hav-
ing a diameter of 3 -5 /^. Hansen and En gel state

3 n$ ® O

$ ^ o^ ^ o

00 Q

O
Cb

FIG. 39. YEAST-FUNGI.

that this yeast is the cause of fermentation in
bread.

Saccharomyces ellipsoideus. — The cells are ellipti-
cal (Fig. 39, 4), mostly 6 //, long, and are isolated or
united in little branched colonies. Two to four

176 A MANUAL OF BACTERIOLOGY

spores are found in a mother cell. It is a low yeast
when grown in beer wort ; but it is really a species
of wine ferment, which produces the spontaneous
fermentation in must.

Saccharomyces conglomeratic. — The cells are al-
most round (Fig. 39, 5), measuring from 5 to 6 ^
in diameter, and united in clusters. This organism
occurs in wine at the beginning of the fermentation,
and on decaying grapes.

Saccharomyces exiguus. — The cells are conical
(5 fju x 2 -5 //,), and are united in slightly branched
colonies (Fig. 39, 6). Spore-forming cells each
contain from two to three spores, which lie in a
row. This organism occurs in the after-fermenta-
tion of beer.

Saccharomyces Pastorianus. — The cells are oval
or elongated (Fig. 39, 7). 'The colonies consist
of primary club-shaped links (18 to 22 //, long),
which build lateral, secondary, round or oval
daughter-cells (5 to 6 //, long).' The spores number
from two to four. This organism occurs in the
after-fermentation of wine, fruit-wines, and fer-
menting beer. It is very common in the air.

Saccharomyces apiculatus. — The cells are lemon-
shaped (Fig. 39, 8) and from 6 to 8 yu, long x from
2 to 3 fju broad, and sometimes slightly elongated.
Gemmation occurs only at the pointed ends. Spore
formation is unknown. It occurs in fermented
wine, in spontaneous fermentations of all kinds of
fruits, and in certain kinds of beer. It is a low yeast,
giving rise to a feeble alcoholic fermentation, and
it does not invert sucrose. When mixed with S.

THE BIOLOGY OF MICROBES, ETC. 177

cerivisice it retards the action of the true beer
ferment.1

Saccharomyces mycoderma. — The cells are oval,
elliptical, or cylindrical (Fig. 39, 9), measuring
about 7 fi long and about 2 //, thick. They are
united in richly-branched colonies ; and the cells
are often elongated, so as to resemble a hyphal
filament. This organism forms the scum on the
surface of beer, wine, sauerkraut, and fruit-juices.
It has nothing to do with the alcoholic fermenta-
tion; and is not identical with Bacterium aceti
(Mycoderma aceti), which is the microbe of the acetic
fermentation in wines and beers.

Saccharomyces vini. — This organism is the true
wine-producing ferment, for it is the cause of the
alcoholic fermentation of grape-juice. Its cells are
elliptical, slightly smaller than those of S. cerevisiw.
It forms spores, and is very common in the atmo-
sphere.2

It should be borne in mind that fermentation is
not a chemical, but a vital process; for the
researches of Pasteur and others have shown that
every fermentation has its specific ferment; in all
fermentations in which the presence of an organised
ferment has been ascertained the ferment is neces-
sary.

1 See Martinand and Reitsch's paper in the Comptes Rendus,
t. 112 (1891).

2 For further information concerning the yeasts see Jorgensen's
Micro-Organisms of Fermentation ; Pasteur's Etudes sur la Biere,
Etudes sur la Vin ; Engel's Les Fermentes Alcooliques ; and the
papers of Hansen.

CHAPTEE VI

INFECTIOUS DISEASES AND MICROBES, ETC.

'THE study of disease-gerrns by the new and
accurate methods of bacteriology has led to a
clearer and better understanding of the manner in
which, at any rate, some of the infectious diseases
spread. While it was understood previous to the
identification of their precise cause that some
spread directly from individual to individual (e.g.
small-pox, scarlet fever, diphtheria), others were
known to be capable of being conveyed from one
individual to another indirectly, i.e. through ad-
hering to dust, or being conveyed by water, milk, or
by food-stuffs (e.g. cholera, typhoid fever). But we
are now in a position to define and demonstrate
more accurately the mode in which infection can
and does take place in many of the infectious
diseases. By these means we have learned to
recognise that the popular distinction between
strictly contagious and strictly infectious diseases —
the former comprising those diseases which spread,
as it were, only by contact with a diseased indi-
vidual, while in the latter diseases no direct contact
is required in order to produce infection, the disease

178

INFECTIOUS DISEASES AND MICROBES, ETC. 179

being conveyed to distant points by the instru-
mentality of air, water, or food — is only to a very
small extent correct. Take, for instance, a disease
like diphtheria, which was formerly considered a good
example of a strictly contagious disorder ; we know
jiow that diphtheria, like typhoid fever or scarlet
fever, can be, and, as a matter of fact is, often con-
veyed from an infected source to great distances by
the instrumentality of milk. In malignant anthrax,
another disease in which the contagium is convey-
able by direct contact, e.g. in the case of an abrasion
or wound on the skin coming in contact with the
blood of an animal dead of anthrax, we know that
the spores of the anthrax bacilli can be, and, in
many instances are, conveyed to an animal or a
human being by the air, water, or food. The
bacilli of tubercle, finding entrance through a
superficial wound in the skin or mucous membrane,
or through ingestion of food, or through the air, can
in a susceptible human being or an animal produce
tuberculosis either locally or generally. The differ-
ence as regards mode of spread between different
diseases resolves itself merely into the question,
Which is, under natural conditions, the most
common mode of entry of the disease-germ into
the new host? In one set of cases, e.g. typhoid
fever, cholera, the portal by which the disease-germ
generally enters is the alimentary canal ; in another
set an abrasion or wound of the skin is the portal,
as in hydrophobia, tetanus, and septicsemia ; in
another set the respiratory organs, or perhaps the
alimentary canal, or both, are the paths of entrance

180 A MANUAL OF BACTERIOLOGY

of the disease-germ, as in small-pox, relapsing
fever, malarial fever ; and in a still further set the
portal is just as often the respiratory tract as the
alimentary canal, or a wound of the skin, as in
anthrax, tuberculosis. But this does not mean that
the virus is necessarily limited to one particular
portal, or that it must be directly conveyed from
its source to the individual that it is to invade.
All this depends on the fact whether or not the
microbe has the power to retain its vitality and
virulence outside the animal or human body.' 1

It must be borne in mind that not all the diseases
described in the present chapter can, at the present
time, be termed true microbian diseases ; yet with
the progress of science, and by following the lines
already laid down, we have not the slightest doubt
that in time the microbes of all the infectious
diseases will be discovered and cultivated.

YELLOW FEVER.

Micrococci (0*6 to 0'7 //, diam.) have been found
in the kidney, spleen, and liver during the course
of yellow fever. They form rosaries and masses,
which greatly distend the blood-vessels and give
rise to hemorrhages. The yellow-fever microbe is
termed Micrococcus amaril by Dr. Domingos Freire.
This microbe grows on gelatine, and reproduces the
disease in rabbits and guinea-pigs. If, however,
the microbe is cultivated in gelatine for six genera-
tions it loses the greater part of its virulence, and

1 From a lecture delivered at the Royal Institution, London
(February 20, 1891), by Dr. E. Klein, F.E.S.

INFECTIOUS DISEASES AND MICROBES, ETC. 181

when this attenuated virus is introduced into the
body by inoculation, it produces a mild type of
yellow fever,-and confers immunity against the fatal
type of the disease. From 1883 to 1890 Freire l has
inoculated 10,881 persons in Brazil with cultures of
M. amaril. The mortality of those so vaccinated
was 0'4 per cent., although the patients lived in
districts infected with yellow fever, whilst the
death-rate of the uninoculated during the same
period was from 30 to 40 per cent.

Yellow fever is distributed (within certain areas)
by moist winds and human intercourse. "Water and
the soil have nothing to do with the spread of the
disease, although it is a disease which clings to the
ground, hence one of the reasons of its endemic
nature. It is always prevalent in the plains near
the sea-coast, and along the courses of the great
rivers. Heat (21° C.) and a certain saturation of
the atmosphere are essential conditions for an
epidemic of yellow fever. Frost puts an end to an
epidemic at once, and storms, heavy rains, or cold
weather check its progress.

HYDROPHOBIA.

Hydrophobia or rabies is a canine disease, which
is communicated by a bite, and the inoculation of
man and other animals by the saliva. The exact
nature of the microbe of this disease is not yet
known. According to Pasteur,2 Fol,3 Babes,4 and

1 Comptes Rendus, 1889 and 1891, 2 Comptes Rendus, 1884.

3 Ibid., 1885, p. 1276 ; Le* Microbes, 1885, p. 41.

4 Les Bacteries, 1890, p. 550.

182 A MANUAL OF BACTERIOLOGY

Dowdeswell,1 the microbe appears to be a micro-
coccus, and it has been observed in microscopical
sections of the spinal cord of animals dead of
rabies. Dr. Fol's preparations were made by
hardening the spinal cord or brain by immersion,
directly after death, in a solution containing 2-5
grammes of potassium bichromate, and 1 gramme of
copper sulphate in 100 cc. of .water. The piece of
tissue is divided so as to be able to take up Weigert's

FIG. 40. MICROCOCCI IN HYDROPHOBIA.

A, In cerebral matter (after Fol). B, In human saliva.

x 1000

solution of hsematoxylin ; then placed in absolute
alcohol, imbedded in paraffin, and cut into sections
not more than -^Q mm. in thickness. The sections
are finally decolorised by a solution containing 2*5
grammes of potassium ferrocyanide, 2 grammes of
borax in 100 cc. of water. In these sections, Fol
found small micrococci (0*2 //, in diarn.) in the
lymph spaces of the neuroglia, and between the

1 Journ. Roy. Microscop. Society, 1886 ; and Lancet, 1886.

INFECTIOUS DISEASES AND MICROBES, ETC. 183

axis cylinder and its medullary sheath. This
microbe (Fig. 40 A) occurs in groups and as
diplococci, but never in chains. According to Fol,
if a cultivation (in bouillon) be made of part of the
brain, there is a deposit which, on inoculation into
healthy animals, produces all the features of rabies.
If, however, the cultivation be more than six days
old there are no marked toxic effects. Fol says
that nothing can be distinctly made out by merely
reducing the nervous tissue of a rabid animal to a
pulp and examining it microscopically, as recom-
mended by Gibier.

Babes states that he has found micrococci in the
brain and spinal cord of rabid animals. These
measure from 0'6 to 0*8 p in diameter, i.e. from three
to four times as great as the microbe described by
Fol. These micrococci are stained in situ by Loffler's
alkaline methylene blue solution. They are culti-
vated on blood serum or agar-agar (at 37° C.), and
on bouillon made with the brain of a rabbit. The
micrococci grow slowly and give rise to grey spots.
' A pure culture of the second, or even of the third
generation, when inoculated into animals occasion-
ally produces hydrophobia, but in most cases the
cultures have no pathogenic properties, and it must,
therefore, be concluded that the microbe has either
lost its virulence or that it is not the actual cause of
the disease.'

The late Mr. G. F. Dowdeswell observed a
microbe, measuring about the same diameter as
Babes' micrococcus, in the central canal of the

1 Comptes Rendus, 1883, p. 1701 ; 1884, pp. 55 and 531.

184 A MANUAL OF BACTERIOLOGY

spinal cord and the medulla oblongata of dogs dead
of rabies.

The author has also observed a micrococcus (Fig.
40B) in the saliva of a woman suffering from
hydrophobia.1 The micrococcus, which is deeply
stained by methylene blue, measures from 0'6 to
0*8 fj, in diameter. This microbe does not occur in
healthy human saliva.

We cannot say that the microbe of rabies has
been isolated with anything like success, for the
above investigations do not fulfil Koch's canons (see
Chapter i.) to ascertain the pathogenic nature of the
microbe or microbes in question. It is probable
that the virus of rabies will not develop in the
absence of a living pabulum, and in all probability
it is not possessed of powers of active resistance to
those injurious influences which act upon it when
exposed to the air, etc. In fact, the virus of rabies
cannot survive the drying, changes of temperature,
etc., it necessarily undergoes when scattered over the
ground, as we often see happen by the slobbering of
a rabid animal.

The saliva of rabid animals does not contain a
ptomaine, for when it is diluted with a small
quantity of sterilised distilled water, and then
heated to 90° C. for a few hours, the saliva loses its
virulent power. This proves that no alkaloid was
present, because it would not have been destroyed
on the application of heat.2 Besides, M. Nocard

1 The saliva was kindly sent to the author by Dr. T. M.
Dolan, of Halifax.
3 Griffiths' Researches on Micro-Organisms, p. 193.

INFECTIOUS DISEASES AND MICROBES, ETC. 185

dialysed the pure saliva of rabid animals, and
proved that its solid constituents were always
virulent, and reproduced the disease when injected
into healthy animals, while the fluid portion, simi-
larly injected, remained inactive. If an alkaloid or
ptomaine had been present it would have been found
in the fluid portion, and would have given rise to
toxic effects when injected into the system. Al-
though a ptomaine has not been discovered in the
saliva of rabid animals, Dr. Anrep1 isolated a
poisonous ptomaine from the brain and medulla
oblongata of rabbits suffering from rabies. This
ptomaine reproduced all the characteristic symp-
toms of the disease, and it is stated that a gradual
habituation of the animal to small doses of the
ptomaine produced a certain degree of immunity.

Babies is not a disease of the blood, for the sup-
posed microbe is not found in the blood system,
and when the blood of a rabid animal is injected
into animals it does not reproduce the disease. In
fact, the virus is located in the nervous system,
especially the medulla oblongata.

The period of incubation of rabies is usually not
less than from four to six weeks, and sometimes
longer. ' At the end of this incubation period the
wound, first of all, becomes slightly uncomfortable ;
there is itching, and the heat becomes almost
intolerable, especially as this is usually accom-
panied by a sharp stinging pain; the patient
becomes feverish and very thirsty; the face is
pallid and has a peculiar anxious expression, the

1 British Medical Journal, 1889, p. 319.

186 A MANUAL OF BACTERIOLOGY

muscles of the face being drawn and restless, and
gradually this expression amounts to one of actual
terror or horror. On the second or third day the
patient becomes much more excited, is restless in
every sense of the word, and a very peculiar feature
is that he has a characteristic habit of giving a
suspicious side-glance as though constantly looking
out for some hidden danger; then as the fever
advances a rambling delirium supervenes ; the
thirst increases, but along with this there is great
difficulty in swallowing — especially fluids — and
after making one or two attempts to swallow, the
very sight of water suggests such horrors that,
thirsty as the patient is, he is anxious to avoid it.
Then muscular tremors are noted ; these become
more and more marked, and violent spasms are
easily stimulated, as in tetanus. A sharp sound, a
touch, a bright light, or even a breath of air, may
give rise to violent muscular convulsions, and
eventually the patient is slowly suffocated as in
tetanus' (Woodhead). Such are some of the tor-
turing symptoms of hydrophobia. But it may be
stated that the symptoms are varied, depending
upon the nature of the region in the nervous
system — encephalon or spinal cord — where the
virus locates itself. The virus is found in every
part of the encephalon. Although the saliva of
rabid animals is virulent, it is not used by Pasteur
in his prophylactic treatment, the reason being that,
as saliva contains various microbes, it may give rise
to septic poisoning, etc., as well as rabies. There-
fore Pasteur has recourse to the central nervous

INFECTIOUS DISEASES AND MICROBES, ETC. 187

system, where the virus is obtained in a pure state.
This pure virus is continually being inoculated on
the surface of the brain of healthy animals; the
object of this is to keep up the supply of the virus,
in question. The virus can be intensified or modi-
fied by passing it through various animals. For
instance, by passing it from the dog to the monkey,
and subsequently from monkey to monkey, the
virus grows weaker at each passage, until its
virulence entirely disappears. Successive passages
from rabbit to rabbit, and from guinea-pig to
guinea-pig, increase the virulence of rabies virus.
The intensified virus comes to a fixed maximum
in the rabbit. If now transferred to the dog it
remains intensified, and shows itself to be much
more virulent than the virus of ordinary street
rabies. So great is this acquired virulence, that
the intensified virus injected into the blood-
system of a dog unfailingly gives rise to mortal
madness. These facts suggested to Pasteur that,
by keeping a set of attenuated viruses of different
strength, some not mortal, he could preserve the
animal economy against the ill effects of more
active ones, and these latter against the effects of
mortal ones.

The sets of attenuated viruses are not obtained by
the passage of the virus through different animals,
for the method now in use at the Pasteur Institute
consists in suspending portions (a few centimetres
in length) of the spinal cords of inoculated rabbits
in a dry atmosphere (i.e. the marrows are desiccated
in sterilised bottles of one litre capacity by means

188

A MANUAL OF BACTERIOLOGY

of caustic potash). By this method the virulent
power gradually diminishes, and finally disappears.
By using attenuated viruses of varying intensities
(prepared by desiccation), Pasteur has successfully
treated numberless animals and human beings which
are now refractory to rabies.

To prepare the inoculating fluid a mad dog is
killed, and the brain and medulla oblongata are
carefully removed with sterilised instruments, etc.
Very small pieces of the medulla oblongata and of

, — — the Central canal are then

placed in a sterilised glass.
They are triturated with a glass
rod, and when reduced to a fine
jelly-like mass sterilised veal
bouillon is added in quantity
to about half a table-spoonful.
This dilute dog- virus is used
for inoculating a rabbit on the
surface of the brain. A full-
grown living rabbit is placed
upon a dissecting board, flat
on its abdomen, and its four limbs secured
by strings to pegs driven in the wood (Fig. 41).
After this the animal is placed under the influence
of chloroform. The hair is cut away, and an inci-
sion, one inch long, is made from a point midway
between the eyes. The operator cuts down to the
skull, which is then trepanned (Fig. 4 la), and a little
circular disc of bone is removed, as far as possible
without injuring the external membrane of the
brain. At this point the operator takes a hypoder-

FIG. 41. TREPANNING
A RABBIT.

INFECTIOUS DISEASES AND MICROBES, ETC. 189

mic syringe (see Fig. 20), filled with the diluted dog-
virus, and inserts it under the dura mater, injecting
two drops of the virus. The disc of bone is then
replaced, and the skin flaps are sewed together by
means of two or three sutures. ' A pad of cotton
wadding, carefully purified by heat, is used to dry
the skin, after which a little of the same wadding is
used as a dressing ; this dressing is kept in position
by a free application of flexile collodin, the two
together forming an air-proof shield, through which
no microbes from the external air can make their
way to the wound, which, as a rule, heals up most
perfectly in less than a couple of days/

After death the brain and medulla oblongata are
removed, and a dilute virus is prepared from them,
as in the case of the dog-virus. This is injected
beneath the dura mater of a second rabbit, the
operation being repeated in fresh rabbits until the
shortest incubation period has been reached. This
incubation period of seven days' duration is reached
by the fiftieth passage, the rabbit taking ill on the
seventh day, and dying on the tenth day or later, is
the one used for human inoculations as well as for
the purpose of perpetuating the disease in other
rabbits. By dealing with a sufficiently large number
of animals it is possible to have a rabbit dying
every day, and thus also to put one spinal cord in a
desiccating bottle every day. By the fourteenth
day there will be a set of fourteen marrows under-
going the desiccation. These marrows vary in
virulence. The marrow of one day's desiccation is
the most virulent, and the virulence of the other

190 A MANUAL OF BACTERIOLOGY

marrows decreases gradually until the fourteenth
day of desiccation, when a minimum is reached. At
the Pasteur Institute, the marrows of more than
fourteen days are thrown away as being inert and
useless.

A person having been bitten by a mad dog is
first injected * with the weakest virus, and on each
successive day or so with gradually stronger viruses
until the more powerful or most powerful virus is
used. After this treatment the patient very rarely
dies of rabies. During the years 1886-9 no less
than 7893 patients were treated at the Pasteur
Institute, and out of this number there were 53
deaths, which represents a mortality of 0'67 per
cent. But since 1889 the mortality has been re-
duced to 0*2 per cent., due, no doubt, to the better
skill in the application of the treatment.2

It may be stated in passing that at the Pasteur
Institute, Paris, there are two rabbits inoculated,
and, consequently, also two dying (of rabies) every
day, ' for fear if one alone were used it might die
from accident, and the series be interrupted. Prac-
tically one animal is found to be quite sufficient,
and the second one is only inoculated for prudence'
sake.'

' The medulla or cord of a rabbit in which the

1 In the hypochondria (i. e. certain abdominal regions).

2 Concerning the interesting statistics of the Pasteur Insti-
tutes of St. Petersburg, Odessa, Moscow, Warsaw, Charkow,
Turin, Bucharest, Naples, and Havannah, the reader is referred
to the latest edition of Cornil and Babes' book — Les Bactdries
(1890).

INFECTIOUS DISEASES AND MICROBES, ETC. 191

incubation has been seven days, when injected intra-
cranially into a dog, develops rabies in the latter
animal in about twelve days. The nervous matter
of this dog, inoculated back by the same process
into rabbits, at once reproduces the malady after an
incubation of seven days, and thus the series is
recovered/

Pasteur's treatment is prophylactic and not cura-
tive, for it is powerless against the disease when the
first symptoms have once made their appearance.
Hence the necessity of early treatment.

The mode of action of the Pasteurian inoculations
has been explained by the two following theories :
(1) Metschnikoff1 states that the white blood-
corpuscles (phagocytes) absorb and digest the living
microbes, and their power of absorption for microbes
is trained and increased by the progressively stronger
inoculations, so that finally the virus deposited by
the rabid animal can also be absorbed and destroyed.
The whole process is carried out, therefore, in the
lymphatic system. (2) Woodhead and Wood be-
lieve that the treatment consists essentially in caus-
ing the tissues to acquire a tolerance before the
microbe has had time to develop. ' The tissue cells
are acted upon by increasingly active virus, each
step of which acclimatises the cells for the next
stronger virus, until at length when the virus formed
by the microbes introduced at the time of the bite
comes to exert its action, the tissues have been so
far altered or acclimatised that they can continue
their work undisturbed in its presence, and, treating

1 Fortschrift der Medicin, 1885.

192 A MANUAL OF BACTERIOLOGY

the microbes themselves as foreign bodies, destroy
them. When the cells are suddenly attacked by a
strong dose of the poison of this virus they are so
paralysed that the microbes can continue to carry
on their poison-manufacturing process without let
or hindrance ; but when the cells are gradually
though rapidly, accustomed to the presence of the
poison by the exhibition of constantly-increasing
doses, they can carry on their scavenging work even
in its presence, and the microbes are destroyed,
possibly even before they can exert their full poison-
manufacturing powers. Some such explanation as
this would account for the interference with the
course of the disease even after the patient has been
bitten. The microbe is localised, it takes some
time to form its poisonous products, and whilst this
is going on the whole of the nervous and other
tissues are being gradually acclimatised by the
direct application of small quantities of the poison
artificially introduced.'1

In concluding our remarks concerning rabies, it
may be stated that the rabid marrows can be pre-
served for several months in pure and neutral
glycerine. Hence the use of this fluid for preserv-
ing the marrows (for inoculation against rabies)
during their transit from France to foreign countries.

For further information the reader is referred to
the undermentioned books and papers on the
subject.2

1 Woodhead's Bacteria and their Products, p. 327.

2 Pasteur in Comptes Rendus, 1881-86; Dolan's Hydrophobia :
M. Pasteur and his Methods ; Gamaleia in Annales de VInstitut

INFECTIOUS DISEASES AND MICROBES, ETC. 193
ERYSIPELAS.

This disease is due to the Micrococcus erysipela-
tosiis (0*4 fj, diam.) which abounds in the lymphatic
vessels of the skin at the margin of an erysipelatous
zone. This microbe, which is smaller than M.
vaccinice, occurs singly and in chains, as well as
zooglcea. The microbe grows on nutrient gelatine,
agar-agar, and solid blood-serum, as a whitish film
on the surface of the nourishing medium. Orth *
and Fehleisen2 have both cultivated the microbe
artificially, and reproduced the disease in rabbits.
But Fehleisen went a step further and reproduced
the disease in man by inoculating three patients
with pure cultivations of the microbe. 'These
inoculations were justifiable because they were
undertaken with a view to cure certain tumours.
Thus one case of lupus, one case of cancer,3 one case
of sarcoma, were considerably affected, and to the
good of the patients.' In the human subject typical
erysipelas was produced in fifteen to sixty hours
after inoculation.

Pasteur, 1887; Reye8 in Gac. Med. Mexico, 1889, p. 344;
Dolan in Provincial Medical Journal, 1890, p. 137 ; Zagari in
Giornale Inter nazionale delle Scienze Mediche, 1890 ; Hime in
Lancet, 1886, p. 184 ; Griffiths' Researcftes on Micro-Organisms,
p. 323.

1 Archivfur Experim. Pathol, 1874.

a Die Aetiolcyie des Erysipels, 1883.

8 If cancer is due to Scheuerlein's Cancer bacillus, it is pro-
bable that the M. erysipelatosus is antagonistic to its growth.

194 A MANUAL OF BACTERIOLOGY

PUERPERAL FEVER.

According to Heiberg,1 micrococci have been found
in the form of chains and zooglcea in all organs
affected in this disease. Heiberg's micrococcus has
not yet been artificially cultivated, consequently we
cannot say that the microbe is the real cause of this
highly infectious disease.2 The infectiousness of
puerperal fever is now well established, although
the microbe or microbes which give rise to the
different symptoms classed under the name of
puerperal fever have not been isolated. Certain
poisonous ptomaines have been isolated by Bourget
from the viscera of a woman who died of puerperal
fever, and subsequently he proved the existence of
the same ptomaines in the urine of patients suffer-
ing from the same disease. Pasteur and others are
convinced that with the possible exception of cases
where, by the presence either of internal or external
abscesses, the body before confinement contains
microbes, the antiseptic treatment ought to be
infallible in preventing puerperal fever from declar-
ing itself. It may be stated that the introduction
of the antiseptic and aseptic methods has produced
not only a remarkable diminution of mortality, but
also of the morbidity or illness incident to the
puerperal state.

1 Die Puerperalen und Pydmiochen Processe.

2 In 1889 a midwife carried the contagion to five different
women, all of whom died of the disease (The Echo, Sept. 17,

INFECTIOUS DISEASES AND MICROBES, ETC. 195

Prof. I. Giglioli1 gives the following statistics
concerning the patients in the Maternity Hospital
at Copenhagen between the years 1850 and 1874
(the antiseptic method being introduced in the year
1870):—

From 1850 to 1864 the mortality was 41'6 per 1000.
„ 1865 „ 1870 „ „ 19-6 „

„ 1870 ,,1874 11-4 „

And in Naples the mortality during the years
1875-78 was 0'12 per 1000 patients.

Dr. W. 0. Priestley gives in his paper, which
was read before the Congress of Hygiene,2 an inter-
esting table showing the maternal deaths in six
lying-in hospitals since the introduction of antiseptic
and aseptic methods. With these he contrasted
the figures of M. Le Fort before the era of anti-
septics : —

MORTALITY IN MATERNITY HOSPITALS FROM ALL

CAUSES IN VARIOUS COUNTRIES OF EUROPE

(LE FORT).

Before the introduction of Antiseptics.

Deliveries. Deaths. Per 1000.

Total, . 888,312 30,394 34'21

1 Fermenti e Microbi, p. 157.

2 Held in London during September 1891.

196 A MANUAL OF BACTERIOLOGY

After the introduction of Antiseptics.

I
Date.

Deliveries.

Deaths which
would have
Deaths, occurred on
basis of Le
Fort's figures.

Vienna, . .

1881-5

15,070

106 516

Dresden, .

1883-7

5,508

57 188

Russia, ...

1886-9

76,646

290 2,622

New York, . .

1884-6

1,919

15 66

Boston, .

1883-6

1,233

27 42

London (General
Lying-in Hospital),

1886-9

2,585

16 88

Total,

102,961

511 3,522

The number of lives saved out of the 102,961
deliveries since the introduction of antiseptics is
the following : —

Expected deaths on Le Fort's basis, . 3522
Actual deaths, . . . 51 11

Saving, . . 3011

From the above figures it will be seen that while
according to M. Le Fort, the maternal deaths in
European lying-in hospitals were 34'21 per 1000
under the old regime, the mortality is now reduced
to somewhat less than 5 per 1000. This computa-
tion, put in another way, indicates that if the former
rate of mortality had been maintained 3522 maternal

1 4-363 per 1000.

INFECTIOUS DISEASES AND MICROBES, ETC. 197

deaths might have been expected, whereas the actual
deaths were only 511. In other words, 3011 lives
of mothers were saved as the result of new and
purely scientific methods of treatment.1

INFLUENZA.

This is a very different disease from the catarrhal
affections known by the same name. It is really
an acute specific disease running a definite course
like scarlatina or measles ; but very little is known
of the cause or nature of this ubiquitous disease
which has attacked humanity in its own violent
fashion at short intervals from probably the earliest
ages. The history of the recorded epidemics of La
Grippe is marvellously complete for centuries.
Every country and every climate in the world is
subject to it, yet it appears to find a permanent
home nowhere as a constant or endemic resident,
but to disappear from the face of the earth for a
series of years. It is, however, probable that the
microbe of this disease has some undiscovered
endemic source.

The symptoms of epidemic influenza follow pre-
cisely the type of the other infective fevers, and
preserve a remarkable uniformity and individuality
in successive epidemics. Sir Morell Mackenzie'2
believes that the disease is due to ' a specific poison
of some kind which gains access to the body, and,

1 For further information on the subject of puerperal fever
see Flessinger's paper in Gaz. Med. de Paris, 1889, p. 313 ; and
Widal's Etude sur V Infection Puerperale (1889).

2 Fortnightly Review, June 1891.

198 A MANUAL OF BACTERIOLOGY

having an elective affinity for the nervous system,
wreaks its spite principally or entirely thereon. In
some cases it seizes on that part of it which governs
the machinery of respiration, in others on that
which presides over the digestive functions ; in
others again it seems, as it were, to run up and
down the nervous key-board, jarring the delicate
mechanism, and stirring up disorder and pain in
different parts of the body, with what almost seems
malicious caprice.' Therefore, according to Mac-
kenzie, the supposed microbe resides in, or acts
on, the nervous tissues of the body.

There are many reasons for thinking that the
contagium of influenza is borne through the air by
winds rather than by human intercourse. One
reason for thinking so is that it does not appear to
travel along the lines of human communications,
and, as is seen in the infection of ships at sea, is
capable of making considerable leaps. Dr. Parsons,
on the other hand, believes that the epidemic is
propagated mainly by human intercourse, though
not in every case necessarily from a person suffering
from the disease.

Concerning the germ of influenza, Klebs thought
that he had discovered this in certain Flagellata
found in the plasma or corpuscles of the blood
during the febrile stage, but no cultivations were
made. Gluber found a micrococcus (in pairs) in the
blood ; and Frankel noticed the same microbe in the
sputum of a patient suffering from influenza. This
microbe may have been Micrococcus pneumonice, as
pneumonia frequently follows an attack of the

INFECTIOUS DISEASES AND MICROBES, ETC. 199

disease. Eibbert thinks that Micrococcus pyogenes is
invariably present, and is the actual cause of influ-
enza ; but Besser has shown that it is common in
healthy men at least during the epidemic. In 1884
Seifert found a micrococcus in the sputa of influenza
patients and of no others.

Although the microbe of influenza has not yet
been isolated, there is little doubt that influenza is
a microbian disease ; for its constancy of type, the
mode of its transmission, its independence of climatic
and seasonal conditions, all suggest that its cause
is 'specific' — i.e. having the properties of growth
and multiplication which belong to a living thing.1

PNEUMONIA.

In this disease large numbers of micrococci are
present in the lungs.

The microbe (Micrococcus pneumonice, Fig. 33, 17)
was discovered by Friedlander,2 and occurs in the
sputa of pneumonic patients, either singly, as diplo-
cocci, short chains, and zooglcea. Sometimes the
microbes are free, while at other times they are
encysted in the lymphatic cells. They are oval,
encapsulated microbes, and have been cultivated in
blood-serum, peptonised gelatine, bouillon, and on
steamed potatoes.

1 See Sisley's Epidemic Influenza (1891) ; Erodie's paper in
Nature, July 23, 1891 ; Parsou's Report on Influenza to the Local
Government Board (1891) ; the Hon. R. Russel's pamphlet, The
Spread of Influenza (1891) ; Cantani's V Influenza (1890) ; Tala-
mon's La Grippe et les Microbes (1890). See also the Appendix.

2 Virchow's Archiv, vol. Ixxxvii.

200 A MANUAL OF BACTERIOLOGY

Griffini and Cambria1 observed the same micro-
cocci in the blood of pneumonic patients. Salvioli
and Zaslein2 cultivated these microbes, derived from
the same source, in bouillon at 37° to 39° C. ; and
when injected into mice and rabbits they gave rise
to pneumonia. Giles3 found the same microbes in
many cases of pneumonia in India ; and pure culti-
vations, when injected into the subcutaneous tissues
of rabbits, produced the disease. Those researches
have been confirmed by Afanassiew.

When the artificially - cultivated microbe is
inoculated in the tissue of the lungs it produces
in animals all the characteristic symptoms of
pneumonia; the lungs become red, solid, and en-
larged, and pieces of them sink in water. In
pneumonia the blood is considerably altered, for
the hsematin, globulin, and the salts are greatly
reduced.

According to Emmerich, the growth of the
micrococcus of pleuro-pneumonia on peptonised
gelatine is similar to the one derived from human
pneumonic sputum; and when this microbe is
injected into rabbits it produces typical pneumonia.
Nolen and Poels 5 injected pure cultivations of the
microbe of human pneumonia into cattle, and pro-
duced pleuro-pneumonia with all its characteristic
symptoms. However, it may be mentioned that

1 Gentralblatt fur d. Med. Wissemch., 1883.

2 Ibid., 1883.

3 British Medical Journal, 1883.

4 Comptes Rendus de la Socidte de Biologie (Paris), t. 5.

5 Centralblattfur d. ;Med. Wissensch., 1884.

INFECTIOUS DISEASES AND MICROBES, ETC. 201

Dr. Klein1 does not accept these statements without
reservation.

Professor Brieger2 has shown that when M.
pneumonia is grown in solutions of glucose or
sucrose, acetic acid is formed along with ethyl
alcohol and formic acid. The same products are
formed when the microbe is grown in solutions of
creatine and calcium lactate.

Dr. P. F. Frankland3 has recently investigated
the action of the same microbe on various carbo-
hydrates, with the following results : —

(a) Micrococcus pneumonice sets up a fermentive
process in solutions of dextrose, sucrose, lactose,
maltose, raffinose, dextrin, and mannitol.

(b) It does not ferment solutions of dulcitol or
glycerol, and has thus the power, like the Bacilhis
ethaceticus (see p. 155), of distinguishing between
the isomers, mannitol, and dulcitol.

6C6HU06 + H20 = 9C2H5HO + 4CH3COOH + 10C02 -j- 8H2.

In other words, the above equation represents the
quantitative decomposition of mannitol into alcohol,
acetic acid, carbon dioxide, and hydrogen.

It would be interesting to ascertain whether
acetic acid and alcohol are formed in human milk
during an attack of pneumonia; for it may be
stated that the lactose is reduced from 43-6 to
30-2 parts per 1000.

1 Micro-Organisms and Disease, p. 77 (3d. ed.).

2 Zeit. Physiol. Chem., vol. viii. p. 306 ; and vol. ix. p. 1.

3 Journal of Chemical Society, 1891, p. 253.

202 A MANUAL OF BACTERIOLOGY

SCARLATINA.

This disease is the result of the action of the
Micrococcus scarlatince, which has been found in
the blood, organs, the exudations and tissues of
the ulcerated throat, and in the desquamating epi-
demic cells of this disease. M. scarlatince has
also been observed in the urine of patients
suffering from scarlatina ; and this fluid contains a
ptomaine represented by the formula C5H12N04.
The same ptomaine has also been extracted from
pure cultivations of Micrococcus scarlatince in pepto-
nised gelatine. In fact, the microbe forms this
ptomaine from the medium in which it lives.1

M. scarlatince^ (0.5 //, diam.) occurs singly, as
diplococci, in chains, and in masses (Fig. 33, 9) ;
and it grows on the surface of nutrient gelatine, as
well as in the depth of that medium. It also grows
on agar-agar and in beef bouillon. On nutrient
gelatine with slanting surface this microbe forms
greyish, circular, flat discs, which ultimately form a
grey film. Gelatine tubes inoculated by stabbing
show some characteristic features. After twenty-
four hours' incubation the surface of the stab
appears sunk, and the depression thus formed
increases in breadth and depth during the next two
or three days, so that by that time there is a
distinct funnel-shaped depression indicating the
upper end of the channel of inoculation marked as
a white streak. Then, commencing at the bottom

1 See Dr. A. B. Griffiths' paper in Comptes Rendus de I'Aca-
ddmie des Sciences, vol. cxiii. (Nov. 9. 1891), p. 656.

INFECTIOUS DISEASES AND MICROBES, ETC. 203

of the funnel, the gelatine becomes liquefied, and
this liquefaction gradually extends in breadth, and
always in depth, along the line of the growth. The
liquefied part of the gelatine is clear, and at the
bottom of it is a whitish precipitate.

In stab-cultivations, using agar-agar or solid
blood serum as the medium, white dots make their
appearance in the streak; but these are of a
brownish colour where thickly packed together.
On the surface of agar-agar or solid blood serum
a continuous film is formed.

In alkaline bouillon the growth forms whitish,
fluffy, or loose masses at the bottom of the tube.
In milk M. scarlatinas grows fairly well, and turns
the milk at first thick, then quite solid ; sometimes
this occurs after two or three days' incubation at
37° C., sometimes a little later.

Drs. Klein and Power have proved that a certain
eruptive disease of the teats and udders of cows
is capable of communicating scarlatina to human
beings through the medium of the milk derived from
such cows.

In certain extensive outbreaks of scarlatina at
Hendon, Wimbledon, etc., Dr. Klein found Micro-
coccus scarlatince in the blood of scarlatina patients
both during life and after death ; and he found the
same microbe in the tissues and organs of persons
dead of scarlatina. These outbreaks of scarlatina
were traced by Power and others to the milk-supply
from certain farms where the cows were suffering
from what is now known as cow -scarlatina. In
both human and bovine scarlatina the same microbe

204

A MANUAL OF BACTERIOLOGY

(M. scarlatince) is always present in the tissues,
organs, and blood; and from both sources sub-
cultures of the microbe, when inoculated into
healthy cows, produce the disease. For instance,
when pure subcultures of the microbe were inocu-
lated into calves and cows, the microbe was found
in the spleen, kidneys, teats, udders, lung, skin, etc.
Fig. 42 represents a section through the skin of
the nostril of a calf that had been experimentally

FIG. 42. SECTION THROUGH SKIN OF THE NOSTRIL OF A CALF THAT HAD

BEEN EXPERIMENTALLY INFECTED WITH M. SCARLATINA DERIVED FROM

A HUMAN SOURCE (Klein).

infected with M. scarlatince derived from a human
source. In this figure it will be observed that the
microbe is present in large numbers.

In fact, Dr. Klein's important researches on the
relationship existing between the cow - disease,
already alluded to, and human scarlatina may be
summarised as follows : —

(a) The disease in man and in the cow alike is
characterised by closely similar anatomical features

INFECTIOUS DISEASES AND MICROBES, ETC. 206

(b) From the diseased tissues and organs of man
and cow alike the same microbe can be separated,
and artificial subcultures be made from it.

(c) These subcultures, no matter whether estab-
lished from man or cow, have the property, when
inoculated into calves, of producing in them every
manifestation of what is known as the Hendon
cow-disease, except the sores or ulcers on the teats
and udders — no doubt for the reason that the milk
apparatus is not yet developed in calves.

(d) Subcultures of the microbe made from human
scarlatina and inoculated into recently calved cows
produced in them, along with other manifestations
of the Hendon cow-disease, the characteristic ulcers
on the teats — ulcers identical in character with
those observed at the Hendon farm.

(e) The subcultures, established either from the
human or the cow disease, have an identical pro-
perty of producing in various rodents a disease
similar in its pathological manifestations to the
Hendon disease of cows and to scarlatina in the
human subject.

(/) Calves fed on subcultures established from
human scarlatina obtain the Hendon disease.

(g) Children fed on milk from cows suffering
from the Hendon disease obtain scarlatina.

Bearing on the same subject, it may be mentioned
that in the parish of St. George's, London, five
persons were attacked with scarlatina on the same
day (in October 1886). They had used a cheap
brand of condensed milk ; and in this milk Dr.
Klein proved (both by cultivation and inoculation)
the presence of Micrococcus scarlatinas.

206 A MANUAL OF BACTERIOLOGY

These investigations prove that cows suffer from
scarlatina ; that the specific microbe circulating in
the blood of the diseased animals contaminates the
milk; and that such milk conveys the disease to
human beings. The disease has also been directly
communicated to man by inoculation with the virus
from the ulcers on the teats and udders. In a
particular case, recorded by Dr. J. Cameron,1 a man
received the virus of scarlatina into a recent scratch
upon his forefinger while milking a diseased cow.

As both human beings and cows are liable to be
attacked with scarlatina, and as the milk of the
latter (when diseased) is capable of producing an
extensive outbreak of the disease in human beings,
it is advisable that milk should be boiled before use.
.This destroys any microbes which may be present.2

LEPROSY.

The microbe of this disease is Hensen's Bacillus
leprce. It measures from 4 to 6 //, long and about 1
/i wide (Fig. 33, 2), and it occurs in masses within
the large leprosy-cells of the nodules of the skin
and organs, as well as of the mucous membrane
of the mouth, palate, and larynx. Two types of
leprosy are described — the anaesthetic and tuber-
cular varieties ; the first variety is more frequently
seen in the tropics, the latter in temperate climates.
In the anaesthetic variety the bacillus is present in

1 Transactions of Epidemiological Society, 1885-6.

2 For full details of Klein's researches see the Reports of
Medical Officer to Local Government Board, 1885-6 ; 1886-7 ;
1887-8; 1887-9.

INFECTIOUS DISEASES AND MICROBES, ETC. 207

the interstitial tissue of the nerves. B. leprce is
sometimes motile and produces spores. It grows
on blood serum and alkaline infusions of meat-
extract ; and Damsch1 has produced the disease in
cats by inoculating them with leprous tissues. The
microbe is absent in the blood of lepers ; therefore
it probably spreads by the lymphatic vessels.

Hansen's discovery2 of B. leprce lias since been con-
firmed by Neisser,3 Cornil,4 Babes,5 Hillis,6 Stevens,7
Thin,8 Rake,9 Kobner,10 Bordoni-Uffreduzzi,11 and
Gianturso ; 12 and during the present Leprosy Com-
mission in India, Drs. Rake, Buckmaster, Thomson,
and Kanthack have also succeeded in rearing B.
leprce13 on blood serum ; but growths of this microbe
are difficult to obtain. Bordoni-Uffreduzzi obtained
' growths from the marrow of a bone in which there
were a number of free leprosy bacilli ; these appeared
on serum (to which a quantity of glycerine had been
added) that was maintained at a temperature of 37°
C. These he described as delicate, thin, slightly

I Virchow's Archiv, vol. xcii.

9 Ibid. vol. Ixxix. 3 Ibid. vol. Ixxxiv.

4 Union Medicate, 1881.

5 Archives der Physiologic, 1883.

• Transactions of Pathological Society, 1883.

7 British Medical Journal, 1885.

8 Med.-Chir. Transactions, vol. Ixix.

9 Transactions of PathoL Soc., 1887.

10 Virchow's Archiv, vol. Ixxx.

II Zeitschrift fur Hygiene, vol. iii. p. 178.

12 Centralblatt fur Bakteriologie und Parasitenkunde, vol. ii.
p. 701.

13 Concerning the differences between the leprosy and tubercle
bacilli, 'see Slater's paper in Quart. Journ. Micros. Science, 1891,

208 A MANUAL OF BACTERIOLOGY

yellow films with irregular borders. On glycerine
agar-agar they are said to have developed as small,
grey, munded, isolated points, usually at the end of
ten days or a fortnight ; secondary cultivations,
however, made their appearance at the end of forty-
eight hours, and after the first few cultivations the
microbe could be grown on serum or on ordinary
gelatine and agar-agar, but much more slowly than
when glycerine had been added.'

Leprosy, or elephantiasis grecorum, is a specific
disease, characterised by the slow development of
nodular growths in connection with the skin,
mucous membranes, and nerves, and by the super-
vention of ansesthesia, paralysis, and a tendency to
ulcerative destruction and gangrene.

Although prevalent in the Middle Ages, leprosy
is very rare in Europe at the present day, being
confined to isolated areas on the shores of Spain,
Portugal, Sweden, Norway, Iceland, and in Italy,
Kou mania, Hungary, and Greece, where it is still
endemic. It is, however, common in Egypt,
Morocco, Cape Colony, Madagascar, Southern Asia
(including Japan), Brazil, United States of Colom-
bia, Guiana, Argentina, New Zealand, and in certain
islands of the Pacific Ocean (especially Hawaii).

In the United States of Colombia leprosy first
made an appearance in 1646, and was introduced
into that country from Spain. It seems to have
spread slowly but surely throughout a great part of
the country during the succeeding two hundred
years; but since 1870 the increase in the number
of cases has been much more rapid, and within that

INFECTIOUS DISEASES AND MICROBES, ETC. 209

period the disease has spread to districts where it
was previously unknown, until now almost every
district in Colombia is more or less infected.
According to a medical authority residing in
Bogota, it is stated that one-tenth of the inhabit-
ants of Santander and Boyaca are lepers. As the
population of these two states is about 1,000,000,
this estimate would give 100,000 lepers in that por-
tion of Colombia alone. Another authority states
that there are only 30,000 lepers in the two states
previously mentioned; but whichever figure is.
correct, it shows that a large percentage of the
inhabitants are suffering from this fell disease.
Marriages constantly take place between non-lepers
and lepers, and children are born of these unions ;
but they generally develop the disease in a few
years. The lepers also marry among themselves,
and their children are almost always lepers. Very
little is done in the way of isolation, consequently
leprosy is bound to spread more and more through-
out Colombia unless some great effort is made to
arrest its progress. It is the universal opinion all
over Colombia that leprosy is both contagious and
hereditary ; but it is probable that the system
requires to be predisposed by bad food, unsuitable
climate, dirty and confined lodging, exposure to
chills and damp, etc., before leprosy can be con-
tracted by contagion. There is no doubt that the
absence of hygienic appliances and personal clean-
liness aid its development immensely.1

So far as is at present known, there is no cure for

1 See the British Consular Report from Boyota,, 1891.

210 A MANUAL OF BACTERIOLOGY

leprosy;1 but no doubt, with growing experience,
leprous vaccine will soon be discovered ; and it is
even possible that, with the experience already
gained, such a result may at once be obtained
(Pasteur).

SYPHILIS.

Syphilis is a specific disease ; and, ' after the local
introduction of the syphilitic poison, some ten to
fifty days elapse before the true Hunterian chancre
first appears, but at the same time indurated buboes
or glands may be detected in the. groins. In a few
weeks the blood becomes tainted by the peculiar
virus, and this interfering with the nutrition of the
blood capillaries and tissues, produces a series of
morbid phenomena, divided by syphilographers into
secondary and tertiary, the term primary being
retained for the manifestations due to local inocu-
lation. Leaving no tissue untouched,2 syphilis is
well known also for the variety of its manifestations
and for its propensity to attack parts of the body
often respected by other forms of skin disease and
blood poisoning. A proneness to leave behind much
dusky, copper-coloured staining of the skin, whilst

1 It is stated that leprosy has been cured by the ' Mattei
remedies ' (Report of the St. Joseph's Asylum at Mangalore,
1891), but these 'remedies' have been proved to be quack pre-
parations, etc., by the medical profession.

2 Hence the reason that Byron called this disease — 'the
great:'—

' I gaid the small- pox has gone out of late ;
Perhaps it may be follow'd by the great. '

(Don Juan, c. i., v. 130.)

INFECTIOUS DISEASES AND MICROBES, ETC. 211

its inflammatory eruptions scarcely cause itching,
are features of diagnostic interest.'

The Bacillus of syphilis was discovered by Dr. S.
Lustgarten 1 in the nucleated cells of various syphi-
litic products, e.g. ' in the discharge of the primary
lesion and in hereditary affections of tertiary
gummata.' He never found the microbe free
between the tissue elements, but always enclosed
in cells. Nevertheless, it may be stated that Eve
and Lingard 2 isolated a bacillus from the blood, as
well as from the diseased tissues in syphilis, which
they cultivated in artificial media.

Lustgarten's bacillus measures from 3 to 4 //, long
and 0.8 //, wide (Fig. 33, 21); it has a swelling at
each end. It is believed that this microbe produces
spores, and, according to Lustgarten, it is the virus
of syphilis. Doutrelepont, De Giacorni, and Schlitz
have confirmed Lustgarten's observations.

TETANUS.

Tetanus or lockjaw is an infectious disease caused
by the Bacillus of tetanus, which inhabits certain
soils; for it was proved by Nicolaier3 that soil
obtained from streets and fields,4 when inoculated
into mice, rabbits, and guinea-pigs, gave rise to the
characteristic symptoms of tetanus. The microbe
of this disease forms spores. ' These spores gaining

1 Med. Jahrbucher der K. K. Gesellsch. d. Aerzte (Vienna), 1885.

2 Lancet, 1886, p. 680.

3 Dissertation (Gottingen), 1885.

4 Soils obtained from cultivated gardens and from woods do
not give rise to tetanus.

212

A MANUAL OF BACTERIOLOGY

access to an abrasion or wound of the skin in man
or animals, are capable of germinating there and
multiplying, and of producing a chemical poison,
which is absorbed into the system, and sets up the
acute complex nervous disorder called lockjaw.'

The tetanus bacillus- (1.2 yu, long) produces spores
only at one end (Fig. 43), and in the spore-bearing
condition is known as the drum -stick -shaped
bacillus. It is motile and anaerobic, growing on
gelatine- plates (containing glucose) in an atmo-
sphere of hydrogen.
In tubes containing
blood serum or nutrient
gelatine, it grows in
the depth of the me-
dium, forming a kind
of cloud. The medium
emits a fusty smell,
which is characteristic
of this microbe.

In obtaining culti-
vations of the tetanus
bacillus, other anaerobic microbes grow, and also pro-
duce spores. But Kitasato found that the tetanus
bacillus produced spores earlier than the other
bacilli present in tetanic pus. Consequently, he
devised the following method for separating the
tetanus bacillus from the other microbes : — As soon
as spore-formation in the tetanus bacilli had com-
menced, the tubes (containing them) were heated
for a considerable time at 80° C., with the result
that all the bacilli were destroyed, but not the

FIG. 43. THE TETANUS BACILLUS.
(x 1000.)

INFECTIOUS DISEASES AND MICROBES, ETC. 213

spores of the tetanus bacilli. These spores after-
wards germinated (at 30° C.), and give rise to pure
cultivations of the tetanus bacillus.

This microbe is localised at the actual point of
inoculation (i.e. in the pus and the walls of the
abscess), and is never present in the internal organs.
The ptomaine, which the tetanus bacillus gives rise
to, is manufactured at the site at which it is actually
introduced, and ' from this point it is absorbed into
the body, and is carried to the special tissues on
which it acts/

Professor L. Brieger1 has succeeded in isolating
four ptomaines from pure cultivations of the tetanus
bacillus. This first is tetanine (C13H30N204), which
produces tetanus in animals ; the second is tetano-
toxine (C5HnN), which produces tremor and
paralysis, followed by violent convulsions; the
third is spasmotoxine (formula unknown), which
produces tonic and clonic convulsions; and the
fourth ptomaine (which has not been named) causes
tetanus, accompanied with a flow of saliva and
tears.2 Tetanine has also been extracted from the
limb of a patient who had died from tetanus.
Brieger looks upon the poisonous substance tetano-
toxine as a toxalbumin; but he may have over-
looked the possibility that this proteid may contain
a ptomaine closely bound to it, or in an isolated
condition within its molecules.

1 Virchow's Archiv, vol. cxii. (1838), p. 549 ; vol. cxv. (1889),
p. 484 ; Berliner Klinische Wochenschrift, 1888 ; and Unter-
suchungen iiber Ptomaine, 1886, p. 89.

2 These tetanic ptomaines do not occur in the urine of
patients suffering from tetanus.

214 A MANUAL OF BACTERIOLOGY

As the tetanus bacillus is localised, there can be
no doubt that tetanus is due to the above poisons
(manufactured indirectly by the bacillus) producing
effects after getting into the blood, by virtue of
some selective action on certain parts of the motor
nerve-centres.

The spores of the tetanus bacillus have an ex-
tremely wide distribution, being found in soils, etc.,
in various parts of the world. According to Bos-
sano,1 soils which contain much organic matter
nearly always contain tetanus bacilli, ' and that
latitude, climate, and special meteorological condi-
tions have far less influence on their development
than defective drainage, imperfect hygienic condi-
tions, and the degree of cultivation of the soil.'

Dr. Kitasato 2 has recently shown how to produce
immunity against tetanus, and he has cured animals
suffering from this disease. Kitasato first renders
an animal immune against tetanus, and then injects
the blood serum of that animal into animals suffering
from the disease. In order to render an animal
immune or unsusceptible, the tetanus bacilli are
first injected ; this injection being followed by in-
jections of iodine trichloride, which are repeated at
intervals of twelve hours. After four days the
animal, which under ordinary circumstances would
have died from tetanus, is not only cured, but ren-
dered immune against the disease. The blood serum
of such an animal has been found in successive

1 Comptes Rendus, tome 107, p. 1172; and Recherches Ex-
pdrimentales sur VOrigine Microbienne du Tetanos (1890).

2 Deutsche Medicinische Wochenschrift, 1890, No. 49, et seq.

INFECTIOUS DISEASES AND MICROBES, ETC. 215

experiments on mice and rabbits to act as a com-
plete cure. Kitasato's experiments prove (a) that
the blood of rabbits which have been rendered un-
susceptible to tetanus possesses properties destruc-
tive of the tetanus virus ; (&) that these properties
are to be observed also in extra-vascular blood and
serum free from cells ; (c) that these properties are
of so permanent a nature that they are still mani-
fested by such serum after it has been injected into
other animals ; consequently, by transfusion of such
blood or serum, important therapeutic actions can
be obtained ; (d) that this power of destroying the
tetanus poison is absent from the blood of such
animals as are not immune against tetanus; and
after such animals have been killed by the tetanus
poison, it can be shown to be present in their blood
and tissues.

If animals (such as mice and rabbits) highly sus-
ceptible to tetanus are cured by this treatment, we
have good reason to believe that it will also cure
human beings, which are far less susceptible to the
disease.

MALARIA.

The discovery of the Bacillus malarice placed
malaria among the acute specific diseases. Concern-
ing the distribution of malaria, moisture and air
have much to do with it, for the disease is more
abundantly developed in wet than in dry years.
Moisture in the soil is essential for the production
of malaria, while clayey, loamy, and marshy soils

216 A MANUAL OF BACTERIOLOGY

favour its development. Professor C. Tommasi-
Crudeli 1 states that the following conditions are
necessary for the Bacillus malaria to produce spores :
(a) ' Une temperature de 20 degres centigrades
environ ; (b) un degre" mode're d'humidite* perman-
ente; (c) 1'action directe de 1'oxygene de Fair sur
toutes les parties de la masse [that is, of the soil].
II suffit que Tune de ces trois conditions fasse
deTaut, pour que le deVeloppement des sporules, et
la multiplication du ferment malarique, soient ar-
retes.' In marshy districts, the larger the amount of
organic matter present in a soil, the greater will be
the prevalence of malaria. The disease is more
prevalent the lower the level of the country, although
in Central Africa a height of 2500 feet is not free
from it. Both air and water may convey the dis-
ease, and there is little doubt that it finds an entrance
into the system by means of air, potable water, and
food.

Bacillus malarice (2 to 7 //. long) gives rise to
leptothrix filaments, and produces spores either at
the ends or in the centre of the cell (Fig. 38, 18).
This bacillus was found in the blood of malarial
patients by Klebs and Tommasi-Crudeli,2 and they
also found it in the spleen, medulla, lymphatic
glands, and venous blood of persons dead of malaria.
On gelatine B. malarice gives rise to a well-developed
growth, and when a drop of the culture is inoculated

1 La Malaria de Rome et VAncien Drainage des Collines
Romaines (Paris), 1881 ; and Atti ddla R. Accademia dei Lincei,
1879.

2 Atti ddla R. Accademia dei Lincei, 1879, 1880, and 1881 ;
and Archivfur Experimental Pathologie, 1879.

INFECTIOUS DISEASES AND MICROBES, ETC. 217

in rabbits it reproduces malarial fever, with all its
characteristic symptoms, the threads and spores of
the bacilli being found in abundance both in the
spleen and the marrow. This microbe grows also
on albumin, urine, and other media in the presence
of air, and at a temperature of about 20° C. B.
malaria was originally discovered in the soil of the
Roman Campagna, and Antonio Ceci 1 obtained pure
cultures of the microbe from this soil. When these
pure cultures were inoculated in animals they pro-
duced malaria or intermittent fever.

Dr. B. Schiavuzzi2 has confirmed Klebs and Tom-
rnasi-Crudeli's discovery of Bacillus malarice, and
that it is the real cause (directly or indirectly) of
malaria. Cohn 3 has also verified the work of the
Italian bacteriologists.

On the other hand, Laveran,4 Richard,5 Marchia-
fava and Celli,6 Golgi,7 Evans,8 and others have dis-
covered certain organisms allied to the Flagellata in
the blood of patients suffering from malaria. These
organisms have been called Plasmodium malarice,
and they are said to give rise to intermittent fever
in man after intravenous injection. The blood cor-
puscles of a person so infected again contain the
plasmodia ; and it is further stated that these or-
ganisms alter the composition of the blood.

1 See Professor Giglioli's Fermenti e Microbi, p. 592.

2 Atti della R. Accademia dei Lincei, 1886.

3 Beitrage zur Biologic der Pflanzen, 1886, p. 245.

4 Comptes Rendus, 1881-2. 5 Ibid. 1882.

6 Annali di Agricoltura (Roma), 1886, p. 4.

7 Archivioper h Scienze Mediche, vol. x. (1886), p. 109.

8 Proceedings of Royal Society, 1891.

218 A MANUAL OF BACTERIOLOGY

In a paper read before the Accademia del Lincei
on May 2, 1886, Professor Tommasi-Crudeli1 says
that he does not accept the statement that the plas-
modia found in the blood of malarial patients are
the cause of malaria. In fact, he says ' la grande
extensione dell' infezione malarica : le varie forme,
ora lente e latenti, ora rapide e intense, nelle quali
questa infezione si manifesta : la lunga persistenza,
anche allo stato latente, della malaria in un terreno :
son tutti forti argomenti contrari alia ipotesi che la
malaria sia dovuta ad un parassita di natura animale ;
e favorevoli all 'opinione che i germi malarici siano
Schizomiceti, simili a quelli delle tuberculosi, e di
altre persistenti infezioni.'

The alteration in the composition of the blood in
patients suffering from malaria (previously alluded
to) may be due to a soluble enzyme secreted by B.
malarias (Schiavuzzi), and certainly this is not im-
probable, for Dr. Lauder Brunton, F.RS.2 has shown
that many microbes have the power of ' manufac-
turing a ferment suited to their needs.'

Bacillus malaria is inhaled into the blood by way
of the lungs, and perhaps it may enter through the
stomach and skin also. It flourishes in marshy
districts, in deltas, on alluvial soils, and on the
banks of tropical rivers — in fact, a proper degree of
porosity, of temperature, and of humidity of soil
favour the growth of this microbe : hence the reason

1 This eminent savant has been obliged to give up his im-
portant investigations. He wrote to the author as follows : ' I
have been compelled to give up microscopical researches since
1886, because my eyes are almost ruined.'

2 Proceedings of Royal Society, vol. xlvi. p. 542.

INFECTIOUS DISEASES AND MICROBES, ETC. 219

that B. malaria has been called 'an earth-born
poison.' 1 This microbe is said to be heavier than
most gases, ' and scarcely floats six feet above the
ground ; it may be wafted some distance by winds,
but mountains hold it back, and belts of trees,
especially the eucalyptus, destroy its efficacy.'

Gubler2 and many others have shown that the
eucalyptus or ' fever-destroying ' tree has consider-
able power in destroying the microbe of malaria,
this being due to the action of the aromatic gases
given off by the tree. One instance may be cited
of the fever-destroying properties of the eucalyptus.
' In a desolate part of the Campagna there stands an
old monastic institution upon a spot consecrated by
tradition as that whereon St. Paul was martyred.
For centuries this part of the Campagna [Tre Fon-
tane 3] was a stronghold of pestilential fever, and
prolonged residence in the monastic institution in
question surely led to death. Some few years ago
a band of Trappist monks planted the eucalyptus in
its cloisters, and the trees have since grown to a
great height. What is more important, however, is
that the place is now once more habitable, and fever,
it is said, reigns there no more.' 4 There are also
plantations of the eucalyptus in Corsica, Algeria,
Italy, California, Australia, and other parts of the
world ; and there is little doubb that these trees are
antagonistic to the spread of malaria, because the

1 Felkin in Proc. Roy. Soc. of Edinburgh, vol. xvi. p. 269.
- Journal de Pharmacie et de Chimie, 1871.

3 Known anciently as Aquae Salvise.

4 Kingzett's Nature's Hygiene (3rd ed.), page 266; see also
Giglioli's Fermenti e Microbi, pp. 247-257.

220 A MANUAL OF BACTERIOLOGY

essential oil secreted by the trees contains a hydro-
carbon— C10H16 ; and as this is vapourised, it is re-
solved in the presence of atmospheric oxygen and
moisture into camphoric peroxide, camphoric acid,
and hydrogen dioxide l : —

(a) 2 010H16+502 = 2C10H1404+2H20,
(/9) C10H1404+2H20 = C10H1604+H202;
and it is the hydrogen dioxide, so produced, which
destroys the microbia of malaria.

In the treatment of malaria certain medicinal
substances are used. (1) Tommasi-Crudeli 2 recom-
mends arsenious acid in small doses ; and, according
to many English authorities, Fowler's solution (con-
taining 1 part of arsenious acid in 120 parts of water)
should be prescribed in 5 to 10 minim doses three
times a day. (2) Quinine salts, in large doses, have
also been recommended, especially by travellers
who have had to pass through malarial districts.

TYPHOID FEVER.

The microbe of this disease has been found in
Peyer's glands, the spleen, larynx, lungs, liver, and

1 Mr. C. T. Kingzett, F.C.S., manufactures these substances
on a large scale. He decomposes the essential oils (principally
turpentine oil), in the presence of water, by passing a current
of air into them, the products being sold as ' Sanitas ' fluid and
oil, both of which are powerful germicides. Kingzett imitates
the decomposition of the essential oils by a similar process as
the one which goes on naturally in the eucalyptus, pine, and
camphor forests. It may be stated that 0'4 gramme of ' Sani-
tas' oil completely destroyed Micrococcus prodigiosus, Bac-
terium allii, Bacillus tuberculosis, and Bacillus subtilis when
grown in various media as tube-cultivations.

" Atti della R. Accademia del Lincei, 1885.

INFECTIOUS DISEASES AND MICROBES, ETC. 221

in the lymphoid follicles of the intestine in fatal
cases. Sometimes the microbe is present in "the
kidneys and urine. Bacilhis typhosus measures
from 2 to 3 ft long, and from 0*3 to 0'5 //, wide ; and
it forms filaments which sometimes measure 50 //,
in length. It (Fig. 33, 4) has rounded ends ; and
it has been stated that spore -formation takes place
at the extremities of the rods. This statement is,
however, doubted by some bacteriologists; because
the so-called spores have never been observed to
germinate, etc. B. typhosus grows on bouillon,
nutrient gelatine,1 steamed potatoes (at 37°C.), and
blood serum ; and it can grow either in the presence
or in the absence of free oxygen. On gelatine-
plates, the microbe gives rise to greyish colonies
with irregular margins, without liquefying the gela-
tine. In tube-cultivations, a growth appears as a
bluish-grey film on the surface, whilst 'in the
needle track there is a delicate zone of the same
bluish-grey colour, surrounded in turn by a peculiar
opalescent milkiness. The most characteristic
growth, however, occurs on sterilised potatoes. It
is characteristic in that, even when there is a most
luxuriant growth of the typhoid bacillus, it cannot
be recognised by the naked eye, even at the end of
three or four days, except by a peculiar moist ap-
pearance of the potato, which, taken along with the
appearances in milk and on gelatine, so far as is at
present known, distinguishes the growth of this
microbe from all others. It will be remembered,
however, that the potato is slightly acid ; and it

1 Gaffky in Mitth. aus dem. k. Getundheitsamte, 1886.

222 A MANUAL OF BACTERIOLOGY

appears that this acidity is necessary for this typical
growth, for on potatoes rendered slightly alkaline
there appears a yellowish or dirty grey growth with
sharply-defined margins — a growth quite different
from that above described.'

Fraerikel and Simmonds1 state that this microbe
is the cause of typhoid fever, for they have pro-
duced the disease in monkeys, mice, and rabbits, by
inoculation, from a pure cultivation of the microbe.
Many other microbes (especially micrococci2)
'appear in the intestines when the disease is ap-
proaching its end, but the bacillus in question is the
only one found in the blood and internal organs [as
well as in the roseolous eruption], so that it is really
characteristic of the disease.'

According to Janowski,3 the action of light is
detrimental to the growth of B. typhosus; and he
has also proved that a temperature of 55°C., con-
tinued for ten minutes, destroyed the microbe.
Although destroyed at 55° C., B. typhosus has been
found alive in ice which had remained continuously
frozen for a period of 103 days;4 and Dornil has
discovered that ice is often a medium for transmit-
ting infectious diseases — especially typhoid fever.
But if ice is a means of transmitting typhoid fever,
potable water is a much more dangerous source of
infection. ' The remarkable instance which occurred
at the Caterham Waterworks (1879), where by the

1 Die Aetioloyische Bedeutung des Typhus-bacillus, 1886.

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

3 Centralblattfur BaJcteriologie, Bel. 8 (1890).

4 F. Davis's Handbook on Potable Water (1891).

INFECTIOUS DISEASES AND MICROBES, ETC. 223

merest accident of one workman suffering from
typhoid fever, who went down into the well and
worked there a few hours, and defiled the well, thus
contaminating hundreds of millions of gallons of
water which were pumped out and distributed to
the townspeople round about, four hundred cases of
typhoid fever followed the next week, and seventy
or eighty deaths occurred in consequence ' (Hogg).
Certainly this instance proves that water is a source
of infection ; but potable water is more frequently
contaminated by the excreta of patients suffering
from typhoid fever ; and when such is the case, an
epidemic of typhoid fever is the result of drinking
such water. In 1874, an epidemic of this disease
broke out at Over-Darwen, when 2035 persons were
attacked, which terminated in 104 deaths. The
outbreak was traced to the water supply. In 1884,
a similar epidemic broke out at Zurich j1 the origin
of which was traced to the water of the river
Limmat having been polluted with sewage contain-
ing typhoid-fever dejecta.

Epidemics of typhoid fever have also occurred at
Florence,2 Vienna, Home, Naples, etc., which have
been traced to potable waters having been contami-
nated with the evacuations of typhoid- fever patients.3

1 Revue d* Hygiene, 1885.

8 Tommasi-Crudeli in Istituto di Anat. Patologico (Turin),
1882, p. 154.

3 See also Thome's Reports to Medical Officer of Local Govern-
ment Board, 1880, et seq. ; Cassedebat in Comptes Rendus de
VAcademie des Sciences, vol. ex., and Annales de VImtitut
Pasteur, 1890 ; Giglioli's Fermenti e Microbi, pp. 268-282 ; Dr.
E. Frankland's Experimental Researches in Pure, Applied, and

224 A MANUAL OF BACTERIOLOGY

As the stools or dejecta of typhoid-fever patients
contain the typhoid bacilli, they are highly infec-
tious ; consequently they should always be disin-
fected before being thrown away. This would
greatly interfere with the spread of the disease.
Several authors have recommended carbolic acid or
mercuric chloride for disinfecting the stools ; but
iron sulphate, according to Jalan de la Croix, is far
more powerful than carbolic acid, and is only
slightly inferior to mercuric chloride : besides, iron
sulphate is a cheap disinfectant, non-poisonous and
inodorous, and therefore may safely be recom-
mended for the purpose of disinfecting the stools of
patients suffering from typhoid fever and other
infectious diseases. The author1 has proved the
high value of iron sulphate as a germicidal and
fungicidal agent ; and this compound readily de-
stroys Bacillus typliosus. It may be stated that
Dr. Proust 2 has used, for a number of years, iron
sulphate to disinfect the stools in cases of typhoid
fever.

Bacillus typhosus forms a ptomaine, which has
been extracted from pure cultures of the microbe,
ill glycerine-bouillon (3: 100), by Brieger.3 This

Physical Chemistry, p. 605 ; and S. T. Griffiths in the Tarn-
worth Herald, August 15 and 22, 1891.

1 Proceedings of fioyal Society of Edinburgh, vol. xv. ; Journal
of Chemical Society, 1883-87 ; Chemical News, vols. xlvii.-lvi. ;
Bulletin de la Societd Chimique de Paris, 1889, p. 667 ; The
Diseases of Crops (G. Bell & Sons).

s Traitd tf Hygiene.

3 Untersuchungen ilber Ptomaine, 1886, p. 85 ; and Virchow's
Archiv, 1889, p. 488. See also Gautier's Chimie Bioloyitjiie
(1892), p. 269.

INFECTIOUS DISEASES AND MICROBES, ETC. 225

base, which has been called typhotoxin (C7H17N02),
dilates the pupil, produces diarrhoea, and rapidly
kills animals. Luff l has also extracted a ptomaine
from the urine of typhoid fever patients ; but no
formula has been given to this base (i.e. it has not
been submitted to quantitative analysis).

Dr. Lauder Brunton says, in regard to typhoid
fever, that 'the symptoms do not point so much
to the c formation of a poison affecting the body
generally, as to the local action of the microbes
upon the intestines, although in some epidemics of
typhoid fever the intestinal symptoms are but
slightly marked, while bronchial irritation is due
to the action of a microbe or to a ptomaine pro-
duced by it on the bronchial mucous membrane/

CHOLERA.

Since the great epidemic of 1832, cholera lias
had a peculiar fascination for those interested in
the subject; for the disease has always been
shrouded in mystery until recent times. ' Before
the three last epidemics (1865, 1873, 1884) cholera
usually came to Europe by what may be called the
Continental routes — the caravan routes through
Persia, Asia Minor, and Russia ; but in the three
last it came by the Mediterranean or maritime
route, first by land through Egypt, brought there
by Mecca pilgrims, and thence to the seaports of
France, Italy, and Spain, whence it gradually made
its way northward and inland, spreading over the

1 British Medici Jwmal, 1889, p. 193.
P

226 A MANUAL OF BACTERIOLOGY

whole of Europe.' The native habitat or the
endemic area of this terrible disease is in India —
especially in the delta of the Ganges. ' It can be
readily understood, after the fearful ravages which
it made in places in which it was not actually
endemic, and after it had decimated the population
in certain parts of India, in Egypt, in the low-lying
portions of Persia, and Asia Minor, and in Europe,
that many observers should be anxious to find out
the ultimate cause of the disease ; and as early as
1848 Virchow, and in 1849 Pouchet, Brittan, and
Swaine found numbers of vibriones in the dis-
charges of choleraic patients, without, however,
being able to assign to them or prove for them any
specific rdle in the causation of the disease.' x Since
1848, many scientists have been at work trying to
establish a specific cause of cholera ; but it was not
until 1884 that Dr. R Koch2 discovered the comma
bacillus in choleraic dejecta, etc. Although many
distinguished pathologists have not accepted Koch's
evidence of the bacillary nature of Asiatic cholera,
there can be no doubt, after the important and
extensive researches of Drs. Macleod and Milles,3
that the comma bacillus of Koch is the cause
(directly or indirectly) of Asiatic cholera.

The comma bacillus or Spirillum cholerce Asiaticce
measures from 1'5 to 2-5 //, long and O6 p broad
(Fig. 33, 3). It occurs singly, in pairs often S~
shaped, in filaments which are screw-shaped, and

1 Woodhead's Bacteria and their Products, p. 151 (W. Scott).

2 Deutsch. Med. Woch., 1884; Berlin Klin. Woch., 1885.

s Proceedings of Royal Society of Edinburgh, vol. xvi. p. 18.

INFECTIOUS DISEASES AND MICROBES, ETC. 227

in zooglcea, and it is motile and aerobic. Numbers
of this microbe are found in the ' rice-water ' stools
formed by the desquamation of the mucous mem-
brane of the intestines. They also occur in the in-
testinal follicles, and in the sub-epithelial spaces,
and probably in the kidneys and urine.

There are several other comma-shaped bacilli, but
these differ in many respects from the microbe
which Koch has so frequently found in choleraic
dejecta. The following is the list of the other
comma-shaped bacilli, with the names of their dis-
coverers : —

(a) Finkler and Prior's bacillus (Spirillum Fink-
leri), found in cholera nostras. It is thicker than
Koch's bacillus ; and the colonies on gelatine plates
are much larger than those of the comma bacillus
of the same age. (6) Lewis's Spirillum sputigenum
was found in the saliva ; it is thicker than Koch's
bacillus, and is quite distinct from the latter
microbe, (c) Miller's bacillus was found in some
cases of caries of the teeth ; it is similar to Tinkler's
bacillus. (d) Kuisl's bacillus, found in human
faeces, is also similar to Tinkler's bacillus, (e)
Spirillum tyrogenum (see Fig. 24) of Deneke is
smaller than Koch's bacillus. It occurs in old
cheeses, and, unlike the comma bacillus, it will not
grow on steamed potatoes. (/) Klein's bacillus
was found in some cases of diarrhoea, especially in
monkeys. It grows differently in gelatine, giving
rise to an offensive smell, (g) Ermengem and
others have found comma-shaped bacilli in the
intestines of guinea-pigs, pigs, rabbits, horses, etc.,

228 A MANUAL OF BACTERIOLOGY

but, unlike Koch's bacillus, they will not grow in
10 per cent, gelatine, (h) Lingard found two kinds
of comma-shaped bacilli in a case of noma, the
smaller of which is said to have been similar to the
choleraic one. (i) Gamaleia's bacillus was found
in a fatal fowl disease which was prevalent at
Odessa, (j) Weibel found various forms in mucus,
but their mode of growth is distinct.

Koch's Spirillum cholerce Asiatics is always
present in Asiatic or malignant cholera, and it has
not been found apart from this disease, and dis-
appears from the body with the disease. Its habitat
is the intestinal canal, and the detection of this
bacillus enables the physician more readily to
diagnose the earliest cases in an epidemic of cholera.
Ermengem,1 Watson Cheyne,2 Koch,3 Nicati and
Kietsch,4 Macleod and Milles,5 and others, have
produced the disease in dogs and guinea-pigs by
inoculation with pure sub-cultures of Koch's comma
bacillus. The last two investigators have arrived
at the following conclusions concerning cholera and
its microbe : —

(a) The comma bacillus (Koch's) is always
present and associated with certain changes in the
small intestine in cases of Asiatic cholera. (&)
There is no evidence to show that it is a normal
inhabitant of the human alimentary canal, and

1 Recherches sur le Microbe du GhoUra Asiatique (1885).

2 British Medical Journal, 1885.

3 ' Etiology of Cholera ' in Laycock's Microparasites and
Disease.

4 Revue d' Hygiene, 1885 ; Archives de Physiologic, 1885.

5 Loc. cit., pp. 18-35.

INFECTIOUS DISEASES AND MICROBES, ETC. 229

therefore no proof of the assertion that it is a result
of the disease, (c) The means used to introduce
the comma bacillus into, and those used to lessen
the peristalsis of, the small intestine of the guinea-
pig, cannot be regarded as causing appearances like
those of Asiatic cholera, or as causing the death of
the animal, far less a mortality of over 60 per cent.
(d) Pure cultivations of the microbe are pathogenic
to the guinea-pig, (e) The contents of the ileum
from those animals killed by injections of pure
cultivations of the bacilli act in the same manner
as pure cultivations of that microbe. (/) The
microbe multiplies in the small intestine of the
animal, and there is associated therewith changes
similar to those in man in Asiatic cholera, (g) As
there are conditions which favour the passage alive
of the comma bacillus through the stomach of the
guinea-pig, and also conditions which favour its
multiplication in the small intestine of that animal;
so in man, as there cannot be a doubt that the
microbe finds conditions favourable to its multipli-
cation in his small intestine, it must have found
conditions favourable to its entrance alive through,
in all probability, the mouth and the stomach
(Macleod and Milles).

The comma bacillus grows in neutral bouillon,
gelatine, agar-agar, milk, and on steamed potatoes.
It grows best if the medium is slightly alkaline,
and at a temperature ranging from 16° to 40° C.
On gelatine plates the colonies (Fig. 44) are evident
in about twenty-four hours, and appear, under a
low power, as small, somewhat irregular pale masses.

230

A MANUAL OF BACTERIOLOGY

These gradually increase in size, and, where near
the surface of the gelatine, a small depression forms
over them, so that, on looking from the side at the
surface of such a cultivation, it presents numerous
little depressions instead of the original smooth
surface of the gelatine, each depression correspond-
ing to a colony of these bacilli. As the colony in-
creases in size it becomes less compact, and the

gelatine in the immedi-
ate vicinity becomes
fluid.1 At this stage
zooglcea are formed.
The colony goes on in-
creasing in size for a
few days, but ultimately
ceases to extend, or
extends only very slow-
ly. Tube-cultivations
are also characteristic.
In twenty-four hours,
at a temperature of 18°

C" gr0wth is evident

along the needle- track
as a whitish line,
broader at the upper part, and gradually tapering
to the lower. At the upper part of the gelatine
there is a slight depression, and during the next
twenty-four hours the growth becomes more marked,
the depression increasing in size so as to look like

1 Dr. Lander Brunton has shown that this liquefaction is due
to a [ferment (enzyme) secreted by the comma bacillus (Proc.
Roy. Soc., vol. xlvi. p. 542).

FIG. 44. COLONIES OF CHOLERA-BACILLI

ON GELATINE-PLATE.

(X80.)

INFECTIOUS DISEASES AND MICROBES, ETC. 231

an air-bubble at the top of the track. In the

following days the gelatine at the top becomes

liquid, and this liquidity extends gradually to the

bottom of the track, thus there is a funnel-shaped

appearance from the greater amount of the fluid at

the top than at the bottom. At the same time, the

mass of bacilli falls to the bottom of the fluid and

assumes a somewhat rosy colour,

so that there is a rose-coloured

convoluted string running down

the lower part of the track. The

fluid at the upper part, which in

about a week has extended to the

sides of the tube, becomes clear,

except a very thin layer at the

top, which remains opalescent,

the top itself being often covered

with a very fine scum. Scattered

over the solid gelatine forming

the sides of the funnel are seen

numerous small irregular •

refracting particles. These are FlG. 45. TUBE-CULTIVATION

the small zooglcea masses which OF CHOLERA-BACILLI.

. . ,, ^ . , , (After Watson Cheyne.)

have fallen to the sides and
bottom of the funnel-shaped cavity (Fig. 45), and
which Dr. Watson Cheyne considers the most typi-
cal appearance during the growth of the comma
bacillus in tube cultivations. On agar-agar the
comma bacillus grows fairly well, but it does not
liquefy this medium. On blood serum (at 37° C.)
this microbe grows most luxuriantly. It also grows
in milk, but gives rise to no noticeable alterations ;

232 A MANUAL OF BACTERIOLOGY

' it may, therefore, be readily understood how deadly
the cholera microbe may become if it once finds a
resting-place in milk.' 1

Brunton, Lewis, and Cunningham, Klebs and
Cantani, and others, have all obtained indications
of a poison or ptomaine in cholera dejecta. Pouchet,
Brieger, and Yilliers have extracted several
ptomaines from cholera dejecta, as well as from
pure cultivations of the comma bacillus (Brieger).

Dr. Lauder Brunton 2 says, c The symptoms occur-
ring in cholera are probably due to the action on
the tissues of a poison [or poisons] generated by
the microbe, and not of the microbe itself, just as
intoxication is due to the alcohol produced by the
yeast plant, and not to the action of the plant itself
on the nervous system and blood.' Besides the
ptomaines produced by the comma bacillus, this
microbe secretes a soluble enzyme.3

Cholera follows the course of rivers. Moisture
in the atmosphere and the soil is needed for its
distribution. Moist winds spread it, but the great
factor in the distribution of cholera, as already
stated, is human intercourse. Although human
intercourse is the chief factor in distributing this

1 Hence milk adulterated with water from districts in which
there are persons suffering from cholora may be the means of
causing an epidemic of the disease. The same may be said of
typhoid fever.

2 Disorders of Digestion (1888), p. 41 ; see also pp. 292 and
263 ; and Practitioner, 1884, et seq.

3 See Dr. Brunton's paper in Proc. Roy. Soc. , vol. xlvi. p. 542 ;
and Dr. G. E. C. Wood's paper in Proc. Roy. Soc. , Edinburgh,
vol. xvii. p. 29.

INFECTIOUS DISEASES AND MICROBES, ETC. 233

disease, potable water is one of the most convenient
vehicles for the distribution of the comma bacillus.
If the dejecta of one or more choleraic patients
contaminate a water supply, the water becomes a
medium for spreading the disease. Such are the
conclusions of Koch, Macnamara,1 and many other
observers. 'In India, in the regions in which
cholera is endemic, the wells, as a rule, are merely
surface tanks into which sewage and surface water
may be drained, and which are frequently on the
same level as, and connected with, the cesspools,
so that even the water supply contains a consider-
able quantity of organic matter in which organisms
of all kinds can flourish most luxuriantly; whilst
these same wells, being merely dug-out pits beneath
the slightly raised houses, are open for the recep-
tion of sewage and excreta of all kinds, especially
in times of illness, when neither patients nor nurses
have strength or time to see these are properly
removed.' The recent epidemics of cholera in
India, Spain, Japan,2 and other countries, have been
traced to the water supply ; 3 and it is stated that
the epidemic of 1884 killed 80,000 persons in Spain
alone.4 But it may be stated ' that with all the
improvements that have been made in the drainage
system and water supply of Lower Bengal, cholera

1 British Medical Journal, 1884, p. 502.

2 An epidemic of cholera or korera-byo (as the Japanese call
it) occurred in Japan in 1890, and there were 13,141 deaths out
of 21,116 cases (vide Sir Edwin Arnold's Seas and Lands
[1891], p. 474).

3 Lancet, 1885, et seq.

4 Giglioli's Fermenti e Microbi, p. 300 *eq.

234 A MANUAL OF BACTERIOLOGY

has only diminished about 60 per cent., so that
there still remain certain factors that favour the
spread of cholera, and every now and again such a
spread or outbreak may take place with extreme
rapidity, and may involve a very wide area.
Cleanliness, however, both general and personal,
may be said to be the most important factor in the
prophylaxis of cholera/

It should be borne in mind that in cases of
cholera, isolation and disinfection are absolutely
necessary to prevent the disease spreading.1 For
further information on the subject of cholera and
its microbe, the reader is referred to the under-
mentioned works.2

GLANDERS.

This contagious infective disease is caused by the
Bacillus mallei (Fig. 33, 12), which has been found
in the lungs, liver, spleen, and nasal membranes of
horses and sheep dead or dying from glanders.

The same microbe has been found in human
glanders or farcy ; and the death of Dr. Hoffmann,
of Vienna, in 1889 is a standing proof of the patho-
genic nature of this microbe, and its being the cause
of the disease known as glanders.3 In man, this

1 Cameron's The Cholera Microbe and How to meet it
(Bailliere & Co.).

2 Klein's Bacteria in Asiatic Cholera ; Brunton's Disorders
of Digestion (1888), p. 262; Thome's 'Sea-Borne Cholera' in
British Medical Journal, 1887 ; Straus, Roux, Nocard, and
Thuillier in Comptes Rendus de la Sotiete de Biologie, 1883 ; and
Bellews's History of Cholera in India (1885).

Griffiths' Researches on Micro-Organisms, p. 15.

INFECTIOUS DISEASES AND MICROBES, ETC. 235

microbe has been found in the blood and pus of the
ulcers. According to Loftier, glanders is essentially
a disease of hot countries, ' where the comparatively
high temperature appears to be extremely favour-
able to the development of the bacillus outside the
body, especially in such materials as fodder, manure,
and stable refuse generally. We have interesting
evidence of this in statistics collected by Krabbe,
who gives the following proportion of horses affected
with glanders per annum per 100,000 horses in the
following countries : — Norway, 6 ; Denmark, 8'5 ;
Great Britain, 14; Sweden, 57; Wurtemburg, 77;
Russia, 78; Servia, 95; Belgium, 138; the French
Army, 1130; and the Algerian Army, 1548.'

B. mallei measures from 2*5 to 5 p long, and
about one-fifth of its own length broad. It grows
on blood serum (at 38° C.), sterilised potatoes (at
37° C.), in neutral solutions of extract of beef (at
37° C.), and in various vegetable infusions. Horses,
asses, cats, rabbits, mice, and guinea-pigs, inoculated
with a few drops of a pure cultivation of this
microbe, have died with the characteristic lesions of
glanders (glanderous ulcers and modules in the
internal organs, and on the nasal septum).

Stables, in which glanders has occurred, should
be thoroughly washed out with a 2-per cent, solu-
tion of carbolic acid or some other equally powerful
disinfectant.

DIPHTHERIA.

Diphtheria is an extremely infectious disease
which attacks man and certain animals.

236 A MANUAL OF BACTERIOLOGY

Two microbes were originally isolated by Klebs
and Loffler from human diphtheritic membranes;
but Dr. Klein1 has shown that the Klebs-Loffler
bacillus No. 1 is not constant in diphtheritic mem-
branes, does not act pathogenically on animals ; and
does not grow on solid gelatine at 20° C. In fact,
this microbe has been termed the pseudo-diphtheria
bacillus. The other species, Klebs-Loffler bacillus
No. 2, is always present in diphtheritic membranes —
in fact, it is present even in the deeper layers of

FIG. 46. BACILLUS DIPHTHERIA (Klein).

A, The Bacillus x 1000. B, Section through the mucous membrane of
pharynx of a child dead of diphtheria. C, Colonies from a plate-cultiva-
tion of B. diphtheria.

the membranes in great masses, and almost in pure
culture. This microbe acts virulently on animals,
and grows on gelatine at 19-20° C. Klein considers
this bacillus to be the true microbe of diphtheria
(Fig. 46 A and B).

On the slanting surface of gelatine in tubes,

1 'Etiology of Diphtheria' in Reports to Local Government
Board, 1889-90, p. 143; Proc. Roy. Soc., 1890; Centralblatt fur
Bakteriologie, Bd. vii. (1890).

INFECTIOUS DISEASES AND MICROBES, ETC. 237

Bacillus diphtherias (No. 2) gives rise to greyish
dots after 36-48 hours' incubation at 20° C. After
three or four days, these appear as white round
convex droplets, which ultimately aggregate together
forming yellowish -brown colonies. Colonies are
also formed when the microbe is grown as a plate-
cultivation (Fig. 46 C). In alkaline bouillon, B.
diphtherice gives rise to a turbidity in twenty-four
hours after inoculation; and afterwards a greyish-
white precipitate is produced at the bottom of the
tube.

In milk kept at 18-20° C., this microbe grows
very rapidly. The milk always remains fluid ; but
in two or three days after inoculation, flakes of
casein separate.

B. diphtherice (from 3 to 6 /z, long) does not pro-
duce spores ; but it gives rise to a soluble enzyme
and one or more ptomaines. Drs. Eoux and Yersin l
isolated an enzyme, from a pure cultivation of the
microbe in question, which produces all the symp-
toms of diphtheria. This is a true enzyme, for
boiling water destroys its action.

The author2 extracted a ptomaine (C14H17N206)
from urine in cases of diphtheria; and the same
ptomaine was also obtained from pure cultures of
B. diphtherice on peptonised gelatine. This ptomaine
is not present in normal urine. Brieger and
Fraenkel3 have also isolated a toxalbumin from

1 Annales de VInstitut Pasteur, 1888.

2 Griffiths in Comptes Rendus, vol. cxiii. p. 656 ; Nature,
vol. xlv. p. 72.

3 Berlin Klin. Woch., Bd. xxvii. pp. 241 and 1133.

238 A MANUAL OF BACTERIOLOGY

pure cultivations of the microbe. This substance is
said to have produced toxic effects when injected
into animals. ' These observers, however, did not
separate from the albumoses that were formed any
enzymes that might be present, consequently they
were working with a mixture of substances. The
products that they obtained gave most of the re-
actions of albumoses ; they were certainly toxic,
but they probably contained both enzymes and
albumoses' (Woodhead).

B. diphtherice (No. 2), which is identical with
those of Koux and Yersin, Zarniko, Escherich, and
Loffler, acts very virulently on guinea-pigs on sub-
cutaneous inoculation : at the seat of the injection
a tumour is produced, which in its pathology and
in microscopic sections, completely resembles the
diphtheritic tissue of the human subject. In
human diphtheria B. diphtherias is present only in
the diphtheritic membrane, but neither in the
blood nor in the diseased viscera ; the same holds
good of the experimental guinea-pigs. In sub-
cutaneous inoculation with artificial culture, though
it causes in these animals acute disease and death
— the lungs, intestine, and kidney are greatly con-
gested— the diphtheria bacillus remains limited to
the seat of inoculation (Klein).

Klein has shown that this microbe also attacks
the cat and cows, as well as man and the guinea-
pig. But, unlike human diphtheria, the disease
locates itself in the lungs of the cat (Fig. 47), i.e.
the lung is the organ in which the diphtheritic
process in the cat has its seat. The domestic cat

INFECTIOUS DISEASES AND MICROBES, ETC. 239

is, therefore, a means of introducing diphtheria into
a household.

Klein has also shown that a definite disease can
be produced in the cow by the B. diphtherice, con-
sisting of a diphtheritic tumour at the seat of
inoculation with copious multiplication of the
bacilli, a severe pneumonia, and necrotic change in
the liver; the contagious nature of the vesicular
eruption on the udder and
excretion of the bacilli
in the milk prove that
in the cow the bacilli
are absorbed as such into
the system. The mor-
phological characters
and the pathogenic ac-
tion of these bacilli from
milk were exactly the

Same as those from FjG 47 BACILLUS DIPHTHERIA

human diphtheria. Ac- (Klein.)

COrding tO the Same ^presents a cover-glass preparation
of fresh lung exudation from a cat that

authority, 1 litre (T76 died of naturally acquired diphtheria in
•m'nftA nf -millr Prmrmnorl a house ^herein diphtheria afterwards
Q attacked the children of the household.

between 30,000 and (x 1000.)

40,000 bacilli; therefore,

there is little doubt that cows suffering from diph-
theria are capable of transmitting the disease to
human beings by means of the milk ; and human
beings suffering from the same disease may also
infect a milk-supply, and so spread the disease
among the consumers of such milk.

Dr. G. Turner1 states that fowls, turkeys, and

1 Reports to Local Governme.nt Board, 1886-7, p. 3.

240 A MANUAL OF BACTERIOLOGY

pheasants also suffer from diphtheria, for he found
the characteristic diphtheritic membranes in these
birds; and he has also seen fowls and pigeons
which had also been inoculated with diphtheritic
membrane from a child's throat attacked with a
disease which in all respects resembled what Turner
regards as natural fowl-diphtheria. Similar accounts
have been received from foreign bacteriologists,1
so that the identity and transmissibility of the dis-
ease from fowls to men seems very probable.

It may be stated, en passant, that Power 2 traced
the outbreaks of diphtheria in 1886 at York and
Camberley to the infectiousness of the milk-
supplies ; and there is no doubt that milk is a
medium in which other diseases besides diphtheria
may be spread over a wide area.

For some years, there has been a serious increase
of diphtheria in this country, which Dr. Thome3
attributes to the increasing aggregation of children
in elementary schools ; and Dr. Seaton,4 to the pre-
sent systems of water-supply and sewerage.

B. diphtherias is possessed of great tenacity of
life. If it is dried and kept at 33° C. it is still
alive after three months ; but at 45° C., this microbe
is killed in four days. ' If a fragment of the false
membrane containing bacilli be removed, wrapped
in sterilised paper, or linen, and be carefully pro-
tected from the action of light, cultivations may be

1 British Medical Journal, 1884; Journal d* Hygiene, 1884.

2 Report to Local Government Board, 1886, p. 311.

3 Diphtheria : its Natural History and Prevention (1891).

4 Report of International Congress of Hygiene, 1891.

INFECTIOUS DISEASES AND MICROBES, ETC. 241

made from it at any time during a period of five
months. If, however, instead of keeping it dry and
in the dark, fragments of these membranes are
exposed to the light and moistened and desiccated
alternately, the virus is destroyed much more
rapidly. From all this, and from the fact that the
bacillus is destroyed by moist heat at 58° C., it is
evident that by far the best method of disinfecting
clothes, the floor, the walls, and furniture, is by the
use of a liberal supply of boiling water ; for although
a temperature of 98° C. (dry), continued for an hour,
is necessary to destroy the vitality of the bacillus,
moist heat at a very much lower degree (acting only
for a minute or two, according to the temperature),
is sufficient to disinfect everything on which it is
allowed to act ' (Woodhead).

Drs. Behring and Kitasato 1 have recently dis-
covered a method of producing immunity against
diphtheria. As this is similar to Kitasato's method
of treating tetanus, which has been already described
(see p. 214), no further remarks are needed.

'Antiseptic throat washes,2 not merely gargles,

1 Deutsche Medicinische Wochenschrift, 1890, p. 1113 ; and
Zeitschrift fur Hygiene, 1890-1.

2 The following is an excellent antiseptic throat wash : —

IJ. Potass, chlor. pulv. , 3 ij.

Acid hydroch. fort, 3 j.

Let stand mixed for 10 minutes,

then add water gradually shaking

each time to . f . 5 vi. I

Syrup, . . f.5j. {

To be used with a spray apparatus or syringe. This fluid not
only loosens the diphtheritic membrane, but also destroys the
bacilli.

242 A MANUAL OF BACTERIOLOGY

plenty of fresh air, and good nourishing food, are
what are required in the treatment of diphtheria.
Kill the germs as far as possible by means of the
antiseptics [germicides], and strengthen the tissue
cells by plenty of oxygen, and by promoting the
excretion of effete products, by food and exercise,
so that the cells shall be able to form their protec-
tive products, and shall also be able to play their
part as phagocytes when called upon to do so.' It
should be borne in mind that in diphtheria the
bacilli are localised in the throat ; but the poisonous
products (ptomaines and enzyme), which the bacilli
form, pass into the system. If the bacilli are
destroyed by germicides,1 these poisonous products
cannot increase in the system ; and if they have not
already accumulated in too large a quantity, they
are readily excreted. ' Another important point is
that the disappearance of the bacilli from the mouth
is not simultaneous with the removal of the false
membrane, and Roux and Yersin have found that
the specific bacillus may persist in the mouth for
several days (in one case fourteen days) after all
traces of the membrane have disappeared, and they
give the good practical advice that diphtheritic
patients who are becoming convalescent should not
be allowed to associate with their school-fellows,
play-mates, or families, for at least a fortnight after
the membrane has disappeared ; and that it is quite
as important to wash out the throat freely three or

1 Dr. Wagner (Jour, fur PraJct. Chemie, vol. xi. ) has success-
fully used a solution of salicylic acid in the treatment of
diphtheria.

INFECTIOUS DISEASES AND MICROBES, ETC. 243

four times a day with disinfecting lotions as that
the clothes and bed linen should be thoroughly
disinfected/

TUBERCULOSIS.

Tuberculosis, in its varied protean guises, is one
of the most widespread and deadly diseases in these
northern latitudes. It has been stated that at any
given time there are 200,000 persons in this country
suffering from phthisis pulmonalis — the commonest
form of the disease — and in each year nearly 70,000
persons die from it. The following tables show the
death-rates per million from tuberculosis at different
ages :—

(a) From Phthisis.

Age 10.

Age 15.

Age 20.

Age 25.

Age 35.

Age 65.

Age 75.

Males, . . .

628

2093

3687

3941

4089

2152

752

Females, . .

1077

3019

3809

4175

3842

1364

546

(b) From other Tubercular Diseases.

Age 5.

Age 10.

Age 35.

Age 75.

Males, ....

5008

641

103

Females,

3942

515

Tuberculosis is known by various names, according
to the parts of the body the disease may happen to
attack, or according to the kind of lesions it pro-

244 A MANUAL OF BACTERIOLOGY

duces, or, finally, according to its general effect on
the body. Thus phthisis or consumption, lupus,
caseous pneumonia, cheesy inflammation of the lungs,
consumption of the intestines, tabes mesenterica,
tubercular pleurisy, ceseous broncho-pneumonia,
scrofula, tubercular meningitis, etc., are all forms
of the same disease, which is produced by a
microbe — Bacillus tuberculosis — discovered by Pro-
fessor R Koch1 in 1882. This bacillus lives in the
blood and tissues, and gives rise to tubercles, which
are small abnormal nodules of newly-formed tissue
studding the diseased organ or organs. Each
tubercle is made up of nucleated cells and tubercle
bacilli, the latter being located chiefly in the giant
cells. As the tubercles are continually being
thrown off from the diseased person or animal,
tuberculosis is an infectious disease. B. tuberculosis
attacks other animals besides man ; among these
may be mentioned cows, fowls, rodents, pigs, etc.
Although tuberculosis is essentially the result of
the action of Koch's bacillus, there are certain
factors which render man and animals liable to
contract the disease, and thereby receive the poison.
These factors are deficiency of oxygen by bad venti-
lation, foods (from tuberculous animals), certain
diseases,2 starvation, inheritance, predisposition, etc.
The last-named factor may be acquired through the
system being of a lower standard than usual, or
may be inherited.

1 Berliner Klin. Wochenschrift, Bd. xv. p. 221.

2 Among the diseases which render man liable to contract
tuberculosis are syphilis, diabetes, measles, whooping-cough, etc.

INFECTIOUS DISEASES AND MICROBES, ETC. 245

Tuberculosis, or that form of the disease known
as phthisis (consumption), runs through certain
families. There are two theories which account for
the inheritance of phthisis — (a) that the tissues of
children born of phthisical parents are especially
favourable to nourish the tubercle bacilli; i.e. the
tissues form a fertile soil for the subsequent growth
of the microbes; (b) that the tubercle bacilli are
actually contained in the ovum or among the sper-
matozoa, and so become a constituent part of the
embryo and foetus which develops within the
uterus. Baumgarten records the fact that he has
observed the tubercle bacilli in the ovum of the
rabbit, and many observers have frequently seen
the bacilli mingled with active spermatozoa. Pro-
fessor Johne, of Dresden, discovered numerous
tubercles in the lungs of a foetal calf of seven
months intra-uterine growth. This proves that if
the ovum had not been inoculated, it received the
virus (i.e. the tubercle bacilli) through the placenta,
which amounts practically to the same thing.
Similar intra-uterine inoculation has been shown
to be more than probable in the human being ; and
Professor Burdon Sanderson1 believes that many
cases of phthisis are congenital, i.e. dependent on
causes which have operated before birth.

Besides being hereditary, tuberculosis is also
infectious, i.e. the disease is capable of being trans-
mitted by direct or indirect infection from one host
to another.

There are four modes in which the tubercle

1 Report of International Congress of Hygiene, 1891.

246 A MANUAL OF BACTERIOLOGY

bacilli enter the body, viz., by pulmonary inhala-
tion (atmospheric infection), swallowing (enteric
infection), direct inoculation, and heredity, (a)
Inhalaton is the commonest mode of infection.
Koch and numerous other observers have proved
that animals, after a few inhalations of phthisical
sputum, disseminated in a spray, readily become
infected with tuberculosis. Eansome l has isolated
the tubercle bacilli from the breath of patients
suffering from advanced phthisis ; and the author 2
has confirmed Kansome's investigations ; therefore
it will be seen that tuberculosis may pass from
husband to wife, and vice versd ; and it may also
affect members of the same family, not because of
any hereditary taint, but through the simple fact of
close companionship.3 The sputa or expectorations
of phthisical patients are highly infectious, even
after being desiccated for several months. Bacillus
tuberculosis is often to be found in places lived in
by consumptives ; and Prausnitz has lately collected
the dust in various compartments of trains which
often convey patients from Berlin to Meran, and
inoculated a number of guinea-pigs with it. Two,
out of five compartments so examined, were found
to contain the bacillus; the dust of one rendered
three out of four guinea-pigs tuberculous, while
that of the other compartment infected two of these

1 Proc. Roy. Soc., 1882.

2 Proc. Roy. Soc. Edinburgh, vol. xvii. p. 268.

3 See Weber's book, The Gommunicability of Consumption
from Husband to Wife ; and Heron's Evidences of the Communi-
cability of Consumption.

INFECTIOUS DISEASES AND MICROBES, ETC. 247

animals. The animals were killed after several
months, and their organs had developed tubercles
containing the characteristic bacilli. (b) Swallowing,
or enteric infection, is a means of introducing the
tubercular virus into the animal economy. Babbits,
guinea-pigs, fowls, pigs, etc., become tubercular when
fed upon tubercular tissues, sputum, saliva, milk,
pure cultivations of the tubercle bacilli, etc. Klebs,
Arloing, Chauveau, Villemin, Gerlach, Baumgarten,
and others have shown, by direct experiment, that
the milk, flesh, etc., ' from cattle affected with tuber-
culosis would, when introduced alone or along with
other food into the alimentary canal of rabbits,
etc., give rise to tuberculosis in the pharynx, in the
lymphatic glands of the neck, the stomach, intestine,
omentum, liver, and spleen, and then, later, in other
organs.' Many authorities state that the flesh of
tuberculous animals (cattle, fowls, pigs, etc.) give
rise to tuberculosis in human beings. On the other
hand, there are authorities which state that there
is not much danger of human beings contracting
tuberculosis from eating meat from tuberculous
cattle; but it is a unanimous opinion among all
competent authorities that the milk of tuberculous
cows is a source of great danger to human beings —
often giving rise to tuberculosis, especially in chil-
dren. It should be borne in mind that 'boiling always
destroys the virulence, even when the milk contains
bacilli, which is the case when the udder of the
affected cow is itself tuberculous ;' and the risk of in-
fection is greatly diminished, if not abolished, when
meat from tuberculous cattle is thoroughly cooked.

248 A MANUAL OF BACTERIOLOGY

The experiments of Galtier, Bang, and others have
proved that the various products derived from milk
— butter, cheese, and butter-milk — may all contain
the tubercle bacilli, and that these retain their
vitality in such products for a period of from four-
teen to thirty days. The majority of these bacilli
may be separated from milk if the cream is first
removed by means of a centrifugal machine, but if
the milk is very rich in bacilli a few usually remain
in the milk, and even in the cream. In order to do
away with this danger, it is necessary to expose the
milk or the cream before churning to a temperature
high enough to kill the tubercle bacilli (85° C. for
about five minutes), (c) Direct inoculation is the
third mode of infection. When tubercular matter
or pure cultivations of the tubercle bacilli are intro-
duced beneath the skin of susceptible animals,
such as rabbits, guinea-pigs, cats, etc., they always
produce, in four or more weeks, the typical
tubercular lesions — swollen lymphatic glands,
tubercles in the spleen, liver, and lungs, and en-
largement and caseation of the bronchial glands.
Besides, there are instances recorded in which sores
on the udder of cows have infected with tuberculosis
the hands of the persons milking them ; and it is
not improbable that the common house-fly may
disseminate the virus of phthisis by inoculating
open sores on the hands and face (Spillman and
Haushalter1).

Bacillus tuberculosis measures from 2 to 8 yu, long
and about 0*2 //, broad. It occurs in phthisical
1 Comptes Rendus, vol. cv.

INFECTIOUS DISEASES AND MICROBES, ETC. 249

sputum (Fig. 48), in the cells of tubercles, and in
the blood,1 tissues, urine,2 faeces, saliva,3 and sweat 4
of tuberculous patients. Watson Cheyne5 and
other observers believe that the microbe is a spore-
producing bacillus ; but this assertion is doubted by
Lankester6 and others. B. tuberculosis has been
cultivated artificially, and it has been proved that
the strength of its virulence is not lessened by suc-
cessive cultivations. When inoculated into various
animals it always produces tuberculosis. The pre-
sence of this microbe in the sputa of patients sup-
posed to be suffering

from phthisis is a ft-^-*

certain diagnosis ;
and it may be men-
tioned that the mi-
crobes are most
numerous in the
small caseous dots
contained in the
sputa. These dots
should be searched
for, then crushed

between two cover glasses, dried, stained, and
examined with high powers.

B. tuberculosis grows on solid blood serum at 37° C.
(i.e. the temperature of the body), and in eight

1 Weichselbaum in Wiener Med. Blatter, 1884.

2 Bates in Centralblatt fur d. Med. Wissemch., 1883, p. 145.

3 Griffiths in Proc. Roy. Soc., Edinburgh, vol. xv. p. 44.

4 Griffiths' Researches on Micro-Organisms, p. 268.

5 The Practitioner, 1883, p. 248.
« Nature, 1884.

FIG. 48. BACILLUS TUBERCULOSIS.
A, From human sputum, a, Bacilli,
b, Nuclei, x 1500. B, Bacilli, x 435.

250 A MANUAL OF BACTERIOLOGY

or ten days after inoculation gives rise to whitish
or yellowish drops or ' scales.' There is no lique-
faction of the medium if the culture is perfectly
pure. The bacillus also grows on the surface of
bouillon (containing glycerine), forming a delicate
thin film. Pawlowsky1 has grown the tubercle
bacillus on sterilised potatoes ; but to succeed with
this medium a considerable quantity of moisture
must be kept in contact with the growing microbe.
Nocard and Koux 2 have shown that most luxuriant
growths of the tubercle bacillus are readily obtained
when the microbe is grown on agar-agar and blood
serum to which 6-8 per cent, of glycerine has been
added ; but after many successive cultivations on
these glycerine media, the virulence of the microbe
becomes distinctly diminished.

B. tuberculosis forms cellulose in the organs and
blood of tuberculous persons ;3 and it has been
recently stated that the microbe, when growing in
glycerine bouillon, produces an albumose.4 The
tubercle bacillus has great tenacity of life, for the
author 6 has shown that it is capable of being dried
up for three or four months at a temperature of
32° C. without losing its vitality : and Cornil was
able to demonstrate that at the ordinary temperature
of the room the tubercle bacillus, kept in water from
the Seine, still retained its vitality after seventy

1 Annales de I'Institut Pasteur, 1888-9.

2 Annales de I'Institut Pasteur, 1887, p. 19.

3 See the author's Researches on Micro-Organisms, p. 155.

4 Crookshank and Herroun in British Medical Journal, 1891,
p. 401.

5 Proc. Roy. Soc. Edinburgh, vol. xv. p. 42.

Y ^r W* AttJB

UFI7BESITT1

INFECTIOUS DISEASES AtfD MICROBES, ETC. 251

days' immersion in that medium. As already stated
the best temperature for the growth of this bacillus
is 37° C. ; at 40° C. its activity is diminished ; and
at a temperature ranging from 50° to 60° C. it is
killed. Boiling or strongly heating cultivations of
all microbes destroys them, or, in other words, the
media so treated become sterilised. Goethe knew
nothing about microbes, yet, with the genius of a
great poet, he makes Mephisto say : —

* Der Luft, dem Wasser, wie der Erden
Entwinden tausend Keime sich,
Im Trocknen, Feuchten, Warmen, Kalten !
Hatt' ich mir nicht die Flamme vorbehalten,
Ich hatte nichts Aparts f iir mich. '

In addition to the action of heat, sulphuretted
hydrogen, ozone, a solution of salicylic acid, and the
electric current (E.M.F. of 2'16 volts), all destroy
the vitality of Bacillus tuberculosis.1

Although it is out of place to discuss the methods
used in the treatment of infectious diseases in a
manual devoted to general bacteriology, we give a
very brief account of what is known as 'Koch's
cure' for tuberculosis. Ever since Dr. Koch dis-
covered the tubercle bacillus (in 1882) he has been
endeavouring to obtain an inoculating fluid which
would kill the bacilli, and bring about a sufficiently
strong and healthy reaction to expel them from the
body without, at the same time, destroying healthy
organs. Such a fluid Koch believes he has dis-
covered in his tuberculin,2 which is a glycerine

1 See the author's book, loc. cit., pp. 176, 182, 184, and 227.

2 Deutsche Medizinische Wochenschrift, Nov. 14, 1890, and
Jan. 15, 1891.

252

A MANUAL OF BACTERIOLOGY

extract from pure cultivations of destroyed tubercle
bacilli. This so-called lymph contains mineral
salts, colouring substances, unknown extractive
matter, besides the dead bacilli. According to
Koch, some of these substances can be removed
from the 'lymph' tolerably easily. The effective
substance is mainly insoluble in absolute alcohol,
and can be precipitated by it, not, indeed, in a pure

condition, but still com-
bined with the other
extractive matter, which
is also soluble in alcohol.
The colouring matter
may also be removed,
so that it is possible to
obtain from the extract
a colourless dry sub-
stance, which contains
the effective substance
in a much more con-
centrated form than
the original glycerine
solution. The effective

FIG. 49. INJECTING KOCH'S ' LYMPH.' Substance appears to be

a derivative from albu-
minous compounds, and is closely allied to them.
It is not a ptomaine ; but appears to be an enzyme ;
and tuberculin contains less than 1 per cent, of this
enzyme.1

The treatment consists in injecting, subcutane-

1 See also Hunter's paper in British Medical Journal, 1891
(ii), p. 169.

INFECTIOUS DISEASES AND MICROBES, ETC. 253

ously, small doses l of diluted (with water) tuber-
culin into the back of patients (Fig. 49) suffering
from certain forms of tuberculosis; and as the
treatment progresses the doses are slowly increased
'as long as there may be bacilli in the body.'
Koch's ' lymph ' does not kill the tubercle bacilli,
but destroys the tuberculous tissues, and thereby
starves the bacilli contained in such tissues. It
also sets up a localised reaction in the vicinity of
the bacilli, by means of which the cells are so
strengthened that they are able to prevent the
extension of the bacilli into the surrounding parts ;
in fact there is a battle between the cells and the
bacilli, and if the former are strengthened, it is
possible for them to destroy the latter ; and this is
what Koch's ' lymph ' is believed to do. •

As to the value of Koch's treatment, there is no
decided opinion among those best capable of judg-
ing ; for some authorities are against, while others
are in favour of, the ' lymph ' as a diagnostic and
curative agent. Professor K. Virchow 2 (the greatest
living pathologist) ' has found, in a number of cases
that have come under his observations,' — a compar-
ative small number when the enormous number
that have been injected is taken into consideration, —
' that the characteristic degeneration of the tissues of
the young tubercle is not always brought about,
that the localisation of the disease is not by any
means perfect, that there is a tendency of tubercu-
lous material that should be thrown off to continue

1 0-0005 to o-oi cc.

2 Berliner Klinische Wochenschrifl, Jan. 21, 1891, p. 49.

254 A MANUAL OF BACTERIOLOGY

the infection and even increase its rapidity of
spreading, especially in the lungs, and that in some
cases the bacilli, instead of being rendered inert,
appear to take on greater activity, and to be carried
in the various currents in the body, even to parts
situated at some distance from the original tubercu-
lous focus.' According to Dr. Cornil, tuberculous
affections of the skin are ameliorated by Koch's
remedy, but it should be sparingly employed in the
incipient stages of phthisis ; and it is useless, and
even dangerous, in advanced and acute cases of
phthisis. Nevertheless, Professor Koch has made a
great advance in the therapeutic treatment of infec-
tious diseases.1

ANTHRAX.

The disease known as anthrax, splenic fever,
splenic apoplexy, or malignant pustule, is a disease
affecting man and animals. ' In some countries the
losses to agriculturists and farmers owing to the
fatal character of the disease in sheep and cattle is
enormous. In man it is chiefly known among wool-
sorters and those engaged in the handling of hides.
This disease has been definitely proved to be due to
the Bacillus anthracis, which, after its entry into the
system of an animal or human being, multiplies very
rapidly in the blood and spleen, and, as a rule, pro-
duces a fatal result, at any rate in sheep and cattle.'

1 Various methods for treating phthisis are detailed in the
author's book: Researches on Micro -Organisms, pp. 286-3] 9
(Bailliere & Co.) ; and see also Dr. Drewitt's paper in Trans.
Clin. Soc., 1887. Drewitt treated a child suffering from lupus
partly by scraping and partly by salicylic acid.

INFECTIOUS DISEASES AND MICROBES, ETC. 255

Bacillus anthracis measures from 5 to 20 /i long,
and from 1 to 1*25 //, broad (Fig. 50), and often
occurs in masses of filamentous threads. It pro-
duces oval spores, and when either the bacillus or
its spores are injected into mice, guinea-pigs, sheep,
rabbits, etc., they die with all the characteristic

FIG. 50. BACILLUS ANTHRACIS.

A, Bacilli (a) forming spores ( x 1200).

B, Convolutions of bacillary threads (x 320).

lesions, etc., of anthrax. Even the inhalation of the
spores is capable of giving rise to anthrax in man
and susceptible animals. B. anthracis has been
found in the blood, spleen, and other organs, also in
the urine and faeces of animals suffering from or

256 A MANUAL OF BACTERIOLOGY

dead of anthrax. This microbe grows in nutrient
gelatine, agar-agar, neutral bouillon, and on steamed
potatoes at all temperatures between 15° and 43° C.,
best between 25° and 40° C. Free access of air is
essential for B. anthracis to produce spores. Suc-
cessive cultivations of this microbe do not weaken
its virulence. On gelatine plates it gives rise to
small white colonies after two or three days' incuba-
.tion. When these colonies are examined under low
power they appear as masses of twisted threads,
but in cover-glass preparations (Fig. 50 B) these
thread-like filaments are readily observed. In tube-
cultivations the bacillus presents a characteristic
appearance. Along the track of the needle there
appear lateral growths which give the culture a
peculiar feather-like appearance. But after a time
the gelatine liquefies, and the growth sinks to the
bottom of the tube, where the bacilli undergo de-
generation. On agar-agar a similar appearance is
presented, but there is no liquefaction of the medium.
B. anthracis grows on steamed potatoes as a creamy-
white granular mass.

It has been stated that the anthrax bacillus pro-
duces a ptomaine called anthracin and an albumose l
from the medium on which it lives.

Klein and Parsons2 have shown that anthrax
bacilli without spores are destroyed in five minutes
when exposed to a temperature of 103° C. (dry heat),
but the spores are not destroyed until they have

1 Hankin in Proc. Roy. Soc., 1890, p. 93; and Martin in
Nature, vol. xlii. p. 118.

2 Report to Medical Officer of Local Government Board , 1884.

IXFECTIOUS DISEASES AND MICROBES, ETC. 257

been exposed to a temperature of 104°C. for four
hours (dry heat). However, boiling in water for
only one minute was sufficient to render inert the
spores of B. anthracis.

According to MM. Chamberllent and Moussons,1
anthrax bacilli have been discovered in the milk of
cows affected with the disease, and not only is milk
a means of giving rise to an outbreak of anthrax,
but polluted drinking water derived from wells may
also spread the disease.

As already stated, successive cultivations do not
weaken the virulence of Bacillus anthracis, but if
the microbe is cultivated in neutral bouillon at 42°
or 43° C. for twenty days an attenuated virus is
obtained. Pasteur's premier vaccin protects animals
against the disease ; but to make them perfectly
refractory, they are inoculated a second time with a
vaccine (deuodeme vaccin) of less strength. Attenu-
ated viruses for the protective inoculation against
anthrax have also been obtained by exposing the
bacilli to a temperature of 55° C., or to an aqueous
solution of carbolic acid (0*5 to 1 per cent.), or sul-
phuric acid in a diluted form, as well as other
chemicals. According to Hankin,2 immunity against
anthrax is obtained by inoculation with the albu-
mose derived from pure cultivations of the bacilli,
and he has also cured animals suffering from anthrax
by injecting the albumose into their bodies.

1 Comptes Rendus, vol. cvii. p. 142.

2 Report of British Association, 1890 ; and British Medical
Journal, 1890.

258 A MANUAL OF BACTERIOLOGY

ACTINOMYCOSIS.

This disease attacks cattle and occasionally man
himself. It is caused by the ray-fungus or Actino-
myces. '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 indura-
tion of the tongue — 'wooden tongue' Occasionally
these tumours occur in the skin and lungs. The
ray-fungus has been cultivated on solid ox-serum,
and when pure cultures are injected into animals
they give rise to actinomycosis.

THRUSH.

This disease is caused by the fungus O'idium albi-
cans. It is found on the mucous membrane of the
mouth of infants, causing white patches on the
tongue, gums, and soft palate. Like the higher
fungi, this plant is composed of hyphse and spores,
which take root in the mucous lining of the mouth.
The spores are produced by the division of the ter-
minal cells, or sometimes by endogenous formation
within the hyphse.

In concluding the present chapter we may say
that most infectious diseases have a microbian
origin, but there are some (e.g. typhus fever, whoop-
ing-cough, mumps, etc.) in which no microbes have
been isolated and cultivated apart from the body ;
and there are other infectious diseases which owe
their origin to small animal organisms, known as

INFECTIOUS DISEASES AND MICROBES, ETC. 259

Protozoa. Dysentery and tropical abscess of the
liver are due to Amcebce,1 and in India a fatal dis-
ease (surra), which attacks horses, mules, and camels,
is caused by one of the Flagellata.1

1 See Dr. A. B. Griffiths' book, The Physiology of the Inverte-
brata (Reeve and Co.).

CHAPTEK VII

THE MICROBES OF THE AIR

'THE solid matter floating in the atmosphere is
every day becoming of greater and greater interest
as we are gradually realising the important part it
plays in the economy of nature, whether viewed
as to its physical, physiological, or meteorological
aspects. One fundamental point on which we have
at present very little information of anything like a
definite character is as to the number of solid par-
ticles present in the atmosphere. We know that
they are very numerous, and it seems probable that
the number varies under different conditions of
weather, but what number of particles are really
present under any conditions, and how the number
varies, we have at present very little idea. In this
field of research the physiologists are far in advance
of the physicists, as they have devised means of
counting the number of live germs floating in the
atmosphere, and already we have a good deal of
information as to how the number varies under
different conditions.'

Before describing the living particles in the
atmosphere we allude to some recent investigations

260

THE MICROBES OF THE AIR

261

on the number of dead or inorganic particles con-
tained in the air. Mr. J. Aitken, F.R.S.,1 has in-
vented an ingenious apparatus by which the number
of dust particles in the atmosphere may be readily
estimated. Among the results obtained are the fol-
lowing : —

No. of Dust Particles in Air.

Source of air.

No. per cc.

No. per cubic in.

Outside (raining)
Outside (fair) .
Room
Room near ceiling
Bunsen flame .

32,000
130,000
1,860,000
5,420,190
30,000,000

521,000
1,119,000
30,318,000
88,346,000
489,000,000

These results indicate that ' there is most dust in
the air during dry weather, and perhaps during
anti-cyclonic conditions, and least during wet
weather, and perhaps in cyclonic areas.'

Aitken has also ascertained the minimum and
maximum number of dust particles per cubic centi-
metre (cc.) in the air of various towns, etc. Among
these results are the following : —

At Hyeres (near Toulon), . from 5000 to 46,000

, Cannes,

1550

, 150,000

Lucerne (mountain air),

210

, 2350

Paris, .

92,000

, 210,000

London,

48,000

, 150,000

Ben Nevis (mountain air),

335

473

Dumfries,

395

, 11,500

Mentone,

1200

, 7200

1 Transactions of Royal Society of Edinburgh, vol. xxxv.
p. 1 ; Proceedings of Royal Socie.ty of Edinburgh, vol. xvii.
p. 193, and vol. xviii. pp. 39 and 259.

262 A MANUAL OF BACTERIOLOGY

Aitken concludes (1) that the earth's atmosphere is
greatly polluted with dust produced by human
agency ; (2) that this dust is carried to considerable
elevations by the hot- air rising over cities, by the
hot and moist air rising from sun-heated areas of the
earth's surface, and by winds driving the dusty air
up the slopes of hills ; (3) that none of the tests
made of the Mediterranean sea air show it to be very
free from dust ; and (4) that the amount of dust in
the atmosphere of pure country districts varies with
the velocity and the direction of the wind : fall of
wind being accompanied by an increase in dust.
Winds blowing from populous districts generally
bring dusty air.

It is stated that a man in the town inhales about
37,500 germs every twenty-four hours, and no
fewer than 2,250,000 inorganic particles every
minute.1 ' Most of these are merely annoying,
though a few are real messengers of disease and
death. If the lungs are warm and moist, they can
repel the particles ; but with cold and dry lungs the
suffering from the clogging must soon begin/

Besides the inorganic or dead particles, the air
is more or less laden with living particles. The
majority of these are of the non-pathogenic or harm-
less kind, but there is plenty of evidence to show
that pathogenic microbes lurk about in the atmo-
sphere, and that many infectious diseases are propa-
gated by means of air-carried microbes. Hence the
reason that the study of aerial microbes is peculiarly

1 A cigarette smoker sends no fewer than 4,000,000,000 of
particles (more or less) into the air with every puff he makes.

THE MICROBES OF THE AIR

263

interesting and attractive. The investigations of
Burdon Sanderson, Tyndall, Lister, and Lankester
have all thrown considerable light upon the condi-
tions of life of these lower organisms ; but Pasteur
was the first investigator who made a systematic

Fio. 51. MIQUEL AND DE FREUDENREICH'S FILTER.

A, Filter-tube (A, enlargement of same, with cap at d).

B, Aspirator. C, Gasometer.

study of the presence and distribution of microbes
in the atmosphere.

It was not, however, until 1879 that Drs. Miquel

264 A MANUAL OF BACTERIOLOGY

and De Freudenreich attempted the quantitative
estimation of aerial microbes. Their method con-
sists in aspirating a known volume of air through a
tube containing previously sterilised plugs of glass-
wool (Fig. 51). The solid particles, including any
microbes, are arrested ; and the plugs of glass-wool
are then thoroughly mixed with a known volume of
sterilised water. The mixture is now sub-divided
into such a number of equal parts that each part
shall contain not more than one microbe. Each of
these sub-divisions is then introduced into a cultiva-
tion tube or flask (see Fig. 17) containing sterilised
bouillon. These tubes or flasks are placed in an
incubator, and any that have received a living
microbe will, in a short time, exhibit the fact by
suffering visible alteration. As an example, sup-
posing the plug through which twenty litres of air
were drawn, by the aspirator (Fig. 51 5), was mixed
with 25 cc. of sterilised water, and twenty-five
tubes of bouillon were then each inoculated with
1 cc. of this mixture, and if, after a suitable incuba-
tion, it was found that only sixteen of them suffered
alteration, it would be concluded that only sixteen
microbes were present in the 25 cc. of water distri-
buted among the twenty-five tubes, or, in other
words, that the twenty litres of air contained sixteen
living microbes.

Miquel and De Freudenreich have since substi-
tuted soluble media (powdered sugar or de-hydrated
sodium sulphate) for the insoluble glass-wool. By
the use of soluble filtering media, there is no chance
of any microbes becoming imprisoned, as is the case

THE MICROBES OF THE AIR 265

when glass-wool is used. Drs. Miquel,1 Fol,2
Gautier,3 and other French bacteriologists use
soluble filtering media ; and bouillon as the medium
for the growth of microbes.

In England and Germany solid cultivation media
have been substituted for the liquid bouillon ; and
when the microbian mixture is introduced into
melted nutrient gelatine, it ' can be evenly dis-
persed throughout the medium by gentle agitation,
and by subsequently allowing it to solidify, the
microbes are not only isolated, but rigidly confined
to one spot. Thus each individual microbe becomes
a centre round which extensive multiplication takes
place, and in a few days definite points of growth
are visible to the naked eye, which are appropriately
described as colonies. Although each colony con-
sists of many thousands, or even millions of in-
dividual microbes, yet, as in the first instance, they
owe their origin to a single organism or indivisible
group of organisms, it is correct to regard the number
of colonies as representing the number of microbes/

One of the best methods for estimating the num-
ber of microbes in a known volume of air, is that
devised by Dr. W. Hesse.4 Hesse's method con-
sists in slowly aspirating a known volume of air
through a glass tube (28 X If in.) which has pre-
viously been coated internally with a film of sterilised
nutrient gelatine. The microbes suspended in the

1 Annuaire, de VObservatcrire de Afontsourix, 1880-92.

2 La Nature, 1885.

3 Rente Scientijique, 1886.

4 Mittheilungen am dem Icaiserliche.n Oesundheitnamte, vol. ii.

266

A MANUAL OF BACTERIOLOGY

air are rapidly deposited within the tube, and on the
surface of the gelatine give rise to colonies. Fig.
52 represents Hesse's aeroscope. At D is an india-
rubber stopper, perforated to admit a small glass

Fig. 52. HESSE'S AEROSCOPE.

tube, plugged with cotton-wool ; and at the opposite
end is a perforated indiarubber cap, which is
covered by an imperforated cap (C) of the same
material. The aspirator consists of two like flasks
(A B); one of which is filled with water. These
flasks are reversible ; but the one containing the

THE MICROBES OF THE AIR 267

water is always fixed uppermost when the air is
passing through the tube. The down-flow of water
causes the air to pass slowly through the tube when
the outer cap (C) has been removed ; and as the
flasks are of known capacity, two, five, ten, or more
litres of air may be aspirated through the tube.
After this the cap is replaced, and the tube is then
removed to a warm situation for several days, in
order that colonies may develop.

Before introducing the nutrient gelatine, the
tube, caps, and plug are sterilised by means of a
solution of mercuric chloride, and finally with

FIG. 53. GRIFFITHS' MODIFICATION OF HESSE'S AEROSCOPE.

alcohol. After this treatment, 50 cc. of melted
nutrient gelatine are poured into the tube, which is
then sterilised in a steamer by the discontinuous
method.

The author has made a modification of Hesse's
apparatus (Fig. 53), by substituting a small exhaust
pump of known capacity for the aspirator. This
modification is far handier and occupies less space
than Hesse's aeroscope ; while it gives results which
agree with those obtained with the original apparatus.
The late Dr. T. Carnelley1 also modified Hesse's

1 Report of British Association, 1887, p. 654.

268

A MANUAL OF BACTERIOLOGY

aeroscope by substituting a flat-bottom flask for the
tube.

Dr. P. F. Frankland1 has devised a method by

FIG. 54. COLONIES IN FLASK.
(After Frankland.)

which a known volume of air is drawn, by means of
an air-pump, through a short glass tube (4 x i in.)

1 Philosophical Transactions, vol. clxxviii. p. 113.

THE MICROBES OF THE AIR 269

containing two small porous plugs placed one in
front of the other. The first plug consists of glass-
wool coated with sugar, whilst the second contains,
in addition, a layer, £ inch in thickness, of fine
sugar-powder. The microbes, suspended in the
aspirated air, are deposited on these plugs, which are
introduced into separate flasks, each containing a
a small quantity of melted nutrient gelatine. Each
flask is then agitated until the plug is disintegrated,
and since the sugar-coating of the glass-wool dis-
solves in the liquid gelatine, the microbes become
immediately detached. The gelatine is now allowed
to solidify, forming a thin film over the inner sur-
faces of the flasks. The flasks are finally placed in
an incubator ; and in a few days colonies derived
from the microbes, which were collected by the
plugs make their appearance and can be counted
and further studied (Fig. 54).

The author1 has examined the air of Lincoln,
Paris, and London. The methods used for estimating
the number of microbian colonies in a known volume
of air were those of Hesse and Frankland. Before
August 6th, 1888, Hesse's method was used, while
after that date Frankland's method was substituted
for that of Hesse. The average number of colonies
in three gallons (fifteen litres) of air are given in
the following tables : —

1 Proceedings of Royal Society of Edinburgh, vol. xvii.
p. 265 ; and Researches on Micro-Organisms, p. 59.

270 A MANUAL OF BACTERIOLOGY

THE Am OF LINCOLN.

EAR

188

PLACE.

•

-J

f>^

">*

tiD

-*f

•?

•5

(1) Top of hill

(near Cathedral),

(2) Base of hill

(Broadgate),

THE AIR OF PARIS.

Place or Part of Paris.

Situation
in Paris.

August, 1887.

(1) Cimetiere du Pere la Chaise,

(2) Boulevard Saint -Germain, .

Centre

104

(3) Forest of Ville d'Avray,

S.W.

(4) Rue de Rennes, . . . .

Centre

(5) Palais du Trocadero, . ' *.

(6) Park of Versailles,

S.W.

(7) St. Cloud, , ..';..

S.W.

(8) Boulevard Voltaire,

100

(9) Cimetiere Montparnasse,

(10) Cimetiere Montmartre,

(11) Parcdes Buttes Chaumont, .

N.E.

THE AIR OF LONDON.

Place or Part of London.

July, 1888.

August, 1888.

(1) Forest Gate (Essex).

(2) City (near Bank), .

110

(3) West End (Piccadilly), .

(4) East End (near Mint), .

106

THE MICROBES OF THE AIR 271

The conclusion drawn from these investigations
are the following : —

(a) There are a larger number of microbes in the V
atmosphere during the summer than either the
spring or winter. They appear to reach a maximum
during the month of August. (&) The number of
microbes found in the atmosphere decreases, the
higher one ascends. Hence near the Lincoln Cathe-
dral there are fewer microbes in the atmosphere
(on any given day) than in the valley of the Witham.
The same remark also applies to the number of
microbes found in the atmosphere at the top of the
Trocadero Palace, Paris, where there are fewer
microbes than in a low-lying but crowded thorough-
fare like the Boulevard Saint-Germain, (c) There
are a" larger number of microbes in the atmosphere
of crowded centres than in less densely-populated
districts, (d) By gradually passing from a large
city towards the country the number of aerial
microbes decreases ; e.g., there are fewer microbes
in the atmosphere of the Forest of Ville d'Avray,
the Park of Versailles, and the village of St. Cloud,
than in the principal thoroughfares of Paris and
London.

Dr. P. Miquel l (who is the greatest authority on
aerial microbes) has published elaborate tables con-
cerning the number of microbes in the air of certain
parts of Paris. During the year 1888, Miquel
obtained the following mean number of microbes in
the air (per cubic meter) at Montsouris, and in the
vicinity of the Hotel de Ville, Paris : —

1 Annuaire de T 'Obner vatoire de Montsouris, 1877-92.

272

A MANUAL OF BACTERIOLOGY

SEASONS.

Montsouris.

Hotel de Ville.

Winter, ....

171

2870

Spring, ....

210

8920

Summer, ....

400

12280

Autumn, ....

185

6800

Annual Means,

242

7720

The mean annual results (for eight years, 1881-88)
of the number of microbes contained in one cubic
metre of the air at Montsouris and in the vicinity
of the Hotel de Ville (i.e. in the centre of Paris) are
given in the following table : —

Montsouris.

Hotel de Ville.

January, . ....

228

2310

February,

170

3140

March, ....

255

3420

April,

358

4340

May,

379

5950

June,

448

5070

July,

676

5200

August, ....

628

5640

September, ....

470

5510

October, ....

332

4335

November, ....

239

3700

December, ....

189

2885

From the above results it will be seen (a) that
there are a larger number of microbes in the atmo-
sphere in the centre of Paris than at Montsouris ;

THE MICROBES OF THE AIR 273

(b) that there are a larger number of microbes in
the atmosphere during the summer than any other
period of the year. Miquel has also shown that as
the number of microbes in the atmosphere increases,
so does the mortality from zymotic or infectious
diseases.

The investigations of Dr. P. F. Frankland on
aerial microbes have added considerably to our
knowledge of this interesting subject. Frankland
has not only examined the air so as to ascertain the
number of microbes present in a known volume, but
he has discovered many new forms.1

Frankland has obtained the following results
concerning the number of microbes present in ten
litres, or two gallons, of air at different places : —

Primrose Hill (top), ... 9

„ (bottom), . . 24

Norwich Cathedral (top of spire, 300 ft. ), . 7

(tower, 180ft.), 9

,, ,, (in the close), . . 18

St. Paul's Cathedral (Golden Gallery), . 11

(Stone Gallery), . 34

' „ „ (Churchyard),. 70

>> £ f Reigate Hill, .

J Heath near Norwich,

q g I Garden at Reigate, . . 25

° I Garden near Norwich, . . 31

§ g [ Kensington Gardens, . . .13

-2^ { Hyde Park, . . 18

^ j I Exhibition Road, . . 554

Frankland has also shown that within doors the

1 Philosophical Transactions, vol. clxxviii. p. 257.

274

A MANUAL OF BACTERIOLOGY

number of microbes suspended in the air depends
upon the number of people present and the amount
of disturbance of the air which is taking place.

Dr. Fischer1 has proved that sea air is almost free
from microbes. Carnelley2 and Pe'tri3 have also
shown that the air of sewers is remarkably free
from microbes. This is due to the moisture on the
walls of these subterranean channels.

The following microbes are always present (more
or less) in the atmosphere : —

Micrococcus citreus conglomerate.
Micrococcus violaceus.
Micrococcus rosaceus.
Bacterium indicum.
Micrococcus prodigiosus.
Bacterium aceti.
Bacterium lactis.
Micrococcus cyaneus.
Bacterium xanthinum.
Bacillus figurans.
Micrococcus carnicolor.
Micrococcus candlcans.
Micrococcus albus.
Sarcina lutea.
Surcina aurantica.
Bacillus Jluorescus.

Micrococcus liquefaciens.
Sarcina liquefaciens.
Micrococcus gigas.
Micrococcus chryseus.
Bacillus aurescens.
Bacillus aureus.
Bacillus citreus.
Bacillus plicatus.
Bacillus chlor'mus.
Bacillus polymorphic.
Bacillus profusus.
Bacillus pestifer.
Bacillus Icevis.
Bacillus cereus.
Bacillus subtilis.

Besides other microbes, there are always present
in the atmosphere an abundance of moulds and
yeast-fungi.

Although the microbes connected with the com-
mon infectious diseases have not been discovered in

1 Zeitschrift fur Hygiene, vol. i.

2 Philosophical Transactions, vol. clxxviii. p. 61.
* Zeitschrift fur Hygiene, vol. iii.

THE MICROBES OF THE AIR 275

air, ' yet there can be no doubt that, in the imme-
diate vicinity of the foci of disease, such microbes
are present, and that their distribution and convey-
ance in the air will take place in just the same
manner as in the case of non-pathogenic microbes.
The investigations on aerial microbia, so far as they
have as yet been carried, are of service in indicating
how, we may escape from all microbes, whether
harmful or harmless ; and secondly, how we may
avoid the conveyance of microbes into the atmo-
sphere from places where pathogenic forms are
known or likely to be present. This acquaintance
with the distribution of microbes in general, and
the power of controlling their dissemination which
it confers, is really of far wider practical importance
than discovering whether some particular pathogenic
form is present in some particular sample of air.
It is this knowledge which has led to the vast
improvements in the construction and arrangement
of hospital wards and of sick-rooms generally, and
which has directed attention to the importance of
avoiding all circumstances tending to disturb and
distribute dust. It is, moreover, this knowledge of
the distribution of microbes in our surroundings
which has formed one of the foundations for the
antiseptic treatment of wounds — that great step in
surgery with which the name of Sir Joseph Lister
is associated.'1

1 For further information see Frankland's papers in Journal
of Society of Arts, vol. xxxv. p. 485; Proc. Roy. Soc., 1885-86;
Miquel's Les Organismes Vivants de I' Atmosphere; Prudden's
Dust and its Dangers ; and Griffiths' Researches on Micro -
Organisms.

CHAPTEE VIII

THE MICROBES OF THE SOIL

SOIL is very rich in microbes, and these insignificant
plants play a most important part in the processes
of putrefaction and nitrification, which are always
at work for man's benefit and welfare.

Among the microbes present (more or less) in soil
are the following : —

Bacillus typhosus.
Bacillus radicicola.
Bacterium septicumagrigenum.
Bacillus cedematis maligni.
Streptococcus septicus.
Bacillus subtilis.
Bacillus toruliformis.
Bacillus floccus.
Bacillus septicus.
Bacterium termo.
Bacterium allii (?).

The Nitrous Bacillus.
The Nitric Micrococcus.
Bacillus tar deer escens.
Bacterium urece.
Bacillus fiuorescens.
Micrococcus cereus.
Bacillus of Mouse Septiccemia.
Bacillus mycoides.
Bacillus anthracis.
Bacillus of tetanus.
Bacillus of malarice.

Spirillum cholerce Asiaticce.

In addition to the above, the spores, etc., of many of
V the higher fungi are present in soil. Some of these
are detrimental to the growth of vegetation, for they
become internal or external parasites, and thereby
produce disease.1 Not only are the higher plants
attacked by parasites present in soil, but man and
animals suffer from diseases, like tetanus, malaria,
etc., which are caused by soil-microbes.

1 Griffiths' Diseases of Crops.

276

THE MICROBES OF THE SOIL

277

To study the microbes present in soil, both solid
and liquid media are used, but the employment of
the former is much more satisfactory. The follow-
ing methods are used by bacteriologists to ascertain
the number of microbes in a known weight, etc., of
soil : — (a) A sample of the dried soil is triturated with
sterilised distilled water, and then a small quantity
of this water is sprinkled on the surface of a gelatine
plate, (b) The soil is introduced into a test-tube
containing liquefied gelatine. After a thorough
shaking the mixture is poured out upon a glass
plate, so as to form a plate- cultivation, (c) When
bouillon is used the soil is first triturated with water,
and then a drop of the water is transferred to a
flask containing sterilised bouillon.

Among the results obtained of the number of
microbes present in various soils are the following :

(A) GRIFFITHS' ANALYSES.

Samples of Soil from—

Number of
Microbes in 1 gram.

Lincoln (Monk's Road),

,, (Castle grounds),
Manchester (Infirmary grounds), .

,, (Plymouth Grove),
London (Forest Gate), ....
,, (Hyde Park), ....
Paris (Forest of Ville d'Avray), .

„ (Near Sevres)

,, (Pare Monceaux),
Dieppe (near Church of St. Jacques), .

,, (near the Casino),
New Zealand (after 14 weeks' desiccation),

611,000

720,000

1,230,000

550,000

430,000

820,000

780,000

880,000

754,000

1,360,000

1,200,000

240,000

278

A MANUAL OF BACTERIOLOGY

(B) MIQUEL'S ANALYSES.

Samples of Soil from —

Number of
Microbes in 1 gram.

Paris (Rue de Rennes), ....

2,100,000
1 300 000

,, (Pare du Montsouris), ....

750,000

Dr. C. N. Dowd 1 has recently ascertained the
number of microbes in soil derived from various
streets in New York. The soil in each case was
obtained during the upturning of the streets for
relaying gas and water-pipes, etc.

Samples of Soil from —

East Fifty-ninth Street, near Third Avenue, .
East Fifty-nine Street, near Park Avenue,
East Fifty-ninth Street, near Fifth Avenue, .
East Fifty-ninth Street, near Madison Avenue,
Eighth Avenue and Fifty-seventh Street, . .
Tenth Avenue and Sixty-fifth Street, . . .
Eighth Avenue, near Fifty -sixth Street, . .
Eighth Avenue, near Fifty-fifth Street, . .
Fifty-ninth Street and Sixth Avenue, . . .
Fiftieth Street and Eighth Avenue, ....
Sixth Avenue, near Fifty-eighth Street, * .
Seventh Avenue and Fifty-fourth Street, . .
Seventy -first Street, near Eighth Avenue, . .
Forty-ninth Street and Eleventh Avenue, . .
Third Avenue, near Forty-second Street, . .
Third Avenue, near Forty -second Street, . .

Number of
Microbes per cc.

17,675

17,950

157,200

131,100

29,700

29,250

8585

3800

10,650

287

33,150

15,250

20,150

24,900

28,850

67,500

American Medical Record, 1890.

THE MICROBES OF THE SOIL 279

To ascertain the number of microbes in a given
sample of soil, the colonies produced on the gela-
tine-plates are accurately counted. This is per-
formed by the apparatus represented in Fig. 55,
which will be described in the next chapter. To
ascertain the characteristics of the microbes, further
cultivations must be made ; the microbes must be
transplanted into various media, and exposed to
different temperatures ; and they must be inocu-
lated into different kinds of animals. The labour
of separating each species and studying it in detail

FIG. 55. WOLFFHUGEL'S APPARATUS.
(For estimating the number of Colonies in a Plate-cultivation.)

would be extremely great ; hence microbian soil
examinations have largely been confined to the
determination of the number of microbes, and not
to the peculiar species. In determining the signifi-
cance of such examinations, we must bear in mind
the following facts: — (1) The number of microbes
present in a soil does not necessarily indicate the
number of pathogenic forms. (2) Small samples
of soil may show marked variations in the number
of microbes ; this is owing to minor local influences.
(3) Surface soil always contains a larger number of

280 A MANUAL OF BACTERIOLOGY

microbes than sub-soil; and at a depth of 8 or
10 feet there are hardly any present. (4) Most of
the microbes of soil are harmless when introduced
into the human or animal body ; but the bacilli of
tetanus, anthrax,1 typhoid fever, malaria, and
cholera have been found in soil.

Dowd's investigations have proved that the ex-
posure of so much soil in the upturning of streets
is detrimental to the health of the surrounding
community. However, it should be remembered
that so long as the soil is wet it cannot spread the
microbes in the air; but the soil does not long
remain wet. It dries beside the trenches, it adheres
to the implements, the clothes and boots of the
workmen, and, in fact, to everything which comes
in contact with the trenches ; and, finally, much of
it is left on the surface when the pavement is relaid.
In all these conditions it may be carried away as
dust. The microbes go with the dust, and access
to the body is then made easy. The amount of
dust in the air is much increased by these trenches ;
but, on the other hand, the deeper layers of soil or
earth from which this dust is derived do not contain
nearly so many microbes as the surface layer.

An important method for dealing with the dust
of streets, especially during epidemics, is to water
them with some germicidal substance, by means of
the Strawsonizer or pneumatic distributor.2 This
machine is capable of distributing one or more

1 Pasteur in Bulletin de VAcademie de Medecine, 1880.

2 Obtainable at Messrs. Strawson and Co., Newbury, Berk-
shire.

THE MICROBES OF THE SOIL 281

gallons of any fluid over an acre of land, and to a
width of 23 feet. Therefore, it would be advan-
tageous to use this machine for watering streets,
cattle markets, etc., with a weak solution of ' sanitas,'
carbolic acid, or any other cheap disinfectant.

Concerning cultivated soils, nitrogen is a most
important element in the growth of crops. Berthe-
lot1 has shown that a fixation of atmospheric
nitrogen takes place in certain vegetable soils by
the action of microbes and other fungi.

Hellriegel and Wilfrath have proved that legu-
minous plants obtain their great supplies of nitrogen
from the air. This power of absorbing free nitrogen
is due to the roots of leguminous plants becoming
inoculated with the microbes present in soil. The
microbes, which give rise to tubercles on the roots
and rootlets, enter into a partnership or symbiotic
relationship with the leguminous plant for mutual
advantage. These microbes have the power of
bringing the free nitrogen into organic combination.

Perhaps the chief soil-microbe which enters into
symbiosis with leguminous plants is Dr Beyerinck's
Bacillus radicicola. This microbe has been isolated
from cultivated soils as well as from the tubercles
on the roots of Vicia faba (the field bean) ; and
Beyerinck has inoculated the roots of seedling-
beans with this microbe, and in each case it
multiplied within the roots, ultimately giving rise
to tubercles.

The process of nitrification or the conversion of
organic and ammoniacal nitrogen into nitrates was

1 Comptes Rendut, vol. cviii.

282 A MANUAL OF BACTERIOLOGY

first shown by Mlintz and Schloesing 1 to be due
to the action of microbes in the soil. Although
these savants had previously described a microbe
causing nitrification, it was not until 1890 that Dr.
P. F. Frankland, F.RS.,2 M. Winogradsky,3 and Mr.
R. Warington, F.RS.,4 simultaneously described the
true cause of nitrification. The nitrifying microbes
were isolated by the fractional dilution method.

(1) Frankland' s researches. — Dr. and Mrs. Frank-
land have isolated a nitrifying microbe from soil.
' Nitrification having been in the first instance in-
duced in a particular ammoniacal solution by means
of a small quantity of garden soil, was carried on
through twenty-four generations, a minute quantity
on the point of a sterilised needle being introduced
from one nitrifying solution to the other. From
several of these generations gelatine-plates were
poured, and the resulting colonies inoculated into
identical ammoniacal solutions, to see if nitrification
would ensue ; but although these experiments were
repeated many times, on no occasion were they
successful.' In other words, the microbe in ques-
tion refused to grow on gelatine. The ammoniacal
solution, already referred to, contained :— 1000 cc.
of distilled water, 100 cc. of salt solution,5 0'5

1 Comptes Rendus, vol. xlviii. p. 301 ; vol. Ixxxv. p. 1018 ;
vol. Ixxxix. pp. 891 and 1074.

2 Philosophical Transactions, vol. clxxxi. pp. 107-128.

3 Annales de FInstitut Pasteur, 1890, p. 213 seq.

4 Journal of Chemical Society, 1891, pp. 484-529 ; Chemical
News, vol. Ixi. (1890), p. 135.

5 This solution contained 1 gramme of potassium phosphate,
0*2 gramme of crystallised magnesium sulphate, and O'l gramme
of calcium chloride (fused) in 1000 cc. of water.

THE MICROBES OF THE SOIL 283

gramme of ammonium chloride, and 5 grammes of
carbonate of lime (pure) ; and in this solution the
microbe grew and multiplied. As this solution con-
tains no organic matter, it will be seen that nitrifica-
tion can take place in purely mineral solutions. This
power of growing in mineral solutions prevented the
development of other microbes (present in the soil
used for inoculation) which require organic matter
for their growth. After proving that the microbe
refused to grow on gelatine, 'experiments were
commenced to endeavour to isolate the microbe by
the dilution method. For this purpose a number
of series of dilutions were made by the addition, to
sterilised distilled water, of a very small quantity
of an ammoniacal solution which had nitrified. It
was hoped that the attenuation would be so perfect
that ultimately the nitrifying microbe alone would
be introduced. After a very large number of
experiments had been made in this direction, the
authors at length succeeded in obtaining an at-
tenuation consisting of about one-millionth of the
original nitrifying solution employed, which not
only nitrified,1 but, on inoculation into gelatine-
peptone, refused to grow, and was seen, tinder the
microscope, to consist of numerous characteristic
bacilli hardly longer than broad, which may be
described as bacillococci.'

The chief characters of the Frankland Bacillus of
nitrification (Fig. 56 A) are the following : —

(a) The solutions in which the isolated microbe

1 The presence of nitrous acid was ascertained by both
diphenylainine and sulphanilic acid.

284

A MANUAL OF BACTERIOLOGY

grows remain perfectly clear, (b) The microbe has
the remarkable capacity of indefinite growth in a
medium devoid of organic matter, (c) It is O8 fju
in length, and hardly longer than broad, hence it
has been called a bacillococcus. It occurs both
isolated, in pairs, and in small irregular groups, (d)
In the living state it exhibits a vibratory movement
only, (e) The microbe cultivated in ammoniacal
solutions converts the ammoniacal into nitrous

nitrogen, and not into
nitric nitrogen. (/)
The same microbe ap-
pears to grow in broth
or bouillon, but not on
solid gelatine-peptone.

(2) Winogradsky 's re-
searches.— Winogradsky
has also obtained a
similar bacillus to that
of Frankland, which
grows in an inorganic
ammoniacal solution, but

not on gelatine - peptone ; and he has shown
that this microbe grows (and may be isolated)
on the surface of gelatinous silica containing the
inorganic ammoniacal salts already mentioned.
This nitrifying microbe gives rise to very charac-
teristic colonies on gelatinous silica. Winogradsky's
bacillus measures from 1-1 to 1-8 ^ long, and does
not exceed 1 //, broad. This microbe occurs singly,
in pairs, rarely in chains of three to four individuals,
and as zooglcea. It converts ammoniacal into

FIG. 58. MICROBES OF NITRIFICATION.

A, Frankland's nitrous bacillus.

B, Warington's nitric micrococcus.

THE MICROBES OF THE SOIL 285

nitrous nitrogen, and can grow in ammoniacal solu-
tions devoid of organic matter. There is little
doubt that Frankland's and Winogradsky's microbes
are the same. Both sets of experiments prove that
the nitrous bacillus of the soil converts ammoniacal
into nitrous nitrogen, and not into nitric nitrogen.

(3) Waringtoris researches. — Mr. Warington has
also isolated, by the dilution method, a microbe
which converts ammonia into nitrous acid only ;
and confirms the investigations of Frankland and
Winogradsky. In addition to this, Warington has
apparently isolated a microbe from soil which con-
verts nitrites into nitrates. This microbe produces
neither nitrites nor nitrates in ammoniacal solu-
tions; in fact, it cannot oxidise ammonia. The
nitric microbe (Fig. 56 B) is a micrococcus, and
grows in a solution of potassium nitrite.

'The nitrification effected by soil is thus ex-
plained as performed by two microbes, one of
which oxidises ammonia to nitrates, while the other
oxidises nitrites to nitrates. The first microbe is
easily separated from the second by successive
cultivations in solutions of ammonium carbonate.
The second is (probably) separated as easily from
the first by successive cultivations in solutions
of potassium nitrite containing monosodium car-
bonate.'

' In soil the nitric microbe is equally active as
the nitrous, since soil never contains any but ex-
tremely weak solutions of ammonia, andsuper-
carbonates are always present.'

CHAPTEK IX

THE MICROBES OF WATER

THE organisms present in water have long been
observed by the aid of the microscope, but it is
only during the last decade that bacteriological
methods have been introduced for the systematic
examination of potable and other waters.

Water is one of the most convenient vehicles for
the distribution of microbes, and unfiltered water
abounds in these small specks of animated matter.
This need not cause any surprise, because, as we
have already seen, the atmosphere and soils are
laden with microbes. In fact, every shower of rain
diminishes the number of microbes suspended in
air. These are then found in puddles, pools, ponds,
rivers, etc., and consequently are carried into well
and other potable waters. Although the majority
of these microbes are harmless, it is always advis-
able to filter water before use. One of the best
filters for this purpose is Maignen's * Filtre Eapide/
Among the various microbes found in water are
the following : —

ILllr*

« -

'§

IlllllrilliJ

^j -K> -K> ?* r* ^ ^ 5$ -»o <«> -»o *»

?^ »d *a "?? *5? ^^f*» S »d »d w 9

•ZJL

288 A MANUAL OF BACTERIOLOGY

In addition to the Schizomycetes, various Protozoa,
etc., are always present (more or less) in water, and
Fig. 57 represents certain animal and vegetal forms
found in some potable waters.

Although the majority of Schizomycetes and Proto-
zoa found in waters are harmless, it has been proved

FIG. 57. INFUSORIA, ETC., IN WATER.

1, Daphnia. 2, Chilodon. 3, Paramcecium. 4, Acineria. 5, Paranema.
6, Cercomonas. 7, Actinophrys. 8, Amoebae. 9, Amoeba diffluens. 10,
Protococcus. 11, Diatoms. 12, Desmids 13, Confervse. 14, Spores of
fungi. 15, Pieces of vegetable tissue. 16, Amoeba (more highly magnified).
17, Cyclops. 18, Cypris. 19, Anguillula.

that certain outbreaks of typhoid fever, cholera,
etc., have been traced to water supplies; and the
microbes of typhoid fever, cholera, tetanus, etc.,
have all been found in drinking waters contami-
nated with sewage. Dysentery and tropical abscess
of the liver are due to certain species of Amcebce,

THE MICROBES OF WATER 289

which enter the system through the medium of
water. In view of these facts the bacteriological
analysis of water is a subject of great importance ;
but the primary object in such analyses is not the
search for pathogenic microbes. Such an investi-
gation is generally fraught with insuperable diffi-
culties, and, for sanitary purposes, is practically
worthless. ' It is obvious that, even if the typhoid
bacillus, or any other pathogenic microbe could be
detected with unerring certainty in any water in
which it was present, a search for this bacillus in
the ordinary course of water examination would
still have only a very subsidiary interest. Waters
are surely not only to be condemned for drinking
purposes when they contain the germs of zymotic
disease at the time of analysis, but in all cases
when they are subject to contaminations which
may at any time contain such germs. Sewage-
contaminated waters must on this account be in-
variably proscribed, quite irrespectively of whether
the sewage is, at the time that the water is sub-
mitted to examination, derived from healthy or
from diseased persons. . . . The real value of these
bacteriological investigations, if judiciously applied,
consists in their power of furnishing us with in-
formation as to the probable fate of dangerous
organisms, should they gain access to drinking
water. It is by their means that we have learnt
that many such organisms can preserve their
vitality, nay, in some cases can actually undergo
multiplication in ordinary drinking water ; that
they are destroyed by maintaining the water at the

290 A MANUAL OF BACTERIOLOGY

boiling point for a short time; and that they are
more or less perfectly removed by some processes
of nitration and precipitation, whilst other pro-
cesses of the same nature are worthless, or even
worse' (Frankland).

Before describing the methods for the bacterio-
logical examination of waters, we must allude to
(a) the collection of the samples, and (b) the trans-
port of the same.

To collect the samples of water accurately stop-
pered bottles (70 cc. capacity) are used. These
must be perfectly clean, and rinsed out with dis-
tilled water. Each bottle is put into a small tin
canister, and the canisters (containing the bottles)
are heated in a steriliser to about 180° C. for at
least three hours. * The bottles thus sterilised can
be easily transported without suffering contamina-
tion by dust to the place where the sample is to be
collected. In collecting the sample of water the
outside of the bottle should be rinsed in the water
before removing the stopper, and when the bottle
is opened the water is at once allowed to enter and
fill the bottle to the extent of four-fifths, the stopper
being immediately replaced and tightly screwed in,
so that the exposure to the air is reduced to a
minimum. The bottle is replaced in the tin
canister, and the lid closed. In collecting samples
of water from rivers, reservoirs, lakes, or ponds, it
is better not to remove the stopper until the bottle
is completely immersed in the water, and to replace
it while still beneath the surface.' After collection
the sample of water should be examined as soon as

THE MICROBES OF WATER 291

possible, for it has been proved by Dr. T. Leone,1
Dr. P. F. Frankland,2 and others, that microbes
multiply very rapidly in water. For instance,
Leone gives the following figures, which show the
rapid increase of microbes in a sample of water kept
for only five days : —

Number of Microbes in 1 cc. of water (at 14° to 18° (7.).

Water on day of collection, . » 5

after 1 day's standing . . „ 100

„ 2 days' „ , . 10,500

„ 3 „ ... 67,000

„ 4 „ „ . . . 315,000

„ 5 „ „ ; . . 500,000

If the water has to be transmitted a considerable
distance, occupying several days in transit, Dr. P.
Miquel 3 recommends the use of a glaciere, or box,
in which the bottle is surrounded with ice.

There are two principal methods in use for the
bacteriological examination of water. The first is
the plate-cultivation process, which consists in
taking a known quantity (say 1 cc.) of the water,
and mixing it with melted nutrient gelatine con-
tained in a test-tube. After shaking, the contents
of the tube are rapidly poured out upon a sterilised
glass plate, then allowed to solidify, and finally
placed in a damp chamber, kept at about 22° C.
After a few days' incubation colonies make their
appearance on and in the layer of gelatine. The
colonies are counted by means of the eye or lens,

1 Gazzetta C/iimica Italiana, voL xv. (1885), p. 385.
3 Proceedings of Royal Society, 1886.

* Manuel Pratique d' Analyse Bactiriologique des Eaux (1891),
p. 26.

292 A MANUAL OF BACTERIOLOGY

with the aid of Wolffhugel's counting apparatus
(see Fig. 55), which consists of a glass plate, ruled
with vertical and horizontal lines into centimetre
squares, which are often sub-divided. The cultiva-
tion-plate is placed on a black background, and the
ruled glass plate placed over the former, without
touching the colonies. 'If the colonies are very
numerous the number in some small divisions is
counted ; if less, in some large ones ; and an average
is obtained from which the number of colonies on
the entire surface is calculated.'

The second method is largely used in France,
and is known as ' fractionnement dans le bouillon.'
The sample is first diluted with sterilised water of
known volume. After this one gramme (1 cc.) of
the water is taken up by means of a sterilised capil-
lary pipette, which is dipped four times into the
water at different points of the liquid mass to
obtain the above-mentioned quantity. By this
means a fair sample of the water is obtained. In
the laboratory of Dr. P. Miquel thirty-six small
flasks (each 15 cc. capacity) are each half filled
with sterilised bouillon. These flasks, having each a
glass cap containing a sterilised cotton-wool plug,
are placed in a divided box. Each flask receives
one, two, or three drops of the sample of water, as
the case may be; all the flasks are placed in an
incubator at 30°-36° C. during a period of at least
fifteen days, when the microbian colonies are
counted.

Before introducing the small quantity of water
into either a solid or a liquid medium, the original

THE MICROBES OF WATER 293

sample should be violently shaken to ensure an even
distribution of the microbes throughout the water.

By using Koch's or the plate-cultivation method,
the author l obtained the following average number
of microbes (colonies) in 1 cc.of a sample of water from
theriverWitham(at Lincoln) during the year 1887 : —

January, . . 2,016

February, . 3,488

March, . . 10,287

April, . . 11,692

May, . . 11,923

June, . . 12,000

July, . . 10,184
August, .

September, . 4,110

October, . . 9,621

November, . 10,211

December, . 9,787

These figures (monthly means) give a yearly
mean of 8665 microbes in 1 cc., or quarterly means
as follows :

Spring, . . - . . . . . 11,300

Summer, .-V . ... . 11,092

Autumn, . . . . . . . 7,980

Winter, . ... .-. ... ... . 5,097

From these results the greater number of microbes
in the Witham were during the spring and summer.
Another series of experiments with water from
certain rivers gave the following results : — Witham,
11,860; Irwell, 9230; Thames, 25,745; and the
Seine, 56,219 microbes per cubic centimetre.

Dr. P. F. Frankland 2 has made periodical exami-
nations of the river and well waters from which
the water-supply of London is derived ; and during
the year 1886, he obtained the following number of
colonies (on gelatine-plates) per 1 cc. of water : —

1 Griffiths' Researches on Micro-Organisms, p. 77.

2 Journal of Society of Chemical Industry, vol. iv. (1885), and
vol. vi. (1887); Transactions of Sanitary Institute, vols. viii. and
ix. ; Proc. Roy. Soc., 1885; and Proc. Inst. of Civil Engineers,
1886.

294

MANUAL OF BACTERIOLOGY

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THE MICROBES OF WATER

295

It will be seen from the above analyses that the
number of microbes is greatly reduced by the
methods of nitration, etc., in use by the various
London water companies. The average reduction
in the number of developable microbes present in
the river waters before delivery by the companies
is from 96*2 to 99'1 per cent.

Dr. Miquel1 has also made periodical examina-
tions of the various waters in and around Paris.
Paris takes its supplies of water from the Seine and
Marne. There are three water-works belonging to
the former, and one belonging to the latter river.
During 1890 Miquel obtained the following results
with the Paris water supply : —

SEINE.

MAKNE.

MONTHS.

Ivry.

Austerlitz.

Chaillot.

St. Maur.

January, .

52,670

41,020

85,350

75,960

February,

43,620

59,590

107,590

58,120

March, .

34,710

46,070

80,920

57,750

April, . .

38,640

29,020

86,760

16,310

May, . .

12,930

30,960

37,920

12,890

June, .

28,150

40,340

90,860

14,270

July, . .

14,130

26,830

84,520

10,450

August,

6,780

21,910

121,430

13,570

. September, .

20,220

76,170

227,400

6,410

October,

22,350

42,390

143,120

11,860

November, .

37,720

45,690

144,200

95,690

December, .

78,950

73,820

129,900

62,470

Yearly means,

32,530

44,490

111,660

36,305

Annuaire de VObservatoire de Montsouris, 1887-91.

296

A MANUAL OF BACTERIOLOGY

The above results represent the monthly means of
the number of microbes (colonies) obtained from 1
cc. of water by the ' fractionnement ' method.

The quarterly means are given in the next
table :—

SEINE.

MARNE.

Ivry.

Austerlitz.

Chaillot.

St. Maur.

Winter,
Spring,
Summer,
Autumn,

43,500
26,570
13,710
46,340

48,890
33,440
41,635
53,965

91,285
71,845
144,250
139,070

63,940
14,490
10,140
56,640

From Miquel's analyses the quarterly means of the
number of microbes contained in 1 cc. of sewer-
water (collected at Clichy and St. Ouen, Paris) are
as follows : —

Winter,
Spring,
Summer,
Autumn,

14,780,000

16,760,000

9,638,000

6,375,000

It appears from the observations of Miquel that the
largest number of microbes found in river and sewer
waters is during the spring months. The self-
purification of rivers polluted with sewage has given
rise to a great deal of discussion ; but there is no
doubt that in some rivers the sewage is rapidly
oxidised by the oxygen dissolved in the water or
separated from plants. Professor von Pettenkofer 1
states that sewage may be permitted to flow into a

1 Chemiker Zeitung, 1891.

THE MICROBES OF WATER 297

river if its volume is not more than -j^th that of
the river water and its rate of flow decidedly greater
than that of the current ; and it has been shown
that the Isar, for example, possesses this self-
purification. The bacteriological investigations of
Prausnitz prove the purifying power of the Isar.
The number of 198,000 microbes per cc. found at
the mouth of the Munich sewer was reduced at
Ismaning to 15,231, and at Freisingto 3602. These
results agree with those of other bacteriologists.
Fraenkel found in the water of the Spree above and
below Berlin 6000 microbes per cc., but in the city
a million. It has been stated that ' the mere number
of microbes found has, however, no sanitary signifi-
cance, since the microbes found in the water are
almost exclusively harmless, and, indeed, destroy the
pathogenic microbes in the struggle for existence.'
But it should not be forgotten that Griiber1 and
Frankland2 have shown that Spirillum cholerce
Asiaticce, Bacillus anthracis, etc., are capable of living
and multiplying in sewage, and that the first-named
microbe retained its vitality for 1 1 months ' in com-
pany with countless numbers of a micrococcus which
had accidentally gained access.'

' It is necessary, therefore, to exercise considerable
caution in judging upon this point in the present
state of our knowledge, and it would be highly pre-
mature to place too much reliance upon this alleged ./
destruction of pathogenic forms by non-pathogenic

1 Wiener Medicinische Wochemchrift, 1887.

2 Proceedings of Royal Society, 1886 ; and Journ. Society of
Chemical Industry, vol. vi. (1887).

298 A MANUAL OF BACTERIOLOGY

ones.' With all due respect to such an authority as
Yon Pettenkofer, 'we learn that ordinary sewage
forms a suitable medium not only for the indefinite
preservation of some pathogenic microbes, but also,
in some cases, for their rapid growth and multipli-
cation.'

It is well known that surface waters (e.g. rivers,
ponds, etc.) are rich in microbial life ; but waters,
derived from deep wells and springs, which have
undergone natural filtration through porous strata,
contain only few microbes. Frankland has shown
that ' this removal of microbes from water also takes
place in a very marked manner when it is submitted
to some kinds of artificial filtration, such as that
through very finely-divided coke or charcoal, as well
as in the filtration of water on the large scale through
sand.' A glance at Frankland's table (p. 294) shows
the great reduction in the number of microbes
present in the water obtained from the Thames and
the Lea, after filtration through fine sand. But,
according to Frankland, the following factors are
calculated to influence the number of microbes
present in the distributed water : —

(a) Storage capacity for unfiltered water.
(6) Thickness of fine sand used in filtration.

(d) Renewal of filter-beds.

(a) Through greater storage capacity, the neces-
sity of drawing the worst water from the river is
avoided, a matter which in the case of a stream like
the Thames, liable to frequent floods, is of great
importance. During the period of storage the water

THE MICROBES OF WATER 299

deposits the greater part of its suspended matter,
including a large proportion of the microbes. Then
a further diminution takes place through degenera-
tion and decay of the microbes, for the number of
microbes in the unfiltered river-waters diminishes
on keeping irrespectively of subsidence, probably
owing to the competition between different forms
hostile to each other, as well as by the production
of chemical compounds inimical to their further
multiplication, (b) That the thickness of the filter-
ing stratum should exercise an important influence
on the number of microbes passing through the filter
must be sufficiently obvious. In estimating the
thickness of such a sand filter the fine sand only
should be taken into consideration, as it is only this
portion of the filter which can have any effect in
the removal of microbes, (c) That the filtration is
the more perfect the slower the rate, (d) That the
complete removal of microbes from water, by filtra-
tion, is unattainable without frequent renewal of the
best filtering materials.

' It is often urged that the bacteriological exami-
nation of water is of little practical importance,
inasmuch as the microbes found are not necessarily
prejudicial to health, and that the method of exami-
nation does not aim at the detection of harmful
forms. A little more mature consideration, how-
ever, will show that the actual detection of harm-
ful or pathogenic forms is a matter of very little
importance; and if methods of water purification
are successful in removing microbes in general, and
more especially those which find a suitable home in

300 A MANUAL OF BACTERIOLOGY

natural waters, there can be no serious doubt that
they will be equally successful in removing harmful
forms, which are not specially adapted for life in
water. Could it be, for instance, reasonably con-
tested that a method of purification which is capable
of removing the Bacillus aquatilis from water would
be incapable of disposing of the Bacillus anthracis,
when suspended in the same medium ? The sup-
position is, on the face of it, absurd, and not a par-
ticle of experimental evidence can be adduced in its
favour. It is, therefore, only rational to conclude
that those methods of water purification, both natural
and artificial, which succeed in most reducing the
total number of microbes, will also succeed in most
reducing the number of harmful forms should they
be present ' (Frankland).

There are three methods by which microbes may
be absolutely removed from water. These are by
the agency of (a) electricity ; (b) heat ; (c) filtration
through porous porcelain.

(a) Electricity. — The author 1 has shown that the
electric current is capable of destroying the vitality
of several microbes when growing in liquid media.
For instance —

An E.M.F. of 2*16 volts destroys Bacillus tuberculosis,
,, ,, 2 '26 ,, Bacterium lactis,

,, ,, 3 '24 ,, Bacterium aceti,

„ ,, 3'3 ,, Bacterium allii,

,, ,, 2'72 ,, Bacillus suUilis ;

and an E.M.F. of 180 volts readily destroys the

i Proceedings of Royal Society of Edinburgh, vol. xv. p. 45 ;
vol. xvii. p. 264 ; and Researches on Micro -Organisms, p. 177.

THE MICROBES OF WATER 301

Protozoa contained in ordinary potable waters. Due
to the author's investigations, Mr. E. Meade Bache l
proposes to sterilise city waters by the agency of
electricity. He says that ' after reading the results
of Dr. Griffiths I gladly reverted to the intention
with which I had set out in my experiments of being
able to suggest means by which water supplied to
cities could be sterilised for drinking purposes.
The means at our command seem to me ample. It
is true that we cannot electrolise successfully a large
reservoir of water, for in that the electricity would
be too diffused to be effective. It is true that, in
pipes from which water is flowing into or out of the
reservoir, its germs would not be subjected to attack
for more than a second. It is true that the resist-
ance that we should have to overcome in water
would be large. But the electro-motive force
(E.M.F.) of a few thousand volts (there are dynamos
that generate ten thousand), thrown athwart a pipe
of proper dimensions, would probably paralyse every
bacterium in its path. ... If, upon issuing from as
well as upon entering a reservoir, the water were
attacked in pipes from poles all but encircling them,
with an electro-motive force of a few thousand volts,
all germs must reach the denizens of cities supplied
from such a source wholly innocuous, because they
would be dead.'

Whether electricity is applicable or not for the
sterilisation of water on a large scale, there is no

1 Proceedings of American Philosophical Society, vol. xxix.
1891), pp. 26-39.

302

A MANUAL OF BACTERIOLOGY

doubt that it is a means by which microbes may be
absolutely removed from water in the laboratory.

(b) Heat. — Heat is a means of destroying microbes
in water, but many microbes require a temperature
above the boiling point before they are destroyed.
Dr. Miquel1 has shown that the number of microbes
or colonies decreases gradually as the temperature
of the water is raised. The water of the Seine,
obtained from two different sources, gave the fol-
lowing results : —

Average

Temperature of water
(centigrade scale).

number
of
colonies

Temperature of water
(centigrade scale).

number
of
colonies

per cc.

At 20° ...

464

At 22° ...

848

45°duringl5mins.

396

43° during 15 mins.

640

55°

j .

50°

}

132

65°

j .

20-8

60°

75°

j .

9-6

70°

27-2

85°

, .

6-6

80°

26-4

95°

, .

2-8

90°

14-4

100°

3-3

100°

•

5-2

Although some microbes are capable of withstand-
ing the action of boiling water for 15 minutes, they
are all destroyed when the temperature is raised to
110°— 115° C. for the same space of time. MM.
Rouart and Geneste-Herscher have devised an ap-
paratus in which large quantities of water may be
sterilised by the action of heat (see Miquel's book,
loc. cit., p. 188).

1 Manuel Pratique d' Analyse Bactdriologique des JEaux,
p. 182.

THE MICROBES OF WATER 303

(c) Filtration through porous porcelain. — This is
the last method for the absolute sterilisation of
water. The Chamberland filter, which is univer-
sally used in bacteriological laboratories, is a device
by means of which water is forced through porous
porcelain. This filter may be attached to an ordinary
water-tap if the pressure of the water-supply is suf-
ficient to force the water through the porcelain ; if
not, a small force pump is required.

Certain bacteriologists classify waters according to
the number of colonies revealed on cultivation. For
instance, Miquel gives the following standard for
the classification of waters : —

A water excessively pure yields from 0 to 10 colonies per cc.

,, very pure ,, , 10 to 100

pure „

ordinary (mediocre) „
impure „

very impure , ,

100 to 1,000
1,000 to 10,000
10,000 to 100,000
100,000 or more

Such standards as this one are of little value, because
a water which reveals only 10 to 100 colonies may
be a worse water for drinking purposes than one
which reveals 1000 colonies. The former may be
contaminated with sewage, and, consequently, would
form a suitable medium for the further development
of microbes, whereas the latter may be free from
sewage; therefore it would be the better water of
the two. The fitness or otherwise of water for
drinking purposes cannot be pronounced from the
number of colonies obtained in a few quantitative
determinations. 'It must never be forgotten that
the sanitary examination of water is surrounded
with such difficulties that it is only by bringing to

304 A MANUAL OF BACTERIOLOGY

bear on such particular case all the evidence that it
is possible to obtain, and then interpreting this
evidence by the light of an extended experience,
that a sound judgment can be arrived at.' l

From what has been said in the last three chap-
ters, it will be seen that we live in a world that is
teeming with life. The air, the soil, the waters of

- ocean, river, and pond swarm with living microbes,
each more or less perfectly adapted to the conditions
of its existence. Many problems arise with regard

f to this world of living things ; but suffice it to say
that almost every drop of water which evaporates
into the air carries with it germs, and there is no
reason to suppose that the germs perish. On the
contrary, there is much to lead us to believe that
the germs have far greater powers of resisting high
temperatures, desiccation, and other adverse con-
ditions than the fully developed microbes. We
may thus see how the air (and probably the soil)
comes to be laden with germs which, should they
fall into an appropriate infusion, or into water, may
give rise to the teeming life which we know to be
so soon developed in it.

1 For further information see Fabre-Domerque's Manuel
Pratique d1 Analyse Micrographique des Eaux (1890) ; Salazar
and Newman's Examen Quimico y Bacteriol6gico de las Aquas
Potables (1890) ; Giglioli's Fermenti e Microbi (1887) j Frank in
Zeitschrift fur Anal. Chemie, vol. xxx. p. 305 ; and Roux's
Precis d' Analyse Microbiologique des Eaux (1891).

CHAPTER X

THE PTOMAINES AND SOLUBLE FERMENTS

THE advancement of organic chemistry has increased
our knowledge of the alkaloids occurring in the
vegetal kingdom — bodies which are of great import-
ance both from a therapeutical and a toxicological
aspect. Since the year 1872 a new source has been
discovered of the natural origin of alkaloids, viz.,
from the animal kingdom, and the knowledge and
investigation of these bodies have proved of great
service in the study of both physiological and
pathological chemical processes.

The ptomaines (TTT&ILCL = corpse) were first dis-
covered in decomposing animal tissues, as their
pseudonym of cadaveric alkaloids implies. Their
presence in these dead tissues introduced a new
factor in the post-mortem search for poisons in
suspected cases of murder. This subject was
brought into prominence by a murder trial in
Rome, in which a man was accused of poisoning
his master by administering delphinine. The
accused was acquitted because the alkaloid ob-
tained from the dead body differed in many of its
reactions from those of delphinine ; in other words,
the poison extracted from the body was a ptomaine
produced by microbes after death. In 1882, G. H.

306 A MANUAL OF BACTERIOLOGY

Lamson was accused of murdering his brother-in-law
with aconitine, a vegetable alkaloid. The defence
set up was that the alkaloid found in the dead body
was one of the ptomaines produced after death.
But it was conclusively proved by Drs. Dupre' and
Stevenson that the dead body contained aconitine
which had been administered during life ; con-
sequently Lamson was executed l for murdering his
brother-in-law. It will be seen from these remarks
that the subject of ptomaines opens up an important
point in all cases of poisoning where the poison is
of an alkaloidal nature.

A more important result of the discovery of
ptomaines has been the explanation of the cases of
poisoning by decayed animal foods, such as sausages,
fish,2 ' tinned' and putrid meats, in which they have
been found.

The ptomaines are produced during the process
of putrefaction, etc., of animal substances. By the
direct action of microbes, the albuminoid molecules
are disintegrated with the formation of ptomaines
among other products. From this remark, it will be
seen that the ptomaines are not secreted or excreted
by microbes, for they are the residua after microbian
action. Not only have putrefactive microbes the
power of giving rise to ptomaines, but certain patho-
genic microbes yield ptomaines, or toxines, as
Brieger calls them, when they are the products of
microbian diseases.

1 At Wandsworth on April 28, 1882.

2 See Dr. Griffiths' paper, ' The Poisoning of a Family by
Mussels,' in Chemical News, vol. Ixii. p. 17,

THE PTOMAINES AND SOLUBLE FERMENTS 307

(a) General properties of the Ptomaines. — All the
ptomaines (cadaveric) are soluble in alcoholic ether.
Many of them dissolve in chloroform and amylic
alcohol. The general reagents which precipitate
the ptomaines are the following: — Myer's and
Nessler's reagents, a solution of iodine in potassium
iodide, the iodide of bismuth and potassium, and
the phosphomolybdate of sodium. Mercuric chlo-
ride sometimes precipitates and sometimes does not
precipitate the ptomaines, but it generally forms
with them a double crystallisable chloride deposited
from boiling water. Auric chloride often gives a
yellow precipitate, soluble in water, or generally a
very soluble aurochloride, which rapidly dissolves.
Picric acid forms slightly soluble picrates. Tannin
produces insoluble, as well as very slightly soluble
tannates. Sulphuric acid diluted with a very small
quantity of water produces a red- violet colour with
the ptomaines. Hydrochloric acid gives a red-
violet colour, which heat develops. Platinic chlo-
ride generally forms crystallisable compounds with
the ptomaines. The majority of the ptomaines are
crystallisable compounds, although a few are liquids.
They are all, more or less, of a poisonous nature.

(b) Extraction of the Ptomaines.— The three most
important methods for extracting ptomaines from
putrefying and other material are those of Gautier,
Brieger, and Luff. (1) Gautier' s method consists in
adding oxalic acid to the warm liquor of bacterial
fermentation. By this means fatty or oily liquors
are liberated, and float on the surface of the liquor.
After separating the fats, the liquor is filtered. The

308 A MANUAL OF BACTERIOLOGY

filtrate is distilled, when pyrrol, skatol, phenol,
indol, the volatile fatty acids, and a portion of the
ammonia, are driven off. Gautier then adds lime
(until alkaline) to the portion which has not been
distilled, separates the precipitate which forms and
which contains the greater portion of the fixed fatty
acids, and he then distils the alkaline liquor to
dryness in vacuo, taking care to condense the
vapours in weak sulphuric acid. The bases are
then distilled with ammonia. After the distillation
is completed, the distillate is neutralised, then
evaporated nearly to dryness, when ammonium
sulphate deposits in the crystalline condition. This
is separated and rejected. Concentrated alcohol is
now added to the mother liquor, which dissolves
the sulphates of the ptomaines. After evaporating
off the alcohol, a small quantity of caustic soda
solution is added. This solution is successively
treated with ether, petroleum ether, and chloroform
(i.e. three different extracts are obtained). As to
the product remaining in the retort with the excess
of lime which had served to separate the bases or
ptomaines, it is treated with ether at 36° C., which
dissolves the fixed bases. By the addition of a
small quantity of acidulated water, the bases are
separated from the ether, and are then easily pre-
cipitated by the addition of an alkali. (2) Briegers
method consists in boiling the putrefying material
with water and then filtering. The filtrate is pre-
cipitated with plumbic acetate. This precipitate is
filtered off a stream of sulphuretted hydrogen is
passed through the filtrate, and the lead sulphide

THE PTOMAINES AND SOLUBLE FERMENTS 309

separated by filtration. This filtrate is evaporated
to a thin syrup, and this is extracted with amylic
alcohol The amylic solution is treated with water,
concentrated by evaporation, then acidulated with
sulphuric acid, and washed several times with ether,
which frees it from the oxy-aromatic acids. The
aqueous-acid liquor is then concentrated to a
quarter of its volume. After standing twenty-four
hours, the precipitate which forms is dissolved in
boiling water and decomposed by sulphuretted
hydrogen. In concentrating the liquors, Brieger
crystallises at once various mineral or organic salts
which are rejected, then the dried residue is treated
with absolute alcohol, which, after concentration,
deposits the putrefactive bases or ptomaines in the
crystalline condition. The various ptomaines are
now separated by the difference of their solubility,
or by fractional precipitation with platinic chloride,
auric chloride, picric acid, etc.1 (3) Luff's method
is used for the extraction of ptomaines contained
in abnormal urines. A considerable quantity of
the urine is made alkaline by a solution of sodium
carbonate, and then agitated with half its volume
of ether. The ethereal solution (after standing) is
filtered and agitated with a solution of tartaric acid.
The tartaric acid combines with any ptomaines pre-
sent, forming soluble tartrates, and the solution of
tartrates forms the lower layer of the liquid mass.
The tartaric acid solution (after being separated
from the ether) is also made alkaline by the

JSee Brieger's Ueber Ptomaine 1885; Welter e Untersuchun-
gen ilber Ptomaine, 1885 ; Untersuchungen uber Ptomaine, 1886.

310 A MANUAL OF BACTERIOLOGY

addition of sodium carbonate, and is once more
agitated with half its volume of ether. The ethereal
solution (after standing) is separated, and the ether
allowed to evaporate spontaneously. The residue
(after drying over sulphuric acid) is finally examined
for ptomaines.

The ptomaines or animal alkaloids are divided
into two groups — the non-oxygenous and the
oxygenous ptomaines.

(A.) The Non-oxygenous Ptomaines.

Parvoline (C9H13N). — This base was isolated by
Gautier and Etard, from mackerel and horse-flesh
after bacterial putrefaction. It is an oily yellow
base, with the odour of hawthorn.

Collidine (C8HnN) or isophenylethylamine

C6H5 — CH was discovered by Nencki in

1876, among the products of the bacterial putrefac-
tion of gelatine and the pancreas of the ox. An
isomeride of this ptomaine was isolated by De
Coninck from the muscular tissues of the cuttle-
fish (Sepia) ; and it is probable that it has the con-
stitutional formula of dihydropyridine. Collidine
is a yellow fluid with an offensive odour ; and it
is soluble in water, alcohol, and ether.

Coridine (C10H15N). — This ptomaine was ex-
tracted by Guareschi and Mosso from the bacterial
putrefaction of fibrin. It is an oily fluid, having a
slight odour of pyridine and conicine. It forms a

THE PTOMAINES AND SOLUBLE FERMENTS 311

crystalline platinochloride, an aurochloride, and a
hydrochloride. De Coninck has extracted a base
with the same formula from the muscular tissues
of the cuttle-fish after bacterial putrefaction.

Hydrolutidine (C7HUN). — This base was extracted
by Gautier and Mourgues from cod-liver oil. It is
a colourless body, which is slightly oily and very
alkaline. It is slightly soluble in water, and forms
crystalline double salts with the chlorides of gold
and platinum.

Hydrocollidine (C8H13N). — This ptomaine was ex-
tracted by chloroform 'from the bacterial putrefac-
tion of mackerel, horse-flesh, and other albuminous
matters. It is a colourless liquid of the odour of
syringa, and has a density of 1*029 at 0° C. This
base forms crystalline double salts with the chlorides
of hydrogen, gold, and platinum. Hydrocollidine
is very poisonous; in fact, Gautier says of this
ptomaine : ' Elle determine du tremblement, des
convulsions tetaniques, 1'animal meurt avec le cceur
en diastole gorge de sang.'

Hydrocoridine (C10H17N). — This base was ex-
tracted by the author1 from pure cultivations of
Bacterium allii on nutrient agar-agar. It is a white
solid, soluble in warm water, alcohol, ether, and
chloroform. It crystallises from water in micro-
scopic needles belonging to the prismatic system.
These crystals are extremely deliquescent, and have

1 See Griffiths' papers in Comptes Rendus de I'Acaddmie des
Sciences, vol. ex. p. 418 ; Centralblatt fur Bakteriologie und
Parasitenkunde, Bd. 7, p. 808 ; Chemical News, vol. xli. p. 145 ;
and Gautier's Chimie Biologique (1892), p. 268.

312 A MANUAL OF BACTERIOLOGY

the odour of hawthorn, especially when heated.
This ptomaine forms crystalline double salts with
the chlorides of platinum and gold.

It appears that the ptomaines just described be-
long to, or are derivatives of, the pyridine series of
organic bases.

Neuridine (C5H14N2). — This ptomaine was dis-
covered by Professor Brieger as a constant product
of the bacterial putrefaction of albuminous sub-
stances. Its hydrochloride, platinochloride, and
aurochloride have been obtained in the crystalline
condition ; but the free base is so unstable that it
has never been obtained pure. A solution of sodium
hydroxide decomposes neuridine hydrochloride into
dimethylamine and trimethylamine.

Saprine (C5H14N2). — This base is isomeric with
the preceding, but differs from it in the solubilities
of its salts, and probably also in chemical constitu-
tion. It was discovered by Brieger in 1883.

Cadaverine (C5H14N2). — Brieger isolated a third
isomeride from albuminous substances subjected to
prolonged putrefaction. This base is also readily
formed in cultivations of Finkler's spirillum. Dr.
Ladenburg l proved that this ptomaine has all the
chemical and physical properties of pentamethyl-
enediamine. It is a non-poisonous liquid with an
alkaline reaction. This base boils at 115° C., and it
has the odour of conicine.

Putrescine (C4H12N2) is usually found accom-
panying cadaverine, but makes its appearance

1 Berichte der deutschen chemischen Gesellschaft, vol. xix.
p. 2586.

THE PTOMAINES AND SOLUBLE FERMENTS 313

rather later. It is a volatile liquid, with a sperma-
tic odour, and boils at 135° C. Baumann and
Udranszky proved that this ptomaine has the con-
stitutional formula of tetramethylenediamine, NH2
(CH2)4NH2. Both cadaverine and putrescine have
been isolated from the fseces and urine in cases of
cystinuria.2

Mydaleine. — This base was isolated (along with
cadaverine and putrescine) by Brieger during the
putrefaction of albuminous substances. It is a
poisonous ptomaine which causes paralysis and
death. Mydaleine is believed to be a diamine,
but it has not been thoroughly examined.

Brieger has also isolated two ptomaines from pure
cultivations of the tetanus bacillus, which are pro-
bably diamines. One is called spasmotoxine, and
produces tonic and clonic convulsions, while the
other (which has not been named) causes tetanus,
accompanied with a flow of saliva and tears.

Tyrotoodcon (C6H5N2). — In 1886, Vaughan isolated
this ptomaine from cheese, milk, and ice-cream
which had undergone putrefaction. Tyrotoxicon
produces nausea, diarrhoea, and acute poisoning, and
is said to be identical with diazobenzene.

Phlogosin (formula unknown) is a ptomaine which
was obtained by Leber in 1888 from pure cultiva-
tions of Staphylococcus aureus. It is probably a
diamine.

Methylguanidine C2H7N3 or NH=C

\NH2

1 See also Dr. Lauder Brun ton's Disorders of Digestion, p. 281.

314 A MANUAL OF BACTERIOLOGY

is a very poisonous base, and it is produced when
Finkler's spirillum is allowed to live upon sterilised
beef along with putrefactive microbes. This sub-
stance is believed to be the cause of cholera
nostras.

Spermine (C2H5N) or dimethylenimide (CH2)2NH
occurs in the seminal fluid of animals, and accord-
ing to Schreiner it is produced in cultivations of the
tubercle bacillus, but the author could not detect
the least trace of this base in pure cultivations of
Bacillus tuberculosis.

EtJiylenediamine (C2H8N2). — In 1885, Brieger ex-
tracted this ptomaine from cod-fish after bacterial
putrefaction. It forms crystallisable double salts
with the chlorides of hydrogen and platinum.

Tetanotoxin (C5HnN). — This ptomaine was ex-
tracted by Brieger from pure cultivations of the
tetanus bacillus. It is a colourless liquid which
boils at 100° C., and possesses a disagreeable odour.
When it is injected into animals it produces tremor
and paralysis, followed by violent convulsions.

Methylamines. — Methylamine CH3NH2, dimethyl-
amine (CH3)2NH, and trimethylamine (CH3)3N,
have been extracted from the tissues of various
animals. Triethylamine (C2H5)3N and propylamine
C3H7NH2 have been obtained from putrified animal
substances. While Gautier and Mourgues extracted
ptomaines, having the composition of butylamine
C4H9NH2, amylamine C5HnNH2, and hexylamine
C6H13NH2 from cod-liver oil. These bases are
poisonous.

Scombrine (C17H38N4). — This base was discovered

THE PTOMAINES AND SOLUBLE FERMENTS 315

by Gautier and Etard1 in certain extracts of
mackerel which had undergone bacterial putrefac-
tion. It has the odour of syringa, and is decom-
posed at 100° C. Scombrine forms a crystallisable
platinochloride which is soluble in water.

Morrhuine (C19H27N3). — This ptomaine was iso-
lated by Gautier and Mourgues from cod-liver oil.
It is a yellowish liquid with the odour of syringa,
and is very alkaline and caustic.

Aselline (C^HggNJ. — This base was also isolated
by the same authorities from cod-liver oil. It is an
inodorous solid, which is soluble in ether and
alcohol, but insoluble in water. In large doses it is
poisonous.

(B.) The Oxygenous Ptomaines.

Propylglycocyamine (C6H13N302.) — This ptomaine
was extracted by the author 2 from the urine in a
case of parotitis or mumps, where the kidneys were
involved. It crystallises in white prismatic needles,
which are soluble in water, ether, and chloroform.
This base has a neutral reaction, a slightly bitter
taste, and forms a yellow crystalline platinochloride,
a pale yellow aurochloride, and a white crystalline
hydrochloride. When boiled with oxidising agents
it yields creatine (methylglycocyamine) and finally
methylguanidine and oxalic acid. This ptomaine

1 See Gautier's Chimie Biologique (1892), p. 268.

2 Comptes Rendu*, tome cxiii. p. 656 ; Chemical News, vol.
Ixi. p. 87 ; and Bulletin de la Societe Chimique de Paris, 3e serie,
tome iv. p. 333.

316 A MANUAL OF BACTERIOLOGY

has the constitutional formula of propylglycocya-

mine HN = C It is poisonous

XN(C3H7).CH2.C02H.

and when administered to a cat it produced
nervous excitement, cessation of the salivary flow,
convulsions, and death. This base is not found in
normal urines, it is therefore produced within the
system during the course of the disease, which is
highly infectious.

Neurine (C5H13NO). — This ptomaine is a constant
product of cadaveric putrefaction. It is a syrupy
base, soluble in water, and has a strong alkaline
reaction. It forms a crystallisable platinochloride,
besides other double salts. It is a poisonous base :
O'Ol gram, of neurine kills a cat, and 0*04 gram.
kills a rabbit. Brieger states that this ptomaine has
the same formula as trimethylvinylammonium
hydroxide (CH3)3(C2H3)N.OH.

Choline (C5H15N02). — Like neurine, this ptomaine
is a constant product of cadaveric putrefaction. It
is a syrupy base, soluble in alcohol and ether, and
has a strong alkaline reaction. It forms double salts
with the chlorides of hydrogen, gold, and platinum ;
and it also forms compounds with carbonic and
sulphuric acids. When heated, choline is decom-
posed into glycol and trimethylamine. Choline in
small doses produces pyrexia: in larger doses it
produces paralysis due to poisoning of the motor
end-plates. This ptomaine has the same constitu-
tional formula as trimethyloxyethyleneainmonium
hydroxide (CH3)3(C2H4— OH)N.OH.

THE PTOMAINES AND SOLUBLE FERMENTS 317

Muscarine (C5H3N02). — Brieger isolated this
ptomaine from putrid fish. It also occurs in the
poisonous mushroom (Agaricus muscarius). Mus-
carine is a crystalline deliquescent substance, which
acts on the muscular tissues.

Gadinine (C7H16N02) was obtained by Brieger,
along with muscarine, from putrefying cod-fish. It
forms double salts with the chlorides of hydrogen,
gold, and platinum.

Mytilotoxine (C7H15N02) was isolated by Brieger
from decomposing mussel, and it is the active agent
in mussel-poisoning.

Typhotoxine (C7H17N02). — This ptomaine was ex-
tracted by Brieger from pure cultivations of the
typhoid bacillus. It has an alkaline reaction, and
forms crystallisable salts with phosphotungstic acid
and the chlorides of hydrogen and gold. It is be-
lieved to be the chemical poison in typhoid fever.

In 1886 Brieger obtained an isomeride of this
base from flesh which had undergone bacterial
putrefaction. Although this substance has the
same empirical formula as typhotoxine, they are
entirely different ptomaines.

Scarlatinine (C5H12N04). — This ptomaine was ex-
tracted by the author * from the urine of patients
suffering from scarlet fever, as well as from pure
cultivations of Micrococcus scarlatince. It is a white
crystalline body, which is soluble in water, and has
a faint alkaline reaction. It forms double salts

1 Griffiths in Comptes Rendus de I'Acaddmie des Sciences.
vol. cxiii. p. 656; Proc. R.S.E., vol. xix. p. 97.

318 A MANUAL OF BACTERIOLOGY

with the chlorides of hydrogen and gold, and it is
precipitated by phosphomolybdic, picric, and phos-
photungstic acids.

Tetanine (C13H22lSr204). — Brieger succeeded in
isolating this ptomaine from pure cultivations of
the tetanus bacillus. This base produces tetanic
convulsions and death. Its hydrochloride is very
deliquescent. In 1888 Brieger1 obtained another
oxygenous ptomaine from pure cultivations of the
tetanus bacillus. It is represented by the formula
C6H13N02, and is non-poisonous.

Diphtherine (CUH17N206). — This base was ex-
tracted by the author 2 from the urine of patients
suffering from diphtheria, as well as from pure cul-
tivations of Bacillus diphtheria (bacillus No. 2 of
Klebs and Loffler). It is a white crystalline base,
and it forms double salts with the chlorides of
hydrogen and gold. It is precipitated by tannic,
picric, and phosphomolybdic acids.

Unknown Base (C5HnNO).— This ptomaine was
extracted by E. and H. Salkowski from putrid fibrin.
It forms double salts with the chlorides of hydrogen
and platinum. Gabriel and Aschan 3 have recently
proved that this ptomaine is &-amidovaleric acid.

Unknown Base (C14H20N"204).— This base, which
is believed to be an amido-acid, was extracted
by Guareschi4 from putrid fibrin. It occurs as

1 Virchow's Archiv, Bd. 112, p. 550 ; Bd. 115, p. 490.

2 Comptes Rendus, vol. cxiii. p. 656.

3 Berichte der deutschen chemischen Gesellschaft, vol. xxiv.
p. 1364.

4 Annali di Chimica e di Farmocologia, vol. Ixxxvii. p. 237.

THE PTOMAINES AND SOLUBLE FERMENTS 319

beautiful shining plates, which melt at about 250°C.
This ptomaine is soluble in water, ether, and
chloroform.

Mydine (C8HnNO). — This ptomaine was ex-
tracted by Brieger from human corpses and pure
cultivations of the typhoid bacillus. It is non-
poisonous, and its picrate melts at 195° C.

Betaine (C5HnN02) or trimethylglycocine was
first isolated from urine by Liebreich in 1869. It
is related to neurine and choline. In 1885, Brieger
extracted the same base from poisonous and non-
poisonous mussels. Betaine is a non-poisonous
ptomaine.

Pyocyanin (C14H14N02). — This is the greenish
pigment produced by Bacillus pyocyaneus. It is
soluble in water, alcohol, and chloroform. Accord-
ing to Ledderhose, this compound appears to be a
derivative of anthracene.

Unknown Bases (CyH^Og and C5H12N204).—
Dr. G. Pouchet extracted both of these bases from
putrid animal substances. They are very poisonous,
and give rise to crystalline hydrochlorides and
platinochlorides.

Sucholotoxine. — This is a ptomaine extracted from
pure cultivations of the microbe of hog cholera. It
forms double salts with the chlorides of platinum
and hydrogen. Yon Schweinitz1 states that this
base is very poisonous.

Suplagatoxine. — This was extracted by Von
Schweinitz 2 from pure cultivations of the microbe
of swine plague. It is also poisonous. The chemical

1 Journ. Amer. Chem. Soc., 1891. 2 Ibid.

320 A MANUAL OF BACTERIOLOGY

formulae of Von Schweinitz's ptomaines have not
been ascertained.

Various bases, of unknown composition, have
been extracted from urine, faeces, and tissues in
certain infectious and contagious diseases; and in
addition to these there is another class of animal
alkaloids which have been termed leucomaines by
Gautier. According to Gautier the leucomaines
are excretory products (like urea, carbonic acid,
etc.) formed by ' vital physiological processes ' from
albuminous substances, consequently they must be
eliminated from, or destroyed in, the system, or
disease will be the result of their poisonous action.
"We resist, therefore, incessant auto-infection by two
distinct mechanisms ; the elimination of the leuco-
maines by means of the excretory organs, and by
the destruction of the leucomaines by means of the
oxygen contained in the blood. 'Some of these
leucomaines are exceedingly poisonous, and when
retained may give rise to very serious toxic symp-
toms. Brieger and others, however, deny that any
such bodies are formed, or at any rate have yet
been found in the tissues of the living body, or
that they owe their existence to the tissues. They
consider that they are simply absorbed from the
intestinal canal where they are formed by bacteria '
(Woodhead).1

It should always be borne in mind that ' the
discovery of ptomaines is complemental, not ant-
agonistic, to the germ theory/

1 A full description of the leucomaines is given in the author's
book, Researches on Micro- Organisms, pp. 121-134.

THE PTOMAINES AND SOLUBLE FERMENTS 321

ID addition to the ptomaines, there are the
enzymes and albumoses, which are chemical prin-
ciples, excreted by microbes and allied fungi, or the
products of the activity of other living cells, e.g.
those of the glands of the stomach, pancreas, etc.
Such soluble ferments or enzymes as pepsin,
ptyalin, trypsin, diastase, invertin, and emulsin are
well known to physiologists and chemists. It is
not, however, these bodies which we intend to
describe, but the albumoses produced by living
microbes. In small doses these albumoses are pro-
tective; and they appear to be the protective
principles in most vaccines.

'The albumoses produced by microbes resemble
those formed during normal digestion in being
poisonous when injected directly into the circula-
tion, although they may not be so greatly absorbed
from the intestinal canal. One of the most remark-
able discoveries in regard to albuminous bodies is
the fact that some of them which are perfectly
innocuous, and, indeed, probably advantageous to
the organism in their own place, become most
deadly poisons when they get out of it. Thus the
thyroid and thymus glands, which are perfectly
harmless and probably useful, were found by
Wooldridge, when broken up in water, to yield a
proteid which instantaneously coagulated the blood
if injected into a vein, so that the animal died as if
struck by lightning; while Schmidt-Mlihlheim, under
Lud wig's directi on, found that peptones had an exactly
opposite effect,and prevented coagulation altogether.'1

1 Dr. Lauder Brunton in Nature, vol. xliv. (1891), p. 330.
X

322 A MANUAL OF BACTERIOLOGY

Dr. Bitter, in 1887, 'furnished rigorous proof
that microbes produce album oses separable from
the organisms which form them. He managed to
kill the microbes by sterilisation at 60°C. without
materially destroying their products, and in this
way demonstrated that two microbes, when grown
on gelatine, produced albumoses which were able,
apart from the microbes, to liquefy gelatine and
peptonise albumin.' In the same year Loffler, after
separating the microbes by means of a Chamber-
land filter, obtained an albumose from pure cultiva-
tions of Bacillus diphtheria?. This albumose is
precipitated by alcohol, and is soluble in water.
Eoux, Yersin, Brieger, and Frankel have obtained a
similar substance from cultivations of the same
microbe. This albumose produces all the charac-
teristic symptoms of diphtheria ; therefore, B. diph-
therice, which excretes this poisonous albumose, or
toxalbumin, as Brieger calls it, is really the cause
of the disease.

Hankin x has extracted an albumose from cultiva-
tions of anthrax bacilli. It is precipitated by
alcohol, and is soluble in water. Martin 2 obtained
two albumoses from pure cultivations of the same
microbe. These albumoses are strongly alkaline;
but they are not so toxic as the ptomaine which
B. anthracis is said to produce.

Among the microbes which excrete albumoses are
the following : —

1 Proc. Roy. Soc.t May 22, 1890.
- Ibid.

THE PTOMAINES AND SOLUBLE FERMENTS 323

Bacillus diphtheria.
Bacillus anthracis.
B<i <• Him tuberculosis.
Spirillum cholerce Asiatics.
Bacillus of hog cholera.
Spirillum tyrogenum.
Bacillus of swine plague.

Bacillus typhosus.
Bacillus of tetanus.
Staphylococcus aureus.
Spirillum Finkleri.
Bacillus urecB.
Bacillus butyric us.
Bacillus malaricB (?).

Drs. Brunton and Macfadyen l have proved that
the albumoses, excreted by certain microbes, have
the power of liquefying gelatine ; and there is every
reason to believe that the liquefaction of gelatine,
during the cultivation of microbes, is due to the
action of albumoses.

V7ery little is known of the composition of the
albumoses ; but their reactions with certain reagents
( Millon's fluid, magnesium sulphate, copper sulphate
and potash, etc.) prove that they are derived from
proteids. They are neither albumins nor globulins ;
in other words, they belong to the albumose group
of bodies.

Two albumoses, sucholoalbumin and suplago-
albumin, which Von Schweinitz 2 extracted from
pure cultivations of the microbes of hog cholera
and swine plague respectively, are white, pulveru-
lent substances, soluble with difficulty in water, and
precipitated from this solution by absolute alcohol.
They can be obtained in crystalline plates by drying
over sulphuric acid in vacuo.

In addition to the ptomaines and albumoses,
other substances are formed by microbes. Among

1 Proc. Roy. Soc.t vol. xlvi. (1889), p. 542.
- Journ. Amer. Chem. Soc., 1891.

324 A MANUAL OF BACTERIOLOGY

these are the various coloured pigments ; but the
chemistry of the microbian pigments is a subject
which has been very little investigated. They are
undoubtedly products formed from the decomposi-
tion of albuminoids by the agency of microbes.1

In concluding the chapter, it may be stated that
the substances which microbes produce put a stop
to their activity; thus the alcohol produced by
yeast, the phenol, cresol. etc., produced by putre-
factive microbes, are themselves germicides, which
ultimately kill the organisms that produce them.

1 Concerning the composition of the red pigment produced by
M. prodigiosus, see the author's paper iii Comptes Rendus, vol.
cxv. p. 321. The green pigment — pyocyanin — has already been
described (p. 319). The author has described ptomaines in
glanders, pneumonia, and puerperal fever ; and also one pro-
duced by M. tetragonus (see Comptes Rendus, vols. cxiv. and
cxv.).

CHAPTER XI

GERMICIDES AND ANTISEPTICS

THE substances which destroy the vitality of
microbes are called germicides or disinfectants ;
while those which simply retard or hinder the
growth of microbes are generally spoken of as
antiseptics. It must be borne in mind that this is
only a conventional classification or division, for a
germicide may become an antiseptic by simply
reducing its strength; and, conversely, an anti-
septic (as a rule) may become a germicide by in-
creasing its strength.

Among the more common salts Mr. 0. T. King-
zett1 has proved that the chlorides, nitrates, and
sulphates of the alkalis exhibit but slight antiseptic
and germicidal effects, and those of the alkaline
earths are not much better. The same salts of
manganese, zinc, tin, iron, lead, and aluminium are
all of more or less pronounced value. As a rule
the chlorides are to be preferred. The same salts
of copper and mercury are comparatively most
effective; the nitrate of mercury is, however, not
so reliable as the chloride, which is, according to

1 Journ. Soc. Chem. Industry, vols. vi. and vii.

326 A MANUAL OF BACTERIOLOGY

Kingzett, the most active antiseptic and germicide
among these classes of substances.

Edington l has shown that mercuric chloride dis-
solved in water (rendered acid) in the proportion of
1 part in 1000 destroys the spores of Bacillus
anthracis in fifteen minutes, for the spores after this
treatment and subsequent washing in sterilised
water refused to grow on nutrient agar-agar. Mer-
curic chloride also destroys Bacterium allii, Micro-
coccus tetragonus, M. prodigiosus, M. violaceus, Sarcina
lutea, Bacillus subtilis, and other microbes.2 Perhaps
a more powerful germicide than mercuric chloride
is mercuric iodide; and Woodhead3 has used a
solution containing ' 1 gramme of mercuric iodide
with a slight excess of potassium iodide in 1000 cc.
of distilled water.'

Chlorine gas and the vapours of bromine and
iodine are powerful germicides, readily destroying
most microbes. According to the author's 4 investi-
gations, the germicidal power of the three halogen
elements is inversely as their atomic weights (Cl =
35*5; Br = 80; I = 127), i.e. chlorine is the most
powerful germicide, then bromine, and finally iodine.
In fact, the germicidal power of these elements
coincides with their chemical affinities ; but this
remark does not apply to the salts containing these
elements. Iodine,5 potassium iodide, sodium iodide,

1 British Medical Journal, 1889.

2 Griffiths' Researches on Micro- Organisms, p. 204.

3 Proceedings of Royal Society of Edinburgh, vol. xv. p. 246.

4 Loc. cit., p. 182.

5 Griffiths in Proc. Roy. Soc. Edinburgh, vol. xv. p. 37.

GERMICIDES AND ANTISEPTICS 327

ethyl iodide, potassium iodate,1 bromine, ethyl
bromide, chlorine, ferric chloride, and sodium fluo-
silicate - (' salufer ') destroy many microbes, includ-
ing Bacillus tuberculosis, Sarcina lutea, Bacterium
allii, B. cedematis maligni, and B. subtilis.

Many of the derivatives of benzene and its horao-
logues are powerful germicides. Among these com-
pounds may be mentioned the following : benzoic
acid, sodium benzoate, sodium benzenesulphinate,
salicylic acid, sodium salicylate, carbolic acid,
sodium carbolate, etc. ; and the late Dr. T. Car-
nelley 3 proved that ' the para-compounds (of
benzene) are usually more powerfully antiseptic
than the corresponding ortho- and meta-compounds.'4
For instance, it has been shown that of the three
sodium nitrobenzoates, it required 101-6 grammes
of the ortho-compound, 12'1 grammes of the meta-
compound, and 7*7 grammes of the para-compound
respectively to sterilise 1 litre of nutrient gelatine.
There are, however, a few exceptions to this rule :
among these are the three sodium hydroxybenzoates:
it required 11 '6 grammes of the ortho-compound
(sodium salicylate), 67'2 grammes of the meta-
compound, and 162'1 grammes of the para-com-

1 Griffiths in Proc. Roy. Soc. Edinburgh, vol. xvii. p. 257.

- Thomson in Chemical News, vol. Ivi. p. 132.

:{ Journal of Chemical Society, 1890, p. 636.

4 It may be stated that a derivative of benzene with a certain
empirical formula may exist in three isomeric modifications.
Although these isomerides have the same empirical formula,
their constitutional formulae, and consequently their properties,
are entirely different. This difference depends upon the relative
positions of the elements or groups of elements introduced into
the molecules. If we represent the orientation of the side-

328 A MANUAL OF BACTERIOLOGY

pound respectively to' sterilise 1 litre of nutrient
gelatine.

The author 1 has shown that a saturated solution
of salicylic acid destroys Sarcina lutea, M. prodigio-
sus, Bacillus tuberculosis, Bacterium allii,M.tetragonust
Bacterium lactis, Bacterium aceti, M. aurantiacus,
Bacillus subtilis, Leptotlirix buccalis, M. urece, Bacillus
butyricus, etc. It should be borne in mind that
salicylic acid is a more powerful germicide than
sodium salicylate; and that the natural salicylic
acid is a more powerful germicide than the artificial
variety.

Koch has proved that a 3 per cent, solution of
carbolic acid completely destroyed the spores of
Bacillus anthracis in seven days, while a 5 per cent,
solution destroyed them in two days. A 1 per cent.

chains in benzene derivatives by numerals, the terms ortho-,
meta-, and para-compounds are readily understood : —

The term ortho- is always applied to the positions 2 and 6 in
relation to 1.

The term meta- is always applied to the positions 3 and 5 in
relation to 1.

The term para- is always applied to the position 4 in relation
to 1.

1 Proc. Roy. Soc., Edinburgh, vol. xiii. p. 527; vol. xiv.
p. 97 ; vol. xv. p. 37 ; vol. xvii. p. 257 ; and Researches on
Micro-Organisms, p. 223 seq.

GERMICIDES AND ANTISEPTICS 329

solution easily destroyed the sporeless bacilli, but
in a '5 per cent, solution they were not destroyed.

As an antiseptic agent carbolic acid, in the shape
of dressings and lotions, and as a spray in surgical (
operations, is of the greatest value ; but, according
to Jalan de la Croix,1 its germicidal properties are
inferior to those of salicylic acid.

Among the oxidising germicides and antiseptics
are the following : — Hydrogen dioxide, ozone, ' sani-
tas oil,' 'sanitas fluid/ potassium permanganate
(Condy's fluid), and turpentine oil. All these sub-
stances have germicidal and antiseptic properties,
which are due (directly or indirectly) to the libera-
tion of nascent oxygen.

The author has shown that when silk threads,
impregnated with tubercle bacilli and the bacilli of
hay fever, were placed in a mixture containing 5 cc.
of 'sanitas oil' and 100 cc. of water for seven days,
the microbes were completely destroyed by this
powerful oxidising agent.

Mr. C. T. Kingzett, F.C.S.,2 has performed a
large number of experiments with ' sanitas oil ' and
' sanitas fluid,' and his experiments prove the high
value of these preparations as germicidal and anti-
septic agents.

In addition to the above-mentioned germicides
and antiseptics there are many others, among these
being the following : — Sulphurous anhydride, alka-
line sulphides and hyposulphites, hydrogen sulphide

1 Arvhiefur Experim. PathoL, vol. xiii.

2 Nature's Hygiene (3d ed.), pp. 319-351.

330 A MANUAL OF BACTERIOLOGY

quinine, a- and yS- naphthol, arsenious acid, sodium
arsenite, potassium arsenite, arsenic acid, alcohols,
boric acid, certain essential oils, etc. Heat,
electricity, and certain gases have also the power
of destroying microbes.

As antisepsis and disinfection play such impor-
tant parts in medicine, surgery, and sanitation, it is
desirable that greater attention should be paid to
the investigation of the action of various chemicals,
etc., on microbes than has hitherto been the case.

CONCLUDING REMARKS.

We have seen that microbes are omnipresent,
being so light in weight they are readily carried
over thousands and thousands of miles by air
currents without losing their vitality. This is not
surprising when we bear in mind that Eome has
been showered with the sands of Sahara, France
with South American diatoms, and that the volcanic
dust from Cotopaxi fell thousands of miles away
from the seat of the eruption. If sands, diatoms,
and volcanic dust are capable of being carried
enormous distances, it is hardly irrational to suppose
that microbes may travel from planet to planet,
especially the anaerobic forms, and even those
which are aerobic are capable of being desiccated
without losing their vitality.

Then again, from these first living germs, in
which the peculiarities of the animal and vegetal
kingdoms are hardly yet separated, the laws of

GERMICIDES AND ANTISEPTICS 331

development, the struggle for existence, natural in-
crease, geographical distribution, and many other
known and unknown forces might have produced
the different forms of the animal and plant world,
which inhabited the earth in the past as they do in
the present. We know that in the maintenance of
such views we' stray far from the boundaries of
biological science ; but we find the biologist, always
remaining conscious of the limitations of his know-
lodge, admitting his ignorance with resignation,
foiled in his experiments and observations, not
always resisting the longing of Faust, ' Zu schauen
alle Wirkungskraft und Samen,' but gladly giving
himself up to the allurement of filling with some
fantasy that blank in which modern investigation
has failed.

APPENDIX

I. THE MEASLES BACILLUS

DRS. CANON and PiELiCKE,1 of Berlin, have recently
discovered the true microbe of measles. They have
examined the blood of fourteen patients suffering from
measles, and in all cases found the same microbe.
Microscopic slides of the blood were stained with
eosin-methylene-blue ; the bacilli being stained blue.
They differ in size, being sometimes as long as the
radius of red blood corpuscle, sometimes as large again,
and sometimes smaller ; similar bacilli were also found
in the expectorations, and in the various secretions of
the patients. They grow in artificial media.

II. MICROCOCCUS TETRAGENUS CONCENTRICUS.

Dr. Schenk,2 of Vienna, has discovered in the
stomach of patients suffering from intestinal catarrh a
new microbe, which he calls M. tetragenus concentricus.
It is not known whether this microbe is identical with
that found in the bodies of those who have recently
died from stomachic influenza.

1 Deutsche Medicinische Wochenschrift, 1892.

2 Wiener Allgemeine Medizinische Zeitung, Feb. 1892

332

APPENDIX

III. THE INFLUENZA BACIL

Drs. Pfeiffer, Kitasato, and Canon have (inde-
pendently of one another) discovered the influenza
microbe. It has been found in the saliva and the
bronchial discharges characteristic of influenza. It
exists in the form of small rodlets, strung together in
threads. It grows in agar-agar and sugar, or in agar-
agar and glycerine. In the saliva of influenza patients,
the bacilli are found in large numbers; they may
penetrate from the pus cells into the tissue of the
lungs, and even pass as far as the surface of the pleura.
This fact explains the rapidity and fatality of lung
complications in influenza. The same bacillus has also
been found in the blood of patients suffering from the
disease.

The knowledge that a bacillus residing in the saliva
causes influenza will not cure the epidemic; but the
prompt and practical application of this knowledge by
complete disinfection of all bronchial and nasal secre-
tions, and the isolation of influenza patients will
arrest the plague. It also indicates the reasonableness
of what is known as the carbolic acid treatment of
influenza, which has been practised with considerable
success, especially in the early stages of the disease.

IV. BACILLUS PLUVIATILIS.

The author1 discovered this microbe in rain-water,
contained in a barrel, and exposed to the air during
certain mild weeks in the winter of 1890. At this
period of the year, the majority of the other microbes
in the water were in an inactive condition, con-

1 Dr. A. B. Griffiths, Bulletin de la Societe Chimique de Pari«,
1892, 3e s^rie, tome vii. p. 332.

334 A MANUAL OF BACTERIOLOGY

sequently the struggle for existence was reduced to a
minimum. It is probable that this rare microbe is
an aerial form, but the author has not found it in the
atmosphere.

E. pluviatilis grows well on gelatine plates, and in
four days forms a small yellow colony, with liquefac-
tion of the gelatine. The growth of this microbe in
gelatine-tubes is also characteristic, and in from thirty-
six to forty-eight hours after inoculation, it forms a
thin yellowish band with a number of small lateral
filaments. On the surface of the gelatine, there is
developed a brilliant yellow colony. In bouillon at
30° C. this microbe forms a yellowish pellicle on the
surface, and ultimately a flocculent deposit of the same
colour settles at the bottom of the tube. It grows
very rapidly on potatoes, giving rise to an orange
growth which extends over almost the whole surface
of the potato.

B. pluviatilis occurs in pairs and threads; and
individual bacilli vary in length from 2 to 4 //,, and in
breadth from 0*6 to 0'8 yu,. This microbe, which
stains well with the aniline colours, does not produce
spores. It forms, in peptonise gelatine, a white
crystalline ptomaine having the formula C9H21N205.

Neither the microbe nor ptomaine possesses any
pathogenic properties,

V. THE CANCER BACILLUS.

Scheuerlein has cultivated a special microbe from
cancerous tissues, which he considers as the veritable
agent in producing cancer. This microbe grows well
on solidified blood serum at 39° C. ; and after three
days' incubation, the whole surface of the medium is
covered with a colourless pellicle. After many days

APPENDIX 335

or weeks, a brownish-yellow colour is developed, and
ultimately the pellicle assumes the appearance of small
liquid drops.

This microbe also grows on agar-agar, nutrient
gelatine, potatoes, and in bouillon. It measures from
1*5 to 2'5 fjb in length and 0'5 p in breadth, and forms
spores. According to Scheuerlein,1 the same microbe
is present in most malignant growths (especially sar-
coma and carcinoma), and when pure cultures of it
were injected into the mammary glands of bitches, they
gave rise to small soft tumours. On no occasion has
Scheuerlein failed to produce these tumours, con-
sequently he believes that he has discovered the true
factor in the astiology of cancer. It may be remarked,
en passant, that Domingos Freire 2 has confirmed the
results of Scheuerlein ; but there is no doubt that they
still require further confirmation.

VI. THE MICROBE OF WHOOPING COUGH.

Afanassieff3 has found a bacillus in the pearly
phlegm of persons suffering from whooping cough.
This microbe, which measures from 0*6 to 2'2 /z, in
length, is readily cultivated on gelatine plates, where
it forms small round 'or oval colonies of a brownish
colour. It is in shape what the French call batonnet;4
and Afanassieff, who injected pure cultures of this
microbe into the trachea of dogs and cats, has produced
the typical symptoms of whooping cough in these
animals.

1 Deutuche Medicinische Wochenschrift, 1887, p. 1033.

2 Society de Me'decine Interne de Berlin, 1887.

3 St. Petersburger Medicinische Wochenschrift, 1887.
* Pointed at both ends, like a tip-cat.

336 A MANUAL OF BACTERIOLOGY

VII. PURE FERMENTATIONS AND MICROBES.

Frankland and Frew1 have recently studied the
action of Bacillus ethacetosuccinicus (which they dis-
covered) on dulcitol and mannitol. The decomposition
of these substances may be regarded as involving two
independent reactions, viz. : —

(a) C6H1406 = 2 C2H60 + C02 + CH202

(b) C6H1406 = C4H604 + C2H402 + 2H2;

but from the proportion which the acetic acid bears to
the alcohol, it appears that two molecules are resolved,
in accordance with equations a, for every one that is
decomposed according to b. Or, in other words, the
decomposition of either dulcitol or mannitol by this
microbe is represented by the following equation : —

3 C6H1406 = 4 C2H60 + 2 CO, + 2 CH202 + C4H604
+ C2H402 +" 2 H2.

The microbe which produces this change measures
from 1*7 to 2'5 //, in length and from 0'5 to 1 fju in
breadth. It occurs generally in pairs, and does not
produce spores.

Frankland and Lumsden 2 have studied the decom-
position of mannitol and dextrose by Bacillus ethaceticus.
The products of the fermentation of both these com-
pounds consists of ethyl alcohol, acetic acid, hydrogen,
carbon dioxide, and traces of succinic acid. When the
fermentations are conducted in a closed <epace, there is
invariably also a considerable quantity of formic acid
produced, whilst in fermentations in an open space
(flasks plugged with cotton wool), formic acid, except
in traces, is a most exceptional product. The propor-

1 Journal of Chemical Society, 1892, pp. 254-277.

2 Proceedings of C/iemical Society, 1892, p. 70.

APPENDIX 337

tions in which the several products are obtained from
mannitol is approximately represented by the equation :
3 CaHM06 + H20 = C2H402 + 5 C9H60 + 2 CH202

+ C02,

whilst in the case of the dextrose, the products occur
in the proportions : —

2-5C2H60 : 1-5C2H402 : 3CH202 : C02.

There is a close resemblance between these fermenta-
tions by B. ethaceticus and those produced through the
agency of Friedlander's micrococcus, which renders it
probable that this ethacetic decomposition is a very
general and typical form of fermentative change (Frank-
land).

VIII. PTOMAINES.

The author 1 has extracted the following ptomaines
from urine in cases of measles, whooping-cough, and
erysipelas : —

(a) From Measles. — The ptomaine produced during
the course of this disease is a white substance which
crystallises in small laminae. It is soluble in water,
and has an alkaline reaction. It is precipitated by
the general reagents used in testing for such bodies.
Analysis of the base itself and of its platinochloride
correspond with the formula C3 H5 N3 0 ; and the
various reactions of this ptomaine, and the products
of its decomposition, prove that it has the constitution
of glycocyamidine :

HN— HC

This ptomaine is very poisonous, and, when adminis-

1 Dr. A. B. Griffiths, Comptes Rendus de VAcaddmie des
Sciences (Paris), tome cxiv. p. 496; Bulletin de la Societe
Chimiqne de Paris, 1892, 3e se"rie, tome vii. p. 250.

338 A MANUAL OF BACTERIOLOGY

tered to a cat, it produced high fever (40° C.), and
death within thirty-six hours.

(b) From Wliooping- Cough. — The ptomaine which
occurs in this highly infectious disease is a white
crystalline substance. It has the formula C5H19N02.

These three ptomaines are not present in normal
urines, consequently they are produced in the system
during the diseases.

ix. BRIEGER'S METHOD FOR ISOLATING PTOMAINES.

This process has been already alluded to, but it
should be stated that after the oxy-aromatic acids have
been driven off, the H2S04 is precipitated by baryta,
and the precipitate removed by filtration. The excess
of baryta is precipitated by C02, and the BaC03 also
removed by filtration. The filtrate is then heated on a
water-bath, cooled, and precipitated with HgCl2. The
precipitate is washed and decomposed by H2S; the
HgS is filtered off, and the filtrate concentrated. The
mineral salts, etc., crystallise out first and are rejected,
then the dried residue is treated with absolute alcohol,
which, after concentration, deposits the hydrochlorides
of the ptomaines. These are separated by fractional
precipitation with platinic chloride, auric chloride, etc.

' In some of his researches, Brieger l has shortened
the process by precipitating the putrid fluids, after
boiling and filtering directly, with HgCl2, i.e. the first
precipitation with lead acetate is omitted. As HgCl2
does not precipitate all ptomaines, both precipitate and
filtrate must be examined.'

1 Die Ptomaine, 1885-1886 (3 parts).

APPENDIX 339

X. MICROBES OF SOIL, WATER, AND AIR.

Concerning our previous remarks on the microbes of
the soil, it may be added that Keimers * has recently
ascertained the number of microbes in soil at various
depths. For instance —

A sample of soil at surface contained 2,564,800 microbes per cc.

,, ,, 2 yards below surface contained 23,100 ,, ,,

>. 3* „ „ „ » 6,170

» 4J „ „ „ „ 1,580

>. 6 „ „ „ „ 0 „

It has been already stated that Bacillus tetani has been
found in soil ; and Mace 2 has recently found B. typhosus
in various samples of soil. B. tuberculosis and B. coli
communis have also been found in soil. On the other
hand, De Giaxa 3 has shown that soil is a bad medium
for the preservation of B. cholerce Asiaticce, this being
due to the large number of saprophytic species present,
whose struggle for existence interferes with the vitality
of the cholera microbe. In fact, this is an important
example of the survival of the fittest, for De Giaxa has
proved that soil per se has no detrimental action on the
cholera microbe.

If soil is a bad medium for the preservation of some
forms, water (especially when polluted) is a better
medium for others. At the recent Congres d' Hygiene
Ouvriere4 M. Gautier exhibited illustrations of the
typhoid, carbuncle, cholera, and diphtheritic microbes,
with many others, in Seine water ; 5 and the dis-
tinguished chemist said to householders and others,

1 Zeitschrift fiir Hygiene, vol. vii.

- Comptes Rendus, vol. cvi. p. 1564 ; and his Traite Pratique
de Bacteriologie (1-891), p. 717.

3 Annales de Microyraphie (1890) vol. ii.

4 Held in Paris during April 1892.

5 Paris drinking-water.

340 A MANUAL OF BACTERIOLOGY

1 Do not fear these foes. If they hurt you, it is because
you drink unfiltered water and eat ill-baked bread.
Filter your water or boil it, and if your bread seems
ill-baked, toast it well or let it stay some time in a hot
oven.'

If householders, corporations, and others would
attend a little more to the ordinary rules of health —
such as filtering water, boiling milk, destroying
unsound food, removing refuse, isolating infectious
persons, disinfecting articles of an infectious nature,
etc. — there would be a considerable decrease in the
number of infectious cases, especially during the time
of epidemic diseases. In fact, these rules would go a
long way towards the prevention of such diseases.

There is no doubt that many of the epidemics of
cholera and other infectious diseases have been largely
due to bad or imperfect sanitation. In densely-
populated centres it is imperative that the most
perfect rules of sanitation should be practised by
corporations, sanitary authorities, householders, and
others. One cannot help but believe that the visita-
tions of epidemic diseases in the past have been
blessings in disguise, because they have taught us that
cleanliness in all things (in person, food, drink, home,
and city) tends directly to prevent and combat the
attacks of such diseases as cholera, typhoid fever,
scarlatina, etc. In past times town authorities and
householders did not heed the voice of the cholera
fiend, as is sung in Mackay's lyric, 'The Cholera
Chant'—

' They will not hear the warning voice.
The cholera comes, — rejoice ! rejoice !
He shall be lord of the swarming town !
And mow them down, and mow them down ! '

Although there is still room for improvement in
sanitary matters, yet no one can be blind to the fact

APPENDIX 341

that, in every direction, sanitation has made rapid
progress in Great Britain. l

If, by observing such rules as those specified, we can
keep in check the obnoxious microbes in water and
food, it is not such an easy matter to deal with those
present in the atmosphere.2 But even aereal microbes
(those spirits of the air) may be, to a large extent,
kept in check by the use of disinfectants.

XI. STATISTICS CONCERNING ZYMOTIC DISEASES.

The Quarterly Report of the Registrar-General, relating
to the deaths in England and Wales from zymotic
diseases, gives the following figures : —

5202 deaths from whooping-cough.
2769 measles.

1306

1078

1361

890

diphtheria.

scarlatina.

diarrhoea.

'fever '(chiefly enteric).

small-pox.

The above figures give a total of 12,682 deaths from
zymotic diseases during the first three months of 1892.

1 For those interested in sanitary matters, the author recom-
mends Dr. A. C. May bury 's excellent Epitome of the Public
Health Act, 1891 (H. Kimpton, 82 Holborn, London).

2 As microbes are always present in air, soil, and water, it
may well be asked, ' Where do they come from ? ' We know
not where ; perhaps from the djinnistan of the Persians.

LIST OF FIRMS WHERE BACTERIOLGICAL
APPARATUS, ETC., CAN BE OBTAINED.

Microscopes, etc.

C. Zeiss, Jena, Germany ; or Zeiss's agent, C. Baker,
244 High Holborn, London.

Incubators, Sterilisers, etc.

F. E. Becker & Co., 33 Hatton Wall, London; K.
Muencke, 58 Luisenstrasse, Berlin ; R. Kanthack,
Imperial Mansions, Oxford Street, London.

Chemical Apparatus and Chemicals.

J. Orme & Co., 65 Barbican, London.

Staining Solutions, etc.

F. E. Becker & Co., 33 Hatton Wall, London; R.
Kanthack, Imperial Mansions, Oxford Street, London.

Agar-agar and Gelatine.

Christy & Co., 25 Lime Street, London ; J. F. Shew &
Co., 89 Newman Street, Oxford Street, London; R.
Kanthack, Imperial Mansions, Oxford Street, London.

Microtomes.

Cambridge Scientific Instrument Co., Cambridge ; R.
Kanthack, Imperial Mansions, Oxford Street, London.

Dissecting Knives, etc.

C. Baker, 244 High Holborn, London.

Mr. Kanthack furnishes estimates of the requirements of a
completely fitted bacteriological laboratory.

INDEX.

Abbe's condenser, 21.
Actinomyces, 82, 258.
Actinomycosis, 258.
Aerobic microbes, 110.
Aeroscopes, 263-269.
Agar-agar, 57.
Agents, cementing, 91.

,, clearing, 91.

,, dehydrating, 90.

,, mounting, 91.

„ washing, 90.
Air, microbes of, 260-275, 330.
Air, number of microbes in,

269-275.

Albumin, egg, 55.
Albumoses, 321-324.
Amoeba, 259.
Anaerobic microbes, 110.
Anthracin, 256.
Anthrax, 255-257.
Antiseptics, 325-330.
Apochromatic lenses, 17.
Apparatus, microphotographic,

Appendix, 332-341.
Aspergillus, 52.
Autoclaves, 32, 52.

Bacilli, 149-170. •
Bacillus alvei, 150.

,, antkracis, 255.

,, arachnoidea, 169.

,, beribericus, 149.

Bacillus butyricus, 82, 156.

,, cavicida, 164.

,, cholera Asiatica, 64,
80, 339.

,, cyanogenus, 159.

,, diphtheria;, 79, 236.

,, diphtheria colum-
barum, 163.

„ diphtheria vitidorum,
163.

,, epidermidis, 166.

,, erythrospoi^us, 159.

,, ethaceticu-s, 155, 201,
336.

,, ethacetosuccinicu$, 336.

,, figurans, 170, 274.

,, Hansenii, 170.

,, ianthinus, 159.

„ lepra, 76, 78, 206.

,, leptomitiformis, 169.

,, malaria, 215.

„ mallei, 77, 234.

,, megaterium, 166.

,, cedematis maligni, 110,
160.

„ of cancer, 193, 334.

„ of conjunctivitis, 169.

,, of grouse disease, 154.

,, of indigo fermenta-
tion, 160.

„ of influenza, 333.

,, of measles, 332.

„ of nitrous fermenta-
tion, 166, 282.

,, of rabbit diphtheria,
164.

,, of rhinoscleroma, 160.
343

344

INDEX

Bacillus of swine erysipelas,

165.

,, of swine plague, 165.
,, of symptomatic an-
thrax, 157.
,, of syphilis, 211.
,, of iilcerative stoma-
titis, 165.
,, of whooping-cough,

335.

,, pellucida, 169.
,, phiviatilis, 333.
, , putrificus coli, 1 66.
,, pyocyaneusj 161.
» pyogenesfcetidus, 164.
,, radicicola, 281.
,, septiccemice, 162-3.
,, septicus, 169.
,, spinosus, 110.
,, subtilis, 52, 65, 108,

110, 154, 274.
„ tetani, 211, 339.
,, tuberculosis, 50, 66.

76, 244-254, 339.
,, typhosus, 79, 221, 339.
,, ulna, 157.
,, violaceus, 159.
Bacteria, 133-149.
Bacteriological laboratory, 8-

48.

Bacteriology, definition of, 1.
Bacterium aceti, 136, 274.
allii, 134, 135.
,, Balticum, 143.
,, brunneum, 146.
,, cholerce gallinarum,

137.

,, chlorinum, 144.
,, coli commune, 139,

339.
,, crassum sputigenum,

145.

,, decalvans, 137.
,, Fischeri, 143.
,, foetidum, 139.
,, indicum, 141, 274.

lactis, 137, 274.
,, lineola, 134.
,, luminosum, 144.

Bacteriummerismopedioides,

141.

,, Neapoianum, 139.
, , oxytocum pernicio-

sum, 141.

,, Pflilgeri, 142.
„ phosphor -escens, 142.
,, photometricum, 145.
,, pneumonicum agile,

145.

,, pseudo • pneumoni-
cum, 138. ,^
,, septicus agrigenum,

139.
,, septicum sputigenum,

140.

,, terrao, 133, 276.
,, violaceum, 145.
,, xanthinum, 139, 274.

^o#/w, 141.
Begyiatoa alba, 168.

,, mirabilis, 168.
,, m'vea, 168.
,, roseo-persicina, 167.
Berberis vulgaris, 101.
Beri-beri, 149.

Biology of microbes, 114, 177.
Blood serum, liquid, 51.
,, ,, solid, 51.

Bouillon, 49.
Bread-paste, 58.
Buffon's theory, 98.

Camera lucida, 23.
Canada balsam, 70-95.
Cancer bacillus, 193, 334.
Canons, Koch's, 2.
Capillary pipettes, 53.
Cementing agents, 91.
Chamberlaiid's filter, 47.
Chemical separators, 76.
Cholera, 225-234, 340.
Classification of microbes, 1 10,

112.

Clearing agents, 91.
Clip, mounting, 94.
Comma-shaped bacilli, 227.

INDEX

345

Concluding remarks, 330.
Condenser, Abbess, 21.
Cover-glass preparations, 68,

76.

Cover -glass testers, 89.
Cultivating microbes, methods

of, 49-68.

Cultivations, fractional, 59, 62.
Cultivations, plate, 59.
Cultivation tubes, 41-47.
Cultures, drop, 65.
Cutting, section, 29-30, 88.

Damp chambers, 56.
Definition of bacteriology, 1.
Dehydrating agents, 90.
Dilution method, 59, 63.
Diphtheria, 235-243.
Diplococcus, a, 109.
Discontinuous heating, method

of, 51.

Disinfectants, 214, 325, 341.
Diseases, microbes and, 178-

259.
Dissecting instruments, 23, 24,

25.

Dissecting microscope, 25.
Dissecting, mode of, 26.
Division of microbes, 109-110.
Drawing by hand, 23.
Dust in air, the, 261.
Dysentery, 259.

E
Edinburgh laboratory, the, 8-

Enzymes, 2, 18, 324.
Equivocal generation, 100.
Erysipelas, 193, 338.
Estimating microbes in air,

methods of, 263-272.
Estimating microbes in soil,

methods of, 277.
Estimating microbes in water,

methods of, 291-292.
Eucalyptus, the, 219.

Fermentation, 1, 174.
Fermentations, pure, 336.
Filter, hot-water, 50.
Fission, 108, 109.
Flagellata, 198, 217, 259.
Flasks, cultivation, 42-47.
Fluids, examination of, 67.
Fluids, staining, 69-70.
Formation of spores, 108-9.
Forms of microbes, 107.
Foul-brood, 150.
Fractional cultivations, 59, 62.
Fresh tissues, examination of,

67.
Fresh tissues, mounting, 91.

Gelatine, nutrient, 57.
Germicides, 325-330.
Glanders, 234, 324.
Ground rice, 58.
Grouse disease, bacillus of,
154.

Hardening agents, 84, 86.
Hay-fever, bacillus of, 154.
Hydrophobia, 181-192.

Identification of microbes, 1 13.

Imbedding mixtures, 86-88.

Incubators, 37-41.

Infectious diseases and mi-
crobes, 178-259.

Influenza, 197-199.

bacillus of, 333.

Infusions, various, 52.

Injection syringes, 53.

Inoculating media, modes of,
58-59.

Inoculating needles, 53.

Instruments, dissecting, 23-25.

Introduction, the, 1-7.

346

INDEX

Kakke, 149.
Klein's bacillus, 227.
Koch's canons, 2.

,, lymph, 252.
Kuisl's bacillus, 227.

Laboratory, the bacteriological,
8-48.

Leprosy, 206-210.

Leptothrix buccalis, 166.
,, innominata, 167.
,, parasitica, 167.

Lifters, 93.

List of firms, 342.

Living animals, methods of in-
troducing microbes into, 94.

Malaria, 215-220.
Measles bacillus, 332.
Measurement, unit of, 95-97.
Media, cultivation, 49-68.
„ fluid, 49-54.
„ solid, 54-61.
Merismopedia, a, 109.
Methods of cultivating mi-
crobes, 49-68.

,, of mounting mi-

crobes, 83-97.

,, of staining mi-

crobes, 68-83.

Microbes and diseases, 178-259.
division of, 109.
of air, 260-275.
of soil, 276-285.
of water, 286-304.
properties of, 4, 5.
reproductive power

of, 6, 7.
size of, 5.
weight of, 5.
which excrete albu-

moses, 323.
Micrococci, 114-132.

,, in pyaemia, 132.

Micrococci in rabies, 182-184.

,, in septicaemia, 132.

Micrococcus amaril, 180.

,, aurantiacus, 116.

,, bombycis, 129.

,, candicans,UO,274.

,, cereus Jlavus, 118.

,, chlorinus, 55, 116,

274.

,, cinnabareus, 117.

,, citreus conglomcra-
tus, 118, 274.

,, cyaneus, 117, 274.

, , endocarditicus, 126.

,, erysipelatosus, 193.

,, Jlavus liquefaciens,
118.

, , Jlavus decidens, 118.

, , Jlavus tardigradus,
118.

,, fulvus, 117.

,, gonorrhoea, 80, 127.

,, hcematodes, 117.

,, in gangrene, 130.

,, luteus, 116, 274.

,, in measles, 126.

,, in pernicious an-

aemia, 132.

,, in purpura, 123.

,, insectorum, 131.

, , intracelhdaris me-

ningitidis, 128.

,, inwhooping-cough,
131.

, , of cattle - plague,

129.

, , of foot-and-mouth

disease, 130.

, , of nitric fermenta-

tion, 132, 285.

„ of tissue necrosis,

131.

,, ovatus, 127.

,, perniciosus, 131.

199.

prodigiosus, 1 14,274.
pyogems, 119, 199.
pyogenes alb us, 120.

INDEX

347

Micrococcus pyogenes auretis,

119.
» pyogenes citreus,

radiatus, 119.

rosaceus, 117, 274.

scarlatina, 82, 202.

septicus, 130.

subflavus, 119.

tetragenus concen-
tricm, 332.

tetragonus, 128.

urece, 120.

variolas et vaccinia,
124.

versicolor, 118.

violaceus, 117, 274.
Microphotographic apparatus,

21.
Microscope, dissecting, 25.

the, 14-22.
Microtomes, 27-29.
Milk, 51.

Miller's bacillus, 227.
Mounting agents, 91.

„ clip, 94.
Mycoprotein, 4.

Needles, inoculating, 53.
Nitric microbe, 285.
Nitrification, 1, 281-285.
Nitrous microbe, 284.
Number of microbes :

in air, 269-275.

in soil, 277-281.

in water, 281-287.

Objectives, 16, 18, 20.
Oculars, 17, 19.
Oldium albicans, 258.
Origin of microbes, 98-107.

Pasteur Institute, 12-14, 190.
Phargocytes, 191.

Phthisis, 243.
Pigments, 319, 324.
Plate-cultivations, 59.
Pleomorphism, 102-107.
Pneumonia, 199-201.
Potatoes, cooked, 56.
Preparations, cover-glass, 68.
Properties of microbes, 4.
Proteus mirabilis, 148.

,, vulgaris, 146-148.

„ Zenkeri, 149.
Protozoa, 259, 288.
Ptomaines, 305-320, 324, 337.
,, extraction of, 307-

310, 338.

,, properties of, 307.
Puccinia graminis, 101.
Puerperal fever, 194-197.
Putrefaction, 1.
Pyocyanin, 161, 319, 324.

Rabies, 181-192.
Regulators, 39-41.
Reproduction of microbes, 6,

108.

Rice, ground, 58.
Rules of sanitation, 340.

Saccharomyces apiculatus, 176.

cerevisice, 174.

conglomerate*,
176.

ellipsoideus, 175.

exiguus, 176.

minor, 175.

mycoderma, 177.

Pastorianu s,176.

vini, 177.
Saccharomycetes, 173.
Sanitas, 220, 329.
Sanitation, rules of, 340.
Sarcina, 109.
Scarlatina, 202-206.
Schizomycetes, 99, 107, 110,
173,288.

348

INDEX

Separators, chemical, 76.
Serum inspissator, 36.
Serum steriliser, 35.
Size of microbes, 5.
Soil, microbes of, 276-285, 339.
,, number of microbes in,

277-281.

Spasmotoxine, 213.
Spirilla, 171-173.
Spirillum attenuatum, 173.
,, cholerce Asiaticce,

171, 226.

concentricum, 173.
FinMeri, 171, 227.
Obermeieri, 81, 172.
Rosenbergii, 173.
sanguineum, 172.
sputigenum, 227.
lenue, 172.
tyrogenum, 61, 171,

227.

undula, 172.
violaceum, 173.
vohttans, 172.
Spirochsetse, 173.
Spirochceta gigantea, 173.
,, plicatilis, 173.
Spontaneous generation, 99.
Staining fluids, 69, 70.
Staining microbes, methods of,

68-83.
Statistics concerning diseases,

341.

Sterilisers, 31-37.
Streptococcus, 109.
Surra, 259.
Syphilis, 210.

Testers, cover-glass, 89.
Tetanine, 213.
Tetanotoxine, 213.
Tetanus, 211, 215.
Throat washes, 241.

Thrush, 258.

Tissues, examination of fresh,

67.

Torula cerevisice, 174.
Torulse, 52.
Tuberculosis, 243-254.
Tuberculous milk, bacilli in,

75.

Tubes, cultivation, 41-47.
Turn-tables, 92.
Typhoid fever, 220-225.

Unit of microscopical measure-
ment, 95-97.

Vibriones, 170-171.
Vibrio rugula, 170.
,, serpens, 170.
Vivisection, 3, 4.

Washing agents, 90.

Water and epidemics, 223, 233.

,, filtration of, 298.

,, microbes of, 286-304,
339.

,, standard of purity of,
303.

,, sterilisation of, 300-304.

,, storage of, 298.
Waters, examination of, 290.
Weight of microbes, 5.

Yeasts, 52, 173-177.
Yellow fever, 180.

Zeiss's microscopes, 15.

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and Northern Regions of Ontario, the Canadian Shores of the Great
Lakes, the Lake of the Woods Region , Manitoba and " The Great
North- West," The Canadian Rocky Mountains and National Park,
British Columbia, and Vancouver Island. By ERNEST INGEKSOLL. With
Maps and many Illustrations. Crown Svo, limp cloth, 6s.

THE GUIDE-BOOK TO ALASKA AND THE NORTH-

WEST COAST, including the Shores of Washington, British Columbia,
South-Eastein Alaska, the Aleutian and the Seal Islands, the Behring
and the Arctic Coasts. By E. R. SCIDMORE. With Maps and many
Illustrations. Crown Svo, limp cloth, 6.y.

THE GENESIS OF THE UNITED STATES. A

Narrative of the Movement in England, 1605-1616, which resulted in the
Plantation of North America by Englishmen, disclosing the Contest
between England and Spain for the Possession of the Soil now occupied
by the United States of America; set fo:th through a series of Historical
Manuscripts now first printed, together with a Re-issue of Rare Contem-
poraneous Tracts, accompanied by Bibliographical Memoranda, Notes,
and Brief Biographies. Collected, Arranged, and Edited by ALEXANDER
BROWN, F.R.H.S. With 100 Portraits, Maps, and Plans. In two volumes.
Royal Svo. buckram, ^3 135. 6d.

fiction.

In Three Volumes.

THE HEAVENLY TWINS. By Madame SARAH GRAND,

Author of " Ideala," &c. Third Thousand.

THE LAST SENTENCE. By MAXWELL GRAY, Author of

" The Silence of Dean Maitland," &c. Second Thousand.

AS A MAN IS ABLE. By DOROTHY LEIGHTON.

THE COUNTESS RADNA. By W. E. NORRIS, Author of
" Matrimony," &c. [/« August.

A COMEDY OF MASKS. By ERNEST DOWSON and

ARTHUR MOORE. \_fn September.

THE HOYDEN. By Mrs. HUNGERFORD. u* October.

A SUPERFLUOUS WOMAN. [In November.

BENEFITS FORGOT. By.WOLCOTTBALESTIER.[/rt«««;y,i8g4.

A/K. WILLIAM UEINEMANN'S LIST. 11

ffictfon,

In One Volume.

FROM THE FIVE FIVERS. By FLORA ANNIE STEEL.

Author of" Miss Stuart's Legacy." Crown 8vo, cloth, (s.

IDEALA. By Madame SARAH GRAND, Author of "The

Heavenly Twins." New Edition. Crown 8vo, cloth, 6s.
THE TOWER OF TADDEO. By OUIDA, Author of "Two
Little Wooden Shoes," &c. New Edition. Crown 8vo, cloth. Illus-
trated. 6s.

CHILDREN OF THE GHETTO. By I. ZANGWILL,

Author of " The Old Maids' Club," &c. New Edition, with Glossary.
Crown 8vo, cloth, 6s.

THE PREMIER AND THE PAINTER. A Fantastic
Romance. By I. ZANGWILL and Louis COWEN. New Edition. Crown
8vo, cloth, 6s.

THE NAULAHKA. A Tale of West and East. ByRuDYARD
KIPLING and WOLCOTT BALESTIER. Crown 8vo, cloth, dr. Second
Edition.

AVENGED ON SOCIETY. By H. F. WOOD, Author of
"The Englishman of the Rue Cain," "The 'Passenger from Scotland
Yard." Crown 8vo. Cloth, 6s.

THE O'CONNORS OF BALLINAHINCH. By Mrs.

HUNGERFORD, Author of "Molly Bawn," &c. Crown 8vo. Cloth, 6s.

PASSION THE PLAYTHING. A Novel. By R. MURRAY

GILCHRIST. Crown 8vo, cloth, 6s.

THE SECRET OF NARCISSE. By EDMUND GOSSE.

Crown 8vo, buckram, 55.

THE PENANCE OF PORTIA JAMES. By TASMA,

Author of " Uncle Piper of Piper's Hill," &c. Crown 8vo, cloth, $s.

INCONSEQUENT LIVES. A Village Chronicle, shewing
how certain folk set out for El Dorado ; what they attempted ; and what
they attained. By J. H. PEARCE, Author of "Esther Pentreath," &c.
Crown 8vo, cloth, 5*.

A QUESTION OF TASTE. By MAARTEN MAARTENS,

Author of "An Old Maid's Love," &c. Crown 8vo, cloth, 5*.
COME LIVE WITH ME AND BE MY LOVE. By

ROBERT BUCHANAN, Author of "The Moment After," "The Coming
Terror," &c. Crown 8vo, cloth, $s.

VANITAS. By VERNON LEE, Author of " Hauntings," &c.

Crown 8vo, cloth, 5* .

THE DOMINANT SEVENTH. A Musical Story. By
KATE ELIZABETH CLARKE. Crown 8vo, cloth, 5*.

In preparation.

ORIOLE'S DAUGHTER. By JESSIE FOTHERGILL, Author

of " The First Violin," &c.

RELICS. By FRANCES MACNAB.

A BATTLE AND A BOY. By BLANCHE WILLIS HOWARD,

Author of " Guenn," &c.

APPASSIONATA: The Story of a Musician, By ELSA
D'ESTERRE KEELING.

MR. BAILEY MARTIN. By PERCY WHITR.

12 MR. WILLIAM HEINEMANN'S LIST.

fbeinemann's Jnternatfonal SLfbrarp.

EDITED BY EDMUND GOSSE.

New Review. — " If you have any pernicious remnants of literary chauvinism
I hope it will not survive the series of foreign classics of which Mr. William
Heinemann, aided by Mr. Edmund Gosse, is publishing translations to the great
contentment of all lovers of literature."

Each Volume has an Introduction specially -written by the Editor.
Price, in paper covers, zs. 6d. each, or cloth, 3$. 6d.

IN GOD'S WAY. From the Norwegian of BJORNSTJERNE

BjORNSON.

Athenceum. — "Without doubt the most important and the most interesting
work published during the twelve months."

PIERRE AND JEAN. From the French of GUY DE MAU-
PASSANT.

Pall Mall Gazette. — "Admirable from beginning to end."

Athenceum, — " Ranks amongst the best gems of modern French fiction."

THE CHIEF JUSTICE. From the German of KARL EMIL

FRANZOS, Author of " For the Right," &c.

New Review. — " Few novels of recent times have a more sustained and
vivid human interest."

WORK WHILE YE HAVE THE LIGHT. From the

Russian of Count LEO TOLSTOY.

Manchester Guardian. — "Readable and well translated; full of high and
noble feeling."

FANTASY. From the Italian of MATILDE SERAO.

Scottish Leader. — " The book is full of a glowing and living realism

There is nothing like ' Fantasy ' in modern literature."

FROTH. From the Spanish of Don ARMANDO PALACIO

VALDES.
Daily Telegraph. — " Vigorous and powerful in the highest degree."

FOOTSTEPS OF FATE. From the Dutch of Louis

COUPERUS.

Gentlewoman. — "The consummate art of the writer prevents this tragedy
from sinking to melodrama. Not a single situation is forced or a circumstance
exaggerated." ,

PEPITA JIMENEZ. From the Spanish of JUAN VALERA.

New Review (Mr. George Saintsbury) : — "There is no douht at all that
it is one of the best stories that have appeared in any country in Europe for the
last twenty years."

THE COMMODORE'S DAUGHTERS. From the Nor-
wegian of JONAS LIE.

Athenceum. — " Everything that Jonas Lie writes is attractive and pleasant ;
the plot of deeply human interest, and the art noble."

THE HERITAGE OF THE KURTS. From the Norwegian

of BJORNSTJERNE BJORNSON.

National Observer. — " It is a book to read and a book to think about, for,
incontestably, it is the work of a man of genius."

LOU. From the German of BARON ALEXANDER VON ROBERTS.

DONA LUZ. From the Spanish of JUAN VALERA.

THE JEW. From the Polish of JOSEPH IGNATIUS KRASZEWSKI.

In the Press.

UNDER THE YOKE. From ihe Bulgaiian of IVAN VAZOFF.

MR. WILLIAM HEINEMANN'S LIST. 13

popular 36* 6&, Novels.

CAPT'N DAVY'S HONEYMOON, The Blind Mother,
and The Last Confession. By HALL CAINE, Author of " The Bondman,"
" The Scapegoat," &c. Sixth Thousand.

THE SCAPEGOAT. By HALL CAINE, Author of "The

Bondman," &c. Eighteenth Thousand.

THE BONDMAN. A New Saga. By HALL CAINE.

Twenty-second Thousand.

DESPERATE REMEDIES. By THOMAS HARDY, Author

of " Tess of the D'Urbervilles," &c.

A LITTLE MINX. By ADA CAMBRIDGE, Author of "A

Marked Man," &c.

A MARKED MAN: Some Episodes in his Life. By ADA
CAMBRIDGE, Author of "Two Years' Time," "A Mere Chance," &c.

THE THREE MISS KINGS. By ADA CAMBRIDGE, Author

of "A Marked Man."

NOT ALL IN VAIN. By ADA CAMBRIDGE, Author of "A

Marked Man," " The Three Miss Kings," &c.

A KNIGHT OF THE WHITE FEATHER. By TASMA,

Author of "The Penance of Portia James," "Uncle Piper of Piper's
Hill," &c.

UNCLE PIPER OF PIPER'S HILL. By TASMA. New

Popular Edition.

THE RETURN OF THE O'MAHONY. By HAROLD

FREDERIC, Author of " In the Valley," &c. With Illustrations.

IN THE VALLEY. By HAROLD FREDERIC, Author of

" The Lawton Girl," " Seth's Brother's Wife," &c. With Illustrations.

PRETTY MISS SMITH. By FLORENCE WARDEN, Author

of "The House on the Marsh," "A Witch of the Hills," &c.

THE STORY OF A PENITENT SOUL. Being the
Private Papers of Mr. Stephen Dart, late Minister at Lynnbridge, in the
County of Lincoln. By ADELINE SERGEANT, Author of " No Saint," &c.

NOR WIFE, NOR MAID. By Mrs. HUNGERFORD, Author

of "Molly Bawn," &c.

MAMMON. A Novel. By Mrs. ALEXANDER, Author of "The

Wooing O't," &c.

DAUGHTERS OF MEN. By HANNAH LYNCH, Author of

" The Prince of the Glades," &c.

A ROMANCE OF THE CAPE FRONTIER. By BERTRAM

MITFORD, Author of "Through the Zulu Country," &c.

'TWEEN SNOW AND FIRE. A Tale of the Kafir War of
1877. By BERT RAM MITFORD,

I4 MR. WILLIAM HE1NEMANWS LIST.

popular 30* 6&* 1Ro\>els.

THE MASTER OF THE MAGICIANS. By ELIZABETH

STUART PHELPS and HERBERT D. WARD.
THE HEAD OF THE FIRM. By Mrs. RIDDELL, Author

of " George Geith," " Maxwell Drewett,"'&c.

ACCORDING TO ST. JOHN. By AM^LIE RIVES, Author
of " The Quick or the Dead."

KITTY'S FATHER. By FRANK BARRETT, Author of

"The Admirable Lady Biddy Fane," &c.

DONALD MARCY. By ELIZABETH STUART PHELPS,

Author of " The Gates Ajar," &c.

PERCHANCE TO DREAM, and other Stories. By MAR-
GARET S. BRISCOE.

THE AVERAGE WOMAN. By WOLCOTT BALESTIER.
With an Introduction by HENRY JAMES.

THE ATTACK ON THE MILL, and other Sketches

of War. By EMILE ZOLA. With an essay on the short stories of M.
Zola by Edmund Gosse.

WRECKAGE, and other Stories. By HUBERT CRACKAN-

THORPE.

MADEMOISELLE MISS, and other Stories. By HENRY

HARLAND, Author of " Mea Culpa," &c.

TRUE RICHES. By FRANCOIS COPP£E. With an Intro-
duction by T. P. O'CONNOR.

WRECKERS AND METHODISTS. Cornish Stories. By
H. D. LOWRY.

LOS CERRITOS. A Romance of the Modem Time. By
GERTRUDE FRANKLIN ATHERTON, Author of "Hermia Suydam," and
" What Dreams may Come."

A MODERN MARRIAGE. By the Marquise CLARA LANZA.

popular Sbillino DBoofes,

MADAME VALERIE. By F. C. PHILIPS, Author of "As
in a Looking-Glass," &c.

THE MOMENT AFTER: A Tale of the Unseen. By
ROBERT BUCHANAN.

CLUES ; or, Leaves from a Chief Constable's Note- Book.

By WILLIAM HENDERSON, Chief Constable of Edinburgh.

MR. WILLIAM HEINEMANWS LIST. 15

H)ramatfc ^literature.

THE MASTER BUILDER. A Play in Three Acts. By
HENRIK IBSEN. Translated from the Norwegian by EDMUND GOSSE
and WILLIAM ARCHER. Small 410, with Portrait, 51. Popular Edition,
paper, i*. Also a Limited Large Paper Edition, au. net.

HEDDA GABLER: A Drama in Four Acts. By HENRIK
IBSEN. Translated from the Norwegian by EDMUND GOSSE. Small 410,
cloth, with Portrait, 5*. Vaudeville Edition, paper, is. Also a Limited
Large Paper Edition, z\s. net.

THE PRINCESSE MALEINE: A Drama in Five Acts
(Translated by Gerard Harry), and THE INTRUDER : A Drama in
One Act. By MAURICE MAETERLINCK. With an Introduction by HALL
CAINE, and a Portrait of the Author. Small 410, cloth, $j.

THE FRUITS OF ENLIGHTENMENT: A Comedy in
Four Acts. By Count LYOF TOLSTOY. Translated from the Russian by
E. ]. DILLON. With Introduction by A. W. PINERO. Small 410, with
Portrait, 5^.

THE DRAMA, ADDRESSES. By HENRY IRVING. 8vo.

With Portrait by ]. McN. Whistler. Second Edition. Fcap. 3*. (xt.
SOME INTERESTING FALLACIES OF THE

Modern Stage. An Address delivered to the Playgoers' Club at St.
James's Hall, on Sunday, 6th December, 1891. By HERBERT BEERBOHM
TREE. Crown 8vo, sewed, 6V/.

THE PLAYS OF ARTHUR W. PINERO. With Intro-
ductory Notes by MALCOLM C. SALAMAN. i6mo, Paper Covers, is. 6d.
or Cloth, zs. 6d. each.

I. THE TIMES : A Comedy in Four Acts. With a Preface
by the Author.

II. THE PROFLIGATE : A Play in Four Acts. With
Portrait of the Author, after J. MORDECAI.

III. THE CABINET MINISTER: A Farce in Four Acts.

IV. THE HOBBY HORSE : A Comedy in -Three Acts.
V. LADY BOUNTIFUL: A Play in Four Act>.

VI. THE MAGISTRATE : A Farce in Three Acts.
VII. DANDY DICK : A Farce in Three Acts.
VIII. SWEET LAVENDER.

To be followed by The Schoolmistress, The Weaker Sex, Lords and
Commons, and The Squire,

16 MR. WILLIAM HEINEMANN'S LIST.

poetry

TENNYSON'S GRAVE. By ST. CLAIR BADDELEY. 8vo,

paper, w.

LOVE SONGS OF ENGLISH POETS, 1500—1800.

With Notes by RALPH H. CAINE. Fcap. 8vo, rough edges, 3^. 6d.
%* Large Paper Edition, limited to 100 Copies, ioy. 6d. Net.

IVY AND PASSION FLOWER: Poems. By GERARD
BENDALL, Author of " Estelle," &c. &c. 12010, cloth, 3*. 6d.

Scotsman. — " Will be read with pleasure."

Musical World. — " The poems are delicate specimens of art, graceful and
polished."

VERSES. By GERTRUDE HALL. I2mo, cloth, -$s. 6d.

Manchester Guardian. — " Will be welcome to every lover of poetry who
takes it up."

IDYLLS OF WOMANHOOD. By C. AMY DAWSON.

Fcap. 8vo, gilt top, 5*.

IbeinemamVs Scientific

MANUAL OF BACTERIOLOGY. By A. B. GRIFFITHS,

Ph.D., F.R.S. (Edin.), F.C.S. Crown 8yo, cloth, Illustrated. 7s. 6d.
Pharmaceutical Journal. — "The subject is treated more thoroughly and
completely than in any similar work published in this country. .... It should
prove a useful aid to pharmacists, and all others interested in the increasingly
important subject of which it treats, and particularly so to those possessing little
or no previous knowledge concerning the problems of micro-biology."
MANUAL OF ASSAYING GOLD, SILVER, COPPER,
and Lead Ores. By WALTER LEE BROWN, B.Sc. Revised, Corrected,
and considerably Enlarged, with a chapter on the Assaying of Fuel, &c.
By A. B. GRIFFITHS, Ph.D., F.R.S. (Edin.), F.C.S. Crown 8vo, cloth,
Illustrated, 7$. 6d.

Colliery Guardian. — "A delightful and fascinating book."
Financial World. — " The most complete and practical manual on everything
which concerns assaying of all which have come before us."

GEODESY. By J. HOWARD GORE. Crown 8vo, cloth, Illus-
trated, 55.

St. James's Gazette. — "The book may be safely recommended to those who
desire to acquire an accurate knowledge of Geodesy."

Science Gossip. — " It is the best we could recommend to all geodetic students.
It is full and clear, thoroughly accurate, and up to date in all matters of earth-
measurements."

THE PHYSICAL PROPERTIES OF GASES. By
ARTHUR L. KIMBALL, of the Johns Hopkins University. Crown 8vo,
cloth, Illustrated, 5$.

Chemical News.—' ' The man of culture who wishes for a general and accurate
acquaintance with the physical properties of gases, will find in Mr. Kimball's
work just what he requires."

HEAT AS A FORM OF ENERGY. By Professor R. H.
THURSTON, of Cornell University. Crown 8vo, cloth, Illustrated, 55.

Manchester Examiner. — " Bears out the character of its predecessors for
careful and correct statement and deduction under the light of the most recent
discoveries."

LONDON: "

WILLIAM HEI NEMAN N,
3? BEDFORP STREET* W.C

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