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http://Awww.archive.org/details/cu31924003200106

THE STRUCTURE AND FUNCTIONS

OF

BACTERIA

HENRY FROWDE, M.A.

PUBLISHER TO THE UNIVERSITY OF OXFORD

LONDON, EDINBURGH, AND NEW YORK

THE

STRUCTURE AND FUNCTIONS

ele

BACTERIA

BY

ALFRED FISCHER

PROFESSOR OF BOTANY AT THE UNIVERSITY OF LEIPZIG

TRANSLATED INTO ENGLISH BY

A. COPPEN JONES

Orford
AT THE CLARENDON PRESS

1900

PRINTED AT THE CLARENDON PRESS
BY HORACE HART, M.A.
PRINTER TO THE UNIVERSITY

AUTHOR'S PREFACE

SEEING that there is at the present time no lack of treatises
upon bacteria, the publication of yet another needs some apology.
Such an apology is expressed in the title, Lectures on Bacteria*.
The lectures are intended to be an introduction to general bac-
teriology. They purpose to give a survey that shall collect and
condense the innumerable special researches into a connected
whole, and indicate in broad outlines the present position and
extent of bacteriological science.

Besides those medical aspects of bacteriology which in other
treatises occupy, rightly enough, the chief place, attention is drawn
to the importance of bacteria in agriculture, and to the parts they
play in the great fundamental processes of life—the circulation of
nitrogen and carbon dioxide. Furthermore, it seemed desirable to
point out and emphasize the advancement that general physiology
has received from bacteriological investigations. Finally, an
attempt has been made to remove the bacteria from the isolated
position to which their morphological and physiological pecu-
liarities had relegated them, and, by comparative studies, to
indicate their relations to other organisms.

A treatment of the subject on these lines that should be at the
same time not too bulky, seemed to me to be wanting. I there-
fore undertook the publication of the course of lectures I have
delivered for some years to students of biology, pharmacy, and
agriculture, with here and there among them—as it were like a
white raven!—a medical student.

ALFRED FISCHER.
LEIPZIG,

22 July, 1897.

* The title of the German work is Vorlesungen tiber Bakterien.

TRANSLATOR’S PREFACE

In offering to English readers a translation of Professor
Alfred Fischer's Vorlesungen iiber Bakterien, an Introduction is
rendered almost unnecessary, in the first place by the favourable
reception of the original German edition, and secondly, by the
fact that no work on Bacteriology of similar scope and mode
of treatment has appeared in England since De Bary’s classical
Lectures on Bacteria, But those Lectures were published in
1887, and the twelve years that have passed since then have
been years teeming with activity in every field of bacteriological
research. The present work epitomizes, in a comparatively small
space, the results of these investigations, and attempts to elucidate
them in their relations to the many-sided problems of Pathology,
and of Technical and Agricultural Chemistry, and the great
chemical processes of nature.

It should be stated that the few variations from the original
that occur have been made with the Author’s sanction, and that,
furthermore, the book has enjoyed the advantage of a proof-
revision by Professor Marshall Ward of Cambridge. The Index
is the work of Dr. Alfred J. Ewart.

ASC).

Davos PLatz,
March 13, 1900.

CONTENTS

CHAPTER I PAGE
INTRODUCTION: MORPHOLOGY

Form, Size, and Structure of the Bacterial Cell, Cell-membrane and Cell-contents
Morphology of the Bacterial Cell . . : : ‘ F a ‘ , ‘ 2 2
Finer Structure of the Bacterial Cell

CHAPTER II
MORPHOLOGY (continued)
Pigments, Intracellular Products ; Movement and Organs of Locomotion; Cell-division; Spore-

formation and Germination . ; é ‘ ‘ : ‘i , : : 4 ae
Movements and Organs of Locomotion . : ‘ : : ; ‘ ‘ 2 ‘ - 14
Reproduction of Bacteria by Fission . ' , ; : ‘ , ‘ » 16
Spores and Sporulation ‘ : : 4 ; ‘ ‘ : : » 19

CHAPTER III

TAXONOMY
The Question of ‘Species’ among Bacteria; Variability; Involution and Attenuation; the
Classification of Bacteria ‘ é : ‘ : ‘ ‘ , ‘ . ‘ « 24
CHAPTER IV
TAXONOMY (continued)
The Systematic Position of Bacteria; Other low Organisms with Pathogenic Properties . - 35
CHAPTER V
' DISTRIBUTION AND ORIGIN
Distribution of Bacteria ; Their modes of life; Spontaneous Generation . , ‘ : Ad
CHAPTER VI
PHYSIOLOGY OF NUTRITION AND GENERAL PRINCIPLES OF CULTURE
Chemical Composition of Bacteria ‘ 4 ‘ : F ‘ : F ‘ : 52
Food Stuffs of Bacteria . 5 : : ‘ Z ‘ 2 : : ‘ i 83

CHAPTER VII
RESPIRATION OF BACTERIA
Aerobiosis and Anaerobiosis; Light-producing Bacteria; Marine Bacteria; Sulphur and Iron
Bacteria : . . - F s 2 : : i ‘ . ; : . 60
CHAPTER VIII
INFLUENCE OF PHYSICAL AGENTS
Light, Electricity; Pressure, Temperature, Dryness and Moisture ; Disinfection by means of
Physical Agents. : s z 2 ' ; . . . : - 71
CHAPTER IX
THE ACTION OF CHEMICALS

Chemotaxis and Chemical Disinfection . ‘ ‘ 7 . : ‘i i ‘ . 78

vili CONTENTS

CHAPTER X PAGE
BACTERIA AND THE CIRCULATION OF NITROGEN IN NATURE

1. Introduction: The Assimilation of Free Nitrogen by the Bacteria of the Root Nodules of
Leguminous Plants, and by Bacteria inthe Soil . ; . i . » + 88

CHAPTER XI

BACTERIA AND THE CIRCULATION OF NITROGEN (continued)
2. The Liberation of Organic Nitrogen by Putrefaction, and its Mineralization by the Nitri-

fying Bacteria : F ; : ; : ‘ ‘ : : , : - 98
CHAPTER XII
THE CIRCULATION OF CARBON DIOXIDE IN NATURE

1. Introduction ; Organized Ferments and Enzymes; Races of Ferment Organisms ; Fermen-
tation of Alcohol and of Acids; Optical Activity and Fermentation : : : » 107

CHAPTER XIII
THE CIRCULATION OF CARBON DIOXIDE IN NATURE (continued)

2. Bacterial Fermentation of Carbohydrates . : ‘ : ‘ ‘ 3 . i . 116
Milk and other Dairy Products. ‘ : : ; : : : ‘ a ee) © 6
Butyric Fermentation . : ‘i ‘ F ‘ : : ; ; : . 12i
Cellulose Fermentation . . : : A : : : ‘ 7 c . 122
Mucilaginous Fermentation ‘ 5 3 . . 123

Fermentations in technical processes. . : ‘ : é : : : . 123

CHAPTER XIV
THE CIRCULATION OF CARBON DIOXIDE IN NATURE (continued)

3. The Yeast Fungi and Alcoholic Fermentation. Theory of Fermentation and Anaerobiosis.
Concluding Remarks on the Circulation of Nitrogen and Carbon in Nature. ‘ . 126
Theory of Fermentation and Putrefaction : ; . 133

CHAPTER XV

BACTERIA IN RELATION TO DISEASE

1. Diseases of Plants; Harmless ‘ Messmates’ in the Human Body; Pathogenic Bacteria ;
Points of Attack and Sources of Infection ‘ 5 é . . . ‘ : » 137

CHAPTER XVI
BACTERIA IN RELATION TO DISEASE (continued)
2. Description of some Pathogenic Species. . . . : : . : F « 147

CHAPTER XVII
BACTERIA IN RELATION TO DISEASE (continued)
3. Mode of Action of Bacteria, and the Reaction of the Organism, Serum Therapeutics and

Immunity. ‘ . ; ' : : , . : : 5 » 158
NOTES. : i 1% BR af wa ek > 6h a . . 169
INDEX ee Re rr) ee 185

*,* The Arabic figures inserted in round brackets in the text refer to the
Bibliographical Notes, pp. 169-184.

CHAPTER I

INTRODUCTION: MORPHOLOGY

Form, Size, and Structure of the Bacterial Cell, Cell-membrane,
and Cell-contents.

THE first historically recorded discovery of bacteria was made more
than two hundred years ago by Anton van Leeuwenhoek, the Dutch
naturalist and pioneer in the world of the infinitely little. Exploring those
then untravelled regions with home-made lenses of wonderful efficacy, he

found in the saliva of the human mouth
minute organisms to which he gave the
name of animalcula on account of their
power of movement,

In the description (1) and in the figures
he gives (reproduced in facsimile in Fig. 1),
curved and straight forms and long and
short rods are plainly recognizable. They
constitute the earliest reliable record of
bacteria, the study of which in later times
has revolutionized medicine and expanded
into a new science. From the year 1683
Leeuwenhoek’s observations stood alone
until, a century later, the Danish savant
Miiller made further investigations on bac-
teria. He classified them with the infusoria
and gave them names that are now familiar
to us all, Vibrio, Spirillum, and Bacillus.

SP Aas

) =——
of ES
—

=~

Fic. 1. Oldest known figures of genuine
bacteria (bacteria of the mouth) from Leeu-
wenhoek. 4 and F represent Bacillus buc-
calis maximus. B is perhaps Vibrio
buccalis; its movements were followed by
Leeuwenhoek from Cto D. Zis a species of
coccus, and G, no doubt, a Spzril/um sputi-
genum (compare with FIG. 26).

In 1838 Ehrenberg described in his great work on infusoria a large number
of bacterial forms, ranking them with his group Vibrionia, and from this
time forward the bacteria have never again drifted entirely out of sight.

It was not, however, until about the seventh decade of this century that

FISCHER B

av

2 INTRODUCTION: MORPHOLOGY

the subject was taken up by pathologists, who from that time to this
have contributed more than any others to the building up of the science of
bacteriology. It was after the publication of Robert Koch’s (2) first studies
on anthrax that the activity of those numerous investigators began, whose
incessant industry has so furthered our knowledge of these most minute
organisms and has accumulated a bulk of material such as the most
voluminous treatises (8) are inadequate to grapple with.

Before this period of expansion and rapid growth, a growth that has
been in some directions too luxuriant for strength or permanence, the
bacteria had been studied chiefly by botanists (Cohn, Nageli). Their work
had been both physiological and structural, and it is on the foundations
which they laid that the science in its modern form has been built up. For
the fundamental labours of these earlier years, that saw, too, Pasteur’s
brilliant researches into the physiology of fermentation, the student may be
referred to Loffler’s lectures on the history of bacteriology (4).

Morphology of the Bacterial Cell.

The vegetative phase of all the smaller bacteria consists of a single cell.
In the simplest types this has the shape of a sphere (coccus). If one diameter
is greater than the other, i.e. if the sphere becomes a cylinder, it is spoken
of as a rod or bacillus. In one group of cylindrical bacteria the cells are
more or less spirally twisted (Vibrio, Spirillum, Spirochaete). In Vibrio
(Fig. 2, ¢c) the twisting comprises only a quarter of a revolution or less, in
Spirillum (Fig. 2, a) one or more widely separated turns, while Spzrochaete
(Fig. 2, ¢) exhibits numerous closely-wound spirals like those of a corkscrew.
The simplest method of permanently fixing the shapes of the bacteria is to
let a small drop of water containing them dry upon a cover-glass. By this
means they are all flattened to the plane of the glass, the vibrio, for instance,
appearing, in spite of its three-dimensional curvature, only as a slightly
curved ‘comma’-shaped rod (Fig. 2, @). It was for this reason that Koch
called the vibrio of Asiatic cholera the comma-bacillus, but as a matter of fact
it has, apart from the curvature, no resemblance to a comma. A spirillum
dries in the form of a semicircle (Fig. 2, 4) and a spirochaete appears as
a sinuous line (Figs. 2,¢; 26, f), The spirochaetes often attain great length,
but it is uncertain whether they then consist of a single cell or of a connected
chain of several cells. The other types of bacteria—cocci, bacilli, vibrios,
and spirilla—are always unicellular. They may be grouped together as
Hfaplobacteria as contrasted with the many-celled true filamentous forms,
the Trichobacteria. In the members of this group (in the sulphur-bac-
terium Beggiatoa (Fig. 17, a), for instance) the vegetative phase is an un-
branched chain or filament of closely united cells, each one having the shape
of a bacillus and becoming free from the others and motile at the period of

MORPHOLOGY OF THE BACTERIAL CELL 3

reproduction. The wxbranched filamentous bacteria without definite sheaths
are generally classed together under the collective name of Leptothrix. The
most complex form is C/adothrix, an aquatic genus with numerous dicho-
tomous branchings. Here the cell-chains are enclosed in a separate sheath
and the lateral shoots arise by single cells pushing sideways through this
envelope and growing out in a new direction. The resulting branch is thus
only superficially connected with the parent stem (Figs. 2, f; 12). This
is spoken of as ‘false branching’
as contrasted with true branching,
such as occurs in the mycelium of
a fungus (Fig. 2,¢). In this case
one of the cells of the main stem
sends out a lateral evagination
which, continuing to grow in the
new direction, forms a branch.
This new member is a /ateral out-
growth of the parent cell itself, and
as intimately connected with the
cells of the stem as they are with
one another. Among the tricho-
bacteria this method of growth is
unknown.

We have now enumerated the
various morphological types occur-
ring among the bacteria, and there
remain to be considered the aggre-
gates and colonies which arise
among the haplobacteria by the

Fic.2. a, Spirillum undutla, living, with spiral twisting ;
, the same dried on the cover glass in semicircular shapes.
¢, Vibrio cholerae, slightly spiral ; d, driedincommaform, ¢,
Spirochaele Ober maieri from the blood in recurrent fever.

| Cladothrix dichotoma, a branched example, with sheath
and so-called ‘false branching’; above “a short branch of
two cells is just pushing through the sheath. g, Penicillium

multiplication of the single cell or
the proliferation of large numbers
in close proximity. The Anthrax
bacteria, for instance, often form

glaucum, a fragment of mycelium with true branching
(from Brefeld). Amplification: a@ and } 1500, ¢ and @
2250, € about 800, 7 600, # 120.

unbranched threads or chains which

are externally undistinguishable from those of the true trichobacteria
(Fig. 28). Unlike these, however, the anthrax filament may at any time
break up into its constituent cells irrespectively of the advent of the repro-
ductive process. Short threads of two or three cells, as well as single cells,
may also occur. These phenomena, both in bacilli and cocci, will be con-
sidered in more detail when the question of species is under discussion,
Very frequently the bacterial cells are found collected in masses of more or
less regular contour held together by a gelatinous substance. To these
masses the name of Zoogloea has been given. They occur both in liquids
and on the surface of solid nutritive substances, such as potatoes or gelatine.
Sometimes in liquids they form a skin or pellicle composed of very dense
BQ

4 INTRODUCTION: MORPHOLOGY

aggregates of individuals. In many cases the zooglocae and pellicles are
merely social aggregates of individuals*, just as a forest or a meadow is,
and, like these, cannot be considered as morphological units. In others, such
as the cloud-like mass shown in Fig. 3, or the delicate ramifying growth of
B. proteus (Fig. 22), we have a true ‘colony’ before us, whose shape is not
accidental, but a result of definite modes of growth and multiplication which
recurs regularly in every culture, the characters thus produced being
often of taxonomic value. In all such zoogloeae and pellicles each single
bacterium is independent of the others. The plant remains a single cell,
and there is never any division of labour such as occurs in the more complex
colonies of the lower plants (volvocineae) or animals (coelenterata).

The bacteria are the smallest of all
known organisms, the most bulky coccus
having a diameter of only about 24 (zg95 mm.).
Among the Staphylococci (the most widely
distributed pus-bacteria) the diameter is often
not morethan 0-8 u,and the volume accordingly
only j75500e000 Cub. mm. Inasmuch as the
cell-substance contains a large proportion of
water, the weight, too, is inconceivably small;
thirty billion would weigh only a gram. In
Fic. 3. Fragment of the botryoidal ag drop of water I c. mm. in size, seventeen

zoogloea of an aquatic bacterium (Zoo-

&lcca ramigera) of older authors. The fumndred million pus-cocci would have room
Tods are thickly aggregated at the peri-

hery, less so in the middle; they are and to spare. Even the comparatively large

eld together by mucus. Magn. 56. : F

compart ne Zoogioeae in Figs.17,4and anthrax bacillus is only 3-10 long and
1-1-2m broad, so that its volume would have

to be increased eight million times before it attained the bulk of a middling-

sized cigarette.

Finer Structure of the Bacterial Cell (5).

It would seem at first sight a hopeless task to attempt to gain an insight
into the architecture of so minute a thing as a bacterium. Nevertheless the
problem has been attacked, and, thanks to the efficacy of modern micro-
scopes, some new facts have been established. We might well suppose that
these organisms, standing as they do upon the very threshold of life, would
be of simpler structure than those elementary units, the cells, of which all the
higher plants and animals are built up. The first question then to be answered
is whether all those parts which constitute a simple cell, such as a plant-cell,

* The word ‘ growth-form’ is often applied to these aggregates. This literal translation of the
German Wuchsform is an unfortunate one, inasmuch as the words growth and Wuchs do not connote
precisely the same ideas, and, furthermore, the expression is used by different authors in slightly
different senses (cf..Goebel, Outlines of Special Morphology of Plants, p. 480).

PLASMOLYSIS 5

can be distinguished in the bacteria. The parts are: the cell-wall (w, Fig. 4,
a), the protoplasm (f, Fig. 4, a), the nucleus (4, Fig. 4, a), and the cell-sap
(s, Fig. 4,@). The cell-sap is contained either in spaces (vacuoles) scattered
through the protoplasm, or in a large central cavity that fills out the greater
part of the cell, the protoplasm being reduced to a thin layer coating the
cell-wall (primordial utricle). Now the relation of this protoplasmic lining of
the cell to the cell-wall is of great importance in the study of the structure
of bacterial cells, and it will be necessary to discuss in some detail the
conditions present in the case of plant-cells in general. It will then be seen
how far the observable facts are competent to elucidate the finer structure
of bacteria. The cell-sap of vegetable cells consists of water in which are
dissolved various substances, organic compounds and mineral salts. The
protoplasm, which forms a sac enclosing
the cell-sap, is during life endowed with
properties similar to those of animal
membranes, being readily permeable to
pure water, but almost or quite imper- ‘°°: }
meable to the substances held in solution
by the cell-sap. Asa result, these bodies
exert a strong pressure (osmotic pressure)
from within outwards upon the proto-
plasmic sac, stretching it and pressing

it tightly against the comparatively un-
yielding cell-wall. An explanation of
this is afforded by the modern Theory
of Solutions which shows that the mole-
cules of a substance dissolved in water
act as though they were in a gaseous

Fic. 4. Plasmolysis of a cell from a fine hair
of Echallium elatertum. a, distribution of the
cell-contents in natural state (mounted in water) >
w, cell-wall; #, protoplasm (primordial utricle); s,
cell-sap in central vacuole; 4, nucleus. 4, in 25%
common salt solution, medium degree of plasmo-
lysis; the protoplasm has rerteated from the cell-
wall and is Being constricted into two parts. c,
same cell after lying about half an hour in the
25 % NaCl; the contents form two separate
spherical masses. Magn. 300.

state and strive to fly outwards away

from each other. Consequently, if the membrane is in the form of a closed
sac surrounded by pure water and containing a solution, the substance
dissolved behaves as a gas, and its molecules, like those of hot air in
a bladder, expand and dilate the sac until the pressure inside is equal
to that without. In the case of the protoplasmic sac of plant-cells this
condition of equilibrium is unattainable, because unlimited dilatation is
prevented by the cell-wall, and the result is that the whole of the cell
is during life in a permanent condition of strain (¢rgor). It will be
observed that osmotic pressure can only originate when there is water on
both sides of the membrane, and this condition is present not only in water-
plants, but also in terrestrial vegetation, inasmuch as the cell-walls are always
saturated with water. If we now reverse the conditions and, instead of
placing the cell in pure water, lay it in a solution of greater osmotic pressure
than that of the cell-sap (5 per cent. saltpetre or 2:5 per cent. common salt)

6 INTRODUCTION: MORPHOLOGY

it is easy to foresee what will happen. The pressure on the outside of the
protoplasmic sac being now greater than that within, the protoplasm is
pressed inward away from the cell-wall. The sac shrinks until the concentra-
tion of the cell-sap is such that the osmotic pressure it exerts is just equal to
that of the fluid outside. Since the shrinking of the protoplasm commences
directly the pressure of the surrounding medium exceeds that of the cell-
sap, we have in this phenomenon, which is known as Plasmolysis, a means
‘of measuring the pressure within the cell. The gist of the whole matter
lies in the fact that the protoplasm is easily permeable to water both within
and without, but presents a more or less impassable barrier to the molecules
of the osmotic substances. As a result, the cells may lie for a long time in
the plasmolysing solution without returning to their original state. If they
be removed from the solution and placed in pure water again, the pressure
of the molecules inside the protoplasm immediately reasserts itself, and the
sac dilates until the original condition of turgescence is restored. All these
phenomena are only observable in the living cell. At death the protoplasm
loses its impermeability, and presents no hindrance to the molecules of
substances in solution. In spherical cells the protoplasm is contracted by
plasmolysis to a ball, but in long cylindrical cells, such as hairs for instance
(Fig. 4), or algal cells, the elongated sac generally becomes constricted
into two or three portions. These are at first connected by strands of
protoplasm (Fig. 4, 4),’but finally separate and form distinct masses. The
commonest case is where two round or oval lumps of protoplasm are left,
one at each end of the cell (Fig. 4,c). When, by transference to pure water,
the process is arrested and reversed, these masses melt together again
as soon as they touch and continuity is restored. This de-plasmolysation
must not be too rapidly performed, or the fragments of the protoplasmic
sac may burst, in which case the protoplasm invariably dies.

The application of plasmolysis to the study of bacterial cell-structure
may now be discussed, but it will be advisable first to consider the results
which other methods of investigation have yielded, notably those of fixing
and staining. If we examine bacteria under a very high power objective
(2,000 diams. and over) very little can be seen. The cell has a fairly sharp
outline, but it is not possible to distinguish a separate membrane enclosing
the cell-contents. The latter appears as a pale homogeneous mass with
occasional granules of stronger refringency, and in the case of some of the
larger bacteria (Spircllum, Cladothrix) sap-vacuoles differentiate themselves
from the protoplasm by their watery appearance. These meagre détails
are all that can be seen in the fresh unprepared state,—of the organs of
locomotion nothing can be seen,—but by the customary methods of fixing and
staining a number of other particulars can be made visible. If a minute.
speck of a pure culture be mixed with a fixing fluid (e.g. osmic or chromic
acid), spread upon a cover-glass, and allowed to dry, the bacteria adhere

FINER STRUCTURE OF THE BACTERIAL CELL 7

strongly to the glass and may by washing be freed from the fixative and
stained with haematoxylin or an anilin dye. Under this treatment all
bacteria, save the most minute forms, show the same structure (Fig. 5). The
membrane appears as a sharp outline enclosing a protoplasmic mass which is
smooth and homogeneous where it lines the membrane, but full of irregular
spaces (vacuoles) towards the centre. The protoplasm is uniformly stained
and shows no trace of finer details. Only the strongly refracting granules
already referred to take on a deeper tinge. Being similar in this respect to
the ‘chromatic substance’ of cell-nuclei, they are sometimes spoken of as
‘chromatin’ granules: a doubtful analogy at best. When the bacterial
cell contains only one such granule (Fig. 5, a, c, d, e) it certainly makes the
impression of a nucleus both as regards its size and its position in the cell.
But there are, just as often as not, several
granules in a single cell (Fig. 5, 4, d, 7), and
apart from the colour-reaction we have no
adequate reason for regarding them as nuclei.
They bear no relation to the process of cell-
division, and it is better to regard them merely
as granules of reserve food-stuff. All efforts to
detect a true nucleus have as yet failed, and it
is safer at present to look upon the bacterial
cell as being devoid of one. There is however
another view of the matter that has gained many

_ Fic. 5. Bacteria fixed with alcoholic
iodine solution and stained in various

adherents. When bacteria are stained with
anilin colours they appear to take up more of
the stain than does the protoplasm of ordinary
plant-cells, and to retain it with greater tenacity
in the face of decolourizing reagents such as
alcohol and weak acids. Since moreover the

ways. a and 4, Cladothrix dichotoma
with sheath and one (a) or several
{?) chromatin granules in each cell
haematoxylin). c, Typhoid bacteria
(methylene blue). d@, Vibrio cholerae
(methyleneblue). 7, Spirillum undula
(haematoxylin), All the figures show
the cell-structure described in the text.
Chromatin granules, black; vacuoles
(sap-vacuoles), white ; protoplasm, stip-
pled. Magn. a-e 2250, f 1500.

nuclei of all cells are distinguished by their

avidity for colouring matters there has arisen the idea of a certain group of
substances, xzclear stains, that possess a peculiar affinity to nuclear substance.
This affinity is mythical. Nuclei take up a// stains with more avidity than
the protoplasm does,.and this behaviour rests probably not on the chemical
nature of the nucleus but on its physical constitution, such as its great density
and power of absorption. It is to the failure to recognize these facts that
the statement must be attributed which we meet with in almost all books on
bacteriology, namely, that the bacteria have a particular affinity to ‘ nuclear
stains’ and that we are therefore justified in regarding them as primitive nuclei
devoid or almost devoid of protoplasm. Speculation followed speculation,
and, the bacteria being the simplest organisms known to us, the hypothesis
has been put forward that the first living things to arise on the earth were
similar naked nuclei, and that the protoplasm is the product of subsequent

8 INTRODUCTION : MORPHOLOGY

evolution. The tingibility of the cell-contents in bacteria is not so great
when one takes into consideration the amount of stain taken up by the cell-
membrane, and even though in some cases an unusual amount may be
absorbed there is certainly nothing that warrants us in assuming the existence
of a specific reaction between the anilin stains and nuclei. The bacterial cell
then, interpreted in the light of the above facts, is a simple protoplast
enclosed within a cell-membrane but devoid of a nucleus.

The phenomena of plasmolysis lend additional strength to this view.
To plasmolyse bacteria it is only necessary to place upon a slide a drop
of water containing bacteria, lay upon it a cover-glass (with a few fibres
of cotton-wool between to prevent crushing), and
allow the plasmolysing fluid to run in at the edge.
Spherical and very short rod-shaped bacteria become
under this treatment more highly refractive. This
increase of density is the outward expression of the
contracting of the cell-contents, and in these very
minute forms it is the only change perceptible. In
the larger bacteria, however, such as typhoid, cholera,
spirillum, cladothrix, &c., the details of the process

‘Fic. 6. Plasmolysis of bac-
teria. a, Vibrio cholerae from
an agar culture (Bouillon+1 7%
peptone+1 (- rape sugar) in
1-25 NaCl. They are plasmo-
lysed but still living ; the proto-
plasm is broken up into refrin-
gentgranules, 6, Thesame more
highly magnified, ¢, Vibrio cho-
Zevae plasmolysed with cilium.
@, Typhoid bacilli in 25 % NaCl,
stained ; cell-contents in various
positions, to the right a cell with
the protoplasm arranged as in
the plant cell (Fig. 4, 4). e,Spiril-
lum undula plasmolysed by the
evaporation of stagnant water ;
the structure of the clumps of

are readily followed. Solutions of 2-5 per cent.
saltpetre or 1 per cent. common salt (fresh blood
serum contains 0-7 per cent. NaCl) are sufficient
to set up plasmolysis. As soon as the solution
reaches the bacteria its effect is visible, and one
sees the protoplasm gradually raised away from the
cell-wall and pushed inward. Just as in elongated
plant-cells, it frequently breaks up into two or three
highly refractive rounded masses which expand and
fuse together again if the plasmolysing solution be

peed gerne ape oe replaced by pure water (Figs. 4 and 6). In short
Magn. a 300, b-e 1500. rods the protoplasm generally contracts to a single

spherical or egg-shaped mass which lies sometimes
in the centre of the cell, sometimes at one end. With a low amplifica-
tion plasmolysed bacteria often look as though they had broken up into
a row of granules (Fig. 6, a), but careful examination under a high power
reveals the delicate membrane of the cell-wall still intact (Fig. 6, 4).
Two facts of fundamental importance ate brought to light by these pheno-
mena. They show in the first place that the protoplasm of the bacterial
cell is not, as is the case with the ‘pellicula’ of infusoria, attached to the
cell-wall, but lies free within it just as does the protoplasm of plant-cells,
and furthermore that the osmotic pressure in the bacterial cell is com-
paratively low, only about half that of the cells of higher plants which need
solutions-twice as strong to plasmolyse them. The pressure inside the bac-

FINER STRUCTURE OF THE BACTERIAL CELL 9

terial cell is nevertheless considerable, namely from two to three atmospheres,
In stronger solutions (5 per cent. saltpetre) the plasmolysis is rapid but
ceases in a few minutes, and the protoplasm expands and fills out the cell
again. ‘This retrogression of the process is caused by the penetration of the
molecules of the salt. Even in weak solutions (2-5 per cent. saltpetre) this
takes place in a few hours, showing that the protoplasm of bacteria is far
more easily permeable than that of the higher plants, a peculiarity which is
shared by other low organisms such as flagellata, cyanophyceae, and florideae.
A most important result of this greater permeability is the ¢ase with which
bacteria are able to adapt themselves to changes of concentration in the
medium they live in, and it must greatly facilitate the absorption of food-
stuffs as well as the excretion of the products of metabolism such as toxins
and enzyms. Finally, it must be mentioned that the motile forms retain their
power of movement during plasmolysis, from which fact certain conclusions
can be drawn as to the nature of the organs of locomotion.

In the usual methods of preparation the liquids in which bacteria are
suspended contain a considerable amount of soluble salts (the ordinary nutri-
tive media hold o-7 per cent. NaCl), and when a drop evaporates upon the
cover-glass the liquid becomes more and more concentrated, so that before
the bacteria become dry they are plasmolysed. It is such plasmolysed cells
which we see in most cover-glass preparations, many species such as
2. typhi, Vibrio cholerae, and Spirillum undula frequently showing strongly
stained granules (Polar-granules) at each end of the cell, the central space
being empty (Fig. 6). The meaning of these appearances is evident. The
general conclusion to which all these observations lead us is that the
bacterial cell-contents are a mass of protoplasm representing an osmotic
system precisely like that of the cells of the higher plants, but, unlike them,
having no nucleus.

The membrane or cell-wall of the bacteria is in most cases thin, delicate,
colourless, and without a perceptible finer structure. Unlike the cell-wall
of plants, it consists of a protein substance (no doubt a modification of the
protoplasm) and not of cellulose *. It would seem therefore that the degree
of division of labour which characterizes the higher plants, where there
is an outer very permeable membrane of cellulose, and an inner more or
less impermeable protoplasmic layer, has not been reached by the bacteria
where communication between the organism and the outer world is regulated
by two layers of medium permeability.

As is the case in many algae and cyanophyceae, the cell-wall of some
bacteria secretes a jelly-like substance which appears optically as a delicate

* Analysis of the cell-wall of the B. ‘udercudosis shows that it contains a large amount of true
cellulose, so large indeed that it has been detected in tuberculous animal tissues, and was thought to
be a product of metabolism (Freund).

10 INTRODUCTION : MORPHOLOGY

clear space, sometimes narrower, sometimes broader than the cell it sur-
rounds (Fig. 7, c and d@). It may be rendered more visible by special
methods of staining. It owes its origin to the swelling up of the outer
layers of the cell-wall by absorption of water, the inner layers being con-
stantly built up anew from within. This gelatinized membrane may become
more and more liquefied until large numbers of cells are buried up in a
common jelly-like mass, and it is in this way that the various forms of
zoogloea arise. The majority of bacteria have no such envelope at all,
or at most a thin invisible mucilaginous coat. The formation is often largely
dependent upon the nature of the food-stuffs available (Fig. 7, 6 and c).
Inasmuch as ‘many bacteria secrete large quantities of mucilage without
forming a definite envelope, the word ‘capsule’ should be sparingly used
and restricted to clearly marked sheaths around the cell. Only in such
cases has it any classificatory value (Lezconostoc). Moreover, the clear
spaces visible around bacteria in
dried preparations are frequently
artificial products. These are par-
ticularly prone to arise in fluids
like blood and lymph, which con-
tain albuminous substances, the
albumen being seemingly precipi-
tated upon the bacteria, around
which it forms a coating that
easily takes up stain. When dry-
ing takes place the cells, being the

Fic. 7. Capsules and mucous sheaths.

a, Bac. an-

thraciswith so-called capsules in a dried streak-preparation
from the liver of a mouse; for the nature of these and of
the capsules of other pathogenic bacteria, see p. 10. dd,
L - :

‘cides (frog spawn fungus); 4, on
non-saccharine media, without sheath; ¢, with mucous
sheath on medium containing sugar (d-c from Liesenberg
and Zopf); @, older zoogloea mass with chains of cells
(from van Tieghem). Magn. a 1500, 4 and ¢ 1200, d 500.

last to give up their moisture,
shrink away from this envelope
and each one appears to be sur-
rounded by a capsule (Fig. 7, a).

That this is, however, nothing but
an artificial structure is shown by the fact that the same bacteria which
in the blood of an animal exhibit a capsule are, with some doubtful
exceptions, both in the tissues and in cultures, quite devoid of one. In
a mouse which has died of anthrax, the bacteria in the blood seem to have
a capsule (Fig. 7, 2), whilst those in sections of the kidney are totally devoid
of one. That this is not due to a difference of the modes of growth in the
blood and in the tissues is shown by the fact that the naked anthrax bacilli
from an agar culture become provided with an apparent ‘capsule’ if they are
rubbed up with a drop of blood from a healthy mouse and allowed to dry
upon a cover-glass.

Among the trichobacteria (Cladothrix, Crenothrix) and some of the
cyanophyceae (Tolypothrix, Lyngbya), the outer layers of the cell-wall
undergo a hardening process which leads to the production of a firm sheath

FINER STRUCTURE OF THE BACTERIAL CELL II

enclosing the cylindrical cells in such a way that they are freely movable
within (Figs. 2 and 5). At the period of reproduction the cells, them-
selves devoid of sheaths, become furnished with cilia, and, escaping from
the open end of the sheath, swim away and give rise to fresh colonies.
In this way whole forests of cladothrix are emptied of their cells and
there remain only the stiff sheaths which finally swell up and become
disintegrated. Occasionally the sheaths become fossilized as it were, by
the deposition upon them of oxide of iron. They are then very resistant
to decay, and accumulate sometimes in enormous quantities in chalybeate
springs and ferruginous marsh water (see Chap. VII). The term ‘sheath’
can only be applied when a distinct tube is formed in which the chain of
cells are enclosed; colourless spaces in stained preparations of filamentous
bacteria do not necessarily imply the existence of a sheath, being often the
result of plasmolysis.

CHAPTER II
MORPHOLOGY—continucd

. Pigments, Intracellular Products; Movement and Organs of Locomo-
tion; Cell-division; Spore-formation and Germination.

Most bacteria are individually colourless, and appear, even when

massed together in a pure culture, either white or of a yellowish tinge.
There are, however, a considerable number of species, the chromogenic or
pigment-bacteria, remarkable for the brilliant colouration of their cultures.
‘Some of the Sarcinae, for instance, have a bright yellow tint, Staphylococcus
pyogenus aureus is golden yellow or orange, B. brunneus yellowish brown,
Micrococcus agilis, Bacillus prodigiosus,and Spirillum rubrum each a different
shade of red, B. cyanogenus (the bacillus of blue milk) blue, B. violaceus
a deep violet ; and many kinds of water-bacteria, as well as the bacillus of
blue pus, give origin to brightly fluorescent pigments. The production of
all these colouring matters is very variable and largely dependent upon the
conditions of growth, upon the composition and reaction of the culture
media, and upon the influence of oxygen, light, and heat.

Most pigment-bacteria appear under the microscope to be colourless,
and the question at once suggests itself whether the colouring matter
is really contained within the cell. In B. prodigiosus, the cause of the
‘bleeding host,’ the pigment can be seen in the form of granules scattered
about between the colourless bacteria, so that in this case there can be no
doubt as to the seat of the colour of the cultures. The various fluorescent
substances also are secretions of the bacteria with which they are associated,
and they diffuse into the culture medium, which often fluoresces brightly
throughout its whole thickness. The pigment of B. cyanogenus also is
dissolved in the culture medium. In most pigment-bacteria similar con-
ditions prevail, that is to say, they are ‘chromoparous’ (6). Some species,
on the other hand, are actually ‘chromophorous, i.e. the protoplasm itself
is coloured. This is the case with the sulphur bacteria Chromatium and
Thiocystis, and with some sap-green species (B. virens). As regards these
last, however, it is undecided whether they are bacteria or minute algae (7).

BACTERIAL PIGMENTS AND CELL-CONTENTS 13

Finally, there are bacteria, B. violaceus for example, in which the pigment
is lodged mainly in the cell-wall; such forms may be termed ‘parachromato-
phorous,

There is conclusive evidence that the red colouring matter of the
sulphur bacteria, ‘ bacteriopurpurin, performs the same functions as the
chlorophyll of higher plants, assimilating carbonic dioxide under the in-
fluence of light and giving off free oxygen (see Chap. VII). Only the outer
layer of the cell contents is charged with pigment, the central mass being
colourless.

In all probability the colouring matter is of importance physiologically
only where it is bound up with the protoplasm. In all the ‘ chromoparous’
bacteria the pigment is an excretion merely, and, as might have been
expected, chemical and spectroscopic analysis fail to show that it has any
connexion with assimilation.

Some of the bacterial pigments are of the nature of lipochromes and
are related to the fats; others have a basic character and resemble the
ptomaines. Many seem to belong to the proteins, and the colouring matter
of B. cyaneo-fuscus has a composition similar to that of indigo.

Differentiated cell-contents, such as the starch or aleurone grains of
higher plants, are wanting in most bacteria. The protoplasm stains a
uniform golden yellow when treated with iodine. Some of the butyric acid
bacteria, however, as well as various species inhabiting the human mouth,
form an exception to this rule, and take on a blue or deep violet tint.
Very little is known about the substance to which this reaction (granulose
reaction) is due; it has been called granulose merely on the strength of
its behaviour towards iodine. Whether its chemical composition is similar
to that of the granulose of starch cannot be decided by this reaction alone.
It seems to be a carbohydrate, however, and carbohydrates as food are
necessary for its growth. The mouth bacteria obtain these of course from
our food, and the butyric acid organisms from the fermenting substances in
which they grow. The‘ granulose’ makes its appearance in the bacterial
cell in the form of very minute grains, so that after treatment with iodine
the yellow protoplasm seems full of black points. These granules become
considerably larger, and then seem to diffuse through the cell contents, so
that finally the whole cell stains blue or violet.

In the butyric acid bacteria granulose is at first absent, but is formed
when the time of sporulation draws near. In that part of the bacterium,
however, where the spore appears, none is deposited, the protoplasm staining
from first to last yellow with iodine. It would seem that we have here an
instance of division of labour, the swollen end of the bacterial cell being
devoted to sporulation, and the cylindrical portion serving as a manufactory
and storehouse for granulose, from which the spore may be nourished

(Fig. 11, c—/).

14 MORPHOLOGY

Absolutely unique in the whole of organic creation is the case of those
bacteria whose protoplasm contains free sulphur (Chap. VII). In these
‘sulphur-bacteria’ the cell is often crammed full of highly refracting
globules, which are soluble in alcohol, xylol, bisulphide of carbon, and
alkalies, and give other reactions of pure sulphur. It is in a non-crystalline,
soft, amorphous state, and when dissolved out by carbon disulphide leaves
delicate clear vacuoles in the protoplasm. Deposits of other substances
within the bacterial cell have not as yet been observed, with the exception
of fat or oil, which sometimes appear in the form of minute globules,
particularly in old cultures.

Movements and Organs of Locomotion.

All bacteria, whether spontaneously mobile or not, are, when suspended
in water, in constant motion. Examined under a high power, they are seen
to be shaken by a rapid oscillatory movement. This, the so-called Brownian
movement (8), is in no sense a vital phenomenon, but is common to all finely-
divided solid particles suspended in liquids. It may be observed in the
particles of gamboge or indian ink rubbed up with a little water. Its cause
is not well understood. As a general rule there is no danger of mistaking
the trembling motion for the spontaneous movements of bacteria.

There are two kinds of independent motion distinguishable among bac-
teria—the ordinary swimming movement, and the rarer ‘ oscillation’ peculiar
to some trichobacteria and similar to the movements of some cyanophyceae.
Among the cocci, Micrococcus agilis is the only one endowed with the loco-
motory powers, but of the bacilli a large number are actively mobile, such as
B. typhi, the butyric bacteria, and the majority of the putrefactive organisms.
The vibriones and spirilla, too, are good swimmers. The bacilli of tuber-
culosis, diphtheria, anthrax, many of the pigment bacteria, and the acetic
and lactic ferments are permanently immobile. When bacteria in active
movement are examined under a high power they seem to shoot across the
field of view with amazing speed; but this velocity is only apparent,
being due to the magnification of the distances they cover. Reduced to
actual figures, the absolute rate of progress is 10 cm. in 15 minutes, or about
3 mm. per second—in proportion to their size a very fair speed.

Locomotion is effected by special organs, the cilia or flagella. In the
fresh state, or in bacteria stained in the ordinary way with anilin colours,
the cilia are not visible, and special methods of preparation are necessary to
render them so. That worked out by Loeffler (9) has the claim to priority and
is the best. He employs in the first place a mordant composed of an iron
salt and tannin, which causes the stain to be thrown down not only within
the cell, but also upon its surface and upon the surface of the cilia, which

LOCOMOTION AND LOCOMOTORY ORGANS 15

then appear deeply coloured. Both bacteria and their cilia are by this
process made to look thicker than they really are, and appear therefore
more clearly.

According to the arrangement of the cilia upon the cell the bacteria may
be divided into three groups—monotricha, lophotricha, and peritricha (0).
The members of the first division bear a single flagellum at one end of the
cell (Figs. 8, 2, and 23); examples are the cholera germ and other vibrios,
and B. pyocyaneus. The lophotrichous bacteria have in place of the
single flagellum a brush or tuft of cilia (Spirdllum, many putrefactive
bacteria; Figs. 8,4; 22, a; 12) and the peritrichous forms have their whole
surface beset with cilia more or less thickly arranged, so that they appear
almost as though they had a shaggy coat (B. typhi, B. coli communis, some
butyric ferments, B. subtilis, B. pro-
zeus, and many others; Figs. 8, c, e, and
II, 13, 22, 24, 28). The arrangement
of the cilia is constant in each species,
and even the number of cilia united to
form the terminal tuft has a taxo-
nomic value. ;

Functionally and structurally the
cilia of bacteria correspond to the
analogous organs in other groups,
such as the swarm-spores of algae and
fungi, the infusoria, and the ciliated

. Fic. 8. Types of ciliation. @, monotrichous

cells of metazoa. They consist of long (1#6rio cholerae) ; 6, lophotrichous(Spirillum un-
< ‘i dula), c, peritrichous (typhoid bacilli); @, develop-
delicate threads of protoplasmatic sub- ment during fission of the cilia tuft of Spiril/uim
‘ a undula; eé, partial and (to the right) complete

stance, which vibrate to and fro and __ looping’of the cilia in Bac. swétilis. Magn. a-e
% 2250. In Figs. a, 4, c, the structure of the cell-con-

propel the bacterium through the water tents has been taken from iodine preparations
: (Fig. 5) in order to illustrate the structure of bac-

asa boat 1S propelled by oars, They teria as far as is known at the present time. In

Figs. dand e the contents are schematically shaded,

ro re) ] ] from the d for in these preparations (stained b effler’s
aed Uk 0! y bo 4 of method) the eecinitatian of colour on the cell-

the cell (Fig. 8, @), and are not re-__ surface conceals the structure. See also Figs. 11,
12, 13, 17, 22, 23, 24, 26 and 28, which give further

tractile. The shrinkage of the cell- examples of various ciliation.

contents during plasmolysis leaves the

cilia unaffected, so that they would seem to be fairly independent organs.

They receive their nourishment and vs viva from the protoplasm, with

which they are connected through minute pores in the cell-wall.

The cilia are very sensitive to injury, unfavourable conditions causing
them to be thrown off. When shed in this way they sometimes become
disintegrated and disappear in a few minutes. As a result a preparation
made from a culture containing actively mobile bacteria frequently shows
not a single cilium, and this is particularly the case when old cultures are
employed, the bacteria they contain being especially sensitive. Not infre-
quently the cilia become rolled up or looped before they are shed, and

16 MORPHOLOGY

peritrichous bacteria then appear to be surrounded by a mass of bubbles
(Fig. 8, ¢).

The speed of movement of the bacteria is subject to great variations,
the cilia being often paralyzed by noxious physiological conditions. Lack of
oxygen, insufficient nutriment, and the accumulation of acid in old cultures
are all factors which bring about this paralysis, and it is only necessary to
remove them (by neutralization of the acid, by removing the cover-glass, by
the addition of sugar, asparagin, &c.) to at once restore movement to the
cilia. That bacteria are motionless is not therefore a proof that they belong
to a non-motile species, and in some cases much experience is necessary for
decision.

The linear progression of bacteria through a liquid is always accompanied
by a rotation around the longer axis of the cell, as is the case with the
swarm-spores of the lower thallophytes. In the flagellate infusoria the end
of the cell bearing the flagellum is always in advance in swimming, and
probably the lophotrichous and monotrichous bacteria move in the same
way, a reversal of the path necessitating a revolution of 180° around the
transverse axis of the cell. The peritrichous bacteria have a similar mode
of progression to that of the members of the other groups, but in addition
they often exhibit curious tumbling movements, the cell hurrying across the
field of view and turning somersaults the whole way.

In Beggiatoa, one of the filamentous bacteria, remarkable slow oscilla-
tory movements of the threads are observed, isolated fragments of which are
also able to glide to and fro as diatoms do. Both these types of movement
occur too among the cyanophyceae, giving the name to one group of forms,
the Oscillariae. The cause of the phenomena is quite incomprehensible,
no organs of locomotion having been discovered, and the cell-wall being
apparently closed all round, so that the protoplasm cannot be perceptibly
extruded anywhere. It is, however, highly improbable that the motion is
independent of the protoplasm. More delicate methods of research will
perhaps enable us to clear up the mystery.

The term /lexile has been applied to filaments which, although fer se
rigid and stiff, are not straight, but twisted and thrown into curves. It is
supposed that such flexile filaments have somewhat less rigid cell-walls,
which yield to the strain exerted on them by the cell contents. These
flexile threads occur in all trichobacteria, but in the haplobacteria the
membrane is always rigid. Kinks and twists in filaments seem to arise
frequently by the mechanical displacement of neighbouring cells, The
subject of flexility needs further investigation.

Reproduction of Bacteria by Fission (11).

Given suitable conditions of nourishment, bacterial cells, like those of
all other organisms, divide into two when they reach a certain size. In

REPRODUCTION OF BACTERIA BY FISSION 17

the filaments of the trichobacteria cell-division means growth and increase
of length of the filament, and the process can only be called multiplication
when a cell detaches itself from its fellows and grows out into a new
filament. Among the unicellular haplobacteria, however, each cell-division
means duplication of the individual. ‘The procedure follows the same course
as it does in the tissues of the higher plants. The cell first increases in
length, and then becomes divided into two by a transverse wall. The
spherical bacteria (cocci) assume an ellipsoidal figure before dividing, and
the two new cells are at first flattened where they are in contact, rounding
off to perfect spheres again as they separate. Of the finer details of the
process nothing has been seen among

the bacteria, nor anything resembling 1
those changes in the arrangement of
the cell-contents which characterize
cell-division in the higher organisms.
The protoplasm is simply abstricted
into two parts separated by the in- |
growing cell-wall, as in Cladophora. | 4G :

In the filaments of this alga cell-
division is ushered in by the deposi- a.
tion of a ring of cellulose on the

Fic. 9. Transverse division of a multinuclear livin,

inner surface of the cell-wall, where
the new partition is to arise (Fig. 9, a),
By constant addition to its inner
edge this ring grows broader, until
at last, cutting through the proto-
plasm, it stretches right across the
lumen of the cell and divides it into

cell of an alga (Cladophora fracta). The new cell-

* wall (as in all multinuclear cells) arises independently

of the division of the nuclei. In Fig. @ the new trans-
verse cell-membrane grows out as a ring at right
angles to the sides of the cell and appears (in optical
section) as rod-like en beige from the latter, the
free ends being surrounded by granular protoplasm,
The large round bodies are starch grains. Fig, 6
represents an older stage, the new membrane is com-

lete with the exception of a small spot in the centre.
The figure is meant to give an idea of what probably
takes place during the fission of bacteria which are
too minute to allow the process to be followed. From

Strasburger. Magn. 600.
two equal parts. There seems little

doubt that division of the bacterial cell takes place in the same way, but
the details of the process are too minute even for our best microscopes to
follow.

Under the most favourable conditions of temperature and nutrition cell-
division takes place in a very short time. B. subtilis, the ‘ hay-bacillus,’
completes the process in half an hour. The cholera vibrio needs only
twenty minutes, so that in one day a single *comma-bacillus’ would rejoice
in a progeny of sixteen hundred trillions. This mass of bacteria would
contain one hundred tons of solid residue, and it would thus be necessary
to make experiments on a gigantic scale to allow even one single cell to
multiply with perfect freedom. In nature, of course, this increase in
geometrical progression can never take place: in the first place, because the
necessary supply of food is never present, not even in the diseased body, and
then again because many of the bacteria soon perish, and the accumulation

FISCHER Cc

18 MORPHOLOGY

of the excreta (bacterial products such as acids, &c.), particularly in pure
cultures, arrests development. As standards of comparison it may be
mentioned that a complete division of nucleus and cell in the staminal
hairs in Tradescantia takes from eighty to one hundred minutes, and that
the process may be effected in Asmoeba in from ten to twenty minutes.
The rate of multiplication among the bacteria is therefore nothing extra-
ordinary, particularly when we consider the simple structure of their cells
and the absence of a complicated karyokinetic process.

When a cylindrical bacterium divides into two it is immaterial, as far as
the result is concerned, whether the division is longitudinal or transverse ;
but, as a matter of fact, we find that it is always transverse, be the cylinder
straight or spirally twisted. This is in harmony with the generally observed
law in cell-division, that the new membrane is always formed in the
most economical manner; that is to say, in such a way that a minimum of
material is required. In cylindrical cells this is manifestly the transversal
position. When the cells resulting from such division remain connected,
chains and filaments arise, particularly in the case of non-motile species.
They occur sometimes even among motile forms, such as the cholera vibrio
(Fig. 28, 2), although in this case the moving cells are more easily separated.
Inasmuch as transverse fission is the only mode of increase among cylindrical
bacteria, it follows that chains or filaments are the only kinds of colony or
‘ growth-form’ that can occur, save where there is a subsequent shifting of
single cells (as in Cladothrix).

In the monotrichous and lophotrichous bacteria it is always the non-
ciliated end of the dividing cell which bears the cilia for the new individual
(Fig. 8, 2), the inner ends of the two rods never bearing them. In all cases
where the cell seems to have cilia at both poles there are in reality two
young cells still united by their inner ends. A curious possibility arises
out of this fact. At every division it is only one individual that is provided
with new cilia, the other cell bearing the old ones, and this process may be
repeated time after time. As a result, it may happen that of two bacteria
swimming about and not as yet disconnected one has brand-new cilia, and
the other a set which has lived through hundreds of generations.

In peritrichous forms it seems probable that, during the elongation of
the cell preparatory to fission, new cilia arise between the old ones along the
side of the cell.

Among the spherical bacteria it is manifestly, as far as the economy of
the cell is concerned, a matter of indifference in which direction the plane
of fission lies, because every plane that passes through the centre of the cell
represents a minimum. A predilection for one particular plane must there-
fore be the outward expression of hereditary morphological characters that
possess a classificatory value. The closest resemblance to the cylindrical
bacteria is shown by those spherical forms in which the planes of fission of

REPRODUCTION OF BACTERIA BY FISSION 19

successive generations are all parallel. In this case, if the cells remain
attached to each other, unbranched chains arise as in Streptococcus pyogenes
(Fig. 10, a), a pus bacterium, or as in Leuconostoc mesenteroides (Fig. 7, a).
If fission takes place alternately in two directions at right angles to one
another, flat tabular groups of cocci are produced, frequently quadrangular,
with four, sixteen, sixty-four cells and so forth. The red sulphur-coccus
————Lhiopedia and Micrococcus (Pediococcus) tetragenus (Fig. 10) are examples
of this mode _ of growth. Finally, the planes of division may be formed in
_ three different directions successively, at right angles to each other, giving
origin, when the cells remain adherent, to cubical packets. The genus
Sarcina (Fig. 10, c) offers a typical instance of these. It is, of course,
possible to determine the manner of fission among cocci only when the
cells remain’ adherent to each other, or where successive generations are
held together ‘by gelatinous exudations. If the cells separate after fission
their grouping gives no indication of the manner of division.

FIG. 10, Modes of fission among the coccaceae. a, Streptococcus pyogenes, planes of fission always parallel,
resulting in chains; 5, Ped/ococcus tetragenus (Micrococcus tetragenus), fission alternately at right angles in two
planes, resulting in plate- -like growths ; c, Savcéxa ludea, fission in three planes at right angles to each other, resulting
in cubes or bale-shaped growths.

There remains yet another mode of increase conceivable, in which the
planes of division arise without definite sequence or relation to previous planes.
This would result in all manner of irregular cell-groups, above all in rami-
fications in one or more planes; but there are no observations that would
prove the existence of such, and we can only conclude that irregular fission
does not take place. It seems likely that even in the great host of micro-
cocci (e.g. the Staphylococci of the pathologists) the splitting up of the
cells follows a fixed rule, but that their prompt severance from one another
prevents the formation of large groups. As far as the Staphylococci are
concerned, division probably occurs in the three dimensions alternately,
but not in regular sequence, so that after a few fissions in one plane a new
direction is taken at right angles to it, to be changed again soon for a
third. In this way we should get short chains, quadrate groups, and minute
cubical clusters side by side, and this is what actually occurs (Fig. 28, a).

Spores and Sporulation (12).

The bacterial cell, although able for a short time to resist the injurious
effect of an unfavourable environment (drought, changes of temperature) or
an insufficient food supply, cannot defy such influences for an unlimited

Ca

20 MORPHOLOGY

period, for months or years. It has, however, the ability to enter into a
resting stage, to assume a shape endowed with great powers of resistance—
the ‘spore.’ This power it shares with all low organisms such as algae or
fungi, which are periodically subject to dearth of nutriment or the inclemency
of the seasons. The term ‘spore’ is commonly applied to all these resting
forms, but must not be supposed to connote anything but a similarity of
function. The word ‘endospore’ has in addition a special morphological
significance, and is applied to the commonest form of bacterial spores.
Taking the sporulation of the anthrax bacillus as a type, we find the
process begins by a contraction of the cell-contents, which ball themselves
together into an egg-shaped mass as yet devoid of a proper membrane,
and lying loose in the otherwise empty rodlet: this is the young spore

Fic. 11. Sporulation and germination. a@, Anthrax bacillus with the cell-contents contracted to form the youn
spore, as yet without membrane; 4, ripe anthrax spore still enclosed in the rod, whose shape has not changed.
cand d, Clostridium butyricum (Prazm.) : ¢, vegetative Benenchacs stage ; d, ripe spore in the swollen spindle-shaped
cell, the contents of whichiare not quite used up in spore-formation. ¢ and f, Plectridium palud : e, unchanged
rod; 7, drum-stick or Lt form with ripe spore in swollen end. Fi Germination of spore in B. anthracis; the
young rod elongates itself in a direction parallel to the longer axis of the ellipsoidal spore (3, 4) (from Prazmowski).
A, Germination of spore of 2. subiz/is; the new rod grows out at right angles to the axis of the spore (3-5), and, as
in anthrax, separates from the spore brane (6) Seon Pr ki). 2B. leptosporus; the spore, surrounded
by a thin mucous coat (dotted 1-3), grows out into a rod without shedding a membrane; simplest form of germina-
tion (from Klein). Magn. a 2250, 5-/ about 1200, g-7 1000.

(Fig. 11, a). This contracts still further, becoming denser and more highly
refractive than when it filled out the cell, and there forms upon its surface
a firm coat, the proper spore-membrane, to the impermeability of which the
durability and resistance of the spore are due.. The spore lies now complete
within the cell-wall (Fig. 11, 4), which gradually decays and sets it free. The
free spore, which may be found abundantly in cultures two to three days old,
is a highly refracting, ellipsoidal, immobile body, considerably smaller than
the cell in which it arose. Under favourable conditions of temperature it
germinates in twenty-four to thirty-six hours. Rags of the cell-membrane
may often be seen still adhering to it (Fig. 11, g, 4, 2). The spores of
B. subtilis are formed in a like manner, the rods, as in B. anthracis, retaining
their cylindrical shape during the process (Figs. 11,4; 13,c). A less simple
type of sporulation is that where the rods change their shape, becoming

SPORES AND SPORULATION 21

spindle-shaped (Fig. 11, c, d), or swollen at one end like a drum-stick
(Fig. 11, ¢, f). The greater part of the cell-contents is withdrawn into this
inflated portion, and forms the spore, but some remains in the cylindrical
section of the cell, where it can be rendered visible by plasmolysis as an
extremely delicate layer lining the membrane. It is no doubt from this
remnant of protoplasm that the cilia derive their nourishment at the time of
sporulation ; for they are not drawn in during the process, but continue to
lash vigorously to and fro until the spore is set free and nothing but the
empty cell remains. The spindle-shaped butyric bacilli and the drum-stick
cells of some bacteria in bog-water form their spores in this manner, and it
seems that in all cases where the spore-bearing part of the cell alters its
shape some of the protoplasm remains in the cylindrical section.

It is certain, in spite of many contradictory statements, that the
alteration in shape has the value of a specific character, and can be used
for classificatory purposes. The significance of the process lies, of course,
not in the change of shape, but in the differentiation of the cell-contents into
two parts, one for the maintenance of life in the cell, the other subservient
to reproduction. It is a foreshadowing of that division of labour which
secures the distribution of spores and their transport to places suitable for
germination.

Endospores are still unknown in a large number of bacteria—in the
whole group of cocci, for instance, and in many pathogenic forms, such as the
bacteria of typhoid fever, tuberculosis, diphtheria, and cholera. That they
do form spores there can be no doubt, but our artificial methods of culture
do not offer them the necessary conditions, the determination of which is
one of the important tasks that bacteriology has before it. In the patho-
genic bacteria mentioned, as well as in many other forms, highly refracting
granules have been described as spores, but proof is wanting ; they have not
been seen to germinate. It is certain that in a number of cases what have
been taken for spores are merely ‘chromatin-granules’ and plasmolysed
lumps of protoplasm in disintegrated or decaying cells.

Ordinary methods of staining leave the spores untinged, and they
appear, so long as they remain within the body of the cell, as clear colourless
spaces. It must be remarked, however, that we are not for this reason
justified in regarding a// clear spaces in stained bacteria as spores. Many
methods have been elaborated for obtaining beautiful double ‘staining of
the spores within the rods, the impermeability of the spores being over-
come by boiling them in the staining solution, or by submitting them to the
action of substances like chromic acid, which render the spore-membrane
more penetrable, either by causing it to swell up, or more probably by
dissolving out certain constituents. The question whether any given
structure is a spore cannot, however, be decided merely by its colour
reaction—germination is the only reliable proof.

22 MORPHOLOGY

As soon as the spores are ripe they are capable of germination, and in
a dried state remain so for many years. This property is not peculiar to the
spores of bacteria: well-dried wheat germinates after being kept for twenty
years, and the spores of the smut-fungus may lie in a herbarium for eight
years and not lose their vitality (see Chap. VIII). Moistening with pure
water is not sufficient to cause germination of bacterial spores. A solution
capable of setting up a nutritive stimulus is necessary, and, of course,
a suitable temperature. .

The first stage of germination is the slow swelling up of the spore,
which gradually becomes less and less refractive (Fig. 11, g 2, # 2, 7 3).
In B. subtilis this phase lasts from one to three hours. Then the spore-
membrane bursts open at one point, and the contents, surrounded by
a delicate cell-wall, are protruded, and elongating soon assume the form
of a rod, the new bacillus (Fig. 11, 4 2-6). The end of this often bears
for a long time the ragged remnants of the spore-membrane. Germination
is now complete, the whole process having taken about four or five hours.
One peculiarity connected therewith is worthy of remark. The spores of
B. subtilis are in shape short ellipsoids, having the longer axis parallel
to the longer axis of the cell that gave them birth. The new rod, on
germination, bursts through the side of the spore, and stretches itself at
right angles to the latter (Fig. 11, % 5), so that the longer axis of the new
generation crosses that of the old. The spores of B. axthracis and of
Clostridium butyricum, on the other hand, burst open at the end, so that the
axes of the new and old generation are continuous. Both these types of
germination are common among bacteria, but they are constant for the
same species, and therefore can be used for purposes of classification.

The simplest germinative process is that shown by some harmless
saprophytic bacteria (e.g. B. leptosporus), where the spore itself grows
out without casting its membrane into the new bacillus (Fig. 11, z). In
this case the unchanged spore-membrane becomes the cell-wall of the young
rod, whereas in B. anthracis, B. subtilis, and Clostridium butyricum the
coat of the spore splits into two layers—an outer, which is left as the
empty membrane, and an inner, which ensheathes the protruding contents
of the spore and becomes the cell-wall of the new-born bacterium. The
spores of many fungi also germinate in this way.

De Bary (18) has described, under the name of arthrospores, structures
which have given rise to a great deal of misunderstanding. He gave this
name to certain cells in filamentous bacteria, such as Cladothrix (Fig. 12),
Thiothrix, &c., which detach themselves from their fellows and swim about
after the manner of swarm-spores, finally growing out into filaments again.
They are certainly reproductive cells (Gonidia), and may perhaps be termed
spores, in so far as a spore is subservient to reproduction. De Bary called
them arthrospores because they are formed from one joint or cell in the

SPORES AND SPORULATION 23

cell-chain, but they possess none of the essential properties of aspore. They
are simply detached vegetative cells without special powers of resistance
and destitute of any lasting germinative faculty. Another kind of arthro-
spore is said to occur in the case of Leuconostoc, where certain cells generally
larger than their neighbours surround themselves with a very thick mem-
brane, and without further preparation enter the ‘resting stage.’ Similar
cases occur among the blue-green algae, but are quite unknown among
ordinary bacteria. What have been described as arthrospores in bacteria (in
cholera cultures, for instance) are in all probability nothing but deeply-
staining granules from the detritus of old cultures. They have never been
seen to germinate. Bacterial arthrospores, if the term were justified, would
be of the same shape as the vegetative cells of the species to which they
belonged; the cholera arthrospore appearing as a curved refringent rod,
that of B. violaceus as a straight cylinder, &c.

Of the causes which bring about sporulation very little can be said.
As with other organisms, it seems to be induced by an unfavourable environ-
ment, arrest of food supply, or the accumulation of the excretory products
of the bacteria themselves. In pathogenic forms, at least in those cases
that have been carefully studied, spores are not produced so long as the
bacteria are enclosed within the diseased tissues. The anthrax bacillus
forms them only in places where there is free access of air—in the excre-
tions of diseased cattle, for instance, or on the surface of the carcase ; never
inside it, either before or after death (see also Chap. XVI).

CHAPTER III

TAXONOMY

The Question of ‘Species’ among Bacteria; Variability; Involution
and Attenuation; the Classification of Bacteria.

DESPITE the morphological sameness of the bacteria, the functions they
perform in the economy of nature are numberless. When their extra-
ordinary versatility first became known, the idea arose that they were beyond
the influence of many of the laws of life which govern all other organisms.
Any opinion regarding bacteria, however absurd it might be, was permitted,
and even the existence of definite species among the bacteria was denied.
The controversy on the species question has been long and heated, and only
in the last few years has it been decided once for all that the conceptions of
genera and species are as justifiable among the bacteria as among other
organisms. The subject-matter of the whole discussion may be summed up
in two words: pleogeny or mutability of function, and pleomorphism or
mutability of shape (14). The pleomorphists maintained that a coccus did
not necessarily remain a coccus all its life long, that it could, under certain
conditions, stretch itself and assume the shape of a bacillus, that this again
could become curved and change into a vibrio, to return again later on to
the coccus form that it commenced with. Words like Micrococcus, Bacillus,
Vibrio, Spirillum, which we now know to have a definite taxonomic value,
were in the eyes of the pleomorphists worthless designations of transient
changes of shape.

As an example of almost inexhaustible versatility the branched aquatic
bacterium Cladothrix dichotoma was advanced. But it has now been proved
to be not truly pleomorphic (15), Only at the season of reproduction, for
the purpose of securing new fields of growth, does the cell undergo change.
It loosens itself from its fellows in a filament, develops a tuft of cilia, and
emerges from the sheath (Fig. 12). The bacillus-like gonidia or swarm-
spores thus produced settle down sooner or later upon some solid body, to
which they adhere, and then grow out into new filaments. Neither coccus,

PLEOMORPHISM : INVOLUTION 25

vibrio, nor spirillum forms appear. The various twistings and distortions of
filaments, and aggregations of motionless gonidia, which have been described
as phases of pleomorphic development, are quite fortuitous.

Pus-cocci (Staphylococci) may be cultivated in any number of different
media, but they appear with unalterable persistence in the form of little
spheres (Fig. 28,2). The cholera vibrio occurs in the form of a slightly
curved rod, and it occurs in this form only. The only observable variation
in growth is that in some cultures isolated cells may prevail, and in others
chains (Fig. 28, 2). In a hay-infusion culture of B. subtilis, for instance,
we meet side by side motile and motionless single cells (Fig. 13, a, 4),
and, especially in the surface pellicles, motile
and motionless chains (Fig. 13, 4). In all these
the individual bacterium remains unchanged, a
unicellular, peritrichous-ciliate, actively motile
rod (Fig. 13, a). The motionless rods arise
from any causes which bring about temporary
paralysis of the cilia, and the motile chains
from the clinging together of successive genera-
tions of cells. In a fresh culture the fluid is
uniformly turbid, with actively moving isolated
rods only. These are afterwards driven by
lack of oxygen to the surface of the culture,
where they grow out into long, motionless, — Fic. 12. Cledothrix dicholoma,
spore-bearing chains devoid of cilia, the aggre- Seat ugg. Of the ele bean

: . i if has opened and gives exit to a swarm
gation of which forms a membrane or pellicle spore; in the right branch a whole
igi *: group of cells has changed into swarm
on the surface of the liquid. This completes spores, each with a lateral tuft of cilia.
in this side too the sheath is swollen

the cycle of forms. up, loosened, Magn. 1000.

These instances will suffice to show that
pleomorphism in its true sense does not exist. In all the simple bacteria
(haplo-bacteria) the ‘growth-form’ only changes. Single cells unite to chains,
clusters, or zoogloea, according to the substratum they live in, but the
form of the individual of the vegetative cell remains the same.

That proper and improper nutriment must have a great effect upon
the size of the cell is evident. Giant and dwarf forms occur among the
bacteria as among all other organisms, and have the same significance.
But for all species of bacteria there is an average size and form, deviations
from which are not more extensive than they are among other organisms :
always assuming that the conditions of existence are suitable, that the
bacteria are in a healthy condition, which is far less frequently the case than
we are apt to think. Imagine a few thousand animals cooped up together
in a confined space, plentifully supplied with food, but without provision for
the removal of their dejecta. A few hours would suffice to bring about

a frightful state of things. And these are just the conditions under which

26 TAXONOMY

the bacteria are living in agar cultures, in all our culture media in fact.
In such circumstances it is no wonder if some of the cells grow into mis-
shapen, feeble involution forms, with their physiological functions, such as
virulence or fermentative power, weakened or suppressed (16).

Bacteria, like all other living things, produce crippled and deformed
individuals when forced to live under unsuitable conditions (Fig.14). These
abnormal bacterial cells are known as ‘involution forms.’ The causes of
involution are of various kinds. Acetic bacteria, for instance (Fig. 14, cd),
produce monstrosities if their own fermentation product (acetic acid) accu-
mulates beyond a certain point, and also if the temperature exceeds the
optimum. In &. sudzzlis involution
forms arise if the relative propor-
tions of nitrogen and carbon in the
culture medium are not suitable—a
solution of o-I per cent. asparagin
and 10 per cent. sugar, for instance.
In other cases a high percentage of
neutral salts induces them. A curious
case of involution is that shown by
the bacteria in the root tubercles of
the leguminosae (see Chap. X).

The shapes that the cells assume
are very varied. Irregular, swollen,
or spindle-shaped rods and twisted
chains are common, and the protru-
sion of short lateral processes from
the cells often gives the growths the

Fic. 13. Bacillus subtilis in hay infusion. The
pale shows the fel cycle of forms. a, Peritri- appearance of a branched system
c ‘od;

ous motile short r: 4, non-motile rods and chains A

a motile chains; ¢, spores in non-motile rods and (Fig. 14). At the same time the
chains that unite on the surface of the infusion to form
2 cic aa pellicle ¢. Magn. a-d 1500, e from cel|-contents are reduced and stain
badly, a few granules being appa-
rently all that is left in the cell. Cells which show a high degree of
involution are dead, and cannot, even in the most favourable conditions,
be revived. They are common in old cultures, and particularly so in those
of strictly parasitic forms, such as the diphtheria and tubercle bacilli, that
even in the most nourishing media do not find quite the conditions they
require.

Such branched involution forms have been thought by some to show
that the bacteria of diphtheria and tubercle (Fig. 14, %, g) are fungi of
a higher order of growth than their ordinary rod form would indicate. It
has been suggested that the rods are merely a stage in the development of
a true filamentous bacterium or even a hyphomycete, and new names have
accordingly been given to the organisms, the tubercle bacteria being placed

INVOLUTION: ATTENUATION 27

in a new genus Mycobacterium or Tuberculomyces, and the diphtheria para-
site in another, Corynebacterium. In the writer’s opinion such an alteration
is not justified, for the branched forms of tubercle bacillus that arise in three
to six months old colonies are by no means common, and agree closely in
appearance with the involution forms in the root-nodules of leguminosae
Fig: 14, e—/, and of the acetic bacteria Fig. 14, c-d.

If the influences which give rise to involution forms be restrained
within certain limits, a general weakening or attenuation of the bacteria is

20 x

!

Fic. 14. Involution forms. a, Bac. sub¢lis from a four-days old culture containing 1 % sal ammoniac, 2% grape
sugar, and o-5 % nutritive salts (weakly acid). 4, Typhoid-like bacilli from water in hay infusion with 4 % sal
ammoniac; they are non-motile, without cilia, and suggestive of the bacteria of root-nodules (e and /). c, Bac-
terium acet? at 39°-41° (from E. Chr. Hansen). d, Bacterium Pasteurianum, 7 hours at 34° (from Hansen). ¢, Bac-
teroids from the root-nodules of Vécéa villosa ; the round spots are the still retainable remnants of cell-contents (from
Morck). a Bacteroids from Lupznus albus (from Morck; the four-armed one is from Vicia villosa). g, Tubercle
bacillus, branched filament from sputum (from Coppen Jones). 4, Diphtheria bacilli, so-called branched forms ;
they are certainly involution forms (from Bernheim and Folger). Magn. @ and 4 1500, c and d@ 100, e and fabout
1500, g 1250, 4 about 100.

effected. This condition arises spontaneously in long-continued cultivation
on artificial media ; virulence, fermentative power, and other functions being
diminished. They may be revived again, however, if the process has
stopped short of involution, by restoring the bacteria to a more natural
environment: in the pathogenic species by repeated passage through the
animal body, in the ferment organisms by giving them renewed oppor-
tunity for active fermentation.

The attenuation induced by prolonged artificial culture can be secured
in a much shorter time by exposure to more noxious agencies. The most
important form of enfeeblement, the loss of infectious power, can be induced

28 TAXONOMY

by almost all operations injurious to life, if only the proper degree of
strength be chosen. Anthrax bacilli are attenuated by a few hours’
exposure to direct sunlight or by the addition to the culture media of o-1-
0-2 per cent. phenol. Trichloride of iodine is effectual in the case of the
diphtheria and tetanus bacilli. By careful exposure to high temperatures,
Pasteur was able to produce a less virulent breed of anthrax bacilli which
retained their acquired characters for a long time (17). The change effected
was the same whether the cultures were maintained at 425°C. for twenty-
eight days, at 43° for six days, at 47° for four hours, or at 52° for fifteen
minutes. The temperature optimum for Bac. anthracis is between 30° and
37°, the maximum about 42° or 43°, and the lethal limit lies between 50°
and 60°, so that it is evident that attenuation is effected the more quickly
the nearer the temperature approaches the lethal limit.

That the decrease of virulence is only one phase of a general weakening
of all the functions is shown by the fact that the bacteria in attenuated cul-
tures have lost the power of forming spores: have become asporagenous (18).
At first sight this seems to be a result of the greatest importance. Spore
formation is one of the principal morphological features of the cell. If we
were really able to suppress it, and to suppress it so successfully that it never
made its reappearance even in the most vigorous cultures, we should indeed
have done a great thing, for we should have obtained nothing less than
the long desired proof of the hereditary transmission of acquired charac-
ters. But appearances, alas, are deceptive. It is no more possible to
obtain in this way a breed of sporeless anthrax bacilli than it is to rear
a tailless race of mice by incessantly cutting off their tails. All we do
is to hinder the production of fully ripe, resistant spores: rudimentary
spores are formed, We attain nothing more than a general enfeeblement
of the cell affecting all its functions equally. This is proved by the fact
that such ‘attenuated’ and ‘sporeless’ cultures get weaker and weaker
and finally die. By the inoculation of the weakened bacteria into suitable
animals and several passages through the body, in short, by a dracing treat-
ment, virulence is restored and resistant, fully ripe spores appear again.
The micro-organisms behave, in fact, just as sickly plants do when removed
to healthy surroundings. The importance of the experimental modification
of virulence for the attainment of artificial immunity will be considered in
a later chapter.

All these considerations show us that external agencies (in the brief period
which our experiments allow) are unable to bring about a lasting, hereditary
change in the structure of bacteria. The morphological characters remain
constant. The variety always throws back to the typical form, the concep-
tion of species and genera having the same value among bacteria as among
the higher organisms. Billroth’s views, according to which all the bacteria
occurring in an infected wound were different developmental stages of the

PLEOGENY 29

same species (Coccobacteria septica), can be no longer held. Like Zopf’s
far-reaching speculations on the cycle of forms among bacteria, they are
matters of history, and they are nothing more. The similar ideas of
Nageli have been unable to hold out in the face of more recent dis-
coveries, and there can be no doubt that the justification of Cohn’s original
conception of the possible systematic classification of the bacteria is now
generally recognized.

Less simple to decide than the question of pleomorphism is the question
of physiological change or pleogeny*. All bacteria possess to a certain
extent the power of living on different kinds of substrata, and the com-
position of these determines in a great measure the nature of the chemical
changes which the bacteria set up. According to the degree to which this
power is developed, we may conveniently divide the bacteria into two
classes, the ~onotrophic and the polytrophic. Among the members of the
first group the requirements of nutrition are specific and clearly defined.
The composition of the substrata may vary only within very narrow limits,
and, as a result, the metabolic products also of these bacteria and the func-
tions they are able to perform are specific and well defined. As typical
examples of the monotrophic group may be mentioned the sulphur bacteria,
the nitrifying organisms, all the truly parasitic bacteria, and those found
in the root-nodules of the leguminosae. Among the great host of ferments
and putrefactive organisms, too, there are monotrophic forms with specific
properties. Such are the bacteria of the acetic, lactic, and some butyric
fermentations, and the bacteria which decompose urine (urea ferment).
Some of the butyric bacteria, on the other hand, are polytrophic, and,
besides possessing specific fermentative powers, are able to break up
albuminous compounds; they are saprogenic as well as zymogenic. Others
of the same class are pathogenic also, and can live in the tissues of the
animal body (malignant oedema, quarter evil). We find, too, forms like
B. vulgaris with pronounced saprogenic (putrefactive) powers, which are yet
able to grow on non-putrefactive substances and cause them to ferment.

Again, many saprophytic bacteria are unable to exist in the animal
body, while others (typhoid, cholera) are decidedly polytrophic in this
respect, and multiply rapidly in the tissues. It is not necessary to give
parallel examples of differences among other organisms.

As might be expected, it is in monotrophic forms that the ‘ specific
characters,’ both morphological and functional, are most sharply marked.
But they undergo no lasting change even in those bacteria which are physio-
logically the most versatile; and the attainment in our cultures of new
varieties, varieties with acquired characters that are permanent and heredi-
tary, is not to be expected. We are able, it is true, to obtain cultures of

* Detailed instances will be found in Chaps. V, XI, XII, XIII, and XV.

3° TAXONOMY

pathogenic species with every desirable gradation of virulence, but they
are not new varieties. They are mere laboratory stocks, which revert at
once to the original type as soon as the original conditions of life are
restored*, It has not been hitherto possible to entirely suppress a single
biological character in any species. The supposed conversion of the anthrax
bacillus into the harmless B. subtilis was never realized, and, like most
reports of a similar character, arose from imperfection in the technique of
pure culture.

Although there is no longer any doubt that the various kinds of bacteria
constitute definite species and genera, it would be futile to minimise the
great difficulty that confronts us as soon as we try to arrange them in a
system of classification. The morphological similarity of the spherical
forms (cocci) and the close resemblance to each other of the rod-shaped
bacilli make a classification based on purely morphological data quite
impossible. Physiological characters have therefore been made use of as
auxiliaries—for instance: growth on various media and need for oxygen
or particular forms of nourishment; specific products such as pigments,
granulose, sulphur, fluorescence, phosphorescence ; specific chemical changes
such as fermentation, putrefaction, disease. A reliable description of the
species of bacteria is a task for the future, and will only be attained by the
united labours of pathologists, physiologists, chemists, and botanists (19).
Much has been done by arranging them in groups according to their most
prominent physiological characters. Examples of such groups are the
saprogenic or putrefactive, the zymogenic or fermentative, the chromogenic
or pigment-producing, the phosphorescent, the thermogenic, the nitrifying,
the iron bacteria, and the sulphur and purple bacteria. Such a classification
is not without value, but it is not justifiable to make generic names on this
principle, and use them as though the groups they indicate were equivalent
to true morphological genera. Such names as Photobacterium, Nitro-
bacter, Nitrosomonas, Granulobacter (for butyric bacteria with the granulose
reaction), Halibacterium (for marine forms), Gonococcus, and Proteus (for
several putrefactive bacteria) are names that should not be admitted in
a systematic classification. As working or trivial names they are useful
enough, but have no claim to equality with genera based on morphological
characters, which must, after all, form the ground-work of all systems of
classification. An attempt must be made, despite the dearth of material,
to obtain clear morphological definitions of the genera at least, even if it
be impossible at present for species. This was Cohn’s guiding principle
(20), and must be adhered to, The very factor to which the progress of
bacteriology is mainly due, namely the number and variety of its students,
has been a hindrance to the proper growth of a classification. Side by

* The various ‘ races’ of brewery yeasts have a different value to these ‘ attenuated’ bacteria.

BAR ec raaay cepa a I a

CLASSIFICATION 3I

side with the medically trained ‘ bacteriologist,; to whom the principles
and meaning of classification were unfamiliar, there have been investigators
from every other branch of biology—brewers, agriculturists, and even
chemists, all vying with each other in the manufacture of species. It is
not argued for a moment that botanists alone have the right to set up
a system; but it cannot be too often repeated that other investigators, if
their taxonomic schemes are to be of any value, must proceed on the
recognized and established principles of classification. As things are, there
is no agreement even as to the value to be attached to the few clearly
marked morphological features which do exist. The only point of unanimity
is the adoption of Cohn’s groups founded on the shape of the cell (coccus,
rod, spiral, or filament). The importance of the distinction between fila-
mentous bacteria and those forms which consist of a single cell deserves
more emphasis than it usually receives. The filamentous bacteria, zricho-
bacteria, constitute an order, separate from the haplobacteria, in which the
colonies of cells are mere ‘growth-forms’ or social aggregates of individuals,
such as, for example, the packets of Sarcinae, or the cobweb-like zoogloea
of B. vulgaris.

The power of movement or its absence has very properly been regarded
as a distinctive feature, but the constancy of the ciliation (mono-, lopho-,
peritrichous) has been undervalued. A cholera vibrio or a bacillus pyocyaneus
has only one flagellum (as a rare exception two); the typhoid bacillus,
B, subtilis, and many others are always peritrichous. Lophotrichous forms
like the spirilla and other aquatic bacteria, or the bacillus of blue milk
(B. syncyaneus), always have a polar tuft of cilia, the number of which is
approximately known and fairly constant. That their numbers in a stained
preparation are often irregular is due to the delicacy of the structures, which
are easily thrown off, and not to any original irregularity. In the motile
bacteria, as in the flagellate infusoria, the number and arrangement of the
cilia are morphological characters of fundamental systematic value. Another
is the form of the sporulating rod, which, although described by some as
variable, possesses in reality the constancy required of systematic characters.
The anthrax bacillus retains its cylindrical shape, the tetanus bacillus
becomes invariably swollen at one end, like a drum-stick (f/ectron), and some
of the butyric bacteria assume the shape of a spindle at the time of spore-
formation. Exceptions to the rule are generally deformed individuals, and
are very rare, not one ina hundred. A careful discrimination of these two
characters, ciliation and sporulation, enables us to arrange the chaotic multi-
tude of rod-shaped bacteria into a few well-defined genera and sub-genera.
It may be argued that in many cases the spores are unknown. This is true,
but it is no reason why we should not classify the better known forms into
‘good ’ genera, relegating the others provisionally, according to the shape of
their cilia, to genera where the sporulation is unaccompanied by change

32 TAXONOMY

of shape in the rod. The names of the genera might be formed con-
veniently in such a way that the root of the word indicated the shape of the
cell, and the termination the arrangement of the cilia. The root-words
might be daktron (rod), kloster (spindle), and plectron (drum-stick), and the
terminations -2zium for monotrichous, -2//um for lophotrichous, and -zdiam
for peritrichous types. The classification of the less numerous spirilla is,
as the table shows, a simpler matter. Among the Coccaceae the manner
of cell-division has already been employed as a generic character, but
greater weight should be attached to the contrast between two more
comprehensive groups into which they naturally fall. These are the
Homoccocaceae, where the planes of fission follow a definite sequence, and
the AJdlococcaceae, in which such regularity is not found (see pp. 18 and 19).

The different meanings attached to the words Bacillus and Bacterium
deserve notice. In two of the most recent systems of classification (21) the
senses in which they are used have little in common. Lehmann and
Neumann apply the word Bacterium to all rod-shaped forms in which spores
are unknown (a point in regard to which any day may bring a change in
our knowledge), the genus Bacilius embracing those in which spores have
been found. Of the manner of ciliation no notice is taken. Migula, on
the other hand, uses the term Bacterium for all non-motile rods, and Bacillus
for peritrichous species, the remaining motile forms, both lophotrichous and
monotrichous, constituting a new genus, Pseudomonas. This classification
very properly regards the presence or absence of spores as unimportant ;
but the shape of the spore-bearing rod is neglected, and no distinction is
made between monotrichous and lophotrichous species. Perhaps the best
plan would be to drop altogether the word Bacterium as a generic term,
seeing that it is now used as a collective name for the whole group of micro-
organisms. The word Bacz//us might then be used, in memory of Koch’s
first work, for all those species which, like the anthrax parasite, are non-
motile and retain their shape during sporulation.

At present we are not justified in regarding the presence or absence of
capsules as sufficiently important to constitute a generic character, although
very useful for the differentiation of species,

The trichobacteria are so few in number that it will suffice at present
to unite them in a single family representative of another order.

Based on the characters that have been under consideration, a classifi-
cation of the bacteria might be arranged as follows :—

Order 1. HAPLOBACTERINAE.

Vegetative phase unicellular, spherical, cylindrical, or spirally twisted; isolated or,
united in chains or clusters.
Family 1. COCCACEAE.

Vegetative cell spherical.

CLASSIFICATION 33

Sub-family 1. Alococcaceae.

Planes of fission without definite sequence; no pronounced colonies or growth-
forms, cells isolated or in short chains or irregular clusters.

Genus Micrococcus, Cohn. Non-motile. Includes most cocci, the pathological
‘staphylococci,’ &c.
Genus PLANOCOCcCUS, Migula. Motile.

Spb-family 2. Homococcaceae.
Planes of fission in definite sequence.

Genus SARCINA, Goodsir. Three planes of division at right angles to each other,
Cubical colonies, non-motile. :

Genus PLANOSARCINA, Migula. Similar to Sarcina, but monotrichous, ciliate, and
motile,

Genus PEDIococcus, Lindner. Two planes of fission, alternate and at right angles.
Micrococcus tetragenus, Thiopedia (a sulphur organism), and probably some species
usually termed micrococcus,

Genus STREPTOCOCCUS, Billroth. Planes of fission parallel, giving rise to chains;
the pathological S¢reptococct and Leuconostoc.

Family 2, BACILLACEAE.

Vegetative cell straight, cylindrical, ellipsoidal or egg-shaped; very short forms
difficult to distinguish from cocci. Fission always transverse.

Sub-family 1. Bacilleae,
Spore-forming rods cylindrical, unchanged.

Genus BACILLUS, Cohn. Non-motile. 2. anthracis, B. diphtheriae, &c.

Genus BACTRINIUM, A. Fischer. Motile, monotrichous, with terminal cilium:
includes provisionally all monotrichous rods whose spores are as yet unknown, e.g.
Bac. pyocyaneus,

Genus BACTRILLUM, A. Fischer. Motile, with lophotrichous ciliation. Includes
provisionally Bac. cyanogenus, and many other sporeless forms.

Genus BACTRIDIUM, A. Fischer. Motile, peritrichous, in some spores as yet un-
known. Very numerous representatives, e.g. B. subtilis, B. megatherium, B. vulgaris
(old genus Proteus), B. tyfhz, and B. colz.

Sub-family 2. Clostridieae.
Rods spindle-shaped during sporulation.

Genus CLOSTRIDIUM, Prazmowski. Motile, peritrichous; includes some of the
butyric bacteria. Genera with monotrichous and lophotrichous ciliation are unknown
as yet.

Sub-family 3. Plectridieae,
Rods drum-stick-shaped during sporulation.

Genus PLECTRIDIUM, A. Fischer. Motile, peritrichous; some butyric bacteria, the
parasite of tetanus and a methane ferment.

FISCHER D

34 TAXONOMY

Family 3. SPIRILLACEAE.
Vegetative cell cylindrical but spirally twisted. Fission always transverse,

Genus VIBRIO, Miiller and Léffler. Very slightly curved rods, ‘comma’ shaped;
motile, monotrichous. Vibr.o cholerae astaticae and numerous other vibrios of fresh and
salt water.

Genus SPIRILLUM, Ehrenberg. Cylindrical cells twisted in an open spiral ; motile,
lophotrichous. Spirillum undula, Sp. rubrum.

Genus SPIROCHAETE, Ehrenberg. Cells long and attenuated, spirally twisted with
numerous turns; cilia unknown; the cell membrane is perhaps yielding. Spirochaete
Obermaieri (remittent fever).

Order 2. TRICHOBACTERINAE.

Vegetative phase an unbranched or branched filament or chain of cells, the indi-
vidual members of which break off as swarm-spores (gonidza).

Family 1. TRICHOBACTERIACEAE.
(2) Filaments non-motile, rigid, enclosed in a sheath.

Genus CRENOTHRIX, Cohn, Filaments unbranched and devoid of sulphur granules,
Genus THIOTHRIX, Winogradsky. The same, but containing sulphur granules,
Genus CLADOTHRIX, Cohn. Filaments branched, false dichotomy (includes
Sphaerotilus).
(4) Filaments motile, with oscillating and gliding movements, and devoid of
a sheath.
Genus BEGGIATOA, Trevisan. Containing sulphur.

For the genus Streptothrix see next chapter. Further particulars as regards the
discrimination of genera and species will be found in the works quoted in Appendix

(No. 3).

CHAPTER IV
TAXONOMY (continued).

Tho Systematic Position of Bacteria; Other low Organisms with
Pathogenic Properties.

A QUESTION often asked is, whether bacteria are animals or plants?
Now the terms ‘animal’ and ‘plant’ are collective terms invented by
laymen to describe familiar living things, insects and elephants, mosses and
oak trees, and they date from a time when such minute beings as bacteria
were quite unknown. It is therefore as superfluous as it is futile to attempt,
as many have done, to detect the distinguishing characters of the ‘ animal’
and ‘vegetable’ kingdoms among organisms for which these terms were
never intended. For this reason Haeckel and others have proposed to
establish a third domain, that of the Protista, which shall include all those
forms in which differentiation has not been pronounced on the lines
of either animal or plant development. The new group would take up
Radiolarians, Flagellata, and Infusoria from the animal side, and the Cyano-
phyceae as well as some low forms of Algae and Fungi from the plants.
The border line between protista on the one hand and plants and animals
on the other, is, it must be confessed, artificial. To these protista, which
embrace approximately all those forms of life we commonly call micro-
organisms or microbes, the bacteria belong.

Another question almost as common as the first is, whether bacteria are
fungi, as the synonym Schizomycetes or fission-fungi would seem to’ imply.
As far as the processes of life are concerned the bacteria and fungi agree
in every detail, for, with very few exceptions *, the members of both groups
are unable to derive their nourishment from inorganic compounds. That is
to say both bacteria and fungi are metatrophic, are restricted in their food
to substances fabricated by the higher organisms. Some of them are even

* The saltpetre bacteria and others.
D2

36 TAXONOMY

paratrophic, that is, are able to exist only upon living animals or plants. But
in spite of the many points of physéological similarity between fungi and
bacteria there are very wide morphological differences. In every fungus, be
it a mushroom, a morel, a mildew (Fig. 15, c), a smut-fungus, or an animal
parasite, like Herpes, we can always recognize two distinct parts, the mycelium
or vegetative section, and, arising from this, the spore-bearing or reproductive
section. The mycelium is in all fungi an irregularly felted mass of branched
filaments, the Ayphae, which in many cases (e.g. Penicillium, Fig. 15, c) consist
of cylindrical cells set end to end, each cell having much the appearance of
a rod-shaped bacterium. The fructification is of varying degrees of com-
plexity, from isolated cells or chains of cells (Fig. 15, c) among the lower fungi

Wi SS
ATS Se
pale wr

Fic. 15. A, Oscéllaria tenuis (one of the Eyenaphiyeras!, fragment of filament; cZ, hollow cylindrical chromato-

phore ; ¢, so-called central body, finely vacuolated protoplasm with deeply-staining granules (black). 3B, Polyfoma

xvella, flagellate with two anterior cilia; v, contractile vacuole; &, nucleus; 4, membrane, cell contents filled with

assimilation products pee) Cc, Penicillium glaucum (true fungus, mycomycete), mycelium has arisen from

te ao @; on aerial hyphae brush-like fruits with chains of conidia. Magn. a 2250, 4 about 600, ¢ (from
refeld) 120.

up to the highly complex sporophores of the toadstools and mushrooms.
Upon a suitable soil the mycelium is able to vegetate for long periods,
continually producing new reproductive cells or fruits. Among the bacteria
such a differentiation is nowhere to be found. Their vegetative phase is
either a single cell or a cell filament on which special reproductive organs
are not developed but which, like Cladothriz, breaks up entirely into
gonidia. When the reproductive organs, the spores, appear, the vegetative
cell as such ceases to exist. As in the myxomycetes and many other
protista, it is entirely used up in the formation of the reproductive cell.
The bacteria may therefore be termed holocarpous, as contrasted with the
fungi which are excarpous, that is to say, able to produce several successive
fructifications from the same vegetative thallus.

Failing to find the kindred of the bacteria among the fungi we must
seek them among those low organisms which we have named collectively

CYANOPHYCEAE 37

the protista. Of these, two groups in particular deserve careful attention,
the blue-green algae or Cyanophyceae, and the Flagellata.

The Cyanophyceae present in their outward configuration many points
of resemblance to the bacteria. We find spherical cells (Chroococcus), rods
(Aphanothece), \ong cell chains or filaments (Oscillaria), and spirally twisted
unbranched forms (Spirulina). As among the bacteria, too, colonies or
‘ growth-forms’ occur; some like sarcina (Glococapsa), others in flat plates
(Merismopoedia), while sheath-bearing bacterial species like Cladothrix find
their parallels among the Scytonemeae (Tolypothrix) with their dichotomous
false branching. But it must not be forgotten that these striking similarities:
to the bacteria are not confined to the Cyanophyceae; the chlorophyll-
bearing algae offer a like series of parallels. The powdery green coating seen
on the north side of tree-trunks, &c., consists of minute, free-living spherical
cells (Pleurococcus), pools and ditches are often coloured green by myriads
of rod-shaped species (Stichococcus), the curved vibrios have their analogy
in the gracefully-formed Raphidia, and examples of sheathed filaments and’
capsulation are not wanting. This is not surprising, for free-living cells
must have the form either of spheres or cylinders, and the simplest cell
unions must be filaments, flat plates, or packets. The similarity of external
configuration is only a superficial similarity and in no way justifies a
systematic approximation of the two groups.

The cells of Cyanophyceae, whether free-living (Chroococcus, Aphano-
thece) or united to form filaments, multiply like all other cells by division,
just as do the bacteria. Just as these, too, do the isolated Cyanophyceae
‘split off’ from their sister cells. And for this reason, supported by the
not less superficial one of resemblance in shape, the bluish-green algae
(‘ Schizophyceae ’) have been placed alongside the ‘ fission-fungi’ (‘ Schizo-
mycetes’) to form a separate class of plants, the ‘Schizophyta.’ But the
‘fission’ of these organisms is not peculiar to them, for it must always occur
when isolated unicellular organisms divide; and the opinion that the bacteria
are a series of colourless organisms parallel to the schizophyceae is not
well founded.

The differences between the two groups are as numerous as the similari-
ties. The Cyanophyceae, if we except the gliding movements of the
Oscillariae, are non-motile, whereas a large proportion of the bacteria are
actively motile and possess special locomotive organs, the cilia. These are
moreover present not only at the time of reproduction, but persist throughout
the life of the cell, The sporulation of the two groups is also different.
The Cyanophyceae form not endospores but arthrospores, which arise by
the differentiation and enlargement of a whole cell. In the finer structure
of the cell again there is but one detail which the Cyanophyceae and bacteria
have in common, the absence of a nucleus. In other respects the cells of the
Cyanophyceae are much more highly differentiated than those of bacteria.

38 TAXONOMY

The bluish-green colouring matter is borne bya special organ, the chromato-
phore (Fig. 15,a@: ch), generally having the shape of a hollow cylinder or
sphere, forming the outermost layer of the cell-body and enclosing the
remaining cell contents (Fig. 15, a,c). The central mass stains deeply, and
thus has somewhat the appearance of a nucleus. We are, however, no more
justified in describing it as a nucleus than in calling the strongly-stained
granules in the protoplasm ‘nuclear chromatin.’ As in the case of the
‘chromatin grains’ of bacteria, the nature of these substances is unknown.
In no bacteria, not even in the chromogenic species, is such a differentiation
of the cell contents to be observed.

If we now turn to the Flagellata we find among them also many points
of similarity to the bacteria. In a species like Polytoma uvella (Fig. 15, 4),
for instance, often found in stagnant waters, we have an egg-shaped cell
enclosed in a definite membrane (%), and provided with a pair of permanent
polar cilia or flagella. Other forms, such as Jonas, are ‘monotrichous,’ others
again, like Tetvamitus, have a brush or tuft of cilia at the anterior end of the
cell. The process of ‘encystment,’ too, closely resembles the formation of
endospores, the cell-contents contracting and secreting a new membrane,
the cyst or spore thus formed being set free by the disintegration of the
membrane of the mother cell just as are the endospores of bacteria. But
notwithstanding these points of likeness the Flagellata differ from the bac-
teria in one fundamental feature: ¢hey possess a definite nucleus (Fig. 15,6: k)
like that of the cells of higher organisms. It is therefore evident that we
have as little right to assume a close genetic relationship between bacteria
and Flagellata as between bacteria and Cyanophyceae. The view most
in harmony with observed facts is that which looks upon the Bacteria as
a distinct group of protista, simpler in structure than the rest, and showing
affinities to both Cyanophyceae and Flagellata. We might regard the bac-
teria as the more direct descendants of the ancestral stock from which these
arose, the differentiation of a chromatophore leading to the Cyanophyceae
and the development of a nucleus as well as the general attainment of motile
power giving rise to the flagellate phylum. Among the arche-bacterial
types there may well have been both motile and non-motile forms which
would have been respectively the starting-points of the two branches, whilst
capsulation, the production of cell-chains or filaments, and other colonial
or growth forms were primitive acquirements which have reappeared
again among the Cyanophyceae and Flagellata and undergone greater
specialization.

The protista (sicro-organisms), the simplest living things, among which
we have tried to indicate the place of the bacteria, are not only manifold
in form, but have very different modes of life and exercise a great variety of
different functions in the economy of nature. Asa general rule, these physio-
logical features are not so conspicuous among the other groups as they are

SACCHAROMYCES: AMOEBAE 39

among the bacteria, but they become so when vigorous growth and suitable
conditions of nourishment allow them to multiply rapidly. Such instances
as the yeasts and the mould-fungi, with their energetic chemical effects,
suffice to remind us that the bacteria are not alone in their performances.

Some of these low organisms (22) are, like the bacteria, capable of pro-
ducing pathological effects, but it is very rarely that such cases present the
characters of a genuine infectious disease. Only within the last few years
has it become known that the yeast-fungi (Saccharomycetes, see Chap. XIV)
are capable of developing pathological properties. Injection into animals
of pure cultures of different species of brewery and distillery yeasts gives
rise to processes having all the characters of true parasitic diseases, ending
in many cases with the death of the animal. The germs spread to distant
parts of the body and are found in all the organs as well as in the blood,
but the lesions produced have not been recognized as those of any known
disease in human beings. Still, an infection by yeast cells is just as con-
ceivable as by any other organisms. It has recently been suggested that
the various forms of cancer and similar neoplasms may be due to parasitic
saccharomycetes, and some investigators have announced the discovery of
such in stained sections. Unimpeachable results have however not been as
yet attained, and it has been shown in some cases that the supposed para-
sites were nothing but fragments of cells and nuclei of the tissues themselves.
The parasitic nature of tumours is indeed denied by some pathologists.

The thrush fungus (Saccharomyces albicans of some authors) found
on the buccal mucous membrane of children belongs possibly to the
saccharomycetes. Its elongated cells have much the appearance of
a yeast and they multiply by budding in the same way, giving rise to
mycelium-like masses which cover the culture fluid with a thick pellicle.
They set up, too, in suitable media (beer-wort) weak alcoholic fermentation.
Whether these masses are true mycelia or not is uncertain, and it is
therefore doubtful whether the fungus should be classed among the yeasts
or with such hyphomycetes as Ozdium and Monilia candida,

Some of the closely allied group of Flagellata occasionally occur as
parasites or messmates in the animal body (e. g. Mastigophora) which, as we
have seen, have alliances to bacteria, but it has not yet been demonstrated
that they are ever pathogenic. Zyrichomonas vaginalis is sometimes found,
associated with other micro-organisms, in the mucous secretion of the
vagina. <A species called Zrzchomonas intestinalis has been found in the
intestine in cases of diarrhcea and cholera, and a similar species occurs
sometimes in the lung (sputum) in cases of bacterial infection. It is highly
probable that all these forms are merely harmless water organisms which
have found their way accidentally into the human body.

Of more importance are some of the Amocbae, that group of naked
protozoa which move about by the protrusion and retraction of portions of

40 TAXONOMY

their protoplasmic substance to which the name pseudopodia has been given.
The amoebae multiply rapidly by simple fission and, like many other
unicellular organisms, undergo from time to time a process of encystment.
The cysts are enclosed in a thick membrane which possesses great
powers of resistance, and from them, under suitable conditions, the new
amoebae emerge. Inasmuch as the amoebae are common in ponds, and
ditches, and in damp earth, it is evident that they can obtain easy access
to the human body. The best known pathological species, Amoeba coli,
has been frequently described in cases of dysentery. But it is not present
in all cases, and it seems to occur sometimes in the healthy intestine,
so that its causal connexion with dysentery has yet to be proved. Pure
cultures, free from bacteria, have not yet been obtained, nor has it been
possible to cause infection by inoculation in animals. An amoeba-like
organism of very doubtful legitimacy has been described associated with
cow-pox (Cytoryctes variolae), but it is certainly not the cause of the
disease. The great prizes for the discovery of the vaccine microbe have
yet to be won.

Closely related to the amoebae is, no doubt, the Plasmodium malariae
(Haemamoeba, Laverania), which occurs in the blood in cases of malarial
fever. It is a very minute amoeboid organism which lives in the plasma,
and (in greater numbers) zzs¢de the red blood corpuscles. At first colour-
less, it becomes pigmented later on from the accumulation of granules of
a black substance (Melanin) which arises from the decomposition of the
haemoglobin. The plasmodia are seen to be most numerous during the
periodical attacks of fever, one, three, or four days as the case may be, which
characterize the disease. They then either become disintegrated or break
‘up into a number of small spheres which have been called spores. Then
the number of amoebae increases again till the next paroxysm. Whether
these are true spores or not, is, like much else written about the malaria
parasites, uncertain. They have not been seen to germinate, nor has it been
possible to obtain pure cultures of the organisms. It seems, however,
certain that they are really the cause of the disease, for its symptoms
can be set up in a healthy body by the injection of blood containing the
plasmodia. How the parasites enter the body, whether by the respiratory
tract or the intestine, or, what is more probable, through minute wounds,
such as insect-stings, is not yet known. Their habitat and mode of life
outside the body are also unknown, but there can be no doubt that they
are saprophytic in malarial districts *.

In the blood of birds, reptiles and amphibia, there are sometimes found

* Researches now in progress on the west coast of Africa and elsewhere seem to show that certain
species of mosquitoes are the bearers of the malarial parasites which are introduced into the human
body by the insects’ probosces.

SPOROZOA. LEPTOTHRIX: ACTINOMYCES 41

parasites similar to the plasmodia, with which they have been associated
to form a new group, the Haemosporidia. Their complete life-histories
are unknown, and clinical descriptions are wanting. The best known
species is Drepanidium ranae, from the blood of the frog. Formerly
described as a Cytozoon, it was at one time regarded with great interest, as
it was thought to be not a parasite, but a tissue element of the frog. Its
parasitic nature is now, however, generally recognized. There may be
mentioned here the numerous kinds of parasitic micro-organisms, which are
classed together as Sporozoa (Gregarinae, Coccidia, Sarcosporidia). They
are all very imperfectly known, as it has not been possible to study them in
pure cultures,

Among the organisms that have been at different times reckoned as
bacteria is a small group of fungi, some of them pathogenic, known as
Streptothrix. They form felted mycelial masses of extremely delicate
branched hyphae. In pure cultures they are in some cases sterile, but in
others bear fructifications in the form of conidia, either single or in short
chains, arising from the hyphae, resembling in this respect the lowest fungi
(Haplomycetes, Hyphomycetes), to which they undoubtedly belong. They
have nothing in common with the bacteria.

There seems to be reason to think that the genus Strepiothrix (Oospora)
will meet the same fate as the so-called Leptomitus. Leptomitus was the
name formerly given to all and every kind of filamentous fungal growth
that made its appearance in neglected solutions in druggists’ shops, in ink,
&c., &c. We now know that these are not the complete forms of any specific
fungus, but merely the sterile mycelia of various moulds growing slowly and
abnormally in more or less unsuitable media. There can be little doubt
that many growths now termed streptothrix will in like manner turn out
to belong to well-known genera of hyphomycetes, which will complete their
life-history when planted on suitable media. Even the best known species
of Streptothrix, S. actinomyces (Actinomyces bovis), the ray-fungus, does not
seem to run through its entire cycle of growth in our artificial cultures.
Its thallus consists of branched filaments made up of cylindrical cells just as
in an ordinary mould-fungus, and it grows upon agar or blood-serum in the
form of thick wrinkled mats with a ‘pile ’-like surface. This velvety coat is
due to the aerial hyphae which rise vertically from the mycelium and break
up at their free ends into rounded cells like conidia. Whether these really
are spores or not remains to be proved.

The ray-fungus gives rise in cattle, and sometimes in man, to peculiar
hard tumours, commonly in the tongue or jaw (‘lumpy jaw’). These tumours
suppurate sooner or later and often spread to other parts. Infection seems
to be effected by the agency of sharp awns of rye and other cereals on which
the fungus probably grows as a mould. They pierce the mucous membrane
of the mouth, and carry the spores into the subcutaneous tissue. Attempts to

42 TAXONOMY

convey the infection by the inoculation of pure cultures or pieces of diseased
tissue have not been successful. Microscopically the actinomycotic nodules
are seen to consist of nests of densely matted mycelium with the surface
layer of hyphae radiating outwards in all directions. The ends of the fila-
ments terminate in club-shaped swellings, whose appearance is characteristic
and unmistakable. They were formerly regarded as sporangia, but accord-
ing to more recent researches are the result of a swelling-up of the cell-wall,
or due to the deposit upon it of a gelatinous substance. They would there-
fore seem to represent rather a degenerative phase of the fungus than
organs of reproduction.

A number of hyphomycetes are associated with various skin-diseases.
Whether they are strictly parasitic, or whether they occur outside the living
body in nature as simple mould-fungi, is uncertain. One of the commonest
forms is Trichophyton tonsurans, the cause of a depilatory skin complaint
known as Heres. The mycelium is found in the epidermal scales and
blisters, and grows well in artificial cultures, giving rise to chains of cylin-
drical gonidia. In Favus,a skin complaint common to man and animals,
another hyphomycete is found, Achorion Schoenleiniz. Some observers
think that this form should be split up into a number of distinct ‘species,
others are content with one. Its morphology (in a botanical sense) has not
yet been fully investigated, but it belongs undoubtedly to the group of the
haplo- or hyphomycetes.

The beautifully formed mould-fungi of the genus Aspergillus also
occasionally invade the human body. Besides the stalked heads with their
radiating chains of conidia, another kind of fructification, the perzthecza, is
known. These perithecia show that Aspergillus belongs to the Perisporiaceae
(Ascomycetes), and for this reason some investigators have looked upon all
haplo- and hyphomycetes as imperfect ascomycetes. According to this
view we must expect to find some day the ascospores and asci of forms
like Achorion, Trichophyton, and countless others. This is undoubtedly
going too far. There undoubtedly exist simple moulds whose whole life-
cycle is comprised in mycelium and conidia. Aspergillus itself lives,
particularly in cultures, for years without forming ascus fruits. The conidia
of the mould-fungi retain their power of germination in the dried state for
very long periods.

Three species at least of Aspergillus, A. fumigatus, A. niger, and
A. flavus (all with coloured spores), are possessed of pathogenic properties.
If a suspension of the spores be injected into a vein in the rabbit, mycelia
grow in all the organs of the body, and the abscesses formed are rapidly
fatal. Among birds, spontaneous infection is not uncommon, commencing
generally in the respiratory tract, the ear or the eye. Human beings, too,
are sometimes attacked, but the disease remains local, not spreading to
other parts of the body. In spite of these facts it is uncertain whether the

MUCORINE FUNGI 43

aspergillus is the original cause of the diseased condition, or whether it
merely finds an entrance in places where the natural resistance of the tissues
is already lowered by injuries such as the attacks of other micro-organisms.
The spores are omnipresent, and accidental invasion ‘is so easily possible
that every case must be judged on its own merits.

There remain to be mentioned as possible pathogenic organisms a few
species of the genus Mucor, a mould-fungus of the order Phycomycetes.
They are much less highly organized than the Aspergilli. The mycelium
consists of branched, ramified, non-septate hyphae, from which rise vertical
branches (aerial hyphae) that bear the spore capsules (sporangia). When
injected into the blood the spores of M. corymbifer and of M. rhizopodi-
formis set up lesions similar to those of aspergillus spores; but only one
case of spontaneous infection has ever been observed in man. It is worthy
of remark that those species of Mucor and Aspergillus which are capable
of growing in the tissues of warm-blooded animals flourish only at high
temperatures (37° C.), whilst those which are satisfied with less warmth grow
in the tissues either badly or not at all.

CHAPTER V
DISTRIBUTION AND ORIGIN

Distribution of Bacteria; Their modes of life;
Spontaneous Generation.

IT would be impossible to give a more concise and, at the same time,
accurate account of the distribution of bacteria in nature than is furnished
by Goethe’s lines in ‘ Faust’ :

‘Der Luft, dem Wasser wie der Erden

Entwinden tausend Keime sich
Im Trocknen, Feuchten, Warmen, Kalten!’

While Mephisto’s haughty words :

‘Hatt’ ich mir nicht die Flamme vorbehalten ;
Ich hatte nichts Apart’s fiir mich’

remind us that fire is the most powerful weapon that mankind possesses
against bacteria. Unfortunately it is an engine of destruction that cannot
always be employed.

In discussing the question of distribution, it is necessary to discriminate
first of all between actively growing bacteria and latent germs. Highly
resistant bacterial spores, and the hardly less resistant dried vegetative cells
of many species, occur everywhere upon the earth’s surface and upon the
animals, plants, and inanimate objects that cover it. They are, in short,
ubiquitous. But living bacteria in active growth and multiplication only
occur where certain conditions necessary to their welfare are fulfilled; above
all, presence of water—the vital element of all organisms—a suitable tem-
perature and a suitable supply of food. In nature such conditions are found
in ponds or rivers contaminated by dead plants or animals, in dung, manure,
and moist soil. In our dwellings it is chiefly in milk and other dairy
produce, stale meat, and victuals of all kinds that bacteria are found. The
majority of them, both in and out of doors, belong to harmless species, but
there is no doubt that occasionally pathogenic forms occur. To find out
the natural haunts of these dangerous neighbours, places where they are

BACTERIA IN EARTH, AIR, AND WATER 45

present not merely as isolated specimens but in large numbers, is one of
the most important tasks bacteriology has before it.

Methods for detecting bacteria in air, water and earth, have been
greatly perfected during the last few years, but only the general principles
can be treated of here: practical details will be found in works especially
devoted to bacteriological technique (28).

Atmospheric germs may be demonstrated in a rough-and-ready way
merely by leaving exposed to the air nutritive culture media, such as agar
or gelatine. The bacteria that fall upon the moist surface soon multiply,
each one forming around itself a small colony visible to the naked eye.
For an exact determination it is necessary to allow a measured quantity of air
to pass slowly through a tube, the inner surface of which is coated with
sterilized gelatine. Upon this the bacteria are precipitated, and the resultant
colonies can then be counted and examined in detail. There are a number
of other methods in which the air is drawn through sterilized glass beads,
wool, sand, or sugar. The bacteria contained in it are thus filtered off, and
the material containing them can be mixed with gelatine or other nutritive
media, and the colonies allowed to develop in the usual way. The air in
our dwellings usually contains more bacteria than that out of doors. Ten
litres of air from a hospital ward, for instance, contained from thirty to
a hundred germs; the same amount of outside air only from one to five,
half of these being the spores of fungi. Air in motion is always far more
laden with bacteria than when at rest, and dusting or sweeping causes their
number to increase enormously. That it must be so is self-evident, for dust
of all kinds is rich in spores and dried but still living vegetative bacterial
cells. They are whirled up into the air with the dust-particles which also
bear adherent germs, and by reason of their lightness settle down through
the atmosphere very slowly.

Where there is no possibility for diseased secretions (e.g. sputum, diph-
theritic membrane) to become dried and pulverized, the bacteria found in
the atmosphere are of a harmless kind, or at most pus-cocci are present.
The air expired from the lungs is absolutely free from micro-organisms, so
that evidently the respiratory tract must act as a germ filter, the bacteria
we inhale being retained within the body. But the majority of them are
harmless, and moreover adhere to the mucous membrane of the nose, mouth,
and pharynx, only a very small number ever getting as far as the lungs.
Still, seeing that we inhale, on an average, five hundred litres of air every
hour and, consequently, some fifty to two hundred and fifty germs, the
danger of dust contaminated by disease products is very great.

Water has always been looked upon as one of the chief agents in the
spread of infectious diseases, and its bacteriological analysis is therefore a
matter of the utmost importance. Most natural waters contain bacteria, and
even the distilled water of our laboratories holds sufficient nutritive substance

46 DISTRIBUTION AND ORIGIN

in solution to allow some to develop. This is, however, not surprising when
we consider that thirty thousand bacteria only contain about z$,>th of
a milligram of solid matter. Rain-water is contaminated with bacteria which
it has taken up in its passage through the air; one sample contained thirty-
five germs per litre. The water of rivers, lakes and springs contains very
varying numbers of micro-organisms, their abundance depending largely
upon the amount of organic matter present. As soon as water is rendered
impure by the influx of such substances as are carried into rivers by sewers
and drainage canals, it ceases to be merely a liquid in which bacteria can
retain their vitality for a long time unimpaired, and becomes a nutritive
medium in which they proliferate with great rapidity. The water of the
Spree for instance contained, above Berlin, on an average 6,140 bacteria
per cubic centimetre, that taken below the city no less than 243,000.

The bacteriological analysis of water is a very simple process, If the
microscope shows that bacteria are not too numerous, a measured quantity
of water (generally 1 c.c.) is mixed with nutrient gelatine and poured out on
to a glass plate or shallow dish. The germs distributed through the gela-
tine are allowed to develop into colonies which can then be counted in the
usual way. The mere enumeration of the bacteria in water is of far less
importance than the determination of their nature and properties. The
majority of forms found in rivers and springs are of an entirely innocent
character ; they are bacteria whose natural habitat is water, and they per-
form a useful task in consuming the dissolved organic impurities. But-when
by any means pathogenic species obtain access to. such waters these may
easily become a source of infection, particularly when, as is the case with
cholera and typhoid organisms, the bacteria belong to species which can
live and multiply side by side with the ordinary saprophytic forms (see
Chaps. XIII and XIV)*. When the water to be analysed contains a very
large number of bacteria it must be diluted, before mixing, with an equal
quantity of sterilized water. For the detection of isolated pathogenic forms
in comparatively pure water, that is to say in water containing very few
bacteria of any sort whatever, a method of ‘nursing’ or ‘enrichment’ is
employed. A sterile nutritive solution (peptone-sugar) is added to the water,
so that the few germs present may multiply before the gelatine is added.
This method involves ofcourse the drawback that the non-pathogenic species
multiply also, and the danger that they may outnumber and overpower the
less abundant bacteria we are looking for.

Numerous aquatic bacteria are able to resist freezing for long periods,
and ice sometimes contains enormous quantities of them (e. g. 2,000 per c.c.).
[Even glacier ice is not free from them.]

Of all the natural habitats of bacteria there is none in which they are

* For marine bacteria, see Chap. VII.

CLASSIFICATION ACCORDING TO NUTRITION 47

found constantly in such numbers as in the soil. They occur both as spores
and in active vegetation (e.g. nitrifying bacteria). In the soil, as in water,
the presence of organic matter is favourable to their increase, and they are
most numerous in cultivated earth. Garden mould seldom contains less than

. 100,000 germs per c.c., among them being almost always some pathogenic
forms such as the bacilli of tetanus and malignant cedema. Putrefactive
and pigment bacteria are also generally present as well as fermentative
and nitrifying organisms. For the ordinary purposes of hygiene the culti-
vation upon nutrient media of the bacteria of earth, water, and the atmo-
sphere, and the detection and determination of pathogenic species, are
sufficient ; the latter task being, however, by no means easy. Still more
difficult and tedious is the systematic investigation of all the species present,
necessitating as it does the employment of a number of different culture
media varying in composition according to the nature of the species to be
cultivated. It is not possible, for instance, to isolate the nitrogen-fixing
bacteria of the soil upon a gelatine suitable for the tetanus parasite. In
each case the character of the culture media must correspond to the require-
ments of the organism to be cultivated.

/ ~~ Tt has been customary to divide the bacteria, according to their mode of
life, into two great physiological classes, the Saprophytes and the Parasites.
Both groups are dependent upon other organisms for their sustenance ; both
are unable either to build up their protoplasm from inorganic bodies, or
to obtain, from the decomposition of such, the energy necessary for their
vital processes. Those bacteria which can live and grow only within the
tissues of living organisms are called parasites, those which are satisfied
with the secretions or excretions of living, or the substance of dead, organisms,
are called saprophytes.

But this classification no longer corresponds to facts. Being a corollary
of two axioms of general physiology, it could be held good only so long
as their validity was unimpaired. The first of these axioms was, that of
all organisms the green chlorophyll-bearing plants alone* are able, with
the aid of sunlight, to assimilate the carbon dioxide of the atmosphere, and
from it to build up carbohydrates. All other living things, that is to say,
plants devoid of chlorophyll, bacteria, and all forms of animal life, were
supposed to be dependent, for their carbon, indirectly or directly, upon the
carbon compounds already formed by green plants. The integrity of this

principle has now been destroyed by the discovery of certain bacteria in the

of the atmosphere. The second postulate was that no organisms of any kind
could fix and utilize the free nitrogen of the air. All organic nitrogen was
thought to be derived from the nitrates of the soil; green plants being

* The red and the brown seaweeds also.

48 DISTRIBUTION AND ORIGIN

again the only forms of life able to take it up in this state, other organisms
receiving it from them at second hand in the shape of highly complex
nitrogenous bodies. But this theory too has had to be abandoned since
bacteria have been found, semi-parasitic in the tissues of plants, that can
assimilate and store up free nitrogen (see Chap. X), The two groups of
bacteria which necessitate a qualification of these two fundamental principles
are, in a sense, complementary to one another; on the one hand, the earth
bacteria which get their nitrogen from soluble nitrates, and their carbon
dioxide from the air; on the other, the bacteria living on certain Legumi-
nosae which derive their carbon dioxide from the tissues of the plant, and
their nitrogen from the air.

These facts force us to recognize the existence among bacteria of a
special group of forms characterized by an extremely primitive metabolism,
a physiological humility which shows them to occupy the very lowest rung
of the ladder of life *.

Such organisms as these cannot possibly be called saprophytes, and
placed side by side with organisms which, like the putrefactive bacteria, have
complicated physiological requirements. A better classification would be
to divide the bacteria, according to their mode of life, into three biological
groups, prototrophic, metatrophic, and paratrophic (24). Prototrophic species
are those which either. require no organic compounds at_all for their
nutrition (nitrifying bacterium), or which, given the smallest quantity of
organic carbon, can derive all their nitrogen from the atmosphere (bacteria
of root-nodules). With them may be classed those forms that are able,
with a minimal supply of organic matter, to break up specific inorganic
bodies and derive energy from the process (sulphur and iron bacteria). In
no case are the chemical changes involved clearly understood, and there
apparently exist forms exhibiting gradations between the purely proto-
trophic and the metatrophic habit.

The metatrophic bacteria, under which heading most known species
must be placed, cannot live unless they have organic substances at their
disposal, both nitrogenous and carbonaceous. They flourish in every place
where these are accessible, in impure water, on foodstuffs of all kinds, and
in all the cavities of the body which are in communication with the exterior
and offer nourishment in the form of secretions or particles of food; the
mouth, for instance, and nose, the alimentary canal, and the vagina. Many
of the metatrophic bacteria bring about profound changes in the chemical
composition of the media in which they grow: fermentation (zymogenic
bacteria), or putrefaction (saprogenic bacteria). Others break up the sub-
stratum in a less conspicuous manner, but develop particularly in media

* Tt seems more than probable that further investigation will result in the discovery of other low
organisms (protozoa) with similar functions.

CLASSIFICATION ACCORDING TO NUTRITION 49

which have already been decomposed by saprogenic species, and contain
a mixture of organic compounds which serve as nutriment. Such bacteria
may be termed saprophile. :

In the case of some metatrophic species the chemical change effected
by their growth varies according to the nature of the medium they grow in
(polytrophism), while the action of others is restricted to some one specific
process (monotrophism, see Chap. III). The ability to grow in the living
tissues is possessed in various degrees by different species, they are faculta-
tive parasites (cholera, anthrax, and perhaps typhoid bacilli), but many are
totally unable to exist under such conditions (obligatory saprophytes, obligatory
metatrophic bacteria). .

Finally, the paratrophic group includes all those bacteria that can
exist only within the living tissues of other organisms. Paratrophic bacteria
occur either not at all outside the animal body (gonococci), or else only as
dust-borne spores or resting cells (tubercle, diphtheria). Their culture is
possible only under conditions closely resembling those of the living tissues
(on serum at blood-heat, for instance), and where there is freedom from
competition with rapidly growing metatrophic species. Some paratrophic
bacteria seem to flourish well even outside the animal body, if their growth
- on culture media is any criterion, but the conditions here are so very
special that no conclusions can be drawn therefrom as to their behaviour
in nature.

Using then the modes .of nutrition as a basis for classification, we
‘distinguish the following groups :—

I. PROTOTROPHIC BACTERIA.

Nitrifying bacteria, bacteria of root-nodules, sulphur and iron bacteria ; occur only in
the open in nature, never parasitic, always monotrophic.

II. METATROPHIC BACTERIA.

Zymogenic, saprogenic and saprophile bacteria; occur in the open and upon the
external and internal surfaces of the body, sometimes parasitic (facultative parasites),
monotrophic or polytrophic.

III. PARATROPHIC BACTERIA.

Occur only in the tissues and vessels of living organisms, true (obligatory) parasites.

’

It is worthy of remark that not only the bacteria but all other organisms
can be arranged in these three biological divisions. All green plants from
the simplest unicellular alga up to the largest forest trees are prototrophic;
all fungi and animals are metatrophic, except parasitic forms, which are
paratrophic.

_ Seeing that the spores of metatrophic bacteria are ubiquitous, it is not
‘surprising that all liquids offering suitable conditions of nourishment should,

if left uncovered and exposed to the air, soon become turbid from the
FISCHER E

50 DISTRIBUTION AND ORIGIN

development of bacteria. But although it seems to us nowadays self:
evident and unavoidable that such turbidity must owe its origin to the
development of the ubiquitous and invisible germs—omune vivum ex ovo—
this occurrence was, until not so very many years ago, a profound mystery.
It appeared absolutely certain that these living ‘animalcula’ must have
arisen spontaneously from the non-living constituents of the liquid. ‘ Spon-
taneous Generation’ (generatio aeguivoca) seemed, on apparently good
evidence, an indisputable fact.

The question as to the origin of life—an older problem even than that
of evolution—is the natural outcome of the Kant-Laplace theory of the
origin of the earth, according to which our planet was at first a molten mass
that had to cool down before living things could exist upon its surface.

Whence came these first forms of life? Were they precipitated from
other planets through space upon our globe, or did they arise de xovo from
the non-living substances that then alone existed? The first hypothesis
has little to support it, and would only throw the question one step further
back. It seems much more probable that the first organisms did really
arise from inorganic matter by ‘spontaneous generation,’ and that, as the
evolutionary theory assumes, all other living things have been gradually
evolved from them in continued series. The doctrine of evolution is,
indeed, incomplete without the assumption of such a commencement. Its
possibility once granted, there is no @ priord reason to deny that it stil]
takes place, and that side by side with the ceaseless evolution of new species
there may actually arise new organisms by spontaneous generation. Since
we can imagine only the very simplest forms of life to be produced in this
manner, it is natural that evidence for their occurrence should have been
sought among bacteria and other fermentative organisms. Experiment
showed that in some cases boiling for many hours did not prevent the
development of bacteria in nutrient solutions, and since it was contrary to
all experience that any living things could have survived such temperatures
spontaneous generation seemed to be beyond dispute. When, as frequently
happened, such organic infusions remained sterile after boiling, the advo-
cates of the theory explained matters by saying that conditions had been
changed, and the fluid was ‘no longer suitable’ for the genesis of life.
They also showed that passing air through it apparently removed these
unsuitable conditions, bacteria appearing again. If, however, the air
had been raised to a high temperature first, or passed through sulphuric
acid, or even through cotton-wool, the sterility of the liquids sometimes
remained unimpaired, but not always. Contradictory results abounded
and difficulties arose both for partisans and opponents of the theory. It was
long before the controversy could be regarded as satisfactorily settled, but
settled it was at last, and we know now that at the present day a spontaneous
generation of organisms does not take place in this way. We know that

SPONTANEOUS GENERATION 51

in all those cases where bacteria appeared in liquids after protracted boiling
they arose from highly resistant spores, whose vitality the heat had been
unable to diminish, and we know further that those experimenters were
right who sought to render air sterile by passing it over sulphuric acid or
through heated tubes, and that it was for this reason that the infusions
sometimes remained clear.

Infusions of organic substances, if they be only boiled long enough, will
remain sterile for years, even in vessels closed only by a plug of cotton
wadding. A spontaneous generation of new life never takes place; for
bacteria, like all other organisms, can only originate from pre-existing
germs. The fantastic theories that spring up from time to time regarding
the derivation of bacteria from the disintegrated protoplasm of the higher
creatures are apocryphal and, like the stories of organisms that consist
only of carbon, oxygen, and hydrogen, must be received with the scepticism
they deserve (26).

Bacteriological research has thrown light from another side also upon
the irrepressible theory of spontaneous generation. Before the discovery
of prototrophic micro-organisms it was inconceivable how the first forms
of life to appear upon the earth could have obtained their nourishment,
for all organisms, including the infusoria and other protozoa, seemed to
be metatrophic. Now, however, that we are acquainted with the meta-
bolism of the prototrophic bacteria, particularly the nitrifying bacteria, we
can form some conception of the mode of nutrition of the earliest terrestrial
organisms,

CHAPTER VI

PHYSIOLOGY OF NUTRITION AND GENERAL PRINCIPLES
OF CULTURE

Chemical Composition of Bacteria (27).

THE substance of bacteria, like that of all other living organisms,
consists for the main part of water, which constitutes about 85 per cent. of
their weight (aman body 65-70 per cent., green plants 60-80 per cent., algae
about go percent.). The high proportion of water (shown also in the abun-
dance of sap-vacuoles) is due to the fact that they are aquatic, not terrestrial
organisms, inhabitants of liquid media. Analysis of the substance of pure
cultures freed as far as possible from the culture medium has given the
following results :—

(NENCKI) (KaPPEs)
Putrefactive bacteria (a Bacillus
mixture of several species). prodigiosus.

Water . . . ° Fi ‘ 83-42 < 7 85-45:
Protein substances . . . «. 1396 . « 10-33
Fat. . ‘ . - F . 1-00 3 3 70
Ash . : : 6 é i é +78 “ - 1-75
Residue (not analysed) . . . 84 8. . 1:57

These two analyses give, of course, only a general idea of the composition
of the bacterial cell. Other cases would probably show greater differences,
for bacteria are, like all other organisms, dependent in their composition
on the nature of the food-stuffs' available. Like these also they are no
doubt capable of selecting their food to a certain degree.

The quantity of the ash will be larger where the surrounding medium
is rich in salts, and there will be a higher percentage of proteids in bacteria
from a nutritious peptone culture than in those nourished on glycerine and
ammonium chloride. Still, on the whole, their composition is evidently not
widely different from that of other organisms, as the above analyses show.

CHEMICAL COMPOSITION OF THE CELL 53

Nencki (27) isolated the proteid constituents of the bacterial cell by pre-
cipitation with boiling hydrochloric acid, removal of fats with ether and
alcohol, solution in caustic potash and final precipitation with chloride of
sodium. The substance thus obtained (mycoprotein) agrees pretty closely
in composition with one prepared by Schlossberger from yeast, and contains
52°39 per cent. carbon, 7°55 per cent. hydrogen, 14-75 per cent. nitrogen,
and about 25 per cent. oxygen. Even if we assume that this body (free
from sulphur and phosphorus) is an unaltered constituent of the bacterial
cell and not a decomposition product from more complex proteins, it is
not necessarily the essential constituent of the protoplasm. If it were we
should have to place the bacteria chemically as well as morphologically
below all other organisms, where, as far as we know, the phenomena
of life are inseparably bound up with the presence of highly complex
bodies like nuclein and nucleo-albumen rich in phosphorus. Since, how-
ever, such compounds do undoubtedly occur in some bacteria, further
investigations are necessary before we can form an opinion as to the nature
and importance of mycoprotein.

Closely related to the proteid bodies (using the word in its widest sense)
are those poisonous substances known as toxalbumines. The constitution
of their molecule is, however, as yet quite unknown *.

Carbohydrates are probably present in all bacteria, but they certainly
do not play such an important part as in the cells of higher plants, for, as
already mentioned, the cell-membrane of bacteria consists not of cellulose
but of a proteid substance. Apart from the not very clear indications
afforded by the ‘granulose reaction’ in some few species (p. 13) there are
no instances of differentiated carbohydrate cell-contents.

The gelatinous substance produced by Leuconostoc (see p. 10) and
by some of the mucilaginous species that occur in wine and beer is probably
a carbohydrate (dextrane, [C,H,,O,],) similar to cellulose and the slimy
secretions of some gelatinous algae. As special temporary cell constituents
must be regarded also those products of fermentation which, although
formed in the cell, are not stored up but excreted as metabolic products (see
Chaps. XI-XIV). The percentage of ash given in the various analyses of
bacteria cannot be taken as indicating in any degree the value of salts in
the economy of the cell, because the nutritive media were not prepared with
this end in view.

Food Stuffs of Bacteria (28).

Mineral substances are as necessary for the Bacteria as for all other
organisms, albeit in a much smaller quantity. One milligram of living
bacteria (i.e. about thirty milliards of individuals) with 1 per cent. of ash

* For the relations of the toxalbumines to infectious diseases, see Chap. XVII.

54 PHYSIOLOGY OF NUTRITION

would contain only ;3, milligram of mineral salts, so that it is evident that
‘very minute quantities of such salts are sufficient for nutritive media, say
from o-I to 0-2 per cent.

Of the elements may be mentioned as indispensable: sulphur, phos-
phorus, calcium, magnesium, potassium, and sodium, with traces of chlorine
and iron. Whether other alkalies such as rubidium or caesium can be
substituted for potassium and sodium, or other alkaline earths, such as barium
or strontium, for calcium, is doubtful. Recent experiments on the metabolism
of mould-fungi render it improbable.

Full details as to the most suitable proportions of mineral salts in nutri-
tive media will be found in practical treatises. In the following brief account.
of the nutrition of bacteria, the words ‘necessary salts’ will be understood
to mean o-r per cent. K, HPO,, 0-02 per cent. Mg SO,, and o-o! per cent.
CaCl, Sodium and iron are also necessary, but are always present in
traces in the other chemicals used unless these have been specially purified ;
they are likewise present in tap-water. For pathogenic forms the addition
of from o-1 per cent. to o-7 per cent. NaCl is advisable ; but if the media
have been prepared with infusion or extract of meat it is not necessary.

These few mineral substances will be found sufficient probably for all
bacteria, be they prototrophic, metatrophic, or paratrophic. The require-
ments in carbon and nitrogen compounds, on the other hand, are widely
different for each group.

The prototrophic nitrifying bacteria grow well in a fluid of the following
composition :—

Water, H,O 2 F . a . F 100-

Potassium Nitrite, KNO,_ : : 05
Potassium Hydrogen Phosphate, K,HPO, F +02
Magnesium Sulphate, MgSO, . - . 3 03
Sodium Carbonate, Na,CO, . ‘i ‘i : 05
Sodium Chloride, NaCl .. ms 4 . 05

The nitrous acid supplies the nitrogen, and the carbon is derived, not from
the Na,CO,, but from the carbonic acid of the atmosphere. Other proto-
trophic soil bacteria are able to fix atmospheric nitrogen, but require some
organic compound as a source of carbon; sugar for instance.

The growth of various metatrophic bacteria upon solutions of different
carbon and nitrogen compounds is illustrated by the following table. All
the solutions contained the same amounts of mineral substances, and, with
the exception of three cases, had an alkaline reaction; free acid, as the
figures show, generally retarding development. The cultures were kept at
the most suitable temperature in each case and were watched for fourteen
days, so that delayed growth might not be overlooked. The symbols
employed have the following meanings :—

FOOD STUFFS 55

+++ = very luxuriant growth, liquid turbid with bacteria or a pellicle upon the
surface (cholera, B, szb¢z/is), or deposit of bacteria at bottom of vessel
(anthrax).

++ = growth moderate, faint but distinct turbidity or a delicate pellicle.
+ = feeble growth, liquid opalescent or turbidity visible only on shaking.
++? = scarcely any growth.
O = no growth.
Source of Source of ~ | Reac- | B. An- B. B. coli vibr. B. B. pyo-
Nitrogen. Carbon. tion. | thracis. | typhi. » CO". | cholerae.| subtilis. | cyaneus.
1| 1% Peptone {1% Grape sugar] alk. |++-+|+-++|+++|+++|+++ [+++
2) ,, Peptone » Peptone alk. | ++ | +4+ | ++ |] ++ + |4+4++
8| ,, Asparagine | ,, Grapesugar| alk. ce) + J+4+4+/4+4+4+/++4+/+4++
4| ,, Asparagine} ,, Grapesugar| acid fo) +? |4+4+4+/] 0 ++ |+4¢+
5| ,, Asparagine | ,, Asparagine | alk. fe) fe) ++) ++ + +
6| ,, Asparagine | ,, Asparagine | acid oO fe) + ° + +
7! ,, Ammonium] ,, Glycerine | alk. fe) fe) ++ + {+++ |) +++
tartrate i
8| ,, Ammonium| ,, Ammonium] alk. ie) ie] +? fe) fo) +?
tartrate tartrate
9| ,, Ammonium| ,, Glycerine alk. (e) o }+4+4+) ++] ++ | ++
chloride
10) ,, Ammonium| ,, Glycerine | acid ° Oo j++4)] 0 ++ +
chloride
11] ,, Potassium » Grapesugar| alk. oO ° + +? |} +4+ |+4++
nitrate
12| ,, Potassium | ,, Glycerine alk. fe) fo) fe) (e) Oo {+++
nitrate
13 none oy Sugar alk, (o) fe) fe) O° fe) Oo?
14| ,, Potassium none alk. O° fe) ° ° oO ce)
nitrate.

.The most conspicuous result that the table shows is the great difference
between B. anthracis and B. pyocyaneus. The former prospers only where
peptone is available as a source of nitrogen (1 and 2), the latter grows just
as well and exhibits its magnificent fluorescence upon nitre (12). In other
words B. anthracis is a ‘peptone bacterium, B. pyocyaneus a ‘nitrate bac-
terium’ approaching the members of the prototrophic group in its physio-
logy. The latter, however, needs a special source of carbon, not being
able to assimilate the carbon dioxide of the air (14). There is another
numerous group of metatrophic forms which, given carbon in a suitable
form, can utilise the nitrogen of ammonia compounds, and thrive as well
as they do upon peptone. B. coli is one of these ‘ammonia bacteria; and
also V. cholerae and B. subtilis. Others, B. typhi for instance, are ‘ amido-
bacteria’; they cannot assimilate the nitrogen of ammonia, but grow fairly
well upon amide bodies, such as asparagine and leucine. One fact must
not be lost sight of if the bacteria are classified in this way, according to

56 PHYSIOLOGY OF NUTRITION

their nitrogen requirements: the power of utilising the nitrogen in different
compounds is largely dependent upon the form in which the carbon is
supplied. Thus, the ammonia bacteria can utilize the nitrogen of saltpetre
if sugar is present, but they are unable to do so if the carbon is presented
to them in the form of glycerine. No bacteria can dispense with nitrogen
entirely, and the feeble growth of B. pyocyaneus in sugar solution alone (13)
was probably due to the presence of impurities, or to the absorption of
ammonia from the air of the laboratory.

The first attempts to characterise bacteria according to their behaviour
towards different nitrogen compounds were made by Nageli, who also
examined yeasts and mould fungi in the same way. The subject has been
still more recently studied by Beyerinck (28).

In medical bacteriology, fluid culture-media are now very rarely ~
employed, having been driven out by the stereotyped recipes for peptonised
gelatine and agar. That this has been disadvantageous there can be no
doubt. The case of 2. typhi and B. coli, the one an amido-bacterium, the
other an ammonia bacterium, shows the value of fluid media of varying
composition for the differentiation of species. Not less important are the
conclusions which may be drawn from such investigations as to the
occurrence of pathogenic bacteria in nature. This subject will be more
fully discussed in later chapters.

The form in which nitrogen is offered affects greatly the influence
which the reaction of the culture medium has upon the growth of the
bacteria. The more assimilable the nitrogen compounds, the less sensitive
are the bacteria to the deleterious effect of free acid. 2B. pyocyaneus, as the
table shows, is, when it gets its nitrogen in the form of asparagine (5-6),
unaffected by the acidity of the medium which palpably retards growth
when ammonium chloride is the only source of nitrogen. The extremely
sensitive V. cholerae is totally overpowered by free acids (4 and 6),
while B. sudtilis, which grows well on weakly acid hay-infusion, is less
marked in its behaviour, and B. colz seems to be quite uninfluenced by the
reaction.

The formation of pigment too, in B. pyocyaneus, as in other chromogenic
bacteria, is largely dependent upon the reaction of the medium and the
nature of the nitrogenous bodies it contains. The organic compounds of
nitrogen, although they all of them contain carbon also, are not in themselves,
sufficient for vigorous growth, as a comparison of Nos. 1, 2, 3 and 5 of the
table shows, The carbon of ammonium tartrate (8), for instance, is
absolutely useless to B. subtilis, and even B. coli and B. pyocyaneus are
half starved when they have no other carbon-containing compound to draw
upon. The value of a special source of carbon, so clearly shown in the
table, is twofold: the excess of organic carbon is used in the building up
of proteids; and, more important still, the breaking down and oxidation of

FOOD STUFFS 57

the carbon compound provides the necessary energy for the full utilisation
of inferior nitrogenous substances (11 and 12).

The organic carbon compounds are of very different degrees of import-
ance as food-stuffs, their value depending mainly but not exclusively upon
their heat of combustion. Grape sugar and sugars in general are the best,
then glycerine and other polyacid alcohols, such as mannite and dulcite,
and finally compounds like tartaric and succinic acid, benzoic acid, and the
monacid alcohols and their derivatives such as fatty acids and amines *.
Compounds, in which an oxygen atom is linked by two valencies to the
carbon atom, as in urea CO(NH,),, or oxalic acid (COOH),, are useless as
sources of carbon, as are also the cyanogen comtpounds. It would appear,
therefore, that the carbon is in its most utilisable condition when it is linked_
with hydrogen in the form Ce being less valuable as CH, still less so as
CHOH, and not at all in the CO or CN radicles. Exceptions to this rule,
however, must not be forgotten.

Speaking generally, the best data for the discrimination of a bacterial
species from the standpoint of nutritional physiology are those afforded by
its behaviour towards nitrogenous compounds, the demands made for carbon
being less narrow and well defined.

The paratrophic bacteria are, as our table shows, able to grow only in
those solutions that contain peptone. They approach in this respect most
closely to the metatrophic forms, but their requirements are often much more
special. The gonococcus, for instance, grows only upon albuminous media,
such as coagulated blood serum. Upon this too the diphtheria bacillus
thrives best. The tubercle bacillus, which we are obliged for the present to
regard as a Strictly parasitic species, is able to grow upon inferior substrata,
even on those of the ammonia bacteria (see Chap. XVI).

For the culture of bacteria there are commonly used, besides bouillon
and other fluids, the so-called solid media prepared with gelatine or agar.
The commonest recipe is an infusion of meat (1 lb. meat to 1 litre water),
with about 1-2 per cent. peptone and sugar, to which Io per cent. gelatine
or 1-2 per cent. agar is added, the whole being boiled and filtered hot.
This gives a clear gelatinous mass, in which the nutritive substances are
equally distributed throughout the non-nutrient gelatine or agar. The
introduction of such culture media (29) has been an important factor in the
rise of bacteriological science, for it is only by the use of easily melted
and easily congealed substrata that we are able to isolate bacteria without
difficulty, and obtain them in pure cultures,

The nutritive fluids mentioned in the table on p. 55 may of course be
mixed with gelatine or agar, and the advantages of several methods united.

* For details, a re-examination of which is desirable, see Nageli ; appendix No. 28.

58 PHYSIOLOGY OF NUTRITION

If it is desirable to exclude all organic compounds, colloid silica’ may be
used as a matrix instead of gelatine. Great latitude is permissible in the
composition of the peptone-sugar-meat solutions that are used. Almost
every laboratory has its own special recipes, dictated either by whim or by
the results of long experience. In many cases the media are unnecessarily
overloaded with nutriment. Besides meat, hay, straw, plums, potatoes and
many other substances may be used to make nutrient infusions. The wort
of beer is often used, and, as an opaque solid culture-medium, potatoes are
often employed. For practical details the technical handbooks must be
consulted.

Apart from characters such as pigmentation or the production of gas,
the modes of growth of different bacteria on the same substratum are
sufficiently varied to be of some use in the differentiation of species. Too
much importance, however, must not be attached to these features. In
fluid media, two chief types of growth are seen. In the one the liquid
remains clear, in the other it becomes turbid. When the fluid remains
clear we know that we have to do with non-motile bacteria with a tendency
to filamentous growth. They collect on the walls and bottom of the tube
in flocculent masses, which may be dislodged by shaking (Anthrax, Strepto-
coccus). When such non-motile forms need much oxygen (B. tuberculosis
for instance), they form a thick membrane, sometimes smooth, sometimes
wrinkled, over the surface of the clear liquid. All isolated, non-filamentous
forms, particularly the motile species (Cholera, Typhoid), cause the medium
to become uniformly turbid. The turbidity varies from a dense milky
opacity to a faint opalescence only visible on shaking. Strongly aerobic
species form a pellicle on the surface of the fluid (V. cholerae, B. subtilis).

By culture upon gelatine we are able to divide the bacteria into two
groups, those which, by means of a peptonising enzyme, liquefy the gelatine
and those which do not. The great majority of known forms belong to the
former class; B. typhi, B. coli, Streptococcus, Lactic bacilli, &c., are examples
of the latter. Further differences of growth on gelatine are brought out by
the methods of ‘plate’ and ‘stab’ culture. In plate culture the melted
gelatine is inoculated with a minute quantity of bacteria, or of substances
containing bacteria, and then poured out in a thin layer upon glass. There
it coagulates, and the germs it contains multiply. The germs being fixed
produce around themselves sharply-defined colonies, each colony having
arisen from a single germ or from a few stuck together. The young
colonies in particular show many characteristic features; colour, form, and
contour, consistence and lustre, and the structure as seen under a low-
power lens are all useful points. A detailed account will be found in
Lehmann and Neumann’s atlas (80), and is needless here. The following
description of the colonies of two well-known bacteria will show the nature
of the differences involved better than an enumeration of all the known

MODES OF CULTURE 59

varieties of growth. The two species selected are the cholera and the
anthrax bacteria.

The anthrax colonies liquefy the gelatine slowly ; they are whitish and
round ; under a low power the edge of the colony appears filamentous, in
‘tresses’ and loops, not smooth; in older colonies an irregular skein or
clump lies in the fluid, almost transparent, gelatine. The cholera colonies
liquefy the gelatine rapidly ; they are yellowish white, and magnified show
a granular texture and an undulating, not filamentous, edge; the liquefied
gelatine immediately round the disintegrating granular clumps is turbid.
It must not be forgotten that many variations occur, and that closely
similar forms, such as the cholera vibrio and the aquatic vibrios or typhoid
bacilli and B. colz, are not distinguishable by their colonies alone.

The gelatine stab-culture is made by thrusting vertically into a tube
of coagulated gelatine a platinum wire charged with the substance of a pure
culture. Species which need oxygen grow only in the upper parts of the
gelatine, anaerobic forms only in the deeper parts. Growth in filaments or
chains shows itself by delicate threads growing out into the surrounding
gelatine so that the path of the needle appears feathery or moss-like,
e.g. Anthrax. Bacteria whose cells are isolated are confined in their
growth to the path of the needle: Among species which liquefy the
gelatine great importance is laid upon the shape of the liquefied tract.
This may be equal all along the path of the needle, and have the appear-
ance of a straight liquid tube in the middle of the solid gelatine, or, if
liquefaction is more rapid towards the surface, a funnel-shaped well is
produced. Here again considerable variations take place, and closely allied
species are not distinguishable by these data alone.

The surface of agar or gelatine that has been allowed to solidify in
tubes in a slanting position is used for ‘streak’ cultures. These are made
simply by smearing the surface of the substratum with a platinum wire
charged with bacteria. The differences in colour, contour, lustre, and con-
sistence are similar to those of the colonies in plate cultures. Agar is not
liquefied by any bacteria.

The modes of nutrition of particular biological groups among bacteria
will be considered in the subsequent chapters.

CHAPTER VII
RESPIRATION OF BACTERIA

Aerobiosis and Anacrobiosis; Light-producing Bacteria; Marine
Bacteria; Sulphur and Iron Bacteria.

THE old and now obsolete term ‘vital air’ applied to oxygen meant
that without it life was impossible. Of all the axioms of physiology, none
has ever seemed to be so unassailable as that which asserted the necessity
of oxygen to every living thing. It appeared to be a law absolutely
without exception that all animals and all plants must ‘breathe, that is
to say, must take up free oxygen in order to oxidise and break up the
organic compounds of their food or of their tissues, and thus obtain the
energy necessary for the processes of life. Deprivation of air seemed
inevitably followed by cessation of life. Here again the study of yeasts
and fermentative bacteria has overturned the old views and revolutionized
our ideas regarding the chemistry of life.

In 1861 Pasteur (31) discovered that many zymogenic bacteria could live
and even set up active fermentation in the absence of oxygen; and he gave
them for this reason the name of anaerobes. His statements, romantic and
improbable as they at first appeared, were soon corroborated, and the
general recognition of their accuracy has resulted in the division of the.
bacteria into two groups, the aerobes and the anaerobes. In the aerobic
bacteria the process of respiration is the same as in all ordinary organisms,
They absorb oxygen, and with it break up non-nitrogenous bodies such as
glycerine or sugar into carbonic acid and water. They are also able, like
plants and animals, to assimilate nitrogenous substances such as peptones
and amido compounds, although with less gain of energy and less easily
than they can carbonaceous bodies. Many of the aerobic bacteria are
totally unable to live without oxygen, and when deprived of it die as would
a mouse in pure hydrogen. They are exclusive or obligatory aerobes. In
rarefied air or in artificial mixtures of gases their vitality is proportional to
the amount of oxygen present. Under an air-pump, for instance, they

AEROBIOSIS AND ANAEROBIOSIS 61

ceas2 to grow long before a vacuum is reached (acetic bacteria, B. subtilis).
Contrasted with the obligatory aerobic bacteria, we have the obligatory
anaerobic forms which thrive only in the absence of oxygen, small traces of
this gas being sufficient to inhibit growth (B. ¢e¢anz, some butyric bacteria,
bacillus of malignant oedema). Between these extremes there is a great host
of bacteria representing every gradation between the two modes of life.
These are the facultative anaerobes, which, while growing best with a
plentiful supply of oxygen, are nevertheless able to exist with a very small
amount, and even with none at all, although in this case their vitality is often
much impaired. Anaerobic bacteria, both obligatory and facultative, are found
everywhere in nature where the air cannot penetrate, or where it is replaced
by other gases—in the deeper layers of the soil, for instance, in the mud of
rivers and standing waters, or the ooze of the sea bottom, and in manure.
In all such places anaerobic bacteria are the principal and often the only forms
of life, and by the fermentative and putrefactive processes they set up they
effect the disintegration and removal Of dead animals and plants.* As be-
longing to the group of facultative anaerobic bacteria may be mentioned the
lactic acid ferment, the bacteria of typhoid and cholera, many staphylococci
and streptococci and most putrefactive bacteria. The power of anaerobic
growth varies, even in the same species, according to the source whence
the germs were derived and the mode of culture. Deprivation of oxygen
affects the bacteria in many ways. Some of the pigment bacteria (B. vio-
laceus, for instance) give rise to colourless cultures if oxygen be excluded ;
others, like Spirdllum rubrum, form pigment most plentifully under these
conditions, though here again, as in so many other cases, exceptions occur.
Many obligatory anaerobic bacteria (e.g. some of the butyric ferments)
are motile, the energy necessary for movement being derived from the
partial breaking-down of the molecules of the fermentable substances on
which they grow. Access of oxygen stops the motion at once, just revers-
ing the conditions found among the aerobic forms, where the cilia work
the more vigorously the more oxygen is present, and become paralyzed at
once if it be cut off. These movements, minute as they are, represent an
expenditure of energy that is considerable when the size of the organisms
is taken into account.

Those aerobic bacteria which possess the power of movement are
attracted by oxygen. If a drop of water containing them be placed upon
a slide and covered with a thin glass slip, they crowd to the edges of the
preparation and around air-bubbles in the water. This property of being
attracted by oxygen has been utilised by Engelmann (82) for the detec-
tion of minute traces of free oxygen. His ingenious method, which

* The chemical aspects of the phenomena of putrefaction as well as the theoretical explanation
of anaerobiosis are discussed in Chap. XIV.

62 RESPIRATION OF BACTERIA

employs aerobic bacteria as living reagents, has given us what has hitherto
been unattainable, a micro-chemical test for the gas. It is a well-known
fact that there is in green plants, going on side by side with the ordinary
respiration of the cells, a second process of gaseous exchange accompany-
ing the assimilation of carbon dioxide and also often incorrectly called
‘respiration.’ The carbon dioxide is split up in the green parts of the
plant by the energy of the sunlight, the carbon being retained, and the
oxygen thrown off into the atmosphere. Now it is not all rays that are
equally powerful to cause this chemical change. The absorption spectrum
of chlorophyll shows that the red rays between Frauenhofer’s lines B and
C are the most strongly absorbed by green plants, and after them that
part of the spectrum just beyond F. It is just these regions that are most
active in causing the evolution of oxygen, and Engelmann demonstrates

this inthe following way. By means
ob ¢ 2 45 i of an arrangement of prisms in the
substage of a microscope he pro-
jects a spectrum upon green algal
filaments or moss-leaves lying in
the field of view, the water in which
= they are mounted containing aerobic
i ll bacteria. If, now, the illumination
be strong, and all extraneous light
be carefully excluded, it can be
seen that the aerobic bacteria crowd
Fic. 16, Detection of oxygen by means of bacteria. sound the filaments just where the

The vertical lines are the Frauenhofer lines of a spectrum :
thrown on the field of the microscope. In the spectrum active rays fall, the chief swarm
a

lies a filament of the alga Cladophora, and around it, at
B, C, and F, the bacteria swarm (see text). Magn. 200. lying between B and C, and an-
other smaller one near F (Fig. 16).
Around these parts of the alga only a few scattered organisms occur, show-
ing that the evolution of oxygen is at a minimum. The application of
this bacterial method for the detection of minute traces of free oxygen is
an operation of great delicacy, and the results which it gives must be
interpreted with great care because the movements of bacteria are very
often rendered more active by other substances beside oxygen, particularly
by food-stuffs, as will be explained in the section on chemotaxis. The
attraction by oxygen is in fact only a special instance of chemotaxis.

The great amount of energy derived from the combustion of food-stuffs,
and from the oxidation of the tissues in higher organisms, is not all expended
in the form of mechanical work, but applied in part to raising the tempera-
ture of the tissues. In animals this is conspicuous in the case of ‘ warm-
blooded’ creatures, and in plants we have instances in the germinating grains
of cereals and in the flowers of the Aroideae, which are often sensibly warm to
the touch. Fermenting and putrefying substances also (hay, manure, cotton-

THERMOGENIC AND PHOSPHORESCENT BACTERIA 63

waste) frequently become heated, the temperature inside the mass being often
raised to 60° or 70°C. This spontaneous heating, which may increase to
actual ignition (spontaneous combustion), is due to the respiratory activity of
aerobic bacteria (thermogenic bacteria of Cohn), which set up fermentation
and putrefaction.

Cohn (83) found in damp cotton-waste a micrococcus which, when well
supplied with air, raised the temperature of the decaying mass to 67° C., if
care were taken to prevent radiation, carbonic acid and trimethylamine
arising as respiratory products.

Light is another form in which the surplus energy of respiration exhibits
itself both in terrestrial and marine plants and animals. The existence of
phosphorescent insects is a matter of common knowledge. The mysterious
light often seen in old willow-trees is given off by the mycelia of certain
parasitic fungi. Among marine organisms there are very many kinds which
are luminiferous. The phosphorescence of the sea is mainly due to light-
producing infusoria, hydrozoa, and ascidia, and in our latitudes above all to
bacteria (34). The various kinds of phosphorescent bacteria have been
collected together to form a ‘ biological’ genus Photobacterium. They include
actively motile straight rods and vibrio-like forms, but very few have been
completely described, and names like B. phosphorescens, B. luminosus, Vibrio
albensis must not be regarded as designating ‘good’ species. The faint
glow seen upon decaying haddocks, mackerel, and other sea fishes, is pro-
duced by these micro-organisms. Whether any phosphorescent bacteria
occur in fresh water is doubtful: but all with which we are as yet well
acquainted are from the sea. Being marine organisms, the phosphorescent
bacteria need in their culture-media from two to three per cent. NaCl,
besides the usual salts and peptone, and to obtain successful cultures
of phosphorescent bacteria the media must contain these substances,
besides some other source of carbon such as sugar, glycerine or asparagine.
Phosphorescent bacteria would seem therefore to be ‘ peptone’ bacteria,
saprophytic in the sea upon dead animals and plants, from which they
are washed off in countless numbers by the waves. The phosphorescent
bacteria of the North Sea and Baltic flourish best at about 18° C., but
can grow quite well at much lower temperatures, even down to o° C.,
resembling in this respect the other inhabitants of northern waters. In the
absence of oxygen they are indeed able to grow slowly, but are then not
phosphorescent, the production of light being an exclusively aerobic pheno-
menon. This is evident, too, when we watch the phosphorescence of the
sea, for perfectly smooth water does not shine at all, while the brilliancy of
the light is always greatest upon the crests of the waves, or where the
water is churned up with air in the wake of a ship. That the respiratory
exchange (oxidation) of the bacteria is the cause of the phenomenon is
proved by the fact that the death of the micro-organisms or exclusion of

64 RESPIRATION OF BACTERIA

oxygen quenches the light at once, whilst a more abundant supply of
highly combustible food-stuffs like carbohydrates increases it. We know
of no specific substance (/uciferinz) to which the luminosity could be due,
and the phenomenon is of quite different nature from that exhibited by phos-
phorescent bodies like calcium sulphide, for in the bacteria the production
of light is quite independent of previous insolation.

Phosphorescent bacteria may easily be obtained by placing the flesh of
fresh haddocks or herrings in a solution of two to three per cent. of NaCl
and keeping at a low temperature (5°-10°C.). In one or two days not
merely the fish, but the whole of the liquid in which they lie, give off
a pale-greenish light which becomes much more brilliant if a little sugar or
glycerine (i. e. respirable material) be added. The bacteria may be readily
isolated by the usual methods upon peptone-sugar-gelatine prepared with
an infusion of fish and common salt. The pure cultures thus obtained are
so strongly phosphorescent that by protracted exposure they may be
photographed in their own light. The rays given off are only the more
refrangible, from DtoG. This is evident, too, from the bluish colour of
the light.

A few words may be said here regarding other marine bacteria (85).
The investigations of the German Plankton Expedition show that the
prevailing forms are actively motile rods and vibrios, cocci being less
numerous. Their distribution is determined mainly by the proximity of
land, for the algal vegetation which is richest near the shore attracts
myriads of marine organisms that provide the bacteria with a rich variety
of nutriment. The influence of the shore extends four or five miles out to
sea. The degree of illumination seems without influence, and no regularity
could be observed in the distribution, but the number of bacteria per c.c.
varies immensely, however far from land the sample be taken, as the
following figures show:

Plymouth Docks (ebb) . . ; - 7 : 13,320 per c.c.
1 mile from shore (flood) 5 : ‘ . < 3,960. +s
240 miles from shore (Gulf Stream) 3 : F 645 is
450 miles from shore (Sargasso Sea) : 20, 200, 206, 168 a

These numbers include the spores of mould-fungi. In 54 per cent. of all
samples about 100 germs per c.c. were found; at depths of from 800 to
1,100 metres below the surface only from eight to twelve bacteria were
present.

Specimens of ooze from the ocean bed at depths varying from 1,523 to
2,406 metres were sterile; others again from 4,099 to 5,250 metres contained
from one to four bacteria per c.c. This paucity is the more remarkable
when we consider that the temperature was from 2° to 5° C., and the
conditions altogether such that foraminifera and radiolaria occurred abun-
dantly. Possibly the culture-medium (peptonized fish-broth gelatine) was

SULPHUR BACTERIA 65

at fault, since it affords nourishment only to metatrophic forms, not to
prototrophic, and it is just these latter that we might expect to find on
the ocean floor. It is not at all unlikely that there are species characterized
by an extremely simple type of metabolism, which would throw new light
upon the mysteries of marine life. Russel has discovered aerobic bacteria
that reduce nitrates in samples of mud from the sea-bottom.

In the aerobic bacteria which we have been considering, respiration
consists essentially in the oxidation of complex organic substances, the
energy thus set free being necessary for the processes of life. In the case
of the anaerobes the energy developed, although smaller in amount, is
likewise derived from the splitting-up of fermentable and putrescible organic
substances. Both classes, aerobes and anaerobes, are metatrophic in this
respect. There is, however, in certain prototrophic bacteria a process going
on comparable to respiration, but consisting in the oxidation of izorganic
compounds. The micro-organisms in question are the nitre bacteria
(see p. 105) and the remarkable
sulphur bacteria, the classical
examples of prototrophic re-
spiration.

The sulphur bacteria (36),
Thiobacteria (p. 13), whose
cells are often crammed full of
‘spherical refringent masses of

pure sulphur, occur in nature

in places where free sulphury gil 17.,.Sclphur Bacteria, sre, Beggiatog al of sul
etted hydrogen is present, s2u%e waters ¢ almost te from sulphur after another day or
Such are sulphur springs where Dectesim; s, piece of a mesh like sooglosa of Lamprsietis
the SH, is principally of mineral ®*84"% 4 ¢ fom Zopf-

origin, and the mud of standing

waters and of the sea-bottom, where it is set free by the putrefaction of dead
plants and animals *. The bacteria were formerly thought to produce the
SH,, and therefore to play an important part in the origination of sulphur
springs, but the splendid researches of Winogradsky have shown that the
micro-organisms need the gas as a food-stuff, and are unable to live without
it. Thiobacteria can be found at any time of the year, but are most
abundant in the early spring and late autumn, periods when the remains
of the summer's vegetation in’standing waters are plentiful, and when the
putrefactive processes set up by other bacteria cause an evolution of SH,.
The sulphur bacteria frequently form a snow-white furry coat on the rotting

vegetation, with here and there pink or pale puce-coloured patches.

SR

oo)
Taye aeK
pean

* The ‘dead-ground’ of the Bay of Kiel and the ‘ Limanes’ of the Black Sea are instances.

FISCHER F

66 RESPIRATION OF BACTERIA

Colourless and coloured species are almost always found in close proximity
to one another—the colourless forms on substrata of all kinds, the coloured
ones in spots exposed to a strong light. The white furry coating generally
consists of filamentous forms, particularly of the unbranched non-motile
filaments of the delicately-sheathed Zhéothrix, anchored by their bases to
the bodies on which they grow. Here and there among them one finds the
free-living, slowly-oscillating filaments of Beggiatoa (Fig. 17 a-c) ; unicellular
colourless species also occur. The coloured forms (Erythrobacteria*)
present a greater variety. The genus Chromatium (particularly C. Okenit)
consists of short, plump, rod-shaped or ovoidal cells which are often present
in such numbers as to give the water a reddish tinge. Thiospirillum is
a corkscrew-shaped form. The dirty pink scum that coats stones and
vegetation in sulphurous waters consists generally of a mixture of non-motile
species, small tablets of spherical cells (TAéopedia), cylindrical species, and
zoogloeae (Lamprocystis, Fig. 17 e). Winogradsky gives descriptions of
nine genera.

The very fact that the red sulphur bacteria collect at those spots where
the illumination is strongest would seem to indicate that there is some
relation between the light and their physiological processes. To understand
this connexion, however, we must first try to gain some conception of the
physiology of the colourless forms. In these it is only the part played by
the sulphuretted hydrogen that is clearly understood, but it is highly
probable that they are prototrophic in habit. The waters in which they
naturally occur contain only traces of organic matter}; they will not grow
at all upon highly nutritious media like peptonised gelatine. Asa source
of carbon, minimal quantities of formic or propionio acid suffice, and for
nitrogen, traces of ammonia, all substances which are common products
of putrefaction. They are strictly aerobic, and do not need light. They
grow best in water containing 100 milligrams SH, per litre, but are killed
by a saturated solution of the gast. If filaments of a sulphur bacterium
already loaded with sulphur be placed in pure water they lose it all in from
twenty-four to forty-eight hours (Fig. 17 @-c), and finally die from ‘ sulphur-
hunger.’ But if, before it be too late, they be replaced in water containing
SH, they recover and continue to grow, the SH, being oxidised and the free
sulphur stored up in the protoplasm of the cells. The disappearance of the
sulphur when the bacteria are placed in pure water is due to a further
oxidatory process by which sulphuric acid is produced. This unites with
the lime dissolved in the water to form gypsum. Other bacteria common
in marshy places, such as Cladothrix and Spirillum, as well as mould-fungi,

* This term is suggested as perhaps the best equivalent of the German word ‘ Purpurbakterien,’
+ In Weilbach water only 0.0048 gram per litre, yet sulphur bacteria grow vigorously in it.
= 4-56 grams SH, per litre. The Stachelberg spring contains 0.073 gram.

SULPHUR BACTERIA 67

are not able to live in water containing SH,; they perish where Chromatium
or Beggzatoa thrives.

Seeing that a solution of SH, in water is very easily decomposed by the
oxygen of the air* and free sulphur deposited, the sulphur bacteria would
be able to take advantage of this oxidation for their life-processes, even if
they merely had the power of existing in H,S solution. The H,S entering
their cells would be oxidised by absorbed atmospheric oxygen, and the
sulphur set free would represent an abundant source of energy for further
oxidation. To explain the facts observed we need only assume further that
the protoplasm increases the oxidizing power of the atmospheric oxygen
and renders it ‘active. The amount of energy gained is very consider-
able. The oxidation of the dissolved SH, gives seventy-one calories, and
the further conversion of the freed sulphur into sulphuric acid no less than
2,109 calories.

The fact that the thiobacteria can dispense entirely with organic com-
pounds which might be burnt up to CO,, together with their inability to
live without sulphur, shows that it is the oxidation of the sulphur alone
which takes the place of the respiratory processes of other organisms.

The two great physiological processes, the assimilation and storage
of respirable material on the one hand, and the liberation of energy by
its oxidation (respiration) on the other, may be represented in a tabular
form. The following scheme shows the processes in green-plants, meta-
trophic bacteria and sulphur bacteria :

ASSIMILATION OF RESPIRABLE MATERIAL.

INTAKE. STORAGE. OUTPUT.
GREEN PLantTs | Carbonicacid avd water | Carbohydrates Oxygen
plus the energy of sun-
light
METATROPHIC Organic substances (e. g.
BACTERIA sugar, which is not
stored but burnt up at
once)
SULPHUR Bac- | Sulphuretted Hydrogen | Sulphur Water
TERIA and Oxygen

* In presence of cotton-wool or some other porous body it is even oxidized to H,SQ,.

68

RESPIRATION OF BACTERIA

RESPIRATION OR LIBERATION OF ENERGY.

SOURCE OF RESPIRATORY GAIN IN
ENERGY. Gas. BEE ENERGY.
GREEN PLants | Carbohydrates Oxygen Carbonic acid | more than 6,000
and water calories
METATROPHIC | Organic sub- Oxygen Carbonic acid | more than 6,000
BACTERIA stances (e.g. and water calories
sugar)
SULPHUR Bac- | Sulphur Oxygen Sulphuric acid | 2,109 calories
TERIA

These two tables give, of course, only a general view of the broader
outlines of the processes. Real and apparent exceptions will occur to the
student, but they do not impair the validity of the schemes as a whole.
The green plants derive from the sun the enormous amount of energy
necessary for the formation of carbohydrates out of carbonic acid and water,
and set it free again in the process of respiration. The metatrophic bacteria
make use of highly combustible organic compounds which they oxidize at
once, and the sulphur bacteria are able at little cost to store up sulphur.
The oxidation of this sulphur represents a very abundant supply of energy,
a supply that is probably far more than sufficient to cover the wants of the
organism living under the conditions already described, and building up its
protoplasm from the minute traces of fatty acids and ammonia contained in
the water it lives in, The question therefore naturally suggests itself whether
the bacteria have not some other use for the surplus energy. We know that
the nitrifying bacteria are able, without sunlight, by means of the energy
they obtain from the oxidation of nitrogen, to seize and assimilate the CO, of
the atmosphere, and perhaps the Thiobacteria are able to do the same with
the much more abundant energy derived from the oxidation of sulphur.
Should this supposition turn out to be true, we should have in the colourless
sulphur bacteria a group transitional as regards their physiology between
the Erythrobacteria and bacteria of ordinary metatrophic habit (37).

The Erythrobacteria or coloured sulphur bacteria are unique in possess-
ing the power of assimilating CO, in the presence of sunlight by means of
a special red colouring-matter (Bacteriopurpurin) which exercises a similar
function to that of the chlorophyll of green plants. Engelmann has shown
that the spectrum of Bacteriopurpurin is of a very peculiar character,
having in addition to an absorption band between B and C another
very broad one in the region of the invisible ultra-red, where the wave
length is from 0-8 to og p. By the bacterial method he was able to
prove that oxygen was evolved in both these regions, showing that the
invisible heat rays are utilized for the assimilation of CO, just in the same

IRON BACTERIA 69

way as those of the visible spectrum. The Erythrobacteria have therefore
a double source of energy, the oxidation of sulphur and the absorption of
sunlight, a great advantage to them in the struggle for existence, inas-
much as they would be able to subsist in places where, from want of
sulphuretted hydrogen, the colourless Thiobacteria would perish. What
products arise from the assimilation of the CO, is not known; starch has
not been detected.

The Erythrobacteria are among the most sensitive phototactic organisms
known, the slightest diminution of brightness repelling, the slightest increase
attracting, them. The actively motile chromatia can be led about under the
microscope at will by suitable manipulation of the light.

The importance of the part which the sulphur-bacteria play in the
economy of nature lies in their behaviour to sulphuretted hydrogen. They
take this gas—a useless and injurious product of organic decay—and work it
up into sulphates, substances which are assimilated by other organisms, and
used to build up new life.

Hardly less remarkable in their nutrition than the sulphur bacteria are
the ‘iron-bacteria’ (ferrobacteria, 38), which resemble them somewhat in
their prototrophic respiration. Our knowledge of these organisms is still
very fragmentary. Stagnant pools in marshy places are often covered by
a greasy-looking brownish scum, that consists mainly of ferric hydroxide,
Fe(HO),, together with organic matter and some phosphate of iron.
The ferruginous matter is deposited as bog iron ore. By the action of
reducing substances arising from putrefaction, the ferric compounds,
particularly ferric hydroxide, are reduced to ferrous compounds, which
are dissolved by and unite with the CO, in the water to form ferrous
carbonate. The atmospheric oxygen alone is sufficient to convert this
back slowly again into ferric hydroxide, and cause its precipitation, but
Winogradsky has shown that the change is not purely chemical, and that
here, too, bacteria step in and accelerate the process. In the iridescent
deposits of the pools enormous numbers of short brittle tubes are found.
These are the broken sheaths of an unbranched filamentous bacterium, that
may be called for the present Lepiothrix ochracea. These yellowish-brown
sheaths are coloured blue by hydrochloric acid and ferrocyanide of
potassium; they contain ferric hydroxide. Specimens of bog iron ore
from Siberia, Sweden, and North Germany contained, in thirty-three
samples, only three with large amounts of these sheaths.

Besides the empty sheaths vigorously growing mats of Leptothrix are
always found, some with the cylindrical cells still 2 sdtz, others where they
have already swum away in the form of gonidia (as in Cladothrix). By
placing the bacteria in water containing CO, the iron may be dissolved out
of the sheaths, leaving them colourless. If these bleached filaments be then
put into a weak solution of ferrous carbonate of a concentration correspond-

7o RESPIRATION OF BACTERIA

ing to that of the pools, the sheaths become coloured anew. It is, however,
only those which contain living cells that take up iron; the empty sheaths
remain colourless. It is evident from this that the living bacterial cell
hastens the oxidation of the ferrous carbonate, just as it does the oxidation
of SH, in the Thiobacteria. In the iron bacteria, too, some energy, albeit
a very small amount, is gained in the process.

Since the sheaths and cell membranes of other aquatic plants (e.g.
Cladothrix and Crenothrix among the bacteria, Conferva (Psichohorrmium)
among the filamentous algae) are also turned yellow by the deposition of
iron oxide, it has yet to be seen whether the iron bacteria form a separate
biological group. Their nutrition as regards carbon and nitrogen also needs
further investigation. They are doubtless prototrophic.

It would be justifiable from some points of view to consider the acetic
bacteria in this section, but they can be more conveniently treated of with
the other fermentation bacteria.

CHAPTER VIII
INFLUENCE OF PHYSICAL AGENTS

Light, Electricity ; Pressure, Temperature, Dryness and Moisture ;
Disinfection by means of Physical Agents.

THE Erythrobacteria are the only forms in which nutrition is influenced
by light, as in the higher plants. It is the predilection for light which
causes Chromatium and other motile species to seek out the brightest parts
of the vessels they are kept in, forming a reddish coating on the side of the
glass.

All other pigment bacteria, most of which are only chromoparous, are
quite destitute of the power to assimilate and split up CO,, although they
naturally absorb certain rays of light. The pigment is in fact only an
‘accidental’ by-product of bacterial life; it is formed just as abundantly in
the dark as in the light, and as a result none of the chromogenic bacteria
become bleached or ‘etiolated’ when light is excluded. This alone shows
that the pigment does not play the part of the chlorophyll of higher plants.
But whilst bacteria grow just as well in darkness as in weak diffused daylight
it is necessary that the illumination should remain within certain limits, the
overstepping of which leads, in our cultures at all events, to the injury and
death of the cells. Very numerous experiments have been made to show
the deleterious effect of light on bacteria (39), but it must not be forgotten
that the conditions under which bacteria occur in nature are not those of
our artificial cultures, be they on solid media or liquid. In the transparent
gelatine and agar it is impossible for them to obtain any shade whatever,
whereas in nature, in ponds and rivers for instance, the minutest algal cell
or particle of mud offers abundant shelter. It seems therefore certain that
diffuse light in nature is innocuous, and even bright sunlight can only
destroy a limited number, and drive the motile forms to seek shelter from

72 INFLUENCE OF PHYSICAL AGENTS

the direct rays. We are certainly not justified in supposing that the
sunshine causes disinfection on such a scale as would lead to the self-
purification of rivers (40).

In cultures, both spores and vegetative bacterial cells are killed by
exposure to direct sunlight for two or three hours, and it is to the actinic
rays, not to the heat, that the deleterious effect is due. All parts of the
spectrum, however, are not equally efficacious, for if the light before falling
upon the bacteria be allowed to pass through coloured solutions, such
as bichromate of potash or ammonium cupric oxide, we find that it is
only the more refrangible rays that have the power of arresting growth.
It has been shown that freshly inoculated broth cultures of typhoid bacilli,
exposed for eight hours to light that had passed through a solution of
bichromate of potash, grew well, the broth becoming turbid with the
organisms. If instead of bichromate an ammoniacal copper solution was
used, the bacilli would not grow at all, the broth remaining clear even after
five days. Leaving aside the question as to whether the culture medium
itself is injuriously changed, it is evidently the blue end of the spectrum
—the photochemically active rays—that destroys the bacteria. These
rays too inhibit the formation of spores in the common mould-fungus
(Botrytis cinerea) during the day. Only at night is it able to develop the
reproductive cells, In some fungi, on the other hand, light is necessary for
fructification. Such are the little (1-2 mm. high) P2/obolus that projects its
ripe sporangia more than a metre into the air, and Coprinus, both forms
common on horse-dung. These become etiolated in the dark just as would
a chlorophyll-bearing plant. Such examples show that it is not possible
to formulate definite laws as to the action of light on colourless fungi (41).
Possibly there are other ‘photophile’ bacteria besides the pigmented
Erythrobacteria and their highly sensitive ally, the B. photometricum of
Engelmann. Asa general rule it is necessary to keep bacterial cultures in
the dark, or protect them at least from intense light. Weak diffused light
does no harm.

For practical purposes of disinfection on a large scale, light (even
bright sunshine) is unsuitable.

Strong electric currents (44) are fatal to bacteria, their protoplasm being
killed and no doubt altered in the same way as that of other plant cells is,
although some of the deleterious effect may be due to the electrolytic
dissociation of the nutrient media, and to the increase of temperature that
the current may cause. These drawbacks are a great hindrance to the
employment of electric currents for the disinfection of food-stuffs.
Attempts have been made (43) in distilleries to destroy by this means
the harmful bacteria without injuring the yeast cells. Currents of about
5 amperes were employed, but the practical difficulties of the process are
very great, and have not yet been entirely overcome.

EFFECT OF HIGH PRESSURE. ON BACTERIA 73

Weak currents act on bacteria presumably in the same way that they
do on infusoria and other motile micro-organisms (44). These are ‘ galvano-
tropic’; they collect around the kathode (negative pole). If the direction
of the current be reversed, the infusoria also turn round 180 degrees, place
themselves parallel to the current, and swim towards the other (now
negative) pole. Experiments with bacteria. have not yet been made,
and they would be very difficult on account of the small size of their
cells.

Very numerous and thorough experiments have been made as to the
effect of Rdxtgen rays upon bacteria, and it has been conclusively shown
that they are not even inhibitory, let alone fatal to the cells (45). The
higher plants behave in the same way, and the hopes that were entertained
of being able to disinfect the diseased body by means of Rontgen rays have
not been realized.

High pressure (46) seems to be.absolutely without influence upon
bacteria. The processes of putrefaction and alcoholic fermentation go on
without interruption under a pressure of from 300 to 500 atmospheres, and
the spores of the anthrax bacillus are unchanged after exposure for
24 hours to 600 atmospheres. In considering such facts as these it must
not be forgotten that the weight supported by a single bacillus (say of
5 « by 1 w) is extraordinarily small (even at 500 atmospheres only about
80 milligrams), so that even at the bottom of the deepest ocean at a depth
of 7,086 metres a coccus of 2 « diameter would only have to withstand
a pressure of go milligrams. In the face of these facts we are hardly
justified in saying that bacteria are less sensitive to pressure than other
organisms, because the behaviour of the larger plants and animals seems to
be in this respect incommensurate with that of bacteria.

The force of gravity exercises upon bacteria no effect comparable with
the geotropism of other plants.

Bacteria, like higher plants and cold-blooded animals, are organisms
whose temperature corresponds to that of their environment, falling and rising
with the external temperature ; they are pockilothermic (47). The relations
of the cell to temperature are expressed by specifying three points on the
thermometric scale, the maximum, minimum, and optimum. These three
cardinal points hold good for all organisms. The different functions of Jife
are performed best at different temperatures, and the temperatures most
favourable to growth, to movement, to fermentative power, to virulence, to
sporulation and to germination are probably different in each case, but
a general average, coinciding with the most rapid multiplication of the cells,
is spoken of as the ‘optimum.’ For all species these three cardinal points
vary slightly within certain limits.

The following cardinal points are those which hold good for growth.
The maximum is that temperature which cannot be exceeded without

74 INFLUENCE OF PHYSICAL AGENTS

cessation of growth, the minimum is the lowest temperature at which
growth even of the feeblest kind is possible. ;

Minimum. Optimum. Maximum,
Germinating wheat . ‘ 5-7°C. 29°C, 42-5°C.
» gourd. =. = 13-7" 33-7° 46-2°

Bacillus anthracis . . 14° 37° 45°

» tuberculosis . 30° 38° “42°

», thermophilus . 42° 63-70° 72°

» subtilis . . 6° 30° 50°

9» fluoresc. liquefac, 5-6° 20-25° 38°

» phosphorescens fe) 20° 38°

The table shows that wheat seedlings and free living metatrophic
bacteria like B. subtilis and B. liguefaciens need approximately the same
temperature, while the gourd plant, an inhabitant of warmer zones,
requires more, as do also the cholera and anthrax germs, 30° to 40° being
the optimum. The phosphorescent bacterium from our cold northern seas
has the lowest minimum of any, and at the other end of the scale we find
the representative of that curious group the thermophile bacteria. The
tubercle bacillus has the narrowest temperature range of all, only twelve
degrees separating maximum and minimum. It is, to use an expression
borrowed from animal physiology, s¢exothermic, as are all the strict parasites
on warm-blooded anjmals. The metatrophic bacteria, on the other hand,
are eurythermic, and can endure great variations of temperature with a
difference of as much as 30° between maximum and minimum.

Bacteria which are pathogenic for warm-blooded animals, and at the
same time eurythermic (e.g. B. anthracis), are very probably metatrophic
in our climate.

The thermophilic bacteria (48), singular as it may seem, are very widely
distributed even in our temperate climate, and a large number of species
have been isolated from the soil and from sewage. It is difficult to see
where these bacteria (mostly non-motile aerobic rods) find the necessary
conditions of existence. Strong sunshine sometimes heats the surface of the.
soil to 70° C. and might permit the development of such forms, but they
must be subject to long periods of quiescence. Manure and other substances
that become heated during fermentation are their most likely habitats,

In B. thermophilus the optimum reaches the temperature of coagulation
of most proteids and the maximum exceeds this. We might be inclined to
regard the protoplasm for this reason as different from that of ordinary cells,
but we must not forget that in all proteid bodies the temperature of coagu-
lation varies very much according to the reaction of the protoplasm, the
amount of salts present and other factors, so that we are not justified in
looking upon the thermophile bacteria as being miracles of nature. Besides,
we know of other organisms that live at very high temperatures. In
the hot springs of Ischia and around the ‘fumaroli’ at Naples micro-

EFFECTS OF HEAT AND COLD ON BACTERIA 75

organisms are found in water at 60°C. and more. In the drainage from
the Carlsbader Strudel (54°) there is a thick growth of Leptothrix with
green Oscillariae, and in other thermal springs crustacea and insect larvae
are known to occur at 60°.

Bacteria may be roughly divided according to the optimum temperature
into two great groups, those which grow best at about 20°C. (B. fluorescens,
B. phosphorescens, B. prodigiosus, and many other metatrophic forms), and
those which need a higher temperature. For the maintenance of a regular
high temperature special incubators are used. These secure great uniformity
of any desired degree of warmth by the use.of some form of thermostat,
and are subject to variations of 0-1 to o-5° only. Still better is a whole room
maintained at a constant temperature. Practical details will be found in
the works cited in No. 3 of Notes (p. 169).

When the temperature at which bacteria are grown approaches either
the maximum or the minimum, not only is growth arrested, but all the
functions of the cell are enfeebled. Continued cultivation under these
circumstances, particularly at temperatures near the maximum, effects
injuries from which the cell recovers only very slowly when the optimal
conditions are restored. (See Lects. III. & XVII.)

Exposure to extreme cold has, apart from arresting growth, very little
ill-effect upon bacteria. In this respect they resemble other poikilothermic
organisms, falling into a lethargic state (Azbernation). Sporeless anthrax
bacilli can endure a temperature of — 26-8° for twelve days without injury,
whilst the spores show no decrease of vitality or virulence after exposure
for twenty hours to —130°. Long imprisonment in ice and even repeated
freezing and thawing can be endured for weeks and months, bacteria be-
having in this respect like algae and other water plants. The cold of our
winters is therefore quite inadequate to kill bacteria, and the application of
artificial low temperature is equally useless as a means of disinfection (49).

In sharp contrast to this indifference to cold is the behaviour of bacteria
towards heat. If the maximum be exceeded death speedily follows (largely
as a result of the coagulation of the protoplasm), and consequently a tem-
perature of 50° or 60° C. for ten minutes is sufficient to kill the sporeless
vegetative cells of all but the thermophile bacteria. At 70° five minutes is
sufficient. It is on this principle that the preservation of tinned foods is
now so largely carried on and the ‘ Pasteurization’ of wine (heating for half
an hour to 70°) is another instance of its application. In ‘fractional sterili-
zation’ as applied to culture media like blood serum, which cannot be heated
to 100°, the same result is aimed at, namely the destruction of the vegetative
cells. Spores which have resisted the first heating are then allowed to ger-
minate and are killed by a second heating, the process being repeated five or
six times if necessary until sterility is secured. At the present time hundreds
of such sterilizers are in daily use in the service of antiseptic surgery.

76 INFLUENCE OF PHYSICAL AGENTS

The spores (47) are always far more resistant than the vegetative cells,
and their resistance is proportional to their dryness. This must not be looked
upon as a peculiarity of bacterial spores, for all resting protoplasm is resis-
tant in consequence of the small amount of water it contains. Grains of
corn rendered as dry as possible in a desiccator can endure from 100° to
110° dry heat for hours without losing the power of germination. They are
in this respect not far inferior to anthrax spores, which succumb to 140° in
three hours.

For many objects disinfection by dry heat is impracticable, as they
would be injured by the high temperature. The method can, however, be
advantageously used for disinfection of the glass ware used in bacteriological
work, but for surgical instruments and dressings boiling water or hot steam
is to be preferred.

Spores are more quickly destroyed by moist heat, although if the
temperature of boiling water be not exceeded some hours are necessary to
destroy such resistant spores as those of B. swbézlzs and its allies. Anthrax
spores are generally killed by boiling water in from two to five minutes, but
one must always reckon on finding a few of much greater resistance that
need ten or twelve minutes.

Moist seeds are much more quickly destroyed, being -killed below
boiling point. The cause of the greater resistance of bacterial spores is not
known, but probably both the impermeability of the membrane and the
durability of the protoplasm playa part. If the impermeability of the
membrane be the cause, it is easy to conceive how the spores can remain
some time in water before the protoplasm can take up moisture. They
would even in boiling water be exposed at first to a dry heat. This view
gains in probability when we consider that the spores of 2. subtilis germinate
very slowly, many hours passing before they swell and lose their lustre by
absorbing water. This stage is passed through more quickly ifthe spores be
boiled for five minutes. It seems as though the spore membrane were at
first very impervious to water. We know too that this is the case with the
resting spores of algae and fungi. The resting cells of other low organisms
such as amoebae, infusoria and flagellata, although not yet investigated,
are doubtlessly similar in this respect to bacterial spores.

The sterilization of fruits and preserves by boiling in closed vessels
is familiar to all and has been in use since the last century. A discussion
of the various kinds of disinfectors, of the Koch steamer, and the autoclave
that by means of superheated steam kills the toughest spores in one minute
at 140° would be out of place here. They are used for the sterilization of
culture media. The uses to which these physical methods of disinfection are
put in public sanitation will be found in text-books of hygiene.

All plants, those of our climates as well as those of the steppes and
deserts, have to protect themselves against dryness in times of drought.

EFFECT OF DESICCATION 77

Mosses and lichens on naked rocks shrivel up to dry friable masses, falling
into a resting condition in which they remain for weeks without losing the
power of growing again as soon as they receive moisture. The algae of
ponds and those that live on soil that is only periodically wetted can also
withstand drought for months. In all these cases it is the whole plant that
falls into a resting-stage, in which it can retain its vitality for long periods,
although not for an unlimited time. Among animals, too, many instances
are known (rotifers, tardigrada, anguillulae) in which the whole organism
can remain dried for weeks and months, and then be recalled to life by
moisture.

Organisms which are provided with a morphologically specialized resting-
stage (spores, cysts, or seeds) can still better resist the effects of dryness.
The spores of the Smut fungus, Ustilago carbo, germinate when placed in
water, even after lying for ten years in an herbarium; and wheat grains
sprout after ten or even twenty years if they have been carefully protected
from moisture. This suspension of vitality cannot, however, go on for
unlimited periods, and the miraculous stories of ‘ mummy wheat’ germinat-
ing after thousands of years are certainly fabulous.

The spores of bacteria (50) can also remain dormant for a very long time ;
those of Anthrax, for example, germinate after ten years’ quiescence. Thus
the resting-stage among bacteria is of similar duration as among other organ-
isms, and is shortened in the same manner by moisture or damp air. Many
bacteria can resist drying for long periods even in their vegetative condition.
Dry tubercle bacilli retain their virulence for weeks, as do also the bacilli of
diphtheria and typhoid fever and pus cocci. True aquatic bacteria, on the
other hand, such as the cholera vibrio, are killed by drying in from two
to five hours *.

For the purposes of practical disinfection drying is not applicable, but
it seems to play an important part in the destruction of bacterial cells in
nature. Still, if dried bacteria, during short periods of moisture such as
continually occur, can find suitable nutriment they revive again, and multiply,
only to fall again into the resting-stage. For this reason it is not to be
expected that disease products, or earth contaminated with organic substances,
will be spontaneously disinfected by mere exposure to the air.

* For the relation of these phenomena to infectious diseases see Chaps. XV. and XVI.

CHAPTER IX

THE ACTION OF CHEMICALS—CHEMOTAXIS AND
CHEMICAL DISINFECTION

EXAMINATION of a drop of stagnant water under a medium-power
objective shows that the bacteria and infusoria it contains are not evenly
distributed through the liquid. They gather in clouds around particles
and flocculi of decaying organic matter, to which they seem to be attracted
as fish are attracted by bait, or ants by aphides. ‘Instinctive’ is the term
(favoured of anthropomorphists) applied to such actions when performed by
the higher organisms, and yet we see them here carried out with unfailing
accuracy by creatures whose whole body consists of but a single cell. Are
then the bacteria too to be credited with instinct? That would indeed be
a surprising conclusion !

This capability shown by unicellular organisms of being attracted by
food-stuffs was first carefully studied by Stahl (51). He showed that the
naked protoplasmic masses which form the plasmodia of the myxomycetes
were affected by a nutritive body placed in their neighbourhood. They turned
towards it on whichever side it was placed, suggesting by this action the
term Trophotropism as a suitable descriptive name for the phenomenon. At
about the same time Pfeffer (52) investigated from a more general chemical
standpoint the effects of such stimuli upon the movements of bacteria,
protozoa, and the spermatozoids of the higher cryptogams, and was able to
show that the nutritive value was not the sole and only determinant, but
that other more recondite factors were involved, factors based on the chemical
constitution of the substance employed asa stimulant. For this reason he
introduced the now generally adopted expression Chemotaxis.

Pfeffer’s method for the study of the chemotaxis of bacteria is simple
and efficient. A capillary tube, sealed at one end and about 5-10 mm. long,
is half filled with the liquid to be tested, e.g. a 5 per cent. slightly alkaline
solution of Liebig’s Extract, or of peptone, in such a way that a bubble of
air remains in the closed end. The outer surface of the glass is carefully
cleaned from any traces of the broth, and it is placed in a drop of water
containing bacteria in such numbers as to be slightly turbid. In five or
ten seconds it can be seen that the bacteria are more thickly congregated
‘around the open end of the capillary than elsewhere, and in a few minutes

CHEMOTAXIS 79

they form a dense swarm there, and begin to enter the tube. The move-
ments of the bacteria in the drop become more lively as soon as the
diffusing peptone reaches them, and at the entrance of the tube they
are a whirling, ‘buzzing’ mass, like hiving bees. The potential energy
of the food-stuff has been converted into the kinetic energy of the
vibrating cilia. If now a cover-glass be laid upon the drop another kind
of chemotaxis can be observed. The bacteria which have entered the
tube move upward, attracted by the air in the blind end, and in about
half an hour that section of the capillary immediately below the bubble
is plugged by a thick mass of organisms. But these phenomena, the
attraction by food and the attraction by air, might as well be called tropho-
tropism as chemotaxis, the latter being shown in

its purest form when solutions of salt are employed. e@ ®
For instance, a 1-9 per cent. solution of KCl has
a powerful effect, and draws the bacteria into the q yl
tube just as peptone does, being still feebly at- mee
tractive even ina dilution of o-o19 percent. Among a;
the alkalies, potassium is chemotactically the most
powerful, then sodium and rubidium. The alkaline ABR
earths are less effective. The influence of a salt ‘ee hat be
is attributable mainly to its electropositive con- Sl a
stituent, the acid radical acting much more weakly. Fic. 18 Chemotaxis. A part
Further details of this interesting subject will $.a0? fronene Moma

efa-

: : ; ill
be found in the works of Pfeffer already cited. eens; and 3 party! fein

: : : ae t.weakly alkaline pept
Among organic substances with a high nutritive [Clution; at Y an airbubble.

eee : About 4 minutes after introduc-
value, asparagin and peptone may be mentioned as __ tion of capillary, the bacilli have
co 5 = collected round the open end of
being strongly chemotactic, whilst sugar, one of the tube, positive chemotaxis.
. 3-4 hr. later, the bacilli, driven
the best food-stuffs and richest sources of energy, by want of oxygen, have collected
5 ‘ 4 a 4 close to the air-bubble. From

has but little attractive power. Glycerine is in nature. Magn. 50.

all cases, as far as is known, inactive.

Contrasting with the phenomena just described, is the power which
certain substances have of repelling bacteria. This is known as negative
chemotaxis. Free acids and alkalies have this effect, and capillaries filled
with their solutions invariably remain empty. Alcohol too is ‘ instinctively ’
despised by micro-organisms. In some salts the action of the acid radical
and that of the base neutralize each other (e. g. carbonate of ammonia 1-76
per cent., phosphate of potash (monobasic) 3-48 per cent.), and the bacteria
then take up a mean position at a certain distance from the mouth of the
tube.

In the case of negatively chemotactic compounds, the poisonousness of
the substance is no more a criterion of its repellent power than the food
value is an index of the attractive power in positively chemotactic bodies.

For instance, a solution of 0-019 per cent. potassium chloride plus o-o1 per

80 CHEMOTAXIS AND CHEMICAL DISINFECTION

cent. mercuric chloride attracts bacteria by reason of the potash it con-
tains, but they rush into the tube only to meet their death from the mer-
cury salt. It would therefore seem that chemotaxis, useful as it must be to
bacteria in the search for food, may lead them to destruction, although of
course they are not exposed in nature to the temptations of such fatally
seductive capillaries.

If the facts gained by these experimental observations are to be
employed to elucidate the life-history of bacteria in their natural habitats,
in water, in the soil, or in the tissues of the diseased body, a number of con-
siderations must be borne in mind. In the first place, chemotaxis can only
take place in media which permit free movement, in liquids. Secondly,
different kinds of bacteria by no means react in the same way to the same
substances. Furthermore, it must be remembered that the sphere of
influence of such a capillary is very small. It is not possible to entice
into it all the bacteria swimming in the drop. It would not be possible,
even if we could renew the solution in the capillary as fast as it diffused so
that it could continually give off a diffusion-stream, as perhaps the decaying
flocculi in stagnant water do. For the bacteria would be already stimulated
by the diffused substance, and to enable them to again react chemotactically
a much higher concentration would be necessary. Pfeffer has shown that
“Weber's Law’ (Psycho-physical law of Fechner), which formulates the
relation between strength of stimulus and intensity of sensation, holds
good for the chemotactic movements of bacteria. Weber’s law is to the
effect that an external stimulus just sufficient to give rise to a sensation
must be increased in a definite ratio in order to awaken that sensation
again. For instance, a weight of one ounce laid upon the hand gives rise
to the sensation of pressure upon a certain spot. If now upon the weight
a further load of #5 oz. be laid no further sensation is aroused. Not
until the original weight has been increased by one-third (i.e. to 1-33 0z.) is
the sensation of pressure aroused again. Had ten ounces been the weight
originally employed it would have had to be raised to 13-33 ounces. For
thermal stimuli the increase must amount to jth, and for visual stimuli to
ziath of the original stimulus in order that it should again cross the threshold
of sensation and enter our consciousness. Precisely the same law governs
the phenomena of chemotaxis. In the case of one of the putrefactive
bacteria the stimulus has to be increased five times before chemotactic
movements are again set up. If the organism be already in a or per
cent. solution of extract of meat the capillary must contain a o-5 per cent.
solution, if it be in 1 per cent. the tube must be filled with 5 per cent.
bouillon before a reaction follows, while to start very active movement we
should have to fill the tube with a broth ten or twenty times as strong as the
surrounding fluid. These facts should not be forgotten when we attempt
to explain by chemotaxis the behaviour of bacteria in the living body, or the

CHEMOTAXIS 81

emigration of leucocytes towards a bacterial focus (53). An exact knowledge
of the conditions involved is impossible, inasmuch as the composition of the
fluids in which the tissues are bathed and the amount of chemotactic
substances they contain are entirely unknown. For this reason considerable
discretion must be exercised in the use of the word chemotaxis if it is not to
become a mere shibboleth (see Ch. XVII).

In the case of some substances, peptone for instance, the absolute
amount sufficient to cause perceptible reaction is very minute. Pfeffer
calculated that a capillary filled with o-or per cent. peptone solution,
a strength just sufficient to attract bacteria in water, contained only
guv0deee0 Of a milligram of peptone. And yet compared with the size of
the bacterial cells this quantity is not disproportional.

Like all other processes that depend ultimately upon the activity of the
living cell, the phenomenon of chemotaxis is extremely obscure. This
much however we know, that the effect of the substances diffusing from
the capillary is to cause the bacteria to turn (hence chemo-/azis) in such
a way that their longer axes are parallel to the direction of the diffusion
stream. They then swim either against the stream (positive chemotaxis) or
with it (negative chemotaxis). Why one substance should act positively
and another negatively is at present quite inexplicable. A more detailed
discussion would be beyond the scope of this treatise, and would be at best
fragmentary. Those substances which in weak solutions exercise a positive
action on bacteria have in some cases (KCl 19 per cent.) the same effect
when the solution is concentrated ; in other cases the bacteria are repelled.
Neutral salts such as KCl and NaCl can be endured in very high concen-
trations. The hay bacillus, for instance, grows well in an infusion containing
9 per cent. NaCl, 5 per cent. sal ammoniac, 11 per cent. KCl, or 10 per
cent. KNO,. Such substances are not poisonous, and arrest growth finally
by the osmotic pressure they set up.

Of great interest are those poisonous compounds which even in minute
quantities destroy the life of the cells. They are not by any means
especially poisonous for bacteria. A one-tenth per cent. solution of corrosive
sublimate (HgCl,) kills tubercle bacteria in ten minutes, and the cells of
algae are destroyed just as soon, if not sooner. In a I per cent. solution
of carbolic acid both tubercle bacilli and ordinary plant cells are killed in
one minute. With few variations and exceptions the protoplasm of all
organisms is destroyed in about the same time by the more powerful poisons.

The destruction of bacteria by poisons—chemical disinfection (54)—is
employed in all cases where the application of the methods described in
the last chapter is impossible on account of the injury caused by the heat.

The resistance of bacteria to chemicals is not only different in different
species, but varies also according to many external circumstances. It is

greatest when the organisms are growing under the most favourable
FISCHER G

82 CHEMOTAXIS AND CHEMICAL DISINFECTION

circumstances, when temperature, food, and other conditions are all
optimal. Bacteria are, in short, like all other organisms, strongest and
most resistant when they are in the best health. There is always a
great difference between spores and the far less resistant sporeless vege-
tative cells, so that a disinfectant can only be considered effective when it is
able to kill spores. In pr.ctice, circumstances may sometimes arise when
this requirement may be dispensed with.

Every disinfectant must be tested on the following three points :

I. In what concentration must it be added to a given substratum in
order to prevent the development and multiplication of bacteria without
killing them? This is the coefficient of inhibition.

11. What is the shortest time in which sporeless bacterial cells in water
at 20°-25° C. are killed by a substance in medium concentration (i.e. not
sufficient to cause plasmolysis or other injury)? This is the zzferdor lethal
coefficient.

111. What is the shortest time in which, under the same conditions, the
spores are killed? This is the superior lethal coefficient.

Laborious investigations without number have been made with the
object of determining these three values for all kinds of organic and
inorganic compounds, and we have now a number of carefully-chosen
substances that are suitable for use as disinfectants. The following tables
give a few examples. Further details will be found in the works cited
in Notes 3 and 54.

I. COEFFICIENT OF INHIBITION FOR ANTHRAX BACILLI IN
OX-BLOOD SERUM. From Behring (54).

The numbers indicate the number of cubic centimetres of serum in
which one gram of the solids or one cubic centimetre of the liquids was
dissolved ; for instance,‘ Corrosive Sublimate, 10,000’ = 1 gram HgCl, in
10,000 c.c, serum :

Cyanin and malachite green. ‘i . : : 40,000
Nitrate of silver . F : ‘ ‘ i if 30,000
Corrosive sublimate . é a ‘ a é : 10,000
Trichloride of iodine ‘ 3 a : z . 1,500
Caustic soda. é . ‘ . . ‘ i 1,500
Cadaverin (bacterial toxine) . F 3 : F 1,500
Quinine hydrochlorate . . . ‘ 5 3 500
Carbolicacid . . . : : . e : 500
Thymol . . Sa 3 . : : 3 250
Salicylate of soda. ‘ ‘ ‘ é 3 é 150
Alcohol. ‘ : ; ‘ : . : 15
Common salt . : . . F : . ‘ 15

The extraordinarily small quantities which are in some cases sufficient to

COMPARATIVE EFFICACY OF DISINFECTANTS 83

prevent putrefaction do not kill the bacteria, they only arrest growth and
multiplication.
II, LETHAL VALUE FOR SPORELESS TUBERCLE BACILLI.

+ Inferior lethal coefficient; the time is given in which tubercle bacilli
(from cultures, not in sputum) are killed. From Yersin (54).

Carbolic acid . . : . . 5% 30 seconds
5 ane 7 ‘ . ‘ 1% 1 minute
Absolute alcohol. , : 7 pie 5 minutes
* Todoform 3 F ‘ 4 é 1% 5 35
Ether . . . . . beaks Io ,,
Corrosive stiblimate 5 ; 0-1% 10,
Thymol . : z 3 . : 03% 3 hours
Salicylic acid . a 7 : s 0-25% 6,

To kill the bacteria in sputum, where they are embedded in mucus,
a much longer time is necessary, e.g. 10 per cent. lysol for twelve hours.
The figures in the table were obtained by mixing tubercle bacilli from
a growing culture with the fluids, taking out samples from time to time,
and sowing them on culture media. The values given may be regarded
as being good for most sporeless bacteria, the sensitiveness of which is
illustrated by this example.

III. LETHAL VALUE FOR ANTHRAX SPORES.
From Paul u. Krénig (55).

Substance. Concentration. Time (Temperature 18° C.).
Corrosive sublimate Z 1-7 % ( 16litres) 12-14 minutes
” . s . 084% ( 32» ) 24-30 ”
” : . : 0-42% (64 5 ) 45-60 ”
af . , o2 % (128 ,, ) 60-80 e
35 ei . or % (256 ,, ) more than 120 minutes
Nitrate of diver : ‘ 425% ( 4 » ) , 15-16 minutes

0-08% (200 ,, ) not in 10} hours

Sulphate ‘of copper . : 16 % ( 1 4 ) notin 10} days
Sugar of lead . ‘ 7 325 % ( I 5 ) notin7 95
Sulphuric acid . é 49 % ( 2 4, ) not in 30 hours
Caustic potash ‘ ‘ 56% ( 1 4 ) 18 hours
Permanganate of potash . 395% ( 4 5 ) 40 minutes
Bichromate of potash. 74% 4 5 ) notin 4 days
Permang. of potash, 8 litres, plus 8 litres HCl 5 minutes
Chlorine water 7 . 022% ( 32litres) 2 ,,
Bromine water : fs 05% (32 » ) 2 55
Carbolic acid : : 5 4 agence not in 24 hours
Formaldehyde . 4 5 # sical 120 minutes

The lethal concentration for spores is determined in the following way.

A suspension of the spores in water is dried upon silk threads, pieces of

glass, or, better still, upon carefully-cleaned garnets, which are then laid in

the disinfectant. Specimens are taken out at intervals and sowed in nutri-
G2

84 CHEMOTAXIS AND CHEMICAL DISINFECTION

tive media. Before this is done, however, the threads or garnets, as the
case may be, must be freed from any adherent disinfectant. Washing with
distilled water is not enough for this, they must be rinsed with ammonium
sulphide, and the soluble metallic salts precipitated and rendered harmless.
Only in this way is it possible to obtain reliable results, for the small
quantity of disinfectant adherent to the spores, although not sufficient to
kill them under ordinary circumstances, might do so during the stage of
imbibition, and would certainly destroy the germinating rod, thus giving
a false idea of the disinfecting power of the substance. In the examples
given in Table III, from 15,000 to 20,000 spores were killed in the stated
times. The concentration is given in per cents. and also in terms of
‘molecular dilution’; for instance, ‘Corrosive Sublimate, 16 litres,’ means
that sixteen litres of the solution contain the molecular weight of HgCl,
(i.e. 271) expressed in grams, so that 100 c.c. of the solution contains
#43 grams =1-7 grams. This method of calculation, so much used-in
modern chemistry, is given because it permits an easy comparison of
different salts—a comparison of molecule with molecule, so to speak.

According to Koch absolute alcohol, concentrated glycerine, concentrated
NaC] solution, and distilled water do not destroy anthrax spores, even after
acting on them for months. The table shows that the halogen elements
(chlorine, bromine, iodine) are the most powerful, and, among the metallic
salts, corrosive sublimate. Nitrate of silver has some little effect, but sulphate
of copper and acetate of lead are powerless. Free acids or free alkalies must
be very strong to act as disinfectants. Chromate of potash, too, although it
is a powerful oxidizing agent, has little germicidal power, whilst potassic
permanganate, far less effective as an oxidizer, is a powerful disinfectant.

Tables II and III show the great difference between spores and vegeta-
tive cells in their behaviour to disinfectants. Five per cent. carbolic acid,
for instance, which kills tubercle bacilli in thirty seconds, does not destroy
anthrax spores in twenty-four hours; with o-1 per cent. sublimate, the times
are ten minutes and sixty or eighty minutes respectively. Absolute alcohol,
powerless against anthrax spores, kills tubercle bacilli in five minutes. The
resistant power of the spores depends, no doubt, principally on the great im-
permeability of the spore wall to dissolved substances of all kinds. This
property is shared by the cysts and membranous coatings of all low organisms
in the resting-stage, and by the seeds of plants. A resting-stage of long
duration is indeed inconceivable without some such protection. In plant
seeds and algal spores the impermeability is caused by the deposition in
the membrane of fats and resinous bodies, and possibly the spore wall in
bacteria is protected in the same way. Another cause of the durability of
spores is the density of the protoplasm, which contains less water than it
does in the vegetative cell.

The great efficacy of corrosive sublimate as a disinfectant seems at first

DISSOCIATION OF SALTS 85

sight to be only a special case of the toxicity of all mercury salts, and formerly
it was thought that they were equally poisonous if only equal amounts of the
metal were present. Equimolecular solutions therefore were supposed to be
necessarily of equal disinfectant power. Recent investigations, however (55),
based upon the modern theory of solutions, have proved this to be wrong,
and rendered it probable that the toxicity of a poisonous salt varies with
the degree of dissociation. The dissociation theory (56) has shown that
in a solution of a salt there are present not only the unaltered molecules of
the dissolved substance, but also a certain number of disintegrated or ‘ dis-
sociated’ molecules. Ina solution of HgCl,, for instance, a certain propor-
tion of the HgCl, molecules are split up into their electrically active
components or ‘ions,’ the positive metallic ions (kations) Hg, and the
negative ions (anions) Cl]. The degree of dissociation, that is to say,
the proportion of dissociated molecules to unchanged HgCl, molecules,
changes with the concentration of the solution, the temperature, and
other conditions, and different salts of the same metal are dissociated in
different degrees. Now many of the physical properties of a solu-
tion, such as electrical conductivity, freezing-point, boiling-point, and
osmotic pressure, depend upon the degree of dissociation, and it seems very
probable that toxicity does also. Since different salts of mercury are very
differently dissociated in watery solutions, we might expect that their toxic
powers would be different also, and as a matter of fact we find this to be
the case. A sixteen-litre solution (1-58 per cent.) of mercury cyanide,
a salt which is but very slightly dissociated, does not kill staphylococci in
three minutes, whilst a solution of corrosive sublimate only a quarter the
strength (i.e. sixty-four litres, 0-4 per cent.) does. Anthrax spores left for
twenty minutes in this solution of sublimate were almost all killed (seven
colonies grew), whilst after a sixteen-litre solution of the cyanide, acting for
the same time, innumerable colonies sprang up.

A comparison of differently dissociated mercury salts shows clearly,
therefore, the relation between dissociation and toxicity. The connexion
becomes more striking still when we compare the same salt in different
degrees of dissociation.

The proportion of the dissociated molecules to the unaltered molecules
in a given solution of a salt is constant. For example in a solution of HgCl,
the proportion of Cl ions to unchanged Hg Cl, molecules is constant. By
adding other chlorine ions, for instance by adding the more strongly disso-
sociated NaCl, the number of dissociated Hg Cl, molecules can be reduced,
the reduction being dependent on the ratio between the amounts of disso-
ciation of HgCl, and of NaCl. Let us suppose a sixteen-litre solution
of Hg Cl, to contain x Cl ions and y unchanged molecules, then, broadly

speaking,” = c (a constant). If, now, there be added enough solid NaCl,

86 CHEMOTAXIS AND CHEMICAL DISINFECTION

which is more readily dissociated than Hg Cl,, to make a sixteen-litre solu-
tion, there will be present ++ Cl ions derived from the NaCl. For the
pure HgCl, solution += cy, but for the HgCl, plus NaCl solution

a+(4+m)=cy or x = aoa In other words, the number of Cl ions

derived from the Hg Cl, is reduced, its degree of dissociation is lessened,
and some of the free Hg and Cl ions are built up again into complete
molecules. The following table shows that as the degree of dissociation
is lessened the toxicity is reduced. The figures give the numbers of
colonies that arose from approximately equal quantities of spores after
these had lain for six minutes in the solutions (55).

Number of Colonies.

16 litre Hg Cl, solution . . : . 8
35 4 plus1 NaCl. 7 32
3 a a 2 es ; ; 124
2” 3 ” 3 0 282
” oe ” 4 ” *: . 382
” ” ” 46 ” . . 410
” ” ” 6 ae x * 803
” ” 33 10 ” . . 1,087

The gradual diminution in germicidal power is unmistakable and needs
no comment.

These facts must be borne in mind in experiments on the disinfection,
by sublimate, of liquids such as serum or bouillon which contain 0-7 per cent.
(eight litres) of NaCl. It is evident that more sublimate will be necessary
than in fluids free from salt. The presence of peptone or albumen will
necessitate a still further addition of sublimate, because some of it will form
insoluble compounds with the proteids whereby its toxicity will be reduced.

Since the degree of dissociation is dependent upon the nature of the
solvent and also upon the temperature of the solution, these factors are of
influence on the disinfecting power. The increase of toxicity which accom-
panies increased temperature is not, however, solely the effect of augmented
dissociation.

Although the technique of disinfection has not as yet been affected by
these recent discoveries, it does not need much penetration to see their high
scientific interest and importance.

There are many other bodies besides those mentioned that could be
used for disinfecting purposes—anilin dyes, such as methy] violet, ethereal
oils, and many aromatic compounds. New disinfectants spring up every day,
and are widely advertised, only to disappear again very soon.

Gases, such as carbonic acid, carbonic oxide, hydrogen, nitrous oxide,
nitric oxide, sulphuretted hydrogen, sulphurous acid, and coal gas, arrest
the growth of bacteria in agar cultures exposed to a slow stream of the
gas, but the method has no practical value. The ozone in the air, too,

DISINFECTION OF THE TISSUES 87

even where most abundant, is in such minute quantities as to be quite
without influence.

The fact that thousands of bacteria pass through our digestive organs
every day suggests the question whether any of the digestive secretions
function as natural disinfectants. The saliva and the pancreatic juice are
alkaline in reaction and unable to injure bacteria, The pancreatic juice, by
reason of the proteids it contains, is even nutritious. The bile acids
inhibit the growth of bacteria, but of all the digestive secretions the free
acid of the gastric juice (2-3 per thousand H Cl) alone is able to kill them.
The action of this even is in any case uncertain, and can only affect sporeless
cells. Normal gastric juice ina test tube destroys in half an hour the
bacteria of cholera, typhoid and glanders, pus cocci, and the sporeless cells
of anthrax and tetanus (57), but spores are not injured by passing through the
stomach. To destroy anthrax spores, they must lie for six hours in 2 per
cent. H Cl, so that it is evident the gastric secretion with only from -2 to
*3 per cent. HCl is quite inadequate, even if allowed to act for days.
Even against sporeless bacteria it is of little effect, as the examples quoted
show. Bacteria given to animals in their food (B. pyocyaneus, blood in-
fected with anthrax, tuberculous tissues) are not entirely destroyed even after
six or eight hours (58).

Chemical disinfection of the diseased body is not possible, for the tissue
cells would be just as much injured by the disinfectant as the bacteria.
Nor is it possible to disinfect by chemical means wounds in which bacteria
have taken up their abode ; an aztisepsis of wounds is not possible. When
a wound cannot be purified by mechanical means the body must be helped
to fight against the invaders, and the only way to do this is to secure clean-
liness, asepsis. sepsis confines itself to the treatment of wounds with
germ-free instruments and dressings without using chemical disinfectants,
and is sufficient to prevent clean fresh wounds, such as are made at
operations, from becoming infected.

As to the cause of the fatal action of disinfectants upon the bacteria we
know but little. We know that the salts of the heavy metals, corrosive
sublimate or nitrate of silver for instance, coagulate protoplasm. They
destroy life probably by precipitating certain substances from the extremely
complex protoplasm of the cells. Other compounds, such as alkalies and
acids, may perhaps act by the separation and solution of proteid bodies,
resulting in a destruction of the protoplasmic structure. Even a change
in reaction might cause the precipitation of certain constituents. In most
cases, however, the matter is beyond any explanation, because we have no
knowledge of the peculiarities of protoplasmic structure on which the
phenomena of life depend.

CHAPTER. X

BACTERIA AND THE CIRCULATION OF NITROGEN
IN NATURE

1. Introduction: The Assimilation of Free Nitrogen by the Bacteria
of the Root Nodules of Leguminous Plants, and by Bacteria in
the Soil.

APART from the activity of organisms like the pigment and phos-
phorescent bacteria, and the remarkable metabolism of the sulphur- and
iron-bacteria, the work of bacteria in nature embraces three great processes :

1. The circulation of nitrogen: effected by putrefaction, the formation of nitrates,
and the assimilation of atmospheric nitrogen.

2. The circulation of carbon by the ‘fermentation ’ of carbohydrates and other non-
nitrogenous products of animals and plants.

3. The causation of disease in other organisms, particularly in man and the higher
animals,
There are in nature five sources of nitrogen open to plants and animals:

1. The atmosphere (79 per cent. by volume of free nitrogen),

2. The nitrates of the soil and the traces of nitrous acid formed in the air during
thunderstorms.

3. Ammonia, which occurs in minute quantities in the air, and is set free abundantly
by the putrefaction and decay of dead organisms.

4. Animal excreta, which contain nitrogen compounds of many kinds, even down to

ammonia; and
5. The tissues of plants and animals.

The first three of the above-named sources are useless to animals, for
these obtain their nitrogen from plants only, either directly (herbivora), or
indirectly through other animals (carnivora). To plants, on the other hand,
nitrogen seemed until comparatively recently to be available only in one
of these three forms. Vegetable physiologists had come to the conclusion
that in nature the nitrates of the soil were the one and only form in which
nitrogen was taken up by plants. For, although it was known that in
experiments plants could be got to take up ammonia salts and even gaseous
ammonia, it was evident that ammonia was not a common source of nitrogen
in nature. And, finally, the atmosphere, the greatest storehouse of nitrogen in
nature, seemed closed to plants. These views, however, were overturned by

ASSIMILATION OF FREE NITROGEN 89

detailed investigations into the nutrition of the Leguminosae. That the Legu-
minosae could grow in soil poor in nitrogen and thrive thereon, even without
nitrogenous manuring, had long been known, and it has now been demon-
strated that they take up nitrogen from the air (59) and convey it to the soil.
This enrichment is particularly evident when the plants are ploughed in.
All other plants, all our cereals and food crops, are, as regards the soil,
merely consumers of nitrogen, since they are unable to take it up in any
form but that of the nitrates of the soil.

The difference is seen even in the amount of nitrogen present in the
tissues of the two groups of plants. Lupine seeds contain 5-7 per cent. N,
wheat grains only 2-1 per cent., lupine haulms 0-94 per cent., wheat straw only
o-5 per cent. An experiment with peas showed that a quantity containing
16 mgr. of nitrogen gave rise to a crop containing 499 mgr., whilst the
quantity of nitrogen in the 4 kilograms of mould they grew in had increased
from 22 to 57 milligrams ; a total gain of 518 mgr. of nitrogen. Calculated
for larger quantities of plants it will be at once seen that these figures
represent an enormous profit. One hectare (=2-4 acres) of lupines is cal-
culated to enrich the earth by 227 kilograms of nitrogen annually. The
amount gained by the meteoric fixation of nitrogen (production of HNO,
and HNO, during thunderstorms) would for the same area be only 0-09 to
1-8 kilogram per annum, so that there is no doubt that it is the atmosphere,
and the atmosphere alone, from which these plants extract the gas.

Seeing that no other cultivated plants* (not even Brassica alba, the
White Mustard) are able to act in this way, we apparently have in the Legu-
minosae a remarkable and unique group of organisms. But we shall be
wrong if we attribute the power of fixing free nitrogen to the tissues of the
plant itself. They are powerless to do this, and behave towards nitrogen in
‘no way differently from the tissues of non-leguminous plants. Not until
they enter into partnership with certain bacteria, the dacteria of the root-
nodules, do the plants act as usurers of nitrogen, gathering and storing it
and growing ever richer. The nodules (60) which arise upon the roots
of the seedling plants when they are a few weeks old are minute white or
pinkish excrescences that soon increase in size and sometimes give the roots
a distorted appearance, as though they were attacked by galls (Fig. 19,
aand 6). At first hard and smooth, they become wrinkled as the foliage
of the plant grows and, by the time the pods are ripe, are shrivelled and
cracked. Their dried remains rot in the ground with the rest of the root.
The nodules are either separate excrescences on the surface of the root,
with whose vessels they are connected by a tiny strand of vascular fibres, or
the root itself swells up in places. In both cases there is always'a close
connexion between the cells of the nodule and the conductive tissue of the

* The nodule-bearing alder and Elaeagnus are perhaps exceptions.

go BACTERIA AND THE NITROGEN CYCLE

plant (Fig. 19,0). Ifa transverse section of a young and firm nodule, which
exudes a turbid milky juice on crushing, be examined, masses of large cells
are seen, distinguished from those that surround them by being filled with
a finely striate or punctate mass. Sometimes there are numerous small
. nests of cells, sometimes they run together to form larger masses. These
cells, which constitute what was formerly known as the ‘bacteroidal tissue’
(Fig. 19,4 and c), are nothing more or less than the enlarged cells of the root
itself crammed full of fine, slender, rod-shaped bodies (Fig. 19d). The nature
of these has been variously inter-
preted. Woronin, the first observer
(1866), took them to be bacteria-like
parasites; other investigators since
then have looked upon them as pro-
teid crystals. If this view were correct
we should have to regard the root-
nodules not as pathological structures,
but rather as a kind of nitrogenous
potato with the albumen in the form
of bacteroids. Recent investigations
have, however, placed it beyond dis-
pute that these rod-shaped bodies are
bacteria, living and growing in the
cells of the root (Fig. 19, c-f). The
normal straight, rod-shaped bacteria,
well tingible with anilin colours, are
only found in the young nodules. As
these grow older the bacteria take
Fic. 19. Root-nodules of Leguminosae. a, root on all manner of distorted shapes ;
nodule of the lupine, nat. size (from Woronin); 4,

longitudinal section through a lupine root and nodale ; indle-shaped, branched, bifurcated
£, fibro-vascular bundle of root ans off fine branches =e ped, 4

to every part of the nodule, and its bacteroidal tissue or inflated forms are common. It is to
(w), low ae, from Woronin); c, a cell from a lupine

odule filled with bacteria (black) between which the i i
finer mime of the pretorlasm is ae. at the an les these deformed bacteria only (Fig. 19,

of the cells intercellular ; from a section (fixed i i i
ot cieels ert Inlae meies rues 2 se foaled 52 and /) that the term bacteroids is still

degrcuieiorm: Cinsvriatetdsheniecvie @PPlied. They are so-called ‘involu-
a pre niles ro OEE, Meee ON, ee ion forms,’ similar tothose found among

many other species of bacteria when
the conditions of growth are unfavourable (the acetic ferment, for instance,
or the bacilli in old tubercle or diphtheria cultures). The cell contents as
well as the shape are affected. They stain badly, and large numbers of the
bacteroids seem to be mere empty shells. The conversion of the bacteria
into bacteroids is, in short, nothing but a sign that the micro-organisms are
dying and yielding up their protoplasmic contents to the plant, which begins
to grow more vigorously as soon as the bacteroids appear. By the time the
seed of the plant is ripe the shrunken empty nodules contain only fragments

ROOT-NODULES OF LEGUMINOSAE gI

of the bacteroids, together with a few intact healthy rods that remain in the
soil and serve as seed material for next year’s nodules.

Root-nodules have been found in all the sub-orders of the Leguminosae
(Papilionaceae, Mimoseae, Caesalpineae), and they always contain bacteria
and bacteroids. Root-nodules without bacteria do not exist, and since Legu-
minosae destitute of nodules behave with regard to nitrogen just as other
plants do, it seems evident that the bacteria are the nitrogen collectors. As
an unproved hypothesis this idea was floating in the air for a long time, until
the classical researches of Hellriegel and Wilfahrt (59) placed the matter
beyond the reach of controversy. Their experiments proved that it was
possible by planting sterilized seeds in sterilized earth to grow leguminous
plants for some months without root-nodules, if they were well protected from
subsequent infection. They showed further that such plants had lost the
power of storing nitrogen, but regained it if the earth they grew in were inocu-
lated with an infusion of soil whereon Leguminosae had previously flourished.
Finally their researches brought to light the marked contrast between
nitrogen-storers like Leguminosae, and nitrogen-consumers such as wheat or
oats. The following table gives a few of the chief results of their investigations.

Amount of Amount of | Gain or Loss
Nitrogen in Nitrogen of Nitrogen
seed and soil. in crop. in crop.
I. NOT STERILIZED AND NOT INOCULATED:
(a) Without nitrogenous manure Grammes. {| Grammes. | Grammes.
Oats. é - is 7 . 0-027 0-007 ~ 0-020
Peas . : ‘ « 5 ‘ 0-041 1-283 + 1-242
(4) Manured with nitrate of calcium
(N =o-112 grammes)
Oats . 7 . . . . 0-139 0-09 - 0-049
Peas . 7 ‘ . F : 0-153 0-700 +0°547
II], INOCULATED WITH SOIL IN WHICH LEGU-
MINOSAE HAD BEEN GROWN, NOT
STERILIZED:
(a) Without nitrogenous manure
Oats . ; i . . ; 0-027 0-007 - 0:020
Peas 4. 6 “Ss oe oa 0-038 0-459 +0-421
(4) With nitrate of calcium
(N =o0-112 grammes)
Oats . és % ‘ é ‘ 0-139 0-088 ~ 0-051
Peas . ‘ * 3 é ‘ O-150 0-220 +0-070
III, INOCULATED AND STERILIZED:
(a) Without nitrogenous manure
Peas . ‘ ‘ - 5 . 0:038 O-O15 — 0:023
(6) With nitrate of calcium
(N =o.112 grammes)
Peas . . . . . . 0-045 O-014 - 0-031

92 BACTERIA AND THE NITROGEN CYCLE

These tables explain themselves. It is clear that the pea nourishes
itself in a similar manner to the oat when cultivated in sterilized soil and
unable to form nodules (III), whereas under natural conditions it bears these
prolifically and stores up nitrogen in large quantities. Furthermore, the
tables show that, whilst oats are rendered much richer in nitrogen by suitable
manuring (Id and II), peas are not affected (III); they cannot utilize
nitrogen offered to them in this form. For oats, on the other hand, it is
a matter of indifference whether the soil is sterilized or not, since they
form no nodules. The advantage gained from non-nitrogenous manure,
such as phosphate of potash, is shared equally by oats and peas.

The next task was to obtain pure cultures of the bacteria of the root-
nodules, and ascertain their behaviour towards atmospheric nitrogen. They
are easy to cultivate on a decoction of leguminous plants to which } per cent.
asparagin and 2 per cent. sugar has been added. In such a medium the
bacteria grow well in the form of slender rods, aerobic and mobile, with
a tendency to produce involution forms or bacteroids. They at first get
their nitrogen from the asparagin, but after about two months’ growth
every litre of the culture has gained from 9 to 18 milligrams of nitrogen
which must necessarily have come from the atmosphere (Beyerinck). Mazé
in similar experiments obtained an increase of 47-5 mgr. nitrogen in fifteen
days, and in another case 23-4 mgr. in eighteen days, so that although further
investigations are desirable there can be no doubt that pure cultures of the
bacteria from root-nodules do actually assimilate and fix the nitrogen of
the air (61).

Morphologically the bacteria from different kinds of Leguminosae,
whether in the nodules themselves or in pure culture, are very similar in
appearance, and the growths on gelatine (which is not liquefied) are all of the
same character. The ‘bacteroid’ forms are likewise of the same kind in
all cases, so that it would seem as if all Leguminosae were inhabited by the
same species of bacterium, to which the name Bacillus radicicola was given by
Beyerinck and Rhizobium leguminosarum by Frank. But as soon as we
investigate the action of these bacteria upon plants we find differences
between them.

If plants of clover, pea, and vetch growing in sterilized earth and
destitute of root-nodules be watered with an infusion of bacteria derived
from the root-nodules of peas, we find that numerous root-nodules arise
both on the pea and on the vetch, whilst the clover forms very few or none
at all. Conversely, clover bacteria are almost useless to the pea and the
vetch. Nobbe and Hiltner (62) as a result of experiments, which although
successful are not beyond criticism, have come to the conclusion that it is
only between members of the same group of Papilionaceae that the bacteria
of the root-nodules are exchangeable, that, for instance, clover bacteria can
be used by other Trifolieae such as lucerne, but not by Phaseolus \upines

SYMBIOSIS: PARASITISM 93

or other Phaseoleae or by Viciae such as Vicia, Ervum, or Pisum, the
bacteria of these again being useless to Trifolieae. We should have, in fact,
if these views be correct, different breeds of one and the same species of
bacterium (B. radicicola), comparable to the different races of brewers’
yeasts or to those described by Eriksson in the wheat rust-fungus Puccinia.

Nobbe and Hiltner have attempted to turn their improved theories to
practical account by the introduction of ‘ Nitragin, a preparation manufac-
tured at the Héchst Chemical Works. Nitragin is a pure culture on gelatine
of bacteria from root-nodules—a kind of living manure to be mixed with the
seed material or soil and strewn over the fields. There are eight different
sorts of nitragin on the market, suitable for peas, lupines, beans, &c., and it
is said to be very advantageous when first planting Leguminosae in virgin
soil, or in soil of poor quality, moors for instance, or where for many years
no Leguminosae have grown and the land is presumably destitute of nodule
organisms. The results are extremely inconstant and frequently very
difficult to judge, so that it is not surprising
that opinions are divided as to its efficacy *.
Instead of nitragin, ‘leguminous earth, i.e.
soil in which Leguminosae thrive well, is
sometimes used with advantage to ‘ inocu-
late’ new ground and bad earth such as
moorland.

The remarkable association of bacteria
with leguminous plants is generally re-
garded as an instance of symbiosis, a :
connexion from which both parties reap Fortis Maire teage a este aad pete
advantage similar to the ‘ mutual’ associa- by in winch Ge ee
tion of alga and fungus in lichens. Here, as icotepaen ee ee ee ee
is well known, the green or brown cells of
the alga are enclosed by the thickly matted filaments of the fungus (Fig. 20).
The alga supplies the metatrophic fungus with organic compounds and the
fungus repays the debt by providing the alga with the necessary moisture
and mineral salts, not to speak of the protection that its embrace involves.
This at least is the opinion of those who, following the ‘symbiotic’ tendency
of the day, look upon a lichen as a co-operative concern. But it is more
than doubtful whether this view is correct. All the algae that live in lichens
are known to be able to live in a free state. They certainly do not need to
be supplied by the fungus with water and mineral salts, and as regards
protection it is difficult to see how the algal cells can be ‘ protected’ by
hyphae which cling round them tightly and send ‘ suckers’ into their interior
(Fig. 20, 6). The fungus behaves in every way as a parasite which lives upon

* (See Dawson, FAi/. Trans. 1899, Vol. 192, pp. 1-28.]

94 BACTERIA AND THE NITROGEN CYCLE

the alga. As a parasite it must establish a close connexion between its
tissues and those of its host, and, since it cannot crawl into the alga as the
tape-worm does into man, it winds its hyphae around the cells, Enclosed
thus in the light, well-ventilated thallus of the fungus, the algae continue to
live, but they draw no nourishment from it. The advantage is entirely on
the side of the fungus, which is a true parasite.

The association between the leguminous plants and the bacteria of
their root-nodules, paradoxical as it appears at first sight, is of a precisely
similar character; the plant is parasitic upon the micro-organism. To
render this view more comprehensible, we will consider in detail the
development of the nodules. The fine root-hairs of a young leguminous
plant, destitute as yet of nodules, push themselves everywhere into the
crannies between the particles of soil, taking up water and mineral salts and
secreting fluids which dissolve and
render assimilable the substances with
which the cell-wall comes into contact.
There can be no doubt that some of
the secretions of the root-hairs act
chemotactically upon many of the
bacteria that swim in the water-filled
crevices of the soil ; and these would be
attracted furthermore by organic sub-
stances issuing from wounds or scratches
upon the root-hairs or upon the epi-
dermis of the root. Asparagin, one of
the most powerfully chemotactic com-
: pounds known, is present in large

Fic. 21. Invasion of leguminous roots by bacteria. quantities in the tissues of germinating
a, Cell from the integument of root of the pea with Bs
steam of tauisie sooges tha passes ea'aey Prants, and must necessarily escape
sroottaroftepea;atnnginpartctectsann, Wherever the cell-walls are injured.
Insidethe halt protoplasm mixed withbacteia whens ¢ #8 quite possible that this substance
rot ay Oth & thin aca upward. ~ acts: asia ‘bait’ to attract the bacteria
which would swarm into a torn root-
hair just as they do into a capillary filled with asparagin (Fig. 21,4). There
are reasons, too, for thinking that the plant facilitates the entrance of the
micro-organisms by a softening of the cell-membrane of the root-hairs.
This much is certain, that the bacteria are chemotactically attracted to the
plant, and that, nourished by the organic substances offered to them, they
multiply rapidly and force their way from the surface of the root inwards.
Here again by softening its cell-walls the plant seems to smooth their path,
and in thick columns the bacteria zoogloea* pushes on from cell to cell

* The so-called ‘ infection-thread,’ first observed penetrating the root-hairs by Marshall Ward
(see Phil. Trans. 1887, Vol. 178, p. 545).

BIOLOGY OF THE ROOT-NODULES 95

into the inner tissues of the root (Fig. 21,¢ & 4). And now the plant begins
visibly to react to the stimulus. Many of the cells of the root become
larger, and carbohydrates and asparagin are brought down from the upper
parts of the plant to be given unstintingly to the micro-organisms, whose
accelerated growth and multiplication soon become manifest in the fast
developing nodules. The root-nodules are nothing else than pure cultures
of bacteria maintained by the plant for its own ends. The microbes, which
grow at first at the expense of the tissues they live in, become before long
more independent and draw their nitrogen from the atmosphere. Carbon
they most likely obtain the whole summer through from the plant, which
probably also secretes the ferment by which the starch is changed into
assimilable sugar.

The root-nodules are now in full activity, the bacteria in the cells
absorbing nitrogen from the air in the intercellular spaces (63), and storing it
up in their own bodies. But this state of exuberant vitality does not last.
From overcrowding and other causes the multiplication of the bacteria
becomes less rapid and bacteroids (involution forms) appear, a sign that the
conditions of life are becoming less favourable. The plant asserts itself, and
begins to absorb the substance of the bacterial cells, conveying the nitrogen
from the root-nodules into the growing seeds. In the lupine at the time of
flowering the nodules contain 5-2 per cent. N, which then becomes gradually
less until, by the time the pods are ripe, only 1-7 per cent. is present. It is the
nodules alone which yield up their nitrogen in this way, the amount contained
in the other parts of the root remaining from first to last unchanged, namely
about 1-6 per cent. The precise way in which the leguminous plant absorbs
the bacterial cells is not clearly understood, but in all probability it dissolves
them by means of a peptonizing enzyme. Only a remnant of the bacteria
return to the soil when the root decays, the greater number are literally
eaten up by the plant. To call this symbiosis is certainly a misapplication
of the term*. The carbohydrates and asparagin that the plant offers the
bacteria in the beginning are nothing but a usurer’s loan, for in the shape of
valuable nitrogen they are demanded back again later on with heavy
interest. Looked at from this standpoint, the view that the leguminous plant
is parasitic on the bacteria no longer appears absurd. It is obliged to ‘ put
itself outside ’ its hosts, just as the fungus is obliged to enclose the algae in
the lichen thallus. But, whilst the lichen fungus is completely parasitic, the
leguminous plant is only partly so. It only derives its nitrogen from its host,
supplying its other needs (carbohydrates, mineral salts, &c.) in the same
way as all ordinary plants, from which it differs indeed only in being unable

* [It should be pointed out that the author's views of the relations between the organisms here
and in the Lichens are not those accepted by botanists generally. See, for example, Pfeffer,
Physiology of Plants, Vol. 1, Engl. ed., pp. 364 and 371.]

96 BACTERIA AND' THE NITROGEN. CYCLE

to assimilate nitrogen from the nitrates in the soil. The table (III) on
p. 91 shows this very clearly. In this semi-parasitic mode of life the
Leguminosae resemble the Rhinanthaceae, Thesium, and some other green
plants, of which the roots graft themselves on to the roots of adjacent
plants of other species. These function as hosts, but it is not known what
substances they yield up to their parasites.

Since it is evident that the soil of every field in which Leguminosae
grow, and probably indeed soil of all kinds, contains the bacteria of root-
nodules, attempts have naturally been made to obtain them direct from
the soil. Up till now these experiments have not been attended with
success, nor is it known whether the bacteria live and multiply in the soil, or
whether they exist merely in the form of spores (as yet unknown) which
are quickened into life by contact with the roots of leguminous plants.

But although it has been hitherto impossible to isolate the bacteria of
root-nodules from soil, Winogradsky has discovered another soil bacterium
which fixes atmospheric nitrogen (64). It has been named Clostridium
Pasteurianum, and belongs to the group of butyric ferments. It can be
isolated in a nutritive solution containing sugar (as a source of carbon) and
mineral salts, nitrates or other nitrogen compounds being of course rigidly
excluded. The sugar is broken up into butyric, acetic, and carbonic acid,
and hydrogen, besides some by-products of uncertain composition, and at
the same time nitrogen is taken from the atmosphere and stored up in the
body of the cells. The more sugar there is present (i.e. the more energetic
the fermentation), the greater is the amount of nitrogen taken up, as the
following figures show:

Dextrose in nutritive N. in nutritive N. in the crop
solution. solution. after fermentation.
Grammes. Milligrammes.
I fo) 3:0
2 {o) 2-9
3 ° 8-1
6 fo) 12-8

Possibly the nitrogen is fixed by nascent hydrogen, and ammonia
formed as the first product. The bacterium in pure culture is an anaerobic
actively motile bacillus which grows in slimy masses. It is considerably
different in appearance from the bacteria of root-nodules, becoming spindle-
shaped during sporulation (hence the name Clostridium) and, like other
butyric bacteria, it stains blue with iodine.

What the conditions are under which Clostridium Pasteurianum exists
in nature, and whence it obtains its carbon, is unknown. Should sugar be
a necessity it could not grow in unmanured soil, but might occur in situa-
tions where the processes of fermentation and putrefaction give rise to a
variety of organic compounds.

NITROGEN-FIXING BACTERIA 97

It is not known whether other butyric organisms are also able to fix
atmospheric nitrogen, but presumably other, perhaps prototrophic, bacteria
of the soil do so. It is highly probable that we shall find such in forests
where the earth is never ‘manured’ and where, notwithstanding, enormous
quantities of nitrogen are fixed and worked up into the living substance of
plants. But all researches in this direction must be received very critically,
for there can be no doubt that the idea now very common in agricultural
circles that a// soil bacteria can assimilate free nitrogen is quite incorrect.
Green and blue-green algae were formerly supposed to be able to do so,
but this opinion is now held to be incorrect (65), and although it is stated
that mould-fungi can fix free nitrogen the experiments on which the state-
ment is based did not take into consideration all the possible sources of
error. The time will perhaps come when ‘Nitragin’ will be replaced by
pure cultures of free-living, nitrogen-fixing bacteria, which will be ‘sown’ so
as to alternate with nitrogen-consuming crops.

FISCHER H

CHAPTER XI

BACTERIA AND THE CIRCULATION OF NITROGEN
(Continued)

2. The Liberation of Organic Nitrogen by Putrefaction, and its
Mineralization by the Nitrifying Bacteria.

ONE of the most conspicuous physiological differences between plants
and animals is the absence in plants of any xitrogen-containing excretions.

Nitrogen, once assimilated by the plant, is built up into proteids,
alkaloids, colouring-matters (chlorophyll, indigo), and other compounds, but
as long as the plant lives is not excreted. It is set into circulation again
only by the death and decay of the tissues, for the living plant can be used
as a source of nitrogen only by parasites and by plant-eating animals,

In the animal body nitrogen is present chiefly in the form of proteids
and their derivatives the albuminoids, e.g. mucin, glutin, keratin, elastin, as
well as other highly complex bodies such as lecithin, haemoglobin, chilin,
and nuclein. In the animal, as in the plant, the nitrogen of these substances
is set free at death, but, in addition, the animal, unlike the plant, gives off in
its secretions (milk) and excretions (dung, urine) a number of nitrogenous
compounds. It is to these substances, enriched still more by the nitrogen
contained in straw, that stable-manure owes its high value as a fertilizer for
plants.

But the nitrogen in fresh stable-manure is in a form that is useless to
plants; they cannot assimilate it. In the urine of herbivora nitrogen is
present mainly as hippuric acid, in human urine mainly as urea and uric
acid. In the excrements it is contained in the remnants of undigested
nitrogenous food, and the products of decomposition set up by the bacteria
of the intestine. These products include indol, skatol, leucin, tyrosin, and
many simpler nitrogen compounds even down to ammonia. But none of
these, not even ammonia, are directly available as nutritive materials for
green plants. Not until the nitrogen has been removed from the organic
molecule by the process of putrefaction, and has been united to a mineral
base by the process of nitrification, can it be taken up by the green plant.

PUTREFACTION 99

Through the tissues of green plants all organic nitrogen must, however,
sooner or later, circulate.

Putrefaction is a purely biochemical process, and can only take place
when the fundamental conditions for all vital action are fulfilled. Ifthe
temperature sink below a certain point, organic substances cannot putrefy,
as was well shown by the frozen Siberian mammoths. When discovered,
their flesh was so little changed that it was eaten by the hunters’ dogs ; yet
it must have lain in Nature’s refrigators for countless centuries. Absence
of moisture acts in the same way as absence of warmth. Dry meat does
not putrefy. Dryness and a low temperature together will preserve organic
matter for any length of time, as is well illustrated by the undecayed
yet unembalmed bodies that have been kept for centuries in the vaults of
certain churches. Other means of preservation by chemical and physical
agencies have been already referred to, but in all methods the funda-
mental principle is the same, namely, to create such conditions that bacteria
cannot live; for putrefaction—the splitting-up of the nitrogenous con-
stituents of organic matter—is the work of bacteria, and of bacteria alone.

The flesh of fruits, being poor in nitrogen and rich in organic acids, is
not attacked by bacteria. The rotting of pears, grapes, oranges, and other
fruits of a like character, is brought about principally by mould-fungi (66)
(Penicillium, Mucor, Botrytis).

The decomposition of dead animal bodies, of vegetable tissues, or of
substances like stable-manure, is far from being a simple putrefactive process.
Side by side with the disintegration of nitrogenous bodies there are going
on a number of fermentative changes by which oz-xitrogenous compounds
are being broken up, besides nitrification and other biochemical processes.
For this reason it is always difficult, and often impossible, to determine the
respective parts played by the different species of bacteria. Putrefactive
processes are going on in all places where nitrogenous organic matter is
under suitable conditions of temperature and moisture, manure heaps for
instance, cesspools, the muddy bottoms of lakes and rivers, and the ocean.
floor.

Proteids are split up by putrefaction into a large number of simpler
compounds both nitrogenous and non-nitrogenous. The substances thus
produced are precisely similar to those resulting from the artificial decom-
position of proteids by fusion with caustic potash, or boiling with hydro-
chloric acid or barium hydrate. Five groups may be distinguished :

1. Albumoses and pepitones: soluble diffusible bodies closely resembling albumen.
They are produced by the action on albumen of bacterial enzymes similar to the enzymes
(pepsin and pancreatin) which give rise to peptones in the digestive tract of man.

2. Aromatic compounds: among others indol and skatol which give the characteristic
odour to human excrement; also some non-nitrogenous substances such as phenol,
phenylacetic acid, and phenylpropionic acid.

3. Amido compounds, all nitrogenous ; leucin, tyrosin, aspartic acid, glycocol.

Hg

100 BACTERIA AND THE NITROGEN CYCLE

4. Fatty and aromatic acids, all non-nitrogenous and therefore having no part in the
circulation of nitrogen ; acetic, butyric, succinic, and valerianic acids.

5. Jnorganic end-products of putrefaction: free nitrogen, ammonia, free hydrogen,

methane, carbonic acid, methylmercaptan, sulphuretted hydrogen. It is probable also,
but*not certain, that phosphuretted hydrogen is formed and is oxidized at once by the
free oxygen of the atmosphere.
Most of these substances are formed also by the chemical decomposition of
proteids, but there is a sixth group which may be termed specific putrefac-
tive products. These are the so-called ptomaines or putrefactive alkaloids
(67). They are nitrogenous compounds (amine-bases), but although a large
number have been described they are, in consequence of the difficulty of
preparing them, still only imperfectly known. From putrid flesh (mammalian,
including human), fish, and gelatine Brieger isolated neuridine (C, Hy, No),
trimethylamine (C,H,N), cadaverine (pentamethylendiamine, C; H,, N,), and
putrescine, a diamine of the methylene series (C,H,,N,). These are all of
them either not poisonous or only poisonous in large doses. Other ptomaines,
derived from putrid foods of various kinds (sausages, cheese), are highly
toxic (Plomatropine, Tyrotoxine). The name toxine was formerly used for
the amine bases only, but it is now applied indiscriminately to all bacterial
poisons irrespectively of their chemical constitution. (See also Ch. XVII.)

As far as the circulation of nitrogen is concerned, we have to consider
only the end-products of putrefaction, free nitrogen and ammonia. All
the more complex intermediate bodies are broken down finally to these.
Leucin, for instance, splits up into valeric acid, ammonia, carbonic acid, and
free hydrogen. Tyrosin yields by aerobic putrefaction hydroparacumaric acid,
paraoxyphenylacetic acid, paracresol, phenol, ammonia, and carbonic acid.
By anaerobic putrefaction it gives rise to indol, carbonic acid, and hydrogen.

The phenomena of putrefaction are so complicated that we do not
know all of the compounds that arise during the process. The course it
runs is, however, greatly modified by the presence of oxygen (68).

This list of putrefactive products is far from being complete, for even
the qualitative investigation of the processes is still unfinished ; quantitative
analyses are at present impossible. We do not know for instance what
determines the predominance of one or the other intermediate product.
The effects of the presence of oxygen are somewhat better understood (68).
If air have free access, putrefaction may go on without any odour at
all, the evil-smelling gases (NH, and SH, for example) being oxidized at
once to form nitrates and sulphates. Aerobic bacteria too, such as the
nitre and sulphur bacteria, bring about this mineralization of organic
nitrogen. Moreover, when air is circulating freely, there is no accumulation
of intermediate products such as skatol or indol. To this kind of aerobic
decomposition, proceeding without offensive smell, the term ‘decay’ may
be applied as distinguished from ‘ putrefaction.’ It occurs on the surface of
manure-heaps, on the outer surface of carcases, and in well-ventilated soil.

PUTREFACTION IOI

In anaerobic decomposition (putrefaction proper), as in anaerobic fer-
mentation, the organic molecules are at first only partly disintegrated, inter-
mediate products such as leucine, tyrosine, skatol, and indol being formed.
In the absence of air these accumulate, and hence it is that the putrefaction
going on in the mud of ponds or ditches, or inside carcases, is accompanied
by such evil odours.

Although the details of the process vary considerably according to the
presence or absence of air, the ultimate products of putrefaction are in both
cases the same: namely, free nitrogen, free hydrogen, ammonia, methane,
carbonic acid, and sulphuretted hydrogen. These also are the end-results of
the disintegration of the human body.

The decomposition of plant tissues, poor in nitrogen but containing
large quantities of cellulose, gives rise to humin compounds. There can be
no doubt that it is a biochemical process, but the nature and functions of
the bacteria concerned in it have not yet been carefully studied (69).

One of the bacteria which was formerly looked upon as a putrefactive
organism par excellence was the Bacterium termo. Cohn described under this
name a short, oval, actively motile bacillus, slightly fluorescent in cultures
(Fig. 22, a). The species is, however, no longer to be identified, for his
description would apply to several common forms. Asa matter of fact there
are many bacteria going by this name which is properly only applicable as
a collective designation for motile bacteria in putrescible substrata.

In the rich bacterial flora of a putrescent fluid (70) two distinct biological
groups must be distinguished, the saprogenic and the saprophile. To desig-
nate all bacteria living upon dead organic matter as saprophytes is insuffi-
cient. As we have already seen, the saprogenic bacteria are able, by the
chemical ferments they secrete, to break up the molecules of proteids. The
saprophile organisms cannot do this, but they find a rich supply of nutri-
ment in the products thus formed. <A large number of metatrophic species
are endowed with this faculty, including some pathogenic forms (cholera and
typhoid), and water bacteria like Spirillum undula. The sulphur bacteria
too must be called saprophile where they form a coating over the rotting
vegetation under water, and the nitrifying organisms also when they oxidize
the NH, evolved from decaying organic matter.

The bacteria of the other division, the true saprogenic organisms,
include very numerous species, some of which are much better known than
others (e.g. Hauser’s B. (Proteus vulgaris), but all of which possess the
power of breaking up the complex proteid molecule more or less quickly
into simpler groups. With the exception of certain toxines in a few cases,
specific products which would serve as classificatory characters are not
known. Indol, for example, the production of which was once thought to
be characteristic of the cholera germ, is produced by all the cholera-like
vibrios, as well as by the &. cold and other forms (Fig. 22, 4). Near to

102 BACTERIA AND THE NITROGEN CYCLE

these pathological bacteria with saprogenic properties many of the fluorescent
and photogenic bacteria would have their place. Bacillus fluorescens lique-
faciens, an actively motile aquatic species, breaks down albumen into
peptones, fatty acids, and other compounds. One of the bacteria of the
intestine, described formerly as Bacillus putrificus colt, yields peptones,
indol, skatol, amides, and ammonia. Similar to this species in its action
on albumens are the toxine-forming B. vulgaris (Proteus vulgaris) and its
allies. B. vulgaris makes its appearance almost always in meat infusions
exposed to the air. It is a slender rod 1-5—4 long by 0-5 » thick, with
a tendency to form chains. It is peritrichous and actively motile (Fig. 22).
Closely related to it are a number of forms (Kurth’s, B. Zopfiz, and others),

Fic. 22. Putrefactive bacteria. a, Bactrillum pseudolermo, corresponds most closely to the old Bacterium teria
of Cohn. 4, Cholera-like vibrio from putrid water. c, Bacillus ureae, the commonest cause of urinous fermentation,
probably identical with Pasteur’s Micrococcus uveae. d-h, Bactridium Proteus (Bacillus Proteus, Bacterium
Zopfit, Proteus vulgaris, &c.). d, peritrichous rod, cobweb-like growth (zoogloea) on gelatine (50 diam.); /,
portion of same more highly magnified (300 diam.); g, tree-like growth on gelatine with thickenings; 4, part of
same magnified 300 diam, to show the structure of thickenings which consist of closely wound threads. Magn. a-d
about 1500, ¢ and g 50, fand & 300.

all placed at one time by Hauser in one genus Proteus, so called on account
of the manifold shapes of their colonies on gelatine*. These colonies are
ramified zoogloeae having under alow power much the appearance of fungus
mycelia, covering the gelatine with a filamentous growth. Each filament
consists of chains of bacteria united by a jelly-like substance (Fig. 22, d-h).-

These apparently specific putrefactive bacteria must not be looked
upon as an exclusive group, such as the sulphur or nitrifying organisms,
whose metabolism is of a circumscribed kind and which can only thrive
when its peculiar conditions can be fulfilled. The various species of Proteus,
for instance, have zymogenic as well as saprogenic properties. They can
ferment carbohydrates, producing gas and acid during the process, as does
also Bacillus coli communis.

* See Annals of Botany, Vol. xiii, 1899, p. 198, for an account of Proteus.

URINOUS FERMENTATION 103

Very careful chemical investigations on pure cultures will be necessary
before the chaos of phenomena presented by the putrefactive bacteria can
be arranged in something like order. In view of the present state of our
knowledge of these organisms, it will be best perhaps to make the definition
of a putrefactive bacterium rather wide, and include all bacteria with
saprogenic properties, whether they possess other pleotrophic properties
(e.g. zymogenic) or not. It is certain that many bacteria, almost all the
cocci for instance, and many pigment bacteria, are destitute of saprogenic
power. Saprogenic bacteria are able to break up albuminous bodies of
every kind and of every morphological character, whatever organs of the
body they be derived from. The influence of these saprogenic properties in
the production of disease will be considered later on.

Just as the Bacterium termo was formerly regarded as the only cause
of putrefaction, so was the Micrococcus ureae discovered by Pasteur regarded
as the specific microbe of urinous fermentation.

Healthy human urine, sterile and acid when passed, becomes sooner
or later alkaline. The urea has been converted by addition of water into
carbonate of ammonia:

CO (NH,),+2H,O = CO, (NH,),.

A similar decomposition is undergone by the uric acid and by the
hippuric acid in the urine of herbivores. It is caused bya bacterial ferment
and, as with the putrefactive organisms, it was formerly thought that one
species alone was at work. This was the Micrococcus ureae of Pasteur,
a short, almost spherical, non-motile cell generally growing in pairs (Fig. 22)
or little chains (71 and 112). We know now, however, that many different
kinds of bacteria are able to cause the ammoniacal fermentation of urine.
No less than sixty species are said to occur in manure and sewage, among
others the B. vulgaris. On the other hand, B. subtilis, B. anthracis, B. typhi,
V. cholerae, the pus cocci, and many saprogenic forms are unable to split up
urea. Conversely, the urea bacteria are incapable of decomposing albu-
minous bodies, which is not surprising, seeing how different the two processes
are chemically.

The resting cells of the urea bacteria’ are everywhere present in urine,
manure, earth, air, and dust. All urine that is left exposed to the air is
sooner or later attacked by them, and its nitrogenous constituents are
converted into ammonium carbonate. In this way the path is prepared for
the nitrifying organisms, by which the nitrogen is made ready for renewed
circulation through the tissues of green plants. :

The amount of nitrogen treated in this way by bacteria is enormous.
The sewers of Leipzig discharge every day, in the form of urine, no less
than 8,400 Ib. of nitrogen.

The two great processes, putrefaction and ammoniacal fermentation, are
going on in nature uninterruptedly. In the soil, in the mud of lakes and

104 BACTERIA AND THE NITROGEN CYCLE

rivers, in all the sewers and refuse heaps of human communities, the two
classes of micro-organisms are incessantly at work, sometimes alone, some-
times together, and the greater part of all organic nitrogen is sooner or later
liberated in the form of ammonia, together with a small amount of free
nitrogen*. The free nitrogen is at once utilisable by the root-nodule
organisms, and by other bacteria in the soil. The ammonia nitrogen, on the
other hand, must be ‘ mineralized’ before it can be used by green plants,
must be oxidized and combined with a base to form a nitric salt ft. This
change, known as nitrification, was formerly thought to be a purely chemical
phenomenon, the oxidation being presumably effected by the free oxygen
of the atmosphere. This view is now known to be incorrect, evidence
having gradually accumulated which proves irrefutably the biochemical
nature of the process. The bacteria to whose activity it is due defied for
many years all attempts at isolation, until Winogradsky, the Russian in-
vestigator, succeeded in separating them from the soil and obtaining pure
cultures (72). His researches too brought to light the unique fact that the
nitrifying bacteria are absolutely prototrophic in their mode of life—and
we owe to him glimpses into the conditions of existence among the lowest
organisms.

The nitrifying bacteria are everywhere present in the soil of our
gardens, fields and meadows, and in the virgin earth of the mountain sides
and plains, untiringly at work preparing the food for plants. They have
been cultivated by us unconsciously for centuries in the ‘saltpetre beds,’
where putrescible material (manure and other animal remains—skins, horns,
glue &c.) is mixed with lime and allowed to ferment, and there can be
little doubt that the Chilian nitre owes its origin to the action of nitrifying
bacteria in a recent geological period. It probably arose from the washing
down of saltpetre that had accumulated around putrefying organisms in the
rainless regions of the coast.

The isolation of the nitrifying bacteria is not difficult; but rather
different methods are necessary from those used for other bacteria. Highly
nutritious culture media are useless, for the nitrifying bacteria are proto-
trophic in the strictest sense of the word. They may be cultivated ex
masse by inoculating with a little garden earth the following solution :

Grammes.
Water . . ’ : . i ‘ 1000+
Potassium phosphate (dibasic) . ‘ 2
Magnesium sulphate . s ‘ i 3
Soda (or Mg CQ) - ‘ ‘ , 5
Sodium chloride. . . . 5

* The nitrogen of crop residues and of the ploughed-in fallow undergoes just the same changes.
+ This applies of course also to the sulphate of ammonia from gas-works, so largely used as
manure.

NITRATE AND NITRITE BACTERIA 105

To these must be added from 20 to 50 mgr. of sulphate of ammonia that
should be renewed in larger doses (1 gr.) at intervals of about a week.
The only source of nitrogen is the sulphate of ammonia; the carbon is
obtained from the CO, of the atmosphere, not from the sodium or mag-
nesium carbonate, which are added only to neutralize the nitric and nitrous
acid that arise from the activity of the bacteria. The sodium chloride
benefits them in some way at present unknown.

The nitrogen of the ammonia is not converted directly into nitric acid,
as was formerly supposed, but oxidized first of all to nitrous acid and then
to nitric. There are two distinct reactions going on, the formation of
nitrites from ammonia, and of nitrates from nitrites. Each is performed by
specific bacteria, which are always present together, so that the compounds
formed by the one kind are immediately taken up by the other and the
end-products alone appear in the soil.

Only in pure cultures is it possible to follow each process separately.
Both are very slow; in a sixteen days’ old culture, 60 mgr. of (NH,),SO,
were converted into nitrous acid daily, and in a six weeks’ old culture 64 mgr.
potassium nitrite daily into nitric acid. Probably, under natural conditions,
the process is more rapid.

To isolate the nitrite from the nitrate .
bacteria, Winogradsky used the customary ¥ wer,
methods of plate culture, substituting colloid ° "AR,
silica for the gelatine *. Very thoroughly a , 2

washed agar may also be used. For the
nitrite organism (NH,),5 O, is added to the
medium, for the nitrate bacteria K N O,. Fic. 23. Nitrifying bacteria (trom Wino-

er : - : dsky). a, Wit ;
The nitrite bacteria are divided by Wino- Fitrite Lactenlais tem Eurleh. & Netooer:

gradsky into two biological genera, Nitroso- Jan eee a ee tiem
coccus and Nitrosomonas, Nitrosococcus is "%" **® Me™ 100.

a non-motile spherical cell (3u in diameter,

and less) found in soil from South America and Australia. Nitrosomonas
is a very short ellipsoidal, motile form, of which two species in particular
may be mentioned. Nitrosomonas europea (Fig. 23, a), ubiquitous in the
soil in Europe, Africa, and Japan, is from o-Qu to 1m wide, and 1-2, to
1-8 long, with a short cilium. MV. javanensis from Buitenzorg (Fig. 23, 4)
is almost spherical (o-5-o-6 u), with a cilium 30 long. This is the longest
cilium known among bacteria. Spores have not been seen. The nitrite
bacteria render the culture turbid as long as they are motile. They also
form zoogloeae, and if crystals of Mg CO, be added the bacteria collect
on them in slimy masses and eat into the mineral just as lichens eat into

a stone.

* For details respecting the composition of the media, see works cited in Notes (pp. 169-184).

106 BACTERIA AND THE NITROGEN CYCLE

The nitrate bacteria (Nitrobacter, Fig. 23, c) are minute non-motile
rods (0-5 4 by 0-25) that grow without causing turbidity in the medium.
They form delicate pellicles on the floor and sides of the culture glass.
Spores are unknown.

As might be expected in the case of organisms with oxidizing functions,
all the nitrifying bacteria are aerobic. They require no light, and yet
in spite of this are able to assimilate the CO, of the atmosphere. In
three experiments, where the nutritive solution contained originally 6 mgr.
CO, in the form of MgCO,, the cultures after seven weeks’ growth showed
37:6 mgr., 26 mgr., and 17-5 mgr. CO, respectively. This was subsequently
proved by Godlewski (73) to have been absorbed entirely from the air
without light and without chlorophyll. As already mentioned, the nitrogen
was derived from the ammonia, or from the nitrous acid. A little nitrogen
is even set free.

The energy necessary for the processes of life is gained by the nitrifying
bacteria from the oxidation of the ammonia, or the nitrous acid. The
materials from which they build up their cells are therefore inorganic
compounds of the very simplest character, carbon dioxide and ammonia or
nitrous acid, with a few mineral salts. They are thus prototrophic in the
strictest sense of the word, for a simpler synthesis of proteids than theirs
is scarcely conceivable.

Facts have recently come to light which show that green plants do
not get the full benefit of all the nitrates formed in the soil by the nitrify-
ing bacteria (74). Nitrate-reducing bacteria have been discovered which
destroy that which the nitrifiers have built up. Several species of these
denitrifying organisms have been isolated from manure. They grow on
a medium containing 0-3 per cent. Na NO,, sugar, and the necessary salts.
Of the nitrogen offered to them, 82-7 per cent. is set free (in some species
as much as 99 per cent.), the rest being used up in their cell-substance.

Although it is probable that the importance of these denitrifying
organisms in agriculture is very small, it cannot be denied that they occur,
and they certainly mar to some extent the clearness of our conception of
the processes by which nitrogen circulates through organic creation.

Just as the nitrifying organisms have their converse in the denitrifying,
so have the sulphur bacteria theirs in the desulphurizing species. A spiril-
lum (S. deszlfuricans) has been isolated from ditches and sewers, which sets
free the SH, from sylphates. The complete life-history of this organism is
not known (75).

There can be little doubt that many other biochemical processes are
going on in nature besides those we have been considering, and mineralogical
chemistry will have to turn its attention to bacteria. Possibly we shall
even find prototrophic species which attack silicates. The reader may be
left to ponder further on the subject.

CHAPTER XII
THE CIRCULATION OF CARBON DIOXIDE IN NATURE

1. Intreduction ; Organized Ferments and Enzymes; Races of Ferment
Organisms; Fermentation of Alcohol and of Acids; Optical Activity
and Fermentation.

THE ultimate source, from which directly or indirectly all organisms
draw their carbon, is the carbonic acid of the atmosphere. In the circula-
tion of this gas bacteria play a not less important part than they do in the
circulation of nitrogen. Animals, it is well known, cannot assimilate CO, ;
they are dependent for their carbon entirely upon plants. Among these again
only the chlorophyll-bearing forms and the red, brown, and blue-green algae
are able, with the energy of the absorbed sunlight, to fix the atmospheric
CO,*. From it the plant builds up the organic compounds, with or without
nitrogen, that constitute the physical basis of all life upon the earth.
Since this assimilating and fixing of carbonic acid is going on without
ceasing, there is an evident necessity, if terrestrial life is to endure, for the
continual return of carbon in the form of CO, to the atmosphere, and such
restitution does as a matter of fact take place. A certain amount is given
back at once by the plants themselves in the process of respiration, and
another portion is being continually poured out into the air in the breath
of man and animals. All the remaining carbon fixed in the tissues of
plants and animals is finally liberated at the death of the organism by the
activity of bacteria and other microbes. Those tissue elements which
contain nitrogen as well as carbon undergo putrefaction, and the carbon
escapes as CO, The innumerable non-nitrogenous constituents on the
other hand—carbohydrates, glucosides, alcohols, organic acids and fats—are

* The prototrophic saltpetre bacteria constitute the sole and only exception to this rule.

108 BACTERIA AND THE CARBON DIOXIDE CYCLE

not putrescible. They are broken up by ‘fermentation,’ carbonic acid
being again the final shape in which the carbon is set free*.

In nature, fermentative organisms are more widely distributed even
than putrefactive. With these they labour to disintegrate and remove dead
animals and plants. But in addition to being indispensable scavengers
they are of prime importance to man in numerous industries—in the pre-
paration of butter-milk, cheese, ‘sauerkraut,’ and bread, and in the
manufacture of alcohol. Some species, on the other hand, are dangerous
enemies to the dairyman and the brewer, and in many technical processes
bacteria play a part useful or injurious as the case may be (77).

There is a diversity of opinion as to what shall be understood by the
word fermentation. Some would apply it to every kind of chemical change
arising from the activity of micro-fungi, and would include putrefaction,
nitrification, and the oxidation of SH, by the thiobacteria, in short all
biochemical processes. From this it is but a short step to regard human
life itself as a fermentation. In the author’s opinion, a more restricted
application is desirable, both in view of the history of the word and as
inducing to clearness. The word ‘ fermentation’ will therefore be used here
to designate the decomposition by means of particular living organisms—the
Sermentation organisms—of non-nitrogenous organic compounds, particularly
carbohydrates.

Given fermentable substances, the necessary conditions for fermentation
are a supply of food-stuffs (above all a special source of nitrogen) for the
micro-organisms and, as in putrefaction, moisture and a suitable tempera-
ture. Even before the nature of fermentation was well understood, we knew
that in addition to the fermentable substance a certain something, a ferment,
was necessary for the production of the phenomenon, and it was thought by
many to be a chemical body. But the classical researches of Pasteur (78)
showed that every fermentation was the work of some living organism, a
fermentum vivum, and not ofa chemical ferment or exzyme. We have there-
fore to distinguish between living or organized ferments and unorganized
chemical ferments or enzymes. The enzymes (79) are substances secreted
by cells (e.g. pancreatin, pepsin, ptyalin), and are capable, even_in the —
absence of the cells that_produce i ific chemical changes
in certain organic substances. The organized ferments are living cells.
They also are able to set up chemical changes in organic bodies, but only so
long as they, the cells, are alive.

Organized ferments and enzymes have much in common. In each case

* In qualification of this somewhat broad statement it should be mentioned that very little is
known as yet concerning the biochemical decomposition of fats (76). Preliminary experiments seem
to have shown that certain bacteria (cholera, typhoid, B. pyocyaneus) are able to split olive-oil or
beef-fat into glycerine and fatty acids, thus making them accessible to fermentation.

FERMENTS AND ENZYMES 109

the chemical change produced is specific, is of one kind, and of one kind
only. In each case too it is effected apparently without any expenditure of
energy, although to produce similar changes in our laboratories we have to
resort to very powerful means such as the employment of high tempera-
tures, intense chemical reactions, and, in many cases, we cannot reproduce
the changes at all. An instance will make these two points clearer. In
plants, starch is converted into sugar by the enzyme diastase without
apparent effort, whilst in the laboratory we can only cause this change
by boiling with mineral acids. But, whereas boiling with acids will
effect very many other changes, diastase is powerless to perform any but
the one alteration of starch into sugar. We can convert sugar into lactic
acid by heating with alkalies; the lactic bacteria can do the same by their
fermentative power, but are unable to produce butyric acid. Some of the
biochemical processes of micro-organisms, the conversion of sugar into
alcohol for instance, cannot be exactly imitated by chemical means.

Finally, neither enzyme nor organic ferment disappears in effecting
such changes, as a reagent disappears in a chemical reaction. There is no
definite relation between the quantity of ferment used and the quantity
of substance fermented, an enzyme like pepsin, or a yeast, being able to
ferment a hundred or a thousand times its own weight of fermentable sub-
stance. ;

The chief and fundamental difference between enzymes and organized
ferments lies of course in the fact that the latter are living beings able to
grow and multiply, increasing the more rapidly the more food and ferment-
able material are placed at their disposal. This the enzymes cannot do, for
they are lifeless chemical compounds, although they have, it is true, many
points of resemblance to the proteids. They are extremely unstable com-
pounds, and when dissolved in water their fermentative power is destroyed
by a short exposure to 50°—60°C., just the lethal temperature of most
sporeless micro-organisms. But towards protoplasm poisons their behaviour
is quite different from that of the organized ferments. Arsenious acid,
phenol, salicylic acid, ether, and chloroform in concentrations which paralyze
the organized ferments leave the enzymes unaffected. Chloroform however
seems to affect some enzymes in a short time.

As regards the nature of the chemical change produced by enzymes
and organic ferments respectively, there is a broad and fundamental difference
between the two. The enzymes exercise solely a hydrolytic influence. That
is to say, they cause the molecules of insoluble compounds to take up
water and to separate into less complex molecules of a different constitution,
the resultant substances being soluble in water. Diastase, for instance, con-
verts one molecule of starch into one molecule of grape sugar:

C,H,,O;+ H,O = CgHj, Og

IIo BACTERIA AND THE CARBON DIOXIDE CYCLE

In the same way, invertase, an enzyme secreted by yeast cells, converts
one molecule of cane sugar into one molecule of glucose and one of fructose :

Cy, Hy,011, + HO = Cg Hy, Og + Cy Hy. Og.

The peptic enzyme (pepsin) of the stomach changes insoluble proteids
into soluble peptones and albumoses. Ad/ these reactions are complete im
themselves, and there are no by-products such as CO, or other gases. The
action of organized ferments is quite different. By them the highly complex
molecules of organic compounds are split up, and numerous substances of a
totally different character arise, generally with the evolution of gases and
other by-products. For this reason it is impossible to represent the reaction
by a simple chemical equation. Ordinary alcoholic fermentation, for in-
stance, cannot be written

C,H,,0, = 2C,H,O+2CO, ;
nor butyric fermentation,
C,H,,0, = C,H,0,+2C0O,+4H,

for, as we shall see presently, numerous other compounds are formed.

Carbonic acid being evolved in almost all instances, it is customary to
designate each process by the name of some other conspicuous product, as
the preceding examples show. <A by-product of one kind of fermentation
may be the chief product of another kind. Acetic acid, for instance, is a
by-product of alcoholic fermentation, but the principal substance formed in
vinegar fermentation.

The organized ferments are often regarded as comprising three -
classes of micro-organisms,—yeasts, bacteria, and moulds. The last-
named, however, are of rare occurrence, e.g. the mucor yeast that causes
the decomposition of wine. The so-called ‘ aspergillus yeast,’ used in China
and Japan in the preparation of soy and saké, works only by means of an
enzyme that it secretes, the alcohol being produced, not by the aspergillus,
but by a true yeast. Similarly in ragz, the ‘ yeast’ of the arrack industry,
there are present both a true yeast and a mucorine fungus (R/Azzopus Oryzae).
The function of this latter is to ‘ invert’ the rice starch and make it accessible
to the yeast (80).

Mould fungi are sometimes employed on an industrial scale in the
preparation of citric acid (81).

The discrimination of the various species of fermentative organisms is
of very recent date. When Pasteur first showed that fermentation was the
work of living cells, we had not the technical means which we now possess
of distinguishing closely related. species from each other. It was sufficient
to assume that every fermentation had its own peculiar fungus, a Bacterium
aceticum in vinegar fermentation, a B. butyricum in butyric fermentation,
and so forth. In the same way it was not until the publication of Hansen’s

RACES AND BREEDS OF BACTERIA III

revolutionary investigations on yeasts that many different species of Saccharo-
myces were recognized. The Saccharomyces cerevisiae of beer-wort, and
the S. ellipsoideus of vinous fermentation, and a few others, were the only
species known. To-day the species S. cerevisiae is divided into hundreds
of ‘races’ or varieties, and the same has happened to the wine ferments. In
these technical processes, some of which are as old as human culture itself,
changes have been going on for thousands of years similar to those changes
we have caused artificially and intentionally in our domesticated plants
and animals. But while we have no difficulty now in distinguishing from
one another the innumerable kinds of cultivated plants, the determination
of racial characters among micro-organisms is a task of great difficulty.
Morphological data are almost useless, and we have to fall back upon physio-
logical peculiarities such as the degree of warmth required (different optima),
the nature and amount of by-products, or the specific fermentative power.

Fic. 24. Fermentation bacteria. a-c, acetic bacteria trom E. Chr. Hansen. a, Bactllus aceti ; 6, B. Pas-
teurianus, ¢ B. Kiitzinglanus; d, B. acid? lacticz, the commonest cause of lactic fermentation ; ¢, Clostridium
butyricum, an anaerobic butyric ferment, ‘ granulose’ bearing ; to the right spure in spindle-shaped rod. /, Plec-
iridium paludosum, anaerobic ferment from marsh water, corresponds in form to the methane bacteria, and to
some of the butyric bacteria. Magn. a~/ 1000.

Moreover, it must not be forgotten that new varieties are continually
arising, old ones dying out and being replaced by others with recently
acquired powers. The shortness of the time necessary for the production
of a new race of organisms is shown by such plants as the chrysanthemum
and the potato. Although the culiure of the potato is only two or three
hundred years old, there are no less than five hundred varieties in exis-
tence, distinguished by shape and colour, by their flavour, by the relative
amount of albumen and starch, and other points. Among bacteria, too,
there are forms that, like the yeasts, have been cultivated from time
immemorial—in cheese-making, for instance, and in vinegar fermentation.
These are now cultivated in pure form in our laboratories.

The difficulty of classification among micro-organisms is further
increased by the fact that many morphologically different species possess

112 BACTERIA AND THE CARBON DIOXIDE CYCLE

similar zymogenic powers. There are a dozen different species of bacteria
that produce butyric acid and lactic acid. And, inasmuch as the descrip-
tions given are often far from exact, the student of these species finds himself
lost in a pathless forest of forms. For this reason it will be impossible here
to describe more than a few species.

Some of the fermentation bacteria can acquire pathogenic properties.
Two of the butyric ferments found in soil are known to be the cause of
disease, Bacillus Chauvoei giving rise to ‘ quarter-evil’ in cattle, and another
form originating malignant oedema. The notorious B. coli communis is
a ferment organism in so far that it splits up grape sugar into lactic acid,
succinic acid, and ethyl and propyl alcohol, carbonic dioxide arising in the
process. By far the greater number of ferment organisms are, however,
harmless, and, considering the millions we take inside us every day in milk,
cheese, and other food, it is satisfactory to know this.

The chemistry of fermentation is clearly understood only in those cases
where the process is oxidatory. The acetic fermentation belongs to this
category, the alcohol being combined with the oxygen of the air to form
aldehyde and water, the aldehyde being further oxidized to acetic acid, and
this again, if the process be allowed to go on, burnt up to carbonic acid and
water. The changes may be represented by equations thus :

CH,CH,HO+0 =CH,COH+H,O
CH,COH +40 =CH,COOH
CH,COOH +40 = 2CO,+2H,O

This fermentation is evidently allied to the respiratory process, and
the similar oxidatory processes of the nitrifying and sulphur bacteria. It
differs from most fermentative changes in the small number of resultant
substances, but is, of course, just as much a phase in the circulation of
carbon as any of the more complex processes, where large numbers of by-
products arise and obscure our view (see Chap. XIV).

Of the fermentations of monacid alcohols only the acetic fermentation
of ethyl alcohol is of industrial importance (82). Alcoholic liquids, such as
beer or wine, if left exposed to the air in a warm place acquire a strongly acid
reaction, and become covered with a white skin or pellicle. This consists
of the ‘ vinegar bacteria, which slowly oxidize the alcohol to acetic acid,
and finally to CO, and water. Sometimes the pellicle consists not of
bacteria but of a yeast (A/Zycoderma aceti), which transforms the alcohol at
once into CO, and water without the intermediate stage of acetic acid.

The old form, Bacillus aceti, has been divided by Hansen into three
different species, B. acett, B. Pasteurianus, and B. Kiitzingianus (Fig. 24).
All three are morphologically very similar, non-motile medium-sized rods
often growing out into chains, of which the pellicle mainly consists. They
are unable to grow in fluids containing more than 14 per cent. of alcohol.

ACETIC FERMENTATION 113

Temperature maxima, and some fine differences of shape, distinguish the
three species from one another. J. ace¢d is stained entirely yellow by iodine,
but in the two other species the gelatinous mass that holds the cells together
in the pellicle takes on a blue tint, the cell remaining yellow. Whether
a carbohydrate is present is doubtful, and must remain so as long as we are
ignorant of the composition of the cell membrane from whose outer layers,
by inhibition of water, the jelly is produced. Cellulose is said not to be
present.

The optimum temperature for acetic fermentation is 34°C., the maxi-
mum 42°, the minimum 4°—7°. Approach to the maximum gives rise in
all three species to involution forms (Fig. 14, ¢ and d, p. 27). The borders
of neighbouring cells become indistinct, the cells themselves swell up to pear-
or spindle-shaped bodies, and short branches arise on the filaments. Even
at the optimal temperature involution forms arise as soon as acetic acid
begins to accumulate, and when about 14 per cent. is present all growth
stops, and the bacteria die. As a concrete example (83) of the activity
of these bacteria it may be mentioned that B. Pasteurianus in 125 c.c. of
lager beer (3-7 per cent. by volume of alcohol) formed in seven days, at
34° C., 4-2 grm. of acetic acid. The alcohol had disappeared, and the acetic
acid was finally oxidized to CO, and water. In a parallel culture, after
twenty-one days, only o-7 grm. acid was present. In the manufacture of
acetic acid the process must, of course, be stopped at the proper time in
order to prevent loss of acid by further oxidation.

In vinegar factories the so-called vinegar plant consists of a zoogloea
mass of acetic bacteria, of which there are doubtless many races.

The usual methods of vinegar-making all aim at bringing the alcoholic
fluid as much as possible into contact with the air, either by running it into
large vats or by letting it flow over wood-shavings in barrels. These
methods originated at a time when the process was supposed to be a purely
chemical oxidation effected by the air. But the bacteria lying upon the
surface of the shavings are the cause of the change; the free access of air
promoting it only because the vitality of the bacteria is increased thereby.

Any alcoholic fluids, such as cider, dilute brandy, or wine, can be used
to make vinegar, but the taste varies according to the nature of those con-
stituents of the liquids that remain unaltered by the process.

As a by-product, acetic acid arises in many other fermentations, the
vinous, the lactic, and the butyric, for example.

In fluids containing no alcohol, however nutritious these may be, the
acetic bacteria cannot thrive. They can make use of ammonia salts as
a source of nitrogen, but obtain this element in the liquids used for vinegar-
making from proteid substances. Whether the alcohol is used for respira-
tion only, or whether its carbon is in part seized upon by the bacteria as
food, is not known.

FISCHER I

114 BACTERIA AND THE CARBON DIOXIDE CYCLE

Other fermentations of monacid alcohols have not been described, but
there can be little doubt that such occur.

Fermentation of higher alcohols (84) has been observed in the case of
a motile bacterium isolated from sheep’s dung, Bacillus ethaceticus. A
culture of this micro-organism in 60 grm. glycerine formed

7-52 grammes Ethyl Alcohol,
3°88 a Acetic Acid,
0:06 3 Succinic Acid,
Carbonic Dioxide,

Free Hydrogen,

Traces of Formic Acid.

24:19 grm. of glycerine was unchanged. The same bacterium causes similar
changes in mannite, but not in its isomer dulcite. The so-called capsule
bacillus Pxeumococcus of Friedlander ferments mannite also (with the same
products), but not dulcite.

Bacillus ethacetosuccinicus (also isolated from dung) ferments both
mannite and dulcite. In cultures eighty-five days old that contained, besides
the necessary nutriment, eight grams of fermentable substance, there was
formed

From Dulcite. From Mannite.

Ethyl alcohol. . . é . LOIl 1-03
Formic acid 5 ‘ F i . 0-128 0263
Acetic acid F F ‘ < ‘ 0-322 0-308
Succinic acid. : ‘ : ‘ 0264 O29
co, . F 5 Fi . . ‘ 1:05 Ir
Free H - . i 3 ° 0-04 0:03
Unfermented residue . s ‘ ‘ 2-62 3:2

These figures are of some interest, showing that the chief products are
alcohol and carbonic dioxide, and proving that the formation of ethyl
alcohol is not a monopoly of the Saccharomyces *.

Glycerine also is capable of fermentation in other ways, butyl! alcohol
and butyric acid arising among the products (B. orthobutylicus, Chap. XIII).

As examples of the fermentation of fatty and aromatic acids, given in
the form of neutral salts, it may be mentioned that acetic acid is oxidized,
by the very organisms that produce it, to CO, and H,O, and that the
dextro-rotatory tartaric acid of wine is converted by various bacteria into
formic, acetic, butyric, propionic, lactic, and succinic acids. It is to the
activity of such bacteria that the diminution of acidity of wine is due,
and probably some of the diseases of wine also. The malic acid in cider is
also oxidized by bacteria to acetic, propionic, and butyric acids, carbonic
dioxide, and water; and similar processes are known in the case of citric,

* B. Fitzianus {rom hay-infusions also produces ethyl alcohol.

SEPARATION OF OPTICAL ISOMERS 115

succinic, and other acids. Lactic acid, itself a product of many carbo-
hydrate fermentations, is converted by butyric bacteria into butyric acid,
carbon dioxide, and free hydrogen. The biochemistry of all these processes
is as yet but little understood (Chap. XIII).

Very remarkable decompositions of the optically ‘inactive’ varieties
of organic acids are effected by some bacteria*. These consist, as is
known, of equal parts of the dextro-rotatory and laevo-rotatory acids,
which twist the plane of polarized light to right and left respectively. In
a solution of the ammonium salt of racemic acid, for instance, only the
dextro-rotatory variety is used by the bacteria, the laevo-rotatory modi-
fication being thereby as it were set free. The inactive varieties of lactic
and mandelic acids are biochemically separable into their components in
the same way (85).

The profound chemical disintegration that these changes seem to show
is only apparent. As a matter of fact they are only selective changes, only
one of the two active compounds of the salt being used up. One of the
mucigenous bacteria, for instance, uses up fumaric acid, but leaves the
stereo-isomeric compound, maleic acid, untouched. A similar phenomenon,
which may be termed the converse of those just described, is exhibited by
a variety of B. coli communis which ferments grape sugar into lactic acids
differing in rotatory power according to the source of nitrogen employed.
If it be supplied with phosphate of ammonia, laevo-rotatory acid is formed,
but if the nitrogen be given to it in the shape of peptone the dextro-
rotatory variety appears; whereas the lactic acid produced by other
fermentations is generally inactive.

We must for the present be content to record these facts—an explana-
tion is not possible. The stereochemical hypotheses, with which modern
chemistry seeks to unravel the molecular structure of these isomeric com-
pounds, throw no light whatever at present on the biochemical phenomena
associated with them.

* Some of the monld-fungi act in a similar way.

12

CHAPTER XIII

THE CIRCULATION OF CARBON DIOXIDE IN NATURE
(continued)

2. Bacterial Fermentation of Carbohydrates.

THE bacteria of lactic fermentation are very widely distributed in
nature, and play an important réle, not only in dairy work, but also in
numerous other industrial processes—often as indispensable auxiliaries,
but sometimes as dangerous enemies and intruders.

Grape sugar, cane sugar, and milk sugar are all accessible to lactic
fermentation. Other sugars (e.g. maltose) are not, nor are carbohydrates
like starch or cellulose. All these must be changed into a fermentable
form before they can be broken up by the lactic acid bacteria, which
themselves produce no such enzymes.

The lactic fermentation is an aerobic process whose optimum lies
between 30° and 35°C. (for some species between 47° and 52°C.). It
can go on for a long period only when basic substances, such as calcic
carbonate, are present to fix and neutralize the lactic acid as it arises,
a very small amount (0-15 per cent.) of free acid arresting the growth of
the bacteria. Eighty per cent. of the fermentable sugar is converted under
favourable circumstances into the so-called ‘fermentation lactic acid,
optically inactive ethylidene lactic acid, besides which are formed varying
amounts of acetic acid, optically active lactic acid, carbon dioxide, and other
by-products.

The power of forming lactic acid from sugar is common to many
bacteria (e.g. the cholera and most other vibrios, B. prodigiosus, various
species from the intestines of infants, and many brewery Sarcinae). But
by lactic acid bacteria are generally understood those forms which are the
regular cause of the acidification of milk in dairies. These micro-organisms
were formerly looked upon as belonging to one species, Bacterium acidi
lactic (86), but more recent investigations have shown that not one species
but many are at work, sometimes one kind, sometimes another, prevailing.

BACTERIA IN MILK 117

A very common form is a short non-motile rod (1-2 » by 0-5 y), facultatively
anaerobic (spores unknown), and not liquefying gelatine (Fig. 24,d@). It
is known by various names, Bacillus aerogenes, B. acidi lactici, &c., and
comprises probably several races of one species. They may be called
the typical bacteria of lactic fermentation. Mingled with them are found,
sometimes in great numbers, the spherical or ovoidal cells which cause
the coagulation of milk. The lactic fermentation of mash in distilleries
is caused by yet another form, B. acidificans longissimus (87). This
bacterium is about 1 broad and something more than 2-5y long. The
number of lactic acid bacteria is great and their determination difficult.
The manifold importance of the lactic ferments can be best illustrated by
some concrete examples.

Milk and other Dairy Products (86), etc.

Cow’s milk, being neutral or slightly alkaline, and containing as it does
from four to five per cent. of milk sugar, 4 per cent. casein, and o-7 per cent.
of the necessary mineral salts, is an excellent nutritive medium for bacteria.
They multiply in it very rapidly, and within a few hours after it is drawn
are present in enormous numbers (from 100 to 6,000,000 per c.c.). As
might be expected, the numbers vary greatly according to the cleanliness
of handling during milking and afterwards. In consequence of the
favourable conditions thus offered for the multiplication of bacteria, the
sterilization of milk, particularly for children, has become a matter of great
importance *, and numerous inventions have been put forward to attain
a thorough and efficient sterilization. But milk, although sterile when
drawn from the udder, becomes infected with micro-organisms at once
on contact with the outside world, and invariably contains spore-
bearing bacteria. Some of the spores are so resistant that even boiling
for an hour, or an hour and a half in a Soxleth boiler, does not destroy
them, so that at present it seems impossible to render milk absolutely
sterile without exposing it to heat so great that its chemical characters are
altered. Fortunately, the majority of the bacteria in milk are sporeless
cells which are killed by boiling for five or ten minutes, and for this reason
expensive sterilizing apparatus is being largely discarded in favour of the
old domestic method of boiling the milk, and then keeping it as cool as
possible, so that the uninjured spores may not germinate (88).

Besides the predominating lactic bacteria, there are always present
species that secrete an enzyme resembling rennet, and frequently chromo-
genic forms in small number.

Since the presence of pathogenic germs in milk may become a source

* In Germany a matter of State and municipal control.

118 BACTERIA AND THE CARBON DIOXIDE CYCLE

of danger (89), experiments have been made to determine their behaviour
in milk. The bacteria of typhoid, anthrax, glanders, tubercle, diphtheria
and cholera grow well, without causing any change in the appearance of
the milk that would suggest contamination—ordinary market milk does
not look as though it contained millions of bacteria—and not until some
time has elapsed does the milk begin to coagulate in consequence of the
acid produced. The anthrax bacillus produces acetic and caproic acids.

Whether the milk of diseased cows contains the specific bacteria
of the infection is not certain in all cases. In tuberculosis, however,
tubercle bacilli have been repeatedly found in the milk.

The acidification of milk for the preparation of cheese made from the
‘acid curd’ is the work of the lactic bacteria already mentioned. The
acid they produce precipitates the casein—the milk ‘coagulates” In
the preparation of ‘rennet curd’ the same end is attained without acid by
the use of rennet, an enzyme prepared from the stomach of calves. In
both cases, the casein freed from the serum of the milk (whey) constitutes
the ‘curds’ from which cheese is prepared.

Numerous ‘diseases’ of milk are caused by bacterial action. Not
infrequently it coagulates without turning acid; this is the work of species
(Zyrothrix, found in cheese) which secrete an enzyme resembling rennet.

Pigment bacteria often make their appearance spontaneously and
cause the milk to have an unnatural colour. Red is produced by B. pro-
digiosus and some Sarcinae, blue by the harmless B. cyanagenus, a small
motile rod that grows on agar in dark-blue or bluish-grey crusts according
to the food-stuffs available. From yellow milk also several pigment
bacteria have been isolated. These coloured milks have generally become
more or less acid. ‘Ropy’ milk is the work of certain mucigenous bacteria
that will be described later on. Bitterness in milk is produced especially
by peptone-secreting bacteria that possess very resistant spores.

Butter is always rich in bacteria. A specimen of Munich butter
contained from six to twenty-five million per gram. Since the butter
contains from o-5 to 1-5 per cent. lactose and other food-stuffs, changes
may be set up by the formation of lactic and butyric acids. The butter
is thereby rendered rancid, but it must be remarked that the rancidity
of butter is in most cases the result of the oxidation of the butter fat
to fatty acids (butyric and lactic) by the free oxygen of the air accelerated
by exposure to light. The peculiar aroma, too, that renders some butters
so tasty, is the product of certain bacteria that are now cultivated in
dairy laboratories and added to the fresh butter (90).

A process of great complication, the details of which are difficult
to follow, is the ripening of cheese. This is the work of many kinds
of bacteria, which are therefore present in cheese in enormous numbers (91).
In a gram of German cheese from five to six million germs have been

BACTERIA AND CHEESE 11g

found, in the same quantity of Swiss cheese one million, in other sorts still
more. In the preparation of Roquefort cheese a mould fungus (Pez?-
cillium glaucum) is introduced with mouldy bread, and forms the well-
known green patches in the cheese. In other cases, forms like Ozdiam
and also yeast fungi play a part.

Not all the bacteria of cheese are equally active in the ripening
process; many are only useless intruders, others perhaps contribute to
the finer shades of flavour. The main part of the process is the work
of the lactic and butyric ferments. Forms that were once supposed to
be specific ‘cheese bacteria’ (Zyrothrix, Duclaux, allied to B. subtilis)
do not play the important part that was attributed to them. How
difficult it is to determine the share which the various species have in the
process, is shown by the fact that in some cheeses no less than nineteen
different bacteria and three yeasts have been found. In other kinds
the flora is still more varied, and it changes, moreover, with the different
stages of the ripening process. This is the reason that, in spite of numerous
and careful investigations, the bio-chemistry of cheese has but few results
to show, and those contradictory. Even the qualitative composition of
cheese varies, and a quantitative analysis is as yet no more possible than
in the case of putrefactive processes. Emmenthaler cheese contains lactic,
butyric, and phenylamidopropionic acids, leucin, tyrosin, ammonia, casein
(partly unchanged, partly in the form of soluble albumoses), fats, and fatty
acids. The fresh curds from which cheese is prepared contain chiefly three
substances, whose gradual alteration constitutes the ripening: 1. Cardo-
hydrates (milk sugar); 2. Proteids (casein and paracasein); 3. Fat. The
milk sugar is converted first by the lactic bacteria, and afterwards by the
butyric bacteria, into organic acids, CO,, and free hydrogen. It is these
gases which cause the cavities in the cheese. The casein is changed, by the
action of the enzyme secreted by the bacteria, into an albumose-like body
(improperly called caseoglutin), and a small portion of it further split up
into tyrosin, leucin, ammonia, and phenylamidopropionic acid; but genuine
putrefactive products, like skatol or indol, are not formed. The ripening
of cheese cannot therefore be regarded as a true putrefaction, but only as
an allied process in which fatty acids arise. The conversion of the casein
goes on very gradually until, by the time the ripening is finished, there
is none left in an unchanged state. It is not known precisely what species
of bacteria are instrumental in effecting this alteration. Fat is not formed
from casein, and the butter remaining in the curds is for a long time
unaffected, but towards the end of the process it is split up into glycerine
and fatty acids. Of these phenomena the two of chief importance are the
fermentation of the milk sugar and the alteration of the casein.

Another derivative of milk, kephir, is produced by the united action
of lactic acid bacteria and a yeast (Saccharomyces). Kephir is a weakly

120 BACTERIA AND THE CARBON DIOXIDE CYCLE

alcoholic effervescent beverage that has been used for centuries in the
Caucasus, where it is usually made from mare’s milk. With us cow’s milk
is generally employed. The ‘kephir grains’ with which it is prepared con-
tain both the bacterium and the yeast. The yeast cells secrete a peculiar
enzyme (lactase) that converts the milk sugar into grape sugar, which is then
fermented to alcohol and CO,. The lactic bacteria, besides giving the
pleasant acid taste to the drink, cause the precipitation of the casein in
a very finely flocculent, easily digestible form. Acetic and succinic acids
arise as by-products (91).

In distillery operations (92) the development of butyric bacteria was
formerly much dreaded. In the preparation of sweet mash from green malt
the heating for two hours at 70° is naturally insufficient to kill the spores.
Experience showed, however, that a certain degree of acidity in the mash
prevented the development of butyric bacteria without injuring the yeast
cells, and careful investigation showed that the acidity was caused by the
lactic ferment. In practice the end is attained by inoculating some of
the sweet mash containing the yeast to be used with pure cultures of a lactic
bacterium, and keeping it at 50° C. (the optimum for these organisms) for
some time before adding it to the main body of the mash. At 50° C. the
lactic bacteria flourish and produce as much as I per cent. of lactic acid, and
the growth of the butyric organisms, whose optimum is 40° C., is inhibited
both by this temperature and by the lactic acid.

This important application of lactic fermentation is now largely super-
seded by Effront’s hydrofluoric acid method. The yeasts are far less sen-
sitive to acids generally than the bacteria, and by continued culture with
increasing quantities of acid they may be’accustomed to such large amounts
that the bacteria are reduced to a minimum, or even quite suppressed.
In a few months the alcohol yeasts may be accustomed to as much as
thirty milligrams of hydrofluoric acid per hectolitre mash, ten mg. sufficing
to kill the bacteria. Other poisons also have been tried; formaldehyde,
for instance, which seems more efficacious even than hydrofluoric acid.
For electrical sterilization see p. 72.

The spoiling and ‘turning’ of beverages and articles of food through
lactic fermentation is a common occurrence. Beer must contain more than
7 per cent. alcohol if it is to be safe from the attacks of the lactic bacteria,
which, if they once get a foothold, soon cause it to ‘turn,’ making it turbid
and giving ita bad taste. Wine, too, is often spoiled by the same organisms,
and may contain as much as 2 per cent. lactic acid, derived from the fructose.
The acidity of wine is, however, more often caused by the acetic bacteria
than by the lactic. Boiled vegetables are not infrequently soured by both
lactic and butyric fermentation.

The various methods of fodder preparation (93), brown hay, sour fodder,
and sweet ensilage, are fundamentally lactic fermentations by which the hay

BUTYRIC ACID BACTERIA 121

is made both durable and more tasty for the cattle. Sauerkraut is another
foodstuff that is the result of such fermentation. In all these cases butyric
fermentation occurs at the same time.

Butyric Fermentation (94).

The butyric fermentation, a strictly anaerobic process, is no less widely
distributed in nature than the lactic. Its importance for the general theory
of fermentation will be considered in the next chapter.

There are several means of obtaining rough, impure fermentations of
butyric bacteria from which, by growing in hydrogen or by other anaerobic
methods, pure cultures may be derived. It suffices to place some peas in
a saccharine nutritive fluid and close the culture flask with a cork through
which passes a tube whose outer end dips under water. In a few days at 30°
or 40° active fermentation sets in, large quantities of gas are given off, and
an odour of butyric acid is apparent. Another method is to boil a mixture of
5 grm. grape sugar and 5 grm. powdered fibrin in 100 c.c. water, and in-
oculate the liquid, whilst boiling, with a little garden earth. After forty-eight
hours at 35° the fermentative process is going on rapidly, and ‘is caused
almost entirely by one species of bacterium, the Granulobacter saccharo-
butyricus of Beyerinck.

Just as was the case with the lactic fermentation, the butyric fermenta-
tion was supposed formerly to be the work of one species of micro-organism
only, the Vibrion butyrique of Pasteur (Amylobacter butyricus, van Tieghem).
There can be no doubt, however, that these terms have both morpho-
logically and physiologically only the value of collective designations, as is
the case with the Clostridium butyricum of Prazmowski. Some twenty
different forms of butyric bacteria have been more or less precisely de-
scribed, and the number could be probably reduced to two or three. Many
are distinguished by the granulose reaction (Beyerinck’s biological genus
Granulobacter), and some are characterized by the alteration in shape of
the cell during sporulation, a process which sets in with great regularity
towards the end of fermentation (Fig. 24, e¢, f). The commonest form of
the spore-bearing cell is the spindle shape (Clostridium), but some species
swell at the end (drum-stick, Plectridium). Almost all are comparatively
large cells (0-5-1 by 3-10) actively motile and peritrichous (Fig. 24, e, /).

Granulobacter saccharobutyricus and G. lactobutyricus (anaerobic Clos-
tridia with granulose), and Bacillus orthobutylicus (an anaerobic Clostridium
with no granulose), all produce considerable quantities of butyric acid, with
some CO,, free hydrogen, acetic, and traces of other fatty acids. JB. ortho-
butylicus ferments carbohydrates and other compounds of many different
kinds ; glycerine, mannite, glucose, cuvert-sugar, cane-sugar, maltose, lactose,
arabinose, starch, dextrine, inuline (not trehalose), erythrite, and gum arabic.

122 BACTERIA AND THE CARBON DIOXIDE CYCLE

In some cases, of course, the fermentation must be preceded by inversion
by enzymes. The other two species mentioned are, as their names imply,
more particular, and need grape, cane, or milk sugar as food.
2-4 grams of glucose fermented by B. orthobutylicus yielded in twenty
days
Grams.

0-842 Normal Butyric acid
0-264 Butylic alcohol
0-229 Acetic acid

besides free hydrogen and CO,. These gases increased by continued fer-
mentation, probably showing that the organism can split up its own pro-
ducts into still simpler compounds down to CO,.

A granulose-bearing species from the soil, described by Beyerinck as
Granulobacter butyricus, is particularly worth notice, since it forms from
maltose, not butyric acid, but butylic alcohol with some CO, and free H.

Not infrequently butyric acid arises as a product in putrefactive pro-
cesses, and it seems that some butyric bacteria (B. butyricus, Hueppe) have
true saprogenic properties, and can break up proteids. Other species, like
B. orthobutylicus, can form butyric acid from peptone only in the presence
of one of the above-mentioned non-nitrogenous bodies.

Milk soured by B. Jactis is often infected by butyric organisms
which disintegrate both the remaining milk sugar and the lactic acid that
has arisen from it. The fermentation of the calcium salt of lactic acid ex-
hibits this change in an uncomplicated form *.

Cellulose Fermentation.

In manure heaps and in the rotting vegetation at the bottom of lakes
and ditches there is going on side by side with the disintegration of
carbohydrates in plant tissues another process, a special fermentation of
cellulose known as methane or marsh gas fermentation (95). The organ-
isms which effect this change first of all ‘invert’ the cellulose (i. e. break it
up into a sugar) by means of an enzyme ,

(Cg Ho Os)n + (He O)n = (Ce Hin Oo)n
and then ferment it to methane CH, and carbon dioxide, fatty acids arising
as by-products. Methane, mixed with free hydrogen and CO,, rises to
the surface when the mud at the bottom of ponds is stirred up with a stick.

There seem to be a considerable number of methane bacteria. The
Vibrio rugula (anaerobic, granulose-bearing) probably is one. Another
species (a delicate anaerobic motile Plectridium) that has been isolated from

* For butyric bacteria in cheese-making see p. 119; their distribution in nature, p. 135 ; their
assimilation of nitrogen in the soil, p. 96.

TECHNICAL FERMENTATION 123

sewer contents is able to ferment filter paper (pure cellulose). The paper
becomes soft and transparent, and is finally completely dissolved. Methane
bacteria are at work also in the intestines of herbivores and of man, and
cause distention by the gases they produce.

Mucilaginous Fermentation (96).

Wine, beer, and milk sometimes become ‘ropy,’ slimy or ‘ stringy’ ;
boiled vegetables, too, sometimes undergo the same change. This is due to
the growth of bacteria, which ferment the carbohydrate constituents of the
liquid, and whose chief product is mucilage, carbon dioxide, free hydrogen,
and the inevitable fatty acids arising as by-products. In some cases the
nascent hydrogen combines with dextrose to form mannite. The mucilage
is a gum-like carbohydrate allied to vegetable gums, and having the same
composition as cellulose (C,H,,0O,),. It is not a fermentation product,
however, in the true sense; that is to say, it is not a direct result of the
protoplasmic metabolism, but arises through the imbibition of water by the
outer layers of the cell-membrane. It is, in fact, similar to the jelly that
holds together the cells in a zoogloea. A useful task would be the investi-
gation of the nature of the unaltered inner layers of the cell-wall, which
consist perhaps of a cellulose-like carbohydrate.

A number of mucilaginous bacteria have been described, and the species
are said to be different for different kinds of sugars. 2B. véscosus sacchari is
said to flourish only in fluids containing cane sugar, another only in grape
sugar (wine), and a third (B. viscosus lactic?) in milk sugar.

Fermentations in technical processes.

Wherever fermentable material is handled on a large scale there is
danger of the invasion of ferment bacteria. And in many processes that
‘go on of themselves’ we must assume that bacteria are at work. Places
where such changes occur, and where the bacteriologist must seek for them,
will occur to every one. Some cases, such as the souring of bark liquor in
tannery, have already been tentatively examined. It will be worth while
to mention a few others.

The ‘Retting’ of Vegetable Fibres (97), such as flax and hemp, is also the
work of bacteria. The tissues are allowed to soak for a long time in water,
and fermentation sets in, the fibres being freed from the enclosing cells by
the solution of the middle layer of the cell-walls. This inter-cellular cement-
substance consists of so-called pectine compounds (pectine salts of calcium)
related to carbohydrates, which are dissolved by the bacteria. The fibres
can then be separated by mechanical means. Up to the present we know
only one of the micro-organisms concerned, an anaerobic Plectridinm
(10z-154 by o-8y), which can use ammonia as a source of nitrogen and

124 BACTERIA AND THE CARBON DIOXIDE CYCLE

ferments pectine substances prepared from linseed, pears, or turnips.
Cellulose and gum arabic are not attacked, but the power of breaking up
other carbohydrates is greatly increased if peptone be supplied instead of
ammonia. As to the products of pectine fermentation nothing is known,
but they are probably, as in other cases, CO, and fatty acids. The old idea
that the process was one of cellulose fermentation is certainly wrong.

In the manufacture of indigo bacteria are considered to play an im-
portant part (98). The indigo plant (Judigofera tinctoria) contains a gluco-
side Judican which by anaerobic fermentation at 25°-35° C. is converted
in from eight to fifteen hours into a sugar (/udigoglucin) and indigo-white.
The fermentation is carried on in large vats, the liquid in which becomes blue
on the surface where it is exposed to the air. By agitation the whole mass
is brought into contact with air, and becomes blue throughout. The micro-
organism which ferments the indican is a short capsuJated bacillus, 2. zndigo-
genus. If the fluid be sterilized and the bacteria killed, no pigment is
formed. The process has not yet been followed in detail.

The Zreparation of tobacco involves, to a considerable extent, the work
of bacteria (99). The dried leaves of the plant are damped and laid in
great heaps to ‘ferment.’ By this process various carbohydrates, nicotine,
and vegetable acids are converted into CO,, butyric acid, succinic acid, and
certain ‘aromatic bodies,’ whose nature is not precisely known. The
proteids are said not to be attacked. Various species of bacteria have
been isolated from the tobacco leaves. The ‘Havannah’ bacteria are
said to differ from those of the German weed, and the inoculation of the
latter with Havannah bacteria has been to some extent successful. The
process is comparable to the improvement of wines by the inoculation of
the grape juice with specific pure yeasts. It is, however, doubtful whether
the real aroma of Havannah tobacco can be transferred in this way, since
the aromatic compounds of the plant itself have to be considered, not
merely those produced zymogenically.

In the beet juice of sugar factories, and also in refineries (100), the
‘ frog-spawn bacterium ’ (Leuconostoc mesenteroides) is sometimes a great pest
(Fig. 7, 6-d). It is a mucigenous bacterium, and the chemical changes it
sets up have been designated as dextrane fermentation, because the mucilage
that appears in such profusion is supposed to be similar to a carbohydrate
of the beetroot, dextrane. The matter requires further investigation, however.
At the optimum temperature (30°-~-35°) the micro-organism grows with
extraordinary rapidity, and whole vats may be filled with it in the course
of a single night*. Besides mucilage, the bacteria produce lactic acid and
CO, The molecules of the fermentable substances are not so completely

‘

* In one case a vat of 49 hectolitres molasses, containing 10 per cent. sugar, was filled in twenty-
four hours with the spawn-like masses,

FERMENTATION OF BREAD 125

disintegrated as in many fermentations. The bacterium is a coccus with
fixed planes of division, and, like the alga Mostoc, forms unbranched rosary-
like chains embedded in jelly. Cane and grape sugar are necessary for
the production of the mucilage. In media containing neither of these
carbohydrates, the organism grows as a simple Streptococcus, without any
capsule.

In bread-making (101), too, we cannot dispense with the help of micro-
organisms, which are important agents in the conversion of the nutritious
wheat meal into a tasty bread. Yeast, as used by bakers, consists of
a mixture of Saccharomyces cells (the alcohol ferment), and of different
species of bacteria. The bacteria, to some extent, prepare the starch for
the yeast by enzymes, and contribute to the taste of the bread by the
organic acids (lactic and acetic) they produce. The fermentation set up by
the yeast gives rise, per kilogram of bread, to about 2-5 grm. alcohol and
2-7 grm. CO, This gas it is which causes the bread to ‘rise,’ and the
inflation is of course increased by the expulsion of alcohol vapour and
steam, which takes place in baking. Some of the fermentation products
finally remain in the bread and affect its taste.

CHAPTER XIV

THE CIRCULATION OF CARBON DIOXIDE IN NATURE

(continued)

3. The Yeast Fungi and Alcoholic Fermentation. Theory of Fermentation
and Anaerobiosis. Concluding Remarks on the Circulation of
Nitrogen and Carbon in Nature.

THE most important of all fermentative processes, from an industrial
point of view, the alcoholic or vinous fermentation, is the work not of
bacteria, but of yeast fungi. Some few bacteria are, as we have seen, able
to produce ethyl alcohol, e.g. B. ethaceticus, but in all the great technical
fermentations, in the manufacture of beer, wine, and spirits, the ferment
organisms are yeasts (Saccharomycetes, Blastomycetes) (102, 103). The yeasts
are unicellular non-motile organisms, not cylindrical or spherical in shape
like the bacteria, but spheroidal (Fig. 25). The shape of the cells, to some
extent of value as a classificatory character, appears at first sight more
irregular than it really is. This is due to the remarkable manner of pro-
pagation. The yeast cells multiply not by fission into two equal halves, as
do bacteria and the cells of higher plants, but by ‘budding.’ The cell-wall
becomes evaginated at one or more points, and the bud-like projections thus
formed are shut off from the mother-cell by a new cell-wall. As the young
cells do not at once break away, and even begin to bud themselves before
connexion with the parent cells is severed, irregular colonial growths arise.
The contrast between this mode of reproduction and the multiplication by
fission of the bacteria is very great. Among the bacteria each new
generation represents half of the parent generation, which in the very
act of multiplication ceases to exist as an individual. In the yeasts a small
part only is separated from the parent, which continues to live and bud off
further descendants. The process of budding, like that of fission, is very
rapid ; a new generation may arise in a couple of hours, so that reproduction
is little less prolific in the yeasts than in the bacteria.

As with bacteria, the successive generations of yeast cells often remain

SACCHAROMYCES 127

in close connexion. But, inasmuch as the budding is irregular, new
individuals sprouting out from any part of the surface of the cell, the
colonies or growth forms that arise are also irregular, branching out not
only in one plane, but in all directions, without any definite sequence of
growth (Fig. 25). The cells are often elongated, sausage-shaped, and the
tangled colonies or ‘growth forms’ have then almost the appearance of

FG. 25. Saccharomyces. a, Saccharomyces cerevisiae,No.1. b, Sacch. Pasteurtanus, No.3. c, Sacch. ellips-
oideus (wine-yeast), No. 1. d, Sacch. ellipsotdeus, No. 2. ¢ and f, pellicle growth (pseudomycelium) of Sacch.
ellips., No. 1; e, at 307-20” or 6°-7?; f, at 15°-30°. _ g-#, spore-bearing cells; g, Sacch. cerevisiae, 1; h, Sacch.

teur., 1; Zand k, Sach. ellips., 1 and 2. 4, germination of two free spores of Sacch. Ludwigit at 18’-20°, from
ee oT right, after 18, 20, 26, 28, 29, 30}, and 33 hours respectively, All cultures in beer-wort, magn. 1000 (from E.
“hr. Hansen).

mycelia (Fig. 25, e¢ and g). Such pseudo-mycelial masses arise both
immersed in liquids and on the surface. In the latter position they often
unite to form membranes or pellicles (‘veils’), as we saw was the case with
some bacteria. Their mode of origin shows them to be, however, not true
mycelia, but only ‘ growth-forms.’

The yeast cells, like those of other plants, consist of a membrane

128 BACTERIA AND THE CARBON DIOXIDE CYCLE

enclosing the protoplasmic cell body, which apparently contains a nucleus *.
Although very minute, the yeast cells are larger than most bacteria, having
on an average a diameter of from 8uto1op. All the yeasts that are of
industrial importance are colourless, and their cultures appear white or of
a yellowish tinge. There is, however, a pink yeast (Saccharomyces glutinis,
weakly fermentative) that frequently crops up as an intruder on culture
plates, and a less common black species is known.

Most yeasts under favourable circumstances { free access of air ; surface
culture, not immersed; temperature about 25° C.) form spores. Instead of
each cell giving rise to one endospore, as in bacteria, several appear, the
protoplasm of the parent-cell dividing into several (generally two to four)
rounded masses, each of which becomes surrounded by a new membrane
(Fig. 25,g, #). The spores are considerably less resistant than those of
bacteria, being killed by a temperature of 62°-70° C. in five minutes.
They are capable of germination as soon as they are formed, but will
withstand drying for long periods. The germinating spore begins to bud
as soon as the spore-wall is thrown off (Fig. 25, 2).

Hansen’s investigations have shown that in the hands of skilled
observers who are familiar with the sources of error, the peculiarities of
sporulation and its relation to certain temperatures, are of great importance
for the differentiation and determination of species and races.

The following table gives the times of sporulation for a ‘high’ yeast
(S. cerevisiae), for a race of wine yeasts (S. edlipsoideus), and for two races
of ‘wild’ yeasts from the air of a brewery (S. Pasteurtianus). Important
points are the optimum and the maximum temperatures, and the time
occupied in forming the spores.

S. Pasteurianus.
Temperature. S. cerevisiae. S. ellipsotdeus.
a é
37°5° no spores
36°-37° 29 hours (Max.)
35° 25 hours
315° no spores 36 hours (Max.)
30° 20 hours (Opt.) | 30 hours (Max.)
27°5° 24 hours (Opt.) | 34 hours (Max.)
25° 23 hours 25 hours (Opt.) | 21 hours (Opt.)
18° 50 hours 35 hours 36 hours 33 hours
11°-12° 10 days (Min.) 77 hours
7° no spores 7 days 7 days It days (Min.)
3°-4° no spores 14 days (Min.) | 17 days (Min.) no spores

* See Wager, ‘ The Nucleus of the Yeast-Plant,” 4zzals of Botany, Vol. X11, Dec. 1898, p. 499.

RACES OF YEASTS 129

The cardinal temperatures for sporulation are given and show the value
of such physiological data. For the differentiation of very closely related
races more subtle distinctions are utilized, such as the shape of the cells,
mode of budding, power of fermentation, and particularly the ability to
break up various sugars of closely allied chemical structure.

Some of the natural species of yeasts are destitute of spores.
Hansen (104), guided by the attempt to cultivate a sporeless breed
of anthrax bacilli, endeavoured to induce the asporogenous condition
among spore-bearing yeasts by similar means, i.e. by cultivation at
high temperature. His experiments were successful; sporulation was
suppressed and did not appear in subsequent generations. At the same
time the fermenting powers of the yeast were slightly altered. It seemed
at first that the attempt to obtain sporeless races had been more successful
among the yeasts than with the bacteria, but subsequent observations
showed that the phenomena were the same in both cases. When mixed
with earth, the sporeless varieties of yeast died out in one year, whereas
the unaltered spore-bearing form of the same species lived for three
years under the same conditions. This fact alone shows that a general
weakening of the organism had set in, and other signs of degeneration
were not wanting. But although the attempt to produce races of yeast
with new morphological characters has not been an unimpeachable success
it is undoubtedly possible to obtain races with more or less permanently
altered physiological functions—races that produce more (or less) alcohol
than the parent form, or in which the by-products of fermentation are
different or present indifferent proportions. In the brewing industries (105)
hundreds of such races have already arisen, and give to each particular
kind of beer its specific flavour, while Hansen’s investigations have led
to the application to yeasts of the methods of pure culture, so that the
most desirable varieties can be artificially propagated.

The wine yeasts also include innumerable races. Almost every kind
of wine has its own species of Saccharomyces. The secondary products
of fermentation are of great importance, particularly the aromatic sub-
stances (ethers) which give the ‘bouquet.’ But, whilst these zymogenic
products are undoubtedly influential in determining the flavour of the
wine, it must not be forgotten that the aromatic constituents of the grape
itself are of still more importance. The pure culture of yeasts of known
fermentative properties is a great step forwards, but it is not everything.
It will never be possible, for instance, to make a sour inferior Rhine wine
into an expensive Johannisberger merely by the use of the Johannisberg
yeast (105).

In the old methods of wine-making the fermentation was left to
‘go on of itself’; in other words, it was left to the yeasts accidentally
present in the grape juice. The grapes hanging on the vines always have

FISCHER K

-130 BACTERIA AND THE CARBON DIOXIDE CYCLE

on their skins numerous yeast cells, which multiply rapidly on burst or
broken grapes and are carried about by insects from one plant to another.
After the grapes have been gathered, millions of yeast cells remain in
the soil, where they pass the winter until next year’s crop supplies
them with fresh food.

When the fermentation is to be made with a pure yeast, it is not
necessary to kill the Saccharomyces already present in the grape juice
by heat; it is sufficient to add a large quantity of the pure culture,
the organisms in which usually easily overcome the less numerous ‘ wild’
yeasts.

The term ‘species’ has the same meaning and value among the
Saccharomyces as among the bacteria, but it must not be forgotten that
the yeast fungi are among the oldest of cultivated plants, and that in the
course of ages innumerable varieties and races have arisen. The various
yeasts of breweries and distilleries must all be looked upon as varieties
of the one species S. cerevisiae, and a few others, those used in wine-
making, as races of S. ellipsoideus (Fig. 25, @, ¢, d), a somewhat smaller,
thinner form than S. cerevisiae.

The peculiar mode of propagation of the Saccharomycetes characterizes
them as a definite and independent group of organisms, whose systematic
value is the same as that of any other order of fungi. This independence
would never have been challenged had not a similar budding-off of cells
been observed in some other fungi (106). Spores of the smut- fungus
(Ustilago) if sown in a decoction of horse-dung germinate and protrude
a small few-celled pro-mycelium, which then buds off laterally rounded
cells just in the same way as yeasts do. The free-lying rounded cells
multiply still further by budding, and give rise to cell groups and colonies
indistinguishable, as far as appearance goes, from true yeasts. They are,
however, quite unable to set up alcoholic fermentation.

Again, the filamentous hyphae of some of the mucorine fungi, if
grown submerged in saccharine solutions, produce cells which sprout and
multiply like yeasts, and even cause a small quantity of alcohol to be
formed (Mucor racemosus, M. erectus, M. circinelloides). Finally, there
are ascomycetes (Exoascus) which give rise to budding cells, and whose
mode of sporulation (ascospores) is suggestive of the sporulation of yeasts.
On these various and insufficient grounds the independence of the Saccharo-
mycetes as a group has been questioned, and the suggestion has been made
that they are nothing but derivatives of higher groups that have lost
the power of repeating the cycle of forms which their ancestors went
through. But none of the Saccharomycetes ever show any indications of
a higher type of growth; they produce vegetative cells, cell groups and
spores, and never anything else. The announcements that have been
made from time to time, that true yeasts have been cultivated from higher

FERMENTATION OF SUGARS 131

fungi, have all proved to be erroneous (106). As to the mode of propaga-
tion by budding, there is no reason why it should not have appeared
independently in several groups, and there are therefore no adequate
‘grounds for refusing the Saccharomycetes the position of a separate self-
contained phylum.

The Saccharomycetes are metatrophic and need the same food-stuffs
as many bacteria. As a source of nitrogen, peptone is by far the best, and
after it asparagine; but ammonia salts suffice if nothing better is to be
had. Carbon is obtained from the fermentable material, sugar, which may
be present in quantities up to 35 per cent. (optimum, 2-4 per cent., or
20-25 per cent.). Where the object is not fermentation of the substratum,
but culture of the organisms, the sugar may be replaced by other sub-
stances, such as glycerine or mannite. In direct contrast to most bacteria,
the yeasts are not affected by an acid reaction of the culture medium,
but are arrested in their growth if free alkali be present. This peculiarity
is of great advantage in brewing and distillery, for by maintaining the
acidity of the liquid (wort) in the vats the development of many injurious
bacteria is prevented, whilst the yeast cells are unhindered in their fer-
mentative power (see p. 120).

Of the carbohydrates only the monosaccharides with the formula
C,H,,0, (e.g. glucose, fructose, galactose) are directly fermentable (107).
The disaccharides (C,,H,.0,,), cane sugar, maltose and lactose must be in-
verted before they can be fermented; that is to say, they must be changed
into monosaccharides (107)*. This is effected, as we have already seen,
by the hydrolytic action of enzymes excreted by the yeast cells themselves.
By an enzyme called zzvertase the beer and wine yeasts change cane sugar
into the so-called invert sugar, and by another, known as madltase, they
change maltose into glucose. Neither of these yeasts can hydrolyze milk-
sugar, and therefore cannot ferment it. This is effected, however, by the
Saccharomycetes found in Kephir grains (p. 120) which secrete an enzyme
(lactase) that inverts the lactose in milk. Every species of yeast has its
own peculiar zymolytic properties.

The polysaccharides other than sugars (cellulose, starch, dextrine,
gums, &c.) are not attacked by the yeasts until they have been converted
into sugars by enzymes of other origin. In the preparation of beer, for
instance, the starch of the barley grains is converted into maltose by an
enzyme (diastase) contained in the grains themselves.

Every fermentation gives rise, apart from the chief products, to
numerous other substances. The following table shows the quantities of
these by-products formed in the fermentation of 1,000 gr. of grape-sugar.

* Probably the various new synthetically-produced sugars will also be arrangeable in accordance
with their structure and ability to be fermented.

K 2

132 BACTERIA AND THE CARBON DIOXIDE CYCLE

(N. B.—The yeast employed was a wine-yeast, but not a ‘pure-culture’
according to modern ideas) (108):

Grammes.
Ethyl Alcohol. 6 www en 506-15
Normal Propyl Alcohol . i 5 . : . 0-02
Iso-butyl Alcohol . F ‘ Z , : O-015
Amy] Alcohol . 5 ‘ 7 4 :. ‘ . O51
Oenanthylic Ether . 3 : , , 0-02
Iso-butylene-glycol . . «wets 1:58
Glycerine “i ‘ ‘ . ‘ 2 5 F 21-2
Acetic Acid. : ‘ : 7 : : . 2:05
Succinic Acid . : i 7 . : . 4°52
Aldehyde . : . a : . ‘ , . traces

In other words, 506 grm. alcohol and about 30 grm. of by-products, to
which is to be added about 450 grm. CO, (estimated). Some one per cent.
of the sugar was used by the yeast as food. It is noticeable that as much
as two per cent. glycerine is present. This is of some importance, for it has
more influence than might be supposed upon the taste of the wine. The
two next most important products are acetic and succinic acids.

The example just given is of course only one of many different kinds
of fermentation ; it must not be taken as a standard. The by-products
particularly are very different both as regards quantity and composition in
the different technical fermentations. In distillation higher alcohols (e.g.
fusel oil) than ethyl alcohol are found. In wine the compound ethers
(esters, compounds of alcohols and organic acids) are of prime impor-
tance because, although present in homoeopathic quantities, they are the
substances which constitute the ‘ bouquet.’

As soon as all the sugar in a fermenting fluid is used up the process
ceases, as it does also in any case when the alcohol has reached 12 or 14 per
cent. It is from this cause that many Spanish wines remain sweet, alcohol
being added to them in order to increase their permanence (?).

Alcoholic fermentation (opt. 25-30°; min, about 0°; max. about 53°)
can progress both aerobically and anaerobically. When air has free access
to the fermenting fluid the yeast cells multiply with great rapidity, but the
power of fermentation, that is to say, the amount of sugar broken up by
unit weight of yeast in unit time, is very small. Conversely, if oxygen be
absent or only very sparingly present, the fermentative power of the organisms
is great, but the rate of growth very slow. This is best illustrated by
a concrete example. To entirely ferment (i.e. to consume all the sugar in)
300 c.c. ‘must,’ twenty-three days were necessary. The time was the same
whether air was present or absent, but the number of cells in each case was
very different. Where complete aeration was carried out the fluid contained
at the end of fermentation 4,454,800 cells per cubic centimetre, whilst if air
was excluded only 50,160 per c.c. were found (109). It is evident that the

THEORIES OF FERMENTATION 133

cells in the last case must have been proportionately more powerful in
effecting fermentation.

All the technical fermentations are, as a matter of fact, anaerobic pro-
cesses, for although at first air is contained in the liquids in the vats, and has
unhindered access to them, the commencement of the fermentative process
alters conditions immediately. The oxygen in the liquid is used up at once
by the organisms, and the CO, they evolve lies in a heavy stratum over
the contents of the vats, completely shutting off the access of atmospheric
oxygen. Under these circumstances the cells are able to develop their full
fermentative power and produce most alcohol. If the object to be attained
is not the fermentation of sugar but the growth of the yeast plant, as in
establishments for the preparation of ‘ pressed’ yeast, plentiful aeration must
be provided for.

Theory of Fermentation and Putrefaction (110).

The fact that many fermentative processes take place in the absence of
oxygen would seem at first sight to give us some clue to the nature of the
phenomena. Comparisons have been drawn between fermentation and the
‘so-called ‘intramolecular respiration’ of higher organisms. Intramolecular
respiration ‘is a term applied by physiologists to the faculty possessed by
plants and animals of existing for a short time in an atmosphere devoid
of oxygen (pure hydrogen, for example), at the same time giving off CO,
and forming in their tissues a minute quantity of alcohol. It seemed as
though all living substance were able to exist for a short time without
oxygen, splitting up respirable material (carbohydrates and perhaps albu-
mens) just as the Saccharomycetes do. The power of fermenting sugar would
indeed be only a special case of intramolecular respiration.

We must not however forget that, even apart from purely oxidatory
changes such as the acetic fermentation, by no means all fermentative
processes are anaerobic. The work of the methane bacteria and of most
butyric organisms is indeed strictly anaerobic, but alcoholic fermentation,
although most vigorous in the absence of oxygen, is not arrested by the
presence of this gas, and most other fermentative processes are similar in
nature, i, e: they are facultative anaerobic.

The term ‘intramolecular respiration’ is intended to indicate a process
in which, although CO, is excreted, the oxygen is taken not from the
atmosphere, but from the complex molecules of the respirable substance
(e.g. sugar), which is broken up into the various less complex bodies we
call ‘ products of fermentation.’ That such reductions do in fact take place.
in anaerobic fermentation (e.g. butyric) may be proved by adding to the
fermenting fluid organic colouring-matters such as indigo or methylene
blue which become decolourized, that is to say they are converted into the
‘leuco’ compound with two additional atoms of hydrogen in the molecule.

134 BACTERIA AND THE CARBON DIOXIDE CYCLE

The bleached solutions become blue again if shaken up with air, showing
that there has been no far-reaching decomposition of the molecule. All
that has happened is that nascent hydrogen has been taken up from the
molecules of the fermenting substance. Whether the hydrogen is set free
in consequence of the organism seizing oxygen from those molecules, or
whether part of it is due to some other disintegration going on in the living
protoplasm, is unknown to us. In any case, it is plain that Pasteur’s dictum,
‘Fermentation is life without oxygen,’ does not quite correspond with facts.
And the circumstance that fermentation (even alcoholic) can go on in the
presence of oxygen is a further argument against such a view, and against
the theory that fermentation is a form of intramolecular respiration.

Another explanation, older even than Pasteur’s, was that proposed by
Traube in 1858, the ‘enzyme’ theory. Traube imagined that ferment
organisms accomplished all their work by means of enzymes excreted by
their protoplasm. Such a view could of course be held only as long as it
was thought that fermentation was a simple splitting-up of complex
molecules into smaller ones: for instance, that alcoholic fermentation took
place according to the formula

C,H, O, = 2 C,H,O+2 CO,,

and that no by-products arose. Since we now know that a large number of
by-products are formed, and since it has not been possible to isolate the
supposed enzymes, Traube’s theory had to be abandoned (111). In one case
only, that of the ammoniacal fermentation of urea, did it seem likely that the
change was effected by an enzyme, for the process is one of simple hydrolysis
(NH,),CO+2 H,O = (NH,), CO.
The enzyme in question, ‘ urase’ (112), has now been isolated. It is a body
of great instability.

The difference between the work of enzymes and organized ferments
must be here again referred to. Enzymes are doubtless active in all
fermentative processes, but the changes they bring about are only prepara-
tory; for example, the inversion of sugars by yeasts, and the peptonizing of
albumens by putrefactive bacteria. The actual fermentative processes with
their numerous by-products are not to be explained by enzymic action,
they are effected by the living protoplasm of the cells *.

Another explanation of the phenomena of fermentation, an explanation
of a totally different character, was proposed by Nageli in 1879. According to
this, the process is entirely extra-cellular, being brought about by vibrations
set up by the molecules of the protoplasm and transmitted to the molecules
. of the substances outside the cell, which are thereby split up into the various
bodies we call products of fermentation. This at first sight very plausible
hypothesis is rendered extremely improbable by the fact that the cell is

' See note No. 111.

FERMENTATION AND ANAEROBIOSIS 135

surrounded by a rigid membrane which would at least very much weaken
the vibrations if it did not quench them. It is true that the explanation is
difficult to disprove, but that is always the case with views based on purely
hypothetical molecular movements.

The view which perhaps more than any other agrees with the physio-
logical or ‘ biochemical’ nature of the phenomena is that which looks upon
them as being the result of chemical processes going on in the living sub-
stance of the cells, The fermentative and putrefactive organisms would
seem to have properties not possessed by other organisms, properties which
enable them to live in places and under circumstances where a complete
combustion of the food-stuffs to CO, and H,O is not possible*. The
energy that all other plants and animals gain by respiration is obtained
by these organisms from a less complete disintegration of the molecules
of the respirable substance, the relative smallness of the amount of energy
set free being compensated by the large quantity of substance decomposed.
In other words, the ordinary respiration of plants and animals is a complete
combustion of a few molecules, that of the ferment organisms an incom-
plete combustion of many molecules. The products of fermentation there-
fore are, unlike the products of respiration, bodies whose heat of combustion
is still high; alcohol, for instance, with 3,246 calories, butyric acid with
3,679 calories.

The degree of adaptation (if the expression be allowed) to anaerobic
habitats is very different in different organisms. The butyric ferments and
the methane bacteria, for instance, are absolutely (obligatory) anaerobic, and
have lost the power of ordinary respiration. The alcohol yeasts, lactic
bacteria, and most other ferment organisms are only facultatively anaerobic.
They are not yet fully weaned from oxygen, which, while not indispensable,
is far from being a poison to them. They can, even in the presence of
oxygen, exercise their remarkable faculty of partly disintegrating the com-
plex molecules of organic compounds, and gaining a small amount of energy
thereby. At the same time these facultative anaerobes can actually respire,
that is to say, can burn up completely some of the food-stuffs given them.
This is proved by the fact that when alcoholic fermentation goes on in the
presence of air a larger amount of CO, is evolved than corresponds to the
alcohol formed. Perhaps it is for this reason that the yeasts grow so quickly
when air is available; the extra energy derived from the combustion of the
sugar to carbon dioxide and water being used to build up new cell-sub-
stance. If oxygen is absent, respiration is not possible ; the other source
of energy alone is available, and growth and multiplication are necessarily
slower.

* E.g. the mud of ponds and ditches, the interior of decomposing carcases, the contents of the
intestine, and below the surface of dung-hills—all places in short where fermentation and putrefaction
are anaerobic.

136 BACTERIA AND THE CARBON DIOXIDE CYCLE

This brief description of the phenomena of fermentation will serve to
indicate the scope and nature of the problems involved.

There remain to be said a few words regarding the by-products of
fermentation, carbon dioxide, free hydrogen, and (in putrefactive processes)
ammonia and free nitrogen. Careful investigation shows that there is a
whole scale of products ranging from compounds whose heat of combus-
tion is high, such as alcohol, down to the gases just mentioned, in which it
is very low or nil. There seems every reason to believe that the combus-
tion of both the chief and the by-products is gradual, by stages. In this way
there must be going on a number of different processes simultaneously in
the cell, the various compounds being decomposed, and their energy tapped
step by step, a process which must necessarily result in the formation of
a large number of bodies of different composition—‘ by-products.’

The description given in the last five chapters of the circulation of the
elements carbon and nitrogen, and of the rédle played therein by bacteria,
would be imperfect and inaccurate if no mention were made of the part
taken in the process by other micro-organisms. The great importance of
bacteria in medicine and in many technical operations has caused them to
receive more attention than other groups, and as a result we too often
regard them as being the only agents in the disintegration of dead organic
matter. This idea is incorrect, for although precise investigations are
wanting there can be no doubt that many other protozoa found in abun-
dance where putrefaction is going on, ciliate and flagellate infusoria and
amoebae, also take part in the work of destruction. They are not always
merely saprophile; they are saprogenic, and assist in the breaking-up of
the molecules of organic substance.

Nor should we forget that the mould-fungi and all other plants devoid
of chlorophyll are entirely dependent for food on decaying organic matter,
the carbon of which they help to dissipate by their respiratory processes.
They furthermore convert stable and resistant bodies (such as wood) into
the easily decayable substance of their own cells. Not a dead twig or
trunk in the forest that does not bear minute Pyrenomycetes silently and
imperceptibly eating up the wood, to be in their turn disintegrated and
dissipated by the putrefactive bacteria to which sooner or later all fungi
fall a prey.

Thus the final destiny of all living substance is sooner or later, directly
or indirectly, to become food for bacteria, and it would seem as though the
earth must at last be suffocated by their numbers. But, apart from the fact
that large quantities of bacteria are consumed by other protozoa (infusoria
and amoebae are often crammed full of them), it must be remembered that
each bacterial cell lives only for a short time and leaves but a limited if
large number of descendants, which in their turn die off and are consumed
by their kindred, only to enter again into new cycles of chemical change.

CHAPTER XV
BACTERIA IN RELATION TO DISEASE

1, Diseases of Plants; Harmless ‘Messmates’ in the Human Body;
Pathogenic Bacteria ; Points of Attack and Sources of Infection.

WITH the exception of the root-nodules of Leguminosae, we know of
no single instance where bacteria invade the closed, living cells of plants.
The sole and only channels of communication between the interior of the
plant and the outside world are the stomata, and these open into a closed
system of air-filled intercellular spaces which are shut off from the cells
themselves. If bacterial spores are blown by the wind, or washed by
the rain into the stomata, they can do no damage, for they can get nowhere
but into the intercellular spaces where they find nothing but moist air.
Without nutriment, bacterial spores cannot germinate, and nutriment is not
present in any form. Even if such microbes as can dissolve cellulose
(methane bacteria) drift into the air passages of a plant, they find no food,
and are therefore unable to injure the cell-walls. The only organisms
whose entrance is accompanied by any danger are those whose spores con-
tain sufficient reserve nutriment to enable them to germinate in pure water,
to grow at first without food, and open the attack on the cell-wall at their
own expense. Such are spores of the parasitic fungi. They contain reserve
food-stuffs, and protrude a hyphal filament which pierces the epidermis
(potato disease), or pushes its way through one of the stomata into the inter-
cellular spaces (ras¢s), and from thence into the cells themselves. The hyphae
of the fungus dissolve the cell-membrane and send in haustoria, or proliferate
in mycelial masses in the cell-cavity.

Bacteria are totally unable to act in this way, and the uninjured plant
is impregnable to their attacks. Even the injured plant offers them nourish-
ment only in the exposed and opened cells of the wound-surface, and this
source is soon shut off by the development of an impenetrable layer of cork
below the wound preventing any further exudation of fluids. Thus the

138 BACTERIA IN RELATION TO DISEASE

wound does not remain moist; the injured cells shrink and dry up, and the
entrance of bacteria is then as difficult as in the case of an uninjured plant.
Infected wounds are therefore dangers that have no existence for plants, and
the spread of a lesion to other parts is not possible.

These considerations enable us to prophesy with considerable certainty
what will be the result of an injection into vegetable tissues of bacteria, even
of a species pathogenic for animals: no development in the intercellular
spaces, and but slight temporary growth on moist wounded surfaces, are
what we should expect, and what experiments show to actually occur (118).

Notwithstanding these well-known facts, there appear time after time
descriptions (generally insufficient enough) of new ‘bacterial’ diseases in
plants. Bacteria, it is true, are often found in diseased plants in enormous
numbers, but they are living metatrophically only, living on tissues that
have already been disintegrated and decayed by parasitic fungi. The
bacteria assist these subsequently in their work of destruction no doubt, and
modify perhaps more or less the character of the disease. But, leaving
aside for the present injuries received from ‘frost or insects, the first attack
on the plant is always made by fungi, which are also very harmful in causing
the spread of accidental wounds, All the cases of so-called ‘ bacteriosis’ in
plants, from the ‘gommose bacillaire’ of the vine down to the ‘ schorf’ of
the potato, are primarily diseases of non-bacterial origin in which the
bacteria are present merely as accidental invaders (114).

As metatrophic organisms, bacteria occur plentifully upon the leaves of
insectivorous plants such as Pinguicula, Drosera,and Nepenthes. The insect-
catching organs are necessarily open to the surrounding world, and bacteria
are blown on to them by wind, and brought by insects and other animals.
The half-digested remnants of captured organisms furnish a supply of rich
nutriment to the bacteria, and it would be extraordinary indeed if they did
not flourish and multiply in such places. Here again attempts have been
made to demonstrate a ‘symbiotic’ relationship between the plant and the
bacteria, whose peptonizing powers were supposed to be of service in dis-
solving the captured insects. Careful investigation has shown these sup-
positions to be incorrect. In the case of Lathraea, whose leaf-chambers
contain hairs often thickly covered with bacteria, it is not certain whether
these are metatrophic or not (115).

Among the lower animals bacterial diseases have been as yet but
little investigated, although they are doubtless very common. The silk-
worm diseases discovered by Pasteur, and the so-called ‘ Foul-brood’ of
bees, are well-known examples. Recently a Bacterium ranicidum has been
described, pathogenic for frogs and fishes (116).

The chief interest as regards disease-producing bacteria is, of course,
concentrated on those associated with man and the higher mammalia.
The symptomatic characters of bacterial diseases being similar in man

BACTERIA OF THE MOUTH 139

and other mammalia, the following description will deal only with the
phenomena of disease in the human subject.

Many bacterial diseases are common to man and the higher animals,
and, as far as we know, there is no parasite of man that is without effect
for some animal or another. This fact represents an inestimable advantage
in the study of disease, since it enables us, by inoculating animals with
pure cultures of bacteria, to put pathological science upon an experimental
basis. The whole of our knowledge of micro-organisms in relation to
disease, including the promising science of sero-therapeutics, is the direct
result of experiments on animals.

Bacteria in the Human Body.—Every human being, even the healthiest,
catries about with him innumerable harmless messmates and guests in
the shape of metatrophic bacteria. All the cavities of the body that are in
communication with the exterior contain them. The intestine, the moist
surface of the mucous membrane of the mouth, nose, pharynx, and
female genitalia, abound with them (117). They do not penetrate the
tissues, but live quite harmlessly in the secretions or excretions, the nature
and composition of which determine the character of the bacterial ‘flora’
in each particular spot. Some species are constant inhabitants, others only
occasional visitors, each kind multiplying and thriving where it finds the most
suitable food, and serving perhaps as a protection against the invasion of
other and perhaps pathogenic species.

Even the relatively dry skin of our bodies always holds spores and
vegetative cells. Their character depends largely upon the occupation of
the individual, and their number, as might be expected, upon the degree
of cleanliness observed.

The bacterial flora of the mouth is very rich in species (118). About
fifty have been described, some as accidental intruders, others as indigenous
organisms. Although never absent, their numbers can be reduced very
much by keeping the mouth scrupulously clean. As we have already
seen (p. 1), their chief forms were known to Leuwenhoek.

Formerly all the bacteria met with in the mouth, whether cocci, rods,
vibrios or spirilla, were classed together under one name. They were
supposed to be different developmental stages of a single species, Lepto-
thrix buccalis, which commonly occurs in the form of Jong unbranched
cell-chains that sprout out from fragments of food, and from the contents
of the tonsillar follicles. Such a view is of course incorrect, and the name
L. buccalis must be used only as a collective designation for mouth bacteria.
Some of these, such as Bacillus maximus buccalis (Fig. 26) and a coccus
form (Jodococcus), give the granulose reaction. Others, such as Vibrio
buccalis (Fig. 26, g), Spirochaete dentium (f), and Leptothrix innominata,
stain yellow with iodine.

Most of the bacteria of the mouth have as yet resisted all attempts

140 BACTERIA IN RELATION TO DISEASE

at culture, and therefore the biochemical characters of the different species
are unknown. This much, however, has been discovered, that the various
kinds present, feeding upon the food particles remaining between the teeth,
produce lactic and other acids, by which the enamel is decalcified in places.
A path to the interior tissues of the tooth being now open, the bacteria
bore their way, by means of their acid secretions, deep into the dentinal
tubules, just as a lichen eats into stones. The organic substance of the
tooth also is destroyed and the tooth becomes hollow and rotten. The
following figures illustrate the effect upon the dental tissues :

Total Weight. Lime. Organic Matter.
187-2 cubic millimetres healthy dentine. . . 0-36 gr. 0-26 gr.=72% OL gr.= 28%
187-2 cubic millimetres carious dentine. . . 0-08 gr. 0-02 gr. = 26% 0-06 gr.= 74%
Loss . . . . 0-28 gr. 0-24 gr. 0-04 pT.

Thus the loss of calcium salts by
the teeth caused by the acid secre-
tion of the bacteria is 92 per cent.,
that of the organic substance 40
per cent.

Dental caries can hardly be
looked upon as a ‘ disease’; it is
rather the necessary consequence,
sooner or later, of the action of
micro-organisms that are normal
inhabitants of the buccal cavity
introduced daily with the food.
The destruction of the teeth is not
the work of any one species in
particular, but due to the activity
of several kinds.

Whence the bacteria of the

mouth come is not at present
pte Damegl mot end tas aoe of known. The typical B, buccal:
a Spirillam spuligchum id Bedlus marinus ucans maximus is not found outside the
on" Vibrio buccalis’ &, rods probably lace aca body, and attempts at culture
ieee ne acidi lactict). Magn. a about 250, 6 400, c-h have been unsuccessful.

The discovery of bacteria,
indistinguishable from those of our own mouths, in the hollow teeth of
Egyptian mummies shows that these micro-organisms have been our
companions from the earliest times.

The healthy stomach is, in consequence of the acidity of the gastric
juice, not suitable for the development of a local bacterial flora. If however,

as a result of illness or disease, the sccretion becomes less acid or neutral, .

BACTERIA OF THE INTESTINE IqI

the bacteria and spores introduced with the food thrive and multiply
abundantly. The Sarcinae which occur in almost every drinking-water
are very.prone to make their appearance. They include several species,
_ which were formerly all classed together as S. ventriculi and supposed to
be slightly pathogenic.

The richest hotbed for bacteria in the animal body is the intestine.
Its contents are alkaline and the prevailing temperature is high and constant,
so that putrefactive and fermentative bacteria of all kinds, both aerobic
and anaerobic, find optimal conditions of existence. Fresh human faeces
contain 75 per cent. water and about 1 per cent. bacteria. These in-
clude spores and rods of all kinds, among which may be often recognized
the large cells of B. buccalis maximus that have been swallowed with food.
It is calculated that from twelve to fifteen milliards of bacteria pass out
of the body daily in the faeces (119). Many of them are dead, and
stain very badly, others are still alive, and in some spores may be seen.
As these spores have not been set free from the rods, there is no doubt
they are of recent origin and formed in the intestine.

The nature of the putrefactive and fermentative changes wrought by
the bacteria on the remnants of undigested food is, of course, dependent
largely on the composition of the food. With a meat diet, putrefactive
changes predominate, and products like tyrosin, leucin, indol, skatol,
sulphuretted hydrogen, and ammonia arise (see Ch. XI). With vegetable
nourishment rich in carbohydrates, fermentation takes the upper hand, par-
ticularly the methane fermentation of cellulose. If the food and habits of
the individual be regular, an intestinal bacterial flora of fairly constant
composition arises, the leading species being generally the pleotrophic
Bacterium coli commune (120) which is both zymogenic and saprogenic
(Ch. XVI). Other species (e. g. B. putrificus cold) occur, but have not yet been
thoroughly investigated. In the interior of the intestine, towards the middle
of the mass that fills it, only anaerobic processes can go on, but at the
sides, in contact with the mucous membrane, which itself is thickly covered
with bacteria, aerobic changes can occur.

It is not necessary to discuss the means by which bacteria obtain
entrance into the alimentary canal. We swallow myriads every day with
our food, and it is only natural that they should multiply in the very
favourable environment that the intestine affords. Their occurrence is
a necessary consequence of their wide distribution in nature, and must
not be taken as indicating a symbiotic connexion between them and
their host. It has been imagined that they were perhaps useful auxiliaries
in the process of digestion, but there is no foundation for such a supposition.
We are fortunately not so miserably dependent upon bacteria as this.
This has been experimentally proved by feeding new-born animals upon
sterilized food (121). In this way it is possible to exclude bacteria, and

142 BACTERIA IN RELATION TO DISEASE

keep the intestine germ-free for a considerable time, and the animals
remain notwithstanding in perfect health. This would certainly not be
the case if a symbiotic relationship subsisted between the bacteria and the
organism. Such a view is contradicted also by the nature of the products
arising from bacterial action. They are quite unfitted for resorption and
utilization by the animal ‘body.

The intestine of the new-born infant is absolutely séeri/e (122), but
bacteria make their appearance in a very few hours after birth, even before
any nourishment is taken. No less than seven different species have been
isolated from the intestine of children before feeding. The first to appear
is the B. coli communé, which takes up its post at once in every mortal
on his entrance into the world, and remains his faithful companion until
death. Whence this ever-present species comes is not known, but it is
in all probability a metatrophic aquatic form. If infants be fed with the
bottle the lactic acid bacteria appear at once in the faeces, and with every
new kind of food new species of bacteria are added to the flora of the
alimentary tract. As long as the mucous membrane is uninjured these
bacteria are quite without danger for the body (123). Once let the continuity
of the epithelium be destroyed however, even at a single point, and grave
pathological changes may be set up, for among the hosts of metatrophic
bacteria inhabiting the intestine some (e.g. B. coli) are facultative parasites
and originators of disease.

It has long been customary to designate as ‘infectious diseases’ (124)
all those in which the entrance of a certain ‘something’ into the body was
a necessary condition for the existence of the complaint, this ‘something,’
the pathological ‘virus,’ playing a part in disease similar to that of the
‘ferment’ in the fermentation processes. Before we knew anything of
bacteria it was usual to speak of a contagium when the disease could be
transferred only by contact with a diseased individual, and of a miasma
when the supposed virus could be carried by the air. And just as the
discovery of the ferment organisms displaced the old conceptions of a
lifeless ferment, so did the discovery, about the middle of the century,
of a ‘contagium vivum’ take the place of the old ideas respecting the
origin of infectious and epidemic disease. We now know that in the
majority of infections the virus animatum is a bacterium.

The demonstration of bacteria in the blood or body fluids of a diseased
animal is an easy matter where large species are concerned, such as the
anthrax parasite. These can be seen even in the fresh blood as pale rods
lying about between the blood-corpuscles; in this way they were discovered
in 1850. More minute forms, particularly cocci, which are easily confounded
with albuminous granules from the blood, must be rendered visible by
staining with aniline or other dyes. Diseased tissues must be fixed,
hardened, cut in sections, and stained in order to render the bacteria in them

PATHOGENIC BACTERIA 143

distinguishable from other cellular elements (Fig. 27). Most of the various
cytological methods used in investigating cell contents are applicable. The
principles of bacteria-staining are not different from those involved in the
staining of other cells, although in particular cases special technical pro-
cesses are necessary to success. For these any of the numerous handbooks
on bacteriological technique may be consulted.

The isolation of pathogenic bacteria from diseased organs is effected

“70 = aga.

ee
x
a
Lane
_
1

Fig. 27. Stained preparations of bacteria, from Ziegler’s Lehrbuch d. allgem. Pathologie, 8th ed., vol. 1. a,
sputum of a consumptive, spread on a coverglass, and dried and stained with fuchsin and methylene blue; the
tubercle bacilli are red, cellular elements ree cells) blue. 4, Gonococci (Micrococcus gonorrhoeae) in fresh
secretion, coverglass preparation : 2, mucus with isolated cocci and pairs; 5 and ¢, pus cells with and without cocci
methylene blue and eosin. , section through an anthrax pustule (stained by Grain's method), methylene blue an
vesuvine. Magn. @ 400, 5 700, ¢ 350.

by the method of plate culture (p. 58). In some cases, tuberculosis for
instance, the centres of disease contain pure cultures of the specific bacteria,
and tubes of nutrient agar or gelatine can be inoculated direct. Much more
labour is entailed where the tissues contain a mixture of bacteria of different
sorts from which the pathogenic species has to be separated and deter-
mined.

The requirements of pathogenic bacteria in artificial cultures differ

144 BACTERIA IN RELATION TO DISEASE

according to the metatrophic or paratrophic habit of the germ. Strictly
parasitic forms, such as the tubercle bacillus, diphtheria bacillus, and gono-
coccus, require the most nourishing media (see Chaps. VI and XVI). Long
continued growth in artificial cultures weakens almost all pathogenic bac-
teria; decrease of virulence and morphological changes (involution) showing
that nutrient media outside the body can never entirely replace the living
tissues. The attenuation can be brought about and increased by many
means (see p. 28) and, within certain limits, can be graduated with great
precision. It is this possibility of varying the virulence of pathogenic
bacteria that underlies a good deal of’ modern sero-therapeutics and pro-
tective inoculation.

The fact that the spores and dried vegetable cells of pathogenic bac-
teria are in many cases possessed of high powers of resistance renders it
possible that the dried secretions and excretions of patients may become
a source of infection for the healthy. The tubercle bacillus, for instance,
is possessed of its full virulence even after lying for two, or even three,
months dried up in dust. As far as the strictly parasitic forms are con-
cerned the dry resting-stage is the only form in which they are met with
outside the animal body. They have never been found growing and
multiplying, for disease products are the only vehicle by means of which
they are spread abroad and, once outside the body, even in situations that
afford abundant food, they invariably succumb to the rapidly-growing
metatrophic species.

But of the many bacteria that are capable of causing pathological
changes in the Jiving tissues a large number are of metatrophic habit and
able to thrive outside the body. Some grow quickly, others slowly, some
have very humble requirements as regards food-stuffs, others are more
pretentious, so that the ability to live saprophytically is very different in
different cases. All of them, however, represent a much greater danger
for mankind than do the obligatory parasites. Not only the pathological
products, such as sputum or pus, are possible sources of infection, but
all substances, such as milk or impure water, that offer a nutrient sub-
stratum and permit these metatrophic forms to multiply, become carriers
of disease.

The detection and isolation of pathogenic germs from among a mixture
of species, such as occur in contaminated drinking-water, is often enough
a task of the greatest difficulty, and can be carried out successfully only by
those who have had long familiarity in the technical details involved. In
many cases experiments on animals are necessary to decide whether a given
form is pathogenic or not.

Infection of the uninjured body by bacteria most frequently takes
place in those organs which communicate with the exterior, particularly
in those that, like the respiratory and alimentary tracts, are the passages

MODES OF BACTERIAL INFECTION 145

or receptacles for substances taken into the body. They are the natural
points of invasion and, as might be expected, they are furnished with means
of defence against it. The epithelium of the stomach and intestine, the
mucous membrane of the mouth, and the epidermis of the outer surface of
our bodies—in short, every intact epithelial coat—seems to be impenetrable
to bacteria. Even when highly virulent germs in large quantities are
brought into contact with uninjured epithelium, no infection takes place
unless a certain unhealthy condition of the tissues, a ‘ predisposition,’ pre-
vails, and for the present this is unexplained. But where there is the
smallest break in the continuity of the cells, there the bacteria can penetrate
into the tissues and do their deadly work. We see this illustrated every
day in wounds on the outer surface of the body, and there can be no doubt
that it takes place in the same manner on the inner surfaces, the mucous
membranes of the bronchi and intestines. And here it is instructive to
recall what happens in plants. These close their wounds from below by
impenetrable layers of cork and thus cut off from bacteria the fluids by
which they are nourished. In animals, on the contrary, the blood and lymph
that exude from a wound form a richly nutritive medium for the growth
of the bacteria, which can then penetrate into the tissues and set up ‘ infec-
tion,’ localized or general as the case may be. In experiments on animals
we imitate the natural course of events by injecting the bacteria beneath
the skin or into a blood-vessel. Insect stings represent a natural form of
this injection and not infrequently inoculate the body with disease germs.

Investigations have yet to be made regarding the number of germs
necessary to cause infection, particularly in the case of natural infections.
In our experiments on animals the smallest doses must contain thousands
of bacterial cells, but it is stated that of the anthrax bacilli ze are enough,
injected hypodermically, to cause death in guinea-pigs (125).

When bacteria have penetrated below the skin there is always the
possibility that they may be taken up by the blood-vessels or the lymphatics
and conveyed to other parts of the body. If they are, they may either
give rise to local lesions elsewhere or else endanger life by setting up
general infection. Anthrax bacilli, for instance, may produce merely a local
abscess (malignant pustule), or may spread and multiply in the blood-
vessels all over the body. Pus cocci may give rise to little pimples or
to the severer form of carbuncle, or scattered abroad by the blood may
induce fatal pyaemia or septicaemia. Some bacteria develop most rapidly
in the blood (anthrax, spirilla of recurrent fever), others principally or
entirely in the tissues (tubercle).

The fundamental phenomena underlying all infections are the same
although the clinical and pathological features of a disease may be very
different in different cases. For these aspects of zymotic complaints medical
literature must be consulted.

FISCHER L

146 BACTERIA IN RELATION TO DISEASE

The relations between the cellular elements of the body and the
invading bacteria are probably similar in all cases. Some bacteria are intra-
cellular, that is to say, they live in the cell-substance, but the majority
are intercellular, the destruction of the cells being a secondary process.
The bacteria multiply in the secretions and exudations of the diseased
tissue, pushing themselves in between the cells and into the spaces caused
by the pathological softening and loosening of connective tissue.

CHAPTER XVI
BACTERIA IN RELATION TO DISEASE (Continued)
2. Description of some Pathogenic Species.

THE following descriptions of some of the more prominent pathogenic
bacteria will be confined to the general biological characters of the organism,
purely medical questions being left to the specialist, who alone is entitled to
discuss them (126).

We have seen that among the Saccharomycetes there has been exercised
by mankind from the earliest epochs of civilization an unconscious process
of selection, which has resulted in the production of innumerable ‘ races’ or
sub-species, distinguishable from each other only by minute physiological
differences. A similar process has taken place among pathogenic bacteria.
Disease and misery have been the lot of man from the dawn of time, and
according to his liability to the attacks of certain species of micro-organisms,
and the degree to which they have adapted themselves to life in the tis-
sues, races and sub-species have arisen. Differing from one another by
physiological peculiarities rather than by morphological ‘characters, these
varieties can often only be determined by resort to experiments on
animals. In the case of some species that are known only from isolated
cases of disease it is impossible even with the most subtle methods to attain
certain results, and the difficulties are often increased by the existence of
laboratory stocks (see Chap. III, p. 28) destitute of hereditary characters.

1. COCCI OF SUPPURATIVE PROCESSES (Figs. 28, a-c; 27,6). By sup-
puration is understood the exudation from injured tissues of a fluid filled with
wandering cells (leucocytes). It has been experimentally shown that sup-
puration can be produced without the agency of micro-organisms—for
instance, by the application of nitrate of silver or corrosive sublimate ; but
there is no doubt whatever that all natural suppurative processes, from
a sloughing wound down to the tiniest pimple on the face, are caused by
micro-organisms. All the commoner pyogenic bacteria are cocci, but in
special cases suppuration may be due to other forms, the typhoid bacillus,

L 2

148 BACTERIA IN RELATION TO DISEASE

for instance, the bacterium of glanders, Actinomyces, and Bacillus pyocyaneus,
the bacterium of blue or green pus. The most widely distributed and at the
same time most harmless form is Staphylococcus pyogenes aureus (Micrococcus
pyogenes). It is a chromogenic species that covers agar with an orange-
yellow growth, and sometimes imparts a strong yellow tinge to pus (127).
The cells are very small, on an average 0-8 in diameter, colourless, and
non-motile. They lie singly, or in pairs, or in short chains ; more commonly
still in little clusters (Fig. 28, a). Besides this orange-yellow form, a paler
kind (S. pyogenes citreus) is known, and also a white one (S. p. albus).
They are apparently distinct species, and, although having the same proper-
ties as S. aureus, are not socommon. In nature the germs are found every-
where, so that they would seem to be metatrophic.

In disease they are most frequent in local suppurative processes, such
as acne (inflammation of the sebaceous glands), sycosis (inflammation of the
hair follicles), carbuncle, osteomyelitis, periostitis. Garré rubbed a pure
culture of S. aureus into his arm, and created thereby a severe carbuncle in
which the bacteria multiplied abundantly. If from such local foci the
bacteria spread, metastatic abscesses arise in other organs and in the joints,
and the condition known as pyaemia results.

Another common pus bacterium is Streptococcus pyogenes (128). Of
this species there are apparently several races very difficult of distinction.
Both in the diseased tissues, and in bouillon cultures particularly, it
forms Jong unbranched chains with cells somewhat larger than those of
Staphylococcus (Fig. 28,4). The chains result from the planes of division
always being parallel to one another. It is regularly present in erysipelas,
and in many other pyogenic diseases. It is sometimes associated with
Staphylococcus, which it exceeds in virulence. Like this, too, it gives rise to
pyaemia and septicaemia if carried abroad by the blood. It is often
present in diphtheria and in phthisis, increasing the severity of the malady,
which then assumes the character of a mixed infection. In cultures,
Streptococcus dies off much sooner than does Staphylococcus, often in a few
weeks. This, and the fact that it is less common outside the body, indicate
perhaps that it is a strictly parasitic organism.

The bacterium of gonorrhoea, the so-called Gonococcus (129), is cer-
tainly of exclusively paratrophic habit. Its artificial culture is only
possible upon blood-serum, all other media, however nourishing, being use-
less. Whence the Micrococcus gonorrhoeae (Fig. 28, c) originally comes is
not known, for it has never been found in nature, and perishes in a few hours
when dried up. But it is a constant associate of the human race, and is
transmissible only by contact. Since the temperature minimum is about
25°C., and as the cells die within five hours in water, it is evident that a
multiplication of the organism in swimming-baths is not possible. There is no
danger of infection from this source. The gonococcus occurs in the inflam-

150 BACTERIA IN RELATION TO DISEASE

organism (probably spores, for the most part) is taken up. The spores pass
uninjured through the stomach, and germinate in the intestine, setting
up intestinal anthrax, which mostly leads to general infection and death. It
is not known whether the bacteria have themselves the power of boring
through the uninjured mucous membrane of the gut, or whether a previous
laceration is necessary, such as might be caused by jagged splinters of food.

In small animals, such as mice, which succumb very quickly (1-3 days),
the disease causes no conspicuous change in the organs, but in sheep and cattle
its effects are more conspicuous. Spores, being formed only where there is
free access of oxygen, never occur in the diseased tissues or the dead body.
They arise under suitable conditions of temperature (18°-30° C.) in the
bloody dejecta of sick animals, and also in carcases that are not buried
deeply underground.

3. TETANUS (182) (Fig. 28, ¢). Lock-jaw or tetanus is caused by a
metatrophic bacterium very widely distributed in the soil, where it probably
gives rise to various putrefactive and fermentative processes. The nature of
these processes is in all probability dependent upon the food that happens to
be available, for the tetanus organism is able both to split up sugar, and in
albuminous substances free from sugar to decompose proteids into CO,, SH,,
CH,, mercaptan, and free hydrogen. The pathogenic properties of the
organism are due, not to any of these substances, but to a violent poison
which has not yet been isolated in a perfectly pure form. Tetanus is-
a typical traumatic infection that arises only by the contamination of
wounds by matter containing the bacillus—earth or the dust of hay or
straw, for example. The bacilli grow only in the wound itself, and even
here but sparingly. They never spread to other parts of the body.

Bacillus (Plectridium) tetani is a slender motile rod 2-4 long by 0-3 —
o-5 w thick. It is inclined to filamentous growth, particularly in anaerobic
cultures. In aerobic cultures the moss-like colonies grow only in the deeper
layers of the gelatine. Before sporulation, which is very constant, the rods
swell up at one end, and in this dilated part the spore appears. The drum-
stick shape, together with the peritrichous type of ciliation, places the
organism in the genus Plectridium.

Two other bacteria of the soil with saprogenic and zymogenic proper-
ties (B. Chauvei and B. oedematis maligni) give rise to quarter-evil and
malignant oedema respectively.

4. DIPHTHERIA (Bacillus diphtheriae, Loeffler; Corynebacterium
diphtheriae, Lehmann and Neumann [188]; Figs. 28, f and 14,2). This
bacterium is found almost without exception in the outer layers of the diph-
theritic false membrane. In consequence of this tendency to superficial
growth, it seldom spreads to other parts of the body, but remains confined
to those cavities which are the usual seat of the disease. Exceptions to this
do, however, sometimes occur. It is often found in the throat in association

ANTHRAX 149

matory secretion of the urethra, both in the cells (Fig. 27, 6) and in the fluid.
Thence it spreads to the epithelium and mucous glands, and finally to the
other parts of the genital system. It may even extend all over the body
(gonorrhoeal rheumatism). The cocci lie usually in pairs, the stained cells
being separated by a clear unstained line where the mutually flattened sides
are in contact. They are non-motile, and about the same size as staphylo-
cocci, from which, however, they are readily distinguishable by the paired
grouping of the cells. None of the above-mentioned cocci are known to
form spores,

A not infrequent cause of inflammation and suppuration in human
beings is the ‘ Pneumococcus’ (Dzplococcus pneumoniae) of Frankel, the usual
cause of pneumonia (180).

2. ANTHRAX (181) (Figs. 28,d@; 27,¢3; 5,¢,73 11, @,23 29). The
anthrax bacillus (8. azthracis) was discovered about the middle of the pre-
sent century (in the early fifties) in the blood of cattle suffering from splenic
fever. It was detected in the form of colourless, motionless rods lying about
between the blood-corpuscles. Although from the first suspected to be
the morbid agent, proof was not available until many years afterwards
(1863). Through Koch’s investigations into the life-history of the organism,
anthrax or splenic fever has found its way into all the text-books as a clas-
sical example of a bacterial infection. Koch followed the process of spore
‘formation, and obtained the first pure cultures of the bacillus, commencing
with this work his brilliant career as the founder of modern bacteriological
technique.

The anthrax parasite is a comparatively large bacillus, cylindrical in
shape, 3-6 » long by 1-5 » thick, varying within certain limits, as all bac-
teria do. Both in the blood and in the tissues single cells and cell-chains
occur (Fig. 27,c). In cultures the growth is chiefly filamentous, the plate
colonies being as a result fringed with shaggy edges, loops, and curls of
filaments stretching out into the gelatine. For the same reason stab cul-
tures have a bristly appearance. The cells are non-motile, but form spores
readily *.

The anthrax bacillus grows well in artificial cultures, but needs good
sources of nitrogen and carbon; it is a peptone bacterium. There is,
however, no doubt that it is not an obligatory parasite, but metatrophic, for
it has been observed to grow well and form spores in cow-dung and earth.
This indicates how the disease may arise in cattle, which are much more
subject to infection than human beings. In man, infection generally takes
place from skin wounds, and the disease remains localized, rarely spreading
to other parts. In cattle, on the other hand, it is with food that the

* With regard to spore formation, see p. 20; germination, p. 22}; resistance to heat, p. 76;
dryness, p. 77; poisons, p. 82 ; degeneration and attenuation, p. 28.

DIPHTHERIA I51

with Streptococci, and in some few cases of apparently indisputable diph-
theria it has seemingly been wanting. It is a true parasite, and in cultures
requires the very best nutriment. Growing best upon blood-serum, to which
has been added bouillon and sugar, it shows a tendency even on this medium
to produce involution forms, although it multiplies rapidly. Irregularly
‘swollen rods arise, and even cells with short branchings (Fig. 27). As was
already mentioned, these have been without sufficient reason looked upon as
representing a higher morphological stage than the normal rods.

In the diphtheritic membrane, and in fresh cultures, the bacillus appears
as a minute, somewhat clubbed or oval, rod-like cell about 1-5-2 » long
by o-5 » thick. It is non-motile, and spores are unknown. In young
cultures the cell-contents stain apparently uniformly. Not infrequently
strongly-tinged granules can be seen, and these when they are large and lie
at the ends of the cell give the impression of spores. They are, however,
nothing but ‘chromatin’ granules, such as have been already described.
The apparently uniformly-stained cell-contents have the same structure

Fic. 28. Pathogenic bacteria. a, ge lococcus pyogenes aureus (Micrococcus pyogenes); 4, Streptococcus
pyogenes; c, Micrococcus gonorrhoeae; d, Bac. anthracis: to the right, spore-bearing cells ; ¢, Bacillus (plectridium)
4etani, motile rods, motionless chains, spores; 4, Bacillus diphtheriae: some of the rods are clubbed, some contain
mtge ‘chromatin’ grains, and others show bands of shrunken protoplasm; g, Bac. tuberculosis, contents of some
rods shrunk to granules as is often seen in sputum prpenoy h, Bac. (Bactridium) typhi; i, Bac. (Bactridium)
colt; k, Vibrio cholerae, single cells and chains. Magn. about 1500.

as in other bacteria, the protoplasm surrounding a sap cavity which, as in
all elongated forms, is divided into chambers by transverse walls of plasma.
In old cultures the protoplasm diminishes in quantity, and the septa
retreat from one another, so that the stained rods then appear transversely
striped (Fig. 28, £), broad, colourless stripes alternating with coloured plasma
streaks, The actual structure of the cell does not change, but becomes
more visible by reason of the changes mentioned.

The diphtheria bacillus is not found in nature, and as yet has been detected
in a living condition only in situations where its derivation from human
patients was certain—on soiled linen, floors and walls of rooms, children’s
toys, and the mouth and nasal cavity of those attending diphtheria cases.
The dry cells remain infectious for many weeks.

152 BACTERIA IN RELATION TO DISEASE

The fact that the diphtheria bacillus deviates in shape from the cylin-
drical renders it easily recognizable, but absolute proof of its identity is
given only by experiment upon animals. The question of toxines and serum
treatment will be considered in the next chapter.

5. TUBERCLE (Figs. 28,2; 27,a; 14,g). The tubercle bacillus discovered
and isolated by Koch is, like the diphtheria organism, a true parasite (184).
Thanks to Koch’s investigations, the demonstration and differentiation of the
bacillus in sputum and diseased tuberculous tissues is now a matter of no
difficulty, but the isolation and pure culture is still an uncertain and delicate
operation. Even on the most suitable media, blood-serum or glycerine agar
at optimal temperature (38° C.), the organisms grow excessively slowly, and
need three or four weeks to form colonies as large as those produced by
other bacteria in three or four days. It seems as though artificial media
can never replace the living tissues of the host, and possibly we shall never
succeed in getting the tubercle bacillus to grow more rapidly in our cultures.
It may be, however, that a happy chance may show us some food, widely
differing from the stereotyped media, that will offer optimal conditions to
the organism. The fact that less ‘ nutritious’ media, media with glycerine
as a carbon, and ammonia as a nitrogen, source, permit slow growth, as do
also potatoes and other vegetable media, may lead to further investigations.

In nature the tubercle bacillus has never been detected growing and
multiplying, nor is it found in any situations where the possibility of
contamination by tuberculous secretions is excluded. The commonest
mode of infection for man is probably by the dried resting-stage, in
which the organisms preserve their virulence for months. For children
the use of the milk of tuberculous cows is a fruitful source of infection ;
the portal of infection in this case being the intcstine. For adults
the inhalation of tuberculous dust and the infection of wounds is the
commoner occurrence. The infection of the lung is doubtlessly in all
cases preceded and conditioned by a ‘ predisposition’ which permits the
inhaled bacteria to develop. Possibly minute lesions play a part in this.
The so-called heredity of tuberculosis is in many cases undoubtedly nothing
but heredity of the ‘ predisposition,’ although infection of the embryo zx
wtero has been observed and induced experimentally. Transportation of
the bacilli by the spermatozoa is impossible, and infection through the
ovum as yet unproven (135). Tuberculosis may attack any part of the
body. The commonest form of the disease is pulmonary phthisis, but it
frequently makes its appearance in the joints, in bones, glands, and other
organs. Nodular inflammatory centres arise, which change later on into
cheesy, necrotic masses. These contain enormous numbers of bacilli, the
proliferation of which in the cells causes the degeneration and the other
pathological changes that precede it.

The tubercle bacillus, like many other much-studied organisms, has been

TUBERCLE 153

fated to receive several names: Bacillus tuberculosis, Koch, 1884, Sclerothrix
Kochit, Metschnikoff, 1889, Mycobacterium tuberculosis, Lehmann and
Neumann, 1896, Tuberculomyces, Coppen Jones, 1896. It is a delicate,
slender rod, often slightly curved, non-motile. The average size is from
15 to 4u long by o2p to o-4p thick, but it is extremely variable in
length and thickness, both in the tissues and in cultures, in both of which
situations it sometimes appears in a filamentous form. On culture media
it gives rise to thick, opaque, scaly or wrinkled membranous colonies of dry
consistence. In consequence of the small size of the cell its finer structure
cannot be seen in detail (136). The cell contents, however, would seem to
be denser than those of other bacilli, for the tubercle bacillus is much more
highly refracting than any other species. It further differs from other
bacteria in containing a large amount of true cellulose in its cell-wall. It is
the greater density of the cell that enables it to retain anilin dyes with such
tenacity, a fact that facilitates immensely the detection of the bacillus in
tuberculous material. In very old cultures, and sometimes in sputum, the
bacilli often appear as dotted, strongly-stained granules alternating with
colourless spaces (Fig. 28,g). We have already seen that this occurs in the
diphtheria bacillus. In both cases it indicates a degeneration, and must
not be regarded as a specific structure—as a sign of sporulation, for
instance. Real spores are not yet known. The tubercle bacillus shows
sometimes also another sign of the discomfort that genuine parasites
undergo in artificial cultures. Involution forms arise—swollen rods and
feebly-branched filaments—reminiscent of the bacteroids of Leguminosae
(Fig. 14, g), which have by some investigators been wrongly regarded as
having morphological and systematic importance *.

Closely resembling the tubercle bacillus is the bacterium which is
presumably the cause of leprosy. It has not been obtained as yet in pure
cultures.

The bacteria already described can all be distinguished by their
morphological characters, doubtful cases being confirmed by experiments

* The translator’s observations do not permit him to agree with this view. According to his
investigations (Centr. f. Bakt., 1895), corroborated since by many other observers, the ramifications
of the tubercle filaments are examples of true branching, and, together with the many other points
of resemblance to Actinomyces, justify us in placing the organism among the filamentous fungi. The
suggestion made in 1895, that a saprophytic form of the bacillus would probably be found outside
the animal body, has also been fulfilled through the discovery, by Moeller (Therapeut. Monatshefte,
Nov. 1898), of a bacillus almost indistinguishable from the tubercle bacillus growing upon Timothy-
grass. Scveral other saprophytic and parasitic organisms having the closest resemblance to Koch's
‘bacillus,’ but growing at low temperatures, have now been described, and leave little room for
doubt that they, together with the organisms of mammalian and avian tuberculosis, are closely
related members of the same family. They show branched filaments in cultures, and when injected
into the living tissues most of them give rise to actinomycosis-like centres of radiating filaments,
terminating in clubbed ends (Zeitschr. f, Hygiene, vol. xxi, 1899).

154 BACTERIA IN RELATION TO DISEASE

upon animals. The following species, on the other hand, are extremely
difficult, and in some individual cases impossible to determine with cer-
tainty.

6. THE COLON BACILLUs (Bactridium [Bacillus] colt commune) is found
constantly, and in large numbers, in the human intestine and in the excreta.
It is in general nothing but a harmless commensal organism, but it may
under certain circumstances become pathogenic both in animals and in
man. The fact that a number of metatrophic bacteria very similar to
B. colt are known makes its recognition very difficult, and it is moreover
almost indistinguishable from the parasite of typhoid fever, Bactridium typhi
(Figs. 28 and 5). Endless pains have been taken to find reliable and
constant points of difference between these two bacteria, and yet even
now the differential diagnosis of the two species is a work of great
delicacy (137). The very circumstance that the colon bacillus grows more
quickly than the typhoid organism only adds to the difficulty, because in
the attempts to isolate the latter from tissues or from water it is over-
powered and choked by the rapidly-multiplying colon bacillus.

Common to both species is the rod-like shape (Fig. 28, 4 and 2).
The dimensions are about the same: B. typhi, 1-4 w long by 0-6-0-8 » thick,
B. coli, 1-34 long, o-4-0-6 pw thick; i.e. B. typhi is rather the larger of the
two. Both are actively motile and peritrichously ciliated. The cilia are
too delicate for their number to be of determinative value. They do not
liquefy gelatine, and spores are unknown in both species. In the absence
of morphological differences, physiological peculiarities have been utilized.
Such are, on the part of B. colz, the fermentative power, the production of
gas, the acidulation and coagulation of milk, and the indo! reaction when
growing in peptone solutions. All these properties are absent in the case
of the typhoid bacillus. Furthermore must be remembered the quicker
growth of &. coli, and its more humble requirements as to food-stuffs. As
was remarked on pp. 55, 56, this last peculiarity is deserving of special atten-
tion in differential diagnosis. It shows also that the 2. colz, an ‘ammonia
bacterium,’ is a very simple metatrophic organism, as is indicated by its
frequent presence in dirty water. The typhoid organism, on the other
hand, is paratrophic, and in all probability never occurs in potable water,
except where introduced in the dejecta of typhoid patients. Such dejecta
carry with them, of course, plenty of nourishment for the bacteria.

These few remarks will suffice to indicate in outline what we know at
the present time concerning the biology of the typhoid parasite. In typhus
abdominalis the bacilli can be demonstrated in all the abdominal organs
(spleen, liver, kidney, lymphatic glands, &c.), generally in little heaps
between the cells, and also in the blood. Infection takes place undoubtedly
from the intestine.

A species allied to the typhoid bacillus is B. typhi murium, that was

CHOLERA 155

recommended by Loffler for the destruction of field mice, and has already
“been used with much success (138).

7. CHOLERA. When Robert Koch returned in 1883 from his famed
journey to India, the headquarters of cholera, having discovered the parasite
that causes the disease, it seemed as though the recognition of the organism
would in all cases be an easy task (139). It was the only pathogenic
organism of its kind then known, a motile, curved, wriggling rod. But the
occurrence of several European epidemics of cholera gave opportunity for
more extended researches, and evidence was soon forthcoming on all sides,
showing that micro-organisms similar to the cholera bacillus were to be
found in almost all kinds of water. This fact must strike every one who
makes a bacteriological examination of stagnant water (Fig. 22,4). Diligent
search for physiological differences was made, but many of these, the growth
in gelatine and the indo] reaction, for instance, were soon found to be
unreliable. Whether the newest method, Pfeiffer’s ‘immunity reaction’
(see p. 167), will fulfil the expectations it has raised, remains to be seen.
Experiments on animals too must be elaborated in other directions than
hitherto. Animals, even in India, are never attacked spontaneously by
cholera. Guinea-pigs can be infected per os with the germs only after
special preparation. If instead of passing through the natural portal of
infection, the mouth, the bacteria be injected into the peritoneum, the
animals die, it is true, but only with the symptoms of general bacterial
poisoning, and afford no clue which could serve to differentiate between the
cholera germ and other aquatic vibrios. Even the bacteriological analysis
of the excreta in suspicious cases is accompanied by great difficulties.
For this reason all public announcements of the discovery of the germ
should be regarded with a considerable amount of scepticism.

In true Asiatic cholera the characteristic dejecta (rice-water stools)
contain enormous numbers of the Koch vibrios, the mucous flocculi
discharged being practically pure cultures of them. They are confined to
the intestine, the wall of which remains generally intact, although it is
occasionally invaded by the proliferating bacilli. They do not attack other
parts of the body, as a rule being able to exert their full toxic power in the
intestine, from which they disappear in non-fatal cases after one or two
weeks. Whilst the experience gained in epidemics is enough to establish
the causal connexion between the bacilli and the disease, the result of
laboratory infections and voluntary experiments afford conclusive proof.
Pettenkofer and Emmerich (140) swallowed pure cultures of vibrios from
the Hamburg epidemic and were attacked, the former slightly, the latter
very severely, with choleraic symptoms. That in cholera, as in all other
infectious diseases, a certain something, a ‘ predisposition, must be present
before infection can take place, is self-evident. In the case of cholera an
important part is played by the diminution of the acidity of the gastric

156 BACTERIA IN RELATION TO DISEASE

juice, and therefore of its antiseptic power, such as occurs from immoderate
eating or drinking, or from climatic causes in summer. Every gastric or
intestinal disturbance will be a predisposing condition.

The Koch ‘comma’ bacillus ( Vibrio cholerae, Figs. 28,23 2,¢3 5,23 6,
a-c) is, like other vibrios, a minute curved, actively motile rod (2 » by 0-4 y),
bearing a cilium at one end. Occasionally two cilia are present, never more.
On the surface of fluid media (bouillon or asparagine + sugar solution)
the organism, being aerobic, forms thin membranes, and at the same time
renders the underlying liquid turbid. Besides the more numerous single
vibrios, long or short motile chains occur. These have been described as
spirilla, but are really nothing but chains of vibrios joined end to end
(Fig. 28, %). True endospores, although doubtlessly produced, are not
yet known. Perhaps sporulation takes place only under tropical con-
ditions of life. The structures found in old cultures that have been
described as spores are only due to involution and granular degeneration
of the cell-contents, as is evident from the extraordinary shapes of the
cells.

The sporeless comma bacilli of our cultures can endure drying only for
a very short time, perishing in a very few hours. A dry resting-stage as in
the tubercle bacillus is therefore wanting, and infection from dry material
impossible. As a metatrophic organism, however, it can live in water for
weeks, and is by no means tied down to peptonized food-stuffs. The table
on p. 55 shows it to be an ammonium bacterium that thrives well in gly-
cerine sal-ammoniac solution if the reaction be suitable. Acidity arrests
growth at once. These facts make it clear how the cholera germ is able to
live and multiply in dejecta, on moist soiled linen, or in water containing
putrescible substances. From albuminous compounds (in peptone broth, for
instance) the cholera bacillus forms indol and other putrefaction products,
while from sugar it can produce lactic acid.

From contaminated river or well-water used for drinking or for house-
hold purposes the bacillus reaches the stomach and intestine by way of the
mouth. It is therefore of the highest importance that all drinking-waters
should be under strict surveillance.

In its morphology and in its metatrophic mode of life the cholera vibrio
resembles our indigenous European aquatic vibrios (141), and in summer it
is, like them, able to live in rivers and ponds. It is, however, originally
a tropical organism, its native place being the East Indies, where it lives in
foul water just as its relatives do with us. Under such circumstances only a
high temperature, 30°-40° C., is necessary to permit it to thrive and develop
its virulence. This condition is present in our climates in summer. Some
of our indigenous vibrios, V. berolinensis and V.danubicus, can develop patho-
genic properties, but it is not known whether they are ever the cause of
disease in human beings. That the tropical species are the more virulent

CHOLERA — 157

is in harmony with the well-known fact that vegetable poisons are always
stronger in the tropics than in temperate climates—the hashish of hemp, for
example.

We see from the above facts that cholera is caused by a tropical aquatic
organism, a metatrophic bacterium, which is not able to obtain a permanent
footing in our climate, but is introduced afresh from time to time through
the various channels of trade and communication.

8. In addition to the well-known species of pathogenic bacteria already
described, there are many with which we are less familiar, the spirochaete
of relapsing fever, the glanders bacillus, swine erysipelas, and other animal
infections. In many diseases, such as rabies, scarlet fever, measles,
whooping-cough, the specific organisms, although supposed to be bacteria,
are not yet known. A few other pathogenic micro-organisms and fungi
were mentioned in Chapter IV.

CHAPTER XVII
BACTERIA IN RELATION TO DISEASE (Continued)

8. Mode of Action of Bacteria, and the Reaction of the Organism.
Serum Therapeutics and Immunity.

WHEN bacteria by one means or another have obtained entrance into
the body, there elapses before the outbreak of the disease a certain interval,
the period of incubation. In the guinea-pig, after infection with Strepto-
cocci, from fifteen to sixty hours pass before symptoms begin ; after infection
with cholera germs, from one to three days ; in human beings the period of
incubation for anthrax is’‘three to seven days, for syphilis, three to four
weeks, for rabies forty days or more. During this time the invading bac-
teria are multiplying and struggling for mastery with the invaded organism.
The struggle may go on without any outward signs at all, or it may be
accompanied by slight disturbances of health. If the tissues conquer at the
very commencement, the disease never breaks out. It is quite possible that
many a temporary indisposition and fleeting local pain may be the expres-
sion of such a conflict, in which the defeat of the bacteria prevents the onset
of grave illness, for it is certain that pathogenic bacteria enter the body far
more often than the number of actual illnesses we are subject to would lead
us to suppose.

But if the first attempts at defence on the part of the body are
futile, and the bacteria are able to multiply, then the disease breaks out
and severe symptoms show that the struggle between host and parasite has
become more intense. Of these symptoms it is now no longer possible to
say which are measures of defence on the part of the tissues, and which are
signs of defeat.

A weakening of the body through the abstraction of nutriment by the
bacteria is hardly possible, for the micro-organisms, even when multiplying

BACTERIAL POISONS 159

abundantly, could take up only a minimal amount of substance. But if
metatrophic ferment or putrefactive bacteria invade the body they would
break up not merely their own bulk of organic substance, but a hundred or
a thousand times as much. Asa secondary aggravating effect, such changes
must not be overlooked in bacterial infections; they may, for instance,
cause destruction of tissue.

Bacteria may, from purely physical reasons, cause local disturbances
of the circulation by accumulating in the capillaries. This happens in
anthrax, where the vessels are for short distances completely plugged by
the bacilli.

These effects of bacterial intrusion, to which formerly great importance
was attached, are now looked upon as merely subsidiary phenomena. The
violent symptoms and serious consequences of a bacterial infection are now
known to be due to the action of ¢oxines, poisons produced by the micro-
organisms.

The contagium animatum that invades the body from outside produces
in the tissues the zrus inanimum, the lifeless poison. The investigation of
these poisons is now in full progress, but, as may be readily conceived, is
attended by great difficulty, for they are all of them extremely unstable
bodies, and many are of proteid nature, and therefore less accessible to the
ordinary methods of chemical research. It is certain that they include
substances of very various chemical composition.

The oldest known group is that of the ptomaines or putrefactive alka-
loids, so called on account of the resemblance of their chemical reactions to
those of vegetable alkaloids. They play, however, only a very small part
in the toxic action of pathogenic bacteria. The isolation of the actual
specific toxines (142) from the cultures has been attended with but little
success as yet, in spite of the great amount of labour that has been spent
upon the subject, and we speak of the ‘ toxines’ without really knowing
them in a pure state. But to have them chemically pure, although very
desirable, is not absolutely necessary, as the following description shows.
To separate the poisons dissolved in the culture fluids from the bacteria,
it is sufficient to filter bouillon cultures through porous earthenware
or kieselguhr. If the poisonous filtrate thus obtained, for instance from
tetanus cultures, be injected into animals, they die with the same sym-
ptoms of lock-jaw as if they had been injected with the bacilli themselves,
In the same way the filtrate of diphtheria cultures kills the animal with
the symptoms of diphtheria. In short, it has been shown that all patho-
genic bacteria produce soluble poisons, that these poisons alone are able to
give rise to the disease, the nature and course of which depend solely upon
the nature of the toxines. Old cultures in particular contain large quanti-
ties of toxines ; they are highly ‘ poisonous,’ whilst the younger growths are
distinguished by the more rapid growth of the micro-organisms—they are

160 BACTERIA IN RELATION TO DISEASE

more ‘virulent’ *. The filtrates can be concentrated by evaporation 77
vacuo, and much more powerful toxic solutions obtained. By treatment
with alcohol or neutral salts, solid precipitates of still greater toxicity can
be gained. These precipitates contain of course a mixture of different sub-
stances, albumens and albumoses from the bouillon, and inorganic consti-
tuents besides the toxines. A complete isolation of the latter has not yet
been possible. They were formerly supposed to be proteid bodies (sox-
albumines), but further purification has shown that they may possibly be of
simpler structure. They are extremely poisonous, one twenty-thousandth
part of a milligramme (=;=;557, gram.) of a concentrated tetanus toxine
being sufficient to kill a mouse. For a human being 0-23 mg. would
probably suffice.

The poisons contained w7thin the bacteria (intracellular toxines) can
also be extracted. The best known example of these is Koch’s tuber-
culin (143), a purified glycerine extract of tubercle bacilli. Pure toxines
have not yet been obtained by this method ; the tuberculin of 1890 con-
tained, in addition to the unknown toxine albumens, albumoses and peptones
from the culture media. The newest tuberculin preparations too, Koch's
TR and TO, are extracts of dried and pounded bacilli from highly virulent
cultures. The finely pulverized bacilli are centrifuged in distilled water.
The first clear yellowish extract is the TO and has properties resembling
those of the old tuberculin. Repeated centrifuging of the sediment of
TO gives the more effective TR. Both TO and TR are mixed with
20 per cent. glycerine to assist their preservation. They are nothing but
virus-containing mixtures of all the soluble constituents of the tubercle
bacilli together with fragments of the crushed cells.

Both the bouillon filtrates and the precipitated toxines retain their
virulence for a long time, but they are very sensitive to high temperatures
and alkalies or acids. Tetanus toxine is destroyed in a few minutes at
65° C., diphtheria toxine in two hours at 58° C. This instability, although
suggestive of the properties of enzymes, must not be regarded as indicating
a relationship of the two classes of compounds.

Just as the poison of serpents or the alkaloids of poisonous plants are
the products of the vital activity of those organisms, so are the toxines
products of bacterial life. They are excreted from the bacterial cell during
life, and are poured into the culture fluids in abundance when the cells die;
hence the great fozsonousness of old cultures, As the microscope shows
us, too, that bacteria often become disintegrated in the tissues, they may
evidently contribute in this way also to the poisoning of the body.

* Translator’s note. These terms have been translated just as they stand in the German, where
the meaning is clear; but they can scarcely be regarded as good English, unless the word virulent
is allowed to connote the power of multiplication possessed by the organisms.

PHAGOCYTOSIS 161

The fact that several species of fossil bacteria are known from the
carboniferous and other periods (144) shows that they are among the oldest
inhabitants of the earth, and were already widely distributed when the
Tertiary Epoch and the evolution of warm-blooded creatures began. We
should therefore expect that these would, in the course of their phylo-
genetical progress, have gradually developed the power of resisting the
attacks of the invading micro-organisms.

The investigation of these protective properties is the subject of ex-
tensive research and has already, in the field of sero-therapeutics, borne
fruit that promises much for the future, although it is true that at present
experience and theory are opposed to each other on many points, and
a long time must elapse before a reliable foundation is reached.

The first of the defensive appliances of the body to which attention
was directed (mainly by Metschnikoff and his followers) were the white
blood-corpuscles or leucocytes, the important part played by which in
physiological and pathological processes has come to be more and more
recognized. These naked cells that
have their origin in the spleen, the
bone-marrow, and other haemopoeic
organs of the body, are often called
‘wandering cells, because they are :
able to leave the blood-vessels and :

Fic. 29. Phagocytosis, from Metschnikoff. a,

lymphatics in which they are distri- leucocyte from pigeon’s blood with anthrax bacilli.
These are in some cases intact and stain well

buted over the whole body and pene- (black), in others degenerate and pale. The oval
ody is the cell nucleus. 4, a living leucocyte from

trate between the fixed cells of the pigeon, devouring anthrax bacilli.

tissues. They are found, for instance,

actively multiplying in the intestinal villi during digestion, and as * pus-

corpuscles’ they constitute the chief part of pus. An increase of them in

the blood-vessels is spoken of as /eucocytosis.

Now in the blood of animals infected with anthrax, and in suppurative
processes of all kinds, the leucocytes frequently (although not always) con-
tain bacteria. These are sometimes present in great numbers, and often
appear to be dead, inasmuch as the cell-contents are changed and stain
badly (Fig. 29, @ and 4). On these phenomena Metschnikoff founded his
theory of Phagocytosis (145). The wandering cells were supposed to
function as phagocytes (devouring cells), to take up and to kill any bacteria
that had penetrated into the tissues. They were supposed, in fact, to
constitute an army of defence that could be mobilized and sent to any
part of the body. This view, the result of direct observations, was further
strengthened when the chemotactic properties of leucocytes (153) were
studied, and, above all, their behaviour towards bacteria and towards the
products of their metabolism contained in pure cultures.

Capillary tubes filled with bacterial substances, introduced into the

FISCHER M

162 BACTERIA IN RELATION TO DISEASE

tissues, were found after a short time to be p:ugged full of leucocytes. The
immigration of these was attributed to chemotaxis, sometimes, it is true,
without due consideration of the necessities of Weber’s law (p. 80). Since
bacterial foci in the tissues are almost always surrounded by a crowd of
leucocytes, and since these are laden with bacteria, Metschnikoff’s theory
seemed well founded.

There was, however, a discovery made respecting the serum of the
blood that seemed to show that phagocytosis was neither the only, nor
the most important, means of defence that the body possesses against
invading bacteria. If bacteria were mixed with blood-serum and _ this
serum examined from time to time by the plate culture method, it was
found that fewer and fewer colonies of bacteria arose, until, after an interval
of some hours, the serum was sterile, the bacteria being all dead. This
bactericidal influence of blood-serum, which is apparently exerted upon
all bacteria, is destroyed by the addition of distilled water or by heating
the serum for an hour to 55° C. According to Buchner it is due to certain
substances, alexines (146) (defensive substances), which, like the toxines, are
very unstable, and have not yet been obtained in a pure state. Seeing that
serum which has been rendered inactive by distilled water can be restored
to bactericidal power by the addition of 0-3 per cent. NaCl or other salts,
there is evidently many a mystery hidden in the ‘alexines.’

In the early eighties, when the alexine theory and the theory of
phagocytosis were in sharp opposition, most weight was laid upon the
destruction of the bacteria. Afterwards, however, the toxine theory of
infection (142) was brought forward, and the question arose as to the
neutralization of bacterial poisons and the presence of ‘antitoxines.’ Since
bacteria of the most various kinds could be destroyed by one and the
same poison, it was not necessary to assume the existence of specific
alexines. The different toxines, on the other hand, seemed to require
different antitoxines, just as almost each different poison requires a specific
antidote. :

Since the animal body is the most delicate, and at present, indeed,
the only reagent we possess for bacterial toxines (which, moreover, we do
not know in a pure state), it is evident that the antitoxic properties of
the blood-serum can only be investigated by means of experiments on
animals,

As in the case of the bacterial poisons, it has not yet been possible
to isolate the antitoxines, They are said to be somewhat more resistant
than the toxines. But the statements of different investigators differ even
as regards the same antitoxines, since they can be examined of course only
in a dissolved state in the serum.

Since the cell-free serum of the blood is a lifeless fluid, we are forced
to regard the leucocytes as the producers and carriers of those substances

TOLERATION AND IMMUNITY 163

which give the serum its bactericidal and antitoxic properties. Alexines
and antitoxines are products of the leucocytes, which fight against the
bacteria more by means of these substances than by virtue of their
phagocytal functions. This way of looking at the matter effects a
compromise, that is already widely recognized, between the theories of
Metschnikoff and the French school, and the German doctrine of anti-
toxines (147).

Another natural means of defence against bacterial poisons possessed
by man and all other organisms is the power of acquiring toleration of
poisons. It is indeed only a special case of the ability possessed by all
living substance to accustom itself to external influences when these increase
slowly and do not exceed a certain degree. It is well known that this
power is highly developed in some organisms and is subject to great
individual variation. We only need recall the acclimatization of animals,
of plants, of the races of mankind, and even of bacteria themselves, living
as these do in pure cultures under conditions totally different from those of
their native haunts. Instances of habituation are familiar enough ; arsenic-
eaters are able in time to endure c-4 gram of arsenic at once, the fatal dose
being ordinarily from 0-1 to 0-2 gram. Morphinomaniacs accustom them-
selves gradually to four times the fatal dose (0-4 gram per os).

By very gradual augmentation of the dose white mice have been made
immune to ricin (the poison of the castor-oil seeds), so that they were able
finally to stand one hundred times the fatal dose without injury (148).

The body can also accustom itself to the toxines of pathogenic bacteria,
and it will be found worth while to review from this standpoint (neglecting
for the time being the antitoxine theory) some of the innumerable observa-
tions that have been made during the last ten years in the fields of immunity
and serum therapeutics. For instance, in order to make a guinea-pig
actively immune to diphtheria poison, one proceeds as follows (149). First
of all the minimal lethal dose of a virulent filtered bouillon culture is
determined ; i.e. the number of cubic centimetres which suffice to kill
a guinea-pig when injected subcutaneously. Perhaps 0-3 c.c. per kilo.
body-weight is enough ; i.e. for a guinea-pig of 250 grammes about 0-08 c.c.
This quantity is naturally not a fixed amount, comparable to a normal
solution used for volumetric analysis; it changes according to the virulence
of the bacilli. One then begins by injecting a guinea-pig with a much
smaller amount, 0-001 c.c. for instance, which perhaps causes only temporary
sickness. Then the dose is gradually and continually increased until
at last an amount far exceeding the fatal dose can be borne without
injury. The animal has become immune to the poison. In this way,
too, horses are immunized for the preparation of Behring’s diphtheria
serum. Instead of minute doses of a highly toxic filtrate, larger doses
may be employed of a filtrate whose virulence has been weakened either’

M 2

164 BACTERIA IN RELATION TO DISEASE

by heat (50°-70°) or by the addition of chemical substances such as
trichloride of iodine or carbolic acid. Finally, inoculation with the at-
tenuated bacteria themselves may be resorted to. All the methods lead to
the same results, and the principle involved in each case is the same,
for the treatment of bacteria with substances that hinder their vegetative
development naturally reduces also the amount of poison they produce.
It was the method of immunizing by means of weakened pure cultures that
formed the starting-point of the treatment with the toxines themselves.
Behring (150), by using tetanus bacilli that were attenuated in different
degrees by iodine trichloride, was able in about seventy-five days to
immunize a horse to such an extent that it endured the injection of 100c.c.
fully virulent tetanus culture. An untreated animal was killed by o-5 c.c.
of the same culture. To attain this degree of immunity several hundred c.c.
of attenuated cultures were necessary. It took to render a horse highly
immune to diphtheria eighty days and eight hundred c.c. of filtered virulent
diphtheria toxine (151). It could then be tapped for curative serum. By
continued treatment the resistance to virus can be still further increased and
persists for a considerable time. In tetanus-immune horses the immunity
lasts two years, and in diphtheria-immune horses about the same time. In
experiments with other bacterial poisons the immunity attained was not
so persistent, although enduring many weeks or months. Variations are
unavoidable.

Assuming that artificial immunity is only the acquired toleration to the
poisons, its disappearance would be explained by the poison gradually leaving
the body and finally disappearing entirely. The cells of the body would
thus gradually lose their power of toleration. The fact that the immunity
lasts two years and more is no argument against this, for we know that
some poisons are excreted from the body with great slowness ; mercury, for
instance, does not disappear for six months or more after administration.
Immunization by injection of toxines must be regarded as an experimental
chronic intoxication.

Such an artificially-induced immunity protects of course not only from
the direct administration of the poison, but also from the virus excreted by
invading bacteria. Tetanus bacteria, for instance, are not arrested in their
growth by the antitoxine, but their poisons are neutralized. The same
takes place in diphtheria (152), while in other diseases it is not yet clear
whether the neutralization of the poison is not accompanied by arrest of
growth of the bacteria (Cholera, p. 167).

The immunity produced is strictly specific; the diphtheria poison, for
instance, imparts immunity only against diphtheria, not against tetanus or
anthrax, or any other infections. This again can be easily explained by
toleration, which is in fact competent to account for all the phenomena
‘which the immunized animal exhibits in its own organism. Towards other

IMMUNITY 165

organisms, however, the immunized animal shows new properties which
underlie the practice of serum therapeutics. By injecting the serum of
immunized animals into other untreated individuals these too can be made
passively’ immune, and be caused to share in some degree the properties
of the serum-giver. The milk also of immunized animals has the same
power as the serum, although to a less extent. The milk of diphtheria-
immune goats, for instance, possesses from one-fiftieth to one-thirtieth the
immunizing strength of the blood-serum of the same animal (153). Nume-
rous experiments upon animals have shown that an immunized mother
can impart her immunity to her offspring. This inherited immunity is not
lasting, but disappears in two or three months. Immunity is not transfer-
able by the father (154),

All the phenomena exhibited by immunized animals can be explained
without the assumption of ‘antitoxines, merely by supposing that the
toxines that have been introduced into the blood leave the body very
slowly, not disappearing entirely for month$ or even years. As long as
minute quantities of toxines remain the animal is immune, although its
immunity is steadily decreasing. And these remainders of toxine in the
serum induce immunity when injected repeatedly into other animals. The
higher the doses to which the serum-yielding animal has been subjected
the greater amount of toxine will its serum contain. The toxine may
accumulate to such an extent that the animal itself may die of poisoning,
although its serum has reached the highest degree of immunizing power.
This hyper-sensitiveness which has been observed by Behring (155) is quite
inexplicable by means of the antitoxine theory, but can be accounted for
without difficulty by the toleration hypothesis.

If the effect of the curative serum depended only upon the introduction
of very dilute toxines, we should expect that it would be greatest defore the
incorporation of the infecting germ, inasmuch as toleration would have been
established before the disease broke out. Wide experience has shown,
however, that good results follow the injection of either the protective serum
blus the cell-free toxines, or the serum f/us the bacteria simultaneously or
one after the other. Here again a plausible explanation is possible without
bringing in the antitoxine theory. It is conceivable that the toxine in
the serum of toxine-tolerant animals is more easily absorbed than the fresh
toxines in the bouillon filtrates, and gets into action before the latter (156).

‘It may be argued that if free toxine were the immunizing agent in
the serum the same symptoms must arise from a serum injection as from
the injection of a toxine. As a matter of fact we find that the injection
of serum is not borne by the organism quite without signs of reaction.
Often enough, as is well known, there are violent secondary symptoms,
ind, inasmuch as the amount of toxine, even in the most effective serum,
must be very small, it is not necessary that distinct reactions should arise.

166 BACTERIA IN RELATION TO DISEASE

These arguments, it is true, do not give anything like a complete
explanation of the phenomena, but they show how far we can go without
assuming the existence of specific antitoxines, using in their place the
phenomena of toleration, phenomena which are, it is true, still unexplained.

As soon as we grant the existence of antitoxines, another mysterious
property of the organism has to be assumed, namely the ability to produce
a specific antitoxine for each kind of toxine, and possibly for every other
poison. Theoretical medicine is at present strongly inclined to explain
immunity and serum therapeutics by such antitoxines. By the incorpora-
tion of poisons the body is stimulated to produce antidotes; these increase
as immunity increases, and are the effective agents in the serum. Serum
therapeutics and serum immunity consist in the introduction into the
organism of ‘anti-substances.’ These substances are, as we have seen, as
yet quite unknown, and opinions are divided as to their manner of action,
whether they neutralize the poison by chemical combination or only
stimulate the body to greater resistance.

The theory of ‘anti-substances’ is also involved in the terminology
which is used by Behring and Ehrlich (158) to designate the various
strengths in which the diphtheria serum is sold. By a ‘normal’ toxine is
understood a filtered virulent diphtheria bouillon culture of which 0-3 c.c.
suffices to kill a one-kilo. guinea-pig when injected subcutaneously. For an
animal of from 200 to 300 grm. o-I c.c. would be enough. A normal anti-
toxic unit is a solution of antitoxine of which 0-1 c.c. destroys the virulence
of 1 c.c. normal toxine, i.e. sufficient to protect a guinea-pig from ten times
the fatal dose. This of course can only be determined by experiments on
animals, toxine and antitoxine being injected together. Therefore to make
a guinea-pig immune against the lethal dose of o-1 c.c. normal toxine,
0-01 c.c. of the normal antitoxine is necessary. Normal serum contains in
Ic.c. one ‘antitoxic unit,’ and therefore 1 c.c.of such a serum is sufficient
to protect ten guinea-pigs against the fatal dose (157).

The therapeutical and clinical questions involved in the serum treat-
ment cannot of course be discussed here. A mature and final judgement
as to its efficacy will not be possible for many years (159).

A theoretical explanation of the phenomena of immunity is rendered
very difficult: by the distinction that has been supposed to exist between
bactericidal and antitoxic action. While it is generally admitted that pro-
tection from diphtheria and tetanus is caused by ‘ antitoxines,’ that is to say,
by substances that: destroy the poison of the bacilli, it is asserted that in
artificial immunity against cholera, typhoid, and some other diseases, the
chief part is played by dactericidal substances. The bactericidal effect is
said to be specific in each case. Pfeiffer’s cholera-serum reaction, the
meaning of which is at present the subject of a good deal of discussion,
may here serve as an example of these phenomena (160).

SERO-THERAPEUTICS 167

To immunize a guinea-pig against cholera, dead bacilli are injected
into the peritoneum, and afterwards living virulent bacilli in gradually
increasing doses. By these means the animal is rendered immune and its
serum can be used for showing the specific reaction. If about 30 milli-
grams of this serum be mixed with a fatal dose of virulent bacilli, and the
mixture be injected into the peritoneum, no ill effects are produced. The
bacilli are destroyed by the serum. They become clumped together, lose
the power of movement, and break up into granules. Outside the body,
too, in a hanging-drop preparation under the microscope the process can
be seen. The bacilli become motionless, stick together, and undergo
granular degeneration. According to Pfeiffer it is strictly a specific reaction
which can be used to differentiate the cholera bacilli, no other bacteria, not
even the vibrios, behaving in this way. The facts have not been received
without some scepticism, and the reliability of the reaction is by no means
universally recognized. The isolation of the active substance in the serum
has not been accomplished any more than in the case of other antitoxines.
Seeing that ‘a serum which had been putrefying for months retained its
specific activity almost unweakened’ (161), it is evident that the ‘ anti-
baeterial’’ bodies must have the stability of mineral substances, and be very
different from antitoxines and all other products of the animal body. The
facts that the cholera bacilli in a hanging-drop culture finally recover from
the paralyzing effects of the serum, and that pure diluted normal serum
of pigeons undergoes a very similar granular degeneration, show us that
we must be cautious in forming an opinion. The whole phenomenon has
a very suspicious similarity to plasmolytic changes, such as might well be
caused by the salts of the serum or of the bouillon (Fig. 6, p. 8).

The observations cited do not, of course, embrace all that a theory of
immunity has to go upon, but they are the chief data) By immunity has
always been meant insusceptibility to a disease—the power of resisting an
invading contagium. According to recent discoveries we must distinguish
between an immunity to the bacteria and an immunity to their toxines.
Furthermore, a distinction must be drawn between natural or congenital,
and acquired immunity. Cold-blooded animals, for instance, are immune
to the diseases of warm-blooded ones, our domestic animals are immune to
cholera, the dog to small quantities of anthrax bacilli. Individual variations
occur as might be expected. We see it in man where a personal immunity
seems often to exist, and must probably be regarded to some extent as
a matter of ‘ predisposition.’ The age of the individual, too, brings changes
in the natural immunity, as is shown by the existence of children’s diseases.
Perhaps these are ‘immunity diseases’ intended to prepare and fortify the
young mortal against bacterial enemies to come, but the question cannot
be discussed here.

Immunity can only be acquired by a pathological change, be it by the

163 BACTERIA IN RELATION TO DISEASE

natural attack of a disease or by artificial inoculation of it. The oldest
protective inoculation, Jenner’s vaccination for small-pox (discovered 1796),
started from the observation that an attack of cow-pox producing only
slight symptoms of disease protected human beings from the dangerous
variola. Up to the present day we know neither the organism of vaccine
nor the active principle of vaccine lymph. In the case of rabies, too, for
which Pasteur (162) introduced a protective inoculation with weakened virus
(dried tissues of rabid animals), the cause of the disease is not known. For
these two kinds of inoculation which formed the starting-point for all
modern investigations on protective inoculation we are still unable to give
any exact explanation.

It was Pasteur, too, who introduced the methods of protective inocu-
lation for anthrax, which have been of such value in. France. The practice
has resulted in reducing mortality from anthrax in cattle from 5 per cent.
to 0-3 per cent., and in sheep from Io per cent. to I per cent. (168).

In spite of garrulous indiscretion on the part of certain people, and
the dishonest cupidity of others, Koch’s tuberculin inoculations (184) must
always have the credit of being the first method in which bacterial products
were used, and not the bacteria themselves, as in Pasteur’s anthrax inocula-
tions, nor an uncertain ‘something,’ as was the case in vaccination. Koch’s
method formed the basis of all the innumerable investigations that have fol-
lowed it, including Behring’s method of serum treatment, which, as we have
seen, is at bottom nothing but the injection of toxine-containing serum.

To formulate a theory of immunity that shall not be lost in clouds of
hypothesis is at present impossible. The alexines, antitoxines, lysines, and
antilysines, that the wordy research of the last few years has given us, are
at present quite unknown (164). The inextricably complex inter-relations
of the antitoxic and antibacterial properties of the body-fluids have not yet
been cleared up, and even the effect of normal blood-serum upon bacteria
is but imperfectly understood. And the greatest difficulty of all is to
explain the long persistence of immunity after recovery from natural attacks
of infectious diseases.

NOTES

1. (p. 1). ANTON v. LEEUWENHOEK, Arcana naturae detecta. The figure dates from
the year 1683, and is taken from a new edition of the Arcana published in
1722 (vol. ii. p. 40).

2. (p. 2). ROBERT KocH, Kreisphysicus in Wollstein, Die Aetiologie der Milzbrand-
krankheit, begriindet auf die Entwickelungsgeschichte des Bacillus anthracis ;
1876. Beitrége z. Biol. der Pflanzen, herausgegeben von Ferdinand Cohn,
vol. ii.

3. (p. 2). Of newer general works may be mentioned: FLUGGE, Die Mikroorganismen
(Engl. transl. publd. by the Sydenham Society), 3rd ed. 1896. LEHMANN and
NEUMANN, Atlas and Outlines of Bacteriology, 1897. For the fermentation
organisms, LAFAR, Technische Mykologie, 1897 (English transl. 1898). Up to
the present only vol. i (Schizomycetes) has appeared (see note 126).

Full references to bacteriological papers will be found in BAUMGARTEN’S
Jahresbericht iiber die Fortschritte in der Lehre von den Garungsorganismen,
and in the Centralblatt f. Bakteriologie. This appears in two sections : (1) medical
and hygienic bacteriology, and (2) general agricultural and technological
bacteriology, physiology of fermentation, and phytopathology.

The literature of the French school will be found in Mack's Traité pratique
de Bactériologie, 2nd ed. 1891.

4. (p. 2). LOFFLER, Vorlesungen iiber die geschichtliche Entwicklung der Lehre von den
Bakterien. Part I, up to the year 1878 (all that has appeared), Leipzig 1887.

5. (p. 4). A synopsis of researches on the structure of the bacterial cell and original
observations will be found in BUTSCHLI’s Weitere Ausfiihrungen iiber den Bau
der Cyanophyceen und Bakterien, Leipzig 1896, and in A. FISCHER’s Unter-
suchungen iiber den Bau der Cyanophyceen und Bakterien, Jena 1897.

6. (p. 12). For the classification of the pigment bacteria here adopted see BEYERINCK,
Die Lebensgeschichte einer Pigmentbakterie, Botanische Zeit. 1891; and also
SCHROETER, Ueber einige durch Bakterien gebildete Pigmente, Cohn’s Beitrage
z. Biol. vol. i.

7. (p.12). Details as to the assimilation of these green bacteria (which are perhaps
minute green algae, Profococcaceae) may be found in ENGELMANN, Zur Biologie
der Schizomyceten, Bot. Zeit. 1882.

8. (p. 14). An acute discussion of the subject is given by N AEGELI, Ueber die Bewegung
kleinster Kérperchen, in Untersuch. ii. niede re Pilze, 882. See also Sitzungsb.
Miinchener Akad., mathem.-phys. Klasse, 1879.

170 NOTES

9. (p. 14). LOFFLER, Centralbl. f. Bakt. vi and vii. Since Léffler’s classical work
the cilia of bacteria have been the subject of numerous researches. Some
details on general morphology and physiology are given in A, FISCHER’S
Untersuchungen iiber Bakt., Jahrb. f. wiss. Bot. xxvii, 1895.

10. (p. 15). This now generally adopted division was first given by MESSEA, Rivista
digiene e sanita publica, 1890, 1.

11. (p.16). Observations of living bacteria during fission are given by BREFELD,
Untersuchungen ii. Schimmelpilze, iv (2. subtilis). [See also MARSHALL WARD,
Proc. R. S., vol. lviii, 1895, p. 24, and Ann. Bot., vol. xiii, 1899, p. 227.] For
determination of the rate of growth of the cholera vibrio by the plate method, see
BUCHNER, LONGARD, and RIEDLIN, Ueber die Vermehrungsgeschwindigkeit
der Bakterien, Centralbl. f. Bakt. vol. ii.

12. (p.19). The spores of bacteria had been already seen and described, but their
properties and development were first accurately described by COHN, Unter-
such. ii. Bakt. iv, in Beitr. z. Biol. d. Pflanzen, vol. ii, 1876. Germination
of spores was seen by BREFELD, note 11. See also PRAZMOWSKI, Untersuch-
ungen iiber d, Entwicklungsgeschichte u. Fermentwirkung einiger Bakterien-
arten, Leipzig 1880; also Biol. Centralbl. iv, 1884 (B. sudtidds and B.
anthracis). The newest observations (corroborating and extending former
ones) on the conditions of sporulation are by SCHREIBER, Centralbl. f. Bakt.,
1. Abt. vol. xx, 1896.

13. (p. 22). On the characters of arthrospores see DE BARY, Vergleichende Morpho-
logie und Biologie der Pilze, Mycetozoen u. Bakterien, Leipzig 1884, pp. 496,
506.

14. (p. 24). For the question of species see BILLROTH, Coccobacteria septica, Berlin.
1874; NAEGELI, Die niederen Pilze in ihren Beziehungen zu den Infektions-
krankheiten und der Gesundheitspflege, Munich 1887; Zopr, Zur Morphologie
der Spaltpflanzen, Leipzig 1882. All these works favour pleomorphism. On
the other side are COHN, Beitr. z. Biol. d. Pfl. i and ii; DE BaRyY, Vergl.
Morphol. und Biol. d. Pilze, 1884, p. 511. These take the view that bacteria
constitute good species and genera. See also HUPPE, Die Formen der Bak-
terien, Wiesbaden 1886.

15. (p. 24). BUSGEN, Kulturversuche mit C/adothrix dichotoma, B. d. deutsch. bot.
Ges. xii, 1894.

16. (p. 26). Involution forms (after NAEGELI, note 14) are described in numerous
works: BUCHNER in NAEGELI’S Untersuchungen ii. niedere Pilze; also
Hwpre, Formen d. Bakterien; PRAZMOWSKI, see note 12; ZoPF, Die Spalt-
pilze, Breslau 1885, 3rd ed. On the so-called branched tubercle-bacillus,
CoOPPEN JONES, Centralbl. f. Bakt. vol. xvii; H. BRUNs, ibid.; Diphtheria
bacilli, BERNHEIM and FOLGER, ibid. vol. xx.

17. (p. 28). PASTEUR, CHAMBERLAND, and Roux, De latténuation des virus et de
leur retour 4 la virulence, Comptes rendus de l’Acad., Paris 1881, vol. xcii ;
also CHAMBERLAND, Le charbon et la vaccination charbonneuse, d’aprés des
récents travaux de M. Pasteur, Paris 1883; CHAMBERLAND and Roux, Sur
Yatténuation de la virulence de la bactéridie charbonneuse sous l’influence des
antiseptiques. Comptes rendus, vol. xcvii, 1883.

18. (p. 28). On asporogenous anthrax and its properties: ROUX, Bactéridie charbon-
neuse asporogéne, Ann. de I’Inst. Pasteur, 1890, vol. iv; PHISALIX in Comptes
rendus, 1892, vol. cxiv. p. 684, and vol. cxv. p. 253.

NOTES 171

19. (p. 30). A very good critical description, both morphological and physiological, of
the known species of bacteria is given by LEHMANN and NEUMANN (note 3).
The pathological forms are treated most fully, but the work may be strongly
recommended to all students.

20. (p. 30). CoHN’s Systematic Classification of the Bacteria in Beitr. z. Biol. d. Pf.
vol. ii.

21. (p. 32). Attempts at new systems introducing new genera have been recently pub-
lished by A. FISCHER (loc. cit., note 9), by MIGULA in Engler and Prantl’s Die
natiirlichen Pflanzenfamilien, Lief. 129, and by LEHMANN and NEUMANN, loc.
cit., note 3. The author offers the arrangement given in the text for further
criticism and consideration.

22. (p. 39). For other pathogenic micro-organisms and fungi see FLUGGE (note 3),
where also figures and literature are given.

23. (p. 45). The methods and technique in the bacteriological analysis of air, water,
earth, food-stuffs, &c., will be found in the works cited in note 3, and in
every text-book of hygiene. The beginner will find a concrete example of
method and results in: HEsSE, Ueber quantitative Bestimmung der in der
Luft enthaltenen Keime, Mitt. aus dem kaiserl. Gesundheitsamte, vol. ii, 1884;
MIQUEL, Des organismes vivant de l’atmosphére, Paris 1883 ; ROUX, Précis
d’analyse microbiologique des eaux, Paris 1892 ; WOLFFHUGEL, Erfahrungen
u. den Keimgehalt brauchbarer Trink- und Nutzwasser, Mitt. a. d. Reichs-
gesundheitsamt, 1886; LOFFLER, Das Wasser u. die Mikro-organismen, Hand-
buch fiir Hygiene, vol. i, 2. Abt., 1896.

\ oa. (p. 48). This mode of classification, as may be seen in these lectures, saves many
a long description.

25, (p. 50). A very readable description of the long conflict over the problem of
spontaneous generation is given in LOFFLER’S lectures (note 4), and also fully
in LAFAR’sS Technical Mycology ; PASTEUR’S classical work on the matter is
Mémoires sur les corpuscules qui existent dans l’atmosphére: examen de la
doctrine des générations spontanées, Annales de Chimie et Physique, 1862,
3. série, vol. Ixiv. It has appeared also in German (by WIELER) in OSTWALD’S
Klassiker der exakten Wissenschaften, No. 39, Leipzig (Engelmann).

\ 26. (p. 51). This rash statement has been made by FERMI, Centralbl. f. Bakt., 2. Abt.
vol. ii, 1896.
27. (pp. 52, 53). NENCKI und SCHEFFER, Ueber d. chemische Zusammensetzung der
Faulnissbakterien, Beitrage z. Biol. d. Spaltpilze, edited by NENCKI, Leipzig
1880 (reprint from the Journal f. prakt. Chemie, neue Folge, vols. xix and xx) ;
also Kapprs, Analysis of Mass-cultures of the Thrush Fungus and some
Bacteria, Leipzig 1889; CRAMER, Die Zusammensetzung der Cholerabazillen,
Archiv f. Hygiene xxii, 1895.
28. (pp. 53, 56). NAEGELI, Ernahrung der niederen Pilze durch Kohlenstoff- und Stickstoff-
verbindungen, Untersuchungen iiber niedere Pilze, 1882 (also in the Sitzungsber.
d. math.-phys. Klasse d. Miinchener Akad. der Wissensch. 1879). See also
particularly BEYERINCK, Over lichtvoedsel en plastisch voedsel van Licht-
bakterien, Versl. en Mededel. der Amsterdamer Akad. d. Wissensch., Naturwiss.
Abt., 2. Serie, vol. vii, 1890; here will be found also the division into pepton,
amido, and ammonia bacteria ; FRANKEL, Beitrage z. Kenntniss des Bakterien-
wachstums auf eiweissfreien Nahrbéden, Hyg. Rundschau iv, 1894. The table
on p. 55 is from personal experiments.

172 NOTES

29. (p. 57). Gelatine was introduced by ROBERT KocH (Zur Untersuchung von
pathogenen Mikroorganismen, Mitt. a. d. kaiserl. Gesundheitsamte, vol. i, 1881).
Agar (prepared from red marine algae, Gracilaria, Eucheuma) was, according
to HUpprE (Methoden d. Bakterienforschung, 5th ed. p. 250), first used by
FRAU HESSE.

30. (p. 58). LEHMANN and NEUMANN (note 3).

81. (p. 60). PAasTEuR, Infusoires vivant sans gaz oxygéne libre, Comptes rendus,
vol. lii, pp. 344 and 1260, 1861. PasTEuR, Etudes sur la biére, Paris 1876,
chap. vi; NENCKI in the works given in note 27, The number of newer
works on anaerobiosis is enormous, see notes 68, 94, 95, 110.

32. (p. 61). ENGELMANN, Neuve Methode zur Untersuchung der Sauerstoffausscheidung
pflanzlicher und tierischer Organismen, Bot. Zeit. 1881, and Ueber Sauerstoff-
ausscheidung von Pflanzenzellen im Mikrospektrum, Bot. Zeit. 1882.

33. (p. 63). F. COHN, Ueber thermogene Bakterien, Berichte d. deutsch. bot. Gesell-
sch. xx. p. 66 (1893).

34. (p. 63). For phosphorescent bacteria see PFLUGER, Ueber die Phosphorescenz ver-
wesender Organismen, Archiv f. d. gesamte Physiologie xi, 1875 ; LuDwiG, Die
bisherigen Untersuchungen iiber photogene Bakterien, Centralbl. f. Bakt. vol. ii,
1887; E. FISCHER, Zeitschrift f. Hygiene, vols. i and ii, 1886, 1887; BEYERINCK,
note 28, and in Archives Néerlandaises des sciences exactes et nat. vol. xxiii,
1889; E. FISCHER, Die Bakterien des Meeres, Plankton-Expedition, vol. iv,
1894; KUTSCHER, Deutsche mediz. Wochenschrift, 1893.

35. (p. 64). From E. FISCHER, Plankton-Exped. vol. iv, 1894.

36. (p. 65). WINOGRADSKY, Ueber Schwefelbakterien, Bot. Zeit. 1887; and in Beitrage
z. Morphologie und Physiologie der Bakterien, Leipzig 1888.

37. (p. 68). ENGELMANN, Die Purpurbakterien und ihre Beziehungen zum Lichte, Bot.
Zeit. 1888; see also previous note.

38. (p. 69). WINOGRADSKY, Ueber Eisenbakterien, Bot. Zeit. 1888; MOoLISCH, Die
Pflanze in ihren Beziehungen zum Eisen, Jena 1892, p. 60.

39. (p. 71). A series of experiments on the influence of light on typhoid bacilli is given
by JANowSKI, Zur Biologie d. Typhusbazillen, Centralbl. f. Bakt. vol. viii, 1890 ;
also BUCHNER, ibid. vols. xi and xii. [See also Phil. Trans., 1894, p. 961.]

40. (p. 72). According to BUCHNER (Centralbl. f. Bakt. vol. xi. p. 782) the influence
of light on bacteria, such as typhoid, cholera, and putrefactive forms, is of
decided importance in the self-purification of rivers. He even suggests white
cemented clarifying or settling basins, in which sewage shall be exposed to sun-
light before’ it is allowed to run into the rivers. Jn such cases, however, it
would be necessary to prevent the organisms finding any shadowed spots; even
the minute cracks in the cement would shield numbers of them from the sun.

Al. (p.72). For the effect of light on fungi see L. KLEIN, Ueber die Ursachen der
ausschliesslich nachtlichen Sporenbildung bei Botrytis cinerea, Bot. Zeit. 1885 ;
also BREFELD, Bot. Untersuch. iiber Schimmelpilze, Heft 3, p. 87 (Coprinus),
Heft 4, p. 76 (Pilobolus).

42. (p. 72). COHN and MENDELSORN, Ueber die Einwirkung des elektrischen Stromes
auf die Vermehrung der Bakterien, Beitr. z. Biol. iji, 1883.

43. (p. 72). MOLLER, references in Centralbl. f. Bakt., 2. Abt., vol. i. pp. 294 and 753,
and original in vol. iii, 1897.

44. (pp. 72, 73). See VERWORN, Psycho-physiologische Protistenstudien, Jena 1889.

47.

48.

49.

50.

51,

52.

53.

54,

55.

NOTES 17:

. (p. 73). WITTLIN, Centralbl. f. Bakt., 2. Abt. vol. ii, 18¢6, p. 676.
. (p. 73). CERTES, De l’action des hautes pressions sur les phénoménes de la putré.

faction et sur la vitalité des microorganismes d’eau douce et d’eau de mer
Comptes rendus, 1884, vol. xcix. p. 385 (reference in Bot. Zeit. 1885).

(pp. 73, 75). The literature concerning bacteria in their relation to temperature i:
enormous, since almost every work dealing with a new species describes it:
behaviour to heat. The foundations of our knowledge of the subject were laic
by Coun (Untersuch. ii. Bakterien iv, in Beitr. z. Biol. vol. ii, 1876). He basec
his work on that of the older investigators in physiology and the problem o
spontaneous generation, and showed first that the spores of the hay-bacillu:
resisted boiling. ROBERT KOCH introduced the methods and results of plan’
physiology into medical bacteriology, Beitr. z. Biol. vol. ii.

(p. 74). MIQUEL, P., Annuaires de l’observatoire de Montsouris 1881, and Mono
graphie d’un bacille vivant au dela de 70 centigrades (Bac. thermophilus), it
Annales de Micrographie i, 1888 (references in Centralbl. f. Bakt. vol. v, 1889)
also GLOBIG, Zeitschrift fiir Hygiene, vol. iii, 1888, and RABINOWITSCH, ibid
vol. xx.

(p. 75). For the lowest artificial temperatures and their effect upon organisms of al
kinds, see WELTER, Die tiefen Temperaturen, ihre kiinstliche Erzeugung, etc.
Krefeld 1895.

(p. 77). Experiments innumerable have been made on the power of bacteria to resis
desiccation; here again the fundamental facts were given by COHN in thi
Beitr. z. Biol.; see also EIDAM, Die Einwirkung verschiedener Temperature1
und des Eintrocknens auf die Entwicklung von Bacterium ¢ermo, Beitr. z. Biol
vol. i, 1875.

(p. 78). STAHL, Zur Biologie der Myxomyceten, Bot. Zeit. 1884 (p. 165, Tropho
tropism).

(p. 78). PFEFFER, W., Lokomotorische Richtungsbewegungen durch chemische Reize
Untersuch. a. d. bot. Institut Tiibingen, vol. i, 1884, and Ueber chemotaktisch:
Bewegungen von Bakterien, Flagellaten und Volvocineen, ibid. vol. ii, 1888
In these two classical works will be found also a full discussion of Weber’.
law and Chemotaxis.

(p. 80). For chemotaxis of leucocytes see: MASSART and BORDET, Recherches su
Virritabilité des leucocytes, Société royale des sc. nat. de Bruxelles, 1890
GABRITSCHEWSRY in the Annales de I’Inst. Pasteur, 1890; BUCHNER, Berl. klin
Wochenschr. 1890; also the description given by RIEDER, Beitrage zu
Kenntnis der Leukocytose, Leipzig 1892.

(pp. 81, 82, 83). On chemical disinfection abundant information will be found in medica
literature; the subject is treated very fully by BEHRING, Bekampfung de
Infektionskrankheiten, 1894. The fundamental principles were given b
R. KocH in Ueber Desinfektion, Mitt. aus dem kaiserl. Gesundheitsamte
vol. i, 1881; see also GEPPERT, Die Wirkung des Sublimates auf Milzbrand
sporen, Deutsche med. Woch. vol. xvii, 1890; YERSIN, De l’action de quelque
antiseptiques et de la chaleur sur le bacille de la tuberculose, Annales de I’Insi
Pasteur, 1888.

(pp. 83, 85, 86). PAUL and KRONIG, Ueber das Verhalten der Bakterien zu chemische
Reagentien, Zeitschr. f. physik. Chemie, vol. xxi, 1896, and Minchener medi:
Wochenschr. 1897 ; also Die chemischen Grundlagen der Lehre von der Gift

174

56.

57,

58,

59.

60,

61.

62.

63.

64,

65.

NOTES

wirkung und Desinfektion, Zeitschr. f. Hygiene xxv, 1897. These works contain
the first exact description of the relation between the poisonousness of a solution
and its degree of dissociation.

(p. 85). For the new theories of dissociation see OSTWALD, Grundriss der allgemeinen
Chemie, 2nd ed., and more fully in the same writer’s Lehrbuch der allg.
Chemie, 2nd ed.

(p. 87). Review of a Russian work by KURLOFF and WAGNER, Ueber die Ein-
wirkung des menschlichen Magensaftes auf krankheits-erregende Keime,
Centralbl. f. Bakt. 1890, vol. vii; also HAMBURGER, Ueber d. Wirkung des
Magensaftes auf pathogene Bakterien, Centralbl. f. klin. Medic. 1880,

(p. 87). CADEAC and BouRNAY, Réle microbicide des sucs digestifs et contagion par
les matiéres fécales (La province médicale, viii, 1893, referred to in Centralbl. f.
Bakt. vol. xvi, 1894, p. 672).

(pp. 89, 91). HELLRIEGEL and WILLFARTH, Untersuchungen iiber die Stickstoff-
nahrung der Gramineen und Leguminosen, 1888, Beilageheft zu der Zeitschr. d.
Vereins f. d. Riibenzucker-Industrie des Deutschen Reiches.

(p. 89). For the anatomy and development of the root-nodules see: WORONIN,
Ueber die bei der Schwarzerle und der gewéhnlichen Lupine auftretenden
Wurzelanschwellungen, Mém. de l’Acad. imp. Petersburg, 7. Serie, vol. x,
1866. BEYERINCK, Die Bakterien der Papilionaceenknéllchen, Bot. Zeit. 1888,
and Centralbl. f. Bakt. vol. xv, 1894; B. FRANK, Ueber die Pilzsymbiose der
Leguminosen, Landwirtsch. Jahrb. 1890; PRAZMOWSKI, Landwirtsch. Ver-
suchsstation, 1890, vols. xxxvii and xxxvili, GONNERMANN, Die Bakterien in
den Wurzelknéllchen der Leguminosen, Landwirtsch. Jahrb. vol. xxiii, 1894.
[See also Dawson, Phil. Trans., vol. cxcii, 1899, p. 1, with references.]

(p. 92). BEYERINCK, Over ophooping van atmospherische stickstof in culturen von
Bacillus radicicola, Akad. d. Wissensch., Amsterdam 1891 ; reviewed in KOCH’S
Jahresber. vol. iii. p. 205, Mazi, Fixation de l’azote libre par le bacille des
nodosités des Légumineuses, Annales Pasteur xi, 1897.

(p. 92). NOBBE, HILTNER, and SCHMID, Versuche iiber die Biologie der Knéll-
chenbakterien der Leguminosen, insbesondere iiber die Frage der Arteinheit
derselben, Landwirtsch. Versuchsst. vol. xlv, 1895. NoBBEand HILTNER, Ueber
die Anpassungsfahigkeit der Knéllchenbakterien ungleichen Ursprungs an ver-
schiedene Leguminosengattungen, ibid. vol. xlvii, 1896. In these papers will be
found the experimental evidence for Nitragin.

(p.95). If FRANK’S assertion that there are no intercellular spaces in the bac-
teroidal tissue and that therefore free nitrogen cannot enter were true, then
it would be equally impossible for oxygen to get into these parts of the root-
nodules, which would be therefore anaerobic! A careful examination of any
transverse section shows just the reverse, as might be expected.

(p. 96). WINOGRADSKY, Sur l’assimilation de V’azote gazeux de l’atmosphére par
les microbes, Comptes rendus der Pariser Akad. 1893, vol. cxvi. p. 1385; 1894,
vol. cxviii. p. 353; and a collective Review in Archives des sciences biologiques,
vol. iii, 1895, St. Petersburg (referred to in Bot. Zeit. 1895).

(p. 97). New light has been thrown on this matter by KossowiTscu, Untersuch-
ungen iiber die Frage, ob die Algen freien Stickstoff assimilieren, Bot. Zeit. 1894.
The experiments of SCHLOESING and LAURENT (Ann. de I’Inst. Pasteur, 1892)
are here critically discussed.

66. (p.

67. (p.

68. (p.

69. (p.

70. (p.

71, (p.

72. (p.

73. (p.

74. (p.

NOTES 17.

99). WEHMER, Untersuchungen iiber die Fiulnis der Friichte, in Beitrigen zu
Kenntnis einheimischer Pilze, 2. Heft, Jena 1895.

100). SELMI in Sitzungsber. d. Akad. zu Bologna, 1872 and 1873; also Alcaloid
cadaverici, Bologna 1881. BRIEGER, Ueber Ptomaine, Berlin 1885-86, Unter
suchungen iiber Bakteriengifte in Berliner klinische Wochenschrift, 1890, anc
many other essays. See also KOBERT, Lehrbuch der Intoxikationen, 1893.

100). HOPPE-SEYLER, Ueber die Einwirkung von Sauerstoff auf die Lebens
thatigkeit niederer Organismen, Zeitschr. f. physiol. Chemie, 1884, vol. viii
NENCKI, Ueber den chemischen Mechanismus der Faulnis, Journal f. prakt
Chemie, vol. xvii. BIENSTOCK, Ueber die Bakterien der Faeces, Zeitschr
f. klinische Medicin, vol. viii. HERFELD, Die Bakterien des Stalldiingers unc
ihre Wirkung, Centralbl. f. Bakt., 2. Abt. vol. i, 1895.

101). WOLLNY, Die Zersetzung der organischen Stoffe und die Humusbildungei
mit Riicksicht auf die Bodenkultur, Heidelberg 1897.

101). Morphology of putrefactive bacteria is treated especially by: COHN
Beitrage z. Biol. vol. i, 1872; HAUSER, Ueber Faulnisbakterien und derer
Beziehungen zur Septikamie, Leipzig 1885; BIENSTOCK (note 68); KUHN
Morphol. Beitrage zur Leichenfaulnis, Archiv f. Hygiene, vol. xiii, 1891
Details as to saprogenic power (particularly the formation of indol) of patho
genic bacteria are given in many medical treatises : see the works of FLUGGI
and LEHMANN and NEUMANN (note 3).

103). PASTEUR and JOUBERT, Sur la fermentation de l’urine. Comptes rendus de
Pariser Akad. 1876, vol. Ixxxiii. MIQUEL, P., Etude sur la fermentation ammonia
cale et sur les ferments de lurée. Annales de micrographie, 1889-1893
vols. i-iii, v. In MIQUEL’s works there are very full descriptions of the distri
bution, species, and functions of the numerous urine bacteria (good reference:
in A. KocuH’s Jahresbericht, vols. i, ii, and iv).

104). The numerous papers of WARINGTON, MUNTZ and others on nitrificatior
have contributed much to our knowledge of the problem, but the key was firs
given by WINOGRADSKY, Recherches sur les organismes de la nitrification, 1°-5
mémoire, 1889-1891, Annales de |’Institut Pasteur iv, v, and Contribution:
4 la morphologie des organismes de la nitrification, Archives de sciences biol
publ. par I’Inst. impér. de méd. expér. & St. Pétersbourg, i, 1892: also Zu
Mikrobiologie des Nitrifikationsprozesses, Centralbl. f. Bakt., 2. Abt. ii, 1896
Quite recently STUTZER and HARTLEB have published new observations on thi
saltpetre ferment (Centralbl. f. Bakt., 2. Abt. iii, 1897). These observations may
well lead astray those not familiar with mycology. The bacteria are said t
grow out into a mycelium, and give rise to a whole host of different form:
—a lapse back into the wildest traditions of pleomorphism. STUTZER anc
HARTLEB'S work is insufficient and full of faults, and is wanting entirely ir
reliable evidence. Such statements as theirs ought not to have appeared afte:
BREFELD’S work was once known. The real ‘saltpetre bacteria’ are anc
remain bacteria, and the reader may rely on their constancy.

106). GODLEWSKI, Ueber die Nitrifikation des Ammoniaks und die Kohlenstoff
quellen bei der Ernahrung der nitrificierenden Fermente. (Polish.) Referrec
to in Centralbl. f. Bakt., 2. Abt. ii. p. 458.

106). BURRI and STUTZER, Ueber Nitrat zerstorende Bakterien und den durch
dieselben bewirkten Stickstoffverlust, Centralbl. f. Bakt., 2. Abt. i, 1895.

79,

80.

81,
82.

83.

84,

85.

86.

. (p.

(p.

(p.

(p.
(p.

(p.
(p.

(p.

NOTES

106). BEYERINCK, Ueber Spirillum desulfuricans als Ursache der Sulfatreduktion,
Centralbl. f. Bakt., 2. Abt. i, 1895.

. (p. 108). v. SOMMARUGA, Ueber Stoffwechselprodukte von Mikroorganismen, iii.

Zeitschr. f. Hygiene, vol. xviii, 1894.

. (p. 108). The newest collective treatise is LAFAR, Technical Mycology ; DUCLAUX,

Chimie biologique, Paris 1883, is also to be recommended.

. (p. 108). PASTEUR’S pioneer work on the ferment organisms began with the investi-

gation of spontaneous generation, and will be found in the works cited in note 25
and in many others. Of works on anaerobiosis (note 31) may be mentioned
Mémoire sur la fermentation acétique (Annales de I’Ecole normale supérieure, i,
1864); Mém, sur la fermentation appelée lactique (Annales de chimie et de
physique, 3. série, vol. lii; numerous investigations on the diseases of wine and
beer in the Etude sur le vin, 1866, and Etude sur la biére, 1876. The first
period of PASTEUR’s brilliant life as an investigator was occupied with the
physiology of fermentation, and laid the foundations of the latter half of his
work, the study of pathogenic organisms.

108). Details and literature concerning enzymes and their réle in the nutrition of
plants and animals will be found in every text-book of physiology or physio-
logical chemistry. [See also GREEN, ‘ The soluble Ferments and Fermentations,’
Pitt Press, 1899.] Theoretical considerations are given by E. FISCHER, Ueber
den Einfluss der Configuration auf die Wirkung der Enzyme, i-iii, Ber. deutsch.
chem. Ges., vols. xxvii and xxviii, 1894 and 1895.

110). On the so-called Aspergillus yeast see Centralbl. f. Bakt., 2. Abt. i,
WEHMER (pp. 150, 565), WENT and PRINSEN GEERLIGS (p. 501) ; ii, WEHMER
(p. 140).

110). WEHMER, Beitr. zur Kenntnis einheimischer Pilze, 1. Heft, 1893.

112). HANSEN, Recherches sur les bactéries acétifiantes, Annales de Micro-
graphie, 1894, also Travaux du laboratoire de Carlsberg, vol. iii (Meddelelser
fra Carlsberg Laboratoriet, Kopenhagen). This work deals chiefly with the
morphological sides of the question. The chemical changes had been already
investigated by PASTEUR, and were known by experience.

113). From LaFar, Physiologische Studien iiber Essiggdrung und Schnellessig-
fabrikation, Centralbl. f. Bakt., 2. Abt. i, 1895.

113). Review in Kocn’s Jahresb. iii, 1892, pp. 230-32, of the work of Frankland
and various collaborators.

115). PASTEUR, Comptes rendus Pariser Akad. 1860, vol. li, p. 298 (racemic
acid) ; LEWKOWITCH, Bericht deutsch. chem. Ges. vol. xvi (amygdalic acid) ;
PERE, Sur la formation des acides lactiques isomériques par |’action des microbes
sur les substances hydrocarbonées (Annales Pasteur, vol. vii, 1893).

(pp. 116, 117). PASTEUR, note 78; HUPPE, Untersuchungen iiber die Zersetzung der

Milch durch Mikroorganismen, Mitteilung a. d. Reichsgesundheitsamt ii, 1884;
ESCHERICH, Darmbakterien des Sduglings, Stuttgart 1886 ; also KRAMER, Die
Bakteriologie in ihren Beziehungen zur Landwirtschaft, 2. Teil, Wien 1892;
DUCLAUX, Le lait, Paris 1887. Bacteria in cheese and milk, see v. FREU-
DENREICH, Ueber den Einfluss der beim Nachwairmen des Kises angewandten
Temperatur auf die Bakterienzahl in der Milch undim Kase, Landwirtsch. Jahrb.
d. Schweiz, vol. ix (referred to in Centralbl. f. Bakt., 2. Abt. vol. i, 1895).

NOTES 177

87. (p. 117). Larar, Die kiinstliche Sduerung des Hefegutes der Brennereien, Centralbl.
f. Bakt., 2. Abt. ii, 1896.

88. (p. 117). FLUGGE, Die Aufgaben und Leistungen der Milchsterilisierung gegeniiber
den Darmkrankheiten der Sduglinge, Zeitschr. f. Hygiene, vol. xvii, 1894.

-89. (p. 117). HEIM, Ueber das Verhalten der Krankheitserreger der Cholera, des
Unterleibstyphus und der Tuberkulose in Milch, Butter, Molken und Kase,
Mitteil. Reichsgesundheitsamt, 1889; OBERMULLER, Ueber Tuberkelbazillen-
befunde in der Marktmilch, Hygienische Rundschau, 1895.

90. (p. 118). CONN, The relation of pure cultures to the acid, flavour, and aroma of
butter, Centralbl. f. Bakt., 2. Abt. ii, 1896; iii, 1897.

91. (p. 118). KRAMER, DUCLAUX, in note 86; also in Centralbl. f. Bakt., 2. Abt.
vol. i, 1895: V. FREUDENREICH, Bakteriol. Untersuchungen iiber den Reifungs-
prozess des Emmenthaler Kases; 1. c. vol. ii, 1896: Vv. KLECKI, Ueber den
Reifungsprozess des Kases; Einen neuen Buttersdiuregirungserreger (Bac. sac-
charobutyricus) und dessen Beziehungen zur Reifung und Lochung des
Quargelkases ; WEIZMANN, Ueber den jetzigen Stand etc. des Kasereifungs-
prozesses ; V. FREUDENREICH, Bemerkungen dazu.

91a. (p. 120). Vv. FREUDENREICH, Bakteriol. Untersuchungen iiber den Kefir, Centralbl.
f. Bakt., 2. Abt. iii, 1897. [For an account of the ‘Ginger Beer Plant,’ a similar
association of a yeast and a bacterium, see MARSHALL WARD, Phil. Trans. 1892,
p. 125.]

92. (p. 120). See note 87, and also LaFar's Techn. Mycology. Numerous papers by
EFFRONT are referred to in A. Kocu’s Jahresbericht; ROTHENBACH, Die
Anwendung spaltpilzfeindlicher Agentien im Brennereibetriebe, mit besonderer
Beriicksichtigung der Kunsthefefiihrung, Zeitschr. f. Spiritusindustrie 1896.

93. (p. 120). Details in Larar’s Technical Mycology, vol. i. p. 259.

94, (p. 121). PASTEUR, note 31; VAN TIEGHEM, Sur le Bacéllus amylobacter, Bullet.

F soc, botan. xxiv. 1877, and Identité du Bacz//us amylobact, et du vibrion butyrique
de Pasteur, Comptes rendus, Paris 1879, vol. lxxxix ; PRAZMOWSKI, note 12 ;
GRIMBERT, Fermentation anaérobique produite par le Bacdl/us orthobutylicus,
ses variations sous certaines influences biologiques, Annales Pasteur vii. 1893 ;
BEYERINCK, Ueber die Butylalkoholgarung u. das Butylferment, Verhandlungen
der kgl. Akad. Amsterdam, 2. Sekt. i. 1893, u. Centralbl. f. Bakt. 2. Abt. ii.
p- 699; Vv. KLEcKI, Ein neuer Buttersduregarungserreger (Bac. saccharo-
butyricus), ibid.; BAIER, Ueber Buttersduregarung (a collective review), ibid.
2. Abt. i. 1895.

95. (p. 122). VAN TIEGHEM, Sur le Bacilius amylobacter et son réle dans la putré-
faction de la cellulose, Comptes rendus, Paris, vol. Ixxxviii. 1879; HOPPE-
SEYLER, Zeitschr. f. physiol. Chemie, vol. x; OMELIANSKI, Sur la fermentation
de la cellulose, Comptes rendus 1895.

96. (p. 123). PASTEUR, Etude sur le vin, 1866; VAN LAER, Note sur la fermentation
visqueuse, Mémoires couronnés der belgischen Akad. Briissel, vol. xliii. 1889 ;
KRAMER, Studien iiber die schleimige Girung, Sitzungsber. Wiener Akad. d.
Wiss. Natur-Cl. 1889; LEICHMANN, Ueber eine schleimige Garung der Milch,
Landwirtsch. Versuchsstat., vol. xliii. [See also MARSHALL WARD and GREEN,
A Sugar Bacterium, Proc. R. S., vol. lxv, p. 65, 1899.]

97. (p. 123). WINOGRADSKY, Sur le rouissage du lin et son agent microbien. Comptes
rendus, Paris 1895.

FISCHER N

178

NOTES

98. (p. 124). ALVAREZ, Sur un nouveau microbe, déterminant la fermentation

indigotique et la production de l’indigo bleu. Comptes rendus, Paris 1887,
vol. cv.

99. (p. 124). BEHRENS, Die Beziehungen der Mikroorganismen zum Tabakbau und zur

100. (p.

101. (p.

102. (p.

103. (p.

104. (p.

105. (p.

106. (p.

107. (p.

108. (p.

109. (p.

Tabakfabrikation. Centralbl. f. Bakt. 2. Abt. ii. 1896.

124). VAN TIEGHEM, Lewconostoc mesenteroides, Annales d. sc. nat., Botanique,
6. série, vol. vii.; LIESENBERG and Zopr, Ueber den sog. Froschlaichpilz, Beitr.
zur Physiol. u. Morphol. niederer Organismen, herausgegeben von Zopf, vol. i,
1892; Kocu, A., and HOSAEUS, Ueber einen neuen Froschlaichpilz der Zucker-
fabriken, Centralbl. f. Bakt. xvi. 1894.

125). LEHMANN, Ueber die Sauerteiggirung und die Beziehungen des Bac.
Zevans wam Bac. coli, Centralbl. f. Bakt. xv. 1894; Poporr, Sur un bacille
anaérobique de la fermentation pannaire, Annales Pasteur 1890; PETERS, Die
Organismen des Sauerteiges und ihre Bedeutung fiir die Brotgdrung, Bot.
Zeit. 1889.

126). An interesting account of the history of alcoholic fermentation, which is
largely identical with the history of the problem of spontaneous generation, will
be found in ADOLF MAvEer’s Lehrbuch der G&hrungschemie, 3rd ed.,
Heidelberg 1879. i

126). REESS, Botanische Untersuchungen iiber die Alkoholgahrungspilze, 1870.
The morphology, physiology, and technical employment of the yeasts have been
furthered in an astonishing degree by the investigation of the Danish savant
EMIL CHRISTIAN HANSEN, published in the Meddelelser fra Carlsberg Labo-
ratoriet in Kopenhagen i.-iii. 1878-94, and in his Untersuchungen aus der
Praxis der Gahrungsindustrie, Heft i, 3rd ed. 18¢5, Heft ii. 1892. HANSEN’S
investigations (mostly on beer yeasts) have been taken up in the case of wine
yeasts by WORTMANN (Untersuchungen iiber reine Hefen, Landwirtsch. Jahrb.
1892, 1894), by ADERHOLD, MULLER-THURGAU and others.

129). HANSEN, E. Cu., Experimental studies on the variation of yeast-cells,
Annals of Botany, ix. 1895.

129). A good idea of the importance of microbiology in technical processes
is given by LINDNER, Mikrobiologische Betriebscontrolle in dem Gahrungs-
gewerbe, Berlin 1895 ; of wine-making a popular account is given by WORTMANN,
Anwendung und Wirkung reiner Hefen in der Weinbereitung; Berlin 1895.
130). BREFELD, Botanische Untersuchung iiber Hefepilze, v. 1883 (Ustila-
gineen); BREFELD, Landwirtsch. Jahrb., v. 1876 (Mucorhefe); DE BarRY,
Vergleichende Morphologie und Biologie der Pilze, 1884, p. 286 (Zxoascus and
its relations to Saccharomyces); KLOCKER and SCHIONING, Experiment.
Unters. iiber die vermeintliche Umbildung verschiedener Schimmelpilze in
Saccharomyces, Centralbl. f. Bakt. 2. Abt. ii. 1896, and Que savons-nous de
Yorigine des Saccharomyces? Meddelelser fra Carlsb. Labor. iv. 1896.

131); For the enzymes and the power of fermentation of various yeasts and
their sub-species (races) see E, FISCHER and LINDNER, Ueber Enzyme einiger
Hefen, Wochenschr. f. Brauerei 1895; also E. FISCHER in note 79;
BEYERINCK, Ueber Nachweis und Verbreitung der Glukase, das Enzym der
Maltose, Centralbl. f. Bakt. 2. Abt. 1895.

131). CLAUCDON and MorIN, Comptes rendus, Paris 1887, referred to in the
Centralbl. f. Bakt. vol. ii.

132). WORTMANN, Untersuchungen iiber den Einfluss des Liiftens sowie den

NOTES 179

der dauernden Garthitigkeit auf den Charakter der Hefe, Weinbau u. Weinhandel,
1895, Nos. 25-27. [But see also A. BROWN, Fermentative Power, Centr. f.
Bakt. u. Paras., 2. Abt., vol. iii. 1897.]

110. (p. 133). The most important works on the theory of fermentation are: TRAUBE,

°

Theorie der Fermentwirkung, Berlin 1858; PasTrurR, Etude sur la biére, 1876,
chap. vi.: Théorie physiologique de la Fermentation, first announced 1861
(note 31); NAEGELI, Theorie der Gahrung, Munich 1879. For intramolecular
respiration see text-books of physiology. The metabolism theory, which is
merely an expression of facts free from speculation, has not yet been carefully dis-
cussed nor considered in the light of the explanation afforded by other theories.

111. (p. 134). H. BUCHNER’s statement (Die Bedeutung der aktiven ldslichen Zell-

produkte fiir den Chemismus der Zelle, Miinch. mediz. Wochenschr. 1897,
No. 12) that an extract (pressure extract) of yeast free from cells can split
up sugar into alcohol and CO, is too incomplete to upset existing theories.
Such a revolutionary statement ought to be supported by chemical analyses,
but these have not been given. In the two papers by Ep. BUCHNER also
(Alkoholische Gahrung ohne Hefezellen) solid proofs of the existence and
activity of the ‘Zymase’ (the alcohol-forming enzyme of the yeast cells) are
wanting. In these papers there is a slight attempt at analytical evidence: the
gas is calculated as CO,, and in two instances the alcohol was determined (Ber.
d. deutsch. chem. Ges. 1897, pp. 117 and 1110). Further communications of
BUCHNER (Ber. d. deutsch. chem. Ges. 1897, p. 2668 ; 1898, pp. 200, 209, 568,
1084, 1531) might seem to support his views more strongly against the objec-
tions raised, had not Abeles (ibid. 1898, p. 2335) recently shown that the amount
of the poisons added to the extract was much too small in proportion to the
amount of yeast from which the extract was obtained. The fragments of
protoplasm in the extract would accordingly not all be killed, and might well
give rise to the fermentative action of the extract, which would be quite inactive
when sufficient amounts of poison are present. BUCHNER’S observations have
shown that the pressure-extract of yeast (free from uninjured cells) speedily sets
up active fermentation in sugar solutions. But further researches are necessary
to show whether a genuine Enzyme (BUCHNER’S ‘Zymase’) is the cause of the
fermentation, or whether it is not rather due to the pressure-extract itself, which
is indeed nothing more or less than a concentrated infusion of protoplasm
squeezed out of the cells and preserved, as it were, in its own juice.

112. (pp. 103, 134). MIQUEL, note 71, and Sur le ferment soluble de l’urée, Comptes

rendus, Paris 1890, vol. iii. Urase decomposes at 50° in from three to four hours,
at 75° in a few minutes, at o° it will keep in bouillon for weeks. The optimal
temperature is said to be 50°-55°. The urase formed in five days by Uvo-
bacillus Schiitzentergii in one litre peptone bouillon converts at 37° thirty-five
grams of urea into carbonate of ammonia.

113. (p. 138). KORNAUTH, Ueber das Verhalten pathogener Bakterien im lebenden

Pflanzengewebe, Centralbl. f. Bakt. xix. 1896; KASPARECK and KORNAUTH,
Ueber die Infektionsfaihigkeit der Pflanzen mit Milzbrandbéden, Archiv f. d.
gesamte Physiol. vol. Ixiii. 1896.

114. (p. 138). FRANK and KRUGER, Untersuchungen iiber den Schorf der Kartoffeln,

Zeitschr. f. Spiritusindustrie 1896; RATHAY, Ueber das Auftreten von Gummi

in der Rebe und iiber die ‘Gommose bacillaire,’ Jahresb. u. Programm der

k. k. dnologischen u. pomologischen Lehranstalt Klosterneuburg, Wien 1896;

MANGIN, Sur la gommose de la vigne, and Sur la prétendue ‘Gommose
N 2

180

115. (p.

116. (p.

117. (p.

118. (p.

119. (p.

120. (p.

121. (p.
122. (p.
123. (p.

124, (p.

125. (p.

126. (p.

127. (p.

128. (p.

NOTES

bacillaire” Revue de viticulture, 1895 (referred to in Centralbl. f. Bakt. 2. Abt.
ii. 1896).

138). TISCHUTKIN, Die Rolle der Bakterien bei der Veranderung der Eiweiss-
stoffe auf den Blattern von Pinguicula, Ber. d. deutsch. bot. Ges., vol. vii. ;
SCHERFFEL, Die Driisen in den Héhlen der Rhizomschuppen von Lathraea
Sguamaria, Mitteilungen des bot. Instit. Graz, vol. i., and Bot. Zeit. 1890. 7
138). WATSON-CHEYNE and CHESHIRE, The pathogenic history under culti-
vation of a new bacillus (Bac. alvez), Journal of the Royal Micr. Society,
London 1885 (foul-brood of bees); PASTEUR, Etude sur la maladie des vers
4 soie, Paris 1879 (Schlafsucht, Flacherie der Seidenraupen); SANARELLI,
Ueber einen neuen Mikroorganismus des Wassers &c., Centralbl. f. Bakt. ix.
1891; ERNST, Beitrage z. patholog. Anat. v. Ziegler, vol. viii. p. 203.

139). MENGE and KRONIG, Bakteriologie des weiblichen Genitalkanales,
Leipzig 1897.

139). MILLER, Die Mikroorganismen der Mundhéhle. Die Ortlichen und
allgemeinen Erkrankungen, welche durch dieselben hervorgerufen werden.
2nd edit., Leipzig 1892. MILLER, Einleitung zum Studium der Bakterio-
Pathologie der Zahnpulpa, Centralbl. f. Bakt. xvi. 1894.

141). GILBERT and DOMINICI, Recherches sur le nombre des microbes du tube
digestif. (La semaine médicale, 1894, referred to in Baumgarten’s Jahresber. x.
p- 608.)

141). ESCHERICH, Darmbakterien des Sauglings, Stuttgart 1886; KIESSLING,
Das Bacterium coli commune, zusammenfassende Uebersicht, Hygienische
Rundschau, 1893; see also note 137.

141), NUTTALL and THIERFELDER, Tierisches Leben ohne Bakterien im
Verdauungskanal, i—ii., Zeitschr. f. physiol. Chemie, xxi. and xxii.

142). SCHILD, Bakterien im Darminhalt Neugeborener, Zeitschr. f. Hygiene,
vol. xix.

142). NEISSER, Ueber die Durchgangigkeit der Darmwand fir Bakterien,
Zeitschr. f. Hygiene, vol. xxii.

142). For the history of our knowledge of infectious diseases see LOFFLER,
note 4. The question as to what is understood by the word ‘infection’ is
discussed by BEHRING, Infektion und Desinfektion, Leipzig 1894.

145). WATSON CHEYNE, Report on a Study of the Conditions of Infection,
British Medical Journal, 1886. :

147). Every text-book of the various branches of medicine gives a chapter to
the bacteriology of that particular branch. It is therefore unnecessary to
mention many works, but BAUMGARTEN’s Pathologische Mykologie 1890, and
CoRNIL and BaBES, Les bactéries (3rd ed. 1893), may be named.

148). ROSENBACH, Mikroorganismen bei den Wundinfektionskrankheiten des
Menschen, 1884; GARRE, Zur Aetiologie akut eitriger Entziindungen (Osteo-
myelitis, Furunkel und Panaritium), Fortschritte d. Med. 1885; PassET,
Untersuchungen iiber die Aetiologie der eitrigen Phlegmone des Menschen,
Berlin 1885 ; LUBBERT, Biologische Spaltpilzuntersuchung, Wiirzburg 1886.

148). ROSENBACH, see previous note; FEHLEISEN, Aetiologie des Erysipeles,
Berlin 1883; PETRUSCHKY, Die verschiedenen Erscheinungsformen der
Streptokokkeninfektion in ihren Beziehungen zu einander, Zeitschr. f. Hygiene,
xviii. 1894.

129. (p.

130. (p.

131. (p.

132. (p.

133. (p.

NOTES 181

148). NEISSER, A., Ueber den Pilz der Gonorrhoe, Centralbl. f. d. ges. Med.
1879 ; BumM, Die Mikroorganismen der gonorrhdischen Schleimhauterkrankung,
Wiesbaden 1887; WERTHEIM, Die ascendirende Gonorrhoe beim Weibe,
Bakteriologische und klinische Studien zur Biologie des Gonococcus ; NEISSER,
Archiv f. Gynakologie, vol. xlii, 1892 ; FINGER, GHON and SCHLAGENHAUFER,
Beitrage zur Biologie des Gonococcus &c., Archiv fiir Dermatologie, xxviii. 1894.

149). FRANKEL, A., Bakteriologische Mitteilung, Zeitschr. f. klinische Med. x.
1886, and Weitere Beitrage zur Lehre von der genuinen fibrindsen Pneumonie,
ibid. xi. 1886; WEICHSELBAUM, A., Ueber die Aetiologie der akuten Lungen-
und Rippenfellentziindungen, Wiener mediz. Jahrb. 1886.

149). KOCH, ROBERT, Die Aetiologie der Milzbrandkrankheit, begriindet auf
die Entwicklungsgeschichte des Bacillus Anthracis, 1876. Beitrage z. Biol. der
Pflanzen, vol. ii, and Mitteilungen aus dem Reichsgesundheitsamte, i.1881. The
Anthrax rods were first observed in the blood by RAYER, Mémoires de la Société
de Biologie, vol. ii., Paris 1851; POLLENDER, Mikroskopische u. chemische
Untersuchung des Milzbrandblutes, Caspers Vierteljahrsschr. f. gerichtl. Med.
viii. 1855 ; BRAUELL, Versuche und Untersuchungen, betreffend den Milzbrand
des Menschen u. der Tiere, Virchow’s Archiv, vol. ix, 1857. The experimental
proof that the rods were the actual cause of the disease was given (as far as
was then possible without pure cultures) by DAVAINE, who inoculated animals
with blood containing the bacilli: see Recherches sur les infusoires du sang
dans la maladie connue sous le nom de sang de rate, Comptes rendus, vol. lvii.
1863, vol. lix. 1864, &c. See also notes 17 and 18.

150). NICOLAIER, Beitrige zur Aetiologie des Wundstarrkrampfes, Dissert.
Gottingen 1885 (also in the Deutsche mediz. Wochenschr. 1884); KITASATO,
Ueber den Tetanusbacillus, Zeitschr. f. Hygiene vii. 1889; KITT, Ueber
Tetanusimpfungen bei Haustieren, Centralbl. f. Bakt. vol. vii. 1890.

150). LOFFLER, Untersuchungen iiber die Bedeutung der Mikroorganismen fiir
die Entstehung der Diphtherie, Mitteilungen aus d. Reichsgesundheitsamte,
vol. ii. 1884; Roux and YERSIN, Contribution a I’étude de Ja diphthérie, Annales
Pasteur, ii., ili, iv., 1888 to 1890; ESCHERICH, Aetiologie u. Pathogenese der
epidemischen Diphtherie, Wien 1894.

134. (pp. 152, 168). Kocu, R., Die Aetiologie der Tuberkulose, Mitteilungen a. d. kaiserl.

135. (p.

Gesundheitsamte, ii. 1884; NOCARD and ROUX, Sur la culture du bacille de la
tuberculose, Annales Pasteur, i. 1887; PROSKAUER and BECK, Beitrage zur
Ernahrungsphysiologie des Tuberkelbacillus, Zeitschr. f. Hygiene, xviii. 1894 ;
CZAPLEWSKI, Die Untersuchung des Auswurfes auf Tuberkelbazillen, Jena 1891 ;
also note 16.

152). GARTNER, Ueber die Erblichkeit der Tuberkulose, Zeitschr. f. Hygiene
xiii, 1893.

136. (p. 153). According to R. Kocu’s latest publication (Ueber neue Tuberkulin-

pradparate, reprinted from the Deutsch. med. Wochenschr. 1897) the stronger
tingibility of the tubercle bacillus is due to the presence of two fatty acids.
After their removal by hot caustic soda solution, the tubercle bacilli are said
to stain just as other bacteria do. In the opinion of the author this agrees
perfectly with the physical theory of staining (p. 7, see A. FISCHER, Untersuch-
ungen iiber den Bau der Cyanophyceen und Bakterien, 1897). For hot caustic
soda undoubtedly lessens the density of the cell contents and membrane, and.
so reduces the power of storing up the colour. This is the explanation of

we

182

187. (p.

138. (p.

139. (p.

140. (p.

141. (p.

NOTES

Kocw’s observation, not the solubility of the fatty acids, which are doubtless
quite inactive.

154). GAFFKy, Zur Aetiologie des Abdominaltyphus, Mitteil. aus dem Reichs-
gesundheitsamt, ii. 1884 ; ESCHERICH and KIESSLING, note 120; EHRENFEST,
Studien iiber die ‘ Bacterium coli Aahnlichen’ Mikroorganismen normaler
menschlicher Faces, Archiv f. Hygiene xxvi. 1896; LOFFLER and ABEL, Ueber
die spezifischen Eigenschaften der Schutzkérper im Blute Typhus- und Coli-
immuner Tiere, Centralbl. f. Bakt. vol. xix. 1896.

155). LOFFLER, Ueber Epidemieen unter den im hygienischen Institute zu
Greifswald gehaltenen Mausen und iiber die Bekimpfung der Feldmauseplage»
Centralbl. f. Bakt. xi. 1892; KORNAUTH, Die Bekampfung der Mauseplage
mittels des Bacillus typhi murium, ibid. xvi. 1894; Gives the results of thirty-six
farmers, of whom thirty were successful, some highly so.

155). Kocu, R., in Bericht iiber die Thatigkeit der zur Erforschung der Cholera
im J. 1883 nach Egypten und Indien entsandten Kommission, Berlin 1887,
bei Springer. VoGEs, Die Cholera-Immunitaét, zusammenfassende Uebersicht,
Centralbl. f. Bakt. xix. 1896.

155). PETTENKOFER, M. v., Ueber Cholera, mit Beriicksichtigung der jiingsten
Choleraepidemie in Hamburg. Miinchener mediz. Wochenschrift, xxxix: 1892.
156). On cholera-like vibrios, e.g. FINKLER and Prior, Forschungen iiber
Cholerabakterien, Bonn 1884. GAMALEIA, Vibrio Metschnikovi et ses rapports
avec le microbe du choléra asiatique, Annales Pasteur, ii. 1888. HEIDER,
Vibrio danubicus, Centralbl. f. Bakt., xiv. 1893. GUNTHER, Vibrio aguatilis,
Deutsche mediz. Wochenschr. 1892. DIEUDONNS, Zusammenfassende Ueber-
sicht tiber die in den letzten zwei Jahren gefundenen ‘ choleradhnlichen’
Vibrionen, Centralbl. f. Bakt. xvi. 1894.

142. (pp. 159, 162). Experiments for obtaining the diphtheria toxines are described by

143. (p.

144, (p.

Roux and YERSIN, Annales Pasteur, ii-iv. 1888-90. LOFFLER, Der gegen-
wartige Stand der Frage nach der Entstehung der Diphtherie, Deutsche
mediz. Wochenschr. 1890. BRIEGER and C. FRANKEL, Untersuchungen iiber
Bakteriengifte, Berliner klinische Wochenschr. 1890. DzIERGOWSKI and
REKOWSKI, Recherches sur la transformation des milieux nutritifs par les
bacilles de la diphthérie et sur la composition chimique de ces microbes,
Archives d. scienc. biol. publ. par I’Inst. imp. de méd. expérim. Pétersbourg,
vol. i. 1892. KOSSEL, Zur Kenntnis des Diphtheriegiftes, Centralbl. f. Bakt.,
xix. 1896.—On tetanus poison: KITASATO, Experimentelle Untersuchungen
iiber das Tetanusgift, Zeitschr. f. Hygiene, x. 1890. GUMPRECHT, Versuche
iiber die physiologische Wirkung des Tetanusgiftes im Organismus, Archiv
f. die ges. Physiol., vol. lix. 1894. BRIEGER and COHN, Untersuchungen iiber
das Tetanusgift, Zeitschr. f. Hygiene, xv. 1893. BRIEGER and BoER, Deutsche
mediz. Wochenschr. 1896, No. 49.—See also notes 143, 145, 147, 152. At the
present time vigorous efforts are being made to isolate the toxines of all the
pathogenic bacteria.

160). KocH, R., Weitere Mitteilungen iiber ein Heilmittel gegen Tuberkulose,
Deutsche mediz. Wochenschr. 1890, No. 46%, 1891, No. 3; KUHNE, Zeitschr.
f. Biologie, Neue Folge, vol. xi. 1893 (Chemische Untersuchung des Tuber-
kulins von 1890) ; Kocu, R., Ueber neue Tuberkulinpriparate, Deutsche mediz.
Wochenschr. 1897, No. 14. :

161). VAN TIEGHEM, Sur le ferment butyrique & I’époque de la houille
(Carboniferous period), Comptes rendus, Paris 1880, vol. xcix., and Annales

145. (p.

146. (p.

147. (p.

H8. (p.

149. (p.

150. (p.

151. (p.

152. (p.

NOTES 183

d. scienc. nat., Botan., 6. série, ix. 1880; also RENAULT, Recherches sur les
bactériacées fossiles, ibid. 8. série, ii. 1896.

161). METSCHNIKOFF, Ueber die Beziehungen der Phagocyten zu Milzbrand-
bazillen, Virchow’s Archiv, xcvii. 1884, Théorie des Phagocytes, Annales
Pasteur, i, 1887, and numberless other papers by METSCHNIKOFF that are
largely devoted to the polemical war that arose over his doctrines, such as:
FLUGGE, Studien iiber die Abschwachung virulenter Bakterien und die erwor-
bene Immunitaét ; BITTER, Kritische Bemerkungen zu Metschnikoffs Phago-
cytenlehre ; NUTTALL, Experimente iiber den bakterienfeindlichen Einfluss des
tierischen Kérpers, Zeitschrift f. Hygiene, iv. 1888 ; BAUMGARTEN, Ueber das
‘Experimentum crucis’ der Phagocytenlehre, Ziegler’s Beitrage zur pathol.
Anat., vii. 1889.—METSCHNIKOFF, Immunitdt, in Weyl’s Handb. d. Hygiene,
vol. ix., 1. Lief. 1897.

162). BUCHNER, Ueber die bakterientétende Wirkung des zellenfreien Blut-
serums, Centralbl. f. Bakt. v. and vi. 1889; Ueber die nahere Natur der
bakterientétenden Substanz im Blutserum, ibid. vi.; Untersuchungen iiber die
bakterienfeindlichen Wirkungen des Blutes und Blutserums, Archiv f. Hygiene,
x. pp. 84-173, 1890. Fopor, Neue Untersuchungen iiber die bakterientétende
Wirkung des Blutes und iiber Immunisation, Centralbl. f. Bakt. vii. 1890. See
also notes 154 to 161 on the specific serum reactions.

163). BORDET, Sur le mode d’action des sérums préventifs, Annales Pasteur,
1896, x.; Les leucocytes et les propriétés actives du sérum chez les vaccinés,
ibid. ix. 1895. Roux, Sur les sérums antitoxiques, ibid. 1894, viii. HAHN,
Ueber die Beziehungen der Leukocyten zur baktericiden Wirkung des Blutes,
Archiv f. Hygiene, xxv. 1895.

163). KOBERT, Lehrbuch der Intoxikationen, 1893, pp. 261, 554; EHRLICH, P.,
Experimentelle Untersuchungen iiber Immunitaét, Deutsche mediz. Wochenschr.
1891, Nos. 32 and 44 (experiments with ricin and with abrin, the poisonous principle
in the seeds of Abrus precatorius).

163). BEHRING, Infektion und Desinfektion, 1894, p. 172, &c., and many other
papers ; see also the subsequent notes. EHRLICH, KOSSEL, and WASSERMANN,
Ueber Gewinnung und Verwendung des Diphtherieheilserums, Deutsche mediz.
Wochenschr. 1894, No. 16; EHRLICH and WASSERMANN, Zeitschr. f. Hygiene,
xvili., 1894.

164). BEHRING, Die Blutserumtherapie. i. Die praktischen Ziele und die
Immunisierungsmethoden zum Zweck der Gewinnung von Heilserum; ii.
Das Tetanusheilserum und seine Anwendung auf tetanuskranke Menschen.
Leipzig, 1892.

164). Roux and MarTIN, Contribution 4 étude de la diphthérie, Annales
Pasteur, viii. 1894.

164). In BEHRING, Bekampfung der Infektionskrankheiten, Infektion und Des-
infektion, 1894, p. 188, and in many other passages, the specific amntitoxic
effect of the serum is emphasized. It has no anti-bacterial (bactericidal) action.

153. (pp. 161, 165). EHRLICH and WASSERMANN, Ueber die Gewinnung der Diphtherie-

154, (p.

antitoxine aus Blutserum und Milch immunisierter Tiere, Zeitschr. f. Hygiene,
xvili. 1894.

165). EHRLICH and HUBENER, Ueber die Vererbung der Immunitit bei
Tetanus, Zeitschr. fiir Hygiene, xviii. 1894. VAILLARD, Sur I’hérédité de
limmunité acquise, Annales Pasteur, x. 1895.

184

155.

156,

158.

159.

160.

162.

163.

164,

(p.

(p.

» (p.

(p.

(p.

(p.

. (p.

(p.

(p.

(p.

NOTES

165). BEHRING, Infektion und Desinfektion, p. 160, and Deutsche mediz.
Wochenschr. 1893, No. 48. WLADIMIROFF, Ueber die antitoxinerzeugende
und immunisierende Wirkung des Tetanusgiftes, Zeitschr. f. Hygiene, xv. 1893.'

165). If this assumption were correct it would explain the well-known fact
that only the earliest stages of diphtheria can be successfully treated by the
serum method. It would account too for Koch’s observation (note 143) that
the new tuberculin T. R. must be.used within one or two weeks of inoculation
if it is to have a really curative effect. In all these cases the toxines introduced
artificially may be imagined to bring about toleration before the body is inundated
with poison from the bacterial centres.

166). Roux, Sur les sérums antitoxiques (Ann. Pasteur 1894), thinks it probable
that the antitoxines act upon the tissue cells and make them insensitive to the
toxines. BEHRING (Infektion und Desinfektion) is inclined to think the toxines
are destroyed by the antitoxines.

166). BEHRING in the works cited; also in Die Geschichte der Diphtherie,
Leipzig 1893, and Gesammelte Abhandlungen zur 4tiologischen Therapie,
Leipzig 1893. EHRLICH, Die staatliche Kontrolle des Diphtherieheilserums,
Berl. klin. Wochenschr. 1896.

166). Of the innumerable works on the value of serum therapeutics that have
already appeared may be mentioned: BEHRING, Die Statistik der Heilserum-
frage, Marburg 1895 ; HEUBNER, Klinische Studien iiber die Behandlung der
Diphtherie mit dem Behring’schen Heilserum, Leipzig 1895; ESCHERICH,
Diphtherie, Croup und Serumtherapie, 1895; GOTTSTEIN and SCHLEICH,
Immunitat, Infektionstheorie und Diphtherieserum, Berlin 1894; GANGHOFNER,
Die Serumbehandlung der Diphtherie, Jena 1897.

166). PFEIFFER, Die Differentialdiagnose der Vibrionen der Cholera asiatica
mit Hilfe der Immunisierung, Zeitschr. f. Hygiene, xix. 1895; PFEIFFER,
Centralbl. f. Bakt., xix. 1896, pp. 191, 385, 593; ibid. xx. 1896, p. 129.
BORDET, Sur le mode d’action des sérums préventifs, Annales Pasteur, 1896.
Dunsar, Zur Differentialdiagnose der Choleravibrionen, Zeitschr. f. Hygiene,
vol. xxi.

167). PFEIFFER and PROSKAUER, Beitrage zur Kenntnis der spezifisch wirksamen
Kérper im Blutserum von choleraimmunen Tieren, Centralbl. f. Bakt., xix.
1896, p. 197.

168). PAasTEUR, Comptes rendus, Oct. 26, 1885, and in many other papers
on this very remarkable inoculation, which is indeed a kind of serum treatment.
For the organs employed (cord and brain) contain not only the postulated
antitoxin, but also the poison which is thus introduced into the bitten animals,
only by a different vehicle. Pasteur’s genius did not need pure cultures of
the unknown hydrophobia germ, and this gives us a right to hope that we may
succeed with his method in the case of other diseases.

168). CHAMBERLAND, Résultats pratiques des vaccinations contre le charbon
et le rouget en France, Annales Pasteur, viii. 1894; see also note 17.

168). GRUBER, Miinchener mediz. Wochenschr. 1896, applies the term ‘ glabri-
ficines’ to the substances in the blood of immunized animals, which cause the
membranes of the bacteria to swell up, and the rods themselves to agglutinate.
‘Lysines’ and ‘antilysines’ were introduced by Krusse (FLUGGE, Mikroorg.,
3rd ed., vol. i. pp. 409, 414). Of all these substances we know only the names.

INDEX

Abeles, criticism of Buchner, p. 179.

Acetic acid, 100; production of by anthrax
bacilli in milk, 118, by bacteria, 113,
114; by butyric —, 121 ; in fermentation
of bread, 125; by kephir, 120; by
yeast, 132.

Acetic bacteria, 111, Fig. 24; literature of,
176; cessation of growth of in partial
vacuum, 61.

Acetic fermentation, 112}; activity of, 113;
equation for, 112.

Achorion Schoenbeinii, occurrence of in
Favus, 42.

Acne, cause of, 148.

Actinomyces, suppuration induced by, 148;
A, bovis, culture and characters of, 41;
supposed sporangia of, 42.

Aerobes, definition of, 60.

Aetherial oils as disinfectants, 86.

Agar media, 57; non-liquefaction of by
bacteria, 59.

Air, presence of bacteria in, 44.

Albumoses, properties of, 99.

Alcohol, heat of combustion of, 135; in-
hibitory percentage of, 83; — for
acetic bacteria, 112; lethal percentage
of, 83; resistance of spores to, 84;
negative chemotaxis of, 79.

Alcoholic fermentation, 126 seq., 178, 1313
by-products of, 132; influence of
oxygen and temperature on, 132.

Alcohols, nutritive value of, 57; fermenta-
tion of, 114.

Aldehydes, production of by yeast, 132.

Alder, root-tubercles of, 89.

Alexines, occurrence and importance of,
162,

Alimentary canal, bacteria of, 180.

Alkalies, influence of on toxines,
negative chemotaxis of, 79.

Allococcaceae, characters of, 32, 33.

Alvarez, on indigo fermentation, 178.

Amido-bacteria, 55 ; — compounds, 99.

Amines, nutritive value of, 57.

160;

Ammonia, occurrence of in cheese, 119;
production of by putrefaction and fer-
mentation, 103.

Ammonia-bacteria, 55.

Ammonium chloride, as source of nitrogen,
55; — tartrate as source of N, 55.
Amoeba, importance of, 39; rapidity of
fission in, 18; A. cofz, connexion of

with dysentery, 40.

Amygdalic acid, bacterial decomposition of
into + and - optical components, 115.

Amy] alcohol, production of by yeast, 132.

Amylobacter butyricus of van Tieghem,
121.

Anaerobes, definition of, 60.

Anaerobic bacteria, distribution and import-
ance of, 61 ; — fermentation, reduction
of indigo during, 133.

Anaerobiosis, literature of, 172, 176; rela-
tion of to fermentation, 133, 135.

Anilin dyes, as disinfectants, 86.

Animalcula, definition of, 1.

Anthrax bacilli, abscesses due to, 145 ; aspo-
rogenous varieties of, 170; facultative
parasitism of, 49; formation of chains
by, 3; germs of, 149; growth of in
fluid media, 58; in milk, 117, 118;
history of, 149; infection by, 145;
inoculation for, 168 ; literature of, 181;
transference of, 150; —, inhibitory per
cent. of poisons for, 82; — lethal per
cent. for spores of, 83; permanence
of, 30; plate and stab-cultures of, 59;
resistance of to low temperatures, 75 ;
size of, 4; — spores, vitality of, 77.

Antilysines, 168, 184.

Antiseptics, 173; — treatment of wounds,
8

rE
Antitoxins, hypothetical character of, 166;
literature of, 183, 184; occurrence and
properties of, 162, 163; theory of, 165.
A phanothece, shape of, 37.
Arsenious acid, influence of on ferments,
109.

186

Arthrospores, 22, 23.

Aseptic treatment of wounds, 87.

Ash, percentage of in bacteria, 52; essential
amounts of, 53, 54.

Asparagin, as source of nitrogen, 55;
chemotactic attraction of, 79.

Aspartic acid, 99.

Aspergillus, parasitism of, 42; patho-

genicity of species of, 42 ; — yeast, 110,
176.
Asporogenous condition, induction of, 28.
Attenuation, 27; influence of on spore
formation and virulence, 28.
Australia, nitrite bacteria of, 105.
Autoclave, use of, 76.

Bacilleae, 33.

Bacillus, 1; definition of, 2, 32; characters
of genus, 33; B. aceté, 111, Fig. 24,
action of I on, 113; &. acd? lacticz,
111, Fig. 24, shape and properties of,
117; &. acidificans longissimus, shape
of, 117; B. aerogenes, shape of, 117 ;
B. anthracis, capsules of, 10, Fig. 7;
characters and life-history of, 149;
formation of spores of, 20, Fig. 113
conditions for — , 23, 150; germina-
tion of, 20, Fig. 11, 22; growth of
in culture media, 55; plugging of
capillaries by, 159; production of
caprionic acid in milk by, 118; tempera-
ture optimum for, 28, minimum and
maximum for, 74; &. drunneus, colour
of, 12; B. dbuccalis maximus, p. 1,
Fig. 13 granulose reaction of, 139;
shape of, 140, Fig. 26; occurrence
of in faeces, 141; B. Chauveti, 150,
pathogenic character of, 112; &. cold,
101, 151, Fig.28; growth of on different
culture media, 55; — influence of

-acidity of on, 56; non-liquefaction of
gelatine by, 58; production of indol
by, 102; B. colt communis, 15; fer-
mentative properties of, 112; fermenta-
tion of carbohydrates by, 102; in-
fluence of peptone and ammonia on
fermentative products of, 115; 2.
cyaneo-fuscus, pigment of, 13 ; B.cyano-
genus, colour of, 12; discolouration
of milk by, 118; B. atphtheriae, 151,
Fig. 28; characters of, 150, 151,
Fig. 28; 2B. ethaceticus, fermentative
activity of, 114; B. fluorescens ligue-
Jaciens, chemotactic attraction of, 79,
Fig. 18; putrefactive properties of,
102; B. indigogenus, fermentation of
indican by,124; B. Kitzingdanus, 111,
Fig. 24; action of I on, 112; B. lepto-
sporus, germination of spore of, 20,
Fig. 11, 22; B. maligni oedematis,
150; B. orthobutylicus, fermentative
activity of, 114; properties of, 121;

INDEX

B. Pasteurtanus, w11, Fig. 24; action
of I on, 112; fermentative activity of,
113; B. phosphorescens, minimum,
maximum, and optimum temperatures
for, 74; B. prodigiosus, colour of, 12 ;
coloration of milk by, 118; percentage
composition of, 52; production of
lactic acid by, 116; &. proleus, 15,
» 102, Fig. 22; B. putrificus colt,

occurrence of in intestine, 141; putre-
factive products of, 102; B. pyocyaneus,
decomposition of fat by, 108; growth
of on culture media, 55; in sugar
solution, 56 ; suppuration due to, 148;
B. radicicola of Beyerinck, 92; &.
subtilis, 15, Fig. 8; cessation of growth
of in partial vacuum, 61; ciliation of,
15; permanence of —, 31; supposed
conversion of anthrax bacilli into, 30;
germination of spore of, 20, Fig. 11,
22; growth of on cuiture media, 55;
influence of acidity of on, 56; in-
volution forms of, 26, 27, Fig. 14; life
cycle of, 25, 26, Fig. 13; minimum,
optimum, and maximum temperatures
for, 74; rapility of fission in, 17;
resistance of spores of to boiling, 76;
B. tetani, 151, Fig. 28; characters of,
150; obligate anaerobiosis of, 61;
B. thermophilus, optimum, minimum,
and maximum temperatures for, 74;
B. tuberculosis, 151, Fig. 28; absence
of spores in, 153; characters of, 153;
culture of, 152; cellulose in cell-wall
of, 9; growth of in fluid media, 58;
minimum, maximum, and optimum
temperatures for, 74; tingibility of,
181; B. ¢yphi, 151, Fig. 28; characters
of, 154; ciliation of, 15; growth of
in culture media, 55; non-liquefaction
of gelatine by, 58; polar granules of,
9; B. typhi murium, use of, 154;
B. ureae, 102, Fig. 22; B. violaceus,
arthrospores of, 23; colour -of, 12;
distribution of pigment in, 133; in-
fluence of oxygen on pigment formation
of, 61; &. virens, distribution of pig-
ment in, 12; B. wiscosus sacchari,
nutrition of, 123; B. vudgarzs, nutritive
requirements of, 29; putrefactive
powers of, 102; zoogloea of, 31.

Bacteria, of mouth and teeth, 140, Fig. 26;
diseases of plants induced by, 138;
non-penetration of plants by, 138.

Bactericidal substances, 83, 166.

Bacteriological analysis, methods and
technics of, 1713; of soils, 47.

Bacterio-purpurin, functions and spectrum
of, 68, 69.

Bacteriosis, 138.

Bacterium, definition of, 32; dimensions
of, 4; 2. acelicum, 110; B. acidt

INDEX

Jactici, definition of, 116; B. butyri-
cum,110; B.coli communz, occurrence
and properties of, 141, 142; 2.
Pasteurtanum, involution forms of,
27, Fig. 14; B. photometricum, photo-
philism of, 72; B. vanicidum, patho-
genic character of, 38; &. dermo,
definition of, 101; ZB. Zopfi, 102,
Fig. 22.

Bacteroids, as involution forms, 27 ; shape
of, 90, Fig. 19.

Bactridium, characters of genus, 33; 2.
colt, 151, Fig. 28; B. coli commune,
154; B. proteus, 102, Fig. 22; colony
of, 4; &. typhi, 151, Fig. 28.

Bactrillum, characters of genus, 33; 2.
pseudotermo, 102, Fig. 22.

Bactrinium, characters of genus of, 33.

Baier, on butyric fermentation, 177.

Raktron, 32.

Barium, substitution of for calcium, 54.

Baumgarten, on phagocytosis, 183; on
pathological mycology, 180.

Beer, mucilaginous ‘fermentation’ of, 123 ;
spoiling of, 120;'— yeast, purification
of, 120.

Bees, bacterial disease of, 138.

Beggiatoa, 65, Fig. 17,66; characters of, 2,
34; oscillating movements of, 16.

Behrens, on tobacco fermentation, 178.

Behring, on antitoxic serum, 184; on dis-
infection, 173, 184; on immunization
of horses to tetanus, 164; on infection,
100; onserum therapeutics, 183.

Behring and Ehrlich, on antitoxins, 166.

Benzoic acid, nutritive value of, 57.

Bernheim and Folger, on involution forms
of diphtheria bacilli, 170.

Beyerinck, on artificial culture of root-
tubercle bacteria, 92; on assimilation
of nitrogen by Bacillus radictcole, 174;
on butyric bacteria, 121; on butyric
fermentation, 177; on glucose, 178;
on nutrition of bacteria, 56, 171; on
pigment bacteria, 169; on phosphor-
escent bacteria, 172; on root-tubercle
bacteria, 174; on Spirillum desulfurt-
cans, 176.

Bienstock, on faecal bacteria, 175.

Bile, action of on bacteria, 87.

Billroth, on bacteria of wounds, 28; on
species of bacteria, 170.

Biochemistry, Chap. X-XIV.

Bitter, on phagocytosis, 183.

Blastomycetes, 126.

Blood, occurrence of bacteria in, 142;
pathogenic bacteria of, 145.

Blood-serum, as nutritive medium, 57.

Bordet, on preventive serums, 183, 184.

Botrytis cinerea, inhibitory action of light
on spore formation in, 72; rotting of
fruits due to, 99.

187

Bouquet of wine, origin of, 129.

Branching, of filamentous bacteria, 3.

Branell, on anthrax, 81.

Bread, cause of rising of, 125.

Brefeld, on division of bacteria, 170; on
fungal yeasts, 178; on germination of
bacterial spores, 170; influence of light
on fungi, 172.

Brieger, on ptomaines, 100, 175; B., Boer
and Cohn, on tetanus poisons, 182;
Pe and Frankel, on bacterial poisons,
182.

a Via water, lethal percentage of, 83,

4.

Brown, on fermentative power, 179.

Brownian movement, 14.

Bruns, on tubercle bacillus, 170.

Buchner, on alexines, 162; on bacteri-
cidal action of blood serum, 183; on
chemotaxis of leucocytes, 173; influence
of light on bacteria, 172; Ed. Buchner
on zymase, 179; H. Buchner, on
alcoholic fermentation without yeast
cells, 179.

Buchner, Langard, and Riedlin, on rapidity
of multiplication of bacteria, 170.

Budding, rapidity of in yeast cells, 126.

Bumm, on micro-organisms of gonorrhoea,
181.

Burri and Stutzer, on denitrifying. bacteria,
175.

Biisgen, on culture of Cladothrix dichotoma,
170.

ica on Cyanophyceae and Bacteria,
169.

Butter, number of bacteria in, 118; causes
of rancidity and of flavour of, 118.
Butyl-alcohol, production of by bacteria,

114, 122.

Butyric acid, 100; heat of combustion of,
135; occurrence of in cheese, 119;
production of by bacteria, 114; — in
rancid butter, 118.

Butyric bacteria, fermentation of lactic
acid and of milk by, 122; spoiling
of beer by, 120; varied nutrition of,
29.

Butyric fermentation, 121; literature of,
177; products of, 122.

Cadaverine, 100; inhibitory percentage of,
82.

Cadeac and Bournay, on action of diges-
tive fluids on bacteria, 174.

Caesalpineae, occurrence of root-tubercles
in, 9I.

Caesium, substitution of for potassium, 54.

Calcium lactate, fermentation of, 122.

Cancer, supposed origin of, 39.

Cane sugar, inversion of, 110.

Capsules, occurrence of, 10.

188

Carbohydrates, bacterial fermentation of,
116 seq.; function and occurrence

ot, 53.

Carbolic acid, inhibitory percentage of, 82 ;
lethal — of, 83.

Carbon, sources of, §5 ; —compounds, nutri-
tive importance of, 56, 57 ; — dioxide,
circulation of, 107, chap. XII; inhibi-
tory action of, 86; — monoxide, in-
hibitory action of, 86.

Carbuncle, causes of, 145 ; production of by
pus bacteria, 148.

Caries of teeth, causes of, 140.

Casein, in cheese, 119; in curds, 119.

Caseoglutin, occurrence of in cheese, 119.

Cattle, anthrax in, 149, 150.

Cell-wall of bacteria, attachment of, 8;
cellulose in, 9; chemical composition
of, 9; permeability of, 8, 9.

Cellulose, fermentation of, 122, 177; occur-
rence of in tubercle bacilli, 153.

Certes, influence of high pressures on
bacteria, 173.

Chamberland, on attenuation,
preventive inoculation, 184.

Cheese, bacteria of, 111, 118; composition
and ripening of, 119 ; literature of, 177.

Chemical composition of bacteria, 52.

Chemotaxis, 78 seq., chap. IX; literature
of, 173 3 application of Weber's law to,
80; character of movement and in-
fluence of concentration, 81; negative,
79; operation of in nature, 80.

Chilian saltpetre, origin of, 104.

Chlorine, necessity of, 54; — water, lethal
percentage of, 83, 84.

Chloroform, action of on ferments, 109.

Cholera, germs of, 155 ; — decomposition of
fat by, 108; facultative parasitism of,
49; growth of in fluid media, 58;
—in plate cultures, §9 ; — in milk, 117,
118; literature of, 182; polytrophism
of, 29; production of indol by, 101;
reproductive activity of, 17; transfer-
ence of, 156; — serum, 167.

Chromatin granules, occurrence of in
diphtheria bacilli, 151 ; in Cladothrix,
7, Fig. 5. rer ;

Chromatium, 66; distribution of pigment
in, 12; C. Okentz, 65, Fig. 17.

Chromic acid, as fixing fluid, 6.

Chromogenic bacteria, 12.

Chromoparous bacteria, 12, 13.

Chromophorous bacteria, 12.

Chroococcus, shape of, 37.

Cider, influence of bacteria on, 114.

Cilia, abscission of, 15; anomalies in con-
nexion with, 18; influence of external
agencies on, 16; constancy and
importance of, 31; forms of, 15, Fig.
8; influence of oxygen on, 61; use
and staining of, 14.

170; on

INDEX

Ciliation, taxonomic importance of, 15.

Citric acid, production of by mould fungi,
Ilo.

Cladonia furcata, 93, Fig. 20.

Cladophora, evolution of oxygen from, 62,
Fig. 16; C. fracta, process of cell-
division in, 17.

Cladothrix, 36; arthrospores of, 22 ; charac-
ters of, 34 ; occurrence of iron in, 70;
‘sap-vacuoles in, 6; sliding growth of,
18; sheaths of, 10; C. dichotoma, 3,
Fig. 2; chromatin granules of, 7,
Fig. 5 ; formation of swarm spores by,

_ 25, Fig. 12; pleomorphy of, 24.

Classification of bacteria, 32, 33, 343
difficulty of, 30; features of, 30, 31.

Claudon and Morin, 178.

Clostrideae, 33.

Clostridium, characters of, forms of, 121 ;
C. butyricum, 111, Fig.24, 121; forma-
tion of spores of, 20, Fig. 11; germina-
tion of, 22; C. Pasteurianum, charac-
ters of, 96; fixation of N by, 96.

Coal gas, inhibitory action of, 86.

Coccaceae, 32.

Cocci of suppuration, 147, 148, 149.

Coccidia, 41.

Coccobacteria septica, 29.

Coccus, definition of, 2.

Cohn, 2; on &. Zermo, tol ; on classification
of bacteria, 29, 30, 31, 171 5; on thermo-
genic bacteria, 63, 172; on putrefactive
bacteria, 175 ; on relation of tempera-
ture to bacteria, 173; on resistance to
desiccation, 173 ; on species of bacteria,
170; on spores of, 170.

Cohn and Mendelssohn, influence of electric
currents on bacteria, 172.

Colloid silica media, 58.

Colon bacillus, 154.

Commaa bacillus, 156; shape of, 2.

Concentration, influence of on bacteria, 9.

Conn, on influence of bacteria on flavour of
butter, 177.

Contagium animatum, 159; — vivum, 142.

Coppen Jones, on branching of tubercle
bacilli, 170.

Coprinus, influence of light on, 72.

Cornil and Babes, 180.

Corrosive sublimate, poisonous action of,

57.

Coryne bacterium, 27; C. diphtheriae, 150,
ISI.

Cow-pox, microbe of, 40.

Cramer, on composition of bacteria, 171.

Crenothrix, characters of, 34; sheaths of,
10; iron in, 70.

Culture, general principles of, 52, chap. VI ;
— media, 54, 55; — fluid, 56; growth
in, 58; for phosphorescent bacteria,
63; — solid, use of, 57, 58; plate, stab
and streak, 58, 59; influence of on per-

INDEX

centage composition, 52; — of acidity
and alkalinity of, 55, 56, 131.

Cupric sulphate, inefficiency of as disin-
fectant, 84; lethal percentage of, 83.

Curds, composition of, 119; influence of
bacteria on, 119.

Cyanin, inhibitory percentage of, 82.

Cyanogen, as source of carbon, 57.

Cyanophyceae and bacteria, relationship
between, 37.

Cytoryctes variolae, association of with cow-
Pox, 40.

Czaplewski, on tubercle bacilli, 181.

Davaine, on anthrax inoculation, 181.

Dawson, on efficacy of nitragin, 93.

De Bary, on arthrospores, 22, 170; on
species in bacteria, 170; on systematic
position of yeast, 178.

Denitrifying bacteria, 176.

Desiccation, influence of on maximal
temperatures, 76; resistance of bacteria
to, 19, 77, 144, 173; of spores to, 19,
44, 45, 128.

Dextrane, occurrence of in slime bacteria,

53-
Diastase, hydrolytic action of, 109, 131.
Dieudonné, on cholera vibrio, 182.
Digestion, relation of bacteria to, 141.
Digestive fluids, action of on bacteria, 87.
Dissacharides, inversion of, 131.
Diseases, relation of bacteria to, 137, chap.
XV.

Disinfection, 84-87, 173.

Dissociation, influence of on toxicity, 85,
174.

Distilled water, resistance of spores to, 84.

Distribution of bacteria, 44.

Diphtheria, cause of, 150; bacilli of, 151,
152; growth of in milk, 117, 118;
involution forms of, 27, Fig. 14;
occurrence of Streffococcus pyogenes
in, 148 ; — toxin, immunity to, 163.

Diplococcus pneumoniae, properties of, 149.

Division, planes of, 18, 19.

Drepanidium ranae, parasitism of, 41.

Drosera, bacteria on leaf of, 138.

Duclaux, on biological chemistry, 176; on
milk, 176.

Dulcite, fermentation of, 114;
value of, 57.

Dunbar, on diagnosis of cholera vibrios,
184.

Dysentery, supposed cause of, 40. __

Dziergowski and Rekowski, on nutrition of
diphtheria bacilli, 182.

nutritive

Ecballium elaterinum, plasmolysis of, 5.

Effront, 177; hydrofluoric method of, 120.

Ehrenberg, on systematic position of
bacteria, 1.

Ehrenfest, on B. coli, 182.

189

Ehrlich, on diphtheria serum, 184; on
immunity, 183.

Eidam, on resistance to desiccation, 173.

Eleagnus, root tubercles of, 89.

Electric currents, germicidal action of, 72 ;
movements induced by, 73.

Elements, essential for bacteria, 54.

Emmenthaler cheese, composition of, 119.

Endospores, 20, Fig. 11, 21.

Engelmann, bacterium method of, 61, 62,
Fig. 16, 172; on green bacteria, 169;
on purple bacteria, 68, 172.

Ensilage, 120.

Enzymes, 176, 178; definition of, 108;
action of, 110; theory of, 134.

Epithelium, impenetrability of by bacteria,
142, 145.

Eriksson, on breeds of Puccinia, 93.

Emst, 180.

Erysipelas, aetiology of, 148, 180.

Erythrite, butyric fermentation of, 121.

Erythrobacteria, 66; photosynthesis of, 68 ;
rays effective in, 68, 69.

Escherich, on diphtheria, 181 ; on intestinal
bacteria of infants, 176, 180; on serum
therapeutics, 184.

Ether, action of on ferments, 109; lethal
percentage of, 83.

Ethyl alcohol, fermentation of, 112; pro-
duction of by bacteria, 112, 1143 by
yeast, 132.

Ethylidene lactic acid, production of, 116,

Eucarpous, 36.

Eurythermic, definition of. 74.

Exoascus, yeast of, 130.

Facultative anaerobes, 61.

Facultative parasites, 49.

Faeces, bacteria in, 141.

Fat, decomposition of by bacteria, 108 ;
— during ripening of cheese, 119;
occurrence of in old cultures, 14; per-
centage of in bacteria, 52.

Favus, cause of, 42.

Fechner, psycho-physical law of, 80.

Fehleisen, on erysipelas, 180.

Fermentation, 108 ; literature of, 176, 179;
biochemical explanation of, 135;
theories of, 133, 134, 135; technical
uses of, 123, 124, 125; oxidatory, 112;
— organisms, relation of to oxygen,
135; — products, heat of combustion
of, 135, 136.

Ferments, 108; action of, 109; organized,
by-products of, 110.

Fermi, on non-nitrogenous bacteria, 171.

Ferro-bacteria, 69.

Fibrin, butyric fermentation of, 121.

Finger, Ghonand Schlagenhaufer, on biology
of Gonococcus, 181.

Finkler and Prior, on cholera vibrios,
182.

190

Fischer, on cilia of bacteria, 170; on
classification of, 170, 171 ; on enzymes,
176; on phosphorescent bacteria, 172;
on plankton bacteria, 172.

Fischer and Lindner, on enzymes of yeast,
178.

Fission of bacteria, 16; rapidity of, 17;
regular and irregular sequence of, 19.

Fixing of bacteria, 6.

Flagellata, relationship of to bacteria, 38.

Flagellum, taxonomic importance of, 31.

Flax, retting of, 123.

Flexile filaments, 16.

Fliigge, on-.decrease of virulence, 183 ; on
micro-organisms, 169; — pathogenic,
171; on sterilization of milk, 177.

Fodder, lactic fermentation of, 120.

Fodor, bactericidal action of blood, 183.

Formic acid, production of by bacteria, 114.

Formic aldehyde, lethal percentage of, 83.

Fossil bacteria, 161; literature of, 182.

Frank, on symbiosis, 174; F. and Kruger,
on potato scale, 179.”

Frankel, on pneumonia, 181 ; germ of, 149;
on nutrition of bacteria, 171.

Freudenreich, on bacteria in cheese and
milk, 176; on kephir, 177.

Freund, occurrence of cellulose in tubercle
bacilli, 9.

Frog spawn bacterium, 124.

Fruits, causes of rotting of, 99.

Fumaric acid, bacterial decomposition of,
115.

Fungi and bacteria, comparison between,

35,36. :
Fusel oil, produ:tion of, 132.

Gabritschewsky, on chemotaxis of leuco-
cytes, 173.

Gaffky, on typhoid fever, 182.

Galvanotropism, 73.

Ganghofner, on serum treatment of diph-
theria, 184.

Garden mould, presence of bacteria in, 47.
Garré, on aetiology of inflammation, 180;
on production of carbuncles, 148.

Gartner, on heredity of tuberculosis, 181.

Gases, toxicity of, 86.

Gastric juice, action of on bacteria, 87, 174 ;
on cholera germs, 156.

Geotropism, absence of in bacteria, 73.

Geppert, on disinfection, 173.

Gelatine media, 57; liquefaction of, 58.

Germination of spores, 20, Fig. 11, 22; of
yeast, 127.

Gilbert and Dominici, on bacteria of intes-
tine, 180.

Glabrificines, 184.

Glacier ice, occurrence of bacteria in, 46.

Glanders germs, growth of in milk, 117, 118.

Globig, on thermophile bacteria, 173.

Gloeocapsa, shape of, 37.

INDEX

Glucase, 178.

Glycerine, chemotaxis of, 79 ; fermentation
of, 1143 nutritive value of, 57; pro-
duction of by yeast, 132; resistance of
spores to, 84; as source of C, 55;
influence of on assimilation of N, 56.

Glycocol, 100.

Godlewski, on nitrate bacteria, 175.

‘Gommose bacillaire’ of vine, 138.

Gonidia, 22.

Gonococci, 30, 49, 180, 143, Fig. 27; media
for, 57 ; properties of, 148.

Gonorrhoea, cause of, 148 ; — rheumatism,
cause of, 149; literature of, 120.

Gonnermann, on root-nodule bacteria, 174.

Gottstein and Schleich, 184.

Gourd, min., max. and optimal temperatures
for germination of, 74.

Granulobacter, 30; G. lactobutyricus, 11;
G. saccharobutyricus, 121.

Granulose, in bacteria, 13, 121, 139.

Gregarineae, 41.

Grimbert, on B. orthobutylicus, 177.

Gruber, on glabrificines, 184.

Gum arabic, butyric fermentation of, 121.

Gumprecht, on tetanus poison, 182.

Giinther, on Vibrio aguatilis, 182.

Haemamoeba, 40.

Haemopoeic organs, 161.

Haemosporidia, 41.

Halibacterium, 30.

Halogens, germicidal action of, 84.

Hamburger, on action of gastric juice on
bacteria, 174.

Hansen, on acetic bacteria, 112, 176; in-
fluence of heat on sporulation, 129;
on /yoteus, 102; on putrefactive
bacteria, 175 ; on yeast, 110, 178.

Haplobacteria, 2; ordinal rank of, 31.

Haplobacterineae, 32,

Haplomycetes, 41.

Havannah tobacco, bacteria of, 124.

Heat, evolution of from seeds, 62, from
bacteria, 63; influence of on ferments,
109 ; — on virulence of anthrax bacilli,
28; production of by Mzcrococcus, 63;
resistance of spores to, 128.

Heider, on Vibrio danubicus, 182.

Heim, on pathogenic bacteria of milk,
butter, and cheese, 177.

Helfeld, on manure bacteria, 175.
Hellriegel and Wilfahrt, on assimilation of
free N, 91, by Leguminoseae, 174.

Hemp, retting of, 123.

Herpes, 36; cause of, 42.

Hesse, on agar culture, 172; on bacterio-
logical analysis of air, 171.

Hibernation of bacteria, 75.

Holocarpous, 36.

Homococcaceae, 33; characters of, 32.

oo

INDEX

Hoppe-Seyler, on fermentation of cellulose,
177; On oxygen and micro-organisms,
175.

Human body, bacteria in, 139.

Hiippe, on involution forms, 170; on lactic
fermentation, 176.

Hydrogen, inhibitory action of, 86 ; produc-
tion of, 122.

Hydrolytic action of enzymes, 109.

Hydrophobia, inoculation for, 184.

Hyphae, 36.

Hyphomycetes, 41.

Immunity, acquired, 163 ; method of pro-
ducing, 163, 164; duration of, 164;
hereditary, 165; literature of, 183, 184;
peculiarities of, 164.

Incubation period, 158.

Incubators, use of, 75.

Indican, fermentation of, 124.

Indigo, production of, 177 ; — fermentation,
177-

Indigofera tinctoria, 124.

Indigoglucine, production of from indican,
124.

_Indol, 99, 100; absence of from cheese,
119; production of by cholera germs,
101, 156; by bacteria, 98 ; — reaction,
155.

Infection, 180; conditions for, 145; mode
of, 144, 145 ; — thread, 94, Fig. 21.

Inhibitory coefficient, 82.

Inoculation, principle of, 144; by insect
stings, 145; methods, toxines, 163,
attenuated cultures, 164 ; for small-pox,
168.

Intestine, bacteria of, 141; sterility of in
new-born infants, 142.

Invertase, action of on cane sugar, I10;
properties of, 131.

Involution forms, 26, 27 ; literature of, 170.

Iodine trichloride, influence of on virulence
of diphtheria and tetanus bacilli, 29;
inhibitory percentage of, 82.

Lodococcus, presence of granulose in, 139.

Iodoform, lethal percentage of, 83.

Iron, necessity of, 54 ; — bacteria, nutrition
of, 69; literature of, 172.

Isobutyl alcohol, production of by yeast, 132.

Isobutyleneglycol, production of by yeast,
132.

Isolation of pathogenic bacteria, methods
of, 143.

Janowski, influence of light on bacteria, 172.
Japan, nitrite bacteria of, 105.

Java, nitrate bacteria of, 105.

Jenner’s vaccination, 168.

Kappes, on composition of bacteria, 171.
Kaspareck and Kornauth, on infection of
plants by anthrax, 179.

IQ1r

Kephir, composition of, 120; inversion of
lactose by, 131; production and pro-
perties of, 119, 120.

Kitasato, on tetanus bacillus, 181; inocula-
tion of, 181; poison of, 182.

Klechi (v.) on B. saccharobutyricus, 177 3
on ripening of cheese, 177.

Klein, on influence of light on fungi, 172.

Klécker and Schidning, on Saccharomyces,
178.

Kloster, definition of, 32.

Kobert, on intoxications, 183; on ptomaines,
175.

Koch, on anthrax, 2, 169, 181; on Bacillus,
32; on B. anthraczs, 149 ; on cholera,
155, 182; on disinfection, 173; on
gelatine culture, 172; method of, 168,
173; steamer of, 76; on tubercle
bacilli, 152, tingibility of, 181; on
tuberculin, 160, 182; on tuberculosis,
181.

Koch and Hosaeus, on slime fungus of
sugar, 178.

Kornauth, on bacteria in plants, 179; on
typhoid inoculation of mice, 182.

Kossel, on diphtheria toxine, 182.

Kossowitsch, on assimilation of N by algae,
174.

Kramer,on economicimportance of bacteria,
176; on mucilage production, 177; on
ripening of cheese, 177.

Krusse, on lysines, 184.

Kuhn, on putrefactive bacteria, 175.

Kiihne, on tuberculin, 182.

Kurloff and Wagner, action of gastric juice
on bacteria, 174.

Kutscher, on phosphorescent bacteria, 172.

Lactic acid, butyric fermentation of, 115,
122; occurrence of in cheese, 119;
production of by bacteria, 115 ; — in
rancid butter, 118; by Leuconostoc, 124;
in fermenting bread, 125; optical
splitting of by bacteria, 115.

Lactic bacteria, non-liquefaction of gelatine
by, 58; in kephir and in beer, 120,
Lactic fermentation, 116, 117; literature

of, 176.

Lafar, on acetic fermentation, 176; on
acidity of yeast, 177; on spontaneous
generation, 171; on technical myco-
logy, 168, 176, 177.

Lamella (middle), solution of by bacteria,
123.

Lamprocystis, 106; L. roseo-persicina, 65,
Fig. 17.

Lathraea, bacteria in leaf-chambers of, 138.

Laverania, 40.

Lead acetate, lethal percentage of, 13; as
disinfectant, 84.

Leguminosae, nitrogenous nutrition of, 89;
partial parasitism of, 94, 95.

192

Lehmann and Neumann, 169; on Bacterium
and Bacillus, 353; on species of bac-
teria, 171; on fermentation of dough,
178.

Leichman, on ‘ mucilaginous’ fermentation,

. . IF:

Leipzig, nitrogen discharged by sewers of,
103.

Leprosy, germ of, 153.

Leptomitus, character of, 41.

Leptothrix, deposition of iron in sheaths
of, 69, 70; occurrence of in hot springs,
75; L. buccalis, 139; L. tnnominata,
139; L. ochracea, 69.

Lethal coefficients, 82.

Leucin, 98, 99 ; in cheese, 119 ; putrefactive
decomposition of, 100; chemotaxis of,
173.

Leucocytes, ingestion of bacteria by, 161,
Fig. 29.

Leucocytosis, 161; literature of, 173.

Leuconostoc, arthrospores of, 23 ; secretion
of mucilage by, 53; LZ. mesenteroides,
capsules of,10,F ig.7; formation of chains
by, 19; growth of in beet-juice, 124.

Leuwenhoek, discovery of bacteria by, 1;
works of, 169.

Lewkowitsch, on production of amygdalic
acid, 176.

Lichen fungi, parasitism of, 93, Fig. 20.

Liesenberg and Zopf, on ZL. mesenteroides,
178.

Life, origin of, 50; primitive character of, 51.

Light, influence of on erythrobacteria and
on pigment formation, 70; — on viru-
lence of anthrax bacilli, 28 ; deleterious
rays of, 71; literature of, 172; produc-
tion of by bacteria, 63; influence of on
spore-formation by, 72.

Lindner, on technics of fermentation, 178.

Lock-jaw, cause of, 150.

Locomotion, organs and rapidity of in
bacteria, 14.

Loffler, 2; on cilia of bacteria, 170; —
method of staining, 14; on diphtheria,
81, 82; on diseases of mice, 155, 182;
on history of bacteria, 169; on micro-
organisms of water, 171.

Léffler and Abel, on typhoid immunization,
182,

Lophotricha, 15; taxonomic importance of,

31.
Liibbert, biology of bacteria, 1&0,
Luciferin, 64.
Ludwig, on phosphorescent bacteria, 172.
Lupinus, percentage of N in, 89; root-
tubercle of, 90; Z. albus, bacteroids
of, 27, Fig. 14; 90, Fig. 19.
Lysines, 168, 184.

Magnesium carbonate, action of nitrite
bacteria on, 165.

INDEX

Malarial fever, microbe of, 4o.

Maltase, properties of, 131.

Mammalia, bacterial diseases of, 138, 139.

Mangin, 179.

Mannite, fermentation of, 1143; nutritive
value of, §7 3 production of by bacteria,
123.

Marine bacteria, 63, 64, 65.

Massart and Bordet, on chemotaxis of
leucocytes, 173.

Mastigophora, parasitism of, 39.

Mayers, on fermentation, 178.

Mazé, 169; on fixation of N, 92, 174.

Melanin, occurrence of in malaria, 40.

Menge and Kronig, on bacteria of vagina,
180.

Mercuric chloride, inhibitory and poisonous
percentages of, 82, 83.

Merismopoedia, 37.

Messea, 170.

Metatrophic bacteria, 54, 55; — fungi, 35;
definition of, 48, 49.

Methane, 100; — bacteria, 123.

Methylmercaptan, Ioo.

Methyl violet, as a disinfectant, 86.

Metschnikoff, on immunity, 183; on phago-
cytosis, 161, 183.

Miasma, 142.

Mice, anthrax in, 150.

Micrococcus, 33; M. agélis, 12 ; movement
of, 14; AZ. gonorrhoeae, 143, Fig. 27;
151, Fig. 28; characters of, 148; forms
of, 149. 4d. pyogenes, 148, 151,
Fig. 28; MW. tetragenus, 33; mode of
division in, 19, Fig. 10; AZ. wreae, 102,
Fig. 22; decomposition of urea by, 183.

Migula, on Bacterium and Bacillus, 32;
on classification of bacteria, 171.

Milk, bacteria in, 117, 118; changes in-
duced by, 118; ropiness of, 118, 123;
butyric bacteria in, 122; — sugar, in
curds, 119.

Miller, on bacteria of mouth, 180.

Mimoseae, root-tubercles of, 91.

Miquel, on bacteriological analysis of air,
171; on LB, thermophilus, 173; on urea
ferments, 175, 179.

Molisch, on iron in plants, 172.

Moller, 172.

Monas, monotrichous character of, 38.

Monilia candida, 39.

Monosaccharides, fermentability of, 131.

Monotricha, 15.

Monotrophism, 29, 49.

Morphology of bacteria, p. 1 seq., Chap. I.

Mosquitoes, relation of to malaria, 40.

Mould fungi, fixation of N by, 97; organic
disintegration by, 136.

Mouth, bacteria of, 139, 180.

Movements of bacteria, 14, 163 influence
of plasmolysis on, 9; taxonomic value
of, 31.

INDEX

Mucigenous bacteria, 15.

Mucilage, 53, 123 ; excretion of, 10; in beet-
molasses, 124.

Mucor, parasitism of, 43; rotting of fruits
by, 99; — yeast, 110, 130; MZ. corym-
bifer, M. rhizopodiformis, parasitism
of, 43.

Miller, 1.

Mummies, bacteria of, 140.

Miintz, on nitrification, 175.

Mycelium, 36.

Mycobacterium, 27; M. tuberculosis, 153.

Mycoprotein, 53.

Myxomycetes, 36.

Nageli, 2, 29; on fermentation, 179; on
involution forms, 170; on molecular
movements, 169; on nutrition of bac-
teria, 56, 171; on species in, 170;
theory of fermentation of, 134.

Neisser, on gonorrhoeal fungus, 181; on
penetration of intestine by bacteria, 180.

Nencki, on anaerobism, 172 ; on isolation of
mycoprotein, 53 ; on putrefaction, 175.

Nencki and Scheffer, on chemical com-
position of bacteria, 171.

Neoplasm, supposed origin of, 39.

Nepenthes, bacteria in pitcher of, 138.

Neuridine, 100.

Nicolaier, on tetanus, 181.

Nitragin, preparation and uses of, 93.

Nitrate bacteria, 55, 105, 106.

Nitrate-reducing bacteria, 106.

Nitrates, as source of N, 55, 88, 94.

Nitric oxide, inhibitory action of, 86.

Nitrification, literature of, 175.

Nitrifying bacteria, culture medium for, 54,
104, 105, Fig. 23.

Nitrite bacteria, 105.

Nitrites, as source of N, 54, production of,
104, 106.

Nitrobacter, 30, 105, Fig. 23, 106.

Nitrogen, 56 ; amount of discharged from
sewers, 103; circulation of, 88, Chap.
X, 98, Chap. XI; fixation of, 96;
liberation of, 98, 99, 104; influence of
combination of on fermentation, 115;
literature on assimilation of, 174;
sources of, 55, 88; supposed absence
of from certain organisms, 51.

Nitrosococcus, 105. 2

Nitrosomonas, 30; N. europea, 105, Fig. 23.
N. javanensis, 105, Fig. 23-

Nitrous oxide, inhibitory action of, 86.

Nobbe and Hiltner, on breeds of root-
tubercle bacteria, 93, ;

Nobbe, Hiltner, and Schmid, on root-
tubercle bacteria, 174. ;

Nocard and Roux, on tubercle bacillus, 181.

Nostoc, 125.

Nuclei, absorption of dyes by, 7, 8. __

Nutrition, 52, Chap. VI; peculiarities of,

FISCHER

ie)

193

47,48; in fungi, 38; varying character
of, 29.
Nuttall, on animals and bacteria, 183.
Nuttall and Thierfelder, on life without
bacteria, 180.

Oats, consumption of combined N by, 91.

Obermiiller, on tuberculous milk, 177.

Obligatory aerobes, 60; — anaerobes, 61 ;
— metatrophes, 49; — saprophytes,49.

Oceanic ooze, bacteria in, 64.

Oedema, malignant, cause of, 150; omni-
presence of germs of, 47.

Oenanthic ether, production of by yeast, 32.

Otdium, 39; influence of on ripening of
cheese, 119.

Olive oil, decomposition of by bacteria, 108.

Omelianski, on fermentation of cellulose,
177.

Oospora, 4l.

Optical decompositions, 115 ; — properties,
relations of to nutrition and fermenta-
tion, 115.

‘Oscillaria, 37; O. tenuis, 36, Fig. 15.

Osmic acid, as fixing fluid, 6.

Osmotic pressure, 5; in bacterial cells, 8.

Oxygen, influence of on alcoholic fermenta-
tion, 132, 178; — on growth of yeast,
133; — on ciliary movement in aerobes
and anaerobes, 61; — on pigment
formation, 61; — on putrefaction, 100;
— on spore formation, 150; relation of
to fermentative organisms, 135.

Ozone, inhibitory action of, 86.

Pancreatic juice, action of on bacteria, 87.
Papilionaceae, root-tubercles of, 91.
Paracasein, occurrence of in curds, 119.
Parachromatophores, 13.
Paraoxyphenylacetic acid, 100,

Parasites, 47 ; bacterial, 144.

Parasitism, influence of temperature on, 43.

Paratrophic, 48, 49; — bacteria, 57; —
fungi, 36.

Passet, on aetiology of Pneumonia, 180.

Pasteur, 2; on anaerobiosis, 60, 133, 172;
dictum of, 134; on fermentation, 110;
influence of heat on anthrax, 29; on
organized ferments, 108 ; on production
of racemic acid, 176; on protective
inoculation for rabies, 168, 184; — for
anthrax, 168; on silkworm disease,
138, 180 ; on spontaneous generation,
171; studies on beer, 179; wine, 177;
works of, 176.

Pasteur, Chamberland, and Roux, on
attenuation, 170. P. and Joubert, on
fermentation of urine, 175.

Pasteurization of wine, 75.

Pathogenic bacteria, 151, Fig. 28 ; isolation
of, 143; culture and attenuation of, 144;
mode of action of, 158, 159; products

194

of, 159, 160; relation of to host, 146;
varieties and races of, 147.

Paul and Krénig, action of chemicals on
bacteria, 173.

Pea, butyric fermentation of, 121; fixation
of N by, 91.

Pectin compounds, action of retting bacteria
on, 123.

Pediococcus, 33; P. tetragenus, 19, Fig. 10.

Pellicles, formation of, 3; character of, 4.

Penicillium, growth of on fruits, 99; P.
glaucum, 3, Fig. 2; 36, Fig. 15;
influence of on ripening of cheese, 119.

Pentamethylendiamine, 100.

Pepsin, action of on proteids, 110.

Peptone, chemotaxis of, 79; minimal
amount for, 81; properties of, 99; as
source of N and of C, 55 ; — bacteria,

55-

Péré, on lactic fermentation, 176.

Perithecia, 42.

Peritricha, 15 ; taxonomic importance of, 31.

Permeability of bacteria, 8, 9.

Peters, on fermentation of dough, 178.

Petruschky, on pathogenic Streptococci, 180.

Pettenkofer, on cholera, 182.

Pettenkofer and Emmerich, on cholera
germs, 155.

Pfeffer, on chemotaxis, 79, 173; minimal
amount of peptone for, 81; on Weber’s
Law, 173; on symbiosis of Lichens, 95.

Pfeiffer, cholera serum reaction of, 166; on
diagnosis of cholera Vzbrio, 184; on
immunity, 155

Pfeiffer and Proskauer, on cholera antitoxin,
184.

Pfliiger, on phosphorescent bacteria, 172.

Phagocytes, ingestion of bacteria by, 161;
chemotropism of, 162.

Phagocytosis, literature of, 182, 183.

Phenol, 99; action of on ferments, 109 ; on
virulence of bacteria, 28.

Phenylacetic acid, 99.

Phenylamido-propionic acid in cheese, 119.

Phenylpropionic acid, 99.

Phisalix, on asporogenous anthrax, 170.

Phosphorescence, of bacteria, 63, 172;
influence of oxygen on, 63; culture of,
63, 64. .

Phosphuretted hydrogen, production of, 100.

Photobacterium, 30, 63.

Phototactic sensitiveness, 69.

Phthisis, occurrence of Streptococcus pyo-
genes in, 148.

Pigments, of bacteria, 12 seq. ; in milk, 118;
distribution of, 12, 13; influence of
medium on, 56; of oxygen on, 61; in
yeasts, 128.

Pilobolus, influence of light on formation of
sporangia of, 72.

Pinguicula, bacteria on leaf of, 138.

Planococcus, 33.

INDEX

Planosarcina, 33.

Plasmodium malariae, 40.

Plasmolysis, 6; of bacteria, 8, Fig. 6;
influence of on movement, 9.

Plate culture, 58.

Plectridiae, 33.

Plectridium, 33; types of, 121; P. paludo-
sum, 111, Fig. 24; formation of spores
of, 20, Fig. 11. P. fetant, 150.

Plectron, 32; of anthrax, 31.

Pleogeny, 24, 29. :

Pleomorphism, 274; literature of, 170; in
haplo-bacteria, 25.

Pleurococcus, 37.

Pneumococcus, 149; fermentative activity
of, 114.

Pneumonia, cause of, 149.

Poikilothermism of bacteria, 73.

Poisons, chemotaxis of, 79, 80 ; modification
of action of, 81, 82; lethal and inhi-
bitory percentages of, 82, 83.

Polar granules, 9.

Pollender, on anthrax in blood, 18r.

Polysaccharides, non-fermentability of by
yeast, 131.

Polytoma uvella, 38.

Polytrophic bacteria, 29.

Polytrophism, 49.

Popoff, on fermentation of dough, 178.

Potash, caustic, lethal percentage of, 83, 84.

Potassium salts, chemotaxis of, 79; —
nitrate as source of N, 55; — perman-
ganate, lethal percentage of, 83, 84.

Potato, bacterial diseases of, 138.

Prazmowski, on development of bacteria,
170; on involution forms, 170; on
root-tubercle bacteria, 174.

Predisposition, 167.

Pressure, influence of on bacteria, 73, 173.

Primitive organisms, theories of, 7.

Propionic acid, production of by bacteria,

114.

Propyl alcohol, production of by yeast, 132.

Proskauer and Beck, on tubercle bacillus, 181.

Proteids, percentage of in bacteria, 52;
decomposition of, 99, 100.

Proteus, 30, 102; P. vulgaris, 101, 102,
Fig. 22.

Protista, 35; place of in nature, 36.

Prototrophic bacteria, 54.

Prototrophism, 48, 49.

Protozoa, importance of in putrefaction, 136.

Pseudomonas, 32.

Pseudopodia, 4o.

Psichohormium, iron in, 70,

Psycho-physical law, 80.

Ptomaines, importance of, 159; literature
of, 175 ; production of, 100,

Ptomatropine, 100.

Puccinia, cultural varieties of, 93.

Pulmonary phthisis, origin of, 152.

Purple bacteria, 172.

INDEX

Pus-bacteria, 148, 149; — cocci, results of
injection of, 145.

Putrefaction, 101; influence of oxygen and
proteids on, 100; of temperature and
water on, 99 ; peculiarities of, 102, 103 ;
liberation of N by, 104; ultimate pro-
ducts of, 101; — in intestine, 141.

Putrefactive bacteria, 102, Fig. 22; litera-
ture of, 175; zymogenic properties of,
102,

Putrescine, Ioo.

Pyaemia, 145; causes of, 148.

Pykotanine, as disinfectant, 86.

Pyogenic diseases, 148.

Pyrenomycetes, disintegration of wood by,
136.

Quarter-evil of cattle, causes of, 120, 150.
Quinine, inhibitory percentage of, 82.
Rabies, inoculation for, 168; Pasteur’s
treatment of, 184.
Rabinowitsch, on thermophile bacteria, 173.
Racemic acid, optical decomposition of by
bacteria, 115.
Radicles, basic and acid, chemotaxis of, 79.
Ragi, symbiotic character of, 110.
Raphidia, 37.
Rathay, on gum-producing bacteria, 179.
Ray-fungus, parasitism of, 41; sporangia
of, 42.
Rayer, on anthrax bacilli in blood, 181.
Red sulphur bacteria, nutrition of, 68.
Rees, on yeasts, 178.
Renault, on fossil bacteria, 183.
Rennet, production of by bacteria, 117, 118.
Reproduction, of bacteria, by fission, 16
seq.; rapidity of, 17; by spore forma-
tion, 19 seq.
Respiration, of bacteria, 60; intramolecular,
133.
Retting bacteria, 123; of flax, 177.
Rhinanthaceae, partial parasitism of, 96.
Rhizobium leguminosarum, 92.
Riedler, on leucocytosis, 173.
Ring-worm, cause of, 42.
Réntgen rays, action of on bacteria, 73.
\ Root-nodules, 89; intercellular spaces in,
174; literature of, 174; percentage of
N in, 95; structure of, 90, Fig. 19;
bacteria of, assimilation of N by, 89,
92; — shape and properties of, go,
Fig. 19, 92, 933 artificial cultivation
of, 92; penetration of, 94; growth and
nutrition of, 95, Fig. 21.
Roquefort cheese, ripening of, 119.
Rosenbach, on infection of wounds, 180.
Rothenbach, on use of pure yeast, 177.
Roux, on antitoxic serums, 183, 184; on
asporogenous anthrax, 170; on hac-
teriological analysis of water, 171.
Roux and Martin, on diphtheria, 183.

195

Roux and Yersin, on diphtheria, 181 ; —
preparation of toxin of, 182. :

Rubidium, chemotaxis of, 79; substitution
of for potassium, 54.

Russel, on nitrate bacteria of oceanic ooze,

65.

Saccharomyces, determination of species in,
128; forms of, 126, 127, Fig. 25;
nutrition of, 131; pathological proper-
ties of, 36; symbiosis of in kephir,
120; systematic position of, 130; S.
albicans, parasitism of, 39; S. cere-
visiae, races of, 111; sporulation of,
128; S. ellipsoideus, 111 ; sporulation
of, 128; S. glutinis, 128; S. mycoderma,
decomposition of alcohol by, 112; S.
Pasteurianus, sporulation of, 128.

Saké, preparation of, Ito,

Salicylate of sodium, inhibitory percentage
of, 82.

Salicylic acid, lethal percentage of, 83;
action of on ferments, 109.

Saliva, action of on bacteria, 87.

Saltpetre beds, 104.

Salt solution, influence of on toxicity of
sublimate, 86.

Sanarelli, on water bacteria, 180.

Suptapenic bacteria, 29; putrefactive power
of, 101.

Saprophile bacteria, definition of, 49; pro-

perties of, 101.

Saprophytes, 47.

Sarcina, 33; mode of division in, 19, Fig. 10;
social aggregates of, 31; S. ventriculi,
I4I.

Sarcosporidia, 41.

Sauerkraut, production of, 121.

Scherffel, on leaf scales of Lathraea, 180.

me on intestinal bacteria of infants,
180,

Schizomycetes, definition of, 35.

Schizophyta, definition of, 37.

Schloesing and Laurent, on fixation of N
by bacteria, 174.

Schlossberger, on mycoprotein in yeast, 53.

Schorf of potato, 138.

Schreiber, spore formation in bacteria, 170.

Schroeter, on bacterial pigments, 169.

Sclerothrix Kochit, 153.

Scytonemeae, relationship of to Cladothrix,
37.

Sea-water, percentage of bacteria in, 64.

Selective power, 115.

Selmi, on ptomaines, 175.

Septicaemia, 145; causes of, 148.

Sero-therapeutics, investigations in, 161;
principles of, 144; literature of, 184.

Serum, bactericidal influence of, 162; cura-
tive, 165 ; — for cholera, 167; — mode
of production of, 164; — treatment,
literature of, 183, 184.

196

Sheaths, occurrence of, 10; ferruginous
modification of, 11.

Silkworm, disease of, 138.

Silver nitrate, inhibitory percentage of, 82 ;
lethal, 83.

Sinapis alba, non-fixation of N by, 89.

Skatol, absence of from cheese, 119; pro-
duction of, 98; properties of, 99.

Small-pox, vaccination for, 168.

Soda, caustic, inhibitory percentage of, 82;
inefficiency of as disinfectant, 84.
Sodium, chemotaxis of, 79; necessity of,
54; — chloride, inhibitory percentage

of, 82 ; — resistance of spores to, 84.

Sommaruga, on metabolism of yeast, 176.

South America, nitrite bacteria of, 105.

Soy, preparation of, 110.

Specific characters, of bacteria, 24, 29;
variations of due to environment,
25-28.

Spirillaceae, 34.

Spirillum, 1,343 sap vacuoles of,6; shape
of, 2; S. desulphuricans, reduction of
sulphates by, 106; S. rubrum, 12;
influence of O on pigmentation of, 61;
S. sputigenum, 1, Fig.1; S. undula, 3,
Fig. 2; 7, Fig. 5; cilia of, 15, Fig. 8;
plasmolysis of, 8, Fig.6; polar granules
of, 9; saprophile character of, 101.

Spirochaete, 2, 34; S. dentium, 139, 140,
Fig. 26; S. Obermaieri, 3, Fig. 2.

Spirulina, shape of, 37.

Spontaneous generation, 50; literature of,
171 ; — combustion, causes of, 63.
Spores, of bacteria, 19 seq.; shapes of, 20,
21, Fig.11; germination of, 20, Fig. 11,
22; influence of heat on, 128; influence
of desiccation on, 76; non-penetration
of plants by, 137; resistance of to
poisons, 83, 84; staining of, 21; litera-
ture of, 170; ubiquitous distribution of,
50; vitality of, 22; of Saccharomyces,

127, Fig. 25.

Spore formation, causes of, 23; influence of
heat on, 129, 150; of oxygen on, 150.

Spore membrane, influence on vitality of
spore, 76.

Sporozoa, parasitism of, 41.

Sporulation, taxonomic value of, 31.

Spree, bacteria of, 46.

Sputum, influence of on action of poisons,
8

3.

Stab-culture, 58; diagnostic value of, 59.

Stahl, on trophotropism, 78, 173.

Staining of bacteria, 6, 7.

Staphylococcus, size of, 4; immutability of,
25; mode of division of, 19; S. pyogenes
albus, 148; S. pyogenes aureus, 151,
Fig. 28; characters of, 148; colour of,
12; S. pyogenes citreus, 148.

Starch, fermentation of, 121.

Stenothermic, 74.

INDEX

Stereo-isomers, production of by bacteria,
115.

Sterilization, 51.

Stichococcus, 37.

Stomach, bacteria of, 140; influence of
acidity of on, 141.

Streak-culture, 59.

Streptococcus, 33; growth of in fluid media,
58; non-liquefaction of gelatine by, 58;
S. pyogenes, 151, Fig. 28; characters
of, 148; chains of, 19, Fig. Io.

Streptothrix, taxonomic position of, 41;
S. actinomyces, 41; supposed sporangia
of, 42.

Stutzer and Hartleb, on nitrifying fungi, 175.

Sublimate, inhibitory percentage of, 82;
lethal, 83.

Succinic acid, 100; nutritive value of, 57;
production of by bacteria, 114;— in
kephir, 120; — by yeast, 132.

Sugar, butyric fermentation of, 121 ; chemo-
tactic attraction of, 79; influence of on
fixation of N; nutritive value of, 57;
— as source of C, 55.

Sulphur, in bacteria, 14, 65, 66.

Sulphur-bacteria, 65, Fig. 17; literature of,
172; nutrition of, 66, 67; respiration
of, 68; saprophile character of, Io1.

Sulphur dioxide, inhibitory action of, 86.

Sulphuretted hydrogen, inhibitory action
of, 86.

Sulphuric acid, lethal percentage of, 83;
inefficiency of as disinfectant, 84.

Sunlight, germicidal action of, 71.

Suppuration, character and causes of, 147,
148,

Sycosis, causes of, 148.

Symbiosis, 93; of bacteria and carnivorous
plants, 35.

Tartaric acid, nutritive value of, 57.

Taxonomy, 24, Chap. ITI.

Teeth, bacterial decay of, 140.

Temperature, influence of an alcoholic
fermentation, 132; on cholera germs,
156; on growth of Leuconostoc, 124; —
on parasitism, 43 ; — on production of
poison, 157; — of yeast spores, 128; — of
bacterial spores, 129, 150 ; — on toxicity,
86; on urase, 179; resistance of bac-
teria to changes of, 19; literature of,
173; high, germicidal action of, 75;
resistance of spores to, 75; influence of
on toxines, 160; on putrefaction, 99;
low, resistance to, 75; maxima, dia-
gnostic value of, 112 ; optimum for acetic
fermentation, 113; relation of bacteria
to, 73-75-

Tertiary epoch, bacteria of, 161.

Tetanus, aetiology of, 181; artificial immu-
nity to, 164; cause of, 150; influence
of antitoxins on, 165; poison of, 182;

INDEX

— bacilli, presence of in soil, 147; —
toxine, 160.

Tetramitus, ciliation of, 38.

Thermogenic bacteria, 63.

Thermogenous bacteria, 172.

Thermophilic bacteria, 74; literature of, 173.

Thesium, partial parasitism of, 96.

Thiobacteria, 65.

Thiocystis, pigment in, 12.

Thiopedia, 66; division of, 19; systematic
position of, 33.

Thiospirillum, 66.

Thiothrix, 34, 66; arthrospores of, 22.

Thrush, origin of, 39.

Thymol, inhibitory percentage of, 82;
lethal, 83.

Tischutkin, on bacterial digestion, 180.

Tissues, pathogenic bacteria of, 145; de-
monstration of in, 142, 143.

Tobacco, fermentation of, 124,

Tolypothrix, 37.

Toxalbumines, 53, 160.

Toxic and antitoxic units, 166.

Toxicity, influence of mixed solutions on,
86; relationship of dissociation to, 85.

Toxines, intracellular, 160 ; preparation and
properties of, 159; poisonous character
of, 160; toleration of, 165; literature
of, 182-184.

Tradescantza, cell-division in, 18.

Traube, theory of fermentation, 134.

Traumatic infection, 150,

Trichobacteria, definition of, 2; ordinal
rank of, 31.

Trichobacteriaceae, 34.

Trichomonas intestinalis, 39; T. vaginalis,

39.

Trichophyton tonsurans, parasitism of, 42.

Trimethylamine, 100.

Trophotropism, 78, 173.

Tubercle, cause of, 152; germs of, 153;—
bacilli, growth of in milk, 117; invo-
lution forms of, 170 ; lethal percentages
of different poisons for, 81, 835 sources
of,118; in sputum, 143, Fig. 27; steno-
thermic character of, 74; vitality of,

144.

Tuberculin, composition and preparation
of, 160; literature of, 182.

Tuberculomyces, 27, 153.

Tuberculosis, aetiology of, 181; influence
of on milk, 118; transference of, 152.

Turgor, definition of, 5. “

Typhoid bacilli, 7, Fig. 5; decomposition
of fat by, 108; growth of in milk, 117,
118; plasmolysis of, 8, Fig. 6; poly-
trophism of, 29; suppuration due to,
147 ; — fever, cause of, 154; aetiology
of, 181.

Tyrosin, 99 ; occurrence of in cheese, 119 ;
production of by bacteria, 98; putre-
faction of, 100.

197

Tyrothrix, in cheese, 119; curdling of milk
by, 118.
Tyrotoxine, 100.

Urase, 178; mode of action of, 135.
Urea, fermentation of, 134; nutritive value
of, 57; — bacteria, 103; literature of,

175.
Urobacillus Schitzenbergdi, 179.
Ustilago, yeast of, 130; U. carbo, resistance
of spores of to desiccation, 79.

Vaccine lymph, 168 ; — microbe, 40.
Vacuoles in bacteria, 6; demonstration of,

7, Fig. 5. ; :

Vaillard, on hereditary immunity, 183.

Valerianic acid, 100.

Valeric acid, 100.

Van Laer, on alcoholic fermentation, 177.

Van Tieghem, on Bac. amylobacter, 177;
on fossil butyric bacteria, 182; on
Leuconostoc mesenteroides, 178.

Vegetables, mucilaginous fermentation of,
123; souring of, 120.

Verworn, on Protista, 172.

Vibrio, 1, 102, Fig. 22; characters of, 34;
shape of, 2; V. albensis, phospho-
rescence of, 63; V. derolinensis, patho-
genicity of, 156; V. duccalis, 1, Fig. 1;
139, 140, Fig. 26; V. cholerae, 3, Fig. 2;
7, Fig. 5; 151, Fig. 28; cilia of, 15,
Fig. 8; culture of, 55,156; plasmolysis
of, 8, Fig. 6; polar granules of, 9;
sensitiveness of to acid, 56; V. danu-
bicus, pathogenicity of, 156; V. rugula,
production of methane by, 121.

Vibrion butyrique of Pasteur, 121.

Vicia villosa, bacteroids of, 27, Fig. 14;
90, Fig. 19.

Vine, bacterial diseases of, 138.

Vinegar, manufacture and fermentation of,
II0, III, 113.

Virulence, definition of, 160.

Virus animatum, 142; — inanimum, 159.

Vital air, 60.

Voges, on immunity to cholera, 182.

Warington, on nitrification, 175.

Water, percentages of in plants and ani-
mals, 52; presence of bacteria in, 45;
bacteriological analysis of, 46.

Watson-Cheyne, on infection, 180; on Bac.
anthracis, 179.

Watson-Cheyne and Cheshire, on Bacillus
alvet, 180.

Weber’s Law, 80.

Wehmer, on Asfergillus yeast, 176; on
decay of fruits, 175.

Weichselbaum, on inflammation of lungs,
181.

Weizmann, on ripening of cheese, 177.

Welter, influence of cold on bacteria, 173.

198

Went and Prinsen Geerligs, on Aspergillus
yeast, 176.

Wertheim, on gonorrhoea, 181.

Wheat, minimum, optimum, and maximum
temperatures for germination of, 74;
percentage of nitrogen in, 74.

Wine, cause of sweetness of, 132; influence
of bacteria on acidity of, 114 ; lactic fer-
mentation of, 120; mucilaginousfermen-
tation of, 123; — yeasts, races of, 129.

Winogradsky, on fixation of N by soil
bacteria, 96, 174; on iron bacteria, 69,
142; nitrifying bacteria, 104, 175; —
plate-culture of, 105; retting bacteria,
177; sulphur bacteria, 65, 172.

Wittlin, 173.

Wiladimiroff, on antitoxin and tetanus
poison, 184.

Wolffhiigel, on bacteriological analysis of
water, I71.

Wollny, on humus, 175.

INDEX

Woronin, on bacteroids of root-nodules,
90; on root-nodules, 174.

Wortmann, on pure yeasts, 178.

Wounds, aseptic treatment of, 87; healing
of in plants and animals, 145; non-
dangerous character of in plants, 138.

Xanthoria parietina, 93, Fig. 20.

Yeasts, 126 seq.; colonies and spores of,
127, Fig. 25; sterilization of, 120; in-
fluence of heat on sporulation of, 129;
literature of, 178; wild varieties of,
1313 variations in, 178.

Yersin, on antiseptics, 173.

Zoogloea, formation of, 3; characters of, 4.

Zoogloea ramigera, 4, Fig. 3.

Zopf, on life cycles of bacteria, 29; on
pleomorphism in bacteria, 170.

Zymase, 179.

THE END

OXFORD:
PRINTED AT THE CLARENDON PRESS
BY HORACE HART, M.A.,
PRINTER TO THE UNIVERSITY

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